Inhibition of platelet-derived growth factor BB-induced expression of glyceraldehyde-3-phosphate dehydrogenase by sodium butyrate in rat vascular smooth muscle cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key regulatory enzyme of glycolysis, which exists in nuclei and functions as a DNA-binding protein as well as a nuclear protein, appears to be modulated by cellular activities. Exposure of quiescent rat smooth muscle cells (SMCs) to platelet-derived growth factor BB (PDGF-BB), which stimulates SMCs proliferation, caused a time-dependent increase in mRNA for GAPDH and its catalytic activity. Treatment of quiescent SMCs with sodium butyrate (SB), which is shown to inhibit PDGF-BB-induced SMC proliferation, caused a time- and concentration-dependent decrease in PDGF-BB-induced GAPDH mRNA expression and its catalytic activity. Nuclear run-on studies revealed that the PDGF-BB-induced rate of GAPDH gene transcription was reduced by about 50% in the presence of 5 mmol/L SB. The protein synthesis inhibitor, cycloheximide, failed to abolish the SB-inhibited PDGF-BB-induced rate of transcription of GAPDH, suggesting that SB is not dependent on ongoing protein synthesis to exert its effects on PDGF-BB-induced GAPDH transcription. Furthermore, measurement of GAPDH mRNA stability at various times after the inhibition of transcription with actinomycin D indicated that 5 mmol/L SB has no significant effect on the half-life of PDGF-BB-induced mRNA. The reduction in PDGF-BB-induced GAPDH expression by SB is probably caused by a cycloheximide-insensitive transcriptional mechanism. Thus, the inhibition of PDGF-BB-induced expression of GAPDH by SB suggests a link between SMC proliferation, energy consumption, and GAPDH gene upregulation.

Sodium butyrate inhibits platelet-derived growth factor-induced proliferation of vascular smooth muscle cells.

Sodium butyrate (SB), a naturally occurring short-chain fatty acid, was investigated for its therapeutic value as an antiproliferative agent for vascular smooth muscle cells (SMCs). At 5-mmol/L concentration, SB had no significant effect on rat SMC proliferation. However, at the same concentration, SB inhibited platelet-derived growth factor (PDGF)-AA-, -AB-, and -BB-induced proliferation of SMCs. Exposure of SMCs to PDGF-BB resulted in activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of beta-PDGF-receptor (beta-PDGFR). The activated beta-PDGFR physically associated and phosphorylated signaling molecules such as ras-GTPase activating protein (GAP) and phospholipase C gamma (PLC gamma). SB, in the absence of PDGF-BB, caused neither beta-PDGFR tyrosine phosphorylation nor phosphorylation and association of GAP and PLC gamma with beta-PDGFR. PDGF-BB-enhanced activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of tyrosine residues of beta-PDGFR were unaffected by SB irrespective of whether SMCs were preincubated with SB before exposure to PDGF-BB plus SB or incubated concomitantly with PDGF-BB plus SB. Likewise, phosphorylation and association of GAP and PLC gamma with PDGF-BB-activated beta-PDGFR were unaffected. In addition, SB did not block PDGF-BB-stimulated, PLC gamma-mediated production of inositol triphosphate. Similarly, PDGF-BB-induced beta-PDGFR degradation was unaffected when SMCs were exposed to PDGF-BB plus SB, and SB by itself had no influence on beta-PDGFR degradation. Unlike beta-PDGFR kinase activity, mitogen-activated protein kinase (MAP-kinase) activity was stimulated by SB by about 2.7-fold. Exposure of SMCs to PDGF-BB caused an approximately 11.4-fold increase in MAP-kinase activity and this increase in activity was not significantly affected when cells were coincubated with PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB abolished most of the PDGF-BB-induced MAP-kinase activity (4.6-fold). Transcription of growth response genes such as c-fos, c-jun, and c-myc were induced by PDGF-BB, and their induction was suppressed, particularly c-myc, by incubating SMCs with PDGF-BB plus SB. Similarly, preincubation of cells with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB diminished PDGF-BB-induced transcription of c-fos, c-jun, and c-myc. However, SB by itself had no significant effect on c-fos, c-jun, and c-myc transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Prediction of carotid stenosis progression by lipid and hematologic measurements.

