Faculty Bibliography

Atherosclerosis and its clinical manifestations (i.e. myocardial infarction, stroke) are major causes of mortality and morbidity in Western countries. Endothelial dysfunction is considered the first step in the cascade leading up to coronary events. Increasing evidence suggests that direct inhibition of thromboxane A2/prostaglandin (TP)-receptors may not only have anti-platelet effects but also impact endothelial dysfunction as well as inflammatory component of atherosclerosis. While TP-receptor involvement in platelet function has received the greatest attention, more recent findings support the critical role of TP-receptor in other pathophysiological aspects of atherothrombosis. Prostanoids (i.e. TxA2, F2-isoprostanes, prostaglandins endoperoxides PGG2/PGH2) are known to promote the initiation and progression of atherosclerosis, not only via platelet activation, but through leukocyte-endothelial interactions and vasoconstriction. Dysfunctional endothelium, characterised by increased COX-activity, releases prostanoids that promote endothelial exposure to adhesion molecules and induce smooth muscle cell contraction. Plaque macrophages synthesise PGH2/PGG2 via COX-2; these potent prostanoids can trigger platelet activation and aggregation despite COX-1 inhibition by aspirin. TP-receptor inhibition has been reported to exert anti-atherosclerotic effects in pre-clinical model of disease. Reduction of plaque burden was associated with plaque stabilisation documented by the reduction in the content of macrophages, apoptotic cells, MMPs and endothelin-1, and the increase in smooth muscle cells content. TP-receptor blockade might have an anti-atherosclerotic and plaque stabilisation effect. The possibility of combining anti-platelet activity with an anti-atherosclerotic effect via selective TP-receptor inhibitors could have important implications especially in clinical conditions associated with increased production of prostanoids, such as diabetes.

OBJECTIVE:Increased arterial stiffness and carotid intima-media thickness (IMT) are considered independent predictors of cardiovascular events. The aim of this study was to compare a system recently developed in our laboratory for automatic assessment of these parameters from ultrasound image sequences to a reference radio frequency (RF) echo-tracking system. METHODS:Common carotid artery scans of 21 patients with cardiovascular risk factors and 12 healthy volunteers were analyzed by both devices for the assessment of diameter (D), IMT, and distension (DeltaD). In the healthy volunteers, analyses were repeated twice to evaluate intraobserver variability. Agreement was evaluated by Bland-Altman analysis, whereas reproducibility was expressed as a coefficient of variation (CV). RESULTS:Regarding the agreement between the two systems, bias values +/- SD were 0.060 +/- 0.110 mm for D, -0.006 +/- 0.039 mm for IMT, and -0.016 +/- 0.039 mm for DeltaD. Intraobserver CVs were 2% +/- 2% for D, 5% +/- 5% for IMT, and 6% +/- 6% for DeltaD with the RF echo-tracking system and 2% +/- 1% for D, 6% +/- 6% for IMT, and 8% +/- 6% for DeltaD with our automated system. CONCLUSIONS:Although B-mode-based devices are less precise than RF-based ones, our automated system has good agreement with the reference method and comparable reproducibility, at least when high-quality images are analyzed. Hence, this study suggests that the presented system based on image processing from standard ultrasound scans is a suitable device for measuring IMT and local arterial stiffness parameters in clinical studies.

