Comments Off on Vegetarians and LDL levels

By admin, April 22, 2015 11:48 am

In epidemiologic studies, a low serum LDL-C of less than 75 mg/dL (≈ 2 mmol/L), typically found in vegetarians and those on non-Western style diets, protects against atherosclerotic cardiovascular disease. Dietary saturated fat and cholesterol generally raise the level of LDL-C in the blood and proportionally raise the coronary risk. The related factors of obesity, metabolic syndrome, and type 2 diabetes may have only modest effects on LDL-C levels, but they increase coronary risk by increasing triglycerides and LDL particle concentration, while lowering HDL-C and causing the production of small, dense LDL particles.
• An HDL level of 50 mg/dL (≈ 1.3 mmol/L) or greater in a man or 60 mg/dL (≈ 1.6 mmol/L) in a woman is associated with a reduced risk of atherosclerosis.
• An HDL level of over 75 mg/dL (≈ 2 mmol/L) or greater in a man or woman is associated with a very low risk of atherosclerosis.
• An HDL of less than 40 mg/dL (≈ 0.8 mmol/L) in a man and less than 50 mg/dL (≈ 1 mmol/L) in a woman increases the risk of atherosclerosis.
• Low HDL levels appear as an independent risk factor for CV events when combined with high LDL levels.

• LDL Cholesterol – Primary Target of Therapy
  

  <100	Optimal
  100-129	Near Optimal/Above Optimal
  130-159	Borderline High
  160-189	High
  190	Very high

• Total Cholesterol
  

  <200	Desirable
  200-239	Borderline High
  240	High

• HDL Cholesterol
  

  <40	Low
  60	High

LDL

The evidence that high LDL concentrations play a causal role in coronary artery disease (CAD)is quite compelling.

• Many investigators have convincing demonstrated a progressive increase in risk that correlates directly with LDL concentrations.
• Treatments that lower LDL cholesterol reduce the risk of CAD.
• Genetic studies analyzing SNPs show that SNPs affecting LDL cholesterol are consistently related to risk of myocardial infarction. In other words, genetic variants associated with elevated LDL levels are consistently associated with increased risk.
• The presence of LDL in atherosclerotic plaques provides additional evidence of its direct involvement.

Statin Drugs

Statins are currently the most powerful cholesterol-lowering drugs available. They act by inhibiting the action of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, which is the rate-limiting enzyme in the sequence of steps by which cholesterol is synthesized in the liver. The liver has two sources of cholesterol: it can take up LDL particles from the blood, or it can synthesis cholesterol using HMG-CoA reductase. The diagram to the right illustrates cholesterol homeostasis in a liver cell. LDL (shown in red) can bind to LDL receptors on the surface of the liver. Binding causes the LDL to be taken up by the liver cell and digested in a lysosome. Fatty acids and amino acids in the LDL are recycled, and the cholesterol enters the cholesterol pool in the liver cell. Note that if the liver cell synthesizes cholesterol, this too will be added to the cholesterol pool. If the concentration of cholesterol in the liver cells exceeds a certain level, HMG CoA reductase is inhibited, and the synthesis of LDL receptors is also inhibited.

Statins act by inhibiting HMG-CoA reductase and shutting off internal cholesterol production. In this way, statins reduce cholesterol concentrations in liver cells, which causes increased production of LDL receptors and increased uptake of LDL by the liver. This ultimately results in a lowering of blood concentrations of LDL cholesterol, and this generally results in a slower progression of atherosclerotic plaques and a reduced risk of plaque rupture. However, observers occasionally report regression of atherosclerotic plaques in patient on statins.

While statins are effective in reducing LDL cholesterol levels, they can be associated with side effects.

• Muscle damage manifest as pain, tiredness, or weakness is the most common side effect and ranges from mild discomfort to difficulty in climbing stairs or walking. Statins very rarely cause severe, life-threatening rhabdomyolysis which can result in severe muscle pain, liver damage, kidney failure, and death.
• Liver damage: Statins can also damage liver cells, resulting in elevated levels of liver enzymes in blood. Liver enzyme levels should be monitored when statins are first started.
• Digestive problems: Some people experience nausea, gas, diarrhea or constipation.
• Rash or flushing
• Increased blood sugar or type 2 diabetes is occasionally seen in patients on statins.

Because of their effectiveness in reducing CAD in patients at high risk, some advocated expanding the use of statins to people who did not necessarily have elevated cholesterol levels. A number of large, well-done clinical trials have demonstrated that statins can reduce the risk of CAD in low-risk individuals, but most experts caution against this because of the cost and the risk of side effects.

Comments Off on ACE inhibitors decrease AMI risk and are antithrombogenic

By admin, April 9, 2015 7:16 pm

As previously described, ACE inhibition may modify not only atherogenesis and plaque vulnerability but also triggering mechanisms responsible for disease onset.²²⁹ For example, the renin-angiotensin system may interact with fibrinolytic function,²⁵⁴ and ACE inhibition may influence endogenous fibrinolysis, resulting in a reduced thrombotic response to plaque disruption.²⁵⁵ Importantly, ACE inhibition also seems to reduce mortality and reinfarction in the presence of β-blocker therapy, suggesting an independent therapeutic effect.²²⁸

ACE activity may contribute to the development of coronary artery disease and myocardial infarction,²²⁵ and ACE inhibition seems to reduce the risk of major ischemic events (reinfarction, cardiac death, and possibly unstable angina) by about 22% in patients with low ejection fractions,^{226 227 228} probably via multiple beneficial mechanisms.²²⁹ ACE inhibitors may influence both atherogenesis (plaque vulnerability) and triggering mechanisms responsible for disease onset (plaque disruption, thrombosis, and/or vasospasm). The latter are discussed below in the section on trigger reduction. The hypothesis that these drugs are antiatherogenic and prevent or slow progression of coronary artery disease is now being tested in clinical trials.

Comments Off on Track Your Plaque

By admin, April 9, 2015 7:03 pm

How to Reverse Heart Disease with the Coronary Calcium Score

by Jeffrey Dach MD

Finally Accepted by the AHA

The AHA (American Heart Association) has steadfastly denied for many years that Coronary Calcium Scoring was a valid marker of heart disease.  Well guess what? They have recanted, and admitted that the amount of calcium in the coronary arteries reliably predicts heart attack risk. This is called the calcium score.(1)

Dr Matt Budoff

UCLA cardiologist, Dr. Matt Budoff, a long-time champion of the Coronary Calcium Scan, and author of the AHA paper says, “The total amount of coronary calcium (Agatston score) predicts coronary disease events beyond standard risk factors.”(1)

Image upper left, courtesy of Wikipedia Rembrandt, The Anatomy Lesson.

Dr. Detrano

Dr. Detrano, in a recent article in NEJM (New England of Medicine), confirms that “The coronary calcium score is a strong predictor of incident coronary heart disease and provides predictive information beyond that provided by standard risk factors”. (31)  The Coronary Calcium Score is a precise quantitative tool for measuring and tracking heart disease risk, and is more valuable and accurate than other traditional markers (such as total cholesterol which is practically worthless as a heart disease risk marker).

What is Coronary Artery Disease?  It’s Plaque Formation.

Coronary Arteries in Cross Section

Age 20-30 years                          Age 50-70 years

Note that even though the right vessel has a larger plaque, the lumen has remodeled so that the inner diameter remains freely open.  Eventually, as we age, the enlarging plaque eventually obstructs blood flow causing a heart attack.  Another common scenario is plaque rupture which exposes the inflammatory debris of the plaque to the circulating blood.  This quickly results in clot formation (thrombosis) resulting in a heart attack and possibly sudden death.

Repeat: The calcified portion of the plaque is consistently 20% of the total plaque volume, allowing use of the calcium score as a marker for total plaque volume.

Heart Attack Rate Associated with Calcium Score

Chart below shows increasing heart attack rate as coronary calcium goes up

Chart Above shows that Calcium Score is Highly Predictive of Heart Attack Risk

What is a Heart Attack?

A heart attack is cell death of heart muscle caused by lack of blood flow with  oxygen deprivation.  AS previously mentioned, this is caused by a arterial blockage by enlarging plaque formation which occludes the lumen, or plaque rupture which causes clot formation which occludes blood flow.  If a small area of heart muscle is involved, the heart attack may be silent with no symptoms.  If a large area is involved, there may be severe chest pain radiating to the left arm or jaw, or other symptoms such as shortness of breath.  If the conduction system is involved, there may be irregular heart rhythm called ventricular tachycardia which can cause sudden death.  Some people have chronic chest pain from diseased arteries and this is called angina pectoris, treated with medicines to dilate the arteries such as nitroglycerine.

