Silvia Paiardi

Effect of Treatment With Candesartan or Enalapril on Subcutaneous Small Artery Structure in Hypertensive Patients With Noninsulin-Dependent Diabetes Mellitus

Structural alterations of subcutaneous small resistance arteries are associated with a worse clinical prognosis in hypertension and noninsulin-dependent diabetes mellitus (NIDDM). However, no data are presently available about the effects of antihypertensive therapy on vascular structure in hypertensive patients with NIDDM. Therefore, we have investigated the effect of an angiotensin-converting enzyme inhibitor, enalapril, and a highly selective angiotensin receptor blocker, candesartan cilexetil, on indices of subcutaneous small resistance artery structure in 15 patients with mild hypertension and NIDDM. Eight patients were treated with candesartan (8 to 16 mg per day) and 7 with enalapril (10 to 20 mg per day) for 1 year. Each patient underwent a biopsy of the subcutaneous fat from the gluteal region at baseline and after 1 year of treatment. Small arteries were dissected and mounted on a micromyograph and the media-to-internal lumen ratio was evaluated; moreover, endothelium-dependent vasodilation to acetylcholine was assessed. A similar blood pressure-lowering effect and a similar reduction of the media-to-lumen ratio of small arteries was observed with the 2 drugs. Vascular collagen content was reduced and metalloproteinase-9 was increased by candesartan, but not by enalapril. Changes of circulating indices of collagen turnover and circulating matrix metalloproteinase paralleled those of vascular collagen. The 2 drugs equally improved endothelial function. In conclusion, antihypertensive treatment with drugs that inhibit the renin-angiotensin-aldosterone system activity is able to correct, at least in part, alterations in small resistance artery structure in hypertensive patients with NIDDM. Candesartan may be more effective than enalapril in reducing collagen content in the vasculature.

Altered structure of small cerebral arteries in patients with essential hypertension.

Objective Structural alterations in the microcirculation may be considered an important mechanism of organ damage. An increased media-to-lumen ratio of subcutaneous small resistance arteries has been demonstrated to predict the development of cardiocerebrovascular events in hypertensive patients. Alterations in the structure of small cerebral arteries have been demonstrated in animal models of experimental or genetic hypertension. However, no evaluation with reliable techniques has ever been performed in humans. Design and methods Twenty-eight participants were included in the present study: they were 13 hypertensive patients and 15 normotensive individuals. All participants underwent a neurosurgical intervention for benign or malign tumors. A small portion of morphologically normal cerebral tissue was excised from surgical samples and examined. Cerebral small resistance arteries (relaxed diameter around 200 μm) were dissected and mounted on an isometric and isobaric myograph, and the tunica media to internal lumen ratio was measured. In addition, cerebral cortical microvessel density (MVD) was also evaluated. The tissue was sectioned and stained for CD31, and MVD was measured with an automated image analyzer (percentage of area stained). Blood pressure values were evaluated, before surgical intervention, by standard sphygmomanometry. Results M/L was significantly greater and MVD significantly lower in hypertensive patients than that in normotensive individuals. No difference between groups in collagen content or mechanical properties of cerebral small arteries was observed. Conclusion Our results indicate that structural alterations of small cerebral vessels are present in hypertensive patients compared with normotensive individuals, similar to those previously observed in subcutaneous small arteries.

Morning rise of blood pressure and subcutaneous small resistance artery structure.

Objectives It has been previously demonstrated that the morning rise (MoR) of blood pressure (BP) may predict major cardiovascular events in hypertensive patients. Structural alterations of small resistance arteries, as evaluated by the tunica media to internal lumen ratio (M/L) of subcutaneous small resistance arteries, may also predict cardiovascular events. Because an increased M/L may amplify the effect of hypertensive stimuli, the present study aimed to evaluate the possible relationships between MoR and M/L in a population of hypertensive patients. Methods Sixty-four patients with essential hypertension were included in the present study. All patients were submitted to a biopsy of subcutaneous fat. Small resistance arteries were dissected and mounted on an isometric myograph, and the M/L was measured. In addition, MoR was calculated from ambulatory blood pressure monitoring (ABPM) according to four previously published different methods (MoR1 to MoR4). Results A statistically significant correlation was observed between M/L and MoR1 (r = 0.52, P < 0.001), MoR2 (r = 0.32, P < 0.01), MoR3 (r = 0.25, P < 0.05) and MoR4 (r = 0.27, P < 0.05), as well as between internal diameter of subcutaneous small arteries and MoR1 (r = −0.45, P < 0.001) and MoR2 (r = −0.28, P < 0.05). Conclusion Our results indicate that subcutaneous small artery structure is related to MoR, possibly because an altered vascular structure may amplify BP changes or, vice versa, because a greater MoR may further damage peripheral vasculature.

