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Retrospective Study of National Danish Registry Sheds Light on Cardiotoxic Risks of Immune Checkpoint Inhibitors
Reporter: Adina Hazan, PhD
Immune checkpoint inhibitors are used to treat over 12 different types of cancers, as a way of encouraging T cells of the immune system to target and kill cancer cells. While this method has proved to be an incredibly important mechanism in fighting deadly cancers, the enhanced immune response has been documented to produce a wide array of side effects, including an increased incidence of cardiovascular events such as arrhythmias and sudden cardiac arrest. Due to the nature of the disease and treatment, most evidence of cardiotoxicity from the treatment comes from small cohorts of patients or individual case studies.
D’Souza and colleagues used four Danish healthcare registries to collect data from over 13,000 patients with malignant melanoma, and over 25,000 patients with lung cancer, starting from 2011 with the introduction of checkpoint inhibitors up until 2017. Each group included patients who received checkpoint inhibitors and those that did not, providing the basis of their retrospective study to determine the risk of developing cardiac events with treatment over a long period of time. The study was published in the European Heart Journal in December 2020.
The authors confirmed the increased risk of cardiac events with the treatment of immune checkpoint inhibitors PD1i and CTLA-4i.
The relative rate of cardiovascular deaths were increased in patients with lung cancer treated with PD1i by three-fold, and
eight times higher in patients with malignant melanoma treated with CTLA-4i.
Moreover, they show increased relative rates of arrhythmias in both diseases treated with PD1i, and malignant melanoma treated with CTLA-4i.
This was also true of the timeframe of treatment, where it was previously believed that most cardiac events would develop within the first six months of checkpoint inhibitor use.
By using data from patients over years, this study shows that these adverse events develop even after the first six months.
The myocardium, or heart muscle tissue, has previously been reported to be damaged with the use of checkpoint inhibitors, suspected to be due to
increasing inflammatory cytokines and enhanced inflammatory pathways. Significantly, previous pharmacovigilance studies have suggested a rate of developing myocarditis at less than 1%, but
data from this study shows that the 1 year risk was 1.8%, “suggesting that the risk may be higher than previously estimated”.
Even though Immune checkpoint inhibitors have been shown to be invaluable tools in the treatment of certain cancers, “the findings [in this study] urge increased awareness of cardiac events in patients receiving ICI”.
Original Research
The risk of cardiac events in patients receiving immune checkpoint inhibitors: a nationwide Danish study
The study aimed to estimate the risk of cardiac events in immune checkpoint inhibitor (ICI)-treated patients with lung cancer or malignant melanoma.
Methods and results
The study included consecutive patients with lung cancer or malignant melanoma in 2011–17 nationwide in Denmark. The main composite outcome was cardiac events (arrhythmia, peri- or myocarditis, heart failure) or cardiovascular death. Absolute risks were estimated and the association of ICI and cardiac events was analysed in multivariable Cox models. We included 25 573 patients with lung cancer. Of these, 743 were treated with programmed cell death-1 inhibitor (PD1i) and their 1-year absolute risk of cardiac events was 9.7% [95% confidence interval (CI) 6.8–12.5]. Of the 13 568 patients with malignant melanoma, 145 had PD1i and 212 had cytotoxic T-lymphocyte-associated protein-4 inhibitor (CTLA-4i) treatment. Their 1-year risks were 6.6% (1.8–11.3) and 7.5% (3.7–11.3). The hazard rates of cardiac events were higher in patients with vs. without ICI treatment. Within 6 months from 1st ICI administration, the hazard ratios were 2.14 (95% CI 1.50–3.05) in patients with lung cancer and 4.30 (1.38–13.42) and 4.93 (2.45–9.94) in patients with malignant melanoma with PD1i and CTLA-4i, respectively. After 6 months, HRs were 2.26 (1.27–4.02) for patients with lung cancer and 3.48 (1.91–6.35) for patients with malignant melanoma and CTLA-4i.
Conclusions
Among patients with lung cancer and malignant melanoma, ICI treated had increased rates of cardiac events. The absolute risks were higher in these data compared with previous pharmacovigilance studies (e.g. 1.8% peri-/myocarditis 1-year risk).
Changes in Levels of Sex Hormones and N-Terminal Pro–B-Type Natriuretic Peptide as Biomarker for Cardiovascular Diseases
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Considerable differences exist in the prevalence and manifestation of atherosclerotic cardiovascular disease (CVD) and heart failure (HF) between men and women. Premenopausal women have a lower risk of CVD and HF compared with men; however, this risk increases after menopause. Sex hormones, particularly androgens, are associated with CVD risk factors and events and have been postulated to mediate the observed sex differences in CVD.
B-type natriuretic peptides (BNPs) are secreted from cardiomyocytes in response to myocardial wall stress. BNP plays an important role in cardiovascular remodelling and volume homeostasis. It exerts numerous cardioprotective effects by promoting vasodilation, natriuresis, and ventricular relaxation and by antagonizing fibrosis and the effects of the renin-angiotensin-aldosterone system. Although the physiological role of BNP is cardioprotective, pathologically elevated N-terminal pro–BNP (NT-proBNP) levels are used clinically to indicate left ventricular hypertrophy, dysfunction, and myocardial ischemia. Higher NT-proBNP levels among individuals free of clinical CVD are associated with an increased risk of incident CVD, HF, and cardiovascular mortality.
BNP and NT-proBNP levels are higher in women than men in the general population. Several studies have proposed the use of sex- and age-specific reference ranges for BNP and NT-proBNP levels, in which reference limits are higher for women and older individuals. The etiology behind this sex difference has not been fully elucidated, but prior studies have demonstrated an association between sex hormones and NT-proBNP levels. Recent studies measuring endogenous sex hormones have suggested that androgens may play a larger role in BNP regulation by inhibiting its production.
Data were collected from a large, multiethnic community-based cohort of individuals free of CVD and HF at baseline to analyze both the cross-sectional and longitudinal associations between sex hormones [total testosterone (T), bioavailable T, freeT, dehydroepiandrosterone (DHEA), SHBG, and estradiol] and NT-proBNP, separately for women and men. It was found that a more androgenic pattern of sex hormones was independently associated with lower NT-proBNP levels in cross-sectional analyses in men and postmenopausal women.
This association may help explain sex differences in the distribution of NT-proBNP and may contribute to the NP deficiency in men relative to women. In longitudinal analyses, a more androgenic pattern of sex hormones was associated with a greater increase in NT-proBNP levels in both sexes, with a more robust association among women. This relationship may reflect a mechanism for the increased risk of CVD and HF seen in women after menopause.
Additional research is needed to further explore whether longitudinal changes in NT-proBNP levels seen in our study are correlated with longitudinal changes in sex hormones. The impact of menopause on changes in NT-proBNP levels over time should also be explored. Furthermore, future studies should aim to determine whether sex hormones directly play a role in biological pathways of BNP synthesis and clearance in a causal fashion. Lastly, the dual role of NTproBNP as both
a cardioprotective hormone and
a biomarker of CVD and HF, as well as
the role of sex hormones in delineating these processes,
should be further explored. This would provide a step toward improved clinical CVD risk stratification and prognostication based on
The presence of any Valvular Heart Disease (VHD) did not influence the comparison of Dabigatran [Pradaxa, Boehringer Ingelheim] with Warfarin
Reporter: Aviva Lev-Ari, PhD, RN
UPDATED on 10/22/2018
Dabigatran (Pradaxa) was no better than aspirin for prevention of recurrent stroke among patients with an embolic stroke of undetermined source in the RE-SPECT ESUS trial reported at the World Stroke Congress.
Pradaxa® (dabigatran etexilate)
Clinical experience of Pradaxa® equates to over 9 million patient-years in all licensed indications worldwide. Pradaxa® has been in the market for more than ten years and is approved in over 100 countries.15
Currently approved indications for Pradaxa® are:16,17
Prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation and a risk factor for stroke
Primary prevention of venous thromboembolic events in patients undergoing elective total hip replacement surgery or total knee replacement surgery
Treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE) and the prevention of recurrent DVT and recurrent PE in adults
Dabigatran, a direct thrombin inhibitor (DTI), was the first widely approved drug in a new generation of direct oral anticoagulants, available to target a high unmet medical need in the prevention and treatment of acute and chronic thromboembolic diseases.18,19,20
REFERENCES
16 Pradaxa® US Prescribing Information, 2018.
17 Pradaxa® European Summary of Product Characteristics, 2018.
18 Stangier J. Clinical pharmacokinetics and pharmacodynamics of the oral direct thrombin inhibitor dabigatran etexilate. Clin Pharmacokinet. 2008;47(5):285–95.
19 Di Nisio M. et al. Direct thrombin inhibitors. N Engl J Med.2005;353:1028–40.
20 Stangier J. et al. Pharmacokinetic Profile of the Oral Direct Thrombin Inhibitor Dabigatran Etexilate in Healthy Volunteers and Patients Undergoing Total Hip Replacement. J Clin Pharmacol. 2005;45:555–63
Event Rate and Outcome Risk, With vs Without Valvular Heart Disease
Outcome
Valvular heart disease, event rate/y, %
No valvular heart disease, event rate/y, %
HR (95% CI)*
P
Stroke, systemic embolic event
1.61
1.41
1.09 (0.88–1.33)
0.43
Major bleeding
4.36
2.84
1.32 (1.16–1.33)
<0.001
Intracranial hemorrhage
0.51
0.41
1.20 (0.83–1.74)
0.32
All-cause mortality
4.45
3.67
1.09 (0.96–1.23)
0.18
*Adjusted using propensity scores
ORIGINAL RESEARCH ARTICLE
Comparison of Dabigatran versus Warfarin in Patients with Atrial Fibrillation and Valvular Heart Disease: The RE-LY Trial
Michael D. Ezekowitz, Rangadham Nagarakanti, Herbert Noack, Martina Brueckmann, Claire Litherland, Mark Jacobs, Andreas Clemens,Paul A. Reilly, Stuart J. Connolly, Salim Yusuf and Lars Wallentin
stroke or systemic embolism (SEE) rates (HR 1.09; 95% CI 0.88-1.33).
For D110 patients, major bleed rates were lower than warfarin (HR 0.73; 95% CI 0.56-0.95 with and HR 0.84; 95% CI 0.71-0.99 without VHD) and
For D150 similar to warfarin in patients with (HR 0.82; 95% CI 0.64-1.06) or without VHD (HR 0.98; 95% CI 0.83-1.15).
For D150 patients stroke/SEE rates were lower versus warfarin with (HR 0.59; 95% CI 0.37-0.93) and without VHD (HR 0.67; 95% CI 0.52-0.86) and similar to warfarin for D110 irrespective of presence of VHD (HR 0.97 CI 0.65-1.45 and 0.85 CI 0.70-1.10).
For intracranial bleeds and death rates for D150 and D110 were lower vs warfarin independent of presence of VHD.
Conclusions—The presence of any VHD did not influence the comparison of dabigatran with warfarin.
Patient was diagnosed with heart disease and pulmonary hypertension in January 2016 and had a triple-bypass operation at age 69. Interview was conducted six months post-surgery.
Author: Gail S. Thornton, M.A.
Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures
Evergreen, Colorado, an idyllic, peaceful community with an elevation of 8,000 feet west of Denver, offers its residents and visitors a beautiful place for arts and culture, summer and winter sporting activities, and scenic beauty. In fact, Ralph Nichols has lived in the town for more than 20 years.
“This past September [2015] was, particularly, challenging for me, where winter begins quite early for us. It became increasingly painful and difficult to breathe in the freezing temperatures. It seemed that my lungs were inflamed and I couldn’t even stand the cold weather. I thought it might be the beginning of a bad cold, and I wasn’t overly concerned that there was anything terribly wrong.”
At that time, Ralph went to his family physician who performed the usual routine examination with no significant results.
“Many years ago, I developed a mild case of scleroderma, a chronic connective tissue disease. I thought that perhaps my symptoms were the result of some type of inflammation in my body that could be managed with prescription medications.”
Scleroderma is known as an autoimmune disease, which adds an inappropriate amount of collagen to various parts of the body, such as the joints, skin, and later stages, various organs, such as the lungs, in Ralph’s case. Scleroderma can cause the organs to shut down and, eventually, cause death.
“I never let this condition stop me from doing anything as it is life-long condition. It was always something I had to tolerate and work through.”
Image SOURCE: Photographs courtesy of Ralph Nichols and Gabriela Contreras. Top left: Ralph today. Top right: Ralph recovering one month after surgery. Bottom left and center: Ralph with his medical team. Bottom right: Ralph in rehabilitation center.
Over the brutal Colorado winter, Ralph’s symptoms were getting worse. He had no idea that his life would dramatically change over the next few months. He went to see his family physician again. During this physical examination, Ralph was referred to pulmonary and cardiovascular specialists for a routine electrocardiogram, echocardiogram and stress test in order to further diagnose his symptoms. He had always been relatively healthy and fit and never been seriously ill or hospitalized.
“On the outside, Ralph was the picture of good health,” said his wife, Gabriela. “On the inside, his body was telling him that something was wrong.”
Three months later in December 2015, Ralph met with Dr. Alexandra Smart, a pulmonologist, who ordered a chest x-ray and other diagnostic tests, including a right heart catheterization. At that point, Ralph’s medical team grew. It was then determined that Ralph needed to see other cardiovascular specialists and undergo more tests. In January 2016, he met with Dr. Sameer Mehta, cardiologist at Cardiac & Thoracic Surgery Associates, in Lakewood, Colorado, who reviewed his tests to date, listened to Ralph’s symptoms, and told him he needed both a right and left heart cardiac catheterization.
“They gave me sedation for the catheterization procedure and went through my neck with a camera to see what was going on with my lungs and heart. We were all singing together on the way to the operating room. During the procedure, my cardiologist found more than he had anticipated.”
The result was not good. Ralph had major blockages in two main arteries that supply blood to his heart muscle compounded by the fact that his lungs were affected by scleroderma.
“The catheterization was alarming. It showed that my arteries were in bad shape. They were both clogged with atherosclerotic plaque; one of them was 99 percent blocked and the other was 85 percent blocked.”
His cardiologist believed that the blockages would not respond to medications quickly or a stent.
“Even though my father had major heart disease and died two years later of cancer at the age of 56, I thought that I would be immune to this particular experience. After all, I was in good health, exercised regularly, lived a reasonable lifestyle and had a great diet.”
Preparing for Life-Saving and Life-Changing Surgery
Unfortunately, surgery was the next step. Ralph was referred to Dr. Mehta’s colleague, Dr. Patrick D. Rudersdorf, cardiothoracic surgeon at Cardiac & Thoracic Surgery Associates.
“I didn’t leave the hospital that day as expected. Instead, I got a visit from Dr. Rudersdorf and couldn’t believe what he was telling me. My only chance to live was having triple bypass surgery which needed to be done immediately. The doctor met with me that same day to explain the procedure, answer my questions and talk through the details of the rehabilitation period after the surgery.”
Dr. Rudersdorf reassured Ralph that he was doing the right thing and calmed my fears.
“He said that I needed this life-saving surgery because I was at high risk for having a major heart attack. I was shocked, at first, at the thought of the intensity of surgery on my body. It’s a situation that no one likes to be in, but I had to make a decision about alleviating the ongoing pain and pressure in my chest along with shortness of breath due to diseased heart arteries. Coronary bypass surgery was my answer to feeling better — and it essentially gave me my life back.”
Dr. Rudersdorf moved his previously planned morning surgery to another day to accommodate me first thing in the morning. Ralph underwent triple bypass surgery at St. Anthony Hospital in Lakewood, Colorado. The procedure was complex and took eight hours. He was in the hospital for a total of 31 days.
“It was an ordeal that I thought I’d never have to experience. I had no time to call anyone, or time to even contemplate life and death…or even being scared. My wife Gabriela spent the entire time in the hospital, supported by our dearest friends, Norma Delaney and Garret Annofsky, in addition to keeping family and friends in other parts of the United States and Mexico updated as well. Once the surgery was over, the medical team woke me up and said the procedure was successful, but I was far from being out of the woods.”
Ralph had some complications because of a condition called pulmonary hypertension, a type of high blood pressure that affects the arteries in the lungs and the right side of the heart. According to the Mayo Clinic’s web site, in one form of pulmonary hypertension, tiny arteries in the lungs, called pulmonary arterioles, and capillaries become narrowed, blocked or destroyed. This makes it harder for blood to flow through the lungs, and raises pressure within the lungs’ arteries. As the pressure builds, the heart’s lower right chamber (right ventricle) must work harder to pump blood through the lungs, eventually causing the heart muscle to weaken and fail. http://www.mayoclinic.org/diseases-conditions/pulmonary-hypertension/home/ovc-20197480
“The pulmonary hypertension limited some of the medications that the doctors would have used during my recovery. It was a tough few days for me in intensive care, hooked up to about 18 monitors. The medical team had to stop and re-start my heart four different times because of atrial fibrillation — finally getting both parts of the heart to dance together in the same rhythm.”
Ralph’s heart was beating abnormally fast and irregular and not functioning the way it should. The doctors restore regular rhythm to the heart by sending an electrical shock to the heart, which is called electrical cardioversion or chemically using antiarrhythmia medications, which is called pharmacologic or chemical cardioversion.
“The doctors shocked my heart first chemically with medications when I was awake. This procedure was the scariest. I was sitting up in bed and felt my heart stop, then the medical team flushed the medication out with saline in order to restart my heart. That procedure was not successful, so that is why the doctors had to shock my heart three more times electrically.
“The reason the doctors stopped my heart was to correct the atrial fibrillation and to get my heart into regular sinus rhythm, which is a wave mode of the heart where everything is synchronized. The doctors did not want me to continue to experience atrial fibrillation because if continued, I would not be able to regain my strength.”
Ralph was finally moved from intensive care to intermediate care after five days and the medical team kept him in intermediate care another 12 days until his heart and lungs got stronger.
