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Posts Tagged ‘Atherosclerosis’


Regulatory MicroRNAs in Aberrant Cholesterol Transport and Metabolism

Curator: Marzan Khan, B.Sc

Aberrant levels of lipids and cholesterol accumulation in the body lead to cardiometabolic disorders such as atherosclerosis, one of the leading causes of death in the Western World(1). The physical manifestation of this condition is the build-up of plaque along the arterial endothelium causing the arteries to constrict and resist a smooth blood flow(2). This obstructive deposition of plaque is merely the initiation of atherosclerosis and is enriched in LDL cholesterol (LDL-C) as well foam cells which are macrophages carrying an overload of toxic, oxidized LDL(2). As the condition progresses, the plaque further obstructs blood flow and creates blood clots, ultimately leading to myocardial infarction, stroke and other cardiovascular diseases(2). Therefore, LDL is referred to as “the bad cholesterol”(2).

Until now, statins are most widely prescribed as lipid-lowering drugs that inhibit the enzyme 3-hydroxy-3methylgutaryl-CoA reductase (HMGCR), the rate-limiting step in de-novo cholesterol biogenesis (1). But some people cannot continue with the medication due to it’s harmful side-effects(1). With the need to develop newer therapeutics to combat cardiovascular diseases, Harvard University researchers at Massachusetts General Hospital discovered 4 microRNAs that control cholesterol, triglyceride, and glucose homeostasis(3)

MicroRNAs are non-coding, regulatory elements approximately 22 nucleotides long, with the ability to control post-transcriptional expression of genes(3). The liver is the center for carbohydrate and lipid metabolism. Stringent regulation of endogenous LDL-receptor (LDL-R) pathway in the liver is crucial to maintain a minimal concentration of LDL particles in blood(3). A mechanism whereby peripheral tissues and macrophages can get rid of their excess LDL is mediated by ATP-binding cassette, subfamily A, member 1 (ABCA1)(3). ABCA1 consumes nascent HDL particles- dubbed as the “good cholesterol” which travel back to the liver for its contents of triglycerides and cholesterol to be excreted(3).

Genome-wide association studies (GWASs) meta-analysis carried out by the researchers disclosed 4 microRNAs –(miR-128-1, miR-148a, miR-130b, and miR-301b) to lie close to single-nucleotide polymorphisms (SNPs) associated with abnormal metabolism and transport of lipids and cholesterol(3) Experimental analyses carried out on relevant cell types such as the liver and macrophages have proven that these microRNAs bind to the 3’ UTRs of both LDL-R and ABCA1 transporters, and silence their activity. Overexpression of miR-128-1 and miR148a in mice models caused circulating HDL-C to drop. Corroborating the theory under investigation further, their inhibition led to an increased clearance of LDL from the blood and a greater accumulation in the liver(3).

That the antisense inhibition of miRNA-128-1 increased insulin signaling in mice, propels us to hypothesize that abnormal expression of miR-128-1 might cause insulin resistance in metabolic syndrome, and defective insulin signaling in hepatic steatosis and dyslipidemia(3)

Further examination of miR-148 established that Liver-X-Receptor (LXR) activation of the Sterol regulatory element-binding protein 1c (SREBP1c), the transcription factor responsible for controlling  fatty acid production and glucose metabolism, also mediates the expression of miR-148a(4,5) That the promoter region of miR-148 contained binding sites for SREBP1c was shown by chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq)(4). More specifically, SREBP1c attaches to the E-box2, E-box3 and E-box4 elements on miR-148-1a promoter sites to control its expression(4).

Earlier, the same researchers- Andres Naars and his team had found another microRNA called miR-33 to block HDL generation, and this blockage to reverse upon antisense targeting of miR-33(6).

These experimental data substantiate the theory of miRNAs being important regulators of lipoprotein receptors and transporter proteins as well as underscore the importance of employing antisense technologies to reverse their gene-silencing effects on LDL-R and ABCA1(4). Such a therapeutic approach, that will consequently lower LDL-C and promote HDL-C seems to be a promising strategy to treat atherosclerosis and other cardiovascular diseases(4).

References:

1.Goedeke L1,Wagschal A2,Fernández-Hernando C3, Näär AM4. miRNA regulation of LDL-cholesterol metabolism. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):2047-2052

https://www.ncbi.nlm.nih.gov/pubmed/26968099

2.MedicalNewsToday. Joseph Nordgvist. Atherosclerosis:Causes, Symptoms and Treatments. 13.08.2015

http://www.medicalnewstoday.com/articles/247837.php

3.Wagschal A1,2, Najafi-Shoushtari SH1,2, Wang L1,2, Goedeke L3, Sinha S4, deLemos AS5, Black JC1,6, Ramírez CM3, Li Y7, Tewhey R8,9, Hatoum I10, Shah N11, Lu Y11, Kristo F1, Psychogios N4, Vrbanac V12, Lu YC13, Hla T13, de Cabo R14, Tsang JS11, Schadt E15, Sabeti PC8,9, Kathiresan S4,6,8,16, Cohen DE7, Whetstine J1,6, Chung RT5,6, Fernández-Hernando C3, Kaplan LM6,10, Bernards A1,6,16, Gerszten RE4,6, Näär AM1,2. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. . Nat Med.2015 Nov;21(11):1290

https://www.ncbi.nlm.nih.gov/pubmed/26501192

4.Goedeke L1,2,3,4, Rotllan N1,2, Canfrán-Duque A1,2, Aranda JF1,2,3, Ramírez CM1,2, Araldi E1,2,3,4, Lin CS3,4, Anderson NN5,6, Wagschal A7,8, de Cabo R9, Horton JD5,6, Lasunción MA10,11, Näär AM7,8, Suárez Y1,2,3,4, Fernández-Hernando C1,2,3,4. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels. Nat Med. 2015 Nov;21(11):1280-9.

https://www.ncbi.nlm.nih.gov/pubmed/26437365

5.Eberlé D1, Hegarty B, Bossard P, Ferré P, Foufelle F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie. 2004 Nov;86(11):839-48.

https://www.ncbi.nlm.nih.gov/pubmed/15589694

6.Harvard Medical School. News. MicoRNAs and Metabolism.

https://hms.harvard.edu/news/micrornas-and-metabolism

7. MGH – Four microRNAs identified as playing key roles in cholesterol, lipid metabolism

http://www.massgeneral.org/about/pressrelease.aspx?id=1862

 

Other related articles published in this Open Access Online Scientific Journal include the following:

 

  • Cardiovascular Diseases, Volume Three: Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics,

on Amazon since 11/29/2015

http://www.amazon.com/dp/B018PNHJ84

 

HDL oxidation in type 2 diabetic patients

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/27/hdl-oxidation-in-type-2-diabetic-patients/

 

HDL-C: Target of Therapy – Steven E. Nissen, MD, MACC, Cleveland Clinic vs Peter Libby, MD, BWH

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/11/07/hdl-c-target-of-therapy-steven-e-nissen-md-macc-cleveland-clinic-vs-peter-libby-md-bwh/

 

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk

Curator: Aviva Lev-Ari, PhD, RN

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/

 

Risk of Major Cardiovascular Events by LDL-Cholesterol Level (mg/dL): Among those treated with high-dose statin therapy, more than 40% of patients failed to achieve an LDL-cholesterol target of less than 70 mg/dL.

