Subcutaneous Resistance Arteries Research Articles

Microstructural Characterization of Resistance Artery Remodelling in Diabetes Mellitus

Introduction: Microvascular remodelling is a symptom of cardiovascular disease. Despite the mechanical environment being recognized as a major contributor to the remodelling process, it is currently only understood in a rudimentary way. Objective: A morphological and mechanical evaluation of the resistance vasculature in health and diabetes mellitus. Methods: The cells and extracellular matrix of human subcutaneous resistance arteries from abdominal fat biopsies were imaged using two-photon fluorescence and second harmonic generation at varying transmural pressure. The results informed a two-layer mechanical model. Results: Diabetic resistance arteries reduced in wall area as pressure was increased. This was attributed to the presence of thick, straight collagen fibre bundles that braced the outer wall. The abnormal mechanical environment caused the internal elastic lamina and endothelial and vascular smooth muscle cell arrangements to twist. Conclusions: Our results suggest diabetic microvascular remodelling is likely to be stress-driven, comprising at least 2 stages: (1) Laying down of adventitial bracing fibres that limit outward distension, and (2) Deposition of additional collagen in the media, likely due to the significantly altered mechanical environment. This work represents a step towards elucidating the local stress environment of cells, which is crucial to build accurate models of mechanotransduction in disease.

MO069SARS-COV-2 RECEPTOR ACE-2, TMPRSS2 AND SOLUBLE ACE-2 IN PATIENTS WITH END STAGE KIDNEY DISEASE

Background and AimsSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic loss of lives due to COVID-19. Individuals with chronic conditions, including patients with kidney failure and/or kidney transplants, are affected more substantially due to multiple comorbidities and altered immune system. The first step of this infection process is the binding of SARS-CoV-2 with angiotensin-converting enzyme 2 (ACE-2) receptor, followed by its priming by transmembrane protease serine 2 (TMPRSS2). We hypothesized that the expressions of ACE-2 and TMPRSS2 are increased in microvasculature, in addition to increased circulating soluble ACE-2 levels in patients with end stage kidney disease (ESKD); i.e. prerequisites to explain to why ESKD patients are susceptible to SARS-CoV-2 infection. Therefore, we assessed if there is a difference in the expression of ACE-2 and TMPRSS2 receptors in the resistance artery and subcutaneous adipose tissue, alongside circulating soluble ACE-2 levels in ESKD patients versus controls.MethodA total of 210 participants were enrolled, representing 80 ESKD and 73 healthy controls for soluble ACE-2 analysis, and 31 ESKD and 26 healthy controls for isolated subcutaneous vasculature bioassay. Immunofluorescence techniques were performed for the detection and evaluation of ACE-2 and TMPRSS2 in isolated subcutaneous resistance artery (200-300 µm of internal âCE€) and adipose tissue. Soluble ACE-2 protein concentration was detected using commercially available ELISA kits.ResultsSoluble ACE-2 levels were significantly higher in ESKD (3.8 ng/mL, IQR 2.4-5.5, n=80) vs control groups (2.7 ng/mL, IQR 2.1-3.7 ng/mL, n=73). There was no difference in soluble ACE-2 between females and males in either group. Soluble ACE-2 was positively correlated with IL-6 (rho=0.257, p=0.02, n=80), while it was negatively correlated with cholesterol (rho= -0.248, p=0.02, n=78) in ESKD patients. The expression of ACE-2 receptor was observed both on endothelium and vascular smooth muscle cells (VSMCs) in arteries from both groups. The expression was higher in ESKD patients (19.1%, n=23) vs controls (15.4%, n=15). Patients with ESKD on ACE-inhibitor/angiotensin receptor blocker treatment showed higher expression of ACE-2 vs. non-treatment group (treatment: 20.2%, n=12 vs non-treatment: 12.8%, n=11) in resistance artery. In subcutaneous adipose tissue the ACE-2 staining was not statistically different among the groups (ESKD: 2.9%, n=10 vs controls: 3.6%, n=10). In addition, TMPRSS2 was expressed both on endothelium and VSMCs in resistance artery, however there was no difference in the expression (ESKD: 8.4%, n=23 vs controls: 10.2%, n=15) between the groups.ConclusionSoluble ACE-2 levels and ACE-2 receptor expression in the vasculature were higher in patients with ESKD as compared to controls. The ACE-2 receptor is present both in the endothelium and VSMCs from arteries in peripheral microcirculation. This supports the suggestion that the uremic milieu induces an optimal environment for SARS-CoV-2 entrance in microcirculation with following consequence on the vasculature during the COVID-19. Similarly, TMPRSS2 expression was observed in vessels from both groups, while increased expression of ACE-2 receptor was observed in those ESKD patients receiving ACE-inhibitor/angiotensin receptor blocker treatment. Further studies are warranted to assess possible sex differences in the target receptor expressions with further elaboration on specific treatment regime(s) for different comorbidities present in patients with ESKD.

