Severin P. Schwarzacher

Systemic endothelial dysfunction is related to the extent and severity of coronary artery disease

Flow-mediated vasodilation (FMD) of systemic arteries, a non-invasive parameter of endothelial function, is correlated with cardiovascular risk factors. The relationship between FMD and morphologically and clinically evident coronary artery disease has not been described. This study was performed to test the hypothesis that an impairment of FMD in the brachial artery is related to the presence and/or extent and severity of coronary artery disease (CAD). We examined 74 patients with angina pectoris and 14 control subjects (age 17 36 years). Angiography revealed coronary artery disease (> or = 30% diameter stenosis) in 44 patients (CAD, age 32 67 years) and smooth coronary arteries in 30 patients (non-CAD, age 22-73 years). Vasodilation following reactive hyperemia and after sublingual nitroglycerin (NTG) was assessed in the brachial artery using B-mode high resolution ultrasound. CAD patients showed markedly impaired FMD compared to the non-CAD group (5.7 +/- 4.8 versus 12.6 +/- 6.7%, P < 0.0001) and to controls (5.7 +/- 4.8 versus 15.7 +/- 3.9%, P < 0.00001). NTG induced similar degrees of vasodilation in the CAD and non-CAD groups but less vasodilation in the CAD patients compared to controls (12.2 +/- 6.3 versus 20.4 +/- 6.9%, P < 0.01). On univariate analysis, impaired FMD in CAD patients and non-CAD patients was related to the extent of coronary disease (1-, 2- or 3-vessel disease; r = -0.67, P < 0.0001), to the maximum percent diameter stenosis in one of the major coronary vessels (r = -0.52, P < 0.0001), brachial artery diameter (r = -0.46, P < 0.0001) and plasma cholesterol level (r = -0.34, P < 0.001). On multiple stepwise regression analysis the extent of coronary disease (r = -0.51, P < 0.0001) and the baseline brachial artery diameter (r = -0.37, P < 0.0001) were independently associated with FMD in CAD and non-CAD patients. The present findings suggest that the impairment of FMD in the brachial artery, a marker of systemic endothelial function, is closely related to the angiographic extent of CAD.

HMG-CoA Reductase Inhibitors Regulate Inflammatory Transcription Factors in Human Endothelial and Vascular Smooth Muscle Cells

Objective—Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. Methods and Results—Simvastatin, atorvastatin, and lovastatin (0.1 to 10 &mgr;mol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-&kgr;B) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay (EMSA). The inhibitory effects of statins on NF-&kgr;B or AP-1–dependent transcriptional activity were examined by transient transfection studies. HMG-CoA reductase inhibitors upregulated I&kgr;B-&agr; protein levels in endothelial cells and decreased c-Jun mRNA expression in smooth muscle cells as analyzed by Western and Northern blotting, respectively. Furthermore, statins inhibited DNA binding of hypoxia-inducible factor-1&agr;. Downstream effects of statins included inhibition of plasminogen activator inhibitor-1 and vascular endothelial growth factor-A mRNA levels in endothelial cells. Conclusions—HMG-CoA reductase inhibitors downregulate the activation of transcription factors NF-&kgr;B, AP-1, and hypoxia-inducible factor-1&agr;. These findings support the concept that statins have antiinflammatory and antiproliferative effects that are relevant in the treatment of atherosclerotic diseases.

Regression of Atherosclerosis Role of Nitric Oxide and Apoptosis

BACKGROUND We have recently found that administration of L-arginine to hypercholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) synthase pathway can induce apoptosis of vascular cells in vitro. Accordingly, the current study was designed to determine if dietary supplementation of L-arginine induces apoptosis of intimal lesions and if this effect is mediated through the NO synthase pathway. METHODS AND RESULTS Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at which time the aortas were harvested for histological studies. L-Arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1. 4 apoptotic cells/mm2, P<0.01). In subsequent studies, aortas were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside (10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal lesion. L-Arginine (10(-3) mol/L) had an identical effect on apoptosis, which was associated with an increase in nitrogen oxides released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP. CONCLUSIONS These results indicate that supplemental L-arginine induces apoptosis of macrophages in intimal lesions by its metabolism to NO, which acts through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.