Reporter: Aviva Lev-Ari, PhD., RN

https://pharmaceuticalintelligence.com/2014/07/29/risk-of-major-cardiovascular-events-by-ldl-cholesterol-level-mgdl-among-those-treated-with-high-dose-statin-therapy-more-than-40-of-patients-failed-to-achieve-an-ldl-cholesterol-target-of-less-th/

 

LDL, HDL, TG, ApoA1 and ApoB: Genetic Loci Associated With Plasma Concentration of these Biomarkers – A Genome-Wide Analysis With Replication

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/18/ldl-hdl-tg-apoa1-and-apob-genetic-loci-associated-with-plasma-concentration-of-these-biomarkers-a-genome-wide-analysis-with-replication/

 

Two Mutations, in the PCSK9 Gene: Eliminates a Protein involved in Controlling LDL Cholesterol

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/04/15/two-mutations-in-a-pcsk9-gene-eliminates-a-protein-involve-in-controlling-ldl-cholesterol/

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/15/artherogenesis-predictor-of-cvd-the-smaller-and-denser-ldl-particles/

 

A Concise Review of Cardiovascular Biomarkers of Hypertension

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2016/04/25/a-concise-review-of-cardiovascular-biomarkers-of-hypertension/

 

Triglycerides: Is it a Risk Factor or a Risk Marker for Atherosclerosis and Cardiovascular Disease ? The Impact of Genetic Mutations on (ANGPTL4) Gene, encoder of (angiopoietin-like 4) Protein, inhibitor of Lipoprotein Lipase

Reporters, Curators and Authors: Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2016/03/13/triglycerides-is-it-a-risk-factor-or-a-risk-marker-for-atherosclerosis-and-cardiovascular-disease-the-impact-of-genetic-mutations-on-angptl4-gene-encoder-of-angiopoietin-like-4-protein-that-in/

 

Excess Eating, Overweight, and Diabetic

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/15/excess-eating-overweight-and-diabetic/

 

Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/12/obesity-issues/

 

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Reversing Heart Disease: Combination of PCSK9 Inhibitors and Statins – Opinion by Steven Nissen, MD, Chairman of Cardiovascular Medicine at Cleveland Clinic

 

Reporter: Aviva Lev-Ari, PhD, RN

 

UPDATED on 5/5/2017

Europeans Mull PCSK9i Post-FOURIER Fallout on Clinical Practice

Patrice Wendling, May 04, 2017

But it was panelist Dr Stephen Nicholls (University of Adelaide, Australia) who took aim at the elephant in the packed auditorium. At an annual cost of about $14,100 for evolocumab and $14,600 for alirocumab (Praluent, Sanofi/Regeneron), the important question facing cardiologists is who will be eligible for these drugs “in a world where we can’t just write a scrip for every FOURIER-type patient; we won’t be allowed to.”

He suggested initially this will include patients with familial hypercholesterolemia and only those with established atherosclerotic CVD whose LDL-C remains unacceptably high despite therapy. Future FOURIER subanalyses may define other eligible high-risk groups.

http://www.medscape.com/viewarticle/879523?nlid=114642_3802&src=WNL_mdplsnews_170505_mscpedit_card&uac=93761AJ&spon=2&impID=1342003&faf=1#vp_2

 

 

UPDATED on 3/14/2017

PCSK9 Inhibitor Access Snarled in Red Tape, Rejections

Patrice Wendling, March 21, 2017

To determine whether this experience is happening nationwide, Navar and colleagues examined first PCSK9 prescriptions in 45,029 patients (median age 66 years; 51% female) between August 1, 2015 and July 31, 2016 in the Symphony Health Solutions database, which covers 90% of retail, 70% of specialty, and 60% of mail-order pharmacies in the US.

Nearly half (48%) of prescribers were cardiologists, and 37% were general practitioners. Most patients (52%) had government insurance, typically Medicare, and 40% had commercial insurance.

In the first 24 hours after being submitted to the pharmacy, 79.2% of prescriptions were rejected. Ultimately, 52.8% of all PCSK9 prescriptions were rejected.

Of special note, 34.7% of prescriptions for the pricy lipid-lowering drugs were abandoned at the pharmacy.

http://www.medscape.com/viewarticle/877515?nlid=113592_3802&src=WNL_mdplsnews_170324_mscpedit_card&uac=93761AJ&spon=2&impID=1314983&faf=1

 

How 2 Drugs Lower Cholesterol Remarkably — and Reverse Heart Disease

Study shows promise for combination of newer drug and statins

How 2 Drugs Lower Cholesterol Remarkably --- and Reverse Heart Disease

Newer cholesterol-lowering drugs combined with more conventional medicine reduces bad cholesterol to incredibly low levels, a new study shows. Perhaps even more important, the combination also reduces the heart-attack-inducing plaque that forms inside the arteries, the study says.

The study was led by cardiologist Steven Nissen, MD, Chairman of Cardiovascular Medicine at Cleveland Clinic. Results appeared recently in the Journal of the American Medical Association (JAMA).

The study looked at the use of a drug called evolocumab by people who took statins to lower the amount of LDL, or bad, cholesterol in their blood. Evolocumab is a drug called a PCSK9 inhibitor. This is a newer kind of medicine that can make LDL cholesterol levels plummet.

The people who took statins and evolocumab had greater reductions in atherosclerosis compared with people who took statins and a placebo.  Atherosclerosis is  a disease in which plaque builds up inside your arteries.  The condition can lead to serious problems, including heart attack, stroke, or even death.

The results are an intriguing indicator — rather than definite proof — that evolocumab may have benefit for patients taking statins, Dr. Nissen says. Researchers are still awaiting the results of large clinical trials investigating whether evolocumab is safe and will prevent heart attack, stroke or death. The first results of these studies are expected in April 2017.

Special ultrasound

In the study, researchers treated for 18 months 968 high-risk people who had extremely high levels of blood cholesterol.

Participants were randomly assigned to take either a statin and a placebo, or a statin and evolocumab.

Researchers monitored the participants’ cholesterol levels. They also used a special ultrasound probe to measure the amount of plaque in their arteries at the beginning and the end of the study. 

“We were able to show that getting the bad cholesterol levels down to really low levels, down to the 20s and 30s, can actually remove plaque from the coronary arteries,” Dr. Nissen says. “This going to levels that we’ve never been able to achieve before.”           

Low cholesterol, less plaque

Results show the group treated with statins and a placebo reduced their LDL cholesterol levels to 93 on average. At the same time, the group treated with the combination of the statin plus evolocumab got down to an average bad cholesterol level of 36.6.

“No one’s ever reached levels that low in a clinical trial,” Dr. Nissen says.

Participants who took evolocumab also had less plaque in their arteries at the end of the study — essentially reversing their heart disease.

“We, for the first time now, have shown that this new class of drugs, the PCSK9 inhibitors, has a favorable effect on the development of plaques on the coronary artery and can actually regress those plaques,” Dr. Nissen says. “And it turns out about two-thirds of patients actually had less plaque at the end of 18 months than they started with.” 

PCSK9 inhibitors, which are expensive, are not for everybody, Dr. Nissen says. Currently, the drug is used in addition to statins for the highest-risk patients with particularly high cholesterol.

SOURCE

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Lipid Metabolism


Lipid Metabolism

Reporter and Curator: Larry H. Bernstein, MD, FCAP 

 

This is fourth of a series of articles, lipid metabolism, that began with signaling and signaling pathways. These discussion lay the groundwork to proceed in later discussions that will take on a somewhat different approach. These are critical to develop a more complete point of view of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  The role of lipids in circulating plasma proteins as biomarkers for coronary vascular disease can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and disease has had much less attention, despite the enormous importance, especially in the nervous system.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism

3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence

  1. Lipid metabolism
  2. Protein synthesis and degradation
  3. Subcellular structure
  4. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

 

Lipid Metabolism

http://www.elmhurst.edu/~chm/vchembook/622overview.html

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidationto produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.

 

metabolism of fats

metabolism of fats

 

http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

 

fattyacidspiral

fattyacidspiral

http://www.elmhurst.edu/~chm/vchembook/images/620fattyacidspiral.gif

 

 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.

 

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2
 NET 12 ATP

 

 

 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP
GRAND TOTAL  130 ATP

 

Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.

 

This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.

 

  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.

 

.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.

 

My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.

 

The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.

 

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1964/lynen-bio.html

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Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

http://science.howstuffworks.com/dictionary/famous-scientists/biologists/feodor-lynen-info.htm

Fyodor Lynen

Fyodor Lynen

 

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
http://dx.doi.org:/10.1172/JCI15593
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).

 

Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

 

Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

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SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.

 

Figure 1

 

When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).

 

Figure 2

 

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

 

 

 

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Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (916). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.

 

Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

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Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

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Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

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We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

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The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

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The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (2224). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

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In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.

 

Acknowledgments

Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.