Common Sites of Plaque Formation – Bifurcations and Mechanical Stress

(image at right courtesy of Wikipedia)

Branching Vessels – Increased Turbulence

Ask any interventional radiologist or invasive cardiologist where they find the plaque formation and obstructions in the arterial tree, and they will say its the same few places over and over again.  These places are the carotid bifurcation, the distal aorta at the bifurcation, the femoral bifurcation, the exit from the adductor canal. And of course, the proximal coronary arteries, and bifurcations of the coronary arteries. A birfurcation is where the vessel branches into two vessels, making a Y pattern.

The bifurcations have maximal turbulance and mechanical stress on the vessel wall.  Remember the blood is flowing under pulsatile pressure, and this mechanical pressure and turbulence, over time, causes little stress cracks in the vessel. The cracks appear at sites of maximal stress.  The coronary arteries are a special case because of the extra motion of the cardiac muscle which moves and stretches the coronary arteries every heart beat, especially as the arteries branch off from the aorta which is relatively stationary, while lower down over the surface of the heart, the vessels move vigorously with each heart beat.  Atherosclerosis is essentially the net result of the healing process for these little cracks in the arterial wall resulting from mechanical stress.

William Davis MD

Advocate of the Coronary Calcium Score

Credit and Thanks is given to William Davis MD at the Track Your Plaque Web site for much of the information in this article.  I have added and embellished some of the information.

Calcium looks White on the CAT scan (red arrows at right).
(Above) CAT scans of the Coronary Arteries.

The left image shows a normal coronary artery (red arrows), while the right image shows a heavily calcified coronary artery (white line outlined by red arrows) indicating high risk for coronary artery disease and heart attack.

All About Coronary Calcium Scoring

 1) Calcium scoring may be superior to angiography as a means to track plaque. That’s because the vast majority of heart attacks are due to plaque rupture and thrombosis at areas of thickened plaque with minimal lumen narrowing.  Over time, the body’s healing process automatically remodels the areas of thickened plaque, and increases lumen size to compensate for the reduced blood flow.

2) Calcium scoring gives a precise number which correlates with the amount of plaque volume. Although only the hard plaque, or calcium in the artery is actually measured, this is useful because it consistently occupies 20% of plaque volume (Total hard and soft plaque).

3) The new 64-slice CAT scanners provide reliable calcium scoring just like any other scanner, both multi-slice and EBT(Electron Beam Cat).

The Track Your Plaque Program, by William Davis MD(2)(3)

1) Quantify plaque with Coronary Calcium Score with CAT scan (or with Electron Beam CT). Obtain your CAT Scan serially, every 12 months to assess response to treatment and lifestyle modification (track your plaque).

2) Use Sophisticated Lipoprotein Panel (Quest-Cario-IQ , LabCorp-VAP)(7-8)) to uncover hidden causes of plaque progression. LDL particle size and number, Lipoprotein (a). Repeat every 6 months.

3) The Main Treatment Goal is the reduction in Coronary Artery Calcium Score, and by inference, reduction in plaque volume and reduction in cardiovascular mortality. The cardiology community still awaits the hard data on these results (CHD mortality and CHD events, treatment arm vs no treatment arm).  These numbers have not been published as far as I know.

How to Measure Success in Halting or Reversing Heart Disease Plaque

According to Dr. Davis, calcium score typically increases at an astonishing rate of 30-35% per year without treatment. Therefore, Dr. Davis considers treatment success to be reduction in this rate from 30 to perhaps only a 5-10 per cent increase in calcium score per year.  An absolute reduction in calcium score on follow up scanning is the optimal outcome, which is difficult to achieve even with strict adherance to the Track Your Plaque program, in Dr Davis’s experience.

Track Your Plaque Program Details – Attain the Following Targets:

a) Reduction of LDL to 60 mg/dl (LDL should be measured directly, not calculated)

b) Reduction of triglycerides to 60 mg/dl.

c) Raising HDL to 60 mg/dl.

d) Correction of hidden causes of plaque on Lipoprotein profile such as total number of small LDL particles, IDL, and Lp(a).

e) Achieving normal blood pressure (<130/80)  Even a small elevation of blood pressure in diseased arteries can cause increased mortality.  Diseased arteries are fragile and plaque rupture can occur easily.

f) Achieving normal blood sugar (=100 mg/dl). Diabetes is a high risk factor for heart disease.

g) Reduction of C-reactive protein to <1 mg/l

Dietary Modification and Supplements to Attain Above Targets:

Niacin

a) Niacin vitamin B3 (Slo-Niacin Upsher-Smith (44) or Niaspan Kos Pharmaceuticals preferred) 500-1500mg. per day (avoid the no-flush niacin which contains inositol).(6)(44)

Buy Niacin  

Slo-Niacin Controlled Release Niacin 500 mg – 175 Tablets

on Amazon.

Omega 3 Fish Oil

b) Fish oil (Omega 3 oils) 4000 mg per day (providing 1200 mg omega-3 fatty acids). (molecular distilled pharmaceutical grade).(36)

Buy Purecaps Super-Critical Co2 Extract Omega 3 Fish Oil

Vitamin D

Vitamin D level restored to above 50 ng/ml (Vitamin D3 2000-5,000 u/day), Vitamin K2 also used.  Low vitamin D is associated with increasing arterial calcification and increased heart disease risk. (26)Consumption of calcium tablets by women increases arterial calcification and heart attack risk.(5)

Buy Vitamin D

Read my article on vitamin D which can be found here.(60)

d) Low Glycemic Diet (avoid Fructose Corn Syrup, avoid wheat products), and eliminate wheat products like Shredded Wheat cereal, Raisin Bran, and whole wheat bagels.

e) Consume foods such as raw almonds, walnuts, pecans; olive oil and canola oil. Beneficial for lipoprotein profile.

f) Increasing protein intake, our major building block for body tissues.  Added benefit of protein intake is that it doesn’t increase blood sugar.  This is low glycemic nutrition.

g) Wine—Red wines contain resveratrol, (don’t exceed two glasses/ day). Bioflavonoids and anti-oxidants have a strong anti-inflammatory effect.

h) Fiber – Gound flaxseed (2 tbsp/day)-Extra fiber aids in detoxifying liver and the entire body  by interrupting the enterohepatic circulation. Psyllium (metamucil). Regulates bowel movements and has favorable effect on lipoprotein profile.

Vitamin C – A Genetic Deficiency Disease

Vitamin C (1000–3000 mg/day), is a key player, as it is the vitamin for strong collagen formation, strengthening the arterial wall.  See Linus Pauling’s patented protocol which includes Vitamin C and amino acids Proline and Lysine, the two amino acids that act as receptors for Lp(a).  By consuming additional Lysine and Proline, the receptor sites on the Lp(a) and other lipoproteins are covered up and made less sticky, resulting in less deposition in the artery wall.  The vitamin C is important not only for strong collagen formation, a major component of the arterial wall, but also for all other structural elements of the body, for that matter. (37)(52)(53)(54)(55)(56)(57)

Humans have a genetic deficiency in Gulano-Lactone-Oxidase (GLO), the final enzyme step in the manufacture of Vitamin C, and therefore unlike all the other animals who make their own Vitamin C, we cannot make this necessary vitamin.  We share with all other primates this genetic disease, the inability to manufacture vitamin C, producing a vitamin C deficiency state in all humans.(58)

Also see Thomas Levy’s two books on Vitamin C. (49-51)

Buy Vitamin C at Tower Labs – Ascorcine a9

j) Exercise and weight loss- improves insulin sensitivity, reduces inflammatory markers, reduces blood pressure, improves lipoprotein profile.

Magnesium

k) Magnesium supplementation is inexpensive and safe. Magnesium deficiency due to dietary deficiency or thiazide diuretics for hypertension is common, and is associated increased heart disease risk.  Magnesium reduces blood pressure, relaxes smooth muscle in arteries, and is needed for normal endothelial function.(41-43)

Buy Magnesium

L-Arginine

L-arginine is converted to nitric oxide, an important substance for arterial health. Research by Furchgott and other showed that nitric oxide (NO) relaxes arterial smooth muscle, dilating coronary arteries by up to 50%.(35)  However, Nitric Oxide (NO) is gone after a few seconds, so it must be replenished at a constant rate to keep the arteries relaxed and open. Lack of NO is associated with constricted arteries, damage to the arterial lining, and accelerated plaque growth. L-arginine shrinks coronary plaque,  corrects “endothelial dysfunction”, improves insulin sensitivity, is anti-inflammatory and shrinks plaque.  Dosage: l-arginine 6000 mg twice a day, best taken on an empty stomach.