Local Intramural Delivery of l-Arginine Enhances Nitric Oxide Generation and Inhibits Lesion Formation After Balloon Angioplasty

BACKGROUND Long-term oral administration of L-arginine has been shown to enhance production of nitric oxide (NO) and to reduce lesion formation. The goal of this study was to determine whether local intramural administration of L-arginine could enhance NO generation and reduce intimal thickening. METHODS AND RESULTS New Zealand White rabbits (n = 27) received a 1% cholesterol diet. For the short-term study, after 1 week of diet, both iliac arteries were balloon injured. Four weeks later, vasoreactivity was assessed angiographically during infusion of acetylcholine (Ach) before and after delivery of L-arginine or saline into the right or left iliac artery (800 mg/5 mL; 0.2 mL/min, 15 minutes) by use of a local drug-delivery balloon. Vessels were then harvested for measurements of NO. For the long-term study, after balloon injury, drugs were delivered as above into the iliac arteries. Two and 4 weeks after L-arginine delivery, vasoreactivity was determined. Subsequently, the iliac arteries were harvested for histomorphometric analysis and measurements of NO. In the short-term study, local delivery of L-arginine restored endothelium-dependent vasodilatation (Ach 10(-5) mol/L; L-arginine +35 +/- 10%; saline -14 +/- 5%; P < .001) and enhanced local production of nitrogen oxides (L-arginine 152 +/- 28; saline 78 +/- 12 nmol/L per milligram of tissue per hour; P < .04). In the long-term study, local administration of L-arginine enhanced vascular NO production as long as 1 week after the injury (L-arginine 394.4 +/- 141.6; saline 86.3 +/- 34.3 nmol/L per milligram of tissue per hour; P < .01) and reduced intimal thickening 4 weeks later (intima/ media ratio: L-arginine 0.56 +/- 0.1; saline 1.40 +/- 0.2; P < .001), largely due to suppression of macrophage accumulation. CONCLUSIONS A single intramural administration of L-arginine enhances vascular NO generation and inhibits lesion formation. Local augmentation of NO production at the site of balloon angioplasty may be a novel strategy to prevent restenosis.

Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells

OBJECTIVES HMG-CoA reductase inhibitors (statins) can modulate the formation of new blood vessels, but the reports on their contribution to angiogenesis are contradictory. Therefore, we investigated whether the effect of statins is dependent either on the concentration of the drug or on the cell type. METHODS AND RESULTS Under basal conditions human vascular smooth muscle cells (HVSMC) and microvascular endothelial cells (HMEC-1) constitutively generate and release vascular endothelial growth factor (VEGF). In contrast, primary macrovascular endothelial cells (HUVEC) produce minute amounts of VEGF. Different statins (atorvastatin, simvastatin and lovastatin, 1-10 micromol/l) significantly reduced basal and cytokine-, nitric oxide- or lysophosphatidylcholine (LPC)-induced VEGF synthesis in HMEC-1 and HVSMC. Interestingly, at the same concentrations statins upregulated VEGF generation in HUVEC. Furthermore, statins exerted dual, concentration-dependent influence on angiogenic activities of HUVEC as determined by tube formation assay. At low concentrations (0.03-1 micromol/l) the pro-angiogenic activity of statins is prevalent, whereas at higher concentrations statins inhibit angiogenesis, despite increasing VEGF synthesis. CONCLUSION Our data show that statins exert concentration- and cell type-dependent effects on angiogenic activity of endothelial cells and on VEGF synthesis. The data are of relevance for elucidating the differential activity of statins on angiogenesis in cardiovascular diseases and cancer.