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  1. Goldstein, JL, Rawson, RB, Brown, MS. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch Biochem Biophys 2002. 397:139-148.

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  1. Shimomura, I, Shimano, H, Horton, JD, Goldstein, JL, Brown, MS. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J Clin Invest 1997. 99:838-845.

View this article via: JCI.org PubMed

  1. Moon, Y-A, Shah, NA, Mohapatra, S, Warrington, JA, Horton, JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem 2001. 276:45358-45366.

View this article via: PubMed

  1. Shimomura, I, Shimano, H, Korn, BS, Bashmakov, Y, Horton, JD. Nuclear sterol regulatory element binding proteins activate genes responsible for entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J Biol Chem 1998. 273:35299-35306.

View this article via: PubMed

  1. Shimano, H, et al. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 1996. 98:1575-1584.

View this article via: JCI.org PubMed

  1. Shimano, H, et al. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 1997. 99:846-854.

View this article via: JCI.org PubMed

  1. Horton, JD, et al. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 1998. 101:2331-2339.

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  1. Korn, BS, et al. Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N). J Clin Invest 1998. 102:2050-2060.

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  1. Shimano, H, et al. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin Invest 1997. 100:2115-2124.

View this article via: JCI.org PubMed

  1. Matsuda, M, et al. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 2001. 15:1206-1216.

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  1. Yang, J, et al. Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene. Proc Natl Acad Sci USA 2001. 98:13607-13612.

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Liang, G, et al. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 2002. 277:9520-9528.

http://www.jci.org/articles/view/15593

 

Structural Biochemistry/Lipids/Membrane Lipids

< Structural Biochemistry‎ | Lipids

Membrane proteins rely on their interaction with membrane lipids to uphold its structure and maintain its functions as a protein. For membrane proteins to purify and crystallize, it is essential for the membrane protein to be in the appropriate lipid environment. Lipids assist in crystallization and stabilize the protein and provide lattice contacts. Lipids can also help obtain membrane protein structures in a native conformation. Membrane protein structures contain bound lipid molecules. Biological membranes are important in life, providing permeable barriers for cells and their organelles. The interaction between membrane proteins and lipids facilitates basic processes such as respiration, photosynthesis, transport, signal transduction and motility. These basic processes require a diverse group of proteins, which are encoded by 20-30% of an organism’s annotated genes.

There exist a great number of membrane lipids. Specifically, eukaryotic cells have a very complex collection of lipids that rely on many of the cell’s resources for its synthesis. Interactions between proteins and lipids can be very specific. Specific types of lipids can make a structure stable, provide control in insertion and folding processes, and help to assemble multisubunit complexes or supercomplexes, and most importantly, can significantly affect a membrane protein’s functions. Protein and lipid interactions are not sufficiently tight, meaning that lipids are retained during membrane protein purification. Since cellular membranes are fluid arrangements of lipids, some lipids affect interesting changes to membrane due to their characteristics. Glycosphigolipids and cholesterol tend to form small islands within the membranes, called lipid rafts, due to their physical properties. Some proteins also tend to cluster in lipid raft, while others avoid being in lipid rafts. However, the existence of lipid rafts in cells seems to be transitory.

Recent progress in determining membrane protein structure has brought attention to the importance of maintaining a favorable lipid environment so proteins to crystallize and purify successfully. Lipids assist in crystallization by stabilizing the protein fold and the relationships between subunits or monomers. The lipid content in protein-lipid detergent complexes can be altered by adjusting solubilisation and purification protocols, also by adding native or non-native lipids.

There are three type of membrane lipids: 1. Phospholipids: major class of membrane lipids. 2. glycolipids. 3. Cholesterols. Membrane lipids were started with eukaryotes and bacteria.

http://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Membrane_Lipids

Types of Membrane Lipids

Lipids are often used as membrane constituents. The three major classes that membrane lipids are divided into are phospholipids, glycolipids, and cholesterol. Lipids are found in eukaryotes and bacteria. Although the lipids in archaea have many features that are related to the membrane formation that is similar with lipids of other organisms, they are still distinct from one another. The membranes of archaea differ in composition in three major ways. Firstly, the nonpolar chains are joined to a glycerol backbone by ether instead of esters, allowing for more resistance to hydrolysis. Second, the alkyl chains are not linear, but branched and make them more resistant to oxidation. The ability of archaeal lipids to resist hydrolysis and oxidation help these types of organisms to withstand the extreme conditions of high temperature, low pH, or high salt concentration. Lastly, the stereochemistry of the central glycerol is inverted. Membrane lipids have an extensive repertoire, but they possess a critical common structural theme in which they are amphipathic molecules, meaning they contain both a hydrophilic and hydrophobic moiety.

Membrane lipids are all closed bodies or boundaries separating substituent parts of the cell. The thickness of membranes is usually between 60 and 100 angstroms. These bodies are constructed from non-covalent assemblies. Their polar heads align with each other and their non-polar hydrocarbon tails align as well. The resulting stability is credited to hydrophobic interaction which proves to be quite stable due to the length of their hydrocarbon tails.

 

Membrane Lipids

Lipid Vesicles

Lipid vesicles, also known as liposomes, are vesicles that are essentially aqueous vesicles that are surrounded by a circular phospholipid bilayer. Like the other phospholipid structures, they have the hydrocarbon/hydrophobic tails facing inward, away from the aqueous solution, and the hydrophilic heads facing towards the aqueous solution. These vesicles are structures that form enclosed compartments of ions and solutes, and can be utilized to study the permeability of certain membranes, or to transfer these ions or solutes to certain cells found elsewhere.

Liposomes as vesicles can serve various clinical uses. Injecting liposomes containing medicine or DNA (for gene therapy) into patients is a possible method of drug delivery. The liposomes fuse with other cells’ membranes and therefore combine their contents with that of the patient’s cell. This method of drug delivery is less toxic than direct exposure because the liposomes carry the drug directly to cells without any unnecessary intermediate steps.

Because of the hydrophobic interactions among several phospholipids and glycolipids, a certain structure called the lipid bilayer or bimolecular sheet is favored. As mentioned earlier, phospholipids and glycolipids have both hydrophilic and hydrophobic moieties; thus, when several phospholipids or glycolipids come together in an aqueous solution, the hydrophobic tails interact with each other to form a hydrophobic center, while the hydrophilic heads interact with each other forming a hydrophilic coating on each side of the bilayer.

http://upload.wikimedia.org/wikibooks/en/b/ba/Liposome_final%2A.png

http://upload.wikimedia.org/wikibooks/en/f/fa/Membrane_bilayer.jpg

 

Liposome_

Liposome_

 

 

Membrane_bilayer

Membrane_bilayer

 

 

 

Evidence Report/Technology Assessment   Number 89

 

Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis

 

Prepared for:

Agency for Healthcare Research and Quality

U.S. Department of Health and Human Services

540 Gaither Road

Rockville, MD 20850

http://www.ahrq.gov

Contract No. 290-02-0003

 

Chapter 1. Introduction

This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.

The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.

This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.

This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.

 

The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1.  Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Dietary Sources and Requirements

Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.

