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Posts Tagged ‘Fatty acid’


Resuscitation From Sudden Cardiac Arrest: Common Variation in Fatty Acid Genes

Reporter: Aviva Lev-Ari, PhD, RN

Common Variation in Fatty Acid Genes and Resuscitation From Sudden Cardiac Arrest

Catherine O. Johnson, PhD, MPH, Rozenn N. Lemaitre, PhD, MPH, Carol E. Fahrenbruch, MSPH, Stephanie Hesselson, PhD, Nona Sotoodehnia, MD, MPH,Barbara McKnight, PhD, Kenneth M. Rice, PhD, Pui-Yan Kwok, MD, PhD, David S. Siscovick, MD, MPH and Thomas D. Rea, MD, MPH

Author Affiliations

From the Departments of Medicine (C.O.J., R.N.L., N.S., D.S.S., T.D.R.), Biostatistics (B.M., K.M.R.), and Epidemiology (D.S.S), University of Washington, Seattle; King County Emergency Medical Services, Seattle, WA (C.E.F.); and Institute of Human Genetics, University of California San Francisco (S.H., P.-Y.K.).

Correspondence to Catherine O. Johnson, PhD, MPH, Department of Medicine, University of Washington, CHRU 1730 Minor Ave, Suite 1360, Seattle, WA 98101. E-mail johnsoco@uw.edu

Abstract

Background—Fatty acids provide energy and structural substrates for the heart and brain and may influence resuscitation from sudden cardiac arrest (SCA). We investigated whether genetic variation in fatty acid metabolism pathways was associated with SCA survival.

Methods and Results—Subjects (mean age, 67 years; 80% male, white) were out-of-hospital SCA patients found in ventricular fibrillation in King County, WA. We compared subjects who survived to hospital admission (n=664) with those who did not (n=689), and subjects who survived to hospital discharge (n=334) with those who did not (n=1019). Associations between survival and genetic variants were assessed using logistic regression adjusting for age, sex, location, time to arrival of paramedics, whether the event was witnessed, and receipt of bystander cardiopulmonary resuscitation. Within-gene permutation tests were used to correct for multiple comparisons. Variants in 5 genes were significantly associated with SCA survival. After correction for multiple comparisons, single-nucleotide polymorphisms in ACSL1 and ACSL3 were significantly associated with survival to hospital admission. Single-nucleotide polymorphisms in ACSL3, AGPAT3, MLYCD, and SLC27A6 were significantly associated with survival to hospital discharge.

Conclusions—Our findings indicate that variants in genes important in fatty acid metabolism are associated with SCA survival in this population.

SOURCE:

Circulation: Cardiovascular Genetics.2012; 5: 422-429

Published online before print June 1, 2012

doi: 10.1161/ CIRCGENETICS.111.961912

 

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Gamma Linolenic Acid (GLA) as a Therapeutic tool in the Management of Glioblastoma

Eric Fine* (1), Mike Briggs* (1,2), Raphael Nir# (1,2,3)

Sefacor, LLC (1); Woodland Pharmaceuticals, LLC (2); SBH Sciences, Inc (3). 

* These authors contributed equally; # Corresponding author (rnir@sbhsciences.com).

 

I. Introduction

 

Glioblastoma multiform is a fast-growing, invasive central nervous system tumor that forms from glial (supportive) tissue of the brain and spinal cord. Glioblastoma multiform also called glioblastoma or glioma along with grade III/IV astrocytoma and abbreviated herein and elsewhere as GBM. It usually occurs in adults and affects the brain more often than the spinal cord.  Brain tumor patients with GBM have a severely major unmet medical need. Current treatment for stage IV glioblastoma provides only 16-month median survival from time of diagnosis.

 

There has been and continues to be a tremendous amount of research with the goal of finding a cure for brain tumors, yet there are only 3 FDA approved drugs for this indication, BCNU in the form of Gliadel® wafers, temozolomide (Temodar®), since 2005 and most recently, 2009 bevacizumab (Avastin®; 10 mg/Kg intra venous) for recurrent GBM. Patients with grade IV glioma undergoing surgical resection of the tumor combined with radiation therapy (RT) to prevent any remaining cancer cells from regrowing have shown historical median survival of 11.5 to 12 months. The first FDA approved glioma treatment was the Gliadel wafer that is placed in the brain tumor bed after surgery, where it degrades, releasing the drug carmustine. This treatment that included surgery and radiation has been shown to extend the median survival of these patients to about 14 months approximately 2 months longer than the group that received placebo wafers (Westphal M, 2003, 2006), (Attenello FJ, 2008). However, the rate of complications, including an increase in cerebrospinal fluid leaks and intracranial hypertension, has limited their use (Nagpal S., 2012).  The current ‘gold standard’ treatment to which all new experimental treatments are compared is temozolomide. Patients with high grade glioma receiving surgery, temozolomide and radiation therapy have a mean survival of 14.5 to 16 months (Stupp R, 2005), (Grossman SA, 2010). Avastin (bevacizumab), is a humanized monoclonal antibody that inhibits vascular endothelial growth factor A (VEGF-A) administered by intravenous infusion and has been approved for treating the recurrence of glioma only after the cancer has become refractory to temozolomide (Cohen MH, 2009), (Chamberlain MC, 2010). Still, GBM remains one of the two worst-case scenarios in the spectrum of cancer, sharing with pancreatic cancer a less than 5% five-year survival rate.

 

Due to the current success of polyunsaturated fatty acid (PUFA) based therapeutics including Lovasa (GlaxoSmithKline/ Reliant Pharmaceuticals) and Vascepa (Amarin) for high triglycerides with mixed dyslipidemia, there seems to be a renewed interest in PUFA’s therapeutic effects in different disease indications, especially cancer.

 

The scientific literature reports various results for the many different PUFA forms and their affects in a wide variety of cancer cell line tests.  The use of PUFA in the clinical setting has shown a slight enhancement of tamoxifen treatment in breast cancer patients when taken as an oral supplement (Kenny FS, 2000). But the lack of clear clinical improvement predominates in most trials such as those for bladder cancer (Harris NM, 2002) and pancreatic cancer (Johnson CD, 2001). Intravenous infusion of the polyunsaturated fatty acid gamma linolenic acid (GLA) for pancreatic cancer patients had met with little success in extending these patients’ lives (Johnson CD, 2001).

We hypothesize that the systemic administration of PUFAs has had limited success in cancer treatment mainly due to their being highly protein bound in the blood upon infusion and the need for an apparently high local concentration in the vicinity of the cancer tissue. In the face of the confounding data for the utility of PUFAs in cancer treatment, our hypothesis has been supported by the promising results found in a small, but uncontrolled pilot clinical trial using a protocol entailing local application of GLA directly into the resected tumor bed of High Grade GBM patients (Das UN, 1995).

 

 

II.  Polyunsaturated fatty acids in Glioblastoma

 

Fatty acids are key nutrients that affect early growth and development, as well as chronic and other diseases. A fatty acid containing more than one carbon double bond is termed polyunsaturated fatty acid (PUFA). PUFA affect the prevalence and severity of cardiovascular disease, diabetes, inflammation, cancer, and age-related functional decline. PUFA are components of the structural phospholipids in cell membranes; they modulate cellular signaling, cellular interaction, and membrane fluidity. The two most important groups of PUFA are the Omega 3 and Omega 6 fatty acids. Alpha-linolenic acid (ALA or 18 : 3n-3) is the parent of Omega 3 fatty acids, and linoleic acid (LA or 18 : 2n-6), the parent of the n-6 PUFA family. The human body is unable to readily synthesize ALA, and LA, classifying them both as essential fatty acids that one must ingest in the diet.    LA and ALA are converted to their respective n-6 and n-3 PUFA families by a series of independent reactions of which both pathways require the same enzymes, Δ6 Desaturase and Δ5 Desaturase, for desaturation and elongation (Sprecher H, 2002).

 

Common polyunsaturated fatty acid forms tested for their anti-tumor effect include gamma linolenic acid (GLA), arachidonic acid (AA) from the n-6 series and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from the n-3 series. One of the most promising PUFA in the development of cancer therapeutics is the GLA.  GLA is a carboxylic acid with an 18-carbon chain and three cis double bonds. Although the cytotoxicity of GLA, AA, EA and DHA is very high in cancer cell-lines, GLA shows the greatest specificity of destroying only cancerous cells and leaving non-cancerous cells intact (Bégin ME, 1986) (Das UN, 1991). For this reason we will narrow the focus of this review to GLA.

 

In-Vitro analysis of GLA on various cancer cell lines

GLA has shown cytotoxicity to a number of cancer cell lines including breast (ZR-75-11), lung (A-549), prostate (PC-3) (Begin ME, 1986), pancreas (Ravichandran D, 2000), liver (Itoh S, 2010).   GLA was the most effective in selectively killing the tumor cells. In a co-culture experiment wherein normal human skin fibroblasts (CCD-41-SK) and human breast cancer cells (ZR-75-1) were grown together in a Petri dish and supplemented with GLA, only human breast cancer cells were eliminated without any effect on normal skin fibroblasts  (Bégin ME, 1986).

