Carolina De Ciuceis

Reduced Vascular Remodeling, Endothelial Dysfunction, and Oxidative Stress in Resistance Arteries of Angiotensin II–Infused Macrophage Colony-Stimulating Factor–Deficient Mice: Evidence for a Role in Inflammation in Angiotensin-Induced Vascular Injury

Objective—Angiotensin (Ang) II-induced vascular damage may be partially mediated by reactive oxygen species generation and inflammation. Homozygous osteopetrotic mice (Op/Op), deficient in macrophage colony-stimulating factor (m-CSF), exhibit reduced inflammation. We therefore investigated Ang II effects on vascular structure, function, and oxidant stress generation in this model. Methods and Results—Adult Op/Op, heterozygous (Op/+), and wild type (+/+) mice underwent 14-day Ang II (1000 ng/kg per minute) or saline infusion. Blood pressure (BP) was assessed by radiotelemetry, mesenteric resistance artery vascular reactivity was studied on a pressurized myograph, and vascular superoxide and NAD(P)H oxidase activity by lucigenin chemiluminescence. Ang II increased BP in Op/+ and +/+ mice but not in Op/Op. Ang II-treated Op/+ and +/+ mice showed reduced acetylcholine-mediated relaxation (maximal relaxation, respectively, 64% and 67% versus 84% and 93% in respective controls; P<0.05), which was unaffected by l-NAME. Ang II-infused Op/Op mice arteries showed significantly less endothelial dysfunction than vehicle-infused counterparts (maximal relaxation 87% versus 96% in shams). Resistance arteries from Ang II-infused +/+ and Op/+ mice had significantly increased media-to-lumen ratio and media thickness, neither of which was altered in Op/Op mice compared with untreated littermates. Vascular media cross-sectional area, NAD(P)H oxidase activity and expression, and vascular cell adhesion molecule (VCAM)-1 expression were significantly increased by Ang II only in +/+ mice (P<0.05). Conclusions—m-CSF–deficient mice (Op/Op) developed less endothelial dysfunction, vascular remodeling, and oxidative stress induced by Ang II than +/+ littermates, suggesting a critical role of m-CSF and proinflammatory mediators in Ang II-induced vascular injury.

Persistent Remodeling of Resistance Arteries in Type 2 Diabetic Patients on Antihypertensive Treatment

Abstract—We hypothesized that resistance arteries from diabetic patients with controlled hypertension have less remodeling than vessels from untreated hypertensive subjects. Eight normotensive subjects (aged 44±3 years, 3 men; values are mean±SEM), 19 untreated hypertensive subjects (46±2 years, 9 men), and 23 hypertensive subjects with type 2 diabetes mellitus under antihypertensive treatment (58±1 years, 15 men) were studied. Resistance arteries dissected from gluteal subcutaneous tissue were assessed on a pressurized myograph. Most diabetic patients (70%) were being treated with angiotensin-converting enzyme inhibitors. Although systolic blood pressure was still above the normotensive range in these patients (144±2 versus 150±3 mm Hg in hypertensive and 114±4 mm Hg in normotensive subjects), diastolic blood pressure was well controlled (83±2 mm Hg) and significantly lower compared with that in untreated hypertensives (100±1 mm Hg; P <0.001) but higher than in normotensives (76±3 mm Hg; P <0.05). Thus, pulse pressure was higher in diabetic patients (P <0.05). The media-to-lumen ratio of resistance arteries was greater in hypertensives (0.083±0.002) compared with normotensive controls (0.059±0.003; P <0.05) and was even higher in diabetic hypertensive subjects (0.105±0.004; P <0.001 versus normotensive controls). The medial cross-sectional area was greater in diabetic and hypertensive patients compared with normotensive controls (P <0.001). Acetylcholine-induced relaxation was impaired in vessels from hypertensive patients and from patients with both diabetes mellitus and hypertension (P <0.05 versus normotensive controls), whereas endothelium-independent vasorelaxation was similar in all groups. Despite effective antihypertensive treatment, resistance arteries from hypertensive diabetic patients showed marked remodeling, greater than that of vessels from untreated, nondiabetic, hypertensive subjects, in agreement with the high cardiovascular risk of subjects suffering from both diabetes and hypertension.