Syntheis and Degradation

Source of Acetyl CoA for Fatty Acid Synthesis

Source of Acetyl CoA for Fatty Acid Synthesis

step 1

step 1

condensation reaction with malonyl ACP

ACP (acyl carrier protein)

ACP (acyl carrier protein)

synthesis requires acetyl CoA from citrate shuttle

synthesis requires acetyl CoA from citrate shuttle

conversion to fatty acyl co A in cytoplasm

conversion to fatty acyl co A in cytoplasm

ACP (acyl carrier protein)

ACP (acyl carrier protein)

FA synthesis not exactly reverse of catabolism

FA synthesis not exactly reverse of catabolism

 

Fatty Acid Synthase

Fatty Acid Synthase

complete FA synthesis

complete FA synthesis

Desaturation

Desaturation

Elongation and Desaturation of Fatty Acids

Elongation and Desaturation of Fatty Acids

release of FAs from adiposites

release of FAs from adiposites

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

Fatty acid beta oxidation and Krebs cycle produce NAD, NADH, FADH2

ketone bodies

ketone bodies

metabolism of ketone bodies

metabolism of ketone bodies

Arachidonoyl-mimicking

Arachidonoyl-mimicking

Arachidonate pathways

Arachidonate pathways

arachidonic acid derivatives

arachidonic acid derivatives

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides

Model for the sterol-mediated proteolytic release of SREBPs from membrane

Model for the sterol-mediated proteolytic release of SREBPs from membrane

hormone regulation

hormone regulation

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

 islet brain glucose signaling

islet brain glucose signaling

 

 

 

 

 

 

 

 

Fish source

Fish source

omega FAs

omega FAs

 

Excessive omega 6s

Excessive omega 6s

omega 6s

omega 6s

diet and cancer

diet and cancer

Patients at risk of FA deficiency

Patients at risk of FA deficiency

PPAR role

PPAR role

PPAR role

PPAR role

Omega 6_3 pathways

Omega 6_3 pathways

n3 vs n6 PUFAs

n3 vs n6 PUFAs

triene-teraene ratio

triene-teraene ratio

arachidonic acid, leukotrienes, PG and thromboxanes

arachidonic acid, leukotrienes, PG and thromboxanes

Cox 2 and cancer

Cox 2 and cancer

Lipidomics of atherosclerotic plaques

Lipidomics of atherosclerotic plaques

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Effect of TPN on EFAD

Effect of TPN on EFAD

benefits of omega 3s

benefits of omega 3s

food consumption

food consumption

 

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Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release


Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN

and 

Content Consultant to Six-Volume e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

This portion summarises what we have covered and is now familiar to the reader.  There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading.  This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology.  It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process.  In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views.  There were surprises, and there were unfulfilled answers along the way.  The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable.  This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified.  In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.

We introduced the content as follows:

1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.

Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.

  • Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
  • PCI for valves vs Open heart Valve replacement
  • PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.

2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.

3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.

4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.

Curation

Curation is an active filtering of the web’s  and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view.  Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale.  One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive.  The same source says “Scoop.it is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.

Healthcare and Affordable Care Act

We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate.  But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system.  There has been a very large rush and an extension to sign up. There are many features that we can take note of:

1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.

2. These changes that are notable are:

  • The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
  • This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
  • This is not  helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
  • Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.

3.  The  medical care system is affected by the issues above, but the complexity is not to be discounted.

  •  The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
  • The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
  • In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
  • Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
  • There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
  • There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
  • Medicaid is expanded substantially under the new ACA.
  • The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
  • The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.

Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.

This portion is outlined as follows:

2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease

2.2 The Role of Calcium in Health and Disease

2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease

Genomics & Genetics of Cardiovascular Disease Diagnoses

actin cytoskeleton

wall stress, ventricular workload, contractile reserve

Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

calcium and actin skeleton, signaling, cell motility

hypertension & vascular compliance

Genetics of Conduction Disease

Ca+ stimulated exostosis: calmodulin & PKC (neurotransmitter)

complications & MVR

disruption of Ca2+ homeostasis cardiac & vascular smooth muscle

synaptotagmin as Ca2+ sensor & vesicles

atherosclerosis & ion channels


It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system.  The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence.  This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”.  The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression.  This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically.  The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.

Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm.  The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction.  The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction.  That is only part of the story.  Ion transport occurs into or out of the cell, the latter termed exostosis.  Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles.  This also involves K+-Na+-ATPase.  The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells.  When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial.  The glass is half full!

 

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Nicotinic Acetylcholine Receptor Genes with Subclinical Atherosclerosis in American Indians: Genetic Variants Study and Gene-Family Analysis

Reporter: Aviva Lev-Ari, PhD, RN

Joint Associations of 61 Genetic Variants in the Nicotinic Acetylcholine Receptor Genes with Subclinical Atherosclerosis in American Indians – A Gene-Family Analysis

Jingyun Yang, PhD*Yun Zhu, MS*Elisa T. Lee, PhD, Ying Zhang, PhD, Shelley A. Cole, PhD, Karin Haack, PhD, Lyle G. Best, BS MD, Richard B. Devereux, MD, Mary J. Roman, MD, Barbara V. Howard, PhD and Jinying Zhao, MD, PhD

Author Affiliations

From the Tulane University School of Public Health and Tropical Medicine, New Orleans, LA (J.Y., Y. Zhu, J.Z.); Center for American Indian Health Research, University of Oklahoma Health Sciences Center, Oklahoma City, OK (E.T.L., Y. Zhang); Texas Biomedical Research Institute, San Antonio, TX (S.A.C., K.H.); Missouri Breaks Industries Research Inc, Timber Lake, SD (L.G.B.); The New York Hospital-Cornell Medical Center, New York, NY (R.B.D., M.J.R.); MedStar Health Research Institute, Hyattsville, MD (B.V.H.); and Georgetown and Howard Universities Centers for Translational Sciences, Washington, DC (B.V.H.).

Correspondence to Jinying Zhao, MD, PhD, Department of Epidemiology, School of Public Health and Tropical Medicine, Tulane University, 1440 Canal St, SL18, New Orleans, LA 70112. E-mail jzhao5@tulane.edu

* These authors contributed equally to this work.

Abstract

Background—Atherosclerosis is the underlying cause of cardiovascular disease, the leading cause of morbidity and mortality in all American populations, including American Indians. Genetic factors play an important role in the pathogenesis of atherosclerosis. Although a single-nucleotide polymorphism (SNP) may explain only a small portion of variability in disease, the joint effect of multiple variants in a pathway on disease susceptibility could be large.

Methods and Results—Using a gene-family analysis, we investigated the joint associations of 61 tag SNPs in 7 nicotinic acetylcholine receptor genes with subclinical atherosclerosis, as measured by carotid intima-media thickness and plaque score, in 3665 American Indians from 94 families recruited by the Strong Heart Family Study (SHFS). Although multiple SNPs showed marginal association with intima-media thickness and plaque score individually, only a few survived adjustments for multiple testing. However, simultaneously modeling of the joint effect of all 61 SNPs in 7 nicotinic acetylcholine receptor genes revealed significant association of the nicotinic acetylcholine receptor gene family with both intima-media thickness and plaque score independent of known coronary risk factors.

Conclusions—Genetic variants in the nicotinic acetylcholine receptor gene family jointly contribute to subclinical atherosclerosis in American Indians who participated in the SHFS. These variants may influence the susceptibility of atherosclerosis through pathways other than cigarette smoking per se.

SOURCE:

Circulation: Cardiovascular Genetics.2013; 6: 89-96

Published online before print December 22, 2012,

doi: 10.1161/ CIRCGENETICS.112.963967

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Triggering of Plaque Disruption and Arterial Thrombosis

Curator and Reporter: Larry H Bernstein, MD, FCAP

 

This discussion is a very interesting experimental model for the elucidation of plaque rupture in acute coronary syndromes.  The knowledge exists that there is a series of steps in develoiping atheromatous plaque.  We also know that platelets and endothelium are the location of this pathological development.  We don’t know exactly the role or mechanism of the contribution of hyperlipidemia, and what triggers plaque rupture.  This work reported is an experimental rabbit model that sheds light on the triggering of plaque rupture.

Triggering of Plaque Disruption and Arterial Thrombosis in an Atherosclerotic Rabbit Model

George S. Abela, MD, MSc; Paulo D. Picon, MD, MSc; Stephan E. Friedl, MEE; Otavio C. Gebara, MD; Akira Miyamoto, MD; Micheline Federman, PhD; Geoffrey H. Tofler, MB; James E. Muller, MD
From the Institute for Prevention of Cardiovascular Disease, Cardiovascular Division (G.S.A., S.E.F., G.H.T., J.E.M.), and the Department of Pathology (C.S.A., M.F.), Deaconess Hospital, Harvard Medical School, Boston, Mass; the Department of Pharmacology, Federal University and University of Passo Fundo (P.D.P.), Rio Grande de Sul, Brazil; the Heart Institute, University of São Paulo (O.C.G.), São Paulo, Brazil; and the First Department of Internal Medicine, National Defense Medical College (A.K.), Saitama, Japan.