“From there, I didn’t go home but instead went to Evergreen Life Center for rehabilitation for two weeks to learn how to walk, climb stairs so that I could access my home on my own, and develop my strength again. The rehab team would let me leave only after making sure I had oxygen in my home.”
After that, Ralph started another phase of his rehabilitation at St. Anthony Cardiac Rehabilitation and Wellness Center. For the next three months, he took part in cardiac rehabilitation three days a week. He passed that with flying colors. Now, he is in another phase of rehabilitation, building his lung capacity two days a week.
Ralph didn’t have the means or even the will to communicate with friends during this tumultuous time, except Gabriela and several close friends who were always at the hospital and rehabilitation center who gave him the strength to continue.
“I finally returned home after many weeks with an enormous feeling of gratitude for each and every one of my friends, as well as the St. Anthony’s hospital team of doctors, nurses, and therapists, who supported me and Gabriela during this exceptional adventure that has certainly changed my life.”
Surely, this experience has been a life-changing experience for Ralph.
Coronary Artery Bypass Facts
Coronary artery bypass grafting (CABG, often pronounced “cabbage”) is a surgical treatment for blocked coronary arteries. Coronary arteries supply blood to the heart muscle and when blockages in these arteries form, chest pain, shortness of breath and heart attacks can occur. Catheter procedures performed by interventional cardiologists address the blockages themselves with stents. Coronary bypass surgery performed by cardiac surgeons reroutes the blood around the blockages to supply better blood supply to the heart muscle and is a better treatment option, although more invasive, for certain patients and more durable for most patients.
Today, Ralph is regaining his strength both in mind and body. He visits the cardiovascular and pulmonary rehabilitation center three times a week for the past few months and walks on their treadmill, lifts weights and pedals the bicycle for one hour, supervised by the therapists. He also sees his medical team for regular check-ups every month, eats healthier with no fat and no salt, and takes a cocktail of medicines daily for his heart and lungs, including amiodarone, furosemide, pitavastatin, and aspirin.
“Almost six months after my surgery, although I am not in the best shape of my life, however, I am in the best spiritual place than ever before. This is a huge milestone for me. I continue to improve my strength, which will make my heart more resilient. There is nothing that I can’t do now, and I am doing everything I can to experience a normal life as far as work and regaining my strength. I find it necessary to move to a warmer climate and lower altitude in order to continue to improve.”
Ralph also is the former lead singer of The Letterman and The Sandpipers, two American easy-listening bands during the 1960-70-80s. He is an entertainer at heart with over 3,000 professional appearances to his credit. He has been performing and recording for over 50 years, traveled the world extensively and performed before members of the Vatican with Pope Pius XII and Royalty with Prince Rainier and Princess Grace Kelly, as well as notables such as Frank and Nancy Sinatra, Tony Bennett, Ronald Reagan, Merv Griffin, Danny Thomas, Shirley Bassey, Rosalind Russell and Bob Hope.
Ralph and his vocal group were dubbed by Billboard Magazine as “the greatest romantic vocal group of all time.” He is also a member of the Vocal Group Hall of Fame, a prestigious honor. He is a true legend as his group has sold more than 20 million recordings, performed live thousands of times, and whose recording of the song “Love” was left by NASA astronauts in a time capsule on the moon.
“I enjoy each and every day and appreciate all that life has to offer.”
Ralph’s next step is to get back to singing and his solo entertainment business, which he holds dear to his heart. That should be a task that he can easily accomplish.
Editor’s note:
We would like to thank Gabriela Contreras, a global communications consultant and patient advocate, for the tremendous help and support that she provided in scheduling time to talk with Ralph Nichols.
Ralph Nichols provided his permission to publish this interview on July 30, 2016.
No evidence to change current transfusion practices for adults undergoing complex cardiac surgery: RECESS evaluated 1,098 cardiac surgery patients received red blood cell units stored for short or long periods
I wish to encourage the e-Reader of this Interview to consider reading and comparing the experiences of other Open Heart Surgery Patients, voicing their private-life episodes in the ER that are included in this volume.
I also wish to encourage the e-Reader to consider, if interested, reviewing additional e-Books on Cardiovascular Diseases from the same Publisher, Leaders in Pharmaceutical Business Intelligence (LPBI) Group, on Amazon.com.
Perspectives on Nitric Oxide in Disease Mechanisms, on Amazon since 6/2/12013
Cardiovascular Diseases, Volume Four: Regenerative and Translational Medicine: The Therapeutics Promise for Cardiovascular Diseases, on Amazon since 12/26/2015
ORLANDO, FL — Patients with type 2 diabetes and established CVD who received the antidiabetic sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (Jardiance, Lilly/Boehringer Ingelheim), as opposed to placebo, had a reduced risk of being hospitalized for heart failure or dying from CVD during a median follow-up of 3.1 years. The finding was strongest in patients without heart failure at baseline[1]. The finding is noteworthy in part because associated heart failure has been a concern, justified or not, with some other diabetes medications.
In these high-risk patients, empagliflozin resulted in a “consistent benefit” in these outcomes, Dr Silvio E Inzucchi (Yale University School of Medicine, New Haven, CT) said, presenting these findings from a prespecified secondary analysis of the EMPA-REG OUTCOME trial at theAmerican Heart Association (AHA) 2015 Scientific Sessions.
Unlike the gasps and applause that greeted him when he presented the trial’s primary outcome results at the European Association for the Study of Diabetes (EASD) 2015 Meeting in Stockholm in mid-September, the audience reaction this time was more measured. The trial had also been published at about the time of its EASD presentation [2].
The principal findings showed that compared with patients who took placebo, those who were randomized to empagliflozin had a 38% (P<0.001) reduced risk of CV death and a 35% P=0.002) reduced risk of hospitalization for HF, at a median follow-up of 3.1 years.
In the current secondary analysis, the 90% of patients who were free of heart failure at study entry showed a steep and significant drop in HF hospitalizations during the trial. There was also a drop in HF hospitalizations with active therapy in the minority who had HF at baseline, but it failed to reach significance.
“I think metformin is likely to remain our first-line oral therapy for patients with type 2 diabetes,” Dr Donald M Lloyd-Jones (Northwestern University Feinberg School of Medicine, Chicago, IL), cochair at an AHA press briefing, told heartwirefrom Medscape. “There is an alphabet soup of diabetes medications,” with multiple agents that effectively lower blood glucose and reduce patients’ risk of retinopathy, nephropathy, and neuropathy.
However, “it was . . . unexpected that [empagliflozin], as reported recently [at the EASD meeting and] in the New England Journal of Medicine [has an] effect on CV death and other CV events.” This is still an early stage of research, he cautioned, and it is not known how the drug exerts its CV effects and whether there is a class effect. “But [this] could be a game changer, because we would love to have [antidiabetic] medications that not only control blood sugar but also reduce death and [other] hard events,” he said.
First CV Outcomes Trial in this Drug Class
Until now, none of the antiglycemic medications has also been shown to improve HF outcomes, Inzucchi explained. “We’ve actually been searching decades for a diabetes medicine that will not only lower blood glucose but also reduce cardiovascular complications,” he said in a press briefing. “And I would remind you that based on the 2008 FDA guidance to industry, all new diabetes medications need to be tested for cardiovascular safety before being allowed on the market,” he added.
EMPA-REG OUTCOME is the first published, large CV-outcome trial of an SGLT-2 inhibitor.
As previously described, the trial randomized 7028 adult patients who had type 2 diabetes and established CVD to receive 10 mg/day or 25 mg/day empagliflozin or placebo. The CVD included prior MI (46.6%), CABG (24.8%), stroke (23.3%), and peripheral artery disease (PAD) (20.8%).
The patients were also required to have an HbA1c level between 7% and 10%, body-mass index (BMI) <45, and, because the drug exerts its effects via the kidney, estimated glomerular filtration rate (eGFR) >30 mL/min/1.73 m2.
“Importantly, study medication was given upon a backdrop of standard care—antihyperglycemia therapy, as well as other evidence-based cardiovascular therapies such as statins, ACE inhibitors, and aspirin,” Inzucchi stressed.
Spotlight on HF Outcomes
The current analysis dove deeper into the heart-failure outcomes in the trial.
The risk of hospitalization for HF or CV death was consistently significantly lower in patients who received empagliflozin vs placebo, in subgroup analyses related to age, kidney function, and medication use (ACE inhibitors/angiotensin receptor blockers [ARBs], diuretics, beta-blockers, or mineralocorticoid-receptor antagonists).
Overall, the patients who received empagliflozin had a 34% reduced risk of being hospitalized for HF or dying from CV causes and a 39% reduced risk of being hospitalized for or dying from HF.
Risk of Hospitalization or Death, Empagliflozin vs Placebo
Outcome
HR (95% CI)
P
Hospitalization for HF or CV death
0.66 (0.55–0.79)
<0.00001
Hospitalization for or death from HF
0.61 (0.47–0.79)
<0.00001
Most patients (90%) did not have HF at baseline.
In the patients without HF at baseline, “as you might expect, [HF] hospitalizations were relatively small in number” (1.8% of patients on the study drug and 3.1% of patients on placebo), said Inzucchi. There was a statistically significant 41% reduced risk of HF hospitalization in patients without HF at baseline on the study drug vs placebo (HR 0.59, 95% CI 0.43–0.82).
In the smaller number of patients who did have HF at baseline, the rate of hospitalizations for HF was much higher (10.4% of patients on the study drug and 12.3% of patients on placebo). But in this case, the difference between patients on the study drug vs placebo was not statistically significant (HR 0.75, 95% CI 0.48–1.19).
The results were similar when the analysis was repeated for the combined outcome of hospitalization for HF or CV death.
“Not surprisingly,” adverse events were more common in sicker patients with baseline HF; genital infections, a well-known adverse event in drugs that increase glucose in the urine, were three times more common in those patients, said Inzucchi.
“I think these are very compelling data, but early days,” said Lloyd-Jones.
Inzucchi receives research grants from Genzyme and honoraria from Boehringer Ingelheim, Merck Sharp & Dome, Sanofi, Amgen, and Genzyme, and he is a consultant on advisory boards for Boehringer Ingelheim, Sanofi, and Amgen. Disclosures for the coauthors are listed in the abstract. Lloyd-Jones has no relevant financial relationships.
The basic functioning of the heart, and the kidney have been covered in depth elsewhere, and pulmonary function less, except in this series. The relationship between them on the basis of endocrine, signaling, and metabolic balance is the focus in this piece.
Icariin attenuates angiotensin II‑induced hypertrophy and apoptosis in H9c2 cardiomyocytes by inhibiting reactive oxygen species‑dependent JNK and p38 pathways
H Zhou, Y Yuan, Y Liu, Wei Deng, Jing Zong, Zhou‑Yan Bian, Jia Dai and Qi‑Zhu Tang
Exper and Therapeutic Med 7: 1116-1122, 2014 http://dx.doi.org:/10.3892/etm.2014.1598
Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)‑induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart‑derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II‑induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B‑type natriuretic peptide, in a concentration‑dependent manner. The cell surface area of Ang II‑treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II‑induced cell apoptosis and protein expression levels of Bax and cleaved‑caspase 3, while the expression of Bcl‑2 was increased by icariin. In addition, 2′,7’‑dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38 in Ang II‑treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II‑induced hypertrophy and apoptosis by inhibiting the ROS‑dependent JNK and p38 pathways.
Short-term add-on therapy with angiotensin receptor blocker for end-stage inotrope-dependent heart failure patients: B-type natriuretic peptide reduction in a randomized clinical trial
OBJECTIVE: We aimed to evaluate angiotensin receptor blocker add-on therapy in patients with low cardiac output during decompensated heart failure. METHODS: We selected patients with decompensated heart failure, low cardiac output, dobutamine dependence, and an ejection fraction ,0.45 who were receiving an angiotensin-converting enzyme inhibitor. The patients were randomized to losartan or placebo and underwent invasive hemodynamic and B-type natriuretic peptide measurements at baseline and on the seventh day after intervention. ClinicalTrials.gov: NCT01857999. RESULTS: We studied 10 patients in the losartan group and 11 patients in the placebo group. The patient characteristics were as follows: age 52.7 years, ejection fraction 21.3%, dobutamine infusion 8.5 mcg/kg.min, indexed systemic vascular resistance 1918.0 dynes.sec/cm5.m2, cardiac index 2.8 L/min.m2, and B-type natriuretic peptide 1,403 pg/mL. After 7 days of intervention, there was a 37.4% reduction in the B-type natriuretic peptide levels in the losartan group compared with an 11.9% increase in the placebo group (mean difference, – 49.1%; 95% confidence interval: -88.1 to -9.8%, p = 0.018). No significant difference was observed in the hemodynamic measurements. CONCLUSION: Short-term add-on therapy with losartan reduced B-type natriuretic peptide levels in patients hospitalized for decompensated severe heart failure and low cardiac output with inotrope dependence.
Development of a Novel Heart Failure Risk Tool: The Barcelona Bio-Heart Failure Risk Calculator (BCN Bio-HF Calculator)
Background: A combination of clinical and routine laboratory data with biomarkers reflecting different pathophysiological pathways may help to refine risk stratification in heart failure (HF). A novel calculator (BCN Bio-HF calculator) incorporating N-terminal pro B-type natriuretic peptide (NT-proBNP, a marker of myocardial stretch), high-sensitivity cardiac troponin T (hs-cTnT, a marker of myocyte injury), and high-sensitivity soluble ST2 (ST2), (reflective of myocardial fibrosis and remodeling) was developed. Methods: Model performance was evaluated using discrimination, calibration, and reclassi-fication tools for 1-, 2-, and 3-year mortality. Ten-fold cross-validation with 1000 bootstrapping was used. Results: The BCN Bio-HF calculator was derived from 864 consecutive outpatients (72% men) with mean age 68.2612 years (73%/27% New York Heart Association (NYHA) class I-II/III-IV, LVEF 36%, ischemic etiology 52.2%) and followed for a median of 3.4 years (305 deaths). After an initial evaluation of 23 variables, eight independent models were developed. The variables included in these models were age, sex, NYHA functional class, left ventricular ejection fraction, serum sodium, estimated glomerular filtration rate, hemoglobin, loop diuretic dose, β-blocker, Angiotensin converting enzyme inhibitor/Angiotensin-2 receptor blocker and statin treatments, and hs-cTnT, ST2, and NT-proBNP levels. The calculator may run with the availability of none, one, two, or the three biomarkers. The calculated risk of death was significantly changed by additive biomarker data. The average C-statistic in cross-validation analysis was 0.79. Conclusions: A new HF risk-calculator that incorporates available biomarkers reflecting different pathophysiological pathways better allowed individual prediction of death at 1, 2, and 3 years.
TNF and angiotensin type 1 receptors interact in the brain control of blood pressure in heart failure
Accumulating evidence suggests that the brain renin-angiotensin system and proinflammatory cytokines, such as TNF-α, play a key role in the neuro-hormonal activation in chronic heart failure (HF). In this study we tested the involvement of TNF-α and angiotensin type 1 receptors (AT1Rs) in the central control of the cardiovascular system in HF rats. Methods: we carried out the study on male Sprague–Dawley rats subjected to the left coronary artery ligation (HF rats) or to sham surgery (sham-operated rats). The rats were pretreated for four weeks with intracerebroventricular (ICV) infusion of either saline (0.25 µl/h) or TNF-α inhibitor etanercept (0.25 µg/0.25 µl/h). At the end of the pretreatment period, we measured mean arterial blood pressure (MABP) and heart rate (HR) at baseline and during 60 min of ICV administration of either saline (5 µl/h) or AT1Rs antagonist losartan (10 µg/5 µl/h). After the experiments, we measured the left ventricle end-diastolic pressure (LVEDP) and the size of myocardial scar. Results: MABP and HR of sham-operated and HF rats were not affected by pretreatments with etanercept or saline alone. In sham-operated rats the ICV infusion of losartan did not affect MABP either in saline or in etanercept pretreated rats. In contrast, in HF rats the ICV infusion of losartan significantly decreased MABP in rats pretreated with saline, but not in those pretreated with etanercept. LVEDP was significantly elevated in HF rats but not in sham-operated ones. Surface of the infarct scar exceeded 30% of the left ventricle in HF groups, whereas sham-operated rats did not manifest evidence of cardiac scarring. Conclusions: our study provides evidence that in rats with post-infarction heart failure the regulation of blood pressure by AT1Rs depends on centrally acting endogenous TNF-α.
Statins in heart failure—With preserved and reduced ejection fraction. An update
HMG-CoA reductase inhibitors or statins beyond their lipid lowering properties and mevalonate inhibition exert also their actions through a multiplicity of mechanisms. In heart failure (HF) the inhibition of isoprenoid intermediates and small GTPases, which control cellular function such as cell shape, secretion and proliferation, is of clinical significance. Statins share also the peroxisome proliferator-activated receptor pathway and inactivate extracellular-signal-regulated kinase phosphorylation suppressing inflammatory cascade. By down-regulating Rho/Rho kinase signaling pathways, statins increase the stability of eNOS mRNA and induce activation of eNOS through phosphatidylinositol 3-kinase/Akt/eNOS pathway restoring endothelial function. Statins change also myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity and inhibit sympathetic nerve activity suppressing arrhythmogenesis. Less documented evidence proposes also that statins have antihypertrophic effects – through p21ras/mitogen activated protein kinase pathway – which modulate synthesis of matrix metalloproteinases and procollagen 1 expression affecting interstitial fibrosis and diastolic dysfunction. Clinical studies have partly confirmed the experimental findings and despite current guidelines new evidence supports the notion that statins can be beneficial in some cases of HF. In subjects with diastolic HF, moderately impaired systolic function, low B-type natriuretic peptide levels, exacerbated inflammatory response and mild interstitial fibrosis evidence supports that statins can favorably affect the outcome. Under the lights of this evidence in this review article we discuss the current knowledge on the mechanisms of statins’ actions and we link current experimental and clinical data to further understand the possible impact of statins’ treatment on HF syndrome.