Angiotensin Receptor Blocker Added to Previous Antihypertensive Agents on Arteries of Diabetic Hypertensive Patients

Lowering elevated blood pressure (BP) in diabetic hypertensive individuals decreases cardiovascular events. We questioned whether remodeling of resistance arteries from hypertensive diabetic patients would improve after 1 year of tight BP control with addition of either the angiotensin receptor blocker (ARB) valsartan or the β-blocker (BB) atenolol to previous therapy, which included angiotensin-converting enzyme inhibitors (ACEIs) and/or calcium channel blockers. Twenty-eight hypertensive type 2 diabetic patients treated with oral hypoglycemic and antihypertensive agents (not receiving ARBs or BBs) were randomly assigned to double-blind treatment for 1 year with valsartan (80 to 160 mg) or atenolol (50 to 100 mg) daily, added to previous therapy. Resistance arteries dissected from gluteal subcutaneous tissues were assessed on a pressurized myograph. After 1 year of treatment, systolic and diastolic BP and glycemia were equally well controlled in the valsartan and atenolol groups. Endothelium-dependent and independent relaxation did not change in the treated groups. After 1 year of treatment, resistance artery media:lumen ratio decreased in the valsartan group (7.9±0.5% after versus 9.8±0.6% before; P<0.05) but not in the atenolol-treated group (9.9±0.9% versus 10.6±1%; P value not significant). Artery walls from atenolol-treated patients became stiffer, with no change in the valsartan-treated patients. In conclusion, similar intensive BP control for 1 year with valsartan was associated with improved structure of resistance arteries in diabetic hypertensive patients, whereas vessels from atenolol-treated patients exhibited unchanged remodeling and a stiffer wall. The addition of ARBs but not BBs to antihypertensive medications that may include angiotensin-converting enzyme inhibitors and/or calcium channel blockers results in an improvement in resistance artery remodeling in diabetic hypertensive patients.

Effect of antihypertensive treatment on microvascular structure, central blood pressure and oxidative stress in patients with mild essential hypertension.

Background: It has been previously demonstrated that dihydropyridine calcium channel blockers may possess antioxidant properties and might improve vascular structure. Combination treatment with an angiotensin-converting enzyme inhibitor may have additional advantages, compared with a thiazide diuretic, in this regard. The aim of the present study was, therefore, to investigate the effects of a short-term treatment with lercanidipine, and to compare two combination treatments: lercanidipine + enalapril vs. lercanidipine + hydrochlorothiazide on structural alterations in retinal arterioles, on skin capillary density and on large artery distensibility. Patients and methods: Twenty essential hypertensive patients were included in the study and treated for 4 weeks with lercanidipine 20 mg per day orally. Then they were treated for 6 months with lercanidipine + enalapril (n = 10) or lercanidipine + hydrochlorothiazide (n = 10) combinations. Investigations were performed in basal condition, after appropriate washout of previous treatments, after 4 weeks of lercanidipine monotherapy treatment, and at the end of the combination treatment. Non-invasive measurements of wall-to-lumen ratio (W/L) and other morphological parameters of retinal arterioles using scanning laser Doppler flowmetry were performed (Heidelberg Retina Flowmeter, Heidelberg Engineering). Capillary density was evaluated by capillaroscopy, whereas pulse wave velocity and central blood pressure were assessed by the Sphygmo-Cor device (AtCor Medical West Ryde, Australia). Results: A significant improvement of W/L and of other indices of retinal artery structure was observed after treatment with lercanidipine alone, with a further improvement after treatment with lercanidipine + enalapril, whereas after treatment with lercanidipine + hydrochlorothiazide the improvement was no longer observed. A similar behaviour was observed for central SBP and DBP. Capillary density was increased only after treatment with lercanidipine + enalapril. Conclusion: Lercanidipine both in monotherapy and in combination with enalapril, was able to improve microvascular structure and to decrease central blood pressure, being thus a useful approach for both reducing blood pressure and improving vascular alterations in hypertension.

Anticontractile activity of perivascular fat in obese mice and the effect of long-term treatment with melatonin.

Aims: It has been demonstrated previously that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction. The aim of this study was to investigate the functional responses of small mesenteric arteries in a hyperphagic animal model of obesity after chronic treatment with melatonin, an endogenous hormone with antioxidant and vasculoprotective properties. Methods and results: Ten obese mice (ob/ob) and 10 control lean mice (CLM) were treated with melatonin 100 mg/kg per day in the drinking water for 8 weeks. Mesenteric small resistance arteries were dissected and mounted on a wire myograph and a concentration-response to norepinephrine was evaluated in vessels with intact PVAT and after PVAT was removed and in the presence of iberiotoxin, a selective blocker of BKCA channels as well as under conditions of induced hypoxia in vitro. The presence of PVAT reduced the contractile response to norepinephrine in both ob/ob and CLM; however, the effect was significantly reduced in ob/ob. The anticontractile effect of PVAT completely disappeared with iberiotoxin preincubation. After melatonin treatment, inflammation was significantly ameliorated, and the contractile response in ob/ob and CLM was significantly reduced when PVAT was removed. Anticontractile effect of PVAT that is lost in obesity can be rescued using melatonin. A reduced expression of adiponectin and adiponectin receptor was observed in perivascular fat of ob/ob, whereas significant increase was observed in ob/ob treated with melatonin. Conclusion: Melatonin seems to exert a protective effect on arteries from both ob/ob and CLM, counteracting the adverse effect of hypoxia and iberiotoxin.