Effects of Melatonin and Pycnogenol on Small Artery Structure and Function in Spontaneously Hypertensive Rats

It was suggested that oxidative stress has a key role in the development of endothelial dysfunction, as well as microvascular structural alterations. Therefore, we have investigated 2 substances with antioxidant properties: melatonin and Pycnogenol. We treated 7 spontaneously hypertensive rats (SHRs) with melatonin and 7 with Pycnogenol for 6 weeks. We compared results obtained with those observed in 7 SHRs and 7 Wistar-Kyoto normotensive control rats kept untreated. Mesenteric small resistance arteries were dissected and mounted on a wire myograph, and a concentration-response curve to acetylcholine was performed. Aortic contents of metalloproteinase 2, Bax, inducible NO synthase, and cyclooxygenase 2 were evaluated, together with the aortic content of total collagen and collagen subtypes and apoptosis rate. A small reduction in systolic blood pressure was observed. A significant improvement in mesenteric small resistance artery structure and endothelial function was observed in rats treated with Pycnogenol and melatonin. Total aortic collagen content was significantly greater in untreated SHRs compared with Wistar-Kyoto control rats, whereas a full normalization was observed in treated rats. Apoptosis rate was increased in the aortas of untreated SHRs compared with Wistar-Kyoto control rats; an even more pronounced increase was observed in treated rats. Bax and metalloproteinase 2 expressions changed accordingly. Cyclooxygenase 2 and inducible NO synthase were more expressed in the aortas of untreated SHRs compared with Wistar-Kyoto control rats; this pattern was normalized by both treatments. In conclusion, our data suggest that treatment with Pycnogenol and melatonin may protect the vasculature, partly independent of blood pressure reduction, probably through their antioxidant effects.

Immunohistochemical evaluation of microvascular rarefaction in hypertensive humans and in spontaneously hypertensive rats

OBJECTIVE No data are presently available about changes in capillary density in the skeletal muscle and in the brain of spontaneously hypertensive rats (SHR) in relation to the development of hypertension. DESIGN AND METHODS We have investigated 4 week-old and 12 week-old SHR and age-matched normotensive Wistar-Kyoto controls (WKY). Microvessel density (MVD) in the cerebral cortex and in a skeletal muscle were evaluated in sections stained for CD31. We also evaluated MVD in the dermal tissue of normotensive subjects and essential hypertensive patients. Subcutaneous small resistance arteries were dissected and mounted in a micromyograph and the media to lumen ratio (M/L) was measured. RESULTS A significant reduction in MVD in the skeletal muscle and in the brain of SHR was clearly observed at 12 weeks of age, after the development of hypertension, but not at 4 weeks of age (pre-hypertensive condition). In hypertensive patients a significant reduction in the dermal MVD and an inverse correlation between M/L and MVD was observed. CONCLUSIONS Our results suggest that, in the brain and skeletal muscle of adult SHR after the development of hypertension, and in the derma of adult essential hypertensive patients microvascular rarefaction may occur.

Effects of losartan and enalapril at different doses on cardiac and renal interstitial matrix in spontaneously hypertensive rats.

We have evaluated the effects of an ACE inhibitor, enalapril (ENA) and of an angiotensin II receptor blocker, losartan (LOS), administered either at hypotensive or non‐hypotensive dosage, on the cardiac and renal structure of spontaneously hypertensive rats (SHR). Forty‐eight rats were included in the study: eight SHR were treated with low‐dose (ld, 1 mg/kg/day) ENA; eight with low‐dose (ld, 0.5 mg/kg/day) LOS; eight with high‐dose (hd, 25 mg/kg/day) ENA; eight with high‐dose (hd, 15 mg/kg/day) LOS; while eight Wistar–Kyoto (WKY) and eight SHR were kept untreated (unt). Treatment was given from the 4th to the 12th week of age. Systolic blood pressure (SBP) was measured non‐invasively every week. The left ventricular weight to body weight (RLVM) and the left + right kidney weight (RKW) to body weight was measured, and the cardiac and glomerular interstitial collagen content was evaluated using sirius red staining and image analysis. In addition, cardiac metalloproteinases activity (43 kDa MMP, MMP‐2, and MMP‐9) was evaluated by zymography. A significant reduction in RLVM was observed in SHR given ENA hd or LOS hd. Cardiac collagen was significantly reduced in SHR ENA hd and SHR LOS hd as well as in SHR LOS ld, but not in SHR ENA ld. The 43 kDa MMP collagenase activity was greater in WKY unt compared with SHR unt, being normalized only in SHR ENA hd. The gelatinase activity of MMP‐9 showed a trend similar to 43 kDa MMP, but differences between SHR and WKY unt were only of borderline statistical significance. No difference among groups was observed in MMP‐2 activity. No significant differences in RKW was observed between groups. However, the collagen content in the glomerular perivascular space was significantly reduced in all treated groups, including those given ld, compared with SHR unt. In conclusion, LOS and ENA showed a similar preventive effect on the increase of RLVM in SHR, but, at least in part, different effects on the extracellular matrix in different organs, being cardiac collagen less sensitive to low dose (ld) ACE inhibition.