OBJECTIVE:The aim of this study was to assess whether patients with primary hyperparathyroidism (PHPT) show reduced endothelial function and to determine the mechanisms involved. The impact of parathyroidectomy (PTx) on endothelial function was also assessed. BACKGROUND:Endothelial dysfunction is reported in patients with PHPT, but the mechanisms involved are unknown. METHODS:We evaluated forearm blood flow changes (strain gauge plethysmography) induced by intraarterial acetylcholine or sodium nitroprusside in 17 PHPT women and 17 age-matched controls. Nitric oxide (NO) availability and oxidative stress were studied by repeating acetylcholine during intraarterial infusion of L-N(G)-monomethyl arginine (L-NMMA, a NO synthase inhibitor) and ascorbic acid (an oxidative stress scavenger). The role of cytochrome P450 epoxygenase (CYP 2C9)-derived endothelium-derived hyperpolarizing factor (EDHF) was assessed by repeating acetylcholine under intraarterial sulfaphenazole. In six PHPT patients, the study was repeated 12 months after successful PTx. RESULTS:Responses to sodium nitroprusside and acetylcholine were similar in PHPT patients and controls. L-NMMA inhibited the response to acetylcholine in controls (P < 0.001), whereas it had no effect in PHPT patients. In both groups, ascorbic acid failed to affect acetylcholine. Sulfaphenazole administration, although not affecting vasodilation to acetylcholine in controls, blunted the response to acetylcholine in PHPT patients (P < 0.005). After PTx, the inhibitory effect of L-NMMA on acetylcholine was restored (P < 0.001), and the inhibitory effect of sulfaphenazole on acetylcholine was abrogated. CONCLUSIONS:PHPT patients show compromised NO availability, whereas oxidative stress generation is not involved. A compensatory CYP 2C9-derived EDHF pathway is activated to sustain endothelium-dependent vasodilation. This PHPT-related endothelial dysfunction is reversed after PTx.

High levels of high-density lipoprotein (HDL) have protective effects against atherosclerosis and cardiovascular diseases. The postulated mechanism of action for these benefits is an enhanced reverse cholesterol transport. Apolipoprotein A-I (ApoA-I) is the major protein of HDL. The clinical benefits of raising ApoA-I/HDL have been clearly established by clinical and epidemiological studies. Despite these observations, there are not very effective pharmacological means for raising HDL. ApoA-I gene delivery by viral vectors seems a promising strategy to raise ApoA-I/HDL levels. Sustained gene expression in animals and humans has been attained using adeno-associated viral (AAV) vectors. The aim of the present study was to determine the efficiency, safety, and biological activity of human ApoA-I intramuscularly delivered using an AAV vector in mice. AAV serotype 8 vectors encoding for human ApoA-I transgene were administered intraportally and intramuscularly in ApoA-I- deficient animals. ApoA-I levels were measured every 2 weeks post administration. The effectiveness of the generated HDL was tested in vitro in cholesterol-loaded macrophages. The administration of the vectors resulted in a significant and sustained increase in ApoA-I and HDL plasma levels for up to 16 weeks at similar extent by both routes of administration. Activity of the generated HDL in removal of cholesterol from cholesterol-loaded macrophages was similar in both groups. Our data suggest that intramuscular AAV8-mediated gene transfer of human ApoA-I results in a significant and maintained increase in ApoA-I and functional HDL.

We investigated the effect of atorvastatin on cyclooxygenase (COX) contribution to endothelial dysfunction in spontaneously hypertensive rat (SHR) mesenteric resistance arteries. Atorvastatin (10 mg/kg per day, oral gavage) or its vehicle was administered for 2 weeks to male SHR or Wistar-Kyoto rats. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In Wistar-Kyoto rats, relaxation to acetylcholine was inhibited by N(G)-nitro-L-arginine methyl ester and unaffected by SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), or ascorbic acid. In SHRs, the response to acetylcholine was attenuated, less sensitive to N(G)-nitro-L-arginine methyl ester, unaffected by SC-560, and enhanced by DuP-697 or SQ-29548 (thromboxane-prostanoid receptor antagonist) to a similar extent. Endothelium-dependent relaxation was normalized by ascorbic acid or apocynin (NADPH oxidase inhibitor), which also restored the inhibition by N(G)-nitro-L-arginine methyl ester. In atorvastatin-treated SHRs, relaxation to acetylcholine was normalized, fully sensitive to N(G)-nitro-L-arginine methyl ester, and not affected by SC-560, DuP-697, SQ 29548, or antioxidants. Dihydroethidium assay showed an increased intravascular superoxide generation in SHRs, which was abrogated by atorvastatin. RT-PCR revealed a COX-2 induction in SHR arteries, which was downregulated by atorvastatin. The release of prostacyclin and 8-isoprostane was higher from SHR than Wistar-Kyoto mesenteric vessels. COX-2 inhibition and apocynin decreased 8-isoprostane without affecting prostacyclin levels. Atorvastatin increased phosphorylated extracellular signal-regulated kinase 1/2, pAkt, peNOS(1177), and inducible NO synthase levels in SHR mesenteric vessels and decreased 8-isoprostane release. In conclusion, COX-2-derived 8-isoprostane contributes to endothelial dysfunction in SHR mesenteric arteries. Atorvastatin restores NO availability by increasing phosphorylated extracellular signal-regulated kinase 1/2, pAkt, peNOS(1177), and inducible NO synthase levels and by abrogating vascular NADPH oxidase-driven superoxide production, which also results in a downregulation of COX-2-dependent 8-isoprostane generation.