Buy Arginine

Reverse Cholesterol Transport and Essential Phospholipid – Phosphatidyl Choline (PC) (38)(39)

James C. Roberts MD FACC, a practicing cardiologist, lectures extensively on his clinical success with Phosphatidylcholine (IV or in Liposomal Oral Format with EDTA):  Reverse Cholesterol Transport and Metal Detoxification.  A DVD of his lectures is available which describes considerable clinical success with oral EDTA and phosphytidylcholine.  This page contains his DVD lecture material complete with clinical case histories.(61)

Essential Phospholipid is available under trade name Phoschol from Nutrasol which increases Lecithin Cholesterol Acyl Transferase activity (LCAT) (Dobiasova M 1988).(40)(40b)

Activating LCAT is beneficial because LCAT is the crucial substance which transports cholesterol from the arterial plaque back to the liver for metabolic breakdown into bile.  This process reverses atherosclerotic plaque formation.  Usual Dosage is 3 softgels Nutrasol Phoschol a day each containing 900 mg Phosphatidyl Choline.(38)(39)

Berberine

Berberine, also called Oregon Grape, is a botanical which has many benefits. Dr Roberts page on Berberine for Preventing and Reversing Heart Disease.

Thyroid Function

Normalize thyroid function. Broda Barnes MD showed that low thyroid function was a significant risk factor for heart disease. This conclusion was based on autopsy data from Graz Austria and detailed in his books, Hypothyroidism the Unsuspected Illness, and his other book, Solved the Riddle of Heart Attacks. Barnes felt that the thyroid lab tests were frequently unreliable, and he used clinical judgement instead. (59)

LipoProtein (a)

All About Reducing Lipoprotein (a)(2)(3)

Lipoprotein little A, also written as Lp(a) is a genetic variant lipoprotein which is associated with a high risk of heart disease, and therefore identification and reduction is essential.  The problem is that the conventional Lipid panels done in your doctor’s office do not include Lp(a).  Only the more sophisticated lipoprotein panels such as the Cardio-IQ (Quest), VAP (Atherotech) or NMR (Liposcience) panels provide Lp(a) data.

Lp(a) and Lipoproteins:

1) Lp(a) is best to measured in (nmol/l), and target  below 75 nmol/l .

2) Lp(a) measured in mg/dl (weight may not be accurate), then target below 30 mg/dl .

3) Measured (not calculated) LDL target 50–60 mg/dl.

4) LDL particle number target (NMR) of 600–700 nmol/l or apoprotein B of 50–60 mg/dl. Reduce small LDL to <10% of total LDL.

Treating Lp(a) with Niacin

Use Niaspan® (Kos Pharmaceuticals) or over-the-counter Slo-Niacin® (Upsher-Smith).   Both are better tolerated than OTC plain niacin, which may cause the hot flushes. Reduce hot flushed by drinking a full glass of water with each gelcap, and some find adding an aspirin tablet to the routine helps to reduce flushing.

Lp(a) and BioIdentical Hormones

Bio-Identical hormones are beneficial for reducing heart disease.  In menopausal females, estrogen preparations such as Bi-Est are used. Estrogens have been shown to reduce coronary artery calcium score.(46)

In males over 50, bio-identical testosterone cream may lowers Lp(a) by as much as 25% (per William Davis MD).  Medical studies show that optimizing Testosterone levels in aging males can reduce risk of coronary artery disease by 60%. (47)(48)

DHEA can promote weight loss, and improve insulin sensitivity.(45)

Lp(a) and L-Carnitine

The supplement L-carnitine can be a useful adjunct; 2000–4000 mg per day (1000 mg twice a day) can reduce Lp(a) 7–8%, and occasionally will reduce it up to 20%.

Buy L-Carnitine

Remember, reduction in calcium score on follow up calcium scan is the goal.

What about Statin-Cholesterol Lowering drugs?

Dr Davis admits that the total cholesterol and the LDL cholesterol numbers are of little value in predicting heart disease risk. And he says that the statin drug side effects, ie. muscle pain and weakness, are more common in actual practice than the drug advertising would suggest, making statin drugs difficult to take for the long term.

In my opinion, statin drugs are not recommended for women as explained in my previous article on Statin Drugs for Women, which can be found here (33)

My other article on Statins, Lipitor and the Dracula of Medical Technology can be found here.  (34)

What about Calcium Supplements for women to prevent osteoporosis?

Dr Davis points out that women who take calcium tablets have double the risk of heart attacks than those on placebo.(5)

Check out my earlier Heart Disease Reversal Page here.

Credit and Thanks is given to William Davis MD at the Track Your Plaque Web Site and Blog for the above information.(2)(3)

Comments Off on Fish oil Omega 3 and heart disease

By admin, April 7, 2015 1:36 pm

Individuals with high intake of Ω-3 polyunsaturated fatty acids,
usually associated with a high fi sh and low red meat diet, have
low rates of heart disease. Fish oil concentrates reduce triglyceride
levels and increase LDL particle size, a benefi cial effect when
administered in high doses (6–12 g/day).

Patients who had
LDL-C levels of less than 70 mg/dL and CRP levels of less than
1 mg/L after statin therapy had the lowest rate of recurrent CV
events.

Comments Off on Effect of Plant Sterol–Containing Fats on Blood Lipid Levels

By admin, April 7, 2015 1:35 pm

Effect of Plant Sterol–Containing Fats on Blood Lipid Levels

Background

In the early 1950s, plant-derived sterols were observed to decrease serum cholesterol levels.^{1 2} The effective dose in humans was reported to be between 5 and 10 g/d when given in divided doses. The efficacy of plant sterols with regard to lowering blood cholesterol levels was soon confirmed,^{3 4 5 6} albeit at somewhat lower doses.⁷ On the basis of these data, plant sterols were briefly used in the reduction of blood cholesterol levels before the introduction of pharmacological agents with higher efficacy and patient acceptance. The resurgence of interest in plant-derived sterols is now coupled with the incorporation of these compounds into fat-containing foods. More recent evidence has shown that esterification of these sterols increases their solubility in fat and their efficacy in lowering low-density lipoprotein (LDL) cholesterol levels.^{8 9}

Comments Off on Modifying Coronary Artery Atherosclerosis

By admin, April 7, 2015 11:18 am

Framingham Heart Study Identifying major CVD risk factors

Over the years, careful monitoring of the Framingham Study population has led to the identification of the major CVD risk factors –
high blood pressure,
high blood cholesterol,
smoking,
obesity,
diabetes, and
physical inactivity

Hypertension
Each 20-mm Hg increase in systolic blood pressure or 10-mm Hg increase in diastolic blood pressure doubles coronary heart disease mortality and stroke mortality. Proposed mechanisms by which hypertension promotes atherosclerosis include damage to endothelial cells, promoting activation and incorporation of lipids into subintima, and stimulation of subintimal smooth muscle cell proliferation.

For prevention of CAD, the NCEP has determined that an LDL-C level of less than 100 mg/dL is optimal for CAD prevention, and a level of 70 mg/dL or less is now recommended for persons with existing CAD plus other risk factors. Ideal blood pressure was set at 120/80 mm Hg or less by

low-dose aspirin, and fish oil—may be considered, depending on the age and estimated CV risk of the patient.

Each 20–mm Hg increase in systolic blood pressure or 10–mm Hg increase in diastolic blood pressure doubles coronary artery disease mortality and stroke mortality.

It is instructive to note that although human LDL-C concentrations tend to cluster from 120 to 200 mg/dL and atherosclerotic disease is the main cause of death in humans, typical nonhuman mammalian LDL-C levels are 10 to 60 mg/dL, and atherosclerosis does not occur unless animals are fed high-fat food.

The risk of a coronary event is thought to increase by 2%for every 1%decrease in plasma HDL

Framingham Heart Study Identifying major CVD risk factors

Over the years, careful monitoring of the Framingham Study population has led to the identification of the major CVD risk factors –
high blood pressure,
high blood cholesterol,
smoking,
obesity,
diabetes, and
physical inactivity

Hypertension
Each 20-mm Hg increase in systolic blood pressure or 10-mm Hg increase in diastolic blood pressure doubles coronary heart disease mortality and stroke mortality. Proposed mechanisms by which hypertension promotes atherosclerosis include damage to endothelial cells, promoting activation and incorporation of lipids into subintima, and stimulation of subintimal smooth muscle cell proliferation.