We followed 19 men and 19 women with asymptomatic carotid stenosis up to 30 months to determine whether hematologic or lipid abnormalities could identify those individuals developing progressing carotid atherosclerosis (defined as an increase in mean percent stenosis greater than or equal to 19% or an increase in a single region of greater than or equal to 23%) on B-mode carotid ultrasonography performed at 2- to 6-month intervals. Our patients demonstrated increased beta-thromboglobulin, platelet factor 4, and fibrinogen compared with age-matched controls. Eight patients developed progression of carotid stenosis, and this group had higher baseline low-density lipoprotein (LDL) and fibrinogen than the 30 nonprogressing patients. Multiple regression analyses of age, sex, smoking, coronary artery disease, peripheral vascular disease, diabetes, hypertension, and baseline high-density lipoprotein (HDL), HDL2, HDL3, LDL, beta-thromboglobulin, platelet factor 4, and fibrinogen identified coronary artery disease and elevated LDL and fibrinogen as the only independent variables significantly associated with the progressing group. We conclude that, in patients with carotid atherosclerosis, a combination of coronary artery disease and elevated LDL and fibrinogen will predict with 88% accuracy whether the patient will have progressing carotid stenosis.

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.

Scavenger activity in monocyte-derived macrophages from atherothrombotic strokes.

Foam cells are lipid-laden macrophages derived primarily from circulating mononuclear cells and are a characteristic feature of atheromatous lesions. The exact role of these foam cells in the pathogenesis of atherosclerotic lesions remains uncertain, but one potential function is to take-up and process excess interstitial arterial lipoproteins, suggested by their extraordinary ability to engulf enormous quantities of modified low density lipoproteins by the so-called "scavenging pathway." To test this possibility, monocytes from 15 atherothrombotic brain infarct patients and age and sex matched controls were isolated and cultured for 7-8 days in 20% normal serum. The monocyte-derived macrophages were investigated for their ability to bind, internalize and degrade both native and modified (acetylated) LDL labelled with 125Iodine. While native LDL was metabolized similarly, stroke macrophages displayed significantly reduced ability to scavenge modified LDL. These findings suggest that insufficient processing of interstitial arterial cholesterol by monocyte-derived macrophages may contribute to the aggravation of atheroma formation. This inadequacy is likely further compromised by reduced levels of serum high density lipoprotein since the absence of a cholesterol-acceptor will promote the slow but continued accumulation of lipids and the formation of foam 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.

Cholesteryl ester hydrolase activity in mononuclear cells from patients with type II hypercholesterolemia.

Cholesteryl ester hydrolase (CEH) activity was measured in freshly isolated mononuclear cells from patients with primary Type II hypercholesterolemia, heterozygous familial hypercholesterolemia (FH) and familial combined hyperlipidemia (CFH). CEH activity was significantly lower in mononuclear cells from Type II patients than in cells from matched normolipidemic individuals. Moreover, the reduced CEH activity in cells from the hypercholesterolemic patients was accompanied by significant accumulation of cholesteryl ester. This pattern of reduced CEH activity and cholesteryl ester accumulation was identical for cells from both the FH and CFH patients. Since low density lipoprotein (LDL) cholesterol concentrations were higher in the Type II patients, we incubated mononuclear cells from normolipidemic individuals with high concentrations of LDL-cholesterol (greater than 150 mg/dl). Under these conditions CEH activity was significantly decreased, cholesteryl ester content increased, and cholesterol linoleate, in particular, accumulated. These data suggest that the intracellular accumulation of cholesteryl esters is determined in part by the extracellular concentrations of LDL-cholesterol and by the activity of CEH within the cells.

The effect of oral contraceptives on mononuclear cell cholesteryl ester hydrolase activity.