The studies outlined below focus on GBM:

Bell et al, (1999) examined the invasion and growth of cell spheroids of human GBM cell lines U87, U373 and MOG-G-CCM.  The spheroids were grown on collagen with up to 1 mM GLA for 5 days. Measurements showed that low concentrations of GLA (< 100uM) increased both apoptosis and proliferation while higher concentrations (>250 uM) significantly impaired spheroid growth. All spheroid preparations showed 100% growth inhibition after 5 days of culture with 500–1000 uM GLA. Similar experiments by Leaver HA et al, [2002a] found that the Lithium  (Li+) salt of GLA was more potent than GLA, most likely due to its increased solubility. Li+GLA showed statistically significant pro-apoptotic and anti-proliferative effects in C6 rat glioma cell line culture at 40 uM PUFA as observed using the MTT assay compared to nontreated controls.  Meglumine gammalinolenate (MeGLA) was also developed for enhancing the water solubility of the PUFA and it showed greater activity than Li+GLA (Ilc K, 1999). Work reported by Scheim (Scheim DE, 2009) on human cell cultures derived from human GBM biopsy treated with 500 uM GLA showed complete cytotoxicity to the cancerous cells, while maintaining complete viability in noncancerous cell organ cultures from human biopsy.

 

 

III. Mechanism of Action for GLA against cancer cells

The mechanisms by which PUFA act on normal and cancerous cells are complex and not well understood. In tumor cells, addition of PUFAs results in the generation of free radicals, enhancement of lipid peroxidation and the suppression of cell rescue proteins and pathways thereby leading to cell apoptosis.  However, in normal cells, supplementation of PUFAs produce adequate amounts of lipoxins, resolvins and protectins that protect the cells from free radicals and reactive oxygen species, suppress inflammation and prevent actions of mutagens and carcinogens (Das UN and Madhavi N, 2011).

 

  1. A.    Free radical generation:

In vitro experiments testing the cytotoxic effects of  PUFA has shown that GLA application induced lipid peroxidation products may have a high affinity to Bcl-2, an integral membrane oncoprotein that is unique in its ability to suppress apoptosis. This interaction prevents Bcl-2 from suppressing apoptosis even in cancer cells. Haldar et al (1995) concluded that Bcl-2 is deactivated upon phosphorylation and Bodur et al (2012), have shown that the exposure to 4-hydroxynonenal (HNE) the main aldehydic product of plasma LDL peroxidation induces Bcl-2 phosphorylation (Haldar S, 1995), (Bodur C, 2012).

To decipher the mechanism of the cytotoxic action of GLA and other fatty acids, cyclo-oxygenase, lipoxygenase inhibitors, and anti-oxidants and free radical quenchers have been added to cancer cell line cultures.  The GLA may induce different cell death pathways in different cell lines. In HeLa cells, indomethacin, a cyclo-oxygenase and inhibitor, and NDGA, a lipoxygenase inhibitor, that were added to cell cultures were ineffective in blocking the cytotoxic action of GLA and DHA (Das UN and Madhavi N, 2011).  However, SOD and Vitamin E, both free radical scavengers blocked the tumoricidal action of GLA on human cervical carcinoma, (HeLa) cells, human leukemia, HL-60 cells, breast cancer, ZR-75-1, cells (Das UN, 1991, 2007), (Sagar PS, 1995).   The increased production of free radicals by GLA treated cancer cells may be one of the reasons for enhanced cytotoxicity of glioma tumors seen in the pilot human clinical trials.

 

  1. B.    GLA influence on Angiogenesis:

Inclusion of GLA in a 3D matrix culture system of the rat aortic ring assay, significantly inhibited angiogenesis in a concentration-dependent manner and a significant reduction of vascular endothelial cell motility was observed (Cai J, 1999).  Localized administration of GLA to orthotopically implanted C6 glioma cell line in the rat brain decreased the tumor cell’s protein expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by 71% (± 16%) and the VEGF receptor Flt1 by 57% (± 5.8%) (Miyake JA, 2009). The GLA treatment reduced the micro vessel density of the tumors by 41% compared to control tumors.  In addition, the GLA treatment caused a significant decrease in ERK1 and ERK2 protein expression of (27 ± 7.7%) and (31±8.7%), respectively. More recently, Miyake et al report that neoangiogenesis is regulated through the ERK1/2 pathway (Miyake M, 2013).

 

  1. C.    GLA influence on cancer related genes:

Miyake et al, [2009] examined the changes in cancer related gene expression in C6 glioma cells growing in rat brains when treated with local GLA brain infusion as compared to vehicle controls. The GLA treatment shows evidence for the upregulation of proteins that would inhibit cell cycle growth and division and induce apoptosis. The expression of p53 was increased (44 ±16%) by GLA as compared to control.

The tumor suppressor protein p53 has many mechanisms of anticancer function, playing a role in apoptosis, genomic stability, and inhibition of angiogenesis. The mechanisms by which p53 works include: activating DNA repair proteins when DNA has sustained damage; arresting growth by holding the cell cycle at the G1/S regulation point if DNA damage is recognized allowing for repair or it can initiate apoptosis, or it can initiate programmed cell death, if DNA damage proves to be irreparable (Liang Y, 2013).  Similarly, the expression of p27 (another tumor suppressor protein) was also increased (27 ± 7.3%) in GLA treated animals (Miyake JA, 2009).

 

  1. D.    Caspase:

Apoptosis is induced by caspase signaling pathways in many cells (Kim R, 2002) (Philchenkov A, 2004). One of the mechanisms of apoptosis involves a mitochondrial signaling pathway, which entails the efflux of cytochrome c from mitochondria to the cytosol (Ge H, 2009). Cytosolic cytochrome c together with Apaf-1 activates caspase-9, which then activates caspase-3 (Cain K, 2002), (Wang X, 2001). Caspase-3 play an important role in apoptosis and degrades proteins such as PARP, which is a nuclear enzyme implicated in many cellular process including apoptosis and DNA repair. Studies by Ge et al, (2009) suggest that GLA treatment induces a dose-dependent increase in cytochrome c and activation of caspase-3 that correlates with the apoptosis of human chronic myelogenous leukemia K562 cells (Kong X, 2006). Further, the apoptosis could be inhibited by a pan-caspase inhibitor (z-VAD-fmk) (Ge H, 2009).

 

  1. E.    Ku Proteins:

The heterodimeric Ku70/Ku80 protein complex is important for DNA repair and plays an important role in double strand breaks especially in gamma irradiation resistant tumor cells where high levels of these proteins are related to hyper proliferation and carcinogenesis (Gullo, 2006). Ku proteins have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation (Yang QS, 2008). GLA treatment of C6 rat glioma cells was accompanied by a 71% reduction in Ku80 protein expression and a 39% increase in the number of micronuclei detected by Hoechst fluorescence, as well as a 49% reduction of cells in S-phase even at concentrations that do not produce significant increases in apoptosis when measured within only a 24 hour exposure (Benadiba M, 2009).

 

 

  1. IV.  In Vivo effect of GLA

As previously discussed, GLA has been reported to have effects in many cancers in vivo with treatments ranging from direct anti-tumor activity in clinical studies with injected GLA to dietary supplementation as an adjuvant to more traditional chemotherapy (Fetrow CW, 1999) (Kleijnen J, 1994). There are a number of anecdotal reports of increased response and duration, but none of these studies have shown convincing evidence to support the continued use of GLA against any specific cancer subtype. In one small clinical pancreatic cancer study using an injectable form of GLA there was some apparent benefit (Fearon KC, 1996), which failed to be reproduced in a larger study (Johnson CD, 2001). Other tumor types for which there have been reports regarding use of GLA in cancer include breast cancer (Kenny FS, 2000, 2001), (Menendez JA, 2004, 2005) bladder cancer (Harris NM, 2002) and even leukemia (Kong X, 2009). In even earlier studies, PUFAs including GLA were shown to have some efficacy against both chemically induced skin carcinogenesis in mice (Ramesh G, 1998) and hepatocarcinoma models in rats (Ramesh G, 1995) although again, these studies were not definitive.  A recurring theme seems to be that for utility, the GLA needs to be present at reasonably high doses in the vicinity of the tumor, indicating the some form of local delivery must be considered, or perhaps some kind of targeted therapy.

 

A. GLA tumorcidal effect on rat glioma:

The Leaver group (Leaver HA, 2002 b) continued their work examining the effects of GLA treatment.  Rats with orthotopically placed C6 glioma tumor in their brains were locally infused with PBS vehicle or GLA solution from 200 uM to 2 mM. The most active was 2 mM, infused at 1 ul/hr over 7 days. In contrast 1mM total dose had no significant difference from the controls.  In the positive response group, tumor regression, increased apoptosis and decreased proliferation were observed. Minimal effects on normal neuronal tissue was detected, with the caveat that their methods were not comprehensive (see discussion on safety, section IV.B. and Conclusion discussion, section VI). Tumor volume was less than 50% of controls in the 2 mM infused rats. However, histology and TUNEL reactivity of the remaining tumor indicated that this may be an under-estimate of residual viable tumor as substantial areas of treated tumors showed characteristics of necrotic tissue and apoptotic cell death. Supporting this hypothesis, tumor tissue sections evaluated by IHC with the proliferative marker Ki67 in the 2mM GLA treated animals showed < 20% of PBS control expression. Note: in these experiments there was no initial debulking surgery of the tumor mass.