Since 1980 when the first 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor or statin was introduced in clinical practice, statins have been extensively used in the treatment of patients with dyslipidemia as well as of those with coronary artery disease (CAD). Importantly, large scale trials and metanalysis have documented their significant benefits in terms of primary and secondary CAD prevention which out-weigh any potential side effects. Statins’ benefits extend, according to recent studies, even in patients with normal or low cholesterol levels and beyond their lipid lowering effects, indicating their multiple protective mechanisms.
Heart failure (HF) is a complex syndrome with different definitions and its diagnosis is based on a combination of symptoms, clinical signs and imaging or laboratory data. different categorization schemes have been used dividing HF in acute or chronic, in systolic or diastolic, and in ischemic or dilated simply reflecting the complexity of the syndrome and the multiplicity of the pathophysiologic mechanisms implicated in the disease development and progression. In addition to the diverse pathophysiology of HF the syndrome is also characterized by high morbidity and mortality. Recent treatment advantages such as angiotensin converting enzyme inhibitors and beta blockers have not yet proven their clinical benefit in subjects with diastolic HF.
As the most common cause of HF is CAD and statins have proven their benefits in a wide spectrum of diseases directly or indirectly associated with atherosclerotic cardiovascular disease, HMG-CoA reductase inhibitors have been tested in subjects with HF. Interestingly, non-randomized, observational and retrospective early studies in subjects with HF of ischemic and non-ischemic etiology have suggested that statins are associated with improved outcomes. Thereafter, two large scale randomized control trials failed to demonstrate any benefits in mortality of HF patients treated with rosuvastatin and subsequently current HF guidelines do not include recommendations for statin use except from when they are indicated for comorbidities, such as established CAD.
Statins inhibit HMG-CoA reductase. This enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to L-mevalonic acid, which is the rate-limiting step in the cholesterol synthesis pathway. Inhibition of the mevalonate pathway and of cholesterol synthesis triggers an increase in LDL receptor activity by stimulating production of mRNA for LDL receptor in liver. The induction of LDL receptors is responsible for the observed increase in plasma clearance of LDL cholesterol. CAD is the cause of approximately two-thirds of cases of systolic HF. The beneficial effects of statins-induced LDL reduction are well established in patients with atherosclerosis and CAD. Nevertheless, the results from statin treatment, even in ischemic HF cases, are not straightforward and several mechanisms have been proposed for this paradox.
multiplicity of HMG CoA reductase inhibitors mechanisms and their effects
The figure demonstrates the multiplicity of HMG CoA reductase inhibitors mechanisms and their effects. ↓: decrease; ↑ increase; FPP: farnesyl pyrophosphate: GGPP: geranylgeranyl pyrophosphate; Ras, Rac, Rho; small GTPases; eNOS: endothelial nitric oxide synthase; ATP: adenosine triphosphate; PI-3 kinase: phosphatidylinositol 3-kinase; AMPK: AMP activated protein kinase; GTP: Guanosine triphosphate; NADPH: Nicotinamide adenine dinucleotide phosphate; ERK: extracellular-signal-regulated kinase; Shadow box represents adverse mechanism and actions of HGM CoA reductase inhibitors.
The anti-inflammatory effects of HMG CoA reductase inhibitors in atherosclerosis have been early recognized. Statins also have a potent anti-inflammatory effect in HF models. Importantly, there is a link between inflammation and HF pathogenesis and is now widely accepted that pro-inflammatory cytokines cause systolic dysfunction, myocardial hypertrophy, activate a fetal gene program in cardiac myocytes, disturb extracellular matrix structure, cause cardiac cachexia etc. In addition, data from the Vesnarinone trial (VEST) in 384 patients with HF demonstrate a decline in survival with increasing TNFα levels confirming the notion that circulating cytokines are associated with adverse prognosis of HF patients.
The proposed, by the aforementioned mechanisms, anti-inflammatory effects of statins have been confirmed experimentally. Indeed, in a rat HF model with preserved ejection fraction (EF), treatment with rosuvastatin resulted in a significant additional improvement in HF and cardiac remodeling, partly due to decreased myocardial inflammation. In rats after acute myocardial infarction simvastatin treatment for 4 weeks beneficially modified the levels of TNFα, interleukin (IL)-1, 6 and 10 in the infarct regions. Importantly, in 446 patients with systolic HF, followed up for a period of 24 months, statins’ treatment was associated with a decrease in serum levels of C-reactive protein (CRP), IL-6 and tumor necrosis factor-alpha receptor II. Recently, in a randomized study of 22 subjects with ischemic HF short term atorvastatin treatment achieved a significant decrease in serum levels of intracellular adhesion molecule-1.
Taken together we can conclude that HMG CoA reductase inhibitors can modify inflammatory status by modulation of PRAP and ERK pathways by down regulating Toll like receptor 4 mRNA expressions and LDL oxidation and by reducing soluble lipoprotein-associated phospholipase A2 mass and activity. Importantly, the theoretical anti-inflammatory properties were confirmed in experimental and clinical HF models.
Endothelial dysfunction contributes to the pathogenesis of HF and can enhance adverse left ventricle (LV) remodeling and increase afterload in subjects with HF. Interestingly, statins have been constantly associated with improved endothelial function in subjects with a variety of cardiovascular diseases. Endothelium derived nitric oxide (NO) is an important determinant of endothelial function and HMG-CoA reductase inhibitors can up regulate endothelial NO synthase (eNOS) by different mechanisms.
Statins induce down regulation of Rho/Rho kinase signaling pathways, increasing the stability of eNOS mRNA and its expression . In addition, in human endothelial cells the Rho-kinase inhibitor, hydroxyfasudil leads to the activation of the phosphatidylinositol 3-kinase/Akt/eNOS pathway. Statins also induce activation of eNOS through the rapid activation of the serine–threonine protein kinase Akt. The beneficial effects of Akt activation are not limited to eNOS phoshorylation but extend to the promotion of new blood vessels growth. HMG CoA reductase inhibitors can further affect endothelial function through their effect on caveolin-1. Caveolin-1 binds to eNOS inhibiting NO production. Incubation of endothelial cells with atorvastatin promotes NO production by decreasing caveolin-1 expression, regardless of the level of extracellular LDL-cholesterol. These effects were reversed with mevalonate highlighting the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.
Although the experimentally confirmed benefits of HMG CoA reductase inhibitors in diastolic dysfunction and left ventricle stiffness, few data exist concerning the underlying mechanisms. As diastolic dysfunction precedes myocardial hypertrophy the anti-hypertrophic pathways mentioned in the previous section (inhibition of RhoA/Ras/ERK, PRAPγ pathways, inhibition of a large G(h) protein-coupled pathway etc.), may also contribute to the restoration of diastolic function. Moreover, in angiotensin II induced diastolic dysfunction in hypertensive mice, pravastatin not only improved diastolic function but also down-regulated collagen I, transforming growth factor-beta, matrix metalloproteinases (MMPs)-2 and -3, atrial natriuretic factor, IL-6 TNFα, Rho kinase 1 gene expression, and upregulated eNOS gene expression. These findings suggest the potential involvement of Rho kinase 1 in the beneficial effects of pravastatin in diastolic HF. Taken together data suggest that HMG CoA reductase inhibitors might be beneficial in patients with diastolic HF, a hypothesis that remains to be confirmed by clinical studies. Nevertheless, mechanistic studies have not fully explored the pathways affecting diastolic function and most data until now are indirect. Therefore efforts should be focus on the underline mechanisms affecting collagen synthesis, MMPs activity extracellular matrix synthesis and overall diastolic function in HF subjects under statin treatment.
Statins through inhibition of small GTPases can modulate MMPs activity in several cell types such as endothelial cells and human macrophages. In rat and human cardiac fibroblasts, stimulated with either transforming growth factor β1 or angiotensin II, atorvastatin reduced collagen synthesis and α1-procollagen mRNA as well as gene expression of the profibrotic peptide connective tissue growth factor 4. This antifibrotic action may contribute to the anti-remodelling effect of statins. In mouse cardiac fibroblasts treated with angiotensin II, the combination of pravastatin and pioglitazone blocked angiotensin II p38 MAPK and p44/42 MAPK activation and procollagen expression-1.
Several studies have documented the impact of statin treatment on arrhythmia potential. The arrhythmic protective effects of statins can be attributed not only to anti-inflammatory properties but also to changes in myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity. Atorvastatin and simvastatin block Kv1.5 and Kv4.3 channels shifting the inactivation curve to more negative potentials following a complex mechanism that does not imply the binding of the drug to the channel pore. Moreover, in hypertrophied neonatal rat ventricular myocytes simvastatin alleviated the reduction of Kv4.3 expression, I(to) currents in subepicardial myocardium from the hypertrophied left ventricle. Furthermore, pravastatin in an animal model attenuated reperfusion induced lethal ventricular arrhythmias by inhibition of calcium overload.
Taking together experimental and cellular evidence supporting an effect of statin treatment in myocardial contractility is spare and for the time being we cannot definitively conclude on the clinical impact of HMG CoA reductase inhibitors in myocardial systolic performance.
Half of the cases of HF are attributed to diastolic dysfunction and the prognosis of HF with preserved EF is as ominous as the prognosis of HF with systolic dysfunction. Unfortunately, no treatment has yet been shown, convincingly, to reduce morbidity and mortality in patients with HF and preserved EF, while this group of patients is usually excluded from large prospective randomized trials and accordingly few data exist for the role of statins in this heterogeneous population.
As there is substantially lack of evidence concerning the effects of HMG CoA reductase inhibitors in subjects with HF and preserved EF the first indirect hypothesis was extrapolated from observational prospective studies in subjects with ischemic heart disease and no evidence of congestive HF. Indeed, in a cohort of 430 consecutive patients with ischemic heart disease and a mean EF of 57% Okura et al. observed that subjects under HMG CoA reductase inhibitors treatment had decreased E/E′ ratio—corresponding to a better diastolic function—and a significantly higher survival rate (Okura et al., 2007). According to the authors those beneficially effects can be attributed to improved endothelial function and vasodilatory response to reactive hyperemia, attenuation of myocardial hypertrophy, and interstitial fibrosis.
Despite the positive results from mechanistic and experimental studies clinical studies have failed to confirm a definitive role of HMG CoA reductase inhibitors in HF. Nevertheless, by extrapolating experimental and mechanistic data in clinical settings we further understand how HMG-CoA reductase inhibitors can beneficially affect subgroups of HF subjects such as those with preserved EF, low B-type natriuretic peptide levels, exacerbated inflammatory response and limited interstitial fibrosis. Nevertheless, as a definitive mechanism is lacking, there is uncertainty about the decisive mode of action and further mechanistic studies are needed to reveal how HMG-CoA reductase inhibitors act in HF substrate.
Pro- A-Type Natriuretic Peptide, Proadrenomedullin, and N-Terminal Pro-B-Type Natriuretic Peptide Used in a Multimarker Strategy in Primary Health Care in Risk Assessment of Patients with Symptoms of Heart Failure
Use of new biomarkers in the handling of heart failure patients has been advocated in the literature, but most often in hospital-based populations. Therefore, we wanted to evaluate whether plasma measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP), midregional pro-A-type atriuretic peptide (MR-proANP), and midregional proadrenomedullin (MR-proADM), individually or combined, gives prognostic information regarding cardiovascular and all-cause mortality that could motivate use in elderly patients presenting with symptoms suggestive of heart failure in primary health care. Methods and Results: The study included 470 elderly patients (mean age 73 years) with symptoms of heart failure in primary health care. All participants underwent clinical examination, 2-dimenstional echocardiography, and plasma measurement of the 3 propeptides and were followed for 13 years. All mortality was registered during the follow-up period. The 4th quartiles of the biomarkers were applied as cutoff values. NT-proBNP exhibited the strongest prognostic information with 4-fold increased risk for cardiovascular mortality within 5 years. For all-cause mortality MR-proADM exhibited almost 2-fold and NTproBNP 3-fold increased risk within 5 years. In the 5e13-year perspective, NT-proBNP and MR-proANP showed significant and independent cardiovascular prognostic information. NT-proBNP and MR-proADM showed significant prognostic information regarding all-cause mortality during the same time. In those with ejection fraction (EF) !40%, MR-proADM exhibited almost 5-fold increased risk of cardiovascular mortality with 5 years, whereas in those with EF O50% NT-proBNP exhibited 3-fold increased risk if analyzed as the only biomarker in the model. If instead the biomarkers were all below the cutoff value, the patients had a highly reduced mortality risk, which also could influence the handling of patients. Conclusions: The 3 biomarkers could be integrated in a multimarker strategy for use in primary health care.
Novel Biomarkers in Heart Failure with Preserved Ejection Fraction
Heart failure with preserved ejection fraction (HFPEF) is a common subtype of congestive heart failure for which therapies to improve morbidity and mortality have been limited thus far.
Numerous biomarkers have emerged over the past decade demonstrating prognostic significance in HFPEF, including natriuretic peptides, galectin-3, soluble ST2, and high-sensitivity troponins.
These markers reflect the multiple mechanisms implicated in the pathogenesis of HFPEF, and future research will likely use these markers to not only help determine heart failure phenotypes but also target specific therapies.
Heart failure (HF) is a global epidemic, defined as an abnormality of cardiac function leading to the inability to deliver oxygen at a rate adequate to meet the requirements of tissues. It is truly a clinical syndrome of symptoms and signs resulting from this cardiac abnormality. Over the past decade, further characterization into 2 entities has occurred: HF with preserved ejection fraction (HFPEF) and HF with reduced ejection fraction (HFREF). HFPEF, previously termed diastolic HF, encompasses the syndrome of HF with a preserved ejection fraction. Cutoffs for this ejection fraction typically are from 45% to 50%. The prevalence of HF is upward of 1% to 2% of the adult population, with an increased prevalence found in elderly and female patients. Multiple studies have shown that the prevalence of HFPEF is actually comparable with the number of patients with HFREF. As expected, most deaths from HFPEF are cardiovascular, comprising 51% to 70% of mortality.
The pathophysiology of HFPEF is controversial and remains poorly understood. Originally, HFPEF was thought to be a primary manifestation of diastolic dysfunction of the left ventricle. However, patients with HFREF are known to also commonly have impaired ventricular relaxation. The primary mechanism of left ventricular (LV) dysfunction is based on structural remodeling and endothelial dysfunction, lending itself to LV stiffness, and increased left atrial pressure. This pressure change is what drives pulmonary venous congestion and subsequent symptomatology. The ventricular stiffness commonly seen in HFPEF is attributed to multiple mechanisms, including fibrosis, excessive collagen deposition, cardiomyocyte stiffness, and slow LV relaxation.
The natriuretic peptides (NPs) are the cornerstone biomarker in congestive HF (CHF). Many of the details of the role of NPs are covered in an article – Florea VG, Anand IS. Biomarkers. Heart Fail Clin 2012;8(2):207–24. The Breathing Not Properly trial originally helped establish the role of B-type natriuretic peptide (BNP) in the diagnosis of CHF. BNP and the N-terminal prohormone BNP (NT-proBNP) have been shown in numerous trials to be an excellent tool for ruling out CHF as a cause of acute dyspnea. Aside from a strong negative predictive value, NPs correlate with HF severity, prognostication, outpatient CHF management, and screening. When attempting to use NPs specifically to distinguish between HFPEF and HFREF, results have shown that NPs do not have a particular cutoff, but are typically elevated in HFPEF in comparison with patients without HF. These levels of NPs in HFPEF are typically lower than levels in patients with HFREF.
Although the role of novel renal biomarkers has not been fully explored specifically in HFPEF, they likely have an impactful role in the assessment and management of acute kidney injury (AKI) and the cardiorenal syndrome. Two biomarkers are briefly discussed here: neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C. NGAL is a 25-kDa protein in the lipocalin family of proteins with a role in inflammation and immune modulation.
The future of biomarkers and their utility in HF is very promising, starting with the potential for using biomarkers as end points in trials. Biomarkers serve as surrogates for various pathophysiologic mechanisms, and there are potential benefits in using them as trial end points. Advantages include the ability to obtain quick and early data, as well as possibly better understand the nature of the disease. However, the counterargument against using biomarkers as trial end points includes whether treatment effects on a biomarker reliably predict effects on a clinically meaningful end point. Reduced cGMP signaling activates NF-κB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A
Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-κB (NF-κB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-κB binding activity was 4-fold greater in the nuclear extract of Npr1-/-mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory κB kinase-b activity and IκB-α protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-κB binding activity associated with hypertrophic growth of mutant mice hearts.
Regulation of guanylyl cyclase/natriuretic peptide receptor-A gene expression
Natriuretic peptide receptor-A (NPRA) is the biological receptor of the peptide hormones atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The level and activity of this receptor determines the biological effects of ANP and BNP in different tissues mainly directed towards the maintenance of salt and water homeostasis. The core transcriptional machinery of the TATA-less Npr1 gene, which encodes NPRA, consists of three SP1 binding sites and the inverted CCAAT box. This promoter region of Npr1 gene has been shown to contain several putative binding sites for the known transcription factors, but the functional significance of most of these regulatory sequences is yet to be elucidated. The present review discusses the current knowledge of the functional significance of the promoter region of Npr1 gene and its transcriptional regulation by a number of factors including different hormones, growth factors, changes in extracellular osmolarity, and certain physiological and patho-physiological conditions.
Atrial natriuretic peptide (ANP), a member of natriuretic peptide family is a polypeptide consisting of 28 amino acids and was discovered as a potent vasodilator and diuretic hormone produced in granules of the atrium. The natriuretic peptide family consists of the peptide hormones ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), each of which is derived from a separate gene. ANP and BNP are cardiac derived peptides, which are secreted and up-regulated in myocardium in response to different patho-physiological stimuli, while CNP is an endothelium-derived mediator that plays an important paracrine role in the vasculature. All of these natriuretic peptides elicit a number of vascular, renal, and endocrine effects mainly directed towards the maintenance of blood pressure and extracellular fluid volume by binding to their specific cell surface receptors. ANP exerts its effects at a number of sites including the kidney, where it produces natriuretic and diuretic responses; the adrenal gland, where it inhibits aldosterone synthesis and secretion; vascular smooth muscle cells, where it produces vasorelaxation; the endothelial cells, where it may regulate vascular permeability; gonadal cells, where it affects synthesis of androgen and estradiol. Each of these target sites of ANP activity has been shown to possess specific high affinity receptors for ANP. To date, three different subtypes of natriuretic peptide receptors have been characterized, purified, and cloned, i.e. natriuretic peptide receptors A, B, and C also designated as NPRA, NPRB, and NPRC, respectively. ANP and BNP specifically bind to NPRA, which contains guanylyl cyclase catalytic activity and produces intracellular secondary messenger cGMP in response to hormone binding.