Abstract

Background

It is now recognized that plaque disruption and thrombosis, a process often triggered by activities of the patient, is generally the cause of the onset of acute coronary syndromes. Understanding of disease onset could be greatly enhanced by the availability of a suitable animal model of plaque disruption and thrombosis. The aim of this study was to replicate and further characterize an atherosclerotic rabbit model of triggering of arterial thrombosis that was introduced by Constantinides and Chakravarti more than 30 years ago but not subsequently used.

  • Aortic plaques were induced by a high-cholesterol diet, by mechanical balloon injury of the artery, or by a combination of the two.
  • Triggering was attempted by injection of Russell’s viper venom (RVV), which is a proteolytic procoagulant, and histamine.

 Methods and Results

A total of 53 New Zealand White rabbits were exposed to one of four preparatory regimens:

  1. rabbits in group I (n=9) were fed a regular diet for 8 months;
  2. rabbits in group II (n=13) were fed a diet of 1% cholesterol for 2 months alternated with 2 months of a regular diet for a total of 8 months;
  3. rabbits in group III (n=5) underwent balloon-induced arterial wall injury, then were given a regular diet for 8 months; and
  4. rabbits in group IV (n=14) underwent balloon-induced arterial wall injury, then were given a diet of 1% cholesterol for 2 months followed by a regular diet for 2 months for a total of 4 months. After completion of the preparatory regimen, triggering of plaque disruption and thrombosis was attempted by injection of RVV (0.15 mg/kg IP) and histamine (0.02 mg/kg IV).
  • In group I, normal control rabbits without atherosclerosis, only one small thrombus was noted in 1 of 9 rabbits.
  • In group II, cholesterol-fed rabbits, thrombosis occurred in 3 of 13 rabbits.
  • Thrombus occurred in all rabbits in group III (5 of 5) and in 10 of 14 rabbits in group IV.
Although the frequency of thrombosis was not significantly different between groups I and II, possibly due to a small sample size, it was significantly different among all four groups (P<.001). Also, the frequency and amount of thrombus formation were significantly different among all four groups (P<.001; P<.0001) but not between groups I and II. Rabbits with atherosclerosis (those in groups II and IV) demonstrated plaque disruption and overlying platelet-rich thrombus formation similar to that observed in patients with acute coronary syndromes. The surface area covered by thrombus was
  1. 2 mm^2 in group I, 1
  2. 5.3±19.2 mm^2 in group II,
  3. 223±119 mm^2 in group III, and
  4. 263±222 mm^2 in group IV.
Rabbits in groups III and IV had the greatest amount of thrombus, and this amount was significantly greater than in rabbits in groups I and II (P<.001 and P<.03, respectively).

Conclusions

A suitable animal model is available for the study of plaque disruption and arterial thrombosis.

  • Hypercholesterolemia and mechanical arterial wall injury seemed to produce plaques vulnerable to triggering of disruption and thrombosis, whereas
  • normal arteries were relatively resistant to triggering.
This model provides a method to evaluate agents that might decrease the occurrence of vulnerable plaques or the amount of thrombus formed after triggering. Most important, the model can be used to identify the features of vulnerable plaques and the pharmacological stressors that trigger plaque disruption and thrombus formation.

Key Words: thrombosis, atherosclerosis, balloon, histamine, RVV

Introduction

Plaque disruption and subsequent arterial thrombosis are now recognized as critical to the onset of acute coronary ischemic syndromes. It is hypothesized that occurrence of thrombotic coronary occlusion has three components.
  1. First, a plaque that is vulnerable to disruption must be present.
  2. Second, acute physiological events are required to induce plaque disruption and thrombosis.
  3. Third, a relatively hypercoagulable state and heightened vasomotor tone increase the likelihood that arterial thrombosis will produce complete lumen occlusion.
 Recent epidemiological studies of human patients with myocardial infarction have demonstrated that in many cases a triggering activity, such as physical exertion, precipitates the acute onset of the disorder. Although a better understanding of plaque vulnerability and triggering would be of great value, knowledge of this process is limited because human studies are difficult and a suitable animal model has not been used.
In human patients, the opportunity to study factors responsible for acute onset of myocardial infarction is limited because coronary angiography performed before the event cannot prospectively identify plaques vulnerable to disruption.(9) After the event, angiography cannot distinguish the features of the plaque responsible for the disruption from those resulting from the disruption.(10) Although findings at autopsy provide detailed information about plaque disruption, these observations may be biased toward more advanced disease, since plaque disruptions producing total vascular occlusion and death may be more severe than those occurring in asymptomatic individuals or in patients with unstable angina or nonfatal myocardial infarction.
These difficulties, inherent in the study of plaque disruption and thrombosis in human patients, create a great need for an animal model of the process. More than 30 years ago, Constantinides and Chakravarti(13) developed such a model in atherosclerotic rabbits. Atherosclerotic plaques were produced in New Zealand White rabbits by intermittent cholesterol feeding. Triggering of plaque disruption and thrombosis was then accomplished by intraperitoneal injection of Russell’s viper venom (RVV, a procoagulant and endothelial toxin) followed by the intravenous injection of histamine, a vasopressor in rabbits. The aortas of the rabbits were then found to have disrupted atherosclerotic plaques with overlying platelet-rich thrombi.
Despite the similarity of these lesions to those observed in human patients, the model has received little attention or use during the past 3 decades. A recent review of the animal models of thrombosis currently in use noted that “thus far, it has not been possible to duplicate in a model the most common clinical cause of thrombosis—an ulcerated atherosclerotic plaque.”(14)
The advantage of the Constantinides model over other animal models used to study thrombosis is that it uses a biological intervention to trigger localized atherosclerotic plaque disruption and formation of platelet-rich arterial thrombi. The model facilitates the study of the process because the investigator determines when disruption and thrombosis will occur.
Disadvantages of the Constantinides model are
  • (1) the low yield of triggering (only about one third of the rabbits developed thrombosis) and
  • (2) the long (8-month) preparatory period. In addition, there is a need to replicate the findings of Constantinides and Chakravarti(13) from 30 years ago because of the biological variability of rabbit strains and RVV.
It cannot be assumed that the rabbits and RVV currently available will produce the results obtained in the 1960s.
In this study, we attempted to reproduce the original model of Constantinides.13 In addition, we wanted to determine whether mechanical injury to the aorta early in the preparatory phase could enhance the development of vulnerable plaques, thereby increasing the yield of disrupted plaques and shortening the preparatory period.

Methods

Fifty-three New Zealand White rabbits weighing between 2 and 3 kg were started on the experimental protocol; 41 survived until the time of attempted triggering. In these 41 rabbits, four dietary and interventional regimens were used in preparation for attempted triggering (Fig 1⇓). The control group, group I, consisted of normal rabbits (n=9) that were fed a regular diet for 8 months. Group II rabbits (n=13) were fed a high-cholesterol diet (1% cholesterol, ICN) for 2 months alternated with 2 months of a regular diet for a total of 8 months.15 Rabbits in group III (n=5) underwent balloon-induced arterial injury and were maintained on a regular diet for 8 months. Rabbits in group IV (n=14) underwent balloon-induced arterial injury, were maintained on a 1% cholesterol diet for 2 months, then were given a regular diet for 2 months for a total of 4 months.
Balloon-induced arterial wall injury of the aorta was performed with a 4F Fogarty catheter introduced through a femoral artery cutdown. The catheter was advanced in a retrograde fashion to the aortic valve and then withdrawn 3 cm. The balloon was inflated with 1.5 cm3 of air, and the catheter was retracted down to the iliofemoral artery. This was repeated three times in each rabbit as cm3 described previously.16 Rabbits were anesthetized with ketamine (50 mg/kg IM) and xylazine (20 mg/kg IM).

Of the 12 rabbits that died during the preparatory period, 5 were in group II, 2 in group III, and 5 in group IV. Seven of the 12 rabbits that died prematurely underwent an autopsy, and none had evidence of plaque disruption or arterial thrombosis. The causes of death included respiratory infection and liver failure from lipid infiltration.

