R. Alam

Receptor-mediated uptake and ‘retroendocytosis’ of high-density lipoproteins by cholesterol-loaded human monocyte-derived macrophages: possible role in enhancing reverse cholesterol transport

Human monocyte-derived macrophages (MDM) are cholesterol-loaded, and the rates of uptake, degradation and resecretion of high-density lipoproteins are measured and compared to the rates in control cells. Results show the binding activity of these lipoproteins is upregulated in cholesterol-loaded cells; the bound and internalized lipoproteins are not degraded to any appreciable extent but primarily resecreted as a larger particle. The enhancement of binding activity for high-density lipoproteins is arrested when cycloheximide is added to the medium, suggesting that protein synthesis is involved. Preliminary evidence also indicates that HDL3 (without apoE) after internalisation is converted intracellularly to a larger apoE-containing HDL2-like particles. Thus, MDM appears to possess specific receptors for HDL3 without apoE that may function to facilitate HDL-mediated removal of excess cholesterol from cells.

Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil.

The effect of the Ca2+ entry blocker, verapamil, on the biosynthesis of cholesterol and the metabolism of low-density lipoprotein (LDL) was studied in cultured human monocyte-derived macrophages. Addition of verapamil (50 microM) of monocyte-derived macrophages enhanced 125I-LDL and 125I-labelled acetyl-LDL binding and internalization, and increased [2-14C]acetate incorporation into cholesterol. Since higher levels of LDL and modified lipoproteins may be implicated in atherogenesis, the more efficient processing of these lipoproteins by monocyte-derived macrophages in the presence of Ca2+ blocker warrants further assessment for its potential as an antiatherogenic agent.

Inhibition of vascular smooth muscle cell proliferation by the calcium antagonist clentiazem: role of protein kinase C.

The proliferation of vascular smooth muscle cells has been implicated as a causative factor in atherogenesis. Calcium channel blockers have been shown to retard the progression of atherosclerosis. To elucidate the mechanism by which these drugs mediate such actions, we studied the effects of a new calcium antagonist, clentiazem, on the in vitro proliferation of vascular smooth muscle cells. PDGF-induced prolifertion of these cells is markedly inhibited by clentiazem. The probable involvement of protein kinase C (PKC) in this cellular response is suggested. Clentiazem appear to cause inhibition of PKC translocation that is induced by phorbol esters and PDGF-BB and the phosphorylation of the 80 kDa protein substrate of PKC in vascular smooth muscle cells. Moreover, treatment with clentiazem leads to a marked decrease in the number of specific phorbol ester binding sites. Analysis of the membrane bound isoenzymes of protein kinase C revealed that the inhibition was specific to delta enzymes. Arterial cholesterol ester hydrolysis is not significantly altered by clentiazem. Our results suggest that clentiazem may inhibit cell proliferation by regulating cytosolic PKC and preventing its membrane translocation and activation.

Restriction fragment length polymorphism of the apoprotein A-I-C-III gene cluster in control and stroke-prone white and black subjects: racial differences.

Background and Purpose: The presence of known restriction fragment length polymorphisms in the apoprotein A-I-C-III gene cluster, which encodes their respective apoproteins, was investigated using the restriction enzymes Sac I and Pst I to determine the potential role of genetic variations for stroke risk in an American population. Methods: Ninety-eight subjects (70 white, 28 black subjects), both normal controls with no carotid stenosis and those with carotid stenosis believed at risk for stroke, defined as showing stenosis focally or diffusely at that site, composed the study population. Results: Sac I polymorphic P2 allele frequency was higher in stroke-risk groups, whereas Pst I polymorphic P2 allele frequency was similar in control and stroke-risk groups. Significantly higher levels of serum cholesterol, triglycerides, and low density lipoprotein (p<0.05) and significantly lower levels of high density lipoprotein (p<0.05) were observed in stroke-risk groups with diffuse stenosis. Results of our study with the two racial groups show the following: the frequency of Sac I polymorphism was significantly higher in American black compared with American white subjects (x2=3.92, p<0.05). Among serum lipids, triglycerides were significantly higher in white compared with black subjects (p<0.05). In white subjects, carotid artery stenosis was associated with significantly elevated total cholesterol and low density lipoprotein (p<0.01) but not with Sac I polymorphism. In black subjects the converse was observed, namely, the Sac I polymorphic S2 allele seemed to be associated with carotid bifurcation stenosis but did not reach statistical significance because of the small number of subjects. In addition, Sac I polymorphism did not correlate with any lipid profile. Pst I polymorphism was not associated with any lipid profile or carotid artery stenosis abnormalities. Conclusions: Our results indicate that carotid artery stenosis identifies white subjects with increased plasma total cholesterol and low density lipoprotein, an atherogenic profile, but not with Sac I polymorphism. These findings suggest that carotid bifurcation stenosis in white subjects is associated with an atherogenic lipid profile but not with apoprotein A-I-C-III restriction fragment length polymorphisms. In black subjects, Sac I polymorphism seems to identify those individuals with significant carotid stenosis, a necessary precursor to atherothrombotic brain infarction, but not those with elevated total cholesterol, elevated low density lipoprotein, and/or reduced high density lipoprotein. These results suggest that Sac I polymorphism may identify black subjects at increased risk for atherothrombotic brain infarctions.