Transglutaminase 2 Inhibitor LDN 27219 Age-Dependently Lowers Blood Pressure and Improves Endothelium-Dependent Vasodilation in Resistance Arteries.

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

Effect of direct renin inhibition on vascular function after long-term treatment with aliskiren in hypertensive and diabetic patients.

We tested the hypothesis that chronic treatment with the direct renin inhibitor aliskiren improves vascular function in resistance and conduit arteries of type two diabetic and hypertensive patients. Sixteen patients with mild essential hypertension and with a previous diagnosis of noninsulin-dependent diabetes mellitus were included in the study. Patients were then randomized to aliskiren (150 mg once daily, n = 9), or ramipril (5 mg once daily, n = 7). Each patient underwent a biopsy of the subcutaneous tissue and small arteries were dissected and mounted on a pressurized micromyograph to evaluate endothelium dependent vasorelaxation in response to acetylcholine ± N omega-nitro-L-arginine methyl ester hydrochloride in vessels precontracted with norepinephrine. Endothelial function has been quantified also in large conduit arteries by flow-mediated dilation. A similar office blood pressure-lowering effect was observed with the two drugs, although changes in DBP were not statistically significant in the ramipril group. Aliskiren significantly improved endothelium-dependent relaxation in subcutaneous resistance arteries, as well as increased flow-mediated dilation in conduit arteries, whereas the effects induced by ramipril did not reach statistical significance. Only aliskiren significantly increased the expression of p1177-endothelial nitric oxide synthase in the endothelium. Both aliskiren and ramipril had a negligible effect on markers of oxidative stress. Aliskiren restored endothelial function and induced a more prompt peripheral vasodilation in hypertensive and diabetic patients possibly through the increased production of nitric oxide via the enhanced expression and function of the active phosphorylated form of endothelial nitric oxide synthase.

Effect of increased potassium intake on the renin-angiotensin-aldosterone system and subcutaneous resistance arteries: a randomized crossover study.

Increased potassium intake lowers blood pressure (BP) in hypertensive patients. The underlying mechanism is not fully understood but must be complex because increased potassium intake elevates circulating concentrations of the BP-raising hormone aldosterone. In a randomized placebo-controlled crossover study in 25 normotensive men, we investigated the effect of 4 weeks of potassium supplement (90 mmol/day) compared with 4 weeks of placebo on the renin-angiotensin-aldosterone system (RAAS), urine composition and 24-h ambulatory BP. Vascular function was also assessed through wire myograph experiments on subcutaneous resistance arteries from gluteal fat biopsies. Higher potassium intake increased urinary potassium excretion (144.7 ± 28.7 versus 67.5 ± 25.5 mmol/24-h; P < 0.0001) and plasma concentrations of potassium (4.3 ± 0.2 versus 4.0 ± 0.2 mmol/L; P = 0.0002), renin {mean 16 [95% confidence interval (CI) 12-23] versus 11 [5-16] mIU/L; P = 0.0047}, angiotensin II [mean 10.0 (95% CI 6.2-13.0) versus 6.1 (4.0-10.0) pmol/L; P = 0.0025] and aldosterone [mean 440 (95% CI 336-521) versus 237 (173-386) pmol/L; P < 0.0001]. Despite RAAS activation, systolic BP (117.6 ± 5.8 versus 118.2 ± 5.2 mmHg; P = 0.48) and diastolic BP (70.8 ± 6.2 versus 70.8 ± 6.3 mmHg; P = 0.97) were unchanged. In the wire myograph experiments, higher potassium intake did not affect endothelial function as assessed by acetylcholine [logarithmically transformed half maximal effective concentration (pEC50): 7.66 ± 0.95 versus 7.59 ± 0.85; P = 0.86] and substance P (pEC50: 8.42 ± 0.77 versus 8.41 ± 0.89; P = 0.97) or vascular smooth muscle cell reactivity as assessed by angiotensin II (pEC50: 9.01 ± 0.86 versus 9.02 ± 0.59; P = 0.93) and sodium nitroprusside (pEC50: 7.85 ± 1.07 versus 8.25 ± 1.32; P = 0.25) but attenuated the vasodilatory response of retigabine (pEC50: 7.47 ± 1.16 versus 8.14 ± 0.90; P = 0.0084), an activator of Kv7 channels. Four weeks of increased potassium intake activates the RAAS in normotensive men without changing BP and this is not explained by improved vasodilatory responses ex vivo.