BACKGROUND:A nitric oxide-independent response, possibly mediated by hyperpolarization, regulates vascular tone, acting as a compensatory mechanism in the presence of impaired nitric oxide availability. Cytochrome P450 2C9 (CYP 2C9) is a source of endothelium-derived hyperpolarizing factors and modulates tissue-type plasminogen activator (tPA) release from endothelial cells; however, no effect of hyperpolarization on fibrinolysis has been documented in humans. We aimed to assess the effect of sulfaphenazole, a specific CYP 2C9 inhibitor, on tPA release in normotensive subjects and patients with essential hypertension. METHODS AND RESULTS/RESULTS:tPA release was measured in the forearm microcirculation of 56 normotensivesubjects and 57 patients with essential hypertension after bradykinin (0.015 microg x 100 mL(-1) x min(-1)) and acetylcholine (1.5 microg x 100 mL(-1) x min(-1)) infusions, with or without sulfaphenazole (0.03 microg x 100 mL(-1) x min(-1)). Bradykinin and acetylcholine infusions were repeated with N(G)-monomethyl-l-arginine (L-NMMA; 100 microg x 100 mL(-1) x min(-1)) and/or sulfaphenazole. tPA release by bradykinin and acetylcholine was higher in normotensive subjects than in patients with essential hypertension (P<0.01). Sulfaphenazole (P<0.01) blunted bradykinin-induced but not acetylcholine-induced tPA release in both groups. In normotensive subjects, L-NMMA infusion reduced tPA release (P<0.01). When L-NMMA was coinfused with sulfaphenazole, tPA release induced by bradykinin, but not by acetylcholine, was further reduced (P<0.01). In patients with essential hypertension, tPA release by both agonists was unaffected by L-NMMA, but only bradykinin-induced tPA release was blunted by sulfaphenazole, alone or with L-NMMA (P<001). CONCLUSIONS:Sulfaphenazole inhibits bradykinin-induced tPA release, which suggests a modulatory role of CYP 2C9-derived endothelium-derived hyperpolarizing factors in tPA release in humans. In patients with essential hypertension, tPA release depends exclusively on endothelium-derived hyperpolarizing factor, which is an ineffective compensatory mechanism in the presence of impaired nitric oxide availability.

OBJECTIVE:The purpose of this report is to describe an automatic real-time system for evaluation of the carotid intima-media thickness (CIMT) characterized by 3 main features: minimal interobserver and intraobserver variability, real-time capabilities, and great robustness against noise. METHODS:One hundred fifty carotid B-mode ultrasound images were used to validate the system. Two skilled operators were involved in the analysis. Agreement with the gold standard, defined as the mean of 2 manual measurements of a skilled operator, and the interobserver and intraobserver variability were quantitatively evaluated by regression analysis and Bland-Altman statistics. RESULTS:The automatic measure of the CIMT showed a mean bias +/- SD of 0.001 +/- 0.035 mm toward the manual measurement. The intraobserver variability, evaluated with Bland-Altman plots, showed a bias that was not significantly different from 0, whereas the SD of the differences was greater in the manual analysis (0.038 mm) than in the automatic analysis (0.006 mm). For interobserver variability, the automatic measurement had a bias that was not significantly different from 0, with a satisfactory SD of the differences (0.01 mm), whereas in the manual measurement, a little bias was present (0.012 mm), and the SD of the differences was noticeably greater (0.044 mm). CONCLUSIONS:The CIMT has been accepted as a noninvasive marker of early vascular alteration. At present, the manual approach is largely used to estimate CIMT values. However, that method is highly operator dependent and time-consuming. For these reasons, we developed a new system for the CIMT measurement that conjugates precision with real-time analysis, thus providing considerable advantages in clinical practice.