Inhibition of PDGF-Mediated Proliferation of Vascular Smooth Muscle Cells by Calcium Antagonists

BACKGROUND AND PURPOSE The mechanism by which calcium antagonists (CAs) inhibit proliferation in vascular smooth muscle cells (VSMCs) is not yet fully understood. We investigated the effects of four CAs (clentiazem, verapamil, diltiazem, and nifedipine) on signal transduction pathways activated by platelet-derived growth factor (PDGF). To determine these effects, the levels of inositol phosphates (IPs), protein kinase C (PKC), and the induction of the transcription factor activator protein-1 (AP-1) were measured. METHODS The mitogenic effect of PDGF on VSMCs was measured by [3H]thymidine incorporated into DNA. IP production was monitored by [3H]myo-inositol incorporation. PKC activation was determined by measurement of myristoylated, alanine-rich C kinase substrate (MARCKS) phosphorylation in digitonin-permeabilized VSMCs. The induction of AP-1 complex was detected by electrophoretic mobility shift assays. RESULTS Each CA significantly inhibited the [3H]thymidine incorporation into DNA in unstimulated cells. Similar significant decreases in [3H]thymidine incorporation by CAs were observed when cells were stimulated by rPDGF-BB. The phosphorylation of MARCKS mediated by rPDGF-BB was significantly reduced by each CA. Clentiazem and verapamil significantly reduced the expression of AP-I induced by rPDGF-BB (P < .01, P < .05). Clentiazem also significantly reduced the expression of AP-1 induced by rPDGF-AB (P < .05). CONCLUSIONS PDGF-mediated proliferation of VSMCs correlates with activation of PKC but not with induction of the AP-1 complexes. In addition, our results suggest that CAs block proliferation of VSMCs by inhibiting DNA synthesis, possibly via PKC.

Low and high density lipoprotein metabolism in atherothrombotic brain infarction.

Background and Purpose: Elevated low density lipoprotein and reduced high density lipoprotein cholesterol may increase the risk of athersothrombotic brain infarction, but the metabolic mechanisms accounting for this relation are poorly understood. Methods: The kinetic parameters of low density and high density lipoprotein were studied in nine subjects with atherothrombotic brain infarction or identifiable (by noninvasive testing) extracranial occlusive disease and in 12 control subjects. Autologous iodine-125-labeled lipoproteins were injected intravenously. Blood samples were drawn 10 minutes after injection and periodically thereafter for 10 days. Kinetic parameters were calculated from the decay curves. Results: The stroke-risk group showed significantly higher triglyceride (p<0.05), total cholesterol (p<0.02), and low density lipoprotein cholesterol (p<0.01). The fractional catabolic rate of low density lipoprotein was significantly lower (p<0.001) and the high density lipoprotein rate higher (p<0.02) in the stroke-risk group than in the control group. Regression analysis (using all subjects) of serum lipoproteins and their respective fractional catabolic rates correlated significantly (for low density lipoprotein, r=0.684, p<0.001; for high density lipoprotein, r=0.595, p<0.002). Mean percent stenosis showed a significant relation with triglyceride level (r=0.678, p<0.01) and low density lipoprotein cholesterol (r=0.535, p<0.02) but not with high density lipoprotein cholesterol. Mean percent stenosis also showed correlation with both fractional catabolic rate of low density lipoprotein (r=0.667, p<0.002) and with serum high density lipoprotein levels (r=0.504, p<0.02). Conclusions: Our study provides insights into the role of altered low and high density lipoprotein metabolism in the pathogenesis of carotid stenosis. The statistically significant association of serum lipoprotein metabolic rates with carotid stenosis, rather than their respective serum concentrations, implies that metabolic parameters may be more important in predicting stroke risk.

Recombinant Tissue Plasminogen Activator and Mutant rt-PA: Binding Kinetics and Cytotoxicity on Brain Endothelial Cells—Relevance to Brain Hemorrhage

The effectiveness of recombinant tissue plasminogen activator (rt-PA) in thrombolytic therapy is theoretically dependent on the rate at which therapeutically administered rt-PA reaches the clot site and the proportion of rt-PA that is enzymatically active. Interaction between rt-PA and its specific plasma inhibitor PAI-1, and between rt-PA and the endothelial cell lining of blood vessels, are two factors that may limit efficacy. While rt-PA can restore blood flow to thrombosed cerebral arteries and revive function of ischemically impaired brain tissue, rt-PA may also provoke brain damage by causing brain hemorrhage. We report on our studies of rt-PA and mutant rt-PA (TNK), assessing the binding kinetics and cytotoxicity on brain endothelial cells (BEC) to determine their action and relevance in brain hemorrhage.

Molecular biological studies in atherothrombotic brain infarction.

Strokes due to atherosclerosis are the most prominent neurological disease affecting adults, and efforts to reduce stroke occurrence, in addition to stroke-risk reduction, will require insights into molecular mechanisms. Our studies showing abnormal metabolism of low and high density lipoproteins (LDL and HDL) in vivo and of RFLP in apoprotein AI, the major protein of HDL, in stroke-prone subjects suggest that greater exploration of fundamental mechanisms of atherothrombotic brain infarction (ABI) should yield preventative strategies, the ultimate treatment for strokes.

Molecular Biology of Atherothrombotic Brain Infarction and Its Role in Vascular Dementia

Dementia due to vascular disease results from ischemic infarctions affecting crucial areas of brain by virtue of their critical nature for memory, such as the hippocampus/dentate gyrus or dorsal medial nucleus of the thalamus; the aggregate mass of infarcts; and the bilateral involvement of cortical damage. However, the most important factor causing ischemic infarction is atherosclerotic vascular disease in both large and small arteries and arterioles. Since the best therapy for both strokes and vascular dementia is prevention, it follows that insights into the fundamental molecular biological mechanisms of atherosclerosis in patients with vascular dementia should provide clues for identifying genetic predisposition and planning rational therapeutic intervention. The hope and expectation is that the offending atherosclerotic changes can be prevented and reversed, thereby averting any ischemic brain injury and the potential for secondary dementia.