Effects of reducing LDL and increasing HDL with gemfibrozil in experimental coronary lesion development and thrombotic risk.

The use of lipid-lowering drugs has been shown to have beneficial effects in primary and secondary prevention of cardiovascular disease. Gemfibrozil has shown beneficial effects as a lipid lowering agent; however, some proactivating effects on platelet function in vitro have been described. We have studied in a porcine model of atherosclerosis if gemfibrozil could prevent the early vascular effects of a cholesterol-rich diet without inducing platelet activation and, hence, mural thrombosis. Pigs were fed for 50 days with a diet rich in saturated fat and cholesterol (cho). The longitudinal follow-up study showed that in control animals LDL-cho increased significantly up to 181.9 +/- 34.2 mg/dl or 79% of total-cho, while HDL-cho was reduced to 19% of total-cho. Gemfibrozil, at average therapeutic plasma levels (peak levels of 28 micrograms/ml) [corrected], induced a significant reduction in the relative amount of LDL (P < 0.05) and increased HDL (P < 0.05). The increase in fibrinogen plasma levels observed in the control group due to the dietary intervention (+25%) was prevented in the treated animals (-5%). In treated animals, vascular lesions were significantly less severe, platelet deposition upon exposure of damaged vessel wall was unchanged and the fibrin layer deposited on the damaged vessel wall was significantly reduced over control animal values. This short term pharmacologic lipid lowering intervention has been able to slow down lesion development and to reduce fibrin formation onto lesioned disrupted vascular substrates without increasing platelet mural thrombosis.

Purification of the porcine platelet GP IIb-IIIa complex and the propolypeptide of von Willebrand factor.

Platelet membrane glycoproteins (GP) are involved in platelet adhesion and aggregation. The glycoprotein IIb-IIa complex (GP IIb-IIIa) is a Ca2+-dependent heterodimer that binds fibrinogen and other adhesive proteins, thereby mediating platelet aggregation and adhesion. We have purified two major glycoproteins from pig platelets by Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl S-300 HR chromatography (Fitzgerald et al. Anal Biochem, 1985): i) the GP IIb-IIIa complex, GP IIb Mr = 140,000 and GP IIIa a single chain of Mr = 95,000-100,000; and ii) a predominant glycoprotein of high molecular weight, the propolypeptide of von Willebrand factor (Mr = 80,000-100,000). Western-blot analysis of the purified GP IIb-IIIa showed that only certain monoclonal antibodies against the human receptor specifically recognize the porcine complex. Differences between the porcine and human GP IIb-IIIa glycoproteins could partially explain the decreased inhibitory effects of GP IIb/IIIa-antagonists (against the human receptor) in porcine platelets.

Porcine platelet von Willebrand antigen II (vW AgII): inhibitory effect on collagen-induced aggregation and comparative distribution with human platelets.

Von Willebrand factor (vWF) is synthesized by human endothelial cells and megakaryocytes as a large precursor, the pre-provWF, which is finally cleaved into the propolypeptide of vWF (pp-vWF) and the mature vWF. We have purified in parallel the pp-vWF and the GP IIb-IIIa from porcine platelets. The N-terminus comparative analysis of porcine pp-vWF with respect to other species revealed more than a 75% and 65% homology with the bovine and human pp-vWF, respectively. Purified pp-vWF inhibited collagen-induced platelet aggregation in porcine platelet rich plasma (PRP) and specifically binds to collagen. Polyclonal antibody pabBp19 against the purified protein was prepared and characterized by ELISA, Western-blot and immunocytochemistry. The distribution of pp-vWF in soluble and membrane fractions from pig platelets has been performed by immunolabeling detection. pabBp19 did not blot human platelet lysates but human pp-vWF was detectable using the antibody Frieda 013091. Cell distribution and quantification studies of human and porcine platelet pp-vWF showed that the protein exists in the soluble and membrane fractions and its pattern is similar in both species.

Effects of statins in thrombosis and aortic lesion development in a dyslipemic rabbit model.