Effect of long-term treatment with melatonin on vascular markers of oxidative stress/inflammation and on the anticontractile activity of perivascular fat in aging mice.

Some reports have suggested that inflammation in perivascular adipose tissue (PVAT) may be implicated in vascular dysfunction by causing the disappearance of an anticontractile effect. The aim of this study was to investigate the effects of chronic melatonin treatment on the functional responses of the small mesenteric arteries and on the expression of markers of inflammation/oxidative stress in the aortas of senescence-accelerated prone mice (SAMP8), a model of age-related vascular dysfunction. We investigated seven SAMP8 and seven control senescence-accelerated resistant mice (SAMR1) treated for 10 months with melatonin, as well as equal numbers of age-matched untreated SAMP8 and SAMR1. The mesenteric small resistance arteries were dissected and mounted on a wire myograph, and the concentration–response to norepinephrine was evaluated in vessels with intact PVAT and after the removal of the PVAT. The expression of markers of oxidative stress, inflammation and aging in the aortas was evaluated by immunostaining. In addition, the adiponectin content and the expression of adiponectin receptor 1 were evaluated in the visceral adipose tissue. In untreated SAMP8 mice, we observed an overexpression of oxidative stress and inflammatory markers in the vasculature compared with the controls. No anticontractile effect of the PVAT was observed in untreated SAMP8 mice. Long-term treatment of SAMP8 mice with melatonin increased the expression of some markers of vasoprotection, decreased oxidative stress and inflammation and restored the anticontractile effect of the PVAT. Decreased expression of adiponectin and adiponectin receptor 1 was also observed in visceral fat of untreated SAMP8, whereas a significant increase was observed after melatonin treatment.

Resistance-Sized Arteries Structure and Capillary Density Changes in Glioblastoma and Meningioma Peritumoral Brain Tissue

Background: Brain vessels play a relevant role in the development of malignant primary tumors. Previous studies performed in preclinical models of brain tumors demonstrated the irregular morphology of vessels in glioblastoma, characterized by multiple abnormalities in shape, permeability and relationship with the contacting structures, such as the basement membrane; however no data about microvascular structural alterations in the brain of patients affected by brain tumors have been previously reported. Methods: We studied small vessels structural alterations, microvascular rarefaction and vascular collagen content in the peritumoral brain tissue of two groups of patients, respectively affected by glioblastoma and by meningioma, who underwent a surgical operation in order to remove the tumor. The two groups of patients were matched for clinical, laboratory variables and risk factors, with the exception of sex. Results: Media to lumen ratio was significantly higher in patients with meningioma compared to patients affected by glioblastoma. Vessels in the brain surrounding glioblastomas show a less organized structure, as demonstrated by the thinner media, by the lower content in collagen and by the lower capillary density. Conclusions: For the first time, we used an in vitro ex vivo technique to analyze structural alterations of resistance sized arteries of peritumoral brain tissue at the radiological tumor-brain interface in patients affected by glioblastoma and meningioma. This approach, together with immunohistochemical evaluation of microvessel density, has supplied new data about the brain microcirculation. These findings can be the result of the aberrant angiogenic process that characterizes glioblastomas and can finally play a role in tumor growth. Further studies are needed to confirm our data, to identify the pathways that lead to this microvascular pattern and to identify possible clinical applications

Carotid stiffness is significantly correlated with wall-to-lumen ratio of retinal arterioles