Changes in extracellular matrix in subcutaneous small resistance arteries of patients with essential hypertension

Background: In the development of hypertensive microvascular remodeling, a relevant role may be played by changes in extracellular matrix proteins. Aim of this study was the to evaluate some extracellular matrix components within the tunica media of subcutaneous small arteries in 9 normotensive subjects and 12 essential hypertensive patients, submitted to a biopsy of subcutaneous fat from the gluteal or the anterior abdominal region.Patients and Methods: Subcutaneous small resistance arteries were dissected and mounted on an isometric myograph, and the tunica media to internal lumen ratio was measured. In addition, fibronectin, laminin, transforming growth factor-beta-1 (TGF-β1) and emilin-1 contents within the tunica media were evaluated by immunofluorescence and relative immunomorphometrical analysis (immunopositivity % of area). The total collagen content and collagen subtypes within the tunica media were evaluated using both Sirius red staining (under polarized light) and immunofluorescence assay.Results: Normotensive controls had less total and type III collagen in respect with hypertensive patients. Fibronectin and TGF-β1 tunica media content was significantly greater in essential hypertensive patients, compared with normotensive controls, while laminin and emilin-1 tunica media content was lesser in essential hypertensive patients, compared with normotensive controls. A significant correlation was observed between fibronectin tunica media content and media to lumen ratio.Conclusions: Our results indicate that, in small resistance arteries of patients with essential hypertension, a relevant fibrosis may be detected; fibronectin and TGF-β1 tunica media content is increased, while laminin and emilin-1 content is decreased; these changes might be involved in the development of small resistance artery remodeling in humans.

Long chain n-3 polyunsaturated fatty acids and vascular function in patients with chronic kidney disease and healthy subjects: a cross-sectional and comparative study

BackgroundPatients with chronic kidney disease have a markedly increased cardiovascular mortality compared with the general population. Long chain n-3 polyunsaturated fatty acids have been suggested to possess cardioprotective properties. This cross-sectional and comparative study evaluated correlations between hemodynamic measurements, resistance artery function and fish consumption to the content of long chain n-3 polyunsaturated fatty acids in adipose tissue, a long-term marker of seafood intake.MethodsSeventeen patients with chronic kidney disease stage 5 + 5d and 27 healthy kidney donors were evaluated with hemodynamic measurements before surgery; from these subjects, 11 patients and 11 healthy subjects had vasodilator properties of subcutaneous resistance arteries examined. The measurements were correlated to adipose tissue n-3 polyunsaturated fatty acids. Information on fish intake was obtained from a dietary questionnaire and compared with adipose tissue n-3 polyunsaturated fatty acids.ResultsFish intake and the content of n-3 polyunsaturated fatty acids in adipose tissue did not differ between patients and controls. n-3 polyunsaturated fatty acid levels in adipose tissue were positively correlated to systemic vascular resistance index; (r = 0.44; p = 0.07 and r = 0.62; p < 0.05, chronic kidney disease and healthy subjects respectively) and negatively correlated to cardiac output index (r = −0.69; p < 0.01 and r = −0.50; p < 0.05, chronic kidney disease and healthy subjects respectively). No correlation was observed between n-3 polyunsaturated fatty acid levels in adipose tissue and vasodilator properties in resistance arteries. n-3 PUFA content in adipose tissue increased with increasing self-reported fish intake.ConclusionsThe correlations found, suggest a role for n-3 polyunsaturated fatty acids in hemodynamic properties. However, this is apparently not due to changes in intrinsic properties of the resistance arteries as no correlation was found to n-3 polyunsaturated fatty acids.