NPRA is considered the biological receptor of ANP and BNP because most of the physiological effects of these hormones are triggered by generation of cGMP or its cell permeable analogs. Recent studies with mice lacking the Npr1 gene, demonstrated that genetic disruption of NPRA increases the blood pressure and causes hypertension in these animals. On the other hand, the effect of ANP was found to be increased linearly in Npr1 gene-duplicated mice
in a manner consistent with gene copy number. All this clearly indicates that the level of NPRA expression determines the extent of the biological effects of ANP and BNP. But the intervention strategies aimed at controlling NPRA expression are limited by the paucity of studies in this area. The cDNA and gene encoding NPRA designated as Npr1 has been cloned and characterized in mouse, rat, bull frog, euryhaline eel, and medaka fish. The primary structure of this gene is essentially same in all the different species and contains 22 exons interrupted by 21 introns. The Npr1 gene sequence has been found to be interspersed with a number of repetitive elements including (SINES), (MER2), and tandem repeat elements in all the different species.
Although the Npr1 gene transcriptional regulation is only poorly understood, the activity and expression of NPRA assessed primarily through ANP stimulated cGMP accumulation are found to be regulated by a number of factors including auto-regulation by natriuretic peptides themselves, other hormones such as endothelin, glucocorticoids, and angiotensin II (ANG II), growth factors, changes in extracellular ion composition, and certain physiological and patho-physiological conditions.
The core molecular machinery of the TATA-less Npr1 gene consisting of SP1 binding sites and the inverted CCAAT box has been authenticated to be indeed functional in rat promoter element. It has been shown that the molecular machinery that regulates the basal expression of Npr1 gene consists of three SP1 binding sites in conjunction with an inverted CCAAT box present in the proximal promoter region. Mutation in any of these SP1 binding sites which
are located within 350 bp upstream of transcription start site in rat Npr1 promoter inhibited SP1 and SP3 binding and decreased the promoter activity by 50–75%, while simultaneous mutation of all the three led to a >90% reduction in promoter activity. The proximal SP1 binding site was much more effective than the distal sites in inducing the expression implying that the proximity to the core transcriptional machinery contributes to the magnitude of the observed effect. The over-expression of either SP1 or SP3 resulted in the induction of the wild type Npr1 promoter, confirming that these transcription factors serve as positive regulators of the Npr1 gene expression.
A number of natriuretic peptides such as ANP, BNP, CNP, and urodilatin (i.e. ANP95–126) can down-regulate ligand dependent NPRA activity after as little as 2 h prior exposure to the ligand, which remains suppressed until 48 h of exposure in cultured cells. The early reduction of NPRA activity is independent of changes in Npr1 gene expression as the pretreatment of cultured cells with actinomycin D (an inhibitor of transcription) for 1 h failed to block the response to ANP implying that ligand acts, at least early on, through a post transcriptional mechanism in reducing NPRA activity. The sustained reduction of NPRA activity, on the other hand, has been shown in fact due to reduction in NPRA mRNA levels (∼50%) by treatment with 100nM ANP for 48 h. This reduction could also be affected by treatment of cultured cells with 8-Br-cGMP with similar kinetic response and was amplified by phosphodiesterase inhibitors, but was not shared by NPRC-selective ligand cANF, suggesting that the down regulation of Npr1 gene expression is mediated by elevations of intracellular cGMP involving either NPRA or NPRB. .. The cGMP regulatory region was pinpointed to position−1372 to−1354 bp from the transcription start site of Npr1 by gel shift assays and footprinting analysis, which indicated its interaction with transcriptional factor(s). Further cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (−1372 bp AaAtRKaNTTCaAcAKTY −1354 bp) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters. The combination of these transcriptional and post-transcriptional ligand-dependent regulatory mechanisms provides the cells with greater flexibility in both initiating and maintaining the suppression of NPRA activity.
The peptide hormone Ang II is an important component of renin-angiotensin system (RAS) and exerts its biological effects such as blood pressure regulation, vasoconstriction, and cell proliferation in many tissues including the kidney, adrenal glands, brain, and vasculature. The two vasoactive peptide hormones, Ang II (vasoconstrictive) and ANP (vasodilatory), interact and mutually antagonize the biological effects of each other at various levels. ANP has been shown to inhibit Ang II-induced contraction of isolated glomeruli and cultured mesangial cells, as well as Ang II-stimulated activation of protein kinase C and mitogen activated protein kinase in vascular smooth muscle cells in a cGMP-dependent manner. Inversely, Ang II has been shown to down-regulate guanylyl cyclase activity of the biological receptor of ANP, NPRA, by activating protein kinase C and/or by stimulating protein tyrosine phosphatase activity, thereby inhibiting the ANP stimulated cGMP accumulation. Ang II also reduces the ANP dependent cGMP levels by stimulating cGMP hydrolysis, apparently
via a calcium dependent cGMP phosphodiesterase.
Endothelin is a vasoconstrictor peptide that was originally isolated from porcine endothelial cells. It is produced as three isoforms (ET1-3) that bind to two receptor subtypes (ETA and ETB). ET is produced in the kidney and subject to regulation by a number of local and systemic factors including immune cytokines and extracellular tonicity. Since, endothelin is avidly expressed in the nephron segment, where NPRA is up-regulated by osmotic stimulus, it was investigated whether endothelin plays a role in the control of basal or osmotically regulated Npr1 gene expression in these cells. The endogenous endothelin and not the exogeneously administered endothelin inhibit the basal but not osmotically stimulated expression of Npr1. The type A (BQ610) and type B (IRL 1038) endothelin receptor antagonists increased the level of NPRA mRNA by two to three-fold, whereas co-administration of exogenous endothelin resulted in partial reversal of this stimulatory effect of receptor antagonists. The increase in extracellular tonicity reduces the endothelin mRNA accumulation (∼15% of control levels) in inner medullary collecting duct cells but this reduction is not found to be linked to the stimulation of NPRA activity/expression in response to osmotic stress.
Glucocorticoids influence the cardiovascular system and induce a rapid increase in blood pressure. Glucocorticoids are known to regulate
transcription in many systems, possibly by interacting with glucocorticoid responsive elements and associated chromatin proteins. These have been shown to affect the atrial endocrine system by regulating both the synthesis and secretion of ANP in vitro and in vivo. Thus, it seems plausible that glucocorticoid may also interact with the atrial endocrine system by modulating ANP receptor levels. The stimulation of vascular smooth muscle cells from rat mesenteric artery with dexa-methasone (a highly specific synthetic glucocorticoid agonist) caused an increase in NPRA mRNA levels in a time dependent manner which reached a plateau after 48 h of glucocorticoid administration. This mRNA increase was mimicked by cortisol and inhibited by glucocorticoid receptor antagonists RU38486. Also cGMP generated by NPRA in dexamethasone treated cells was higher than in control cells and this production was mimicked by cortisol and blocked by RU 38486. These results suggest that glucocorticoids exert a positive effect on NPRA transcription in rat mesenteric arteries.
Previous studies have shown that guanylyl cyclase activity of NPRA is either activated, or inhibited by an increase in extracellular tonicity. Though none of these studies were definitive in terms of elucidating the mechanisms involved, they suggested that the activation predominates with longer exposure (∼24 h), while the inhibition with short-term exposure (minutes) to the osmotic stimulus. More recently, the mechanism(s) underlying the activation of NPRA expression by osmotic stimulus has been investigated. The NaCl (75 mM) or sucrose (150 mM), but not osmotically inert solute, urea (150 mM) increased NPRA activity, gene expression, and promoter activity after as early as 4 h reaching a maximum at 24 h in inner medullary collecting duct cells. The osmotic stimulus also activated extracellular signal regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 mitogen activated protein kinase- (p38 MAPK-β). The inhibition of p38 MAPK-βwith SB20580 completely blocked the osmotic stimulation of receptor activity and expression, and caused a dose-dependent reduction in promoter activity, whereas inhibition of ERK with PD98059 had no effect.
The expression of NPRB, the biological receptor of CNP, has been shown to be regulated by a number of factors including natriuretic peptide ligands, intracellular cAMP levels, water deprivation, TGF-1, dexamethasone treatment, as well as renal sodium status, as its mRNA levels were upregulated in the renal cortex of sodium depleted animals. NPRB expression has also been found to be regulated by alternative splicing. Three isoforms of NPRB have been identified of which NPRB1 is the full length form and responds maximally to CNP, NPRB2 isoform contains a 25 amino acid deletion in protein kinase homology domain and NPRB3 contains a partial extracellular ligand binding domain and fails to bind the ligand. The relative expression levels of the three isoforms vary across different tissues. Since, the smaller splice variants of NPRB act as dominant negative isoforms by blocking formation of active NPRB1 homodimers, these isoforms might play important role in the tissue specific regulation of receptor, NPRB.
The NPRC expression has also been found to be down-regulated by its ligands and their secondary messenger, cGMP, hormones, growth factors, dietary salt supplementation, β-adrenergic blocker, and physiological as well as patho-physiological conditions. On the other hand, NPRC expression gets augmented by TGF-β1, 1,25-dihydroxy VitaminD3 and during conditions like chronic heart failure.
Hypertension is the leading cause of human deaths in today’s world. The natriuretic peptide system plays a well defined role in the regulation of blood pressure and fluid volume. The cellular and physiological effects of natriuretic peptides (ANP, BNP, and CNP) are mediated by their specific receptors NPRA, NPRB, and NPRC. The transcriptional regulation of these receptors has been studied since their identification, but still remains poorly understood. Better understanding and the elucidation of different molecular mechanisms responsible for the regulation of NPRA expression would provide us the framework to develop the therapeutic strategies to manipulate the expression levels of this receptor and to control the biological actions of ANP and BNP during different patho-physiological conditions.
Inhibition of Heat Shock Protein 90 (Hsp90) in Proliferating Endothelial Cells Uncouples Endothelial Nitric Oxide Synthase Activity
Jingsong Ou, Zhijun Ou, AW Ackerman, KT Oldham, & KA Pritchard, Jr.
Free Radical Biol Med 2003; 34(2):269–276
PII S0891-5849(02)01299-6
Dual increases in nitric oxide (•NO) and superoxide anion (O2•-) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of •NO generation to offset the increase in O2•- that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase •NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of •NO and O2•-. Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 µM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated •NO production and increased Lω-nitroargininemethylester (L-NAME)-inhibitable O2•-generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O2•- generation, confirming that during proliferation eNOS generates •NO. Our findings demonstrate that hsp90 plays an important role in maintaining •NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit •NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.
Natriuretic Peptides, Ejection Fraction, and Prognosis – Parsing the Phenotypes of Heart Failure
Since the first pivotal studies introduced the natriuretic peptides as biomarkers for the diagnosis of heart failure (HF), use of both B-type natriuretic peptide (BNP) and its N-terminal equivalent (NT-proBNP) has grown not only for this indication, but also for establishing HF prognosis as well. Indeed, a vast array of studies has established the natriuretic peptides as the biomarker gold standard to prognosticate risk for a wide array of relevant complications in HF (ranging from ventricular arrhythmias to pump failure). In these studies, the prognostic information provided by BNP and NT-proBNP in HF was independent of a number of relevant covariates, including left ventricular ejection fraction (LVEF).
It has been known for quite a while that patients with heart failure and preserved ejection fraction (HFpEF) typically have lower natriuretic peptide values than do those with heart failure and reduced ejection fraction (HFrEF). A conundrum is thus present: whereas both BNP and NTproBNP tend to be lower in HFpEF, when these peptides are elevated in this setting, they remain prognostic; this intriguing circumstance has been relatively poorly studied. It is in this setting that van Veldhuisen et al. examined the impact of LVEF on the prognostic merits of BNP in the COACH (Coordinating Study Evaluating Outcomes of Advising and Counseling in Heart Failure) study in the present issue of the Journal. The investigators found—as expected—that BNP levels were lower in HFpEF, but for a given BNP concentration, prognosis of those with HFpEF in COACH was just as poor as those with HFrEF at matched BNP values. Stated differently, a high BNP in a patient with HFpEF imparted similar prognostic information as it would in someone with HFrEF. Actually, whereas LVEF was not obviously prognostically impactful, when considered across the range of ventricular function, an elevated BNP concentration in the most normal range of LVEF seemed to be associated with a higher risk than at the lower ranges of pump function. Although it is previously established that BNP or NT-proBNP are prognostic independently of LVEF, the well-executed analysis by van Veldhuisen et al. (van Veldhuisen DJ, Linssen GCM, Jaarsma T, et al. B-type natriuretic peptide and prognosis in heart failure patients with preserved and reduced ejection fraction. J Am Coll Cardiol 2013;61:1498–506.) allows for a more in-depth examination of this phenomenon and raises some important questions.
Phenotypic Definition of the Patient With Heart Failure
Phenotypic Definition of the Patient With Heart Failure
Natriuretic Peptides in Heart Failure with Preserved Ejection Fraction
Threshold values of B-type natriuretic peptide (BNP) and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) validated for diagnosis of undifferentiated acutely decompensated heart failure (ADHF) remain useful in patients with heart failure with preserved ejection fraction (HFPEF), with minor loss of diagnostic performance.
BNP and NT-proBNP measured on admission with ADHF are powerfully predictive of in-hospital mortality in both HFPEF and heart failure with reduced EF (HFREF), with similar or greater risk in HFPEF as in HFREF associated with any given level of either peptide.
In stable treated heart failure, plasma natriuretic peptide concentrations often fall below cut-point values used for the diagnosis of ADHF in the emergency department; in HFPEF, levels average approximately half those in HFREF.
BNP and NT-proBNP are powerful independent prognostic markers in both chronic HFREF and chronic HFPEF, and the risk of important clinical adverse outcomes for a given peptide level is similar regardless of left ventricular ejection fraction.
Serial measurement of BNP or NT-proBNP to monitor status and guide treatment in chronic heart failure may be more applicable in HFREF than in HFPEF.
The bioactivity of atrial NP (ANP) and B-type NP (BNP) encompasses short-term and longterm hemodynamic, renal, neurohormonal, and trophic effects. The relationship between cardiac hemodynamic load, plasma concentrations of ANP and BNP, and the cardioprotective profile of NP bioactivity have led to investigation of both biomarker and therapeutic potential of
NPs in HF.
PlasmaBNPandNT-proBNP thresholds (100pg/mL and 300 pg/mL, respectively) used in the diagnosis of undifferentiated ADHF retain good diagnosticperformance for acute HFPEF
Plasma NPs are related to multiple echo indicators of cardiac structure and function in both HFREF and HFPEF.
Box 1Causes of increased plasma cardiac natriuretic peptides
Cardiac
Heart failure, acute and chronic
Acute coronary syndromes
Atrial fibrillation
Valvular heart disease
Cardiomyopathies
Myocarditis
Cardioversion
Left ventricular hypertrophy
Noncardiac
Age
Female sex
Renal impairment
Pulmonary embolism
Pneumonia (severe)
Obstructive sleep apnea
Critical illness
Bacterial sepsis
Severe burns
Cancer chemotherapy
Toxic and metabolic insults
BNP and NT-proBNP fall below ADHF thresholds in stable HFREF in approximately 50% and 20% of cases, respectively. Levels in stable HFPEF are even lower, approximately half those in HFREF.
Whereas BNPs have 90% sensitivity for asymptomatic LVEF of less than 40% in the community (a precursor state for HFREF), they offer no clear guide to the presence of early community based HFPEF.
Guidelines recommend BNP and NT-proBNP as adjuncts to the diagnosis of acute and chronic HF and for risk stratification. Refinements for application to HFPEF are needed.
The prognostic power of NPs is similar in HFREF and HFPEF. Defined levels of BNP and NT-proBNP correlate with similar short-term and long-term risks of important clinical adverse outcomes in both HFREF and HFPEF.
Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely
Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely. a In the acute setting, mid-regional pro–atrial natriuretic peptide may also be used (cutoff point 120 pmol/L; ie, <120 pmol/L 5 heart failure unlikely). b Other causes of elevated natriuretic peptide levels in the acute setting are an acute coronary syndrome, atrial or ventricular arrhythmias, pulmonary embolism, and severe chronic obstructive pulmonary disease with elevated right heart pressures, renal failure, and sepsis. Other causes of an elevated natriuretic level in the nonacute setting are old age (>75 years), atrial arrhythmias, left ventricular hypertrophy, chronic obstructive pulmonary disease, and chronic kidney disease. c Exclusion cutoff points for natriuretic peptides are chosen to minimize the false-negative rate while reducing unnecessary referrals for echocardiography. d Treatment may reduce natriuretic peptide concentration, and natriuretic peptide concentrations may not be markedly elevated in patients with heart failure with preserved ejection fraction. BNP, B-type natriuretic peptide; ECG, electrocardiogram; NT-proBNP, N-terminal prohormone of B-type natriuretic peptide. (From McMurray JJ, Adamopoulos S, Anker SD, et al. The task force for the diagnosis and treatment of acute and chronic heart failure 2012 of the European Society of Cardiology. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012. Eur Heart J 2012;33:1787–847; with permission.)
Natriuretic Peptide Receptor-A Negatively Regulates Mitogen-Activated Protein Kinase and Proliferation of Mesangial Cells: Role of cGMP-Dependent Protein Kinase
peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65–75% in cells overexpressing NPRA, whereas in vector transfected cells, its inhibitory effect was only 18–20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF stimulated [3H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20–25% inhibition in vector-transfected cells. These
results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.