For prevention of CAD, the NCEP has determined that an LDL-C level of less than 100 mg/dL is optimal for CAD prevention, and a level of 70 mg/dL or less is now recommended for persons with existing CAD plus other risk factors. Ideal blood pressure was set at 120/80 mm Hg or less by

low-dose aspirin, and fish oil—may be considered, depending on the age and estimated CV risk of the patient.

Each 20–mm Hg increase in systolic blood pressure or 10–mm Hg increase in diastolic blood pressure doubles coronary artery disease mortality and stroke mortality.

It is instructive to note that although human LDL-C concentrations tend to cluster from 120 to 200 mg/dL and atherosclerotic disease is the main cause of death in humans, typical nonhuman mammalian LDL-C levels are 10 to 60 mg/dL, and atherosclerosis does not occur unless animals are fed high-fat food.

The risk of a coronary event is thought to increase by 2%for every 1%decrease in plasma HDL

Diet and exercise

Certain changes in diet and exercise may have a positive impact on raising HDL levels:^[29]

• Decreased intake of simple carbohydrates.^{[30][31][32][33]}
• Aerobic exercise^[34]
• Weight loss^[35]
• Magnesium supplements raise HDL-C.^[36]
• Addition of soluble fiber to diet^[37]
• Consumption of omega-3 fatty acids such as fish oil^[38] or flax oil^[39]
• Increased intake of cis-unsaturated fats^[40]
• Consumption of medium-chain triglycerides (MCTs) such as caproic acid, caprylic acid, capric acid, and lauric acid.
• Removal of trans fatty acids from the diet^[41]

Most saturated fats increase HDL cholesterol to varying degrees and also raise total and LDL cholesterol.^[42] A high-fat, adequate-protein, low-carbohydrate ketogenic diet may have similar response to taking niacin (vitamin B3) as described below (lowered LDL and increased HDL) through beta-hydroxybutyrate coupling the Niacin receptor 1.^[43]

HDL levels can be increased by smoking cessation,^[35] or mild to moderate alcohol intake.^{[44][45][46][47][48][49]}

Pharmacologic (1- to 3-gram/day) niacin doses increase HDL levels by 10–30%,^[53] making it the most powerful agent to increase HDL-cholesterol.^[54][55] A randomized clinical trial demonstrated that treatment with niacin can significantly reduce atherosclerosis progression and cardiovascular events.^[56] However, niacin products sold as “no-flush”, i.e. not having side-effects such as “niacin flush“, do not contain free nicotinic acid and are therefore ineffective at raising HDL, while products sold as “sustained-release” may contain free nicotinic acid, but “some brands are hepatotoxic”; therefore the recommended form of niacin for raising HDL is the cheapest, immediate-release preparation.^[57]

AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides

As above, but instead of a placebo, patients were given 1,500 to 2,000 mg/day of extended-release niacin.

After two years the niacin group, as expected, had experienced a significant increase in plasma HDL-C (along with some other benefits like a greater reduction in plasma triglycerides).  However, there was no improvement in patient survival.  The trial was futile and the data and safety board halted the trial.  In other words, for patients with cardiac risk and LDL-C levels at goal with medication niacin, despite raising HDL-C and lowering TG, did nothing to improve survival. This was another strike against the HDL hypothesis.

CETP. – Dalcetrapib

By 2008, as the AIM-HIGH trial was well under way, another pharma giant, Roche, was well into clinical trials with another drug that blocked CETP.  This drug, a cousin of torcetrapib called dalcetrapib, albeit a weaker CETP-inhibitor, appeared to do all the “right” stuff (i.e., it increased HDL-C) without the “wrong” stuff (i.e., it did not appear to adversely affect blood pressure). It did nothing to LDL-C or apoB.  This study, called dal-OUTCOMES, was similar to the other trials in that patients were randomized to either standard of care plus placebo or standard of care plus escalating doses of dalcetrapib.   A report of smaller safety studies (called dal-Vessel and Dal-Plaque) was published a few months ago in the American Heart Journal, and shortly after Roche halted the phase 3 clinical trial.  Once again, patients on the treatment arm did experience a significant increase in HDL-C, but failed to appreciate any clinical benefit.  Another futile trial.

Mendelian randomization

On May 17 of this year a large group in Europe (hence the spelling of randomization) published a paper in The Lancet, titled, “Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study.”  Mendelian randomization, as its name sort of suggests, is a method of using known genetic differences in large populations to try to “sort out” large pools of epidemiologic data.

In the case of this study, pooled data from tens of studies where patients were known to have myocardial infarction (heart attacks) were mapped against known genetic alterations called SNPs (single nucleotide polymorphisms, pronounced “snips”).  I’m not going to go into detail about the methodology because it would take 3 more blog posts., But, the reason for doing this analysis was to ferret out if having a high HDL-C was (only) correlated with better cardiovascular outcome, which has been the classic teaching, or if there was any causal relationship.  In other words, does having a high HDL-C cause you to have a lower risk of heart disease or is it a marker for something else?

This study found, consistent with the trials I’ve discussed above, that any genetic polymorphism that seems to raise HDL-C does not seem to protect from heart disease.  That is, patients with higher HDL-C due to a known genetic alteration did not seem to have protection from heart disease as a result of that gene. This suggests that people with high or low HDL-C who get coronary artery disease may well have something else at play.

Comments Off on Inhibition of Arterial Thrombus Formation by ApoA1 Milano

By admin, March 29, 2015 10:47 pm

• Original Contributions  http://atvb.ahajournals.org/content/19/2/378.full
• http://en.wikipedia.org/wiki/ApoA-1_Milano

Inhibition of Arterial Thrombus Formation by ApoA1 Milano

1. Dayuan Li,
2. Sharon Weng,
3. Baichun Yang,
4. Dani S. Zander,
5. Tom Saldeen,
6. Wilmer W. Nichols,
7. Saeed Khan,
8. Jawahar L. Mehta

+ Author Affiliations

1. 
   
   From the Departments of Medicine (D.L., S.W., B.Y., W.W.N., J.L.M.) and Pathology (D.S.Z., S.K.), University of Florida College of Medicine; the VA Medical Center (D.L., S.W., B.Y., D.S.Z., W.W.N., J.L.M.), Gainesville, Florida; and the Department of Forensic Medicine (T.S.), University of Uppsala, Uppsala, Sweden.

1. Correspondence to J.L. Mehta, MD, PhD, Department of Medicine, University of Florida, College of Medicine, Box 100277, JHMHC, Gainesville, Florida 32610. E-mail mehta@medmac.ufl.edu

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Abstract

Abstract—The mutant form of human apoA1, known as apoA1 Milano, is formed as a result of arginine 173 to cysteine substitution and inhibits experimental atherosclerosis in cholesterol-fed animals. This study was designed to determine if apoA1 Milano would modify arterial thrombogenesis. Sprague Dawley rats were intravenously administered the carrier alone (n=8) or apoA1 Milano (20 mg · kg⁻¹ · d⁻¹ for 4 to 10 days, n=17). The abdominal cavity was opened, and the abdominal aorta was isolated. Whatman paper impregnated with 35% FeCl₃ was wrapped around the surface of the aorta, and aortic flow was recorded continuously. In carrier-treated rats, an occlusive platelet-fibrin-rich thrombus was formed in 21.2±4.1 (mean±SD) minutes. Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation (38.8±11.9 versus 21.2±4.1 minutes, P<0.01), inhibited platelet aggregation (25±7% versus 50±11%, P<0.01), and reduced weight of the thrombus (18.5±1.8 versus 23.7±2.3 mg/cm, P<0.01). Total cholesterol and HDL levels remained similar in both groups of rats, but plasma apoA1 Milano levels were elevated in apoA1 Milano–treated rats. In in vitro studies, incubation of platelets with apoA1 Milano reduced ADP-induced platelet aggregation by about 50%, but apoA1 Milano had no direct effect on vasoreactivity. This study provides further evidence for critical role of platelets in thrombosis. Use of apoA1 Milano offers a novel approach to inhibit arterial thrombosis.

Key Words:

• apoA1
• apolipoproteins
• lipoproteins, HDL
• thrombosis

• Received April 17, 1998.
• Accepted July 21, 1998.