Further studies by Miyake JA et al, (2009) showed that increasing the concentration of GLA delivered to the implanted C6 cell glioma in rat brains by treating them with 5 mM GLA/d in cerebrospinal fluid (CSF) caused an even greater decrease in C6 tumor growth in vivo. The average tumor area was reduced by 75 ± 8.8% in comparison with CSF alone.  VEGF protein expression was reduced 77 ± 16%. GLA had an inhibitory effect on vessel number causing a 44 ± 5.4% reduction in tumor micro vessel density.

While the in vivo data have a mixed response when looking at different tumor types and delivery methods, it appears that there may be some utility in GBM, particularly when the drug is delivered locally.  Further exploration of delivery methods for GBM and other tumor types need to be explored including the use of more targeted therapies such as targeted nano-particle delivery and even antibody-drug conjugates (ADC).  The research models also need to reinforce and support if possible the clinical observation of efficacy seen with direct intratumoral (or resected cavity) delivery noted in previous studies carried out in India.

 

B. Safety Studies in the Canine Model:

A safety study in 3 healthy dogs showed that daily injection of 0.25 mg in 1ml of saline for six days into the brain parenchyma under aseptic conditions was found to be safe (Das U N, 1995). CT scan and gross examination of the meninges and subarachnoid space as well as histopathological exams showed no abnormality and no difference between injected side and non-injected side. None of the animals developed any side effects or complications due to the procedure or GLA injection. Note that humans were given 1 mg GLA per day (see next section).  These are at best preliminary findings and further evaluation of safety in normal brain tissues and CSF need to be considered.

 

  1. V.            Clinical application of GLA for Glioma Patients

The most compelling argument for the usefulness of GLA in the treatment of glioblastoma comes from a series of open label, non-randomized trials that were run in India by Drs. Das and Reddy nearly 2 decades ago.  In these studies, summarized below, they found that direct administration of the GLA to the tumor site via infusion over several days provided no observable toxicities or side effects although there were not complete cognitive or behavioral studies done on the patients.  It remains to be shown that there are no significant liabilities to the administration of GLA to brain cancer patients to provide both an extension of life (overall survival benefit) as well as not impinging on the quality of life for the patient.

 

  1. A.    Recurrent glioma patients:

The initial study treating patients with local administration of GLA was performed on patients with recurrent GBM. GLA was injected directly into the tumor and/or an Ommaya reservoir was used to deliver the GLA to the tumor bed after surgical tumor resection followed by standard RT (see Naidu MR , 1992).  This procedure not only showed substantial efficacy but also there were no drug related side effects. Although only a small group of 6 patients, 3 of the 6 were alive at their last follow-up check-in 2 yrs 4 months to 2 yrs 8 months. These patients with recurrent glioma when administered the GLA therapy were in critical condition with life expectancy of 9 months or less. A 50 % survival at ~ 2.5 yrs is much better than historic average of 27% survival at 2 years in primary glioma patients with what is now the “gold standard” treatment of radiation and temozolomide and thus warranted further study.

 

  1. B.    GLA treatment of primary tumor patients:

The next study performed was on patients with grade III Astrocytoma and Grade IV glioblastoma receiving their first intervention. Patients underwent neurosurgery to remove as much of tumor as possible. Before closure of the dura, 1 mg GLA was instilled into the tumor bed and cerebral catheter and reservoir were positioned for subsequent injections. On day 7 post operation, a baseline CT brain scan was taken. One mg daily of GLA in 2-3 ml of sterile saline was instilled for 10 days before a repeat CT scan was taken for comparison  This procedure not only showed substantial efficacy but also there were no drug related side effects. Surgery plus RT supplemented with GLA treatment extended patient survival for 80% of treated patients (12/15) to 34 months with very limited drug-related side effects (Das U N, 1995).

 

  1. VI.           Conclusion

As some of the patients (Trial B, above) were alive and apparently well more than 2 years after receiving treatment, it is rather incredible that this treatment has not been more widely tested in the west in the last 18 years.  It is likely due to the fact that no robust and reproducible preclinical studies have come forward and that more standard GLP toxicology studies were not done.  Safety needs to be the first concern and whether in rats, dogs or monkeys, if direct delivery of GLA to the brain cavity is the best treatment, then it is imperative to have these studies carried out with a full analysis of both histopathological findings as well as the more indirect cognitive and behavioral studies that will be very important in human therapy.  As direct delivery to the brain is not a typical therapeutic approach, it remains to be seen what the regulatory agencies will demand for this kind of novel treatment.  The most pressing need is to have a thorough assessment of normal brain tissue exposure at the doses that are likely to be administered to a human and to include some surgical intervention (slicing through the brain) to mimic the surgical resection of the glioma.  Thus just delivering to the cerebrospinal fluid, while an intermediate assessment tool, may not have full predictive value for the adjuvant application of GLA in the treatment of glioblastoma.  For true safety studies, multiples of the minimum efficacious dose would ideally be done to ensure that there is a safety margin for dose administration errors.  These studies are enabled by Alzet mini-pump technologies as well as direct cannulation and a sterile port for the daily administration of drugs to the test subject.

As systemic exposures will be minimized from direct brain delivery of small amounts such as the 1-2 mg per day in the referenced trials, there would be almost no way to evaluate for typical toxicology organ effects, coupled with the fact that GLA is an endogenous component of fatty acid metabolism.  With drugs such as Gliadel® having been used, with its poor safety profile (Based on Pharmacy Codes: The oral LD50 in rat and mouse are 20 mg/kg and 45 mg/kg, respectively. Side effects include leukopenia, thrombocytopenia, and nausea.) Toxic effects include pulmonary fibrosis and bone marrow toxicity). Moreover, recent studies showing combining carmustine with temozolomide reduces survival time compared to temozolomide alone (Prados MD, 2004). The safety hurdle is fairly low for this devastating and fast growing tumor, however, that is not an excuse to forgo the safety studies that apparently were casually done previously and have kept this potential therapy out of the mainstream medicine for the past 18 years.

Taken together, these reports from the intriguing conundrum provided by the various outcomes of the animal efficacy studies to the patient feeding studies and the various delivery routes tested suggest that there is some rationale for utility of GLA in the treatment of cancer. Disciplined and well-controlled studies need to be undertaken with GLA / GLA salt or derivative forms of GLA that may have better pharmaceutical properties coupled with optimal delivery of the agent to the tumor with or without another therapy (chemotherapy or electrical field therapy ).

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Miyake JA, Benadiba M, Colquhoun A. “Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression.” Lipids Health Dis. 8 (2009): 8.

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Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

This article is the THIRD in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

  • Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

  • Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

  • Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Mitochondrial Metabolism in Impaired Cardiac Function

Mitochondrial Dysfunction and the Heart

Chronically elevated plasma free fatty acid levels in heart failure are associated with
  • decreased metabolic efficiency and cellular insulin resistance.
The mitochondrial theory of aging (MTA) and the free-radical theory of aging (FRTA) are closely related.
They were in fact proposed by the same researcher about 20 years apart. MTA adds
  • the mitochondria and its production of free radicals
  • into the concept that free-radicals damage DNA over time.
Tissue hypoxia, resulting from low cardiac output with or independent of endothelial impairment,
This dysfunctional state causes loss of mitochondrial mass. Therapies aimed at protecting mitochondrial function
  • have shown promise in patients and animal models with heart failure that will be the subject of Chapter III.

Myocardial function in hypertension

Genetic variation in vitamin D-dependent signaling
  • is associated with congestive heart failure in human subjects with hypertension.
Functional polymorphisms were selected from five candidate genes:
  1. CYP27B1,
  2. CYP24A1,
  3. VDR,
  4. REN and
  5. ACE.
Using the Marshfield Clinic Personalized Medicine Research Project,
  • 205 subjects with hypertension and congestive heart failure,
  • 206 subjects with hypertension alone and
  • 206 controls (frequency matched by age and gender) were genotyped.
In the context of hypertension, a SNP in CYP27B1 was associated with congestive heart failure
(odds ratio: 2.14 for subjects homozygous for the C allele; 95% CI: 1.05–4.39).
Genetic variation in vitamin D biosynthesis is associated with increased risk of heart failure.
RA Wilke, RU Simpson, BN Mukesh, SV Bhupathi, et al. Genetic variation in CYP27B1 is associated