Triglyceride (TG)-rich chylomicrons and very low density lipoproteins (VLDL) distribute fatty acids (FA) to various tissues by interacting with the enzyme lipoprotein lipase (LPL). The protein angiopoietin-like 4 (Angptl4) is under sensitive transcriptional control by FA and the FA-activated peroxisome proliferator activated receptors (PPARs), and its tissue expression largely overlaps with that of LPL. Growing evidence indicates that Angptl4 mediates the physiological fluctuations in LPL activity, including the decrease in
adipose tissue LPL activity during fasting. This review focuses on the major ambiguities concerning the mechanism of LPL inhibition by Angptl4, as well as on the physiological role of Angptl4 in lipid metabolism, highlighting its function in a variety of tissues, and uses this information to make suggestions for further research.
Box 1. LPL and TG metabolism
LPL belongs to a family of lipases that also includes hepatic lipase, pancreatic lipase, and endothelial lipase. Because LPL is essential in the lipolytic processing of chylomicrons and VLDL, LPL is primarily expressed in tissues that either require large amounts of FA as fuel or are responsible for TG storage, which include heart, skeletal muscle, and adipose tissue. Upon production by the underlying parenchymal cells, LPL is released into the subendothelial space and is transported to the luminal side of the capillary endothelium by the GPI-anchored protein GPIHBP1, which after transport continues to anchor LPL to the capillary endothelium. The essential role for LPL in the clearance of plasma TG is well-demonstrated by the severe hypertriglyceridemia of patients carrying homozygous mutations in the LPL gene. Generalized deletion of LPL in mice results in severe hypertriglycer-idemia, resulting in the premature death of pups within 24 h after birth. Analogous to the deletion of LPL, the mislocalization of LPL to the subendothelial spaces due the absence or misfolding of GPIHBP1 also results in severe chylomicronemia and hypertriglyceridemia. The LPL enzyme is catalytically active as a non-covalent head-to-tail dimer with a catalytic N-terminal domain and a non-catalytic C terminal domain. Folding of LPL into its dimer conformation occurs in the endoplasmic reticulum, chaperoned by lipase maturation factor 1, calreticulin, and calnexin. In its active 3D conformation, the catalytic site of LPL is postulated to be covered by a lid, which can be opened by the binding of chylomicrons and VLDL to the C terminus. The active LPL dimers rapidly exchange subunits, indicating that a dynamic equilibrium exists between LPL dimers and dimerization-competent monomers. Dimerization-competent monomers have, however, not yet been isolated, and it is unclear whether this monomer is catalytically active. The enzymatic activity of LPL is lost when the LPL dimer is converted into inactive, folded monomers. This conversion to inactive monomers is mainly regulated via post-translational mechanisms and is dependent on nutritional state. Enzymatic activity of inactive monomers can be regained in vitro by the addition of calcium, indicating that inactivation of LPL is a reversible process.
One of the key questions is whether (patho)physiological variations in LPL activity are mediated via regulation of Angptl4 cleavage and/or oligomerization, and which factors are involved in modulating Angptl4 in vivo. Recent biochemical evidence suggests that FA may be able to promote dissociation of oligomers, which, by destabilizing the protein, would impair its ability to inhibit LPL. Destabilization of Angptl4 by FA is, however, seemingly at odds with the marked stimulatory effect of FA on Angptl4 production observed in vitro and in vivo.
The currently accepted molecular model for the inhibition of LPL by Angptl4 is that Angptl4 stimulates the conversion of catalytically active LPL dimers into inactive monomers – following in vitro studies showing that coincubation of LPL and Angptl4 increases the abundance of LPL monomers. Subsequent studies revealed that the proportion of LPL dimers is reduced in post-heparin plasma of mice that overexpress Angptl4 in favor of LPL monomers, providing in vivo support for the dimer-to monomer conversion. The elucidation of the purported biochemical mechanism has strengthened the status of Angptl4 as a LPL inhibitor, but several questions related to the in vivo mechanism remain unanswered. Whereas the original in vitro experiments favored the hypothesis that Angptl4 enzymatically and irreversibly catalyzes the LPL dimer-to-monomer conversion, an in vivo study of Angptl4 transgenic mice suggested that Angptl4 is physically bound to LPL monomers, thereby driving the LPL dimer–monomer equilibrium towards inactive monomers. The latter study also revealed that the relative decrease in post-heparin plasma LPL activity upon Angptl4 overexpression is much more pronounced than the relative decrease in heparin-releasable LPL dimers, pointing to an additional or alternative mechanism. In support, a recently published study suggests that Angptl4, instead of acting as a catalyst, functions as a conventional, non-competitive inhibitor that binds to LPL to prevent the hydrolysis of substrate LPL and Angptl4 are regulated by changes in nutritional state in a tissue-specific manner, reflecting the different functions of these tissues and the corresponding variations in physiological requirements for lipids. Below, we discuss current knowledge on the regulation of Angptl4 and LPL in response to various physiological stimuli and address the importance of Angptl4 in lipid uptake. An overview of the role of Angptl4 in physiological regulation of lipid metabolism is presented in Figure 2.
model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4.
Figure 1. Hypothetical model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4. Angiopoietin-like 4 (Angptl4) and LPL are expressed in the parenchymal cells of muscle, heart, and adipose tissue. Following secretion of LPL and Angptl4 into the subendothelial space, transport of LPL to the capillary lumen is mediated by two mechanisms. The principal transport mechanism (1) relies on GPIHBP1 [glycosylphosphatidylinositol (GPI)-anchored high density lipoprotein-binding protein] picking up LPL from the subendothelial space and transporting it to the capillary lumen. This action by GPIHBP1 is opposed by Angptl4, which is bound to extracellular matrix (ECM) proteins and which retains and inhibits LPL. In the presence of GPIHBP1, high expression levels of Angptl4 are needed to overcome the competition with GPIHBP1. Angptl4 secreted into the capillary lumen, primarily as N-terminal truncation fragment generated by cleavage by proprotein convertases (PCs), inhibits LPL activity on the endothelium by promoting the irreversible conversion of LPL dimers into inactive monomers and/or via a reversible mechanism that requires binding of Angptl4 to LPL. The second transport mechanism involves a so far unidentified carrier and can be disrupted by Angptl4. In the absence of GPIHBP1, Angptl4 fully retains LPL in the subendothelial space (a). The additional loss of Angptl4 liberates LPL and allows it to be transported to the endothelial surface via the unidentified carrier (b). This model suggests that Angptl4 and LPL start interacting before arrival in the capillary lumen, either in the parenchymal cells or in the subendothelial space. Abbreviation: HSPG, heparan sulfate proteoglycan.
Regulation and role of angiopoietin-like 4 (Angptl4)
Figure 2. Regulation and role of angiopoietin-like 4 (Angptl4) in lipid metabolism. Angptl4 is expressed in parenchymal cells of white adipose tissue (WAT), liver, intestine, heart and muscle, as well as in macrophages, where it is subject to cell- and tissue-specific regulation. Angptl4 is a sensitive target of peroxisome proliferator-activated receptor (PPAR) transcription factors in several tissues. In WAT the expression of Angptl4 is induced during fasting and by the transcription factors PPARg, glucocorticoid receptor (GR), and hypoxia inducible factor 1a (HIF1a). In WAT Angptl4 stimulates lipolysis of stored triglycerides (TG) and inhibits lipoprotein lipase (LPL) activity. Expression of Angptl4 in liver is stimulated by PPARa, PPARd, and GR. Because the liver does not express LPL, Angptl4 is mainly released into the blood, affecting LPL activity in peripheral tissues. Angptl4 may also impact upon hepatic lipase activity in liver. Expression of Angptl4 in heart and skeletal muscle is potently induced by fatty acids (FA) via PPARd activation. Angptl4 inhibits LPL activities in cardiac and likely skeletal muscle. FA also stimulate Angptl4 expression in macrophages via PPARd, leading to local inhibition of LPL activity. We hypothesize that macrophage LPL enables uptake of remnant particles containing lipid antigens, which are subsequently presented to natural killer T cells. In the intestine, FA stimulate Angptl4 expression via one of the PPARs. Angptl4 produced by enterocytes may be released towards the lumen and inhibit pancreatic lipase activity. Angptl4 produced by enteroendocrine cells is released towards the blood and may inhibit LPL in distant tissues.
Box 2. Outstanding questions
What is the importance of Angptl4 cleavage and oligomerization to Angptl4 function in vivo?
What is the precise biochemical mechanism behind the inhibition of LPL activity by Angptl4?
At which cellular location(s) does the inhibition of LPL by Angptl4 occur and, if at multiple locations, what is the relative contribution of both tissue-produced Angptl4 compared to circulating Angptl4 with respect to inhibition of tissue LPL activity.
What is the interplay between GPIHBP1 and Angptl4 in the regulation of LPL activity?
What is the protein structure of Angptl4 and LPL?
Does Angptl4 also regulate LPL activity in brown adipose tissue and skeletal muscle and, if so, how is the expression of Angptl4 regulated in these tissues?
What is the potential of Angptl4 as a biomarker in the context of disorders of lipid metabolism?
In the past decade, angiopoietin-like proteins have been demonstrated to regulate plasma TG levels powerfully in mice and humans. The elucidation of these proteins as inhibitors of LPL activity has led to a paradigm shift in how clearance of circulating TG and thereby tissue uptake of FA are regulated. Most of our understanding of angiopoietin-like proteins has resulted from detailed study of Angptl4.
A major portion of the physiological variation in LPL activity in various tissues can be attributed to regulation of Angptl4 production. We predict that Angptl4 will turn out to be equally important for governing LPL activity in muscle during exercise, in brown adipose tissue during cold, and in several tissues during fasting.
Besides the increasing recognition of the pivotal role of Angptl4 in lipid metabolism as an inhibitor of LPL, major insight has been gained into the molecular mechanism of action of Angptl4. Key questions remain, however, especially related to the interaction between LPL, GPIHBP1, and Angptl4 on the endothelium and in the subendothelial space. Several points of interest have been highlighted throughout the text; these include the elucidation of the molecular structure for LPL and Angptl4 by X-ray crystallography and the clarification of in vivo Angptl4 cleavage and oligomerization.
Native Low-Density Lipoprotein Induces Endothelial Nitric Oxide Synthase Dysfunction: Role of Heat Shock Protein 90 And Caveolin-1
Kirkwood A. Pritchard, Jr., Allan W. Ackerman, Jingsong Ou, et al.
Free Radical Biol & Med 2002; 33(1):52–62 PII S0891-5849(02)00851-1
Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (•NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O2•-), which inactivates •NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for •NO, O2•-, phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O2•-by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by [1]34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (-85%) and cytosol (-15%). Only 3–6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O2•-, which scavenges •NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O2•-.
In conclusion, n-LDL decreases the association of hsp90 with eNOS, increases phospho-eNOS levels, and increases eNOS-dependent O2•-generation. These findings suggest that activation of eNOS without adequate levels of hsp90 may signal eNOS to switch from •NO to O2•-generation. Such changes in eNOS radical product generation may play an important role in impairing endothelial and vascular function.
Insulin-like growth factor 1 (IGF-1) signaling regulates contractility, metabolism, hypertrophy, autophagy, senescence, and apoptosis in the heart. IGF-1 deficiency is associated with an increased risk of cardiovascular disease, whereas cardiac activation of IGF-1 receptor (IGF-1R) protects from the detrimental effects of a high-fat diet and myocardial infarction. IGF-1R activates multiple pathways through its intrinsic tyrosine kinase activity and through coupling to heterotrimeric G protein. These pathways involve classic second messengers, phosphorylation cascades, lipid signaling, Ca2+ transients, and gene expression. In addition, IGF-1R triggers signaling in different subcellular locations including the plasma membrane, perinuclear T tubules, and also in internalized vesicles. In this review, we provide a fresh and updated view of the complex IGF-1 scenario in the heart, including a critical focus on therapeutic strategies.
The hormone insulin-like growth factor 1 (IGF-1) is a small peptide of 7.6 kDa, which is composed of 70 amino acids and shares 50% homology with insulin. IGF-1 plays key roles in regulating proliferation, differentiation, metabolism, and cell survival. It is mainly synthesized and secreted by the liver in response to hypothalamic growth hormone (GH); its plasma concentration is finely regulated (Box 1). However, other tissues also produce IGF-1, which acts locally as an autocrine and paracrine hormone. IGF-1 exhibits pleiotropic effects in many organs and is also involved in the development of several pathologies.
Box 1. IGF-1 synthesis and biodisponibilityInsulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide
hormone with endocrine, paracrine, and autocrine effects. It shares
>60% structure homology with IGF-2 and 50% with pro-insulin. IGF-
1 is mainly synthesized in the liver in response to hypothalamic
growth hormone (GH). In the peripheral circulation it exerts negative
feedback on the somatotrophic axis suppressing pituitary GH
release. IGF-1 can also be generated in almost all tissues, but liver
synthesis accounts for nearly 75% of circulating IGF-1 levels. As a
hormone with a wide range of physiological roles, IGF-1 circulating
levels must be strictly controlled. Around 98% of circulating IGF-1 is
bound to insulin-like growth factor binding protein (IGFBP). Six
forms of high affinity IGFBP have been described, with IGFBP3
binding approximately 90% of circulating IGF-1. Also, IGFBP1–6 and
their fragments have significant intrinsic biological activity independent
of IGF-1 interaction.
Canonical and noncanonical IGF-1 signaling pathways Activation of IGF-1R requires the sequential phosphorylation of three conserved tyrosine residues within the activation loop of the catalytic domain. From these phosphorylated motifs, tyrosine 950 contained in an NPXY motif provides a docking site for the recruitment of adaptor proteins, such as insulin receptor substrate-1 (IRS-1) and Shc, as an obligatory step to initiate signaling cascades. Two canonical pathways are activated by IGF-1R in cardiomyocytes – the phosphatidylinositol-3 kinase (PI3K)/Akt pathway and the extracellular signal-regulated kinase (ERK) pathway. Both pathways have been extensively studied, and their involvement in the pro-hypertrophic and pro-survival actions in cardiomyocytes is well established. Interestingly, a noncanonical signaling mechanism for IGF-1R in cardiomyocytes has been described in several recent studies. These studies show that some of the effects of IGF-1 are inhibited by the heterotrimeric Gi protein blocker Pertussis toxin (PTX) in several cell lines, suggesting that IGF-1R is a dual-activity receptor that triggers tyrosine-kinase-dependent responses as well as Gi-protein-dependent pathways. This duality has been reported in cultured neonatal cardiomyocytes; IGF-1R can activate ERK and Akt but also phospholipase C (PLC), which increases inositol 1,4,5 triphosphate (InsP3; IP3) leading to nuclear Ca2+ signals.
The cardiac effects of IGF-1 are mediated by activation of the plasma membrane IGF-1R, which belongs to the receptor tyrosine kinase (RTK) family. IGF-1R comprises a α2β2 heterotetrameric complex of approximately 400 kDa. Structurally, IGF-1R has two extracellular a-subunits that contain the ligand-binding sites. Each α-subunit couples to one of two membrane-spanning β-subunits, which contain an intracellular domain with intrinsic tyrosine kinase activity. Both subunits of IGF-1R are the product of one single gene, which is synthesized as a 180 kDa precursor. The immature IGF-1R full peptide is further glycosylated, dimerized, and proteolytically processed for assembly of the mature receptor isoforms a and b. In neonatal and adult rat cardiomyocytes, the IGF-1R precursor peptide and the processed α and β receptor subunits have been detected. Binding of IGF-1 to its receptor initiates a complex signaling cascade in cardiomyocytes.
Figure 1. not shown. Canonical and noncanonical signaling pathways activated by insulin-like growth factor 1 (IGF-1) in cardiomyocytes. Binding of IGF-1 to plasma membrane IGF-1 receptor (IGF-1R) leads to receptor autophosphorylation in the intracellular β-subunits. Docking of Grβ2 to the phosphorylated IGF-1Rβ subunits leads to extracellular signal-regulated kinase (ERK) phosphorylation through the Ras/Raf/Mitogen-activated protein kinase (MEK) axis. Phosphorylated ERK can translocate to the nucleus to control gene expression. Phosphorylated β-subunits also provide docking sites for insulin receptor substrate-1 (IRS-1), which mediates phosphatidylinositol-3 kinase (PI3K) activation and Akt phosphorylation. Downstream targets of activated Akt are mechanistic target of rapamycin (mTOR), which suppresses autophagy and promotes protein synthesis by activating S6K and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Akt also phosphorylates and inactivates Bad, thus inhibiting apoptosis. IGF-1R activation also promotes its interaction with a Pertussis-toxin-sensitive heterotrimeric Gi protein, which mediates the activation of phospholipase C (PLC) and hydrolysis of plasma membrane phosphatidylinositol 4,5 biphosphate (PIP2) to form inositol 1,4,5 triphosphate (InsP3; IP3) which activates InsP3 receptors located at the endoplasmin reticulum (ER)/nuclear envelope Ca2+ store, producing nucleoplasmic and cytoplasmic Ca2+ increases. The former is involved in the regulation of specific target genes and the latter promotes mitochondrial Ca2+ uptake, which increases mitochondrial respiration and metabolism, further preventing apoptosis and regulating autophagy. Canonical signaling pathways include the ERK and Akt axes, and are shown in red, whereas the noncanonical G protein pathway is shown in blue. Both pathways interact as Ca2+ contributes to ERK activation and additionally both Akt and ERK can compensate each other’s activation. Abbreviations: MEK, Mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; PIP2, phosphatidylinositol 4,5 biphosphate.