The influence of sex steroids on mononuclear cell cholesteryl ester hydrolase (CEH) activity in premenopausal women and women on combined estrogen-progestin oral contraceptives has been studied. In addition, plasma and mononuclear cell cholesterol and esters were measured along with plasma estrogen and progesterone levels. Mononuclear cell CEH activity in control women is highest on Day 20 of their menstrual cycle. The control women had significantly higher CEH activities than women on oral contraceptives. Plasma esters were higher in the oral contraceptive group. However, in mononuclear cells, free cholesterol but not cholesteryl esters were higher in women on oral contraceptives.

PIP: Premenopausal women, 1 control group (n=9) taking no medication or using no oral contraceptives (OCs) and 1 treated group (n=10) receiving OCs for contraception, were studied to determine any effects OCs have on mononuclear cell cholesteryl ester hydrolase (CEH) activity. 9 of the 10 medicated women were taking Ortho Novum 1/50 and the other person was receiving Norlestrin 1/50. Normally menstruating women (controls) showed a significant rise in CEH levels on Day 20 of the menstrual cycle (P .05). The enzyme activity in women on OCs was significantly lower than control women in 3 of 4 testing periods. In addition, plasma and mononuclear cell cholesterol and esters were measured along with plasma estrogen and progesterone levels. Although free cholesterol levels in normal cycling (control) women and in the OC group did not vary significantly during the menstrual cycle between the 2 groups, the women on OCs had significantly higher ester levels than the control women in 3 of the 4 test periods P .05-.005). When paired ratios of plasma cholesterol to esterified cholesterol were compared between control and OC groups, the ratio of free/esterified was significantly higher in the control group in 3 of 4 tests. In the mononuclear cells, on the other hand, the cholesterol/cholesteryl ester ratio was significantly lower in the control group during the 4 test periods. No association between levels of endogenous sex hormones (estradiol, progesterone) and CEH activity were found. CEH levels may be related to incidence of atherosclerosis, and women taking OCs may have increased chances of developing this disease.

The effect of oral contraceptives on mononuclear cell cholesterol ester hydrolase activity in premenopausal women taking oral contraceptives: relevance to atherosclerosis.

PIP: Among the many, multifactorial etiologies of atherosclerosis is excessive filtration and deposition of lipids, particularly cholesterol esters, in arterial walls; furthermore, the monoclonal theory purports that the artheroma is an uncontrolled proliferation of cells similar to a benign tumor. These 2 aspects of atherosclerosis pathogenesis were studied in 5 healthy women on birth control pills by investigating the level of mononuclear cell cholesterol ester hydrolase (CEH). Control subjects underwent identical investigation. Mononuclear CEH activity was signficantly lower in women on oral contraceptives than in controls in 4 of the 5 test intervals and showed no signficant fluctuation in activity. Average value of CEH in 5 women on birth control pills was 927+ or -81 pmol/mg of protein/hour. In 5 men followed at 5-day intervals, no significant fluctuation of CEH activity was found. Mean average was 2373+ or -92. Total cholesterol and its ester in both plasma and mononuclear cells showed no signficant differences at the 5-day intervals between men and women. However, plasma cholesterol/cholesterol ester ratios were significantly higher in women than men at each of the 5-day intervals from Days 5-25. An additional link between female hormones and atherosclerosis is suggested by the finding that women on oral contracepitves, known to be predisposed to premature atherosclerosis, show reduced and nonfluctuating levels of mononuclear cell CEH.^ieng

Energy metabolism during brain ischemia. Stability during reversible and irreversible damage.

The permissible duration of brain ischemia without sustaining damage is short. Less clear are the mechanisms accounting for the vulnerability of brain to ischemic insults. Neurochemical factors implicated include impairment of energy synthesis by mitochondria and of energy-dependent processes such as synaptic transmission, ATPase activity, membrane conductance and altered protein and lipid synthesis. To clarify the vulnerability of energy metabolism, we investigated energy availability and synthesis in our model of global cerebral ischemia. Our studies evaluated in vitro mitochondrial ATP synthesis and the in vivo quantitation of the cortical adenylate pool. Results of our investigations support a growing body of evidence showing the energy state to be relatively stable to ischemia. We conclude that an energy-dependent process of brain is primarily vulnerable to ischemia.