Thrombus formation in the atherosclerotic coronary artery is the immediate cause of acute myocardial ischemia. Coronary artery thrombosis often is initiated by abrupt disruption of the atherosclerotic plaque and deposition and activation of platelets on the subendothelial layers in the disrupted plaque.^{1 2} Experimental and clinical studies have shown reduction in atherosclerosis and cardiac events with aggressive lowering of serum total and LDL cholesterol levels.^{3 4 5} Epidemiological studies also show an inverse relationship between the plasma levels of HDL cholesterol and atherosclerosis.^{6 7} Plasma levels of apoA1, the major protein component of HDL cholesterol, are reduced in patients with premature coronary artery disease.⁸ Whether increased HDL cholesterol or apoA1 is a marker for protection from atherosclerosis or has a direct vasoprotective effect is not known; however, studies in transgenic animal models that overexpress apoA1 suggest that elevated levels of apoA1 are antiatherogenic.⁹

In 1980, Franceschini et al¹⁰ described a family from Limone sur Garda in Northern Italy with a lipoprotein disorder characterized by a striking absence of atherosclerosis despite very low plasma levels of HDL cholesterol. Subsequent studies by this group¹¹ showed that the absence of atherosclerosis in this family was associated with the presence of a mutant form of apoA1 with a single amino acid substitution, arginine 173 to cysteine, which favors the formation of dimers. This mutant form of apoA1, thereafter termed apoA1 Milano, constitutes a large component of the apolipoprotein content in the affected family. All subjects in this family were found to be heterozygous for the mutant allele and had very low levels of HDL cholesterol, high triglyceride levels, and yet no atherosclerosis. The apoA1 Milano has a shortened residence time and causes rapid catabolism of apoA1 in these subjects.¹² The substitution of cysteine for arginine appears to alter the amphipathic nature of the α-helical fragment of apoA1, increasing exposure of its hydrophobic residues.¹³ This structural modification is associated with high affinity of apoA1 Milano for lipids in the lipid-protein complexes and their easy removal. The gene for apoA1 Milano has been cloned by Pharmacia, and the genetically engineered version of the mutant protein has been used in experimental studies. In a study by Ameli et al,¹⁴ administration of the genetically engineered apoA1 Milano caused a marked reduction in the magnitude of intimal lesions and regression of preexisting lesions in cholesterol-fed rabbits.

Because HDL cholesterol fraction per se decreases platelet aggregation and negates the stimulatory effect of oxidized LDL cholesterol on platelet aggregation,¹⁵ and apoA1 Milano in particular facilitates removal of LDL cholesterol, we hypothesized that administration of recombinant apoA1 Milano would inhibit platelet-dependent thrombus formation. This hypothesis was tested in a rat model of arterial thrombosis.

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Materials and Methods

Study Protocol

Twenty five Sprague Dawley (Harlan, Indianapolis, IN) rats weighing 250 to 350 g were fed standard rat chow. Seventeen rats received an intravenous injection of 20 mg/kg of apoA1 Milano in a carrier (or vehicle) of phosphatidylcholine-choline complex (dissolved in saline) in the tail vein every day for 8 to 10 days. Eight rats received an intravenous injection of the carrier of phosphatidylcholine-choline complex. Recombinant apoA1 Milano used in this study was supplied by Pharmacia and Upjohn.

An arterial thrombus model described by Kurz et al¹⁶ was used in this study. All animals were anesthetized with pentobarbital (30 mg/kg). Abdominal cavity was opened and approximately 1.2 cm length abdominal aorta was isolated. The aortic blood flow was recorded continuously by an ultrasonic Doppler flow probe (Crystal Biotech). The signal from the Doppler flow probe was calibrated against an electromagnetic flow probe, and the flow was expressed in mL/min as described earlier.¹⁷ Whatman paper soaked in 35% FeCl₃ was wrapped around the external surface of the aorta. When thrombus was formed, thrombus along with the exposed aorta was taken out and weighed.

About 3 mL of blood was collected for platelet aggregation, measurement of plasma lipids, and apoA1 Milano levels. A segment of the thrombus along with aorta was saved in 2.5% glutaraldehyde, and another segment was saved in 10% neutral buffered formalin. ApoA1 Milano–treated rats were studied in parallel with carrier-treated rats.

Platelet Preparation and Aggregation

Blood was mixed thoroughly with 3.8% sodium citrate (9:1). Blood was centrifuged at 1200 rpm for 10 minutes at room temperature to obtain platelet-rich plasma (PRP) and centrifuged again at 3000 rpm for 15 minutes to obtain platelet-poor plasma (PPP). Platelet count in PRP was counted and kept at about 2×10⁸ to 3×10⁸ cells/mL. ADP (final concentration 20 μmol/L) was used as stimulus for platelet aggregation as described previously.¹⁸ All platelet aggregations were performed in a 4-channel Chronolog aggregometer. Every time platelet aggregation was performed in an apoA1 Milano–treated rat, platelet aggregation was also done in a vehicle-treated rat. In other in vitro studies, PRP was incubated with A1 Milano (0.6 mg/mL) or the vehicle for 60 minutes at 37°C and platelet aggregation in response to ADP determined.

Plasma HDL and Total Cholesterol Measurement

The blood samples were centrifuged at 1400g for 10 minutes, and the supernatant was collected. Total serum cholesterol was determined by enzymatic technique, and serum HDL-cholesterol was measured after precipitation of apoB-containing lipoproteins with phosphotungstic acid.

ApoA1 Milano Levels

Plasma levels of apoA1 Milano were determined by an ELISA by Dr P.K. Shah of the University of California, Cedar-Sinai Medical Center, Los Angeles, CA. The ELISA uses 2 different mouse monoclonal antibodies (MAbs) developed against recombinant apoA1 Milano. The first MAb was used to coat the microtiter wells. Standard recombinant apoA1 Milano and appropriate dilution of the serum samples were incubated. Bound apoA1 Milano was detected with the other MAb, which was biotinylated. The plate was developed using alkaline phosphatase, and the absorbance was read at 405 nm.

Morphologic Analysis of Thrombus

The method for scanning electron microscopy was similar to the method described previously by us.¹⁷ Essentially, aortic segments were fixed in 2.5% glutaraldehyde and then placed in 1% osmium tetroxide in 1% cacodylate buffer (pH 7.2). After several washes in cacodylate buffer, aortic segments were dehydrated in graded alcohols and 1% acetone and then refrigerated overnight in amyl acetate. Specimens were dried to the critical point and coated with silver in a Hummer 5 sputter coating system (Anatech Ltd). Under a dissecting microscope, tissues were cut with a razor blade for full exposure of the intimal surface. Specimens were examined with a Hitachi S450 scanning electron microscope (Hitachi Ltd).

For light microscopic examination, tissues from carrier and apo ApoA1 Milano–treated rats were fixed in 10% neutral buffered formalin for 3 days, processed routinely through alcohols and xylene, and then embedded in paraffin. Five micron thick sections were cut at 2 levels in the paraffin block and stained with standard hematoxylin-eosin stain. Additional sections were stained with a Prussian blue (iron) stain. Other sections were stained to illustrate fibrin and platelets using Carstairs’ method^{19 20} ; in brief, 5 μm thick paraffin sections were hydrated to water, placed in 5% ferric alum for 5 minutes, rinsed in running tap water, stained in Mayer’s hematoxylin for 5 minutes, and then rinsed again in running tap water. Slides were placed for 1 hour in picric acid-orange G solution (composition: 20 mL saturated aqueous picric acid, 80 mL saturated picric acid in isopropanol, and 0.2 g orange G) and then rinsed in distilled water. For 5 minutes, slides were placed in ponceau-fuchsin solution (composition: 0.5 g acid fuchsin, 0.5 g ponceau 2R, 1 mL acetic acid, and distilled water; final volume 100 mL) and then rinsed in distilled water. Slides were treated with 1% phosphotungstic acid until the muscle appeared red and the background pale pink and then rinsed in distilled water. Slides were stained with aniline blue solution (1 g aniline blue in 100 mL 1% acetic acid) for 30 minutes, rinsed in several changes of distilled water, dehydrated, cleared, and then mounted. With a fixation time >48 hours, Carstairs’ method for fibrin and platelets produces differential staining of fibrin (bright red), platelets (gray-blue to navy), collagen (bright blue), muscle (red), and red blood cells (yellow).