Figure 2. not shown. Classical versus proposed models of nuclear Ca2+ signaling in cardiomyocytes. The insulin-like growth factor 1 receptor (IGF-1R) can specifically regulate nuclear Ca2+ signaling independently of the role of Ca2+ on excitation–contraction coupling. On the classic model, inositol 1,4,5 triphosphate (InsP3; IP3) produced after IGF-1R activation travels from the peripheral plasma membrane to the nucleus, where it activates InsP3 receptors. In this model InsP3 bypasses its receptors present on the sarcoplasmic reticulum, which would lead to cytosolic Ca2+ signals. The novel model that we propose is based on recent findings, where the IGF-1R signaling complex is present in T-tubule invaginations toward the nucleus. In these compartments, IGF-1R activation leads to locally restricted InsP3 production that allows nuclear Ca2+ signals to regulate gene expression of genes associated with the development of cardiomyocyte hypertrophy. Abbreviations: RyR, ryanodine receptor; ECC, excitation–contraction coupling; PLC, phospholipase C; DHPR, dihydropyridine receptor.
The beneficial roles of IGF-1 in the cardiovascular system largely explain the interest in the development of new IGF-1-based treatments for cardiovascular disease. So far the FDA has approved two drugs for the treatment of IGF-1 deficiency: mecasermin (Increlex1), a human recombinant IGF-1 analog; and mecasermin rinfabate (IPLEX1), a binary protein complex of human recombinant IGF-1 and human recombinant IGBP-3. The safety of a chronic systemic IGF-1 therapy is open to question because it could promote severe adverse effects, such as an increased risk of cancer. To avoid these problems, several researchers have selectively overexpressed IGF-1 and IGF-1R in the heart.
Box 2. Outstanding questionsInsulin-like growth factor 1 (IGF-1) is an old friend of the heart. Despite the well-known protective effects of IGF-1 on cardiac function and the antiapoptotic effects of this peptide, novel evidence opens new questions to this longstanding relationship.
· How do the multiple signaling pathways triggered by IGF-1 receptor (IGF-1R) interact with each other?
· What lies further than extracellular signal-regulated kinase (ERK)/Akt/Ca2+ activation toward heart function?
· Do these signaling pathways regulate cardiac fibroblast or endothelial cell function?
· Which are the specific downstream signaling pathways of the different pools of IGF-1R and their role in regulating cardiomyocyte survival, hypertrophy, metabolism, proliferation?
· What drives IGF-1R to such specific subcellular compartments?
· What is the relevance of the hybrid IGF-1R/insulin receptors on cardiovascular disease?
· Does a crosstalk exist between insulin receptor and IGF-1R in the heart under physiological and pathological conditions?
· Is one pathway more beneficial than the other?
· Will stem cell therapy of cardiac progenitors be able to provide concrete treatment opportunities?
· Is IGF-1 a key regulator of this outcome?
Abundant evidence supports the key physiological roles of IGF-1 in the heart. In cardiomyocytes, IGF-1 activates multiple downstream signaling pathways for controlling cell death, metabolism, autophagy, differentiation, transcription, and protein synthesis (Figure 1). Of great interest are the findings that the entire IGF-1R complex is strategically located in perinuclear sarcolemmal invaginations that locally control nuclear Ca2+ signaling and transcriptional upregulation (Figure 2). This novel evidence changesmthe classical paradigm of IGF-1 signaling and adds a new level of complexity that may be relevant for other signaling receptors in the heart: interorganelle communication between plasma membrane invaginations and the nucleus.
The strategic localization of IGF-1R in these structures and the association with heterotrimeric G proteins may explain the differences in the phenotypic response induced by IGF-1 and others agonists, like endothelin-1 and angiotensin II, that also signal through intracellular Ca2+. By activating a noncanonical, selective mechanism of nuclear Ca2+ release, IGF-1 can regulate the expression of a specific set of cardiac genes via the generation of a particular signal-encoding pattern, leading to adaptive cardiac hypertrophy, antiapoptotic effects, and metabolic adaptation.
Pulmonary Hypertension in Heart Failure with Preserved Ejection Fraction – any Pathophysiological Role of Mitral Regurgitation
read with interest the study by Lam et al. (1) as an important contribution to the pathophysiological and clinical impact of pulmonary hypertension (PH) in hypertensive patients with heart failure and preserved left ventricular ejection fraction (HFpEF). Recent guidelines on arterial PH recognize HFpEF as a growing cause of left-sided PH, but a definitive appreciation of its true prevalence and prognostic relevance is lacking. The present study provides some new important information on this subject.
It is noteworthy that HFpEF was associated, in a high rate of cases (83%), with a typical hemodynamic pattern of precapillary PH, and a strong correlation was found between pulmonary artery systolic pressure and pulmonary capillary wedge pressure. Most important, pulmonary artery systolic pressure, rather than other echocardiography-derived measures of diastolic dysfunction, was the only significant multivariate predictor of mortality, a finding that was confirmed even when combined comorbid diseases potentially contributing to PH development, such as chronic obstructive pulmonary disease, were taken into account.
In patients with systolic heart failure, a major determinant of PH development is mitral regurgitation. Whether mitral regurgitation could be a putative factor in the pathogenesis of PH in HFpEF patients remains an open and intriguing question.
Accordingly, it would be of interest if the authors could provide some details on how many HFpEF patients exhibited mitral regurgitation, especially in comparison with control hypertensive patients without HFpEF.
Lam CSP, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MM. Pulmonary hypertension in heart failure with preserved ejection fraction: a community-based study. J Am Coll Cardiol 2009; 53:1119–23.
Midregion Prohormone Adrenomedullin and Prognosis in Patients Presenting with Acute Dyspnea Results from the BACH (Biomarkers in Acute Heart Failure) Trial
Objectives The aim of this study was to determine the prognostic utility of midregion proadrenomedullin (MR-proADM) in all patients, cardiac and noncardiac, presenting with acute shortness of breath.
Background The recently published BACH (Biomarkers in Acute Heart Failure) study demonstrated that MR-proADM had superior accuracy for predicting 90-day mortality compared with B-type natriuretic peptide (area under the curve: 0.674 vs. 0.606, respectively, p < 0.001) in acute heart failure. Methods The BACH trial was a prospective, 15-center, international study of 1,641 patients presenting to the emergency department with dyspnea. Using this dataset, the prognostic accuracy of MR-proADM was evaluated in all patients enrolled for predicting 90-day mortality with respect to other biomarkers, the added value in addition to clinical variables, as well as the added value of additional measurements during hospital admission. Results Compared with B-type natriuretic peptide or troponin, MR-proADM was superior for predicting 90-day all-cause mortality in patients presenting with acute dyspnea (c index = 0.755, p < 0.0001). Furthermore, MR-proADM added significantly to all clinical variables (all adjusted hazard ratios: HR=3.28), and it was also superior to all other biomarkers. MRproADM added significantly to the best clinical model (bootstrap-corrected c index increase: 0.775 to 0.807; adjusted standardized hazard ratio: 2.59; 95% confidence interval: 1.91 to 3.50; p < 0.0001). Within the model, MR-proADM was the biggest contributor to the predictive performance, with a net reclassification improvement of 8.9%. Serial evaluation of MR-proADM performed in patients admitted provided a significant added value compared with a model with admission values only (p< 0.0005). More than one-third of patients originally at high risk could be identified by the biomarker evaluation at discharge as low-risk patients. Conclusions MR-proADM identifies patients with high 90-day mortality and adds prognostic value to natriuretic peptides in patients presenting with acute shortness of breath. Serial measurement of this biomarker may also prove useful for monitoring, although further studies will be required. (Biomarkers in Acute Heart Failure [BACH]; NCT00537628)
Invasive Hemodynamic Characterization of Heart Failure with Preserved Ejection Fraction
Invasive hemodynamic assessment in heart failure with preserved ejection fraction (HFpEF) was originally a primary research tool to advance the understanding of the pathophysiology of HFpEF.
The role of invasive hemodynamic assessment in HFpEF is expanding to the diagnostic arena where invasive assessment offers a robust, sensitive, and specific way to diagnose or exclude HFpEF in patients with unexplained dyspnea and normal ejection fraction.
In future years, invasive hemodynamic profiling may more rigorously phenotype patients to individualized therapy and, potentially, deliver novel device-based structural interventions.
The circulatory system serves to deliver substrates to the body via the bloodstream while removing the byproducts of cellular metabolism. Hemodynamics broadly refers to the study of the forces involved in the circulation of blood, which are governed by to the physical properties of the heart and vasculature and their dynamic regulation by the autonomic nervous system.
Afterload represents the forces opposing ventricular ejection and can be quantified by systolic left ventricular (LV) wall stress and aortic input impedance or its individual components (resistance, compliance, characteristic impedance). Wall stress is inconvenient because it depends on heart size and geometry, whereas impedance is cumbersome because it is a frequency-domain parameter that cannot be easily coupled with time-domain measures of ventricular function. Effective arterial elastance (Ea), defined by the ratio of LV end-systolic pressure (ESP) to stroke volume, provides a robust measure of total arterial load. Ea is not a directly measured parameter but, instead, a net or lumped stiffness of the vasculature that incorporates both mean and oscillatory components of afterload (Fig. 1). Preload reflects the degree of myofiber stretch before the onset of contraction, which, in turn, dictates the force and velocity of contraction according to the Frank-Starling principle. In everyday practice, preload is often conceptualized as equivalent to LV filling pressures. However, in fact, preload is most accurately reflected by the LV volume at end-diastole volume (EDV). Filling pressures are related to EDV by the LV diastolic chamber stiffness, which differs in healthy volunteers and subjects with HFpEF.
Fig. 1. Not shown. Ventricular-arterial coupling in the pressure-volume plane. Pressure volume loop at steady state is shown in dark black. The area subtended by the loop (shaded) represents the stroke work. Stroke volume is the difference between end-diastolic volume (EDV) and end-systolic volume (ESV). Ea is defined by the negative slope connecting the ESP and ESV coordinates with EDV and pressure = 0. With acute preload reduction (dotted line loops) there is progressive reduction in EDV, ESV, and ESP. The linear slope of the endsystolic pressure volume relationship (ESPVR) is LV end-systolic elastance (Ees). The curvilinear slope of the end diastolic pressure–volume relationship (EDVPR) is derived by fitting pressure volume coordinates measured during diastasis to the equation shown. The exponential power or stiffness constant (b) obtained is a measure of LV diastolic stiffness. (Adapted from Borlaug BA, Kass DA. Invasive hemodynamic assessment in heart failure. Heart Fail Clin 2009;5(2):217–28; with permission.)
Fig. 3. Not shown. Left ventricular diastolic reserve in HFpEF. In the normal healthy adult, the rate of LV pressure decay during isovolumic contraction (t) is rapid and increases markedly during exercise in association with a reduction in LVmin, allowing for suction of blood into the LV, with no increase in left atrial pressure or LV end-diastolic pressure (LVEDP) despite an increase in LV end-diastolic volume and marked shortening of the cycle length. In HFpEF, relaxation is prolonged at baseline (increased t) with inadequate hastening (shortening of t) during exercise, contributing to an inability to reduce LVmin and, consequently, a complete lack of suction effects. LV filling then completely depends on left atrial hypertension, which develops in tandem with marked elevation in LVEDP. (Data from Borlaug BA, Jaber WA, Ommen SR, et al. Diastolic relaxation and compliance reserve during dynamic exercise in heart failure with preserved ejection fraction. Heart 2011;97(12):964–9.)
Fig. 4. Preload and filling pressures in HFpEF. (A) Cumulative distribution plot shows that acute changes in stroke volume with nitroprusside infusion are lower in HFpEF (black) compared with HFrEF (red). Because afterload (Ea) is lowered, any acute reduction in SV must be related to reduction in preload volume (EDV) and nearly 40% of HFpEF patients experienced stroke volume reduction with nitroprusside, despite high filling pressures (PCWP 20–25 mm Hg), indicating increased reliance on high pressures to achieve adequate EDV. *p<0.0001 compared with HFrEF. (B) LVEDP in a healthy adult (blue) and in a HFpEF patient with increased LV diastolic stiffness (green). At the same preload (EDV), pressure is more than twofold higher in HFpEF. In contrast, at the same LV diastolic pressure (15 mm Hg), LV volume is much lower in HFpEF, indicating decreased LV diastolic capacitance. V15, volume at end-diastolic pressure = 15 mm Hg; LVEDP. (Adapted from Schwartzenberg S, Redfield MM, From AM, et al. Effects of vasodilation in heart failure with preserved or reduced ejection fraction implications of distinct pathophysiologies on response to therapy. J Am Coll Cardiol 2012;59(5):442–51; with permission.)
Updated Clinical Classification of Pulmonary Hypertension
The aim of a clinical classification of pulmonary hypertension (PH) is to group together different manifestations of disease sharing similarities in pathophysiologic mechanisms, clinical presentation, and therapeutic approaches. In 2003, during the 3rd World Symposium on Pulmonary Hypertension, the clinical classification of PH initially adopted in 1998 during the 2nd World Symposium was slightly modified. During the 4th World Symposium held in 2008, it was decided to maintain the general architecture and philosophy of the previous clinical classifications. The modifications adopted during this meeting principally concern Group 1, pulmonary arterial hypertension (PAH). This subgroup includes patients with PAH with a family history or patients with idiopathic PAH with germline mutations (e.g., bone morphogenetic protein receptor-2, activin receptor-like kinase type 1, and endoglin). In the new classification, schistosomiasis and chronic hemolytic anemia appear as separate entities in the subgroup of PAH associated with identified diseases. Finally, it was decided to place pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis in a separate group, distinct from but very close to Group 1 (now called Group 1=). Thus, Group 1 of PAH is now more homogeneous. (J Am Coll Cardiol 2009; 54: S43–54) Updated Evidence-Based Treatment Algorithm in Pulmonary Arterial Hypertension
Robyn J. Barst, J. Simon R. Gibbs, Hossein A. Ghofrani, et al.
J Am Coll Cardiol 2009; 54(1), Suppl S,
Uncontrolled and controlled clinical trials with different compounds and procedures are reviewed to define the risk benefit profiles for therapeutic options in pulmonary arterial hypertension (PAH). A grading system for the level of evidence of treatments based on the controlled clinical trials performed with each compound is used to propose an evidence-based treatment algorithm. The algorithm includes drugs approved by regulatory agencies for the treatment of PAH and/or drugs available for other indications. The different treatments have been evaluated mainly in idiopathic PAH, heritable PAH, and in PAH associated with the scleroderma spectrum of diseases or with anorexigen use. Extrapolation of these recommendations to other PAH subgroups should be done with caution. Oral anticoagulation is proposed for most patients; diuretic treatment and supplemental oxygen are indicated in cases of fluid retention and hypoxemia, respectively. High doses of calcium-channel blockers are indicated only in the minority of patients who respond to acute vasoreactivity testing. Nonresponders to acute vasoreactivity testing or responders who remain in World Health Organization (WHO) functional class III, should be considered candidates for treatment with either an oral phosphodiesterase-5 inhibitor or an oral endothelin-receptor antagonist. Continuous intravenous administration of epoprostenol remains the treatment of choice in WHO functional class IV patients. Combination therapy is recommended for patients treated with PAH monotherapy who remain in WHO functional class III. Atrial septostomy and lung transplantation are indicated for refractory patients or where medical treatment is unavailable. (J Am Coll Cardiol 2009;54:S78–84)
Inhibition and down-regulation of gene transcription and guanylyl cyclase activity of NPRA by angiotensin II involving protein kinase C
The objective of this study was to investigate the role of protein kinase C (PKC) in the angiotensin II (Ang II)-dependent repression of Npr1 (coding for natriuretic peptide receptor-A, NPRA) gene transcription. Mouse mesangial cells (MMCs) were transfected with Npr1 gene promoter-luciferase construct and treated with Ang II and PKC agonist or antagonist. The results showed that the treatment of MMCs with 10 nM Ang II produced a 60% reduction in the promoter activity of Npr1 gene. MMCs treated with 10 nM Ang II exhibited 55% reduction in NPRA mRNA levels, and subsequent stimulation with 100 nM ANP resulted in 50% reduction in guanylyl cyclase (GC) activity. Furthermore, the treatment of MMCs with Ang II in the presence of PKC agonist phorbol ester (100 nM) produced an almost 75% reduction in NPRA mRNA and 70% reduction in the intracellular accumulation of cGMP levels. PKC antagonist staurosporine completely reversed the effect of Ang II and phorbol ester. This is the first report to demonstrate that ANG II-dependent transcriptional repression of Npr1 gene promoter activity and down-regulation of GC activity of translated protein, NPRA is regulated by PKC pathways.
Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene
Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional regulation of Npr1 gene (coding for NPRA).Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Over-expression of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold. However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50% and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level.
Several agents that are known to upregulate Ets-1 transcription, include RA, TNF-alpha, VEGF, and TPA. Ets-1 is upregulated at exposure to agonists such as serum in vitro and is expressed in injured vasculature. MAPK-mediated phosphorylation positively regulates the transcriptional activation functions of Ets-1 by recruiting CBP/p300. Not much is known about Ets-1 expression or regulation in mesangial cells. A temporal increase of mesangial cell Ets-1 expression has been reported which correlates with mesangial cell activation
in mesangioproliferative glomerulonephritis suggesting involvement of PDGF-B. There might be a possibility that during glomerulonephritis increased Ets-1 expression upregulates Npr1 gene as a protective mechanism. Npr1 gene has been shown to negatively regulate mitogen-activated protein kinase and proliferation of mesangial cells.
In conclusion, our results demonstrate that the precise control of Npr1 gene transcriptional activity is achieved through a synergy of activators and repressors in which Ets-1 plays an integral role as a transcriptional activator. Comparatively, Lyf-1 and GATA-1 act as repressors, inhibiting and regulating the transcriptional activity of Npr1 gene promoter. The present findings suggest that Ets-1 plays a critical role in enhancing Npr1 gene transcription and may have an important influence in hypertension and cardiovascular homeostasis at the molecular level.