The triggering agents RVV (Sigma Chemical Co) and histamine (Eli Lilly) were administered according to the method of Constantinides and Chakravarti.(13) RVV (0.15 mg/kg) was given by intraperitoneal injection 48 and 24 hours before the rabbits were killed. Thirty minutes after each RVV injection, histamine (0.02 mg/kg) was administered intravenously through an ear vein. Rabbits were killed by an overdose of intravenous pentobarbital and potassium chloride. The aorta and iliofemoral arteries were dissected and excised, and the intimal surface was exposed by an anterior longitudinal incision of the vessel.

The total surface area of the aorta, from the aortic arch to the distal common iliac branches, was measured. The surface area covered with atherosclerotic plaque and the surface area covered with antemortem thrombus were then determined. Images of the arterial surface were collected with a color charge-coupled device camera (TM 54, Pulnix) and digitized by an IBM PC/AT computer with a color image processing subsystem. The digitized images were calibrated by use of a graticule, and surface areas were measured by use of a customized quantitative image analysis package.

Tissue samples (1 cm in length) were taken from the thoracic aorta, 3 and 6 cm distal to the aortic valve; from the abdominal aorta, 7 and 4 cm proximal to the iliac bifurcation; and from the iliofemoral arteries. and prepared for and examined by light microscopy and they were examined by quantitative colorimetric assay.  Electron microscopy was also carried out with a Hitachi 600 microscope.

Biochemical analysis was done for tissue cholesterol. Free cholesterol and cholesteryl esters in the aorta were determined by high-performance liquid chromatography (HPLC) on the basis of the method of Kim and Chung. Each sample of aorta was ground to a fine powder with anhydrous sodium sulfate and extracted twice with 5 mL chloroform: methanol (2:1). The extract was dried under nitrogen and redissolved in 5 mL isopropanol.   Serum cholesterol, plasma fibrinogen, and platelet counts were done.

Overall comparison among the four groups was conducted with Fisher’s exact test and the Kruskal-Wallis test for discrete and continuous data, respectively. Comparisons between any two groups of rabbits were made by an exact Wilcoxon midrank test.23 P<.05 was considered statistically significant, and measured data were reported as mean±SD.

Results

Extent of Thrombosis After Triggering

In the 41 rabbits that underwent attempted triggering, the frequency of plaque disruption and focal thrombosis varied markedly depending on the type of preparatory regimen. In group I, only 1 of 9 control rabbits developed a thrombus. This was a small white thrombus with a surface area of 2 mm^2. Three of the 13 rabbits in group II on a 1% cholesterol diet developed white thrombi, all of which were small but were larger than that observed in group I (mean surface area, 15.3±19.2 mm^2). In group III, each of the 5 rabbits that had balloon-induced arterial wall injury developed large white thrombi (mean surface area, 223.0±119 mm^2). Ten of 14 group IV rabbits, with combined arterial wall injury and a high-cholesterol diet, developed white thrombi, all of which were large (mean surface area, 263.0±222 mm^2).

Both the frequency of occurrence and the amount of thrombus formation were significantly different among all four groups (P<.001 and P<.0001, respectively). However, the frequency and the amount of thrombus formation tested individually between groups I and II were not statistically different. The average surface area covered by thrombi in rabbits from groups III and IV was significantly greater than that observed in group II (P=.03 and P=.02) or group I (P=.001 and P=.001) rabbits. The average surface area covered by thrombi did not significantly differ between rabbits in group III versus those in group IV.

No white thrombi were noted in the ascending aorta or the aortic arch. In the non–balloon-treated rabbits in groups I and II, only 1 of 5 thrombi was in the abdominal aorta. In the balloon-injured rabbits in groups III and IV, the thrombi were almost evenly distributed between the thoracic and abdominal aorta (48 versus 66). There were more thrombi in the balloon-injured rabbits than in the non–balloon-injured rabbits (P<.002).

Extent of Plaque Covering the Arterial Surface

 The plaque surface area was significantly different among the four groups (P<.0001). Plaque was present in all the rabbits that were maintained on a high-cholesterol diet or that had balloon-induced arterial injury. The plaque distribution for each group is shown in Fig 4⇓. (not shown) Individual comparisons showed a larger amount of plaque in rabbits from groups III and IV than in those from group II (P=.04 and P=.001, respectively). There was no significant difference in the amount of the plaque in group III versus group IV rabbits. The Table demonstrates the relations of the various groups regarding frequency of disruption with the amount of thrombus formation and plaque surface area.
 The intima in group I rabbits appeared normal by gross inspection. In group II rabbits, white-yellow plaque was widely distributed over the arterial surface, with focal punctate ulceration occasionally noted under a dissecting microscope. In group III rabbits, the intima was smooth and widely covered with white plaque. Group IV rabbits had extensive sheets of elevated white-yellow plaque. By gross visualization, ulceration of the surface was present without superimposed thrombus in two rabbits in group IV.

Histological Features of Plaque Disruption and Thrombosis

 Over 4500 tissue sections were prepared and evaluated. Light microscopy of arterial samples from group I showed normal vascular histology. Group II samples had a predominance of foam cell infiltration of the intima surrounded with connective tissue. Group III samples had fibromuscular plaque composed mostly of muscular cell elements and minimal fibroconnective tissue. This was confirmed by Masson’s trichrome stains showing mostly red muscle cells and minimal blue fibrous tissue. Group IV samples had extensive plaque with an infiltration predominantly composed of foam cells.

Light microscopic examination of adjacent serial sections from thrombosis sites revealed platelet-rich thrombi with interrupted but long adhesion sites to the arterial wall over most of their length. Early organization and inflammatory cell infiltration were present within the thrombi. In sections from groups II and IV, some areas of plaque directly adjacent to the thrombi had marked thinning of the connective tissue cap and areas of dehiscent foam cells,. These observations were rare and were noted in <0.5% of the examined lesions. In most cases, the arterial thrombus was not located at a site of obvious plaque rupture. Foam cell infiltration was also noted adjacent to sites of thrombosis.

Figure 6.

A, Light micrograph shows that degenerated foam cells are present in a large cavity below a cap separating the cavity from the intimal surface of thoracic aorta from a rabbit in group IV (Movat’s pentachrome, magnification ×40). B, Light micrograph of large thrombus attached to the luminal surface of the thoracic aorta in the same rabbit shown in A. The cavitation is seen below the thrombus, and the intimal surface is markedly thinned (Masson’s trichrome, magnification ×16). C, Light micrograph of thrombus overlying a region of plaque with a large accumulation of foam cells from a rabbit in group IV. The free edges of the thrombus correspond to the underlying contour of the plaque, which suggests that the thrombus became detached during fixation (Masson’s trichrome, magnification ×25). D, Light micrograph of thrombus from the abdominal aorta in a rabbit from group IV, 48 hours after triggering. The thrombus is firmly attached and becoming organized. The yellow stain represents red blood cells, and the fibrin and platelets appear pink (Carstair’s stain, magnification ×25).
The degree of blue staining indicative of fibrous tissue in Masson’s trichrome–prepared slides was greatest in group II samples, as represented by values closer to the pure blue region (0.0) on CIE coordinates. Group II samples (0.33±0.046, mean±SD) were more blue than group III (0.43±0.06, P<.001) or group IV samples (0.38±0.05, P<.001). The degree of blue staining was not statistically different between samples from groups III and IV.
Scanning electron microscopy demonstrated fissures of various lengths below areas from which overlying thrombi were removed. Endothelial cells could be seen lining the intimal surface of the aorta in the rabbits that had undergone balloon-induced arterial wall injury 8 months earlier. Surface blebs and focal endothelial breakdown with ulcer formation, without grossly visible thrombosis, were occasionally seen in samples from groups II and IV. The base of these ulcers was layered with platelets, fibrin, and red blood cells. Transmission electron microscopy of areas with thrombosis confirms that the thrombi were platelet rich.