Local l-Arginine Delivery After Balloon Angioplasty Reduces Monocyte Binding and Induces Apoptosis

BACKGROUND Local administration of L-arginine after balloon angioplasty has been shown to enhance NO generation and inhibit lesion formation. In this study, we assessed the mechanisms by which local delivery of L-arginine inhibits lesion formation. METHODS AND RESULTS New Zealand White rabbits (n=56) were fed a 1% cholesterol diet. After 1 week, both iliac arteries were balloon-denuded, and a local drug delivery catheter was introduced into both iliac arteries to deliver either L-arginine (800 mg/5 mL with and without 100 microCi L-[2,3-(3)H]-arginine) or saline. Monocyte-endothelial interaction was assessed by functional binding assay; NO activity was measured by chemiluminescence. Intramural administration of radioactively labeled L-arginine led to significantly higher counts in comparison to the contralateral segment for up to 1 week after delivery (676+/-223 versus 453+/-93 cpm/mg; P<0.02); this was associated with significantly higher NO levels in the L-arginine-treated segments (394.4+/-141.6 versus 86.3+/-34.3 nmol/mg; P<0.01). Even after 2 to 3 weeks, monocyte binding was significantly decreased by treatment with L-arginine as compared with saline infusion (P<0.01). After 4 weeks, there was a 9-fold greater number of apoptotic cells in the vessel wall of L-arginine as compared with the saline-treated segments (P<0.05). CONCLUSIONS Intramural delivery of L-arginine immediately after angioplasty causes a sustained increase in tissue L-arginine levels associated with enhancement of local NO synthesis. The local increase in NO synthesis is associated with an attenuation of monocyte binding and increased apoptosis of resident macrophages. This treatment strategy could be valuable for the prevention and management of restenosis.

Morphologic rather than functional or mechanical sonographic parameters of the brachial artery are related to angiographically evident coronary atherosclerosis

OBJECTIVES The purpose of this study was to determine the relationship among coronary atherosclerosis and functional, morphologic, and mechanical parameters assessed noninvasively within the brachial artery (BA). BACKGROUND Flow-mediated vasodilation (FMD) of the BA, intima-media thickness (IMT) of the carotid artery, and distensibility of the aorta have been correlated with the presence of coronary artery disease (CAD). METHODS The BA was examined with high-resolution ultrasound (13 MHz) in 117 male patients, in whom coronary angiography was performed. Coronary artery disease (> or =30% diameter stenosis in > or =1 major branch) was found in 84 patients, and 33 patients had smooth coronary arteries (non-CAD). Wall cross-sectional area (WCSA) was calculated from resting diameter and IMT. RESULTS The BA-WCSA (5.3 +/- 1.5 mm(2) vs. 4.4 +/- 1.4 mm(2), p = 0.002) and IMT (0.37 +/- 0.07 mm vs. 0.31 +/- 0.07 mm, p < 0.001) were significantly greater in patients with CAD compared with non-CAD patients. Flow-mediated vasodilation and distensibility were similar among groups. Using logistic regression analyses adjusting for age, positive family history, hypertension, hypercholesterolemia, smoking, FMD, and distensibility, only WCSA (p < 0.01) and IMT (p < 0.001) correlated independently with the presence of CAD. CONCLUSIONS Morphologic but not functional and mechanical parameters of the BA are associated with the presence of CAD. Among BA sonographic parameters, IMT and WCSA seem to be the most accurate ones for the estimation of coronary atherosclerotic risk.

Determinants of Coronary Remodeling in Transplant Coronary Disease A Simultaneous Intravascular Ultrasound and Doppler Flow Study

BACKGROUND Coronary remodeling plays a significant role in lumen loss in transplant allograft vasculopathy (TxCAD), but the determinants of remodeling are unknown. We assessed the relationship between remodeling and plaque topography, coronary compliance, and blood flow in TxCAD. METHODS AND RESULTS One artery in each of 27 transplant patients was investigated with simultaneous intravascular ultrasound and coronary flow measurements (basal and hyperemic by Doppler flow wire). At 4 to 8 different cross sections (mean 5.1+/-1. 2), plaque topography (concentric or eccentric) was determined, and total vessel area, lumen area, and intimal/medial area (IMA) were measured. Mean remodeling ratio (vessel area/IMA) in eccentric lesions (E, n=28) was significantly larger than that in concentric lesions (C, n=70) (E 5.87+/-0.93 versus C 3.58+/-0.62; P<0.001), despite similar IMA (E 3.89+/-0.68 versus C 3.90+/-0.41; P=NS) and distribution of imaged segments. Remodeling ratio was consistently larger in eccentric lesions in all 3 vessel segments when analyzed separately, and mean remodeling ratio for each artery was larger in vessels with predominantly eccentric lesions. Coronary compliance ([Delta lumen area/diastolic lumen area]/Delta mean arterial pressure x 10(3)) was also significantly greater in eccentric lesions versus concentric lesions (proximal 1.00+/-0.39 versus 0.22+/-0.04; mid 0.71+/-0.17 versus 0.21+/-0.10; distal 0.43+/-0.13 versus 0. 01+/-0.08; all P<0.01). Coronary flow reserve was also significantly higher in coronary arteries with primarily eccentric lesions (E 2. 49+/-0.64 versus C 1.87+/-0.28; P<0.01). CONCLUSIONS Vessel remodeling in transplant vasculopathy is significantly greater in eccentric lesions than in concentric lesions, possibly due to greater coronary compliance and resistive vessel function.