Krüppel-like transcription factor 11 (KLF11) overexpression inhibits cardiac hypertrophy and fibrosis in mice
The Krüppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins. The KLF family member KLF11 is originally identified as a transforming growth factor b (TGF-b)-inducible gene and is one of the most studied in this family. KLF11 is expressed ubiquitously and participates in diabetes and regulates hepatic lipid metabolism. However, the role of KLF11 in cardiovascular system is largely unknown. Here in this study, we reported that KLF11 expression is down-regulated in failing human hearts and hypertrophic murine hearts. To evaluate the roles of KLF11 in cardiac hypertrophy, we generated cardiac-specific KLF11 transgenic mice. KLF11 transgenic mice do not show any difference from their littermates at baseline. However, cardiac-specific KLF11 overexpression protects mice from TAC-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observe lower expression of hypertrophic fetal genes in TAC-challenged KLF11 transgenic mice compared with WT mice. In addition, KLF11 reduces cardiac fibrosis in mice underwent hypertrophy. The expression of fibrosis markers are also down-regulated when KLF11 is overexpressed in TAC-challenged mice. Taken together, our findings identify a novel anti-hypertrophic and anti-fibrotic role of KLF11, and KLF11 activator may serve as candidate drug for heart failure patients.
Complex Models of Signaling: Therapeutic Implications
Curator: Larry H. Bernstein, MD, FCAP
Updated 6/24/2019
Fishy Business: Effect of Omega-3 Fatty Acids on Zinc Transporters and Free Zinc Availability in Human Neuronal Cells
Damitha De Mel and Cenk Suphioglu *
NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Waurn Ponds, Victoria, Australia.
Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,
DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.
DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.
On the other hand,
zinc is the most abundant trace metal in the human brain.
There are many scientific studies linking zinc, especially
excess amounts of free zinc, to cellular death.
Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between
omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.
Many other studies have also suggested a possible
omega-3 and zinc effect on neurodegeneration and cellular death.
Therefore, in this review, we will examine
the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.
Moreover, we will evaluate the collective understanding of
mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.
Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to
reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in
Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as
cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.
Despite the significance of these
two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both
omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].
the reverse process can be seen with increased omega-3 fatty acids in the body.
Many other factors, such as
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,
which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.
Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in
immune responses,
gene expression and
intercellular communications.
omega-3 fatty acids are known to be vital in
the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels
.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.
The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).
In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between
increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels
Table 1. Total percentage of omega-3 fatty acids in common foods and supplements.
Food/Supplement
EPA
DHA
ALA
Total %
Fish
SalmonSardine
Anchovy
Halibut
Herring
Mackerel
Tuna
Fresh Bluefin
XX
X
X
X
X
X
X
XX
X
X
X
X
X
X
>50%>50%
>50%
>50%
>50%
>50%
>50%
>50%
Oils/Supplements
Fish oil capsulesCod liver oils
Salmon oil
Sardine oil
XX
X
X
XX
X
X
>50%>50%
>50%
>50%
Black currant oilCanola oil Mustard seed oils
Soybean oil
Walnut oil
Wheat germ oil
XX
X
X
X
X
10%–50%10%–50%
10%–50%
10%–50%
10%–50%
10%–50%
Seeds and other foods
Flaxseeds/LinseedsSpinach
Wheat germ Human milk
Peanut butter
Soybeans
Olive oil
Walnuts
XX
X
X
X
X
X
X
>50%>50%
10%–50%
10%–50%
<10%
<10%
<10%
<10%
Table adopted from Maclean C.H. et al. [18].
In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that
only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline
In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown
a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.
A trial of subjects with mild memory complaints demonstrated
an improvement with 900 mg of DHA.
We review key findings on
the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.
DHA is the most abundant fatty acid in neural membranes, imparting appropriate
fluidity and other properties,
and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated
in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.
In adult rats’ brain, DHA comprises approximately
17% of the total fatty acid weight, and
in the retina it is as high as 33%.
DHA is believed to have played a major role in the evolution of the modern human –
in particular the well-developed brain.
Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.
Analysis of human cadaver brains have shown that
people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals
Furthermore, studies in mice have increased support for the
protective role of omega-3 fatty acids.
Mice administrated with a dietary intake of DHA showed
an increase in DHA levels in the hippocampus.
Errors in memory were decreased in these mice and they demonstrated
reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.
Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary
DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.
Zinc
Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to
growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process
and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as
a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.
Approximately 3% of all proteins contain
zinc binding motifs .
The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;
catalytic,
coactive and
Indeed, it is the only metal found in all six different subclasses
of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.
Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated
through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.
Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and
its release is dependent on neural activity.
Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.
The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to
form insoluble amyloid in the presence of
high physiological levels of zinc.
Insoluble amyloid is thought to
aggregate to form plaques,
which is a main pathological feature of AD. Further studies have shown that
chelation of zinc ions can deform and disaggregate plaques.
In AD, the most prominent injuries are found in
hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.
All of these neurons are known to favor
rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.
This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of
inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).
High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.
If the human diet is deficient in zinc, the body
efficiently conserves zinc at the tissue level by compensating other cellular mechanisms
to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and
redistribution of available zinc to more zinc dependent cells or organs.
A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the
assessment of the zinc status of individuals and populations.
In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting
as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters
Zinc Transporters
Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur
via intermembrane proteins and zinc transporter (ZnT) proteins
These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).
Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).
The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where
it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,
Figure 1. Putative cellular localization of some of the different human zinc transporters (i.e., Zip1- Zip4 and ZnT1- ZnT7). Arrows indicate the direction of zinc passage by the appropriate putative zinc transporters in a generalized human cell. Although there are fourteen Zips and eight ZnTs known so far, only the main zinc transporters are illustrated in this figure for clarity and brevity.
Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg
zinc transporters
Early zinc signaling (EZS) and late zinc signaling (LZS)
Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].
DHA and Zinc Homeostasis
Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in
increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.
Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,
a significant reduction in plaque load
and the presence of insoluble amyloid were observed.
In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant
reduction in free zinc availability in the hippocampus
and cerebral cortex.
Collectively, the findings from this study are very interesting and indicate a clear connection between
zinc availability and amyloid plaque formation,
thus indicating a possible link to AD.
DHA supplementation has also been reported to limit the following:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.
In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.
In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.
When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible
neuroprotective mechanism of DHA,
which we believe is exerted through
a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.
DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and
free zinc levels in the cytosol were significantly low compared to the control
This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had
low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.
These findings are consistent with previous published data and further strengthen the possible
correlation between zinc, DHA and neurodegeneration.
On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of
ZnT3 in memory and AD pathology.
For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of
ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory
Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.
Collectively from these studies, the following possible mechanism can be proposed (Figure 2).
possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter
Figure 2. Proposed neuroprotection mechanism of docosahexaenoic acid (DHA) in reference to synaptic zinc. Schematic diagram showing possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter expression levels in human neuronal cells, which results in a reduction of zinc flux and thus lowering zinc concentrations in neuronal synaptic vesicles, and therefore contributing to a lower incidence of neurodegenerative diseases (ND), such as Alzheimer’s disease (AD).
More recent data from our research group have also shown a link between the expression levels of histone H3 and H4 proteins in human neuronal cells in relation to DHA and zinc. Following DHA treatment, both H3 and H4 levels were up-regulated. In contrast, zinc treatment resulted in a down-regulation of histone levels. Both zinc and DHA have shown opposing effects on histone post-translational modifications, indicating a possible distinctive epigenetic pattern. Upon treatment with zinc, M17 cells displayed an increase in histone deacetylase (HDACs) and a reduction in histone acetylation. Conversely, with DHA treatment, HDAC levels were significantly reduced and the acetylation of histones was up-regulated. These findings also support a possible interaction between DHA and zinc availability.
Conclusions
It is possible to safely claim that there is more than one potential pathway by which DHA and zinc interact at a cellular level, at least in cultured human neuronal cells. Significance and importance of both DHA and zinc in neuronal survival is attested by the presence of these multiple mechanisms.
Most of these reported studies were conducted using human neuroblastoma cells, or similar cell types, due to the lack of live mature human neuronal cells. Thus, the results may differ from results achieved under actual human physiological conditions due to the structural and functional differences between these cells and mature human neurons. Therefore, an alternative approach that can mimic the human neuronal cells more effectively would be advantageous.
Sphingosine-1-phosphate signaling as a therapeutic target
E Giannoudaki, DJ Swan, JA Kirby, S Ali
Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
Cell Health and Cytoskeleton 2012; 4: 63–72
S1P is a 379Da member of the lysophospholipid family. It is the direct metabolite of sphingosine through the action of two sphingosine kinases, SphK1 and SphK2. The main metabolic pathway starts with the hydrolysis of sphingomyelin, a membrane sphingolipid, into ceramide by the enzyme sphingomyelinase and the subsequent production of sphingosine by ceramidase (Figure 1). Ceramide can also be produced de novo in the endoplasmic reticulum (ER) from serine and palmitoyl coenzyme A through multiple intermediates. S1P production is regulated by various S1P-specific and general lipid phosphatases, as well as S1P lyase, which irreversibly degrades S1P into phosphoethanolamine and hexadecanal. The balance between intracellular S1P and its metabolite ceramide can determine cellular fate. Ceramide promotes apoptosis, while S1P suppresses cell death and promotes cell survival. This creates an S1P ceramide “rheostat” inside the cells. S1P lyase expression in tissue is higher than it is in erythrocytes and platelets, the main “suppliers” of S1P in blood. This causes a tissue–blood gradient of S1P, which is important in many S1P-mediated responses, like the lymphocyte egress from lymphoid organs.
S1P signaling overview
S1P is produced inside cells; however, it can also be found extracellularly, in a variety of different tissues. It is abundant in the blood, at concentrations of 0.4–1.5 μM, where it is mainly secreted by erythrocytes and platelets. Blood S1P can be found separately, but mainly it exists in complexes with high-density lipoprotein (HDL) (∼60%). Many of the cardioprotective effects of HDL are hypothesized to involve S1P. Before 1996, S1P was thought to act mainly intracellularly as a second messenger. However, the identification of several GPCRs that bind S1P led to the initiation of many studies on
extracellular S1P signaling through those receptors.
There are five receptors that have been identified currently. These can be coupled with different G-proteins. Assuming that each receptor coupling with a G protein has a slightly different function, one can recognize the complexity of S1P receptor signaling.
S1P as a second messenger
S1P is involved in many cellular processes through its GPCR signaling; studies demonstrate that S1P also acts at an intracellular level. Intracellular S1P plays a role in maintaining the balance of cell survival signal toward apoptotic signals, creating a
cell “rheostat” between S1P and its precursor ceramide.
Important evidence that S1P can act intracellularly as a second messenger came from yeast (Saccharomyces cerevisiae) and plant (Arabidopsis thaliana) cells. Yeast cells do not express any S1P receptors, although they can be affected by S1P during heat-shock responses. Similarly, Arabidopsis has only one GPCR-like protein, termed “GCR1,” which does not bind S1P, although S1P regulates stomata closure during drought.
Sphingosine-1-phosphate
In mammals, the sphingosine kinases have been found to localize in different cell compartments, being responsible for the accumulation of S1P in those compartments to give intracellular signals. In mitochondria, for instance,
S1P was recently found to interact with prohibitin 2,
a conserved protein that maintains mitochondria assembly and function. According to the same study,
SphK2 is the major producer of S1P in mitochondria and the knockout of its gene can cause
disruption of mitochondrial respiration and cytochrome c oxidase function.
SphK2 is also present in the nucleus of many cells and has been implicated to cause cell cycle arrest, and it causes S1P accumulation in the nucleus. It seems that nuclear S1P is affiliated with the histone deacetylases HDAC1 and HDAC2,
inhibiting their activity, thus having an indirect effect in epigenetic regulation of gene expression.
In the ER, SphK2 has been identified to translocate during stress, and promote apoptosis. It seems that S1P has specific targets in the ER that cause apoptosis, probably through calcium mobilization signals.
Sphingosine 1-phosphate (S1P) is a small bioactive lipid molecule that is involved in several processes both intracellularly and extracellularly. It acts intracellularly
to promote the survival and growth of the cell,
through its interaction with molecules in different compartments of the cell.
It can also exist at high concentrations extracellularly, in the blood plasma and lymph. This causes an S1P gradient important for cell migration. S1P signals through five G protein-coupled receptors, S1PR1–S1PR5, whose expression varies in different types of cells and tissue. S1P signaling can be involved in physiological and pathophysiological conditions of the cardiovascular, nervous, and immune systems and diseases such as ischemia/reperfusion injury, autoimmunity, and cancer. In this review, we discuss how it can be used to discover novel therapeutic targets.
The involvement of S1P signaling in disease
In a mouse model of myocardial ischemia-reperfusion injury (IRI), S1P and its carrier, HDL, can help protect myocardial tissue and decrease the infarct size. It seems they reduce cardiomyocyte apoptosis and neutrophil recruitment to the ischemic tissue and may decrease leukocyte adhesion to the endothelium. This effect appears to be S1PR3 mediated, since in S1PR3 knockout mice it is alleviated.
Ischemia activates SphK1, which is then translocated to the plasma membrane. This leads to an increase of intracellular S1P, helping to promote cardiomyocyte survival against apoptosis, induced by ceramide. SphK1 knockout mice cannot be preconditioned against IRI, whereas SphK1 gene induction in the heart protects it from IRI. Interestingly, a recent study shows SphK2 may also play a role, since its knockout reduces the cardioprotective effects of preconditioning. Further, administration of S1P or sphingosine during reperfusion results in better recovery and attenuation of damage to cardiomyocytes. As with preconditioning, SphK1 deficiency also affects post-conditioning of mouse hearts after ischemia reperfusion (IR).
S1P does not only protect the heart from IRI. During intestinal IR, multiple organs can be damaged, including the lungs. S1P treatment of mice during intestinal IR seems to have a protective effect on lung injury, probably due to suppression of iNOS-induced nitric oxide generation. In renal IRI, SphK1 seems to be important, since its deficiency increased the damage in kidney tissue, whereas the lentiviral overexpression of the SphK1 gene protected from injury. Another study suggests that, after IRI, apoptotic renal cells release S1P, which recruits macrophages through S1PR3 activation and might contribute to kidney regeneration and restoration of renal epithelium. However, SphK2 is negatively implicated in hepatic IRI, its inhibition helping protect hepatocytes and restoring mitochondrial function.
Further studies are implicating S1P signaling or sphingosine kinases in several kinds of cancer as well as autoimmune diseases.
Figure 2 FTY720-P causes retention of T cells in the lymph nodes.
Notes: C57BL/6 mice were injected with BALB/c splenocytes in the footpad to create an allogenic response then treated with FTY720-P or vehicle every day on days 2 to 5. On day 6, the popliteal lymph nodes were removed. Popliteal node-derived cells were mixed with BALB/c splenocytes in interferon gamma (IFN-γ) cultured enzyme-linked immunosorbent spot reactions. Bars represent the mean number of IFN-γ spot-forming cells per 1000 popliteal node-derived cells, from six mice treated with vehicle and seven with FTY720-P. **P , 0.01. (not shown)
Fingolimod (INN, trade name Gilenya, Novartis) is an immunomodulating drug, approved for treating multiple sclerosis. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by over half. Fingolimod is a sphingosine-1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from contributing to an autoimmune reaction.
The S1P antagonist FTY720 has been approved by the US Food and Drug Administration to be used as a drug against multiple sclerosis (MS). FTY720 is in fact a prodrug, since it is phosphorylated in vivo by SphK2 into FTY720-P, an S1P structural analog, which can activate S1PR1, 3, 4, and 5. FTY720-P binding to S1PR1 causes internalization of the receptor, as does S1P – but instead of recycling it back to the cell surface, it promotes its ubiquitination and degradation at the proteasome. This has a direct effect on lymphocyte trafficking through the lymph nodes, since it relies on S1PR1 signaling and S1P gradient (Figure 2). In MS, it stops migrating lymphocytes into the brain, but it may also have direct effects on the CNS through neuroprotection. FTY720 can pass the blood–brain barrier and it could be phosphorylated by local sphingosine kinases to act through S1PR1 and S1PR3 receptors that are mainly expressed in the CNS. In MS lesions, astrocytes upregulate those two receptors and it has been shown that FTY720-P treatment in vitro inhibits astrocyte production of inflammatory cytokines. A recent study confirms the importance of S1PR3 signaling on activated astrocytes, as well as SphK1, that are upregulated and promote the secretion of the potentially neuroprotective cytokine CXCL-1.
There are several studies implicating the intracellular S1P ceramide rheostat to cancer cell survival or apoptosis and resistance to chemotherapy or irradiation in vitro. Studies with SphK1 inhibition in pancreatic, prostate cancers, and leukemia, show increased ceramide/S1P ratio and induction of apoptosis. However, S1P receptor signaling plays conflicting roles in cancer cell migration and metastasis.
Modulation of S1P signaling: therapeutic potential
S1P signaling can be involved in many pathophysiological conditions. This means that we could look for therapeutic targets in all the molecules taking part in S1P signaling and production, most importantly the S1P receptors and the sphingosine kinases. S1P agonists and antagonists could also be used to modulate S1P signaling during pathological conditions.
S1P can have direct effects on the cardiovascular system. During IRI, intracellular S1P can protect the cardiomyocytes and promote their survival. Pre- or post-conditioning of the heart with S1P could be used as a treatment, but upregulation of sphingosine kinases could also increase intracellular S1P bioavailability. S1P could also have effects on endothelial cells and neutrophil trafficking. Vascular endothelial cells mainly express S1PR1 and S1PR3; only a few types express S1PR2. S1PR1 and S1PR3 activation on these cells has been shown to enhance their chemotactic migration, probably through direct phosphorylation of S1PR1 by Akt, in a phosphatidylinositol 3-kinase and Rac1-dependent signaling pathway. Moreover, it stimulates endothelial cell proliferation through an ERK pathway. S1PR2 activation, however, inhibits endothelial cell migration, morphogenesis, and angiogenesis, most likely through Rho-dependent inhibition of Rac signaling pathway, as Inoki et al showed in mouse cells with the use of S1PR1 and S1PR3 specific antagonists.