Heart Failure and Coronary Circulation

There is a decrease in resting and peak stress myocardial function in chronic heart failure patients,
  • with recovery of skeletal muscle phosphocreatine following exercise induced by perhexiline treatment.
This suggested that mitochondrial deficiencies, caused by excessive free fatty acids (FFAs)
  • underlie a common cardiac and skeletal muscle myopathy in heart failure patients.
Tissue hypoxia in chronic heart failure from inadequate circulation in heart failure
  • increases the oxidative stress in lean body mass and in the heart itself.
The heterodimeric transcription factor hypoxia-inducible factor (HIF)-1
  • induces changes in the transcription of genes that encode proteins involved in the adaptation to hypoxia.
HIF-1 activity depends on levels of the HIF-1a subunit, which has a short half-life.
HIF-1a increases in rats with experimentally induced myocardial infarction together with elevated levels of
  • GLUT1 and haemoxygenase-1 in the peri-infarct region of the heart
The cardiac metabolic response to hypoxia is considered to be
  • a return to a pattern of fetal metabolism, in which
  • carbohydrates predominate as substrates for energy metabolism.
The reliance on carbohydrate energy source is thought to be a result of  the downregulation of PPARa with a decreased activity of
The sarcolemmal fatty acid transporter protein (FATP) levels are also decreased with palmitate oxidation,
  • transitioning away from fatty acid metabolism proportional to the degree of cardiac impairment.
The hypoxic changes of heart failure drives a switch toward
  1. glycolysis and glucose oxidation
  2. restriction of myocardial fatty acid uptake.
Nevertheless, late in the progression of heart failure, substrate metabolism is insufficient to support cardiac function, because
  • the hypoxic failing heart is no longer able to oxidize fats and may also be insulin resistant.
The author surmises that mitochondrial dysfunction caused by tissue hypoxia might be mediated by the
  • proapoptotic protein BCL2/adenovirus E1B 19kDa interacting protein (Bnip)3.
It  is strongly upregulated in response to hypoxia. In the isolated, perfused rat heart, Bnip3 expression was
  • induced after 1h of hypoxia, with Bnip3 integrating into the mitochondria of hypoxic ventricular myocytes.
This resulted in mitochondrial defects associated with
  1. opening of the permeability transition pore, leading to
  2. loss of inner membrane integrity and
  3. loss of mitochondrial mass.

Mitochondrial Dysfunction caused by Bnip3 Precedes Cell Death.

Experimentally induced myocardial ischemia had evidence of contractile dysfunction but preserved viability. A progressive
  • decline in circulating levels of endothelial progenitor cells was documented 3 months following instrumentation (P<0.001).
Quantitative polymerase chain reaction analysis revealed that
  • chronic myocardial ischemia produced a biphasic response in both
    • hypoxic-inducible factor 1 and
    • stromal-derived factor 1 mRNA expression.
While initially unregulated, a gradual decline was observed over time (from day 45 to 90), in
  • hypoxic-inducible factor 1 and
  • stromal-derived factor 1 mRNA expression .
On serial assessment, endothelial progenitor cell migration was progressively impaired in response to chemo-attractant gradients of:
  1. vascular endothelial growth factor (10-200 ng/mL)
  2. and stromal cell-derived factor-1 (10-100 ng/mL) .
Decreased circulating levels and migratory dysfunction of bone marrow derived endothelial progenitor cells
  • were documented in a reproducible clinically relevant model of myocardial ischemia.

Nitric Oxide (NO) in Myocardial Ischemia and Infarct

Nitric oxide (NO) is a free radical with an unpaired electron; it is an important physiologic messenger,
  • produced by nitric oxide synthases, which catalyze the reaction l-arginine to citrulline and NO.
The constitutive isoforms exists in neuronal and endothelial cells and is calcium dependent. Calcium binds to calmodulin and
  1. the calcium calmodulin complex activates the constitutive NO synthase that releases NO,
  2. relaxing smooth muscle cells through activation of guanylate cyclase and the production cGMP.
Therefore, the NO produced has a negative inotropic effect on the heart and is instrumental in the autoregulation of the coronary circulation.
The inducible form of NO synthase (iNOS), mostly produced in macrophages, is activated by cytokines and endotoxin. It eliminates intracellular pathogens,
damaging cells by inhibiting
  1. ATP production
  2. oxidative phosphorylation
  3. DNA synthesis.
In infection, lipopolysaccharide released from bacterial walls, stimulates production of iNOS. The large amount of NO produced
  • causes extensive vasodilation and hypotension.
We sought to assess whether oxidation products of
  • nitric oxide (NO), nitrite (NO2−) and nitrate (NO3−), referred to as NOx,
  • are released by the heart of patients after acute myocardial infarction (AMI) and
  • whether NOx can be determined in peripheral blood of these patients.
Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells
  • (macrophages) in the myocardium 3 days after onset of  ischemia.
NOx is formed in heart muscle from NO; It originates through the activity of the inducible form of nitric oxide synthase (iNOS).
Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by
coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured.
Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms.
The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3− was reduced to NO2−
by nitrate reductase before determination of NO2− concentration by chemiluminescence.
The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased,
  • as well as the coronary arterial–venous difference.
Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma.
The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of
  • production of NO by inflammatory cells (macrophages) in the heart.
The appearance of oxidative products of NO (NO2− and NO3−) in peripheral blood of patients with acute MI is
  • the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia.
Further studies are needed to define the clinical value of these observations.
K Akiyama,  A Kimura, H Suzuki, Y Takeyama, …. R Bing.  Production of oxidative products of nitric oxide in infarcted human heart.  J Am Coll Cardiol. 1998;32(2):373-379.   http://dx.doi.org/10.1016/S0735-1097(98)00270-8
OPA1 Mutation and Late-Onset Cardiomyopathy
No cardiac disorders have been described in patients with OPA1 or similar mutations
  • involving the fission/fusion genes as seen in inherited maladies like Charcot–Marie–Tooth disease.
Our results indicate that, at least for OPA1, cardiac abnormalities are not completely
  • manifest until the development of blindness.

The OPA1-mutant mice survived more than 1 year and appeared healthy.

In patients with these diseases, reduced cardiac function may go undetected
secondary to reduced physical activity secondary to loss of vision.
It would be expected that patients with such mutations would have impaired cardiac reserve with
  • reduced ability to respond to high-stress disease states such as myocardial infarction and sepsis.
The OPA1-mutant mice have reduced cardiac reserve, as shown by
  • the lack of response to isoproterenol or to ischemia/reperfusion injury,
This suggests that patients with OPA1 and related inherited mitochondrial diseases
  • should be screened for abnormalities of cardiac function.
Le Chen; T Liu; A Tran; Xiyuan Lu; …AA. Knowlton. OPA1 Mutation and Late-Onset Cardiomyopathy:
Mitochondrial Dysfunction and mtDNA Instability.  http://jaha.ahajournals.org/content/1/5/e003012.full

Oxidative Stress and Mitochondria in the Failing Heart

The major problem in tissue hypoxia in the failing heart is oxidative stress. Reactive oxygen species (ROS), including
  • superoxide,
  • hydroxyl radicals and
  • hydrogen peroxide,
are generated by a number of cellular processes, including
  • mitochondrial electron transport,
  • NADPH oxidase and
  • xanthine dehydrogenase/xanthine oxidase.
The low availability of oxygen, the final receptor of mitochondrial electron transport (ET), results in
  • electron accumulation in the ET chain as the complexes become highly reduced.
A number of experimental and clinical studies have suggested that ROS generation is
  • enhanced in heart failure because of electron leak, and complexes I and II
  • are implicated as the primary sites of this loss.
Prolonged oxidative stress in cardiac failure results in damage to mitochondrial DNA.
The continued ROS generation and consequent cellular injury leads to functional decline.
Thus, mitochondria are both the sources and targets of a cycle of ROS-mediated injury in the failing heart.
Mice with a cardiac/skeletal muscle specific deficiency in the scavenger enzyme superoxide dismutase
  • developed progressive congestive heart failure
  • with defects in mitochondrial respiration.
Oxidative stress in these mice also caused specific morphological changes in cardiac mitochondria
  • characterized by decreased ATP levels,
  • impaired contractility,
  • dramatically restricted exercise capacity and
  • decreased survival.
This was in part corrected by treatment with the antioxidant superoxide dismutase mimetic, namely
  • manganese5,10,15,20-tetrakis-(4-benzoic acid)-porphyrin.
EUK-8, a superoxide dismutase and catalase mimetic improved survival and contractile parameters in a mutant mouse model
  • of pressure overload-induced oxidative stress and heart failure and in wild-type mice subjected to pressure overload.
In addition, mitochondria show
  • functional impairment and
  • morphological disorganization
in the left ventricle of Hypertrophic Cardiomyopathy (HCM)  patients without baseline systolic dysfunction.
These mitochondrial changes were associated with impaired myocardial contractile and relaxation reserves.
A strategy to protect the heart against oxidative stress could lie with
  • the modulation of mitochondrial electron transport itself.
Mild mitochondrial uncoupling may offer a potential cardioprotective effect by decreasing ROS production
  • preventing electron accumulation at complex III and
  • the Fe–S centres of complex I, and may therefore

mtDNA, Autophagy, and Heart Failure

Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA.
Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes.
Mitochondrial DNA (mtDNA) that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated
  • inflammatory responses in cardiomyocytes and
  • is capable of inducing myocarditis and dilated cardiomyopathy.
Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions,
but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload.
Early in the pathogenesis, DNase II-deficient hearts showed
  • infiltration of inflammatory cells
  • increased messenger RNA expression of inflammatory cytokines
  • accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium.
Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9
  • attenuated the development of cardiomyopathy in DNase II-deficient mice.
Furthermore, Tlr9 ablation
  • improved pressure overload-induced cardiac dysfunction and
  • inflammation even in mice with wild-type Dnase2a alleles.
These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.
T Oka, S Hikoso, O Yamaguchi, M Taneike, T Takeda, T Tamai, et al.  Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure.