The relationship between adrenergic stimuli and NO in modulating tissue-type plasminogen activator (t-PA) release from endothelial cells was investigated in normotensive subjects and essential hypertensive patients. Sympathetic activation, a well-known stimulus for endogenous fibrinolysis, is also involved in the determination of cardiovascular risk in essential hypertension. However, the existence of cross-talk between adrenergic stimuli and NO availability in modulating t-PA release is not well established yet. We assessed the release of t-PA in the forearm microcirculation of 58 normotensive subjects (mean age: 47+/-9 years) and 44 essential hypertensive patients (mean age: 48+/-11 years) under specific intra-arterial adrenergic stimuli. Intrabrachial infusion of epinephrine (0.1 to 0.3 microg/100 mL per minute) induced greater t-PA release in normotensive subjects as compared with essential hypertensive patients (P<0.05). However, inhibition of NO synthase with N(G)-monomethyl-L-arginine (100 microg/100 mL per minute) infusion blunted epinephrine-induced t-PA release in normotensive subjects (P<0.05) but not in essential hypertensive patients. In normotensive subjects, t-PA release by epinephrine was not affected by phentolamine (8 microg/100 mL per minute) coinfusion and was abolished in the presence of propanolol (10 microg/100 mL per minute). Intrabrachial isoproterenol (0.03 microg/100 mL per minute) induced a significant increase in t-PA release (P<0.01), an effect blunted by N(G)-monomethyl-L-arginine (P<0.05). In essential hypertensive patients, the response to isoproterenol was impaired as compared with normotensive subjects and was unaffected by N(G)-monomethyl-L-arginine coinfusion. In conclusion, the results of the present study demonstrate that adrenergic-induced t-PA release is mediated by beta-adrenoreceptors via a mechanism involving the NO pathway. Our results show an impaired adrenergic-stimulated t-PA release among essential hypertensive patients, probably mediated via a reduced NO availability. This impaired fibrinolytic activity might contribute to the increased cardiovascular risk associated with hypertension.

OBJECTIVE:Metabolic syndrome is associated with a high risk of cardiovascular disease in hypertension. We evaluated whether metabolic syndrome is associated with early vascular alterations, such as increased peripheral wave reflections and endothelial dysfunction, in untreated essential hypertensive patients. METHODS:Augmentation index and pulse wave velocity were determined with applanation tonometry in 391 untreated hypertensive patients and 166 controls. Endothelium-dependent response was assessed as flow-mediated dilation of the brachial artery. Metabolic syndrome was defined according to the latest National Cholesterol Education Program Adult Treatment panel III. RESULTS:Hypertensive patients showed significantly (P < 0.001) increased augmentation index and central pulse wave velocity, as well as reduced flow-mediated dilation, compared with controls. No further impairment in augmentation index or flow-mediated dilation was observed between patients with or without the metabolic syndrome components and with increasing number of metabolic syndrome. Age and systolic blood pressure, but no other single factor of the metabolic syndrome, resulted as significant predictors of augmentation index and flow-mediated dilation. Female gender was associated with increased augmentation index (P < 0.05) in the presence of the metabolic syndrome. Central pulse wave velocity was selectively impaired by metabolic syndrome in both genders. Finally, reactive hyperemia was reduced in patients with the metabolic syndrome and decreased significantly with the increasing number of metabolic syndrome. CONCLUSIONS:In untreated hypertensive patients, the presence of metabolic syndrome, which selectively impairs central pulse wave velocity, does not account for a further deterioration of peripheral wave reflection and conduit artery endothelial function. Our results suggest that blood pressure, age and gender are important determinants of peripheral vascular structural and functional parameters in these subjects, irrespective of the metabolic syndrome.