Similar Adiponectin Levels in Obese Normotensive and Obese Hypertensive Men and No Vasorelaxant Effect of Adiponectin on Human Arteries.

Obesity is a strong risk factor for hypertension, but the mechanism linking obesity to hypertension is not fully elucidated. In obesity, circulating concentrations of adiponectin are decreased and hypoadiponectinaemia has in some but not all studies been associated with increased risk of hypertension. Due to this inconsistency, we decided to study adiponectin from two aspects in a cross-sectional in vivo study and in an experimental in vitro study. In the cross-sectional study, 103 men with body mass index (BMI) ≥ 30.0 kg/m(2) were studied; 63 had 24-hr ambulatory blood pressure (ABP) ≥ 130/80 mmHg (ObeseHT) and 40 had 24-hr ABP < 130/80 mmHg (ObeseNT). As controls, we studied 27 men with BMI between 20.0 and 24.9 kg/m(2) and 24-hr ABP < 130/80 mmHg (LeanNT). Serum concentrations of adiponectin and body composition using dual-energy X-ray absorptiometry scanning were determined. In vitro, the direct vasomotor response of adiponectin was tested on subcutaneous resistance arteries from human abdominal adipose tissue. The two obese groups had lower adiponectin concentrations compared with LeanNT (p < 0.01) [median (interquartile range)]: ObeseHT 6.5 (5.1-8.3) mg/L; ObeseNT 6.6 (5.2-7.8) mg/L; and LeanNT 9.4 (6.7-12.4) mg/L, with no significant difference in adiponectin concentrations (or body composition) between ObeseHT and ObeseNT (p = 0.67). In vitro, adiponectin did not have any direct vasodilatory effect and adiponectin did not affect angiotensin II-stimulated vasoconstriction. In conclusion, obese hypertensive men have similar serum concentrations of adiponectin as obese normotensive men. In combination with the in vitro data, these findings question a pathogenic role of adiponectin in human hypertension.

Abstract P255: Age and Structural Alterations in Subcutaneous Small Resistance Arteries of Hypertensive Patients

Background: It was proposed that early vascular ageing may be an important mechanism of vascular damage in large conductance arteries. However it is not known whether aging may also affect small resistance artery morphology. Patients and Methods: For this reason, we investigated 100 patients with essential hypertension. Secondary forms of hypertension were excluded according to standard clinical evaluations and biochemical or instrumental assessments. In all patients, an evaluation of small resistance arteries morphology was performed by a wire micromyographic approach (Mulvany’s technique). A small amount of subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. Results: The age range of our populaton was 22-81 years, with a mean value of 57±12 years; 14% of them were current smokers, 32% had alterations in lipid patterns, none of them had diabetes mellitus, 58 were males and average blood pressure values were 156/95±19/12 mmHg. We found a significant correlation between M/L and age (r=0.30, p=0.002): the statistical significance of the correlation persisted after correction for counfounding variables (gender, serum cholesterol, smoking status, serum glucose, systolic or diastolic blood pressure values). A statistically significant inverse correlation was also observed between internal diameter and age (r=-0.20, p=0.046), while the correlation beween age and media thickness did not reach statistical significance (r=0.09, p=0.37). Conclusion: Our data suggest that age may affect microvascular structure in hypertensive patients. It is also possible that hypertension may anticipate the effects of physiological aging, and this should be explored in a relatively large population of normotensive subjects.

Abstract 024: The Direct Renin Inhibitor Aliskiren Improves Vasodilation and Endothelial Function in Resistance Arteries from Diabetic and Hypertensive Patients

