Faculty Bibliography

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.

The methods commonly used for noninvasive ultrasound assessment of endothelium-dependent flow-mediated dilation (FMD) require an electrocardiogram (ECG) signal to synchronize the measurements with the cardiac cycle. In this article, we present a method for assessing FMD that does not require ECG gating. The approach is based on temporal filtering of the diameter-time curve, which is obtained by means of a B-mode image processing system. The method was tested on 22 healthy volunteers without cardiovascular risk factors. The measurements obtained with the proposed approach were compared with those obtained with ECG gating and with both systolic and end-diastolic measurements. Results showed good agreement between the methods and a higher precision of the new method due to the fact that it is based on a larger number of measurements. Further advantages were also found both in terms of reliability of the measure and simplification of the instrumentation. (E-mail: gemi@ifc.cnr.it).

We evaluated the possible association between early vascular abnormalities and the metabolic syndrome (MS) in 77 normotensive subjects (mean age: 50 years) at risk of developing diabetes for family history of diabetes, obesity, or impaired fasting glucose. Fifty healthy subjects were recruited as controls. MS was defined according to the ATP III criteria. Brachial artery endothelium-dependent and -independent vasodilation were assessed as flow-mediated dilation (FMD) and response to glyceryl trinitrate (GTN, 25 microg sublingual), respectively, by automatic computerized edge detection system. Carotid-femoral pulse wave velocity (PWV) and radial augmentation index (AIx) were assessed by applanation tonometry. PWV was significantly (P<0.01) higher in subjects with MS (n=29, 9.0+/-1.9 m/s) as compared with those without MS (n=48, 7.7+/-1.2 m/s) and controls (7.2+/-1.5 m/s). FMD was significantly (P<0.05) reduced in both subjects with (5.8+/-2.7%) and without MS (6.1+/-3.7%) as compared with controls (6.9+/-2.5%). No significant differences were found for response to GTN and AIx. PWV and FMD were significantly (P<0.05) affected by increasing number of MS components. Among the components of the MS, only blood pressure significantly affected PWV, whereas blood pressure and fasting glucose influenced FMD. Logistic regression analysis showed that MS was associated with increased risk of altered PVW (odd ratio: 7.95, confidence limits: 1.06 to 69.11), whereas only blood pressure component was significantly related with increased risk of impaired FMD (odd ratio: 3.60, confidence limits: 1.01 to 12.78). In conclusion, in normotensive subjects at risk of developing diabetes mellitus, the presence of MS is associated with a selective alteration of central PWV.