Backgroud and purpose: Wall-to-lumen ratio of retinal arterioles (W/L ratio) might serve as an in-vivo parameter of microvascular damage. No study has investigated the relationship between carotid stiffness and W/L ratio of retinal arteries. Therefore, the aim of the current study was to assess the correlation between local carotid stiffness, as assessed by echotracking technique, and W/L ratio of retinal arterioles, as assessed by noninvasive flowmetry in normotensive patients and in patients with primary hypertension. Methods: Two hundred and twenty-seven patients underwent renal arteries W/L ratio and local carotid-pulse wave velocity (carotid PWV) measurement. One hundred and fifteen patients had a diagnosis of primary hypertension, whereas 112 were normotensive patients. Results: W/L ratio and carotid PWV were both related with clinic SBP (r = 0.17, P < 0.05; r = 0.50, P < 0.001), clinic pulse pressure (r = 0.22, P < 0.001; r = 0.55, P < 0.001), carotid SBP (r = 0.18, P < 0.05; r = 0.51, P < 0.001) and carotid pulse pressure (r = 0.24, P < 0.001; r = 0.56, P < 0.001). W/L ratio was correlated with carotid PWV (r = 0.18, P < 0.005). At multivariate analysis, carotid PWV remained independently associated with W/L ratio. Conclusion: In hypertensive and normotensive patients, carotid stiffness is significantly correlated with W/L ratio of retinal arteries, independently of possible confounders.

MPS 16-04 EVALUATION OF LOCAL CAROTID STIFFNESS AND WALL-TO-LUMEN RATIO OF RETINAL ARTERIOLES IN NORMOTENSIVE SUBJECTS AND HYPERTENSIVE PATIENTS

Objective: Wall-to-lumen ratio of retinal arterioles (W/L) might serve as an in-vivo parameter of microvascular damage. Few data are available on the correlation between local stiffness, evaluated at carotid artery level, and the presence of retinal abnormalities, assessed by arteriolar/venular ratio. Aim of the study was to analyse the correlation between carotid stiffness (CS) and W/L of retinal arterioles. Design and Method: Methods: 227 subjects (56% female, age 55 ± 4 years, 48% hypertensives, 30% treated) underwent laboratory examinations, clinic BP measurement, carotid-femoral pulse wave velocity (aoPWV) and W/L ratio measurement. CS was determined from the relative stroke change in diameter (measured with a high-resolution echotracking system) and carotid pulse pressure (measured with applanation tonometry). Results: both W/L and CS were significantly related with clinic SBP (r = 0.17, p < 0.05 and r = 0.50, p < 0.001), clinic PP (r = 0.22, p < 0.001 and r = 0.55, p < 0.001), carotid SBP (r = 0.18, p < 0.05 and r = 0.51, p < 0.001) and with carotid PP (r = 0.24, p < 0.001 and r = 0.56, p < 0.001). W/L was not significantly related with age and laboratory data while a positive correlation was observed between W/L and CS (r = 0.18, p < 0.005). At multivariate analysis CS, but not aoPWV, remained independently associated with W/L. Conclusions: In this large group of hypertensives and normotensives local carotid stiffness represents the main determinant of wall to lumen ratio of retinal arterioles.

[PP.21.05] RELATIONSHIP BETWEEN MICROVASCULAR STRUCTURE AND T REGULATORY LYMPHOCYTES OF SMALL RESISTANCE ARTERIES

Objective: Recently it has been demonstrated a role for adaptive immunity, particularly for T regulatory lymphocytes (Tregs), in the development of hypertension and in preventing of angiotensin II–induced vascular injury and inflammation in animal models (Barhoumi T et al, Hypertension 2011;57:469–476). However, no data are presently available in human beings about possible relationships between Tregs and microvascular structural alterations. Design and method: In the present study we enrolled 11 normotensive subjects and 8 hypertensive patients undergoing an election surgical intervention. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance artery structure was assessed by wire myography and media to lumen ratio (M/L) was calculated. W/L of retinal arterioles was obtained by Scanning Laser Doppler Flowmetry. Functional (basal) and structural (total) microvascular density were studied by capillaroscopy before and after venous congestion. No sign of local or systemic inflammation was present in any subjects or patients. We extracted genomic DNA from small resistance arteries and analyzed methylation status of the FoxP3 gene promoter involved in Treg lymphocytes activation. Unmethylated FoxP3 has been demonstrated to be specific for Treg lymphocytes. A peripheral blood sample was obtained before surgery for routine chemistry Results: Results are summarized in the Table. Figure. No caption available. A significant positive correlation was detected between Tregs in small resistance arteries and basal, total and delta gain capillary density in the forearm, whereas no correlations were observed with small resistance artery M/L and retinal arteriole W/L. In addition, a significant inverse correlation was detected between Treg in subcutaneous small vessels and C reactive protein. Conclusions: Our data suggest that Treg lymphocytes detected in subcutaneous small resistance artery wall are related with capillary density and inversely related with inflammatory markers suggesting a protecting role of Treg also, probably, in terms of angiogenetic properties.