ApoA1 Milano and Vascular Reactivity

To determine the direct effect of apoA1 Milano on vascular reactivity, rat aortic rings (4 to 5 mm) were obtained from 6 different rats. Care was taken to avoid any unnecessary manipulation of vessels. The rings were then mounted onto wire stirrups connected to force transducers (Kistler Morse) and placed in custom-designed tissue-organ baths filled with oxygen-saturated (95% O₂+5% CO₂) Krebs-Ringer buffer (composition in mmol/L: NaCl 118, KCl 4.7, CaCl₂ 1.3, KH₂PO₄ 1.2, MgCl₂ 1.2, NaHCO₃ 12.5, Na-EDTA 0.01, and glucose 11.1, pH 7.4). The rings were then stretched to and maintained at a preload tone of 2 g for approximately 60 minutes. During the period of equilibration, rings were incubated with apoA1 Milano (0.6 mg/mL) or carrier for 1 hour at 37°C. Buffer was changed every 30 minutes and continuously bubbled with 95% O₂+5% CO₂. After equilibration, rings were exposed to cumulative concentrations of norepinephrine (NE, 10⁻⁹−10⁻⁶ m) to determine the vasoconstrictor response. Other rings were contracted with NE (10⁻⁷−10⁻⁶ m) to obtain 60% to 70% of maximal contraction, then exposed to the endothelium-dependent receptor-mediated vasorelaxant acetylcholine (Acetylcholine (ACh), 10⁻⁹−10⁻⁶ m) to determine endothelium-dependent vasorelaxation.^{21 22 23}

Statistical Analysis

All data are given as mean±SD. ANOVA was used to compare the 2 experimental groups followed by unpaired t-test with Bonferroni’s correction. A P value of ≤0.05 was considered significant.

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Results

Blood Total Cholesterol, HDL, and ApoA1 Milano Levels

Both group of rats showed no significant change in body weight during the course of the study and did not differ significantly in their plasma levels of total cholesterol. The HDL cholesterol levels were slightly lower (P, NS) in the apoA1 Milano–treated rats. Plasma apoA1 Milano levels were identified only in the apoA1 Milano–treated rats (Table⇓).

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Table 1.

Plasma Total Cholesterol, HDL Cholesterol, and ApoA1 Milano Levels

Platelet Aggregation in ApoA1 Milano–Treated Rats

Treatment with apoA1 Milano markedly inhibited platelet aggregation in each of the treated rats (mean 25±7% versus 50±11% in vehicle-treated rats, n=17 and n=8, respectively; P<0.01). Representative examples of platelet aggregation patterns in the 2 groups of rats are shown in Figure 1⇓. There was no difference in platelet counts in the 2 groups of rats.

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Figure 1.

Representative examples of FeCl₃-induced thrombus formation and ADP-induced platelet aggregation in rats treated with vehicle alone or apoA1 Milano. Note the prolongation of time to thrombosis in apoA1 Milano–treated rats and concurrent decrease in platelet aggregation.

Time to Thrombosis and Weight of Thrombus

Application of FeCl₃ in vehicle-treated rats resulted in oscillations in aortic blood flow for about 15 minutes. This was followed by rapid decrease in blood flow and eventually total cessation, indicating occlusive thrombus formation. Once the flow had totally ceased, there was no spontaneous return of flow over 1 hour of observation in all vehicle-treated animals. A typical pattern of thrombus formation in a vehicle-treated rat is shown in Figure 1⇑.

Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation, and the mean value increased 86% to 38.8±11.9 minutes (compared with 21.2±4.1 minutes in the vehicle-treated group) (Figure 2⇓). Following cessation of blood flow, the thrombus was often unstable as evident from return of flow transiently in all apoA1 Milano–treated rats. A representative example of markedly delayed time to thrombus formation and unstable thrombus in an apoA1 Milano–treated rat is shown in Figure 1⇑. The weight of the thrombus in all apoA1 Milano–treated rats was much less than in the vehicle-treated rats (Figure 2⇓).

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Figure 2.

Prolongation of time to thrombus and reduction in the weight of the thrombus in apoA1 Milano–treated rats compared with vehicle-treated rats. Number of rats in each group is 17 and 8, respectively. Data in mean±SD.

Morphology of the Thrombus

Scanning electron microscopic examination of the aortic region with thrombus revealed endothelial disruption, deposition of platelets on the intimal surface, fibrin strands, and red blood cells, especially at the base of the thrombus (Figure 3A⇓). Cross-sectional view of the aortas of vehicle-treated animals showed extensive platelet-fibrin deposition along the entire intimal surface. The main body of the thrombus consisted of large number of red blood cells tethered with fibrin but few platelets (Figure 3B⇓). There was no discernible difference in the morphology of the thrombus in the 2 groups of rats.

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Figure 3.

Morphology of the thrombus in a carrier-treated rat as determined by scanning electron microscopy. Beginning of the thrombus is characterized by endothelial disruption, large number of platelets adherent to the intimal surface, and red blood cells tethered with fibrin bands (A). Cross-section of the aorta through the body of the thrombus shows very large number of platelet-fibrin aggregates adherent to the intimal surface of the entire vessel wall. The core of the thrombus consists primarily of red blood cells with inter-twined fibrin strands (B).

Light microscopic examination of the thrombi (Figure 4⇓) revealed irregular masses of platelets with focal connections to the intimal surfaces, and variable numbers of red blood cells, accounting for the majority of the thrombus. Fibrin occupied spaces between the platelet aggregates and the red blood cells, particularly adjacent to the arterial wall. Subintimal deposits of crystalline material were present and stained blue with the Prussian blue stain, consistent with transvascular penetration of FeCl₃ (Figure 4⇓). Again, there were no discernible differences in the morphology of the thrombi in the carrier-treated and apoA1 Milano–treated groups.

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Figure 4.

Light microscopic morphology of the aortic thrombus in a carrier-treated rat. A and B, Carstairs’ method for fibrin and platelets. Irregular aggregates of platelets (P) demonstrate connections to the intimal surface of the aorta. The center of the thrombus consists primarily of red blood cells (yellow). Fibrin (red) is interspersed between the platelet aggregates and red blood cells. Original magnification ×40 (A) and ×200 (B). C, Prussian blue (iron) stain. Subendocardial FeCl₃ deposits (dark blue). Original magnification ×40.

Direct Effect of ApoA1 Milano on Platelet Aggregation

Incubation of PRP with apoA1 Milano markedly decreased ADP-induced platelet aggregation in each of the 6 rats (mean platelet aggregation at 5 minutes: 22.8±10.7% versus 54.5±20.9%; P<0.01).

ApoA1 Milano and Vasoreactivity

Incubation of aortic rings with apoA1 Milano had no effect on vasoconstrictor response to NE (EC₅₀: 5.5±0.4×10⁻⁷ m versus 5.6±0.4×10⁻⁷ m; P, NS). Likewise, incubation of aortic rings with apoA1 Milano had no effect on relaxant response to ACh (IC₅₀: 5.1±0.4×10⁻⁸ m versus 5.0±0.2×10⁻⁸ m; P, NS).

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Discussion

Cholesterol and Thrombosis

It is generally presumed that high levels of LDL cholesterol lead to enhanced thrombus formation, probably a result of reduced vasomotion and enhanced platelet aggregability. This has been attributed in part to the loss of NO release and its activity in cholesterol-rich platelets and vascular tissues.^{15 24} Most episodes of acute myocardial infarction are a result of formation of occlusive platelet-rich thrombus in the atherosclerotic narrowed coronary arteries.^{1 2} There is also evidence that myocardial injury is greater in hypercholesterolemic animals subjected to coronary artery occlusion; this is related to diminished vasomotion of the coronary artery and enhanced platelet aggregation in the coronary arterioles.²⁵ Sawa et al²⁶ described a 2-fold increase in plasma levels of plasminogen activator inhibitor-1 (PAI-1) as well as PAI-1 mRNA in hypercholesterolemic rabbits with indwelling catheters. Abela et al²⁷ showed that hypercholesterolemia causes markedly large thrombi in rabbits subjected to balloon injury. However, there are no data on the influence of alteration in lipid components on in situ platelet-fibrin rich thrombus formation that is akin to human arterial thrombus. Also, there are no data on the influence of modification of HDL cholesterol on thrombogenicity.

Observations in the Current Study

The major finding in this study was that administration of recombinant apoA1 Milano produces a substantial inhibition of platelet aggregation and delays formation of arterial thrombus in rats. In vitro incubation of platelets with apoA1 Milano for a short period also decreased ADP-induced platelet aggregation. However, there was no significant effect of apoA1 Milano on vasoconstriction or endothelium-dependent relaxation.

Model of Arterial Thrombosis

The model of arterial thrombosis used in these studies has been used to study the effect of a variety of thrombolytic effect of a variety of agents.^{16 28} The in situ thrombus induced in the rat aorta by external application of FeCl₃ is akin to human intracoronary thrombus in its cellular composition and fibrin content.¹⁶ Light microscopy showed penetration of FeCl₃ across the vessel wall (Prussian blue stain), adherence of large numbers of platelets to the subendothelial layers at several points along the circumference of the vessel wall, and the central mass of the thrombus consisting predominantly of red blood cells with interspersed fibrin. These are also characteristics of arterial thrombi in human coronary arteries.¹⁶ The rapidity with which the thrombus is formed in this model depends on the concentration of FeCl₃ applied on the external surface of the blood vessel. The thrombus, once formed, is stable and not subject to spontaneous dissolution. We chose the 35% FeCl₃ concentration because this concentration causes occlusive thrombus formation in 18 to 24 minutes in all rats weighing about 300 g. Because of the relatively low cost of animals and the ability to study formation of occlusive thrombus, vascular reactivity, and platelet aggregation, this model may be considered useful in evaluation of different pharmacologic agents that modulate platelet function, vascular reactivity, and lipid profile.