Nifedipine reduces the incidence of myocardial infarction and transient ischemia in patients undergoing coronary bypass grafting.

A randomized study was performed on 104 patients undergoing elective coronary artery bypass grafting to examine whether the infusion of nifedipine (n = 53) reduces the incidence of perioperative myocardial ischemia and necrosis in the early postoperative period. Continuous hemodynamic and three-channel Holter monitoring was performed for 24 hours and serial assessment of serum enzymes and 12-lead electrocardiography were performed for 36 hours postoperatively. Nifedipine (minimum dose, 10 micrograms/kg/hr for 24 hours) was applied from the onset of extracorporal circulation. The control group (n = 51) received nitroglycerin (minimum dose, 1 micrograms/kg/min for 24 hours). Using the combined analyses of electrocardiography and Holter recordings, myocardial ischemia was defined as being either a transient ischemic event (TIE), transient coronary spasm (TCS), or myocardial infarction (MI). The two groups did not differ with respect to preoperative New York Heart Association classification, age, history of myocardial infarction, extracorporal circulation and aortic cross-clamp time, number of distal anastomoses, or systemic and pulmonary hemodynamics. The incidence of perioperative myocardial ischemia was substantially lower in the nifedipine than in the nitroglycerin group [TIE: three of 53 patients (6%) versus nine of 50 patients (18%), p less than 0.001; MI: two of 53 patients (4%) versus six of 50 patients (12%), p less than 0.001; and TCS: none of 53 patients (0%) versus two of 50 patients (4%), p = NS].(ABSTRACT TRUNCATED AT 250 WORDS)

Atorvastatin decreases vascular endothelial growth factor in patients with coronary artery disease.

OBJECTIVES The aim of this study was to test a possible influence of atorvastatin on the production of vascular endothelial growth factor (VEGF) in patients with coronary artery disease (CAD) and in vitro. BACKGROUND Vascular endothelial growth factor is suggested to be involved in the growth of atherosclerotic plaque by inducing its neovascularization. Hepatic hydroxymethyl glutaryl-coenzyme A reductase inhibitors (statins) are known to have atheroprotective effects beyond lipid lowering. METHODS Blood was collected from 14 male hypercholesterolemic patients with angiographically confirmed CAD at baseline and after two months of atorvastatin therapy (20 mg/d) and from eight male control patients. In an ex vivo assay, human coronary artery smooth muscle cells (HCASMC) were incubated with the patient plasma collected before and after atorvastatin therapy. To test the direct effect of atorvastatin on VEGF synthesis in vitro, HCASMC were treated with atorvastatin (1, 3 and 10 microM). The VEGF concentration was measured by enzyme-linked immunosorbent assay. RESULTS Atorvastatin therapy reduced VEGF plasma levels in CAD patients (from 31.1 +/- 6.1 to 19.0 +/- 3.6 pg/ml; p < 0.05). The VEGF plasma concentration tended to be higher in CAD patients before treatment compared to control patients (31.1 +/- 6.1 vs. 23.4 +/- 3.6 pg/ml; p = NS). Plasma collected before therapy induced significantly more VEGF in HCASMC compared to the plasma collected after treatment and compared to control cells. In vitro, atorvastatin decreased both the basal and the interleukin-1beta-induced VEGF release in HCASMC. CONCLUSIONS These data suggest that atorvastatin may lower the plasma level of VEGF in CAD patients, which could represent a novel beneficial effect of this and perhaps other statins.