Regarding permeability of the vascular endothelium and endothelial barrier integrity, S1P receptors can have different effects. S1PR1 activation enhances endothelial barrier integrity by stimulation of cellular adhesion and upregulation of adhesion molecules. However, S1PR2 and S1PR3 have been shown to have barrier-disrupting effects in vitro, and vascular permeability increasing effects in vivo. All the effects S1P can have on vascular endothelium and smooth muscle cells suggest that activation of S1PR2, not S1PR1 and S1PR3, signaling, perhaps with the use of S1PR2 specific agonists, could be used therapeutically to inhibit angiogenesis and disrupt vasculature, suppressing tumor growth and progression.
An important aspect of S1P signaling that is being already therapeutically targeted, but could be further investigated, is immune cell trafficking. Attempts have already been made to regulate lymphocyte cell migration with the use of the drug FTY720, whose phosphorylated form can inhibit the cells S1PR1-dependent egress from the lymph nodes, causing lymphopenia. FTY720 is used as an immunosuppressant for MS but is also being investigated for other autoimmune conditions and for transplantation. Unfortunately, Phase II and III clinical trials for the prevention of kidney graft rejection have not shown an advantage over standard therapies. Moreover, FTY720 can have some adverse cardiac effects, such as bradycardia. However, there are other S1PR1 antagonists that could be considered instead, including KRP-203, AUY954, and SEW2871. KRP-203 in particular has been shown to prolong rat skin and heart allograft survival and attenuate chronic rejection without causing bradycardia, especially when combined with other immunomodulators.
There are studies that argue S1P pretreatment has a negative effect on neutrophil chemotaxis toward the chemokine CXCL-8 (interleukin-8) or the potent chemoattractant formyl-methionyl-leucyl-phenylalanine. S1P pretreatment might also inhibit trans-endothelial migration of neutrophils, without affecting their adhesion to the endothelium. S1P effects on neutrophil migration toward CXCL-8 might be the result of S1PRs cross-linking with the CXCL-8 receptors in neutrophils, CXCR-1 and CXCR-2. Indeed, there is evidence suggesting S1PR4 and S1PR3 form heterodimers with CXCR-1 in neutrophils. Another indication that S1P plays a role in neutrophil trafficking is a recent paper on S1P lyase deficiency, a deficiency that impairs neutrophil migration from blood to tissue in knockout mice.
S1P lyase and S1PRs in neutrophils may be new therapeutic targets against IRI and inflammatory conditions in general. Consistent with these results, another study has shown that inhibition of S1P lyase can have a protective effect on the heart after IRI and this effect is alleviated when pretreated with an S1PR1 and S1PR3 antagonist. Inhibition was achieved with a US Food and Drug Administration-approved food additive, 2-acetyl-4-tetrahydroxybutylimidazole, providing a possible new drug perspective. Another S1P lyase inhibitor, LX2931, a synthetic analog of 2-acetyl-4-tetrahydroxybutylimidazole, has been shown to cause peripheral lymphopenia when administered in mice, providing a potential treatment for autoimmune diseases and prevention of graft rejection in transplantation. This molecule is currently under Phase II clinical trials in rheumatoid arthritis patients.
S1P signaling research has the potential to discover novel therapeutic targets. S1P signaling is involved in many physiological and pathological processes. However, the complexity of S1P signaling makes it necessary to consider every possible pathway, either through its GPCRs, or intracellularly, with S1P as a second messenger. Where the activation of one S1P receptor may lead to the desired outcome, the simultaneous activation of another S1P receptor may lead to the opposite outcome. Thus, if we are to target a specific signaling pathway, we might need specific agonists for S1P receptors to activate one S1P receptor pathway, while, at the same time, we might need to inhibit another through S1P receptor antagonists.
Evidence of sphingolipid signaling in cancer
Biologically active lipids are important cellular signaling molecules and play a role in cell communication and cancer cell proliferation, and cancer stem cell biology. A recent study in ovarian cancer cell lines shows that exogenous sphingosine 1 phosphate (SIP1) or overexpression of the sphingosine kinase (SPHK1) increases ovarian cancer cell proliferation, invasion and contributes to cancer stem cell like phenotype. The diabetes drug metformin was shown to be an inhibitor of SPHK1 and reduce ovarian cancer tumor growth.
Mol Cancer Res. 2019 Apr;17(4):870-881. doi: 10.1158/1541-7786.MCR-18-0409. Epub 2019 Jan 17.
SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.
The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.
Nrf2:INrf2(Keap1) Signaling in Oxidative Stress
James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD
Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that
controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins.
This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for
Cul3/Rbx1 mediated degradation of Nrf2.
In response to oxidative/electrophilic stress,
Nrf2 is switched on and then off by distinct
early and delayed mechanisms.
Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure
to switch on nuclear import of Nrf2.
This is followed by activation of a delayed mechanism that controls
switching off of Nrf2 activation of gene expression.
GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to
nuclear localization of Fyn.
Fyn phosphorylates Nrf2tyrosine568 resulting in
nuclear export of Nrf2,
binding with INrf2 and
degradation of Nrf2.
The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.
NPRA-mediated suppression of AngII-induced ROS production contributes to the antiproliferative effects of B-type natriuretic peptide in VSMC
Pan Gao, De-Hui Qian, Wei Li, Lan Huang
Mol Cell Biochem (2009) 324:165–172
Excessive proliferation of vascular smooth cells (VSMCs) plays a critical role in the pathogenesis of diverse vascular disorders, and inhibition of VSMCs proliferation has been proved to be beneficial to these diseases.
In this study, we investigated the antiproliferative effect of
B-type natriuretic peptide (BNP), a natriuretic peptide with potent antioxidant capacity,
on rat aortic VSMCs, and the possible mechanisms involved. The results indicate that
BNP potently inhibited Angiotensin II (AngII)-induced VSMCs proliferation,
as evaluated by [3H]-thymidine incorporation assay. Consistently, BNP significantly decreased
AngII-induced intracellular reactive oxygen species (ROS)
and NAD(P)H oxidase activity.
8-Br-cGMP, a cGMP analog,
mimicked these effects.
To confirm its mechanism, siRNA of natriuretic peptide receptor-A(NRPA) strategy technology was used
to block cGMP production in VSMCs, and
siNPRA attenuated the inhibitory effects of BNP in VSMCs.
Taken together, these results indicate that
BNP was capable of inhibiting VSMCs proliferation by
NPRA/cGMP pathway,
which might be associated with
the suppression of ROS production.
These results might be related, at least partly, to the anti-oxidant property of BNP.
Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics
A Alleaume-Butaux, C Dakowski, M Pietri, S Mouillet-Richard, Jean-Marie Launay, O Kellermann, B Schneider
1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris, 3Public Hospital of Paris, Department of Biochemistry, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland
Cell Health and Cytoskeleton 2013; 5: 1–12
Neuritogenesis is a complex morphological phenomena accompanying neuronal differentiation. Neuritogenesis relies on the initial breakage of the rather spherical symmetry of neuroblasts and the formation of buds emerging from the postmitotic neuronal soma. Buds then evolve into neurites, which later convert into an axon or dendrites. At the distal tip of neurites, the growth cone integrates extracellular signals and guides the neurite to its target. The acquisition of neuronal polarity depends on deep modifications of the neuroblast cytoskeleton characterized by the remodeling and activation of focal adhesions (FAs) and localized destabilization of the actin network in the neuronal sphere.Actin instability in unpolarized neurons allows neurite sprouting, ie, the protrusion of microtubules, and subsequent neurite outgrowth. Once the neurite is formed, actin microfilaments recover their stability and exert a sheathed action on neurites, a dynamic process necessary for the maintenance and integrity of neurites.
A combination of extrinsic and intrinsic cues pilots the architectural and functional changes in FAs and the actin network along neuritogenesis. This process includes neurotrophic factors (nerve growth factor, brain derived neurotrophic factor, neurotrophin, ciliary neurotrophic factor, glial derived neurotrophic factor) and their receptors, protein components of the extracellular matrix (ECM) (laminin, vitronectin, fibronectin), plasma membrane integrins and neural cell adhesion molecules (NCAM), and intracellular molecular protagonists such as small G proteins (RhoA, Rac, Cdc42) and their downstream targets.
Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps:
(1) neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma;
(2) neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and
(3) the stability and plasticity of neuronal polarity.
In neuronal stem cells, remodeling and activation of focal adhesions (FAs) associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis
by acting on intracellular signaling effectors,
notably small G proteins such as RhoA, Rac, and Cdc42,
which are involved in actin turnover and the dynamics of FAs.
The cellular prion protein (PrPC), a glycosylphosphatidylinositol
(GPI)-anchored membrane protein
mainly known for its role in a group of fatal
neurodegenerative diseases,
has emerged as a central player in neuritogenesis.
Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the
signaling pathways fine-tuned by PrPC
to promote neurite sprouting, outgrowth, and maintenance.
We emphasize that PrPC-dependent neurite sprouting is a process in which PrPC
governs the dynamics of FAs and the actin cytoskeleton
via β1 integrin signaling.
The presence of PrPC is necessary to render neuronal stem cells
competent to respond to neuronal inducers and
to develop neurites.
In differentiating neurons, PrPC exerts
a facilitator role towards neurite elongation.
This function relies on the interaction of PrPC with a set of diverse partners such as
elements of the extracellular matrix,
plasma membrane receptors,
adhesion molecules, and
soluble factors that control actin cytoskeleton turnover through Rho-GTPase signaling.
Once neurons have reached their terminal stage of differentiation and acquired their polarized morphology, PrPC also
takes part in the maintenance of neurites.
By acting on tissue nonspecific alkaline phosphatase, or
Association of heart rate variability and inflammatory response in patients with cardiovascular diseases: current strengths and limitations
V Papaioannou, I Pneumatikos and N Maglaveras
Front Phys 2013. http://dx.doi.org:/10.3389/fphys.2013.00174
A few clinical studies have assessed the possible inter-relation between neuro-autonomic output, estimated with heart rate variability analysis, which is the variability of R-R in the electrocardiogram, and different inflammatory biomarkers, in patients suffering from stable or unstable coronary artery disease (CAD) and heart failure. Moreover, different indices derived from heart rate signals’ processing, have been proven to correlate strongly with severity of heart disease and predict final outcome. In this review article we will summarize major findings from different investigators, evaluating neuro-immunological interactions through heart rate variability analysis, in different groups of cardiovascular patients. We suggest that markers originating from variability analysis of heart rate signals seem to be related to inflammatory biomarkers. Atrial Natriuretic Peptide Frameshift Mutation in Familial Atrial Fibrillation
We mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide–cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability. Impact of anemia on clinical outcome in patients with atrial fibrillation undergoing percutaneous coronary intervention: insights from the AFCAS registry. M Puurunen, T Kiviniemi, W Nammas, A Schlitt, A Rubboli, K Nyman, et al.
BMJ Open 2014; 4:e004700. http://dx.doi.org:/10.1136/bmjopen-2013-004700
The study adds to our knowledge on the prevalence and impact of anemia in patients with AF undergoing PCI and thus requiring combination antithrombotic medication. It shows that anemia is a frequent finding and that even mild anemia has an adverse impact on outcome. Atrial Natriuretic Peptide Single Nucleotide Polymorphisms in Patients with Nonfamilial Structural Atrial Fibrillation. P Francia, A Ricotta, A Frattari, R Stanzione, A Modestino, et al.
Clinical Medicine Insights: Cardiology 2013:7 153–159 http://dx.doi.org:/10.4137/CMC.S12239
We report lack of association between the rs5065 and −G664C ANP gene SNPs and AF in a Caucasian population of patients with structural AF. Further studies will clarify whether these or other ANP gene variants affect the risk of different subpheno-types of AF driven by distinct pathophysiological mechanisms. Gene Expression and Genetic Variation in Human Atria.
We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function.
A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.
Global Left Atrial Strain Correlates with CHADS2 Risk Score in Patients with Atrial Fibrillation SK. Saha, PL. Anderson, G Caracciolo, A Kiotsekoglou, S Wilansky, et al.
Global longitudinal LA strain was reduced in patients with AF compared with controls (P < .001) and was a predictor of high risk for thromboembolism (CHADS2 score > 2; odds ratio, 0.86; P = .02). LA strain indexes showed good interobserver and intraobserver variability. In sequential Cox models, the prediction of hospitalization and/or death was improved by addition of global LA strain and indexed LA volume to CHADS2 score (P = .003).
In this paper, changes in ANS activity are quantified by means of frequency and time domain analysis of R-R interval variability. Electrocardiograms (ECG) of 16 patients suffering from DM and of 16 healthy volunteers were recorded. Frequency domain analysis of extracted normal to normal interval (NN interval) data indicates significant difference in very low frequency (VLF) power, low frequency (LF) power and high frequency (HF) power, between the DM patients and control group. Time domain measures, standard deviation of NN interval (SDNN), root mean square of successive NN interval differences (RMSSD), successive NN intervals differing more than 50 ms (NN50 Count), percentage value of NN50 count (pNN50), HRV triangular index and triangular interpolation of NN intervals (TINN) also show significant difference between the DM patients and control group.
Power Spectral Density of the RR interval of a 55 year old healthy volunteer
Power Spectral Density of the RR interval of a 55 year old healthy volunteer
Power Spectral Density of the RR interval of a 62 year old woman suffering from diabetes for the last 15 years.
Power Spectral Density of the RR interval of a 62 year old woman suffering from diabetes for the last 15 years.
Time domain and frequency domain analysis of the RR interval variability of diabetic and normal subjects shows that there is significant difference in these measures for DM patients with respect to normal subjects. Variation of the HRV parameters indicates changes in ANS activity of DM patients. This can provide valid information regarding autonomic neuropathy in people with diabetes. It may be noted that these methods can detect changes before clinical signs appear.
It is established that the instantaneous heart rate (HR) of healthy humans keeps on changing. Analysis of heart rate variability (HRV) has become a popular non invasive tool for assessing the activities of autonomic nervous system. Depressed HRV has been found in several disorders, like diabetes mellitus (DM) and coronary artery disease, characterised by autonomic nervous dysfunction. A new technique, which searches for pattern repeatability in a time series, is proposed specifically for the analysis of heart rate data. These set of indices, which are termed as pattern repeatability measure and pattern repeatability ratio are compared with approximate entropy and sample entropy.
The cardioautonomic reflexes of 82 diabetic subjects and 40 age and sex matched healthy controls were studied using blood pressure and heart rate variation in response to standing, deep breathing, isometric exercise, cold pressor test and determination of QTc interval. Among the 82 patients, 68 patients were found to have cardiac autonomic neuropathy (CAN). Results showed that diabetics had significantly impaired cardioautonomic reflexes compared to non-diabetics, which increases with the duration of diabetes. Out of 68 patients with CAN, QTc prolongation was observed in 64 patients. In conclusion the autonomic nervous system integrity is appeared to be greatly affected by diabetes mellitus and the degree of impairment was dependent on duration of the disease.
A new index of heart rate variability – HRF Fraction – was developed and its value for risk stratification was evaluated in 480 patients with coronary heart disease. The main purpose to introduce the HRVF was to overcome one of the most important constraints – cardiac arrhythmia, especially atrial fibrillation – that limits use of HRV measurement as a routine clinical tool. In 384 patients with sinus rhythm (SR) and 96 with AF HRV measurements from 24h ambulatory ECG were performed. Patients were followed for a median period of 28 months. The HRV indices in those who died were compared to those who survived. Authors found that HRV Fraction and- among standard time-domain indices- only SDANN, possessed properties that allow HRV measurement to be applied for risk stratification studies in unselected population of patients with cardiac arrhythmia.
Using Pearson correlation, the time domain indicators of parasympathetic activity demonstrated very strong correlations at 3 and 6 months compared with baseline, with good correlations at 1 year. The average SD of all 5-minute RR intervals maintained a very strong correlation for the entire year (r >0.94). In the frequency domain, the measures of parasympathetic and sympathetic activity maintained a solid correlation for the entire study period. Reproducibility of HRV was also examined using repeated-measures analysis of variance. The time and frequency domain parameters demonstrated very little variation over the study period of 12 months. Thus, our investigation demonstrated that HRV in long-term diabetics using 24-hour ambulatory recordings is abnormal and reproducible over a 12-month interval; very little variation in all HRV parameters, especially in parameters of parasympathetic activity, occurred during the study period.
We studied 592 HFpatients who had been hospitalized for HFand were followed
for 18 months. The primary end-point was a composite of all-cause mortality and
HF hospitalization. A doubling of galectin-3 levels was associated with a hazard
ratio (HR) of 1.97 (1.62–2.42) for the primary outcome (P = 0.001). After
correction for age, gender, BNP, eGFR, and diabetes the HR was 1.38 (1.07–
1.78; P = 0.015). Galectin-3 levels were correlated with higher IL-6 and CRP
levels (P = 0.002). Changes of galectin-3 levels after 6 months did not add
prognostic information to the base-line value (n = 291); however, combining
plasma galectin-3 and BNP levels increased prognostic value over either
biomarker alone (ROC analysis, P = 0.05). The predictive value of galectin-3
was stronger in patients with preserved LVEF (n = 114) compared to
patients with reduced LVEF (P = 0.001).
Galectin-3 in Ambulatory Patients with Heart Failure: Results
from the HF-ACTION Study GM Felker, M Fiuzat, LK. Shaw, R Clare, ,DJ. Whellan, et al.
Circ Heart Fail. 2012 Jan; 5(1): 72–78. http://dx.doi.org:/10.1161/CIRCHEARTFAILURE.111.963637
Galectin-3 is a soluble ß-galactoside-binding lectin released by activated
cardiac macrophages. Elevated levels of galectin-3 have been found to
be associated with adverse outcomes in patients with heart failure. We
evaluated the association between galectin-3 and long-term clinical
outcomes in ambulatory heart failure patients enrolled in the HF-ACTION
study.
Galectin-3 is elevated in ambulatory heart failure patients and is
associated with poor functional capacity and other known measures of
heart failure severity. In univariate analysis, galectin-3 was significantly
predictive of long-term outcomes, but this association did not
persist after adjustment for other predictors, especially NTproBNP.
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
FrasonFrancis
Gail S Thornton
Irina Robu
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jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
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Dr. Sudipta Saha
tildabarliya
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zs22