We previously demonstrated that the direct renin inhibitor aliskiren (ALK) significantly reduced the remodeling of subcutaneous resistance arteries of hypertensive patients as compared to the angiotensin-converting enzyme inhibitor ramipril (RAM). Here we questioned whether endothelial function of resistance arteries would improve after 1 year of blood pressure (BP) control with ALK or RAM. Sixteen diabetic patients with mild essential hypertension were randomized to ALK (150-300 mg once daily, n=9) or RAM (5-10 mg once daily, n=7). Subcutaneous resistance arteries were dissected from gluteal biopsy and mounted on a pressurized micromyograph. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) and sodium nitroprusside (10 nM to 1 mM) respectively, in arteries pre-contracted with norepinephrine (10 μM). Carotid-femoral pulse wave velocity (PWV) was assessed by applantion tonometry. Forearm flow mediated dilation (FMD) was assessed by ultrasounds. The expression of P-eNOS/e-NOS and the markers of oxidative stress nitrotyrosine and LOX-1 were assessed by immunohistochemistry in resistance arteries. Patients were similar for age, sex, BMI and glycemic and metabolic control. Systolic BP was significantly and equally reduced by both ALK and RAM (153±8.9 mmHg reduced to 128±7.3 mmHg and 151±10.6 mmHg reduced to 121±12.1, respectively, P&lt;0.01) whereas diastolic BP was significantly reduced only in ALK-treated but not in RAM-treated patients (94.2±7.17 mmHg reduced to 81.4±6.31 mmHg, P&lt;0.01; and 84.7±12.22 reduced to 78.6±7.48, NS, respectively). PWV and FMD were similar in both groups before and after treatment. Endothelium-dependent vasodilation was improved only by ALK (max dilation 92.5±3.8% vs 50.5±14%, P&lt;0.05) but not by RAM (max dilation 72.9±5.6% vs 61.8±14.5%, NS). Endothelium-independent vasodilation was similar in all the groups. Only ALK increased P-eNOS/eNOS expression (+44% vs before treatment, p&lt;0.05). The markers of oxidative stress were similar in both groups before and after treatment. In conclusion ALK improved endothelial function and induced vasodilation in resistance arteries from diabetic and hypertensive patients.

Abstract P256: Structural Alterations of Subcutaneous Small Resistance Arteries in Resistant Hypertension

Background: It is was suggested that, in resistant hypertension, the presence of particularly pronounced microvascular alterations may contribute to explain the relative lack of response to treatment Patients and Methods: We investigated a population of 94 treated essential hypertensive patients. Secondary forms of hypertension were excluded, and in all patients a 24-hour blood pressure monitoring was performed in order to exclude a white coat effect. In all patients, we evaluated small resistance arteries morphology by a wire micromyographic approach. Subcutaneous tissue was obtained by local biopsy or during election surgery and subcutaneous small resistance arteries were dissected and mounted on a myograph; the media to lumen ratio (M/L) was then measured. We subdived our patents according to the presence or not of resistant hypertension (clinic blood pressure values above 140/90 mm Hg despite administration of three antihypertensive agent including a diuretic and 24-hour blood pressure values &gt;130/80 mm Hg). Sixteen patients had true resistant hypertension, and were compared with the remaining 78 patients with non-resistant hypertension. Results: are summarized in the Table, The two groups were also different in terms of mean age (57±12 vs. 67±7 years, p=0.016 and pulse pressure/stroke volume, a rough index of large artery distensibility: 0.63±0.31 vs. 0.90±0.33, p=0.02). Conclusion: Hypertensive patients with true resistant hypertension have greater microvascular structural alterations compared with non-resistant hypertensive patients. This could partly explain the resistance to treatment and the high cardiovascular risk observed in these patients.