Mitochondrial Dysfunction Increases Expression of Endothelin-1 and Induces Apoptosis

We developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied
  • preproendothelin-1 gene expression and apoptosis.
Rotenone greatly increased the gene expression of preproendothelin-1 in cardiomyocytes.
This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction.
Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked
  • increase in DNA laddering, an indication of apoptosis.
In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro,
  • mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction
    • with impairment of mitochondria in the failing heart.

Summary

This review focused on the evidence accumulated to the effect that mitochondria are key players in
  • the progression of congestive heart failure (CHF).
Mitochondria are the primary source of energy in the form of adenosine triphosphate that fuels the contractile apparatus,
  • essential for the mechanical activity and the Starling Effect of the heart.
We evaluate changes in mitochondrial morphology and alterations in the main components of mitochondrial energetics, such as
  • substrate utilization and
  • oxidative phosphorylation,
in the context of their contribution to the chronic energy deficit and mechanical dysfunction in HF.
REFERENCES
Zachman AL, Page JM, Prabhakar G, Guelcher SA, and Sung HJ, “Elucidation of adhesion-dependent spontaneous apoptosis in macrophages using phase separated PEG/polyurethane films.”
Acta Biomater. 2012 Nov 2.    http://dx.doi.org/pii: S1742-7061(12)00530-2. 10.1016/j.actbio.2012.10.038.    http://www.ncbi.nlm.nih.gov/pubmed/23128157

Other Related articles published on this Open Access Scientific Journal, include the following:

Perspectives on Nitric Oxide in Disease Mechanisms: The Nitric Oxide Discovery, Function, and Targeted Therapy  Opportunities, 2013, Aviral Vatsa, PhD and Larry H Bernstein, MD, FACP, Editors, Amazon e-Books (forthcoming). https://pharmaceuticalintelligence.com/biomed-e-books/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

Mitochondria: More than just the “powerhouse of the cell” Ritu Saxena, Ph.D. Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD 7/9/2012

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondrial dynamics and cardiovascular diseases, Ritu Saxena, PhD 11/14/2012
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress, Larry H Bernstein, MD, FACP 10/28/2012
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation, Larry H Bernstein, MD, FACP 9/26/2012

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Ca2+ signaling: transcriptional control, Larry H Bernstein, MD, FACP 3/6/2-13
https://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified, Aviva Lev-Ari, PhD, RN 2/3/2013
https://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function, Larry H Bernstein, MD, FACP 9/16/2012
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis, Larry H Bernstein, MD, FACP 2/14/2013
https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar, PhD 11/9/2012
https://pharmaceuticalintelligence.com/2012/11/09/low-bioavailability-of-nitric-oxide-due-to-misbalance-in-cell-free-hemoglobin-in-sickle-cell-disease-a-computational-model/

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure, , Larry H Bernstein, MD, FACP 8/20/2012

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination? Aviva Lev-Ari, PhD, RN 10/19/2012

https://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation, Aviva Lev-Ari, PhD, RN 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography, Aviva Lev-Ari, PhD, RN 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN  3/7/2013

https://pharmaceuticalintelligence.com/2013/03/07/genomics-genetics-of-cardiovascular-disease-diagnoses-a-literature-survey-of-ahas-circulation-cardiovascular-genetics-32010-32013/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Aviva Lev-Ari, PhD, RN 10/30/2012

https://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/

Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD, Aviva Lev-Ari, PhD, RN 4/7/2013

https://pharmaceuticalintelligence.com/2013/04/07/cholesteryl-ester-transfer-protein-cetp-inhibitor-potential-of-anacetrapib-to-treat-atherosclerosis-and-cad/

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013

https://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/

Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013

https://pharmaceuticalintelligence.com/2013/04/03/fight-against-atherosclerotic-cardiovascular-disease-a-biologics-not-a-small-molecule-recombinant-human-lecithin-cholesterol-acyltransferase-rhlcat-attracted-astrazeneca-to-acquire-alphacore/

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013

https://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012

https://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-and-hyperhomocusteinemia/

Mitochondrial metabolism and cardiac function, L H Bernstein, MD, FACP
Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects, L H Bernstein, MD, FACP
Lp(a) Gene Variant Association, L H Bernstein, MD, FACP

Predicting Drug Toxicity for Acute Cardiac Events, L H Bernstein, MD, FACP

Amyloidosis with Cardiomyopathy, L H Bernstein, MD, FACP

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Reversal of Cardiac mitochondrial dysfunction, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

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Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP

This article is the SECOND in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

The mitochondrion serves a critical role as a platform for
  • energy transduction,
  • signaling, and
  • cell death pathways
relevant to common diseases of the myocardium such as heart failure. This review focuses on the molecular regulatory events involved in mitochondrial energy metabolism.
This is followed by the derangements known to occur in the development of heart failure.

 Cardiac Energy Metabolism

All cellular processes are driven by ATP-dependent pathways. The heart has perpetually high energy demands related to
  • the maintenance of specialized cellular processes, including
    • ion transport,
    • sarcomeric function, and
    • intracellular Ca2+ homeostasis.
Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux. Thus, ATP-generating pathways must

  • respond proportionately to dynamic fluctuations in physiological demands and fuel delivery.
In order to support contractile activity, the human heart requires
  • a daily synthesis of approximately 30kg of ATP, via
    • oxidative phosphorylation at
    • the inner mitochondrial membrane.
These metabolic  processes are regulated, involving
  • allosteric control of enzyme activity,
  • signal transduction events, and
  • the activity of genes encoding
    • rate-limiting enzymes and proteins.
Catabolism of exogenous substrates ,such as
  • fatty acids,
  • glucose,
  • pyruvate,
  • lactate and
  • ketone bodies,
generates most of the reduced compounds,
  • NADH (nicotinamide adenine dinucleotide, reduced) and
  • FADH2 (flavin adenine dinucleotide, reduced),
which are necessary for mitochondrial electron transport (Fig. 1).
Fig 1  Fatty acid beta-oxidation and the Krebs cycle produce
  1. nicotinamide adenine dinucleotide, reduced (NADH) and
  2. flavin adenine dinucleotide, reduced(FADH2),
which are oxidized by complexes I and II, respectively, of
Electrons are transferred through the chain to the final acceptor, namely oxygen(O2).
The free energy from electron transfer
  1. is used to pump hydrogen out of the mitochondria and
  2. generate an electrochemical gradient across the inner mitochondrial membrane.
This gradient is the driving force for ATP synthesis via the ATP synthase. Alternatively,
H can enter the mitochondria by a mechanism not coupled to ATP synthesis, via
  • the uncoupling proteins(UCPs), which results in the dissipation of energy.

[ANT, adenine nucleotide translocase; CoA, coenzymeA; FAT, fatty acid transporter; GLUT, glucose transporter;

NAD, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid].

Cardiac Energy Metabolic Pathways

 Oxidation of free fatty acids (FFAs) and glucose in mitochondria
  • accounts for the vast majority of ATP generation in the healthy adult heart.
FFAs are the preferred substrate in the adult myocardium,
  • supplying 70-90% of total ATP.
FAs derived from circulating triglyceride-rich lipoproteins and albumin bound nonesterified FAs
  • are oxidized in the mitochondrial matrix by the process of beta-oxidation (FAO), whereas
pyruvate derived from glucose and lactate
  • is oxidized by the pyruvate-dehydrogenase (PDH) complex,
    • localized within the inner mitochondrial membrane.
Acetyl-CoA, derived from both pathways,
  • enters the tricarboxylic acid (TCA) cycle.
Reduced flavin adenine dinucleotide (FADH2) and NADH are generated, respectively, via
  • substrate flux through the
The reducing equivalents enter the electron transport (ET) chain,
  • producing an electrochemical gradient across the mitochondrial membrane
  • that drives ATP synthesis in the presence of molecular oxygen (oxidative phosphorylation).
The relative contributions of each of these substrates are determined
  • by their availability
  • cardiac workload and
  • hormonal status
In the healthy, normal heart, the ATP requirement is largely met in the actively metabolic mitochondria by
  • the catabolism of free fatty acids (FFAs) via beta-oxidation,
  • the tricarboxylic acid cycle and
  • oxidative phosphorylation
giving rise to a greater ATP yield per C2 unit than with glucose.
The relative contribution of glucose to the mitochondrial acetyl-coenzyme A (CoA) pool increases
  • during the postprandial period,
    • when the heart is insulin stimulated, and
  • during exercise
  • hypoxia, or
  • ischemia
when glucose is favored as a more oxygen-efficient substrate than
  • FFAs (greater ATP yield per oxygen molecule consumed).
Substrate switching in the heart can also be achieved by
  • acute alterations in transcriptional regulation of key metabolic enzymes
  • in response to alterations in substrate levels and oxygen availability, or
  • indeed by the intracellular circadian clock.
This continual process of fine adjustment in fuel selection
  • allows cardiac mitochondria to function
  • under a range of metabolic conditions to meet the high energy demands of the heart.
Mitochondrial enzymes are encoded by both nuclear and mitochondrial genes.
All of the enzymes of
  1. beta-oxidation and the TCA cycle, and
  2. most of the subunits of Electron Transfer/Oxidative Phosphorylation,
    • are encoded by nuclear genes.
The mitochondrial genome is comprised of
  • 1 circular double-stranded chromosome that encodes
  • 13 ET chain subunits within complexes I, III, and IV.
Since mitochondrial number and function require both nuclear and mitochondrial-encoded genes,
  • coordinated mechanisms exist to regulate the 2 genomes and
  • determine overall cardiac oxidative capacity.
In addition, distinct pathways exist to coordinately regulate
  • nuclear genes encoding component mitochondrial pathways.