Biochemical Findings

 Baseline serum cholesterol for all rabbits was 50±25 mg/dL and did not differ among the four groups. In rabbits in groups II and IV, which received cholesterol feeding, serum cholesterol rose to an average peak level of 2500± 1200 mg/dL.
In the two groups that received cholesterol feeding, the total cholesterol content in tissue samples pooled from the thoracic and abdominal aorta was significantly higher in group IV (16±7.2 mg/g) than in group II (2.8±1.6 mg/g) (P<.0001). Rabbits that were maintained on a regular diet (groups I and III) had equally low levels of tissue cholesterol (0.05±0.04 versus 0.06±0.02 mg/g, P=NS).

Hematological Changes Accompanying Triggering

The average fibrinogen level before triggering in the 27 rabbits in which fibrinogen was measured was 210±119 mg/dL; it rose to 403±168 mg/dL 48 hours after triggering (P<.001). Plasma fibrinolytic activity did not change after triggering (85.5±37.8 versus 94.8±33.5 arbitrary units). Platelet counts (measured in only 19 rabbits in groups II and IV) decreased from 350±84×103 to 215±116×103 per cubic millimeter after triggering (P<.001). White blood cell count did not decrease after triggering (12.8±13.0 versus 12.8±7.1×103 cells per cubic millimeter). However, the hematocrit dropped from 35.7±3.8% to 32.0±5.8% (P<.0002).

Discussion

The results demonstrate that vulnerable plaques can be produced and that plaque disruption and platelet-rich arterial thrombus formation may be triggered pharmacologically in an animal model of arterial plaque. This finding documents that the New Zealand White rabbit strains and the RVV currently available can be used to obtain the same results observed by Constantinides and Chakravarti(13) more than 30 years ago.
The frequency of successful triggering was dependent on the type of preparatory regimen used. In control rabbits maintained on a regular diet, only 1 of 9 developed a small thrombus after injection of the triggering agents. Although rabbits fed a high-cholesterol diet had more thrombosis after triggering, the values were not statistically different between rabbits in groups I and II. In other studies of triggering of cholesterol-fed rabbits, a total of 7 of 30 rabbits have developed thrombi, but this also does not achieve statistical significance (unpublished data, 1994). The number of rabbits studied may have been too low to demonstrate a moderate difference of thrombus occurrence. However, earlier work by Constantinides and Chakravarti(13 24) demonstrated a frequency of thrombi in 1 of 22 rabbits not fed cholesterol versus 22 of 77 rabbits fed cholesterol, which does achieve statistical significance (P<.02). This indicates that a larger sample may demonstrate a difference between groups I and II and that cholesterol feeding increases the likelihood of the disruption and thrombosis process in the rabbit model. Thus, our results in conjunction with those of Constantinides and Chakravarti suggest that thrombosis triggered by RVV and histamine may be facilitated in the presence of atherosclerosis. However, these observations do not preclude the possibility of thrombosis in a normal artery, which can be induced by injury from various triggers.
Rabbits subjected to arterial balloon injury developed extensive thrombosis only after triggering, as did rabbits subjected to both arterial injury and a high-cholesterol diet. Thus, a high-cholesterol diet especially in the presence of mechanical injury is capable of producing a plaque vulnerable to disruption and thrombosis by triggering with RVV and histamine.

Production of Vulnerable Plaque by Cholesterol Feeding

The technique of pulsed cholesterol feeding used in this study has been shown to be an effective method of producing experimental atherosclerosis, as have continuous cholesterol feeding regimens. Recently, it has been demonstrated that cholesterol feeding induces an upregulation of vascular cell adhesion molecule-1 in rabbit endothelium. This may predispose a site to monocyte adhesion and migration into the subendothelial space. Continued macrophage accumulation may make the site particularly vulnerable to disruption and thrombosis.
Autopsy studies in humans have led to the hypothesis that a lesion with a lipid pool beneath a thin cap is particularly vulnerable to disruption and thrombosis.4 5 This morphology has been shown to generate stress concentrations that would predispose a plaque to disrupt.  Although sites with lipid pools and thin caps were noted in the present study, their occurrence was too limited to permit studies to determine whether these were sites particularly prone to thrombosis. Cholesterol feeding for 2 years may be required to produce a sufficient number of such lesions to determine their vulnerability to disruption.

Production of Vulnerable Plaque by Balloon-Induced Injury

An important finding of this study is that vulnerability to disruption and thrombosis was present 8 months after deendothelialization with balloon-induced arterial wall injury in rabbits on a regular diet (group III). This occurred in the presence of a regenerated endothelium overlying a diffuse fibromuscular plaque. Previous reports have demonstrated that endothelium that regenerates after balloon deendothelialization is physiologically dysfunctional for a prolonged period. From our study, it appears that endothelial function is compromised in its role as a thrombosis-resistant surface over a long period as well. An important factor that may contribute to the altered function is the presence of underlying plaque.

Triggering Agents RVV and Histamine

Among its numerous constituents, RVV contains proteases that activate factors V and X. Such activation leads to thrombosis, which is most likely to occur at sites of cell injury. In addition to this procoagulant effect, RVV is a direct endothelial toxin.31 However, in the absence of arterial abnormalities produced by cholesterol feeding or other means, RVV alone or in combination with a vasoconstrictor agent rarely produces thrombosis.4 The increase in fibrinogen levels and the stability of hematological factors during triggering indicate that RVV does not act by producing a disseminated coagulopathy. The localization of thrombus at focal arterial sites is further evidence that this model does not merely produce a nonspecific thrombotic effect.
Histamine is an arterial vasoconstrictor in rabbits. This effect is mediated by an H1 receptor that regulates release of norepinephrine at the presynaptic norepinephrine sites. Histamine may contribute to plaque disruption by raising the arterial pressure and stress on the plaque and/or by the development of vasospasm. Other, similar agents, thromboxane A2 and serotonin, also have been shown to result in severe vasoconstriction of epicardial coronary arteries that is mediated by platelet deposition at stenosed sites.

Comparison With Other Models

This is a unique model that combines features of several other animal models that have been used to study atherosclerosis and thrombosis. With regard to thrombosis, the model provides the opportunity to extend observations previously made in other animal models of thrombosis to the special conditions surrounding triggering of acute cardiovascular syndromes. While the model of Folts et al has been invaluable in assessing enhanced platelet deposition in dog and pig coronary arteries, it requires both endothelial injury and the production of a 60% to 70% lumen stenosis. Moreover, it does not use an atherosclerotic artery with a vulnerable plaque.
Badimon et al used a flow chamber to evaluate platelet deposition on activated arterial surfaces. They demonstrated that deep arterial injury results in more thrombus formation than superficial injury. However, their model does not recreate the in vivo environment or provide an opportunity for evaluation of various thrombogenic sites, as does the model presented in this study.

Relation of the Model to Human Coronary Thrombosis

Certain features of the lesions seen in this model are similar to those of human lesions seen at autopsy of patients with fatal myocardial infarction, ie, a lesion with a fissured collagen cap overlying a lipid mass of amorphous and crystalline lipid. However, most of the lesions in the model did not have these features and were more consistent with a recent pathological study of fatal coronary thrombosis, which revealed that in approximately half the cases, the plaque was relatively intact but an inflammatory infiltrate was present.36 Perhaps the incidence of plaque rupture causing thrombus may be even lower in patients with nonfatal coronary thrombosis, as suggested from angioscopic studies of coronary arteries that have shown plaque ulceration of various severities.
Although the model we used produced lesions with many similarities to the nonruptured lesions described in patients, extension of this preparation for a 2-year period has been documented to produce lesions with deep fissures similar to those observed in many patients with fatal coronary thrombosis. Also, use of balloon injury in this model to enhance plaque development resulted in plaques that were morphologically similar to advanced plaques induced by the alternating high-cholesterol diet.
Analyses of human plaques have demonstrated that disrupted plaques have significantly less collagen, glycosaminoglycans, and smooth muscle cells and more extracellular lipid and macrophages than do nondisrupted plaques. This is consistent with findings in our study that rabbits in group II had more connective tissue and a lower rate of disruption and thrombosis than those in groups III and IV.
Perhaps the major limitation of this study is that it used a complex pharmacological mixture as the trigger, which makes speculation on the mechanism of action difficult. Further studies will be necessary to determine which components of RVV and histamine are responsible for the focal thrombosis.