Hypertrophic Remodeling of Subcutaneous Small Resistance Arteries in Patients with Cushing’s Syndrome

OBJECTIVE Structural alterations of small resistance arteries in essential hypertensive patients (EH) are mostly characterized by inward eutrophic remodeling. However, we observed hypertrophic remodeling in patients with renovascular hypertension, in those with acromegaly, as well as in patients with non-insulin-dependent diabetes mellitus, suggesting a relevant effect of humoral growth factors on vascular structure, even independent from the hemodynamic load. Cortisol may stimulate the renin-angiotensin system and may induce cardiac hypertrophy. However, presently no data are available about small artery structure in patients with Cushings syndrome. SUBJECTS We have investigated the structure of sc small resistance arteries in 12 normotensive subjects (NT), in 12 EH subjects, and in eight patients with Cushings syndrome (CS). Small arteries from sc fat were dissected and mounted on a micromyograph. The normalized internal diameter, media thickness, media to lumen ratio, and the media cross-sectional area were measured, as well as indices of oxidative stress. RESULTS Demographic variables were similar in the three groups, except for clinic blood pressure. The media to lumen ratio was significantly greater in EH and CS, compared with NT; no difference was observed between EH and CS. The media cross-sectional area was significantly greater in CS compared with EH and with NT. An increased vascular oxidative stress was present in CS, as demonstrated by increased levels of superoxide anions, cyclooxygenase-1 and endothelial nitric oxide synthase in the microvessels. CONCLUSION Our results suggest the presence of hypertrophic remodeling in sc small resistance arteries of CS, probably as a consequence of growth-promoting properties of circulating cortisol and/or increased vascular oxidative stress.

Lack of prognostic role of endothelial dysfunction in subcutaneous small resistance arteries of hypertensive patients

Objective The presence of endothelial dysfunction in the coronary circulation or in the brachial artery has been found to be associated with a greater incidence of cardiovascular events. However, no data are presently available about the prognostic role of endothelial dysfunction in human small resistance arteries. Design and methods Ninety subjects were included in the present study. They were: 10 normotensive subjects, 36 patients with essential hypertension, 10 patients with phaeochromocytoma, 11 patients with primary aldosteronism, 10 patients with renovascular hypertension, and 13 normotensive patients with non-insulin-dependent diabetes mellitus (NIDDM). All subjects were submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region. Small resistance arteries were dissected and mounted on an isometric myograph, and the concentration–response curves to acetylcholine (from 10−9 to 10−5 mol/l) (endothelium-dependent vasodilatation) and sodium nitroprusside (from 10−9 to 10−5 mol/l) (endothelium-independent vasodilatation) after precontraction of the vessels with norepinephrine were evaluated. The subjects were re-evaluated (by clinical visits or telephone interviews) after an average follow-up time of 5.5 years. Results Twenty-nine subjects had a documented fatal or non-fatal cardiovascular event (5.87%/year). The endothelium-dependent vasodilatation in the subcutaneous small arteries was similar in subjects with or without cardiovascular events. Also, endothelium-independent vasodilatation to sodium nitroprusside was similar in the two groups. Similar results were obtained by subdividing patients in the different subgroups (essential hypertension, secondary hypertension, etc.). Conclusions Our results indicate that endothelial dysfunction in the microcirculation does not predict cardiovascular events. It is possible that a prognostic role of endothelial dysfunction may be observed when other vascular districts prone to atherosclerosis are evaluated, or it might be detected only in patients at low to medium cardiovascular risk, in whom endothelial dysfunction is less advanced.