Early Postnatal Low-protein Nutrition, Metabolic Programming and
the Autonomic Nervous System in Adult Life.

JC de Oliveira, S Grassiolli, C Gravena, PCF de Mathias  Nutr Metab. 2012;9(80)

The developmental origins of health and disease (DOHaD) hypothesis stipulates that adult metabolic disease

  • may be programmed during the perinatal stage.

A large amount of evidence suggests that the etiology of obesity is not only related to food abundance

  • but also to food restriction during early life.

Protein restriction during lactation has been used as a rat model of metabolic programming

  • to study the impact of perinatal malnutrition on adult metabolism.

In contrast to protein restriction during fetal life, protein restriction during lactation did not appear to cause

  • either obesity or the hallmarks of metabolic syndrome, such as hyperinsulinemia, when individuals reached adulthood.

Protein restriction provokes body underweight and hypoinsulinemia.
Hypoinsulinemia programs adult rats to maintain normoglycemia,

  • pancreatic β-cells are less sensitive to secretion stimuli:
  1.  glucose and
  2. cholinergic agents.

These pancreatic dysfunctions are attributed to an imbalance of ANS activity

  • recorded in adult rats that experienced maternal protein restriction

Several studies have reported that the ANS activity is altered in under- or malnourished organisms. After weaning,

  • rats fed a chronically protein-deficient diet exhibited low activity of the vagus nerve,
  • whereas high sympathetic activity was recorded

These data were in agreement with a low insulin response to glucose.
Pancreatic islets isolated from protein-restricted rats showed

  • weak glucose and cholinergic insulin tropic responses
  • suggesting that pancreatic β-cell dysfunction may be attributed to altered ANS activity

Food abundance or restriction with regard to body weight control involves changes in

  • metabolic homeostasis and ANS balance activity.

Although the secretion of insulin by the pancreatic β-cells is increased in people who were overweight,

  • it is diminished in people who were underweight.

Changes in the ANS activity may constitute the mechanisms underlying the β-cell dysfunction:

  • the high PNS tonus observed in obese individuals constantly potentiates insulin secretion,
  • whereas the low activity reported in underweight individuals is associated with a weak cholinergic insulin tropic effect.

Under Nutrition Early in Life and Epigenetic Modifications, Association With Metabolic Diseases Risk

relevant to this issue is the role of epigenetic changes in the increased risk of developing metabolic diseases,

  • such as type 2 diabetes and obesity, later in life.

Epigenetic mechanisms, such as DNA methylation and/or nucleoprotein acetylation/methylation, are

  • crucial to the normal/physiological development of several tissues in mammals, and
  • they involve several mechanisms to guarantee fluctuations of enzymes and other proteins that regulate the metabolism.

The intrauterine phase of development is particularly important for the genomic processes related to genes associated with metabolic pathways.
This phase of life may be particularly important for nutritional disturbance. In humans who experienced the Dutch famine Winter in 1944–1945 and
in rats that were deprived of food in utero, epigenetic modifications were detected in

  • the insulin-like growth factor 2 (IGF2) and
  • pancreatic and duodenal home box 1 (Pdx1),

the major factors involved in pancreas development and pancreatic β-cell maturation.
The pancreas and the pancreatic β-cells develop during the embryonic phase, but the postnatal life is also crucial for

  • the maintenance processes that control the β-cell mass:
  1. proliferation,
  2. neogenesis
  3. apoptosis.

Nutritional Restriction to the Fetus: A Risk of Obesity Onset

If an abundant diet is offered to people who have been undernourished during the perinatal life,

  • this opportunity induces a metabolic shift toward the storage of energy and high fat tissue accumulation

The concept of Developmental Origins of Health and Disease extends to any type of stressful situations that may

  • predispose babies or pups to develop metabolic disorders when they reach adulthood.

Programmed Metabolism and Insulin Secretion-coupling Process

What are the mechanisms involved in the low glucose insulin tropic response observed in low protein-programmed lean rats?
The pancreatic β-cells secrete insulin when stimulated mostly by glucose. However, several nutrients, such as

  • amino acids,
  • fatty acids,
  • and their metabolites,

stimulate cellular metabolism and increase ATP production.

ATP-sensitive potassium channels (KATP) are inactivated by an increased ATP/ADP ratio. This provokes

  • membrane depolarization and
  • the activation of voltage-dependent calcium channels.

These ionic changes increase the intracellular calcium concentration, which is involved in

  • the export of insulin to the bloodstream.

Glucose may also stimulate insulin secretion by alternative pathways involving KATP channels.

Programmed Metabolism and Insulin Tropic Effects of Neurotransmitters

Insulin release is modulated by non-nutrient secretagogues, such as neurotransmitters, which

  • enhance or inhibit glucose-stimulated insulin secretion.

Pancreatic β-cells contain several receptors for neurotransmitters and Neuropeptide, such as

  • adrenoceptors and cholinergic muscarinic receptors (mAChRs).

These receptors are stimulated by efferent signals from the central nervous system, including the ANS,

  • throughout their neural ends for pancreatic β-cells.

During blood glucose level oscillations, the β-cells receive inputs from

  • the parasympathetic and sympathetic systems to participate in glycemic regulation.

Overall, acetylcholine promotes the potentiation of glucose-induced insulin secretion,

  • whereas noradrenaline and adrenaline inhibit this response.

Functional studies of mAChR subtypes have revealed that M1 and particularly M3 are the receptors that are involved in

  • the insulin tropic effect of acetylcholine.

Interestingly, it was reported that M3mAChR gene knockout mice are

  • underweight,
  • hypophagic and
  • hypoinsulinemic,

as are adult rats that were protein-restricted during lactation.
The pancreatic islets from M3mAChR mice (-/-) showed a reduced secretory response to cholinergic agonists.
In studies using transgenic mice in which the pancreatic β-cell M3mAChRs are chronically stimulated,

  • an improvement of glycemic control has been observed

Adult male rat offspring from whose mothers were protein-restricted during lactation

  • exhibit a low PNS activity.

Evidence suggests that ANS changes may contribute to the impairment of glycemic homeostasis in metabolically programmed rats.

Pathways involved in cardiac energy metabolism.

FA and glucose oxidation are the main ATP-generating pathways in the adult mammalian heart.
Acetyl-CoA derived from FA and glucose oxidation is
  • further oxidized in the TCA cycle to generate NADH and FADH2, which
  • enter the ET/oxidative phosphorylation pathway and drive ATP synthesis.
Genes encoding enzymes involved at multiple steps of these metabolic pathways
  1. uptake,
  2. esterification,
  3. mitochondrial transport,
  4. and oxidation
are transcriptionally regulated by PGC-1a
  • with its nuclear receptor partners, including PPARs and ERRs .
Glucose uptake/oxidation and electron transport/oxidative phosphorylation pathways are also regulated by PGC-1a via
  • other transcription factors, such as MEF-2 and NRF-1.

[Cyt c, cytochrome c]

 Fetal metabolism of carbohydrate utilization

This reviewer poses the question of whether the fetal cardiac metabolism, which is characterized by a (facultative) anaerobic glycolysis,
  • results in lactate production that is not redirected into the TCA cycle.
An unexamined, but related question is whether there is an associated change in the ratio of
  • mitochondrial to cytoplasmic malate dehydrogenase isoenzyme activity (m-MDH:c-MDH).
The fetal heart operates without oxygenation from a functioning lung, bathed in amniotic fluid.
An enzymatic feature might be expressed in a facultative anaerobic cytplasmic glycolytic pathway characterized by
  • a decrease in the h-type lactate dehydrogenase (LD) isoenzyme(s) (LD1, LD2) with a predominance of
  • the m-type LD isoenzymes (LD3, LD4, LD5).
The observation here is that the heart muscle is a syncytium, and it functions at a highly regulated rate,
  • not with the spurts of activity seen in skeletal muscle.
In another article in this series, there are morphological changes that occur in the heart mitochondria, and
  • there are three locations, as if the organelle itself were an organ.
The normal functioning myocardium can utilize lactic acid accumulated in the bloodstream during extreme exercise as fuel.
This is a virtue of mitochondrial function.  There is a significant functional difference between the roles of the h- and m-type LD isoenzymes.
The h-type is a regulatory enzyme that forms a complex as NADH is converted to NAD+ between the
  • LD (H4, H3M; LD1, LD2),
  • oxidized pyridine nucleotide coenzyme, and
  • pyruvate
The complex forms in 200 msec as observed in the Aminco-Morrow stop-flow analyzer.  This is not the case for the m-type isoenzyme.
I presume that it is not a factor in embryonic heart.  It would become a factor after birth with the expansion of the lungs.
This would also bring to the discussion the effect of severe restrictive lung disease on cardiac metabolism.