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Objective: Obesity is a strong risk factor for hypertension but the mechanism linking obesity to hypertension is not fully elucidated. In obesity, circulating concentrations of adiponectin are decreased and hypoadiponectinamia has in some but not all studies been associated with increased risk of hypertension. Due to this inconsistency, we decided to study adiponectin from 2 aspects in a cross-sectional in vivo study and in an experimental in vitro study. Design and method: In the cross-sectional study, 103 men with body mass index (BMI) > = 30.0 kg/m2 were studied; 63 had 24-hr ambulatory blood pressure (AMBP) > = 130/80 mmHg (OHT) and 40 had 24-hr AMBP < 130/80 mmHg (ONT). As controls, we studied 27 men with BMI between 20.0 and 24.9 kg/m2 and 24-hr AMBP < 130/80 mmHg (LNT). Serum concentrations of adiponectin and body composition using dual energy X-ray absorptiometry scanning were determined. In the in vitro study, the direct vasomotor response of adiponectin was tested on subcutaneous resistance arteries from human abdominal adipose tissue. Results: The 2 obese groups had lower serum adiponectin concentrations compared with LNT (P < 0.01) (median (interquartile range): OHT 6.5 (5.1–8.3) mg/L; ONT 6.6 (5.2–7.8) mg/L; and LNT 9.4 (6.7–12.4) mg/L, but there was no significant difference in adiponectin concentrations (or body composition) between OHT and ONT (P = 0.67), although OHT had much higher 24-hr systolic AMBP (mean ± SD: 137 ± 11 mm Hg vs. 117 ± 6 mm Hg, P < 0.001) and much higher 24-hr diastolic AMBP (mean ± SD: 83 ± 6 mm Hg vs. 73 ± 4 mm Hg, P < 0.001) compared with ONT. In the in vitro study, adiponectin neither had any direct vasodilatory effect nor did adiponectin affect angiotensin II stimulated vasoconstriction. Conclusions: Despite large differences in 24-hr AMBP (and comparable body compostion), obese hypertensive men had similar serum concentrations of adiponectin as obese normotensive men, and furthermore adiponectin did not show any vasodilatory properties in human subcutaneous resistance arteries. Thus overall, our findings do not support a major role, if any, for adiponectin in human hypertension.

Abstract 379: Circulating T Regulatory Lymphocytes and Microvascular Structural Alterations in Hypertensive Patients and Normotensive Subjects

Background and Methods. Different components of the immune system, including innate immunity and adaptive immunity (T effector lymphocytes and T regulatory lymphocytes: Tregs) may be involved in the development of hypertension. In addition, it was recently demonstrated that Tregs may prevent angiotensin II-induced vascular injury/inflammation, while Th1, Th2 and Th17 may play a role in the progression of vascular remodeling. However, no data are available in human beings about relationships between T-lymphocyte subtypes and microvascular structure. In the present, preliminary study, we enrolled 7 normotensive subjects and 3 hypertensive patients undergoing an election surgical intervention. No sign of local or systemic inflammation was present in any subjects or patients. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph, morphological parameter were measured, in particular media to lumen ratio (M/L) was calculated. A peripheral blood sample was obtained before surgery for assessment of T lymphocytes subpopulations by flow cytometry. Results are summarized in the Table. RTE: recent thymus emigrant; naïve: no contact with antigens, TDEM: highly antigen-experienced cells that assume pro-apoptotic properties. Additional significant positive correlations were observed between M/L and Th17 effector lymphocytes: r=0.67, p&lt;0.05). Conclusion Our data suggest that some lymphocytes subpopulations may be related to microvascular remodeling, and open the possibility to regress small artery remodeling in human by specific drug modulation of lymphocyte subtypes.

Effects of Recombinant Human Erythropoietin on Resistance Artery Endothelial Function in Stage 4 Chronic Kidney Disease

BackgroundRecent studies have raised concern about the safety of erythropoiesis‐stimulating agents because of evidence of increased risk of hypertension and cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients. In the present study, we investigated the effects of recombinant human erythropoietin (EPO) on endothelial function of gluteal subcutaneous resistance arteries isolated from 17 stage 4 patients (estimated glomerular filtration rate 21.9±7.4 mL/min per 1.73 m2) aged 63±13 years.Methods and ResultsArteries were mounted on a pressurized myograph. EPO impaired endothelium‐dependent relaxation in a concentration‐dependent manner. The maximal response to acetylcholine with EPO at 1, 10, and 20 IU/mL was reduced by 12%, 34%, and 43%, respectively, compared with the absence of EPO (P<0.001). EPO‐induced endothelial dysfunction was significantly associated with carotid stiffness and history of cardiovascular events. EPO had no effect on norepinephrine‐induced vasoconstriction or sodium nitroprusside–induced relaxation. ABT‐627, an endothelin type A receptor antagonist, and tempol, a superoxide dismutase mimetic, partially reversed the altered endothelial function in the presence of EPO (P<0.01). Increased expression of endothelin‐1 was found in the vessel wall after incubation with EPO.ConclusionsEPO alters endothelial function of resistance arteries in CKD patients via a mechanism involving in part oxidative stress and signaling through an endothelin type A receptor. EPO‐induced endothelial dysfunction could contribute to deleterious effects of EPO described in large interventional trials.