Effects of olmesartan and enalapril at low or high doses on cardiac, renal and vascular interstitial matrix in spontaneously hypertensive rats

We have evaluated the effects of different doses of an angiotensin‐converting enzyme (ACE) inhibitor, enalapril (ENA) and of an angiotensin II type 1 receptor blocker olmesartan (OLM), on extracellular matrix of the heart, kidney, aorta and mesenteric artery of spontaneously hypertensive rats (SHR). Forty SHR and eight Wistar–Kyoto controls (WKY) were included in the study. Eight SHR were treated with high‐dose OLM 15 mg/kg per day, eight with high‐dose ENA 25 mg/kg per day, eight with low‐dose OLM 1 mg/kg per day and eight with low‐dose ENA (2 mg/kg per day). Eight SHR and eight WKY were kept untreated as controls. Treatment was from age 4 to 12 weeks. Systolic blood pressure (SBP) was measured non‐invasively every week. The left ventricular weight to body weight (RLVM) was measured, and the cardiac, aortic and glomerular interstitial collagen content was evaluated using Sirius red staining and image analysis. Mesenteric small arteries were dissected and mounted on a micromyograph, and the media:lumen ratio (M/L) was calculated. Collagen subtypes were evaluated by polarized light microscopy. The SHR treated with high‐dose OLM or ENA showed a normalization of SBP. The RLVM was significantly increased in untreated SHR compared with untreated WKY, whereas significantly lower values were observed in the groups of SHR treated with high‐dose OLM or ENA. A significant increase in cardiac and glomerular collagen content was observed in untreated SHR. Both high‐ or low‐dose OLM and ENA normalized collagen content in the heart and the kidney. Both high‐dose OLM and high‐dose ENA normalized M/L ratio; however, OLM proved to be more effective than ENA in normalizing collagen pattern. In fact, aortic collagen content was normalized by both high‐dose and low‐dose OLM, but only by high‐dose ENA. In conclusion, both OLM and ENA were significantly and equally effective in the prevention of cardiac and renal damage in SHR, whereas OLM was more effective than ENA in terms of effects on vascular extracellular matrix.

Angiotensin receptor blockers improve insulin signaling and prevent microvascular rarefaction in the skeletal muscle of spontaneously hypertensive rats

Objective Spontaneously hypertensive rats are an example of an animal model of genetic hypertension with insulin resistance. The aim of this study was to investigate insulin signaling in the heart and in the skeletal muscle of spontaneously hypertensive rats, as well as to evaluate the effects of renin–angiotensin system blockade. Design and Methods We investigated eight untreated spontaneously hypertensive rats of 12 weeks of age and eight age-matched normotensive Wistar–Kyoto controls. In addition, eight spontaneously hypertensive rats were treated for 8 weeks with the angiotensin receptor blocker olmesartan, and eight spontaneously hypertensive rats with the angiotensin-converting enzyme inhibitor enalapril. The heart and a skeletal muscle (quadriceps femoris) were promptly dissected and frozen. Insulin signaling was evaluated by Western blot analysis of involved proteins; in addition, microvessel density was indirectly evaluated by immunohistochemistry. Results Blood pressure values were normalized by both olmesartan and enalapril. In the heart, no statistically significant difference in the expression of proteins involved in insulin signaling was observed between untreated spontaneously hypertensive rats and Wistar–Kyoto controls. On the contrary, in the skeletal muscle of untreated spontaneously hypertensive rats, we noted a significant reduction of insulin receptors, of insulin-receptor substrate-1, and of phosphorylated-mammalian target of rapamycin. The treatment with olmesartan normalized insulin signaling, including expression of glucose transporter-4, whereas the treatment with enalapril was ineffective for the insulin receptor and less effective than olmesartan on the insulin-receptor substrate-1, phosphorylated-mammalian target of rapamycin and glucose transporter-4. There was a significant reduction in microvessel density in the skeletal muscle of spontaneously hypertensive rats compared with Wistar–Kyoto controls, and this was completely prevented by both olmesartan and enalapril. Conclusion These results suggest that changes in insulin signaling occur in the skeletal muscle but not in the heart of untreated spontaneously hypertensive rats. In the skeletal muscle, insulin signaling was restored by olmesartan, whereas enalapril was less effective. Effective antihypertensive treatment with olmesartan or enalapril was associated with prevention of microvascular rarefaction.