Related References:

LH Bernstein,  patents: Malate dehydrogenase method,  The lactate dehydrogenase method
LH Bernstein, J Everse. Determination of the isoenzyme levels of lactate dehydrogenase. Methods Enzymol 1975; 41 47-52    ICID: 825516
LH Bernstein, J Everse, N Shioura, PJ Russell. Detection of cardiac damage using a steady state assay for lactate dehydrogenase isoenzymes in serum.   J Mol Cell Cardiol 1974; 6(4):297-315  ICID: 825597
LH Bernstein, MB Grisham, KD Cole, J Everse . Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases. J Biol Chem 1978; 253(24):8697-8701. ICID: 825513
R Belding, L Bernstein, G Reynoso. An evaluation of the immunochemical LD1 method in routine clinical practice. Clin Chem 1981; 27(10):1027-1028.   ICID: 844981
J Adan, L H Bernstein, J Babb. Lactate dehydrogenase isoenzyme-1/total ratio: accurate for determining the existence of myocardial infarction. Clin Chem 1986; 32(4):624-628.  ICID: 825540
MB Grisham, LH Bernstein, J Everse. The cytoplasmic malate dehydrogenase in neoplastic tissues; presence of a novel isoenzyme? Br J Cancer 1983; 47(5):727-731. ICID: 825551
LH Bernstein, P Scinto. Two methods compared for measuring LD-1/total LD activity in serum. Clin Chem 1986; 32(5):792-796.   ICID: 825581

PGC-1a: an inducible integrator of transcriptional circuits

 The PPAR³ coactivator-1 (PGC-1) family of transcriptional coactivators is involved in regulating mitochondrial metabolism and biogenesis.
PGC-1a was the first member discovered through its functional interaction with the nuclear receptor PPAR³ in brown adipose tissue (BAT).
There are two PGC-1a related coactivators,
  1. PGC-1² (also called PERC) and
  2. PGC-1–related coactivator (PRC).
PRC coactivates transcription in mitochondrial biogenesis, with PGC-1a and PGC-1² . Both are expressed in tissues with high oxidative capacity, such as
  1. heart
  2. slow-twitch skeletal muscle, and
  3. BAT
They serve critical roles in the regulation of mitochondrial functional capacity. PGC-1a  also regulates
  • hepatic gluconeogenesis and
  • skeletal muscle glucose uptake.
PGC-1² appears to be important in regulating energy metabolism in the heart, but
  • PGC-1a is distinct from other PGC-1 family members, indeed from most coactivators, in its broad responsiveness to
  1. developmental alterations in energy metabolism and
  2. physiological and pathological cues at the level of expression and transactivation.
In the heart, PGC-1a expression increases at birth coincident with an increase in cardiac oxidative capacity and
  • a perinatal shift from reliance on glucose metabolism to the oxidation of fats for energy.
PGC-1a is induced by physiological stimuli that increase ATP demand and
  • stimulate mitochondrial oxidation, including
  1. cold exposure,
  2. fasting, and
  3. exercise.
Activation of this regulatory cascade increases cardiac mitochondrial oxidative capacity in the heart. In cardiac myocytes in culture, it
  1. increases mitochondrial number,
  2. upregulates expression of mitochondrial enzymes, and
  3. increases rates of FA oxidation and coupled respiration.
Thus, PGC-1a is an inducible coactivator that coordinately regulates
  • cardiac fuel selection and
  • mitochondrial ATP-producing capacity.
 PGC-1a activates expression of nuclear respiratory factor-1 (NRF-1) and NRF-2 and
  • directly coactivates NRF-1 on its target gene promoters.
NRF-1 and NRF-2 regulate expression of mitochondrial transcription factor A (Tfam),
  • a nuclear-encoded transcription factor that binds regulatory sites on mitochondrial DNA and is essential for
  1. replication,
  2. maintenance, and
  3. transcription of the mitochondrial genome.
Furthermore, NRF-1 and NRF-2 regulate the expression of nuclear genes encoding
  • respiratory chain subunits and other proteins required for mitochondrial function.
PGC-1a  also
  • coactivates the PPAR and ERR nuclear receptors, critical regulators of myocardial FFA utilization.
  • regulates genes involved in the cellular uptake and mitochondrial oxidation of FFAs.
  • is an integrator of the transcriptional network regulating mitochondrial biogenesis and function.
Numerous signaling pathways, by increasing either PGC-1a expression or activity, such as –
  • Ca2+-dependent,
  • NO,
  • MAPK, and
  • beta-adrenergic pathways (beta3/cAMP),
    • activate the PGC-1a directly
Additionally, the p38_MAPK pathway
  • selectively activates PPARa, which may bring about synergistic activation in the presence of PGC-1a,
  • whereas ERK-MAPK has the opposite effect.
These signaling pathways transduce physiological stimuli to the PGC-1a pathway:
  1. stress
  2. fasting
  3. exercise
PGC-1a, in turn, coactivates transcriptional partners,which regulate mitochondrial biogenesis and FA-oxidation pathways:
  • NRF-1 and -2,
  • ERRa, and
  • PPARa,
 Insights into the physiological responsiveness of the PGC-1a pathway come from
  • identification of signal transduction pathways that modulate the activity of PGC-1a and its downstream partners.
PGC-1a is upregulated in response to beta-adrenergic signaling, consistent with the involvement of this pathway in thermogenesis.
The stress-activated  p38_MAPK activates PGC-1a by increasing PGC-1a protein stability and promoting dissociation of a repressor.
p38 increases mitochondrial FAO through selective activation of the PGC-1a partner, PPARa. Conversely, the ERK-MAPK pathway
  • inactivates the PPARa/RXRa complex via direct phosphorylation.
Therefore, distinct limbs of the MAPK pathway exert
  • opposing regulatory influences on the PGC-1a cascade.
Recently, NO has emerged as a novel signaling molecule proposed to integrate pathways involved in
  • regulating mitochondrial biogenesis by inducing mitochondrial proliferation.

 A Paradox

Mitochondria are like little cells within our cells. They are the energy producing organelles of the body. The more energy a certain tissue requires
  • such as the brain and the heart
    • the more mitochondria those cells contain.
Conventional transmission electron microscopy of mammalian cardiac tissue reveals mitochondria to be
  1. elliptical individual organelles situated either in clusters beneath the sarcolemma (subsarcolemmal mitochondria, SSM) or
  2. in parallel, longitudinal rows ensconced within the contractile apparatus (interfibrillar mitochondria, IFM).
The two mitochondrial populations differ in their cristae morphology, with
  1. a lamelliform orientation in SSM, whereas
  2. the cristae orientation in IFM is tubular.
The morphology of mitochondria is responsive to changes in cardiomyocytes.
 Mitochondrial oxidative phosphorylation relies
  • not only on the activities of individual complexes, but also on
  • the coordinated action of supramolecular assemblies (respirasomes) of the electron transport chain (ETC) complexes
in both normal and failing heart.
Mitochondria have their own set of DNA and
  • the more energy they generate,
  • the more DNA-damaging free radicals they produce.
Mitochondrial DNA damage is incurred by generation of energy in ATP production, so that
  • the process that sustains life also is the source of toxic damage that causes the dysfunction and mitogeny in the cell.
In human mtDNA mutant cybrids with impaired mitochondrial respiration, the recovery of mitochondrial function
  • correlates with the formation of respirasomes suggesting that
  • respirasomes represent regulatory units of mitochondrial oxidative phosphorylation
    • by facilitating the electron transfer between the catalytic sites of the ETC.
We recently reported a decrease in mitochondrial respirasomes in CHF that fits in the category of a new mitochondrial cytopathy.
 ATP utilized by the heart is synthesized mainly by means of oxidative phosphorylation in the inner mitochondrial membrane,
  • a process that involves the coupling of electron transfer and oxygen consumption with phosphorylation of ADP to ATP.
The catabolism of exogenous substrates (FAs, glucose, pyruvate, lactate, and ketone bodies) provides the reduced intermediates,
  1. NADH (nicotinamide adenine dinucleotide, reduced) and
  2. FADH2 (flavin adenine dinucleotide, reduced),
as donors for mitochondrial electron transport.
The contribution of glucose to the acetyl CoA pool in the heart is
  • increased by insulin during the postprandial period and during exercise.
 All cells and tissues require
  • adenine,
  • pyridine, and
  • flavin nucleotides for energy
by way of Krebs cycle metabolism of fatty acids and carbohydrate substrates.
If DNA holds the blueprint for the proper function of a cell, then any change in the blueprint will change how the cell functions.
If the mitochondria do not function properly, then they cannot fulfill their role in producing energy:
  •  the cell will lose its ability to function adequately.