Comparison of the Intrinsic Vasorelaxant and Inotropic Effects of the Antiarrhythmic Agents Vernakalant and Flecainide in Human Isolated Vascular and Cardiac Tissues

This study explored the intrinsic vasorelaxant and inotropic effects of the mixed potassium and sodium channel blocker atrial antiarrhythmic vernakalant and the class IC antiarrhythmic agent flecainide in human isolated subcutaneous resistance artery and in ventricular trabecular muscle preparations. At test concentrations encompassing free plasma concentrations associated with clinical efficacy for conversion of atrial fibrillation, vernakalant (1-10 μM) displayed no significant direct effects on human resistance artery tone or ventricular contractility. In contrast, tested at equimolar concentrations, flecainide significantly reduced peak isometric contractile force (10 μM) and maximal rates of force development and decline (3 and 10 μM) in the human ventricular muscle preparation while displaying no significant effect on human resistance artery tone. The lack of effects of vernakalant on human resistance artery tone and ventricular muscle contractile function suggests that direct vasorelaxant and inotropic effects do not underlie the rare hypotensive events observed clinically with vernakalant, raising the possibility that secondary (eg, reflex) effects may mediate these events. The demonstration of negative inotropic effects with flecainide in the human ventricular muscle preparations in the absence of an effect on resistance artery tone suggests that the hemodynamic effects of flecainide observed clinically result primarily from direct negative inotropic effects.

Circulating endothelial progenitor cells, microvascular density and fibrosis in obesity before and after bariatric surgery

It is not known whether, in obesity, the capillary density or the number of circulating endothelial progenitor cells (EPCs) are reduced, or whether fibrosis of small vessels is also present. In addition, possible effects of weight reduction on these parameters have never been evaluated. Therefore, we investigated EPCs and capillary density in 25 patients with severe obesity, all submitted to bariatric surgery, and in 18 normotensive lean subjects and 12 hypertensive lean patients as controls. All patients underwent a biopsy of subcutaneous fat during bariatric surgery. In five patients, a second biopsy was obtained after consistent weight loss, about 1 year later, during a surgical intervention for abdominoplasty. EPCs and capillary density were reduced in obesity, and EPCs were significantly increased after weight reduction. Vascular collagen content was clearly increased in obese patients. No significant difference in vascular collagen was observed between normotensive obese patients and hypertensive obese patients. After pronounced weight reduction, collagen content was nearly normalized. No difference in stress–strain relation was observed among groups or before and after weight loss. In conclusion, our data suggest that microvascular rarefaction occurs in obesity. EPCs were significantly reduced in obese patients. Pronounced weight loss induced by bariatric surgery seems to induce a significant improvement of EPC number, but not of capillary rarefaction. A pronounced fibrosis of subcutaneous small resistance arteries is present in obese patients, regardless of the presence of increased blood pressure values. Consistent weight loss induced by bariatric surgery may induce an almost complete regression of microvascular fibrosis.

Mechanisms of endothelial dysfunction in resistance arteries from patients with end-stage renal disease.

The study focuses on the mechanisms of endothelial dysfunction in the uremic milieu. Subcutaneous resistance arteries from 35 end-stage renal disease (ESRD) patients and 28 matched controls were studied ex-vivo. Basal and receptor-dependent effects of endothelium-derived factors, expression of endothelial NO synthase (eNOS), prerequisites for myoendothelial gap junctions (MEGJ), and associations between endothelium-dependent responses and plasma levels of endothelial dysfunction markers were assessed. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxation was impaired in uremic arteries after stimulation with bradykinin, but not acetylcholine, reflecting the agonist-specific differences. Diminished vasodilator influences of the endothelium on basal tone and enhanced plasma levels of asymmetrical dimethyl L-arginine (ADMA) suggest impairment in NO-mediated regulation of uremic arteries. eNOS expression and contribution of MEGJs to EDHF type responses were unaltered. Plasma levels of ADMA were negatively associated with endothelium-dependent responses in uremic arteries. Preserved responses of smooth muscle to pinacidil and NO-donor indicate alterations within the endothelium and tolerance of vasodilator mechanisms to the uremic retention products at the level of smooth muscle. We conclude that both EDHF and NO pathways that control resistance artery tone are impaired in the uremic milieu. For the first time, we validate the alterations in EDHF type responses linked to kinin receptors in ESRD patients. The association between plasma ADMA concentrations and endothelial function in uremic resistance vasculature may have diagnostic and future therapeutic implications.