Mechanism of Antithrombotic Effect of ApoA1 Milano

It has been suggested that apoA1 Milano provides relative protection against vascular disease related to its ability to remove cholesterol from tissues.^{13 14} The apoA1 mutant appears to substantially alter the amphipathic nature of the α-helical fragment of apoA1 , thus increasing the exposure of its hydrophobic residues. This structural modification is associated with a higher kinetic affinity of apoA1 Milano for lipids and an easier dissociation from lipid-protein complexes, which could contribute to its accelerated catabolism and increased efficiency for uptake of tissue lipids.¹² The protective effect of HDL against atherosclerosis has accordingly been attributed in large part to the ability of HDL to facilitate reverse cholesterol transport from peripheral tissues to the liver for removal or revitalization.²⁹ Badimon et al^{30 31} have shown that HDL cholesterol can reverse atherosclerotic lesions in cholesterol-fed rabbits. Burkey et al³² showed that elevated apoA1 fractions reduce aortic smooth muscle cell proliferation, a key early feature of atherosclerosis.

Recent studies from our laboratory have indicated that it is the oxidized LDL cholesterol fraction that enhances platelet aggregation and downregulates NO synthase expression.^{16 33} Studies from other laboratories have shown that HDL inhibits oxidation of LDL cholesterol.^{34 35} We have also shown that HDL cholesterol blocks the platelet stimulatory affect of LDL cholesterol,³⁶ and this effect of HDL cholesterol appears in large part mediated by reversal of the suppressive effect of LDL cholesterol on NO synthase in platelets.³³ Stimulation of NO synthesis in the endothelial cells and platelets has a powerful antithrombotic effect. This phenomenon has been adequately demonstrated in animal models of thrombosis, wherein NO donors³⁷ were used. The current studies showed that administration of apoA1 Milano or in vitro incubation of PRP with apoA1 Milano markedly reduced platelet aggregation. Whether platelet NO production or its activity is enhanced by apoA1 Milano cannot be discerned from this study. However, vascular endothelium-dependent relaxation was not altered when aortic rings were incubated ex vivo with apoA1 Milano. It is, nonetheless, reasonable to postulate that platelet inhibition and/or modulation of vascular reactivity are operative in delaying thrombus formation in apoA1 Milano–treated animals.

Activation of endogenous fibrinolytic pathways by apoA1 fraction of HDL cholesterol has also been well characterized.³⁸ A similar activation of fibrinolytic pathway in apoA1 Milano–treated rats may relate to unstable thrombus and its tendency to spontaneously dissolve.

The light and scanning electron microscopy findings showed in a semiquantitative fashion that the composition of the thrombus in carrier- and apoA1 Milano–treated rats was similar, which indicates that platelets are able to aggregate and form an occlusive thrombus in the presence of extensive arterial endothelial injury despite treatment with apoA1 Milano. This suggests that the inhibitory effects of these agents are quantitative rather than qualitative.

Limitations of the Study

There are some important limitations of the study. First, it would have been ideal to conduct similar studies with wild-type apoA1 to determine if prolongation of time to thrombosis and inhibition of platelet aggregation are unique to the administration of apoA1 Milano. It is possible that wild-type apoA1 can decrease platelet aggregation and hence prolong time to thrombosis. Second, the study lacked information on apoA1 levels in rats treated with carrier or apoA1 Milano. Because apoA1 Milano has been characterized to induce catabolism of apoA1 as well as HDL cholesterol,^{12 13} it is possible that the apoA1 levels were low in apoA1 Milano–treated rats. This concept gains support from the observation of lower HDL cholesterol levels in apoA1 Milano–treated rats (41±8 versus 49±4 mg/dL in carrier-treated rats). Similar observation was made by Ameli et al¹⁴ in rabbits fed high cholesterol diet and administered apoA1 Milano. Third, the apoA1 Milano levels were somewhat lower in this study compared with another study wherein a similar dose was given to rabbits.¹⁴ The differences may relate to differences in species (rat versus rabbit), diet (regular diet versus high cholesterol diet), and study end-point (thrombosis versus atherosclerosis). In the original report on apoA1 mutant, a wide range of apoA1 levels (35 to 116 mg/dL) were identified in the index family.¹⁰ In the transgenic mice, saturated fat diet increased apoA1 levels markedly from 38 to 58 mg/dL.³⁹ Our studies were conducted in fasting rats fed regular chow. In further studies, all these issues will need to carefully evaluated.

Conclusion

This study confirms a critical role of platelet activation in arterial thrombosis. The use of apoA1 Milano—or possibly use of modalities that may increase apoA1 only—offers a novel approach directed to the inhibition of platelet-mediated thrombosis. However, the precise dose and duration of administration of apoA1 mutant needs to be determined and the consistency of these findings across species assessed.

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Acknowledgments

Supported by a Merit Review grant from the VA Central Office (to J.L.M.), a grant-in-aid from the American Heart Association, Florida Affiliate, St. Petersburg, Florida (to B.Y.), and support from the Division of Sponsored Research, University of Florida, Gainesville, Florida (to J.L.M.), and Swedish Medical Research Council, Stockholm, Sweden (to T.S.). We thank Jerry Santiago, HTL, (ASCP) for his technical assistance.

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Comments Off on ApoA1 Milano

By admin, March 29, 2015 10:42 pm

In the end I knew that statins would be overthrown. Their time, like the dinosaurs would come to an end.  I also knew it would be nothing to do with me, or any of the other critics. Nor would it have anything to do with the fact that their terrible burden of adverse effects finally came to light.

No, it would be because their patents ran out, which meant that the big pharmaceutical companies could not make eye-watering profits from them anymore. Inevitably, therefore, they would be replaced. Indeed, for years I have watched, with varying degrees of amusement, the unending efforts to unearth the ‘new’ statins. The wonder drugs to be taken by everyone, for the rest of their lives. The ones that will make billions for the pharmaceutical companies, and several millions for the key opinion leaders promoting them.

Steven Nissen had a go with apoA-1 Milano. This is a little story you may want to look up on Wikipedia.  Basically, a small village was found in Italy (near Milan) where people had very low rates of heart disease, and they also had very low levels of HDL ‘good’ cholesterol.

Turning science on its head, it was therefore decreed that their HDL must be especially ‘good’ at preventing heart disease. [Rather than accept that HDL had bugger all to do with heart disease]. So attempts were made to synthesize their form of HDL, then inject it into patients. This was done in small scale clinical studies.

So brilliantly did Nissen sell the results of these studies that a start-up company called Esperion, which funded the trials, was sold to Pfizer for over a billion dollars on the back of the results. If you want to find out more, the HDL synthesized was called ETC-216, not apoA-1 Milano.

This is what Steven Nissen had to say about the trials.

“The fact that you can actually pull major amounts of plaque out of the artery in five or six weeks is an epiphany,” Nissen says. H. Bryan Brewer, chief of the molecular disease branch at the National Heart, Blood & Lung Institute, adds: “Quite to everyone’s surprise, this indicates that we might be able to be much more successful in a shorter period of time than we thought possible.”

By golly it all sounds very exciting, does it not.  Pulling significant amounts of plaque out of artery walls, in six weeks! This is quite amazing, what a brilliant drug. In truth, though, that quote was from 2003. Has anything since happened since to this magical plaque munching HDL? Is it now on the market, saving lives? Well, as you ask, the answer is no.

However, several other HDL raising agents have also been developed in the last few years. Unfortunately, they also raised the risk of heart attacks and strokes – then disappeared. Torcetrapib, dalcetrapib, anacetrapib…. A yes, anacetrapib, not quite gone yet.

‘Anacetrapib has a knock-your-socks-off effect on HDL and a jaw-dropping effect on LDL,” said Chris Cannon in an AHA press release.  He also said “The lipid effects are jaw dropping in both directions,” Yes, indeed, they were.  Although I am not sure that ‘jaw dropping’ and ‘knock your socks-off’ are truly scientific terms.

But then Forbes ran this headline on the 22^nd October

‘Merck Heart Drug Runs Into New Worry.’ ¹

As an aside, you can find out far more about drugs in development by reading Forbes magazine than you will ever get from the scientific journals.