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 References

Mitochondrial dynamics and cardiovascular diseases    Ritu Saxena
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/
Mitochondrial Damage and Repair under Oxidative Stress   larryhbern
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Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation   larryhbern
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http://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/
Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function    larryhbern
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/
Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis  larryhbern
http://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/
Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar
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The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure    larryhbern
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Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis. Aviva Lev-Ari, PhD, RN 10/30/2012
https://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/
Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD, Aviva Lev-Ari, PhD, RN 4/7/2013
https://pharmaceuticalintelligence.com/2013/04/07/cholesteryl-ester-transfer-protein-cetp-inhibitor-potential-of-anacetrapib-to-treat-atherosclerosis-and-cad/
Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013 https://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/
Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013
https://pharmaceuticalintelligence.com/2013/04/03/fight-against-atherosclerotic-cardiovascular-disease-a-biologics-not-a-small-molecule-recombinant-human-lecithin-cholesterol-acyltransferase-rhlcat-attracted-astrazeneca-to-acquire-alphacore/
High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013 

https://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/
Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012
https://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/
Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-and-hyperhomocusteinemia/

 

 

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Larry H. Bernstein, MD, FCAP

 

Evaluation of Antioxidant Potential of “Maltese Mushroom” (Cynomorium coccineum) by Means of Multiple Chemical and Biological Assays

Zucca P , Rosa A ,  Tuberoso CIG , Piras A ,  Rinaldi AC , Sanjust E , et al.   Nutrients 2013, 5(1), 149-161; doi:10.3390/nu5010149  (just published by Libertas)

https://pharmaceuticalintelligence.com/2013/01/27/antioxidant-po…rium-coccineum/

Cynomorium coccineum is an edible, non-photosynthetic plant widespread along the coasts of the Mediterranean Sea.

The medicinal properties of Maltese mushroom  have been kept in high regard since ancient times.

We evaluated the antioxidant potential of fresh specimens of C. coccineum picked in Sardinia, Italy.

Both aqueous and methanolic extracts were tested by using multiple assay systems (DPPH, FRAP, TEAC, ORAC-PYR).

Total phenolics and flavonoids were also determined.

Gallic acid and cyanidin 3-O-glucoside were identified as the main constituents and measured.

Both extracts showed antioxidant capacities; ORAC-PYR assay gave the highest antioxidant value in both cases.

The methanolic extract was further investigated with in vitro biological models of lipid oxidation;

  • it showed a significant activity in preventing cholesterol degradation and
  • exerted protection against Cu2+-mediated degradation of the liposomal unsaturated fatty acids.

Results of the present study demonstrate that

  • the extracts of C. coccineum show a significant total antioxidant power and also
  • exert an in vitro protective effect in different bio-assays of oxidative stress.

Therefore, Maltese mushroom can be considered a valuable source of antioxidants and phytochemicals

  • useful in the preparation of nutraceuticals and functional foods.

Keywords: plant-based foods; antioxidant; nutraceuticals; Cynomorium coccineum; fungus melitensis; Maltese mushroom

Cynomorium coccineum

Cynomorium coccineum (Photo credit: Alastair Rae)

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Reporter: Aviva Lev-Ari, PhD, RN

 

The role of the saturated non-esterified fatty acid palmitate in beta cell dysfunction

J. Proteome Res., Just Accepted Manuscript
DOI: 10.1021/pr300596g
Publication Date (Web): November 21, 2012
Copyright © 2012 American Chemical Society

Abstract

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels.

The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/l palmitate and combined or not with 25 mmol/l glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. 83 differentially expressed proteins (P < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery.

 

SOURCE:

http://pubs.acs.org/doi/abs/10.1021/pr300596g?elq=7a326578ab424110aabf8de481b35633

 

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struktura infliximabu

struktura infliximabu (Photo credit: Wikipedia)

Larry H Bernstein, MD, FCAP, Reporter

GI Disease, inflammation, elastase-inhibitor, membrane junctions and fatty acids

Sci Transl Med 2012; 4(158): 158ra144

Sci. Transl. Med. DOI: 10.1126/scitranslmed.3004212

RESEARCH ARTICLE
INFLAMMATORY BOWEL DISEASES
Food-Grade Bacteria Expressing Elafin Protect Against Inflammation and Restore Colon Homeostasis
Jean-Paul Motta1,2,3,*, Luis G. Bermúdez-Humarán4,*, Céline Deraison1,2,3, Laurence Martin1,2,3, Corinne Rolland1,2,3, Perrine Rousset1,2,3, Jérôme Boue1,2,3, Gilles Dietrich1,2,3, Kevin Chapman5, Pascale Kharrat4, Jean-Pierre Vinel3,6, Laurent Alric3,6, Emmanuel Mas1,2,3,7, Jean-Michel Sallenave8,9,10, Philippe Langella4,* and Nathalie Vergnolle1,2,3,5,†

1INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse F-31300, France.
2CNRS, U5282, Toulouse F-31300, France.
3CPTP, Université de Toulouse, Université Paul Sabatier (UPS), Toulouse F-31300, France.
4Institut National de la Recherche Agronomique (INRA), UMR 1319 Micalis, Commensal and Probiotics-Host Interactions Laboratory, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
5Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
6Pôle Digestif, CHU Purpan, Toulouse F-31059, France.
7Division of Gastroenterology, Hepatology and Nutrition, Children’s Hospital, Toulouse F-31059, France.
8Institut Pasteur, Unité de Défense Innée et Inflammation, Paris F-75015, France.
9INSERM U874, Paris F-75724, France.
10Universite Paris Diderot, Sorbonne Paris Cite, Cellule Pasteur F-75013, France.

ABSTRACT

Elafin, a natural protease inhibitor expressed in healthy intestinal mucosa, has pleiotropic anti-inflammatory properties in vitro and in animal models. We found that mucosal expression of Elafin is diminished in patients with inflammatory bowel disease (IBD). This defect is associated with increased elastolytic activity (elastase-like proteolysis) in colon tissue. We engineered two food-grade strains of lactic acid bacteria (LAB) to express and deliver Elafin to the site of inflammation in the colon to assess the potential therapeutic benefits of the Elafin-expressing LAB. In mouse models of acute and chronic colitis, oral administration of Elafin-expressing LAB decreased elastolytic activity and inflammation and restored intestinal homeostasis. Furthermore, when cultures of human intestinal epithelial cells were treated with LAB secreting Elafin, the inflamed epithelium was protected from increased intestinal permeability and from the release of cytokines and chemokines, both of which are characteristic of intestinal dysfunction associated with IBD. Together, these results suggest that oral delivery of LAB secreting Elafin may be useful for treating IBD in humans.

Copyright © 2012, American Association for the Advancement of Science
Citation: J.-P. Motta, L. G. Bermúdez-Humarán, C. Deraison, L. Martin, C. Rolland, P. Rousset, J. Boue, G. Dietrich, K. Chapman, P. Kharrat, J.-P. Vinel, L. Alric, E. Mas, J.-M. Sallenave, P. Langella, N. Vergnolle, Food-Grade Bacteria Expressing Elafin Protect Against Inflammation and Restore Colon Homeostasis. Sci. Transl. Med. 4, 158ra144 (2012).

Cytokines involved in IBD

Cytokines involved in IBD (Photo credit: Wikipedia)

Metabolism

Front. Physio., 10 October 2012 | doi: 10.3389/fphys.2012.00401
Outlook: membrane junctions enable the metabolic trapping of fatty acids by intracellular acyl-CoA synthetases
Joachim Füllekrug*, Robert Ehehalt and Margarete Poppelreuther
Molecular Cell Biology Laboratory, Internal Medicine IV, University of Heidelberg, Heidelberg, Germany
The mechanism of fatty acid uptake is of high interest for basic research and clinical interventions. Recently, we showed that mammalian long chain fatty acyl-CoA synthetases (ACS) are not only essential enzymes for lipid metabolism but are also involved in cellular fatty acid uptake. Overexpression, RNAi depletion or hormonal stimulation of ACS enzymes lead to corresponding changes of fatty acid uptake. Remarkably, ACS are not localized to the plasma membrane where fatty acids are entering the cell, but are found instead at the endoplasmic reticulum (ER) or other intracellular organelles like mitochondria and lipid droplets. This is in contrast to current models suggesting that ACS enzymes function in complex with transporters at the cell surface. Drawing on recent insights into non-vesicular lipid transport, we suggest a revised model for the cellular fatty acid uptake of mammalian cells which incorporates trafficking of fatty acids across membrane junctions. Intracellular ACS enzymes are then metabolically trapping fatty acids as acyl-CoA derivatives. These local decreases in fatty acid concentration will unbalance the equilibrium of fatty acids across the plasma membrane, and thus provide a driving force for fatty acid uptake.

English: Acyl-CoA from the cytosol to the mito...

English: Acyl-CoA from the cytosol to the mitochondrial matrix. Français : Transport de l’Acyl-CoA du Cytosol jusqu’à la matrice mitochondriale. (Photo credit: Wikipedia)

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English: The mechanism for Long Chain Fatty Acyl-CoA Synthetase (Photo credit: Wikipedia)

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