HMG-CoA reductase inhibitors (statins) are effective in primary and secondary prevention of coronary heart disease. The mechanism of action is mainly attributed to their plasma cholesterol lowering activity, although additional effects have been suggested. Our objective was to study whether atorvastatin and simvastatin exhibited an inhibitory effect on platelet deposition onto a triggering damaged vessel wall in addition to an antiatherosclerotic effect in the dyslipemic rabbit model. Statins were administered at identical doses of 2.5 mg/kg/day with a hyperlipidemic diet during 10 weeks. Both drugs similarly lowered total cholesterol and, moderately, triglycerides. Mural platelet deposition on damaged vessel wall placed in an ex-vivo flow perfusion system was reduced in atorvastatin treated animals (39.7+/-6.2 X 10(6) PLT/cm2) vs. controls (94.8+/-15.9 x 10(6) PLT/cm2, p <0.02). Simvastatin reduced aortic fatty streak surface coverage (31,7+/-5.3%) vs. controls (47.9+/-4.1%, p <0.005) and intimal thickening in thoracic aorta (0.15+/-0.05 intima to total area ratio in simvastatin treated animals vs. 0.36+/-0.03 in control animals, p <0.05). Atherosclerotic fatty streak coverage correlated positively with total cholesterol, tryglicerides and LDL-cholesterol levels in all groups. HMG-CoA reductase inhibitors similarly lowered plasma lipids but exhibited significantly different effects in the modulation of atherosclerotic development and platelet response at the tested dose. Therefore, the effect of statins on the progression and manifestation of cardiovascular disease might be also mediated by regulating platelet response to vessel injury.

A sudden increase in plasma epinephrine levels transiently enhances platelet deposition on severely damaged arterial wall--studies in a porcine model.

Epidemiologic evidence has shown that sympathoadrenal activation plays a triggering role in the onset of acute coronary syndromes. However, its mechanism is not yet clearly understood. The aim of this study was to assess the effect of a sudden increase in epinephrine on platelet deposition on severely damaged vessel wall at shear rate conditions modelling stenotic vessels in the porcine model. The selected epinephrine concentrations (0.5 micromol/l-1 mmol/l) alone or in combination with collagen or ADP did not affect platelet aggregation in vitro either in whole blood or in PRP, although porcine platelets express alpha2-adrenergic receptors as assessed by PCR. In vitro and ex vivo perfusion experiments were performed using the Badimon chamber at high shear rate conditions (1690 s(-1)). In vitro, epinephrine (130 nmol/l) increased platelet deposition on severely damaged vessel wall (exposing tunica media; approximately 1.6-fold, p <0.05) or immobilized collagen (2.2-fold, p <0.01). Ex vivo perfusion experiments were performed from animals that received intravenous epinephrine infusion for one hour at a low (0.3 microg/kg/min; approximately 17 nmol/l in plasma, at 20 min of the infusion) and a high dose (1.0 microg/kg/min; approximately 106 nmol/l in plasma, at 20 min of the infusion). Only the low dose temporarily increased platelet deposition on severely damaged vessel wall during the first 30 min of infusion [2.4-fold (p <0.05) and 4.2-fold (p <0.01) at 10 and 30 min of the infusion respectively] declining afterwards. Thus, in flow conditions typical of atherosclerotic arteries, a sudden physiological release of epinephrine can temporarily enhance platelet deposition on severely damaged vessel wall while an extensive exposure leads to refractoriness.

A mimetic of the RGDF-peptide [arginine-glycine-aspartic acid-phenylalanine] blocks aggregation and flow-induced platelet deposition on severely injured stenotic arterial wall. Effects on different animal models and in humans.

8-Guanidino-octanoyl-aspartic acid-phenylalanine (SC-49992), a mimetic of the tetrapeptide arginine-glycine-aspartic acid-phelylalanine, inhibits fibrinogen and vitronectin binding to GP IIb/IIIa. SC-49992 effects on impedance and optical aggregation were compared in different species (human, porcine and dog), SC-49992 induced significant inhibition both in whole blood and PRP aggregation, in all species; however, porcine platelets had a SC-49992 IC50 = 2.5 mM while human and dog platelets had a significant lower IC50 (1 microM and 1.5 microM, respectively). Inhibition of flow-mediated platelet deposition on severely injured vessels was studied in porcine blood at high and low shear rates for 5 minutes. Additionally, studies were performed in vessels with 80% stenosis. SC-49992 reduced platelet deposition both at high and low shear rate in parallel streamlined flow and in stenotic conditions. The lower affinity of porcine platelets for SC-49992 seems to be due to a species difference at the GPIIb/IIIa receptor RGD-sequence level.

Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages.

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.

Platelet deposition on eroded vessel walls at a stenotic shear rate is inhibited by lipid-lowering treatment with atorvastatin.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase are widely used in the treatment of dyslipemias and have shown beneficial effects in the primary and secondary prevention of cardiovascular diseases. However, regression studies with lipid-lowering drugs have not shown significant lesion reduction associated with the improvement in clinical events. Therefore, our objective has been to study whether treatment with a lipid-lowering drug of this family, atorvastatin, could reduce platelet deposition on the damaged vessel wall at different shear stress conditions, simultaneously with retardation of the development of atherosclerotic lesions. Using cholesterol-fed swine as the model, we found that atorvastatin significantly diminished platelet deposition on the mildly damaged vessel wall at high shear rates (50%, P<0.01), but it did not have any effect in preventing platelet deposition triggered by a severely injured vessel wall. Development of coronary lesions was also reduced by treatment. These findings suggest that atorvastatin may prevent platelet attachment to eroded vessels and hence, contribute to reducing the thrombotic risk associated with the erosions of the luminal surface and the platelet-dependent progression of atherosclerotic plaques.

Effect of gemfibrozil on peripheral atherosclerosis and platelet activation in a pig model of hyperlipidemia.

BACKGROUND: Gemfibrozil has been shown to have beneficial effects in the primary and secondary prevention of atherosclerosis. However, a platelet pro-activating effect induced by the drug has been reported. MATERIAL AND METHODS: We analysed the effect of hyperlipidemia and its treatment with gemfibrozil on platelet-fibrinogen binding and the development of early fibrinogen-rich vascular lesions in a porcine model of atherosclerosis. Polyclonal antibodies were raised against purified pig fibrinogen and intact platelets. Two groups of animals were fed a cholesterol/saturated fat-enriched diet for 50 days; one group was treated with gemfibrozil and the other with placebo. RESULTS: The hyperlipidemic diet induced a significant increase in total cholesterol; this was prevented by gemfibrozil (P<0.05). The increase in platelet-fibrinogen binding induced by hypercholesterolemia was mildly reduced in the gemfibrozil treated animals. Histological analysis of aortic vascular wall (abdominal aorta at the iliac bifurcation) from hyperlipidemic animals showed early lesions with fibrinogen infiltration; the lesions were reduced in the fibrate-treated animals. CONCLUSIONS: Gemfibrozil delayed the development of peripheral atherosclerotic plaque, normalised the impaired lipid profile induced by the hyperlipidemic diet and did not show a functionally detectable platelet pro-activating effect able to increase platelet-fibrinogen binding.

Regulation of cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase mRNA levels by L-tri-iodothyronine.

In hypophysectomized and thyroidectomized rats, cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase activity, immunoreactive protein and mRNA levels were all considerably decreased. Administration of L-tri-iodothyronine (T3) resulted in a large increase in all three in hypophysectomized rats and in only a 2-fold increase (reaching the values of control rats) in thyroidectomized rats.

Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein by starvation, fat feeding, and diabetes.

We have determined the levels of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase under different metabolic situations to examine its potential role as a regulatory protein in the ketogenic pathway. We used specific antibodies directed against a peptide of the amino acid sequence of the protein as deduced from the cDNA sequence. The amount of mitochondrial HMG-CoA synthase protein rapidly increased in response to cyclic AMP, dexamethasone, starvation, fat feeding, and diabetes, whereas it was decreased by insulin and refeeding. Insulin was also able to counteract the increase in mitochondrial HMG-CoA synthase levels observed under the diabetic condition. Furthermore, the finding that quantitative changes in HMG-CoA synthase protein were less marked than those in the corresponding mRNA in starved and diabetic rats suggests either translational control or increased degradation of either mRNA or protein. All these results indicate that mitochondrial HMG-CoA synthase is a regulatory element in the ketogenic process.