Anyway, I wouldn’t bet the house on anacetrapib as I suspect it will soon follow the other ‘trapibs’ into an early grave. But never mind, Lilly has yet another HDL raising agent waiting in the pipeline, evacetrapib. What is it with these unpronounceable names, and why don’t these companies just give up? However, perhaps this one has a better shot than most, because as Steven Nissen said of dalcetrapib…

“Dalcetrapib was always a long shot,” said Steven Nissen, chairman of cardiovascular medicine at the Cleveland Clinic. “The worry all along was that it wouldn’t have enough effect to produce a benefit,”

Then, as he went on to say of evacetrapib

“We got everything we could hope for from this drug, and maybe more,” remarked study co-author Steven Nissen, adding that “we are going to move evacetrapib forward as rapidly as possible” into a late-stage trial.” Gosh, he is running a study on evacetrapib too.  He was also lead investigator for Torcetrapib².

That Steven Nissen, what a busy chap he is?  Wasn’t he the one who said about the new cardiovascular prevention guidelines.  “The evidence was never there” for the LDL targets, he said. Past committees “made them up out of thin air,” he added.  Why yes, I think he was.

Anyway. They have tried raising HDL…fail. They have tried a different way of lowering LDL using ezetimibe. It lowered LDL, and has had absolutely no effect on cardiovascular disease…fail. They have tried combing different HDL raising agents with statins….fail.

You know, you might even begin to think that raising HDL wasn’t that good an idea. Billions have been thrown this target, everything has miserably failed. It is also interesting that LDL lowering agents, other than statins, have failed to have any impact on cardiovascular disease. Has this had any effect on the lipid hypothesis? You must be joking.

Given this litany of disastrous drugs trials, why do I now believe that statins are about to be overthrown? What agents could be out there that are going to sweep them into the dustbin of history.

Clearly it cannot be an agent that lowers LDL even more than statins. Can it? Well, Steven Nissen has recently said the following about LDL lowering. This quote is in connection to new drugs being developed³.

 “….it matters how you lower cholesterol, not just by how much.”

It matters how you lower cholesterol, not just by how much….. Interesting.  Well, if cholesterol does cause heart disease, then it doesn’t really matter how you lower it, does it? The only question is, by how much? Or is Steven Nissen trying to say something else?

As a further aside, if you want to know where the really big bucks are being spent in cardiovascular research, and what is in the pipeline, all you need to do is follow Steven Nissen’s pronouncements. He is the weather vane. You don’t need to be a weather man to know which way the wind is blowing.

…… (to be continued)

1: http://www.forbes.com/sites/matthewherper/2013/10/22/merck-heart-drug-runs-into-new-worry/

2: http://online.wsj.com/news/articles/SB10001424052702304363104577389353232022644

3: http://www.forbes.com/sites/johnlamattina/2013/09/05/esperions-novel-approach-to-lowering-cholesterol-will-it-be-successful/

Comments Off on Dogs Smell Cancer

By admin, August 10, 2013 9:15 am

University of Pennsylvania training dogs to sniff out ovarian cancer

Jonathan Ball trainings Ohlin to detect ovarian cancer in tissue samples. / University of Pennsylvania’s Working Dog Center

Studies have shown that dogs might have the ability to sniff out cancer. If true, the method could provide a cheap and potentially non-invasive way to detect the disease.

Now, researchers at the University of Pennsylvania’s Working Dog Center are training three dogs how to sniff out samples that contain ovarian cancer markers.

“We’re really excited about it,” Dr. Cynthia Otto, director of the Working Dog Center and associate professor of critical care at Penn Vet, told CBSNews.com.

About 20,000 women in the U.S. get ovarian cancer each year, according to the Centers for Disease Control and Prevention. It causes more deaths than any other cancer of the female reproductive system, even though it only accounts for 3 percent of all women-related cancers.

Play Video

Dogs Can Smell Skin Cancer?

Dogs make an ideal cancer detector because they have a higher density of olfactory or smell sensors and more olfactory genes than humans, Otto explained. In addition, their olfactory bulbs — the part of the forebrain that processes information gleaned from smells — is larger than ours.

“Mice can do a better job at sniffing out things (than dogs),” Otto explained. “But, there is an ability to communicate between a dog and a human so they can tell us what they are finding.”

“I also find working with dogs more rewarding than working with mice,” she added.

There are some theories for why dogs may be able to smell diseases on people. Otto said when she started the project, she had many people e-mail her about how their canines started acting differently when they were diagnosed with cancer. She believes there’s something innate about dogs that allows them to recognize different odors, and it may be tied to how animals tend to shun sick ones from their pack in the wild.

The research project, which was funded by an $80,000 grant from the Kaleidoscope of Hope Foundation, started about a year ago when researchers identified 16 puppies that had the potential to be working dogs. The dogs were selected because they were hunting breeds, and some of them had been bred from generations of working dogs.

The first round of training involved seeking and finding a leather toy and training the dogs how to find live humans. This gave the researchers a closer look at the dogs’ behavior and helped them select candidates.

In this Thursday, Aug. 1, 2013 photo, Jonathan Ball (right) introduces Marta Drexler, an ovarian cancer patient, to McBaine who is in the first round of training for a study that will eventually involve detecting cancerous tissue at Penn Vet Working Dog Center in Philadelphia. Drexler donated ovarian cancer tissue for the study.

/ AP Photo/Matt Rourke

Two of the three finalists for the ovarian cancer training were chosen because they naturally sat down when they found the smell they were seeking.  The third dog, a Labrador, tended to bark when he found his target. However, he worked best when he was in an environment that had fewer distractions, which was perfect for the laboratory and not for other tasks like bomb sniffing.

The cancer training has only been going on for a week, but Otto is surprised at how quickly the pupils are picking up their skills. Right now, the dogs are being trained to sniff out ovarian cancer in tissue samples.

“They’ve already been showing such amazing recognition,” she remarked.

The Penn program isn’t the only research being done on dogs sniffing out disease.

A study in Germany in 2011 showed that dogs were able to detect people with lung cancer in 71 percent of the cases and determine that people didn’t have lung cancer in about 93 percent of cases. Recently, a West Hills, Calif. dog trainer has been working with researchers at the Pine Street Foundation to teach dogs how to detect ovarian cancer using breath samples.

Dr. Leonard Lichtenfeld, deputy chief medical officer for the American Cancer Society, told the Associated Press that canine cancer detecting research is promising but is not yet at the point where it can be used consistently for humans.

“We’re still looking to see whether something could be developed and be useful in routine patient care, and we’re not there yet,” said Lichtenfeld, who is not involved in the University of Pennsylvania research.

Otto hopes that the dogs will be able to detect cancer in blood samples one day. Eventually, she would like to use the dogs on less invasive samples, such as saliva or urine.

“This is just the pilot study,” she said. “We’re going to get more funding… This could provide cancer screening for millions of women.”

Comments Off on Dr Thomas Challenger – Doctors on Call Medcall

By admin, June 26, 2013 3:06 pm

Record demand for house doctors

FIRST ON 7: On-call doctors are gearing up for flu season, which is about to strike Queensland. Kylie Hodge reports.

Enlarge photo

On-call doctors are gearing up for the flu season about to strike Queensland.

There’s record demand for free GPs who visit patients in their own homes at night, on weekends, and holidays.

Every night hundreds of Gold Coast patients have MedCall doctors come to them for free.

DrTom Challenger started the service alone 13 years ago. He now employs 33 other GPs.

“My grandfather was in a nursing home and he was pretty ill and we couldn’t get a doctor out late at night and he ended up dying as a result,” Dr Challenger said.

In Brisbane, Family Care Friendly Medical Services answers calls from across the south east and interstate.

“We’ve got some very bad influenzas down south – some very sick children, and also some of the influenzas are impacting on elder members of the community,” spokesperson Stuart Tait said.

Every patient the doctors treat, is one fewer waiting in the emergency departments.

“They significantly assist with reducing the number of presentations to the emergency departments.” Dr Christian Rowan from AMAQ said.

“One day Telstra disconnected our phones in the office and all the hospitals on the Gold Coast went on bypass with the load of the patients we weren’t seeing.”

But the AMAQ says recent changes to federal funding for GPs are making it more difficult for practices to provide doctors on call.

“The federal government has made after hours viability more difficult,” Dr Rowan said.

Gold Coast:
Medcall, 55 311 224
Free, bulk-billing on-call GP service

Mon-Fri 6pm-8am, Saturdays from noon, All day Sundays & public holidays