Potential Utility of the Model to Study Plaque Disruption and Thrombosis

The observation that large, platelet-rich thrombi can be obtained by triggering in animals with underlying plaques produced by cholesterol feeding or by balloon injury broadens the types of plaque that can be studied for vulnerability. Various types of preparatory regimens could be studied for their ability to promote or retard the development of vulnerable plaque.
The model also can be used to test pharmacological agents that may reduce the development of vulnerable atherosclerotic plaques, such as lipid-lowering agents, antioxidants, calcium channel blocking agents, and angiotensin-converting enzyme inhibitors. Antiplatelet and other antithrombotic drug therapies can be tested for the ability to reduce the amount of thrombus complicating plaque disruption. Finally, the model can be used to characterize the biochemical and cellular bases for plaque vulnerability by comparing the features of sites that do and do not develop thrombi soon after triggering.

 References

3 Friedman M, van den Bovenkamp GJ. The pathogenesis of a coronary thrombus. Am J Pathol. 1966;80:19-44.
4 Constantinides P. Plaque fissures in human coronary thrombosis. J Atheroscler Res. 1966;6:1-17.
5 Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction causing sudden ischaemic death, and crescendo angina. Br Heart J. 1985;53:363-373. FREE Full Text
8 Tofler GH, Stone PH, Maclure M, Edelman E, Davis VG, Robertson T, Antman EM, Muller JE, and the MILIS Study Group. Analysis of possible triggers of acute myocardial infarction (the MILIS Study). Am J Cardiol. 1990;66:22-27. CrossRefMedline
9  Little WC, Constantinescu M, Applegate RJ, Kutcher MA, Burrows MT, Kahl FR, Santamore WP. Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery disease? Circulation. 1988;78:1157-1166. Abstract/FREE Full Text
10 Ambrose JA, Winters SL, Arora RR, Eng A, Riccio A, Gorlin R, Fuster V. Angiographic evolution of coronary artery morphology in unstable angina. J Am Coll Cardiol. 1986;7:472-478. Abstract
11 Davies MJ, Bland MJ, Hartgartner WR, Angelini A, Thomas AC. Factors influencing the presence or absence of acute coronary thrombi in sudden ischemic death. Eur Heart J. 1989;10:203-208. Abstract/FREE Full Text
12  JH, Fuster V, Badimon L, Taubman M, Badimon J, Cheseboro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990;15:1667-1687. Abstract
13 Constantinides P, Chakravarti RN. Rabbit arterial thrombosis production by systemic procedures. Arch Pathol. 1961;72:197-208. Medline
14  Runge RS, Haber E. Animal models for the study of thrombolysis in vivo. Circulation. 1991;83(suppl IV): IV-1-IV-2. Abstract.
15 Constantinides P, Booth J, Carlson G. Production of advanced cholesterol atherosclerosis in the rabbit. Arch Pathol. 1960;70:80-92.

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Carotid Ultrasound more sensitive for Detecting Subclinical Atherosclerosis in patients with rheumatoid arthritis (RA) than CT with calculation of Coronary Artery Calcification Scores

Reporter: Aviva Lev-Ari, PhD, RN

Ultrasound Predicts CVD Risk in Arthritis

Published: Oct 8, 2013 | Updated: Oct 8, 2013

By Nancy Walsh, Staff Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner

 

Carotid ultrasound was more sensitive for detecting subclinical atherosclerosis in patients with rheumatoid arthritis (RA) than CT with calculation of coronary artery calcification scores, Spanish researchers found.

Among a group of 60 patients classified as being at moderate cardiovascular risk on a conventional scoring system, the presence of severe abnormalities on ultrasound reclassified 51 as being at high or very high risk, according to Miguel A. Gonzalez-Gay, MD, of Universitario Marques de Valdecilla in Santander, and colleagues.

And of those 51 reclassified patients, only 12 would have been reclassified as being at high or very high cardiovascular risk using a coronary artery calcification score,the researchers reported in the NovemberAnnals of the Rheumatic Diseases.

Patients with RA are at markedly increased risk for cardiovascular disease (CVD), both from conventional risk factors and the ongoing systemic inflammation associated with RA.

Comprehensive management of these patients therefore should include risk assessment and appropriate interventions, but “adequate stratification of the CV risk in patients with RA is still far from being completely established,” Gonzalez-Gay and colleagues noted.

The insensitivity of conventional risk assessments such as the Systematic Coronary Risk Evaluation (SCORE), even when modified by the European League Against Rheumatism(mSCORE) to account for the increased background risk in RA, has been confirmed byreports of ischemic heart disease among patients not considered to be at elevated risk on these measures.

These researchers previously suggested that carotid ultrasonography be added to the overall risk assessment of RA patients, particularly those with moderate SCORE risk, but whether other noninvasive approaches such as coronary artery calcification also could be useful has been uncertain.

Therefore, they enrolled 95 rheumatoid arthritis patients with no history of cardiovascular events and no diabetes or chronic renal disease.

Most were women, mean age was 59, and mean disease duration was 11 years.

Rheumatoid factor and/or anticyclic citrullinated peptide was present in 72%, and extra-articular manifestations in 16%.

All patients had carotid ultrasonography to assess for plaque and multi-detector CT scanning to detect coronary artery calcification.

Carotid intima-media thickness of 0.90 or the presence of plaque was considered predictive of CVD on ultrasound.

A coronary artery calcification score of zero was considered normal, and a score over 100 indicated a high likelihood of coronary artery disease.

Patients also were given conventional SCORE ratings, based on factors such as age, sex, smoking, blood pressure, and atherogenic index, as well as mSCORE ratings, to estimate the 10-year risk for a fatal cardiovascular event.

The mean SCORE was 2.30, and the mean mSCORE was 2.78.

Cardiovascular risk according to mSCORE was low in 21, moderate in 60, and high or very high in 14.

Most patients with low mSCOREs also had scores of zero for coronary artery calcification, and none of the low mSCORE patients had calcification scores above 100.

But 57% of patients with calcification scores of zero had carotid plaques identified on ultrasound, as did 76.3% of patients with calcification scores between 1 and 100.

While calcification scores above 100 weren’t much more sensitive than mSCOREs for detection of high risk (23.6% versus 19.4%), almost all (70 of 72) patients with high or very high risk were identified with carotid ultrasound, for a sensitivity of 97.2% (95% CI 90.3-99.7).

And when the ultrasound model of intima-media thickness above 0.9 mm and/or carotid plaque also included mSCOREs above 5%, all 72 were correctly identified, for a sensitivity of 100% (95% CI 95-100).

This lack of sensitivity for calcification scores likely reflects the finding that arterial calcification is a later vascular development, and its absence doesn’t rule out the presence of the more vulnerable noncalcified plaques, the researchers explained.

“These results support the use of carotid ultrasonography as the imaging technique of choice for detection of high/very high CV risk in RA patients with moderate mSCORE,” they said.

In an editorial accompanying the study, Patrick H. Dessein, MD, of the University of Witwatersrand in Johannesburg, South Africa, and Anne G. Semb, MD, of Diakonhjemmet Hospital in Oslo, Norway, noted that the use of ultrasound more than tripled the number of patients considered to be at high risk.

If only mSCORE was used for risk stratification, they pointed out, many patients “in routine clinical settings” would be unlikely to receive preventive treatments, “with the serious consequences this has.”

Dessein and Semb also noted that there were certain limitations to this study, including its cross-sectional design and inclusion of patients with long disease duration.

“It remains to be clarified whether carotid ultrasound is as helpful among patients with early disease versus those with longstanding disease in enhancing CVD risk stratification,” the editorialists wrote.

The authors reported no conflicting interests.

From the American Heart Association:

http://www.medpagetoday.com/Rheumatology/Arthritis/42138?xid=nl_mpt_DHE_2013-10-09&utm_content=&utm_medium=email&utm_campaign=DailyHeadlines&utm_source=WC&eun=g99985d0r&userid=99985&email=avivalev-ari@alum.berkeley.edu&mu_id=5099207

 

 

 

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