Diurnal rhythm of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase.

Rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase exhibits a diurnal rhythm of enzyme activity which coincides with the diurnal rhythm of HMG-CoA synthase protein. The peaks of activity and protein (determined by SDS/PAGE and immunoblotting) both occur at D10 (the tenth hour of the daily 12 h dark cycle). The peak of mRNA levels (measured by slot-blot hybridization of liver RNA) is slightly advanced with respect to that of protein, by about 4 h, and shows a maximum at D6. Cytosolic HMG-CoA synthase activity and protein in rats fed on a normal diet were approx. 2-fold higher during the peak at D10 than in the nadir at D2. HMG-CoA synthase mRNA levels were approx. 4-fold higher during the peak at D6 than in the nadir at D2. These results point to a transcriptional and translational regulation of the cytosolic HMG-CoA synthase.

Testis and ovary express the gene for the ketogenic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase.

Ketogenesis has been thought to occur exclusively in the mitochondrial compartment of liver cells. After analysis of five different rat tissues, it was shown that the gene for mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, one of the major control points in the pathway (1992. Casals et al. Biochem. J. 283: 261-264) was expressed only in liver (1990. Ayté et al. Proc. Natl. Acad. Sci. USA. 87: 3874-3878). However, exhaustive analysis of organs and tissues has shown that, in addition to liver cells, testis and ovary express this committed gene in levels similar to those of liver, not only as mRNAs but also as immunodetectable mitochondrial HMG-CoA synthase protein. Immunocytochemical studies locate the mitochondrial HMG-CoA synthase protein in Leydig cells, theca interna cells of ovarian follicle, corpus luteum cells of ruptured ovarian follicle, and epidermal cells of the oviduct. The development of gonadal function appears to be accompanied by mitochondrial HMG-CoA synthase gene expression, as hypophysectomy reduces the expression pattern in gonads. Changes induced in mitochondrial HMG-CoA synthase levels after the depletion of lipoprotein levels in blood closely mimic those of the cholesterogenic cytosolic HMG-CoA synthase and HMG-CoA reductase. These results suggest that mitochondrial HMG-CoA synthase could perform a function similar to that of cytosolic HMG-CoA synthase in de novo cholesterogenesis in gonads, at variance with its ketogenic role in liver.

Immunolocalization of mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase in rat liver.

We report the preparation of specific polyclonal antibodies raised against two synthetic peptides deduced from the cDNA sequence for the rat liver mitochondrial 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) synthase gene. Immunoelectron microscopy using these antibodies on hepatic cryoultrathin sections confirms the mitochondrial localization of this protein in hepatocytes. Immunofluorescence microscopy on frozen sections of adult rat liver revealed fluorescence inside all hepatocytes, with no evidence of zonation, indicating that ketogenesis may not be limited to specific regions of rat liver but is extended to all hepatocytes.

Cell biology of restenosis post-angioplasty.

PTCA is a well established intervention to reduce the severity of atherosclerotic coronary stenosis. In spite of a primary success rate of 90 - 95%, late restenosis occurs in 30 - 50% of patients within 3/6 months of the procedure. Angioplasty in swine induces similar events to those found in humans, thus providing a model for studying strategies for intervention. Blood interaction to the damaged vessel wall occurs with reperfusion after the intervention. Therefore, the in vivo characterization of the interaction of cellular elements (platelets and white cells) and blood proteins with the exposed vascular cells in the vessel wall post-angioplasty may be necessary to identify early triggers of restenosis. Angioplasty was performed simultaneously in the coronary and carotid arteries of swine by fluoroscopy assisted standard techniques. Angiography was performed acutely post-dilatation and residual lumen diameter evaluated. Dilated vessels from 30 min to 6 h postintervention were processed to prepare RNA and preserved to perform immunohistochemistry. Dilatation injury induces maximal expression of c-fos 30 min and c-myc from 2 to 4 h postdilatation. Platelet deposition is initiated immediately post-dilatation as well as infiltration of fluid phase proteins on the damaged areas.