Measurement of oxidative stress and endothelial dysfunction in patients with hypopituitarism and severe deficiency adult growth hormone deficiency

Patients with adult GH deficiency (AGHD) have a high cardiovascular risk and probably an alteration of the oxidative balance, although evidence is lacking. To evaluate the presence of endothelial dysfunction and oxidative stress in patients with AGHD. Biochemical parameters of oxidative stress and endothelial dysfunction were compared in 25 patients with previously untreated AGHD and 25 healthy controls matched by age and sex. Multivariate analysis was performed to identify independent predictors of oxidative stress. Vascular function of subcutaneous resistance arteries was also analyzed by means of wire myography in 7 patients with untreated AGHD and in 7 healthy controls with similar characteristics. The values of C-reactive protein, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α), were higher in the AGHD group (4.6 vs. 0.2 μg/L, P = 0.02; 5.6 vs. 1.2 pg/mL, P = 0.001; 6.7 vs. 2.1 pg/mL, P = 0.04; respectively). The levels of type-1 vascular cell adhesion molecule, total anti-oxidant state, oxidized LDL (LDL-ox) were also greater in AGHD patients (678 vs. 423 ng/mL, P = 0.004; 1235.6 vs. 1002.3 μmol/L, P = 0.01; 172.2.5 vs. 42.3 ng/mL, P = 0.02; respectively). Nitric oxide (NO), reduced glutathione (GSH) and reduced/oxidized glutathione ratio (GSH/GSSG) values were lower than controls (18.7 vs. 31.6 mmol/mg protein, P = 0.01; 372.2 vs. 756.2 μmol/L, P = 0.03; 17.2 vs. 38.4, P = 0.04; respectively). Multiple regression analysis showed that AGHD was an independent predictor of increased LDL-ox (P = 0.002) and decreased GSH (P = 0.000). Furthermore, the degree of vascular relaxation to repeated exposure of acetylcholine was lower in AGHD (P = 0.025). Patients with AGHD have an increased degree of oxidative stress and endothelial dysfunction that could already be present in early stages of the disease. Studies with a greater number of patients are needed in order to confirm our findings.

Recombinant human erythropoietin alters subcutaneous resistance artery endothelial function through a mechanism involving oxidative stress and endothelin-1 in patients with stage 4 chronic kidney disease

Recombinant human erythropoietin alters subcutaneous resistance artery endothelial function through a mechanism involving oxidative stress and endothelin-1 in patients with stage 4 chronic kidney disease

Impaired recovery of intracellular calcium and force after activation in isolated myometrial and subcutaneous resistance arteries from women with preeclampsia

Preeclampsia is a major cause of maternal and perinatal morbidity and mortality, but its cause is poorly understood. This study investigated whether there is an abnormality of intracellular calcium ([Ca2=]i) and tension during recovery from activation in isolated resistance arteries in preeclampsia and investigated the underlying mechanisms. Subcutaneous and myometrial resistance arteries from preeclamptic, normotensive pregnant and nonpregnant women were mounted on an isometric myograph and loaded with fura-2 to allow simultaneous measurement of force and [Ca2+]i. Arteries were activated by a high-potassium solution or noradrenaline, and the rate of decline in force and [Ca2+]i examined following washout. Basal tone and [Ca2+]i and rise in force and [Ca2+]i induced by high-potassium solution did not differ between groups but the rate of decline after washout was significantly slowed in both subcutaneous and myometrial arteries from preeclamptic women as compared with normotensive pregnant or nonpregnant women. The rate of decline in force after noradrenaline was also slowed in arteries from preeclamptic women. In subcutaneous resistance arteries from nonpregnant women, removal of the endothelium did not affect the rate of decline in force after high-potassium solution. However, inhibition of the plasma membrane Ca ATPase with carboxyeosin mimicked the findings seen in preeclampsia. In contrast, inhibition of the sarcoplasmic endoreticulum Ca ATPase with cyclopiazonic acid had no effect on the rate of decline in force or [Ca2+]i. The rate of relaxation and decline in [Ca2+]i in resistance arteries are impaired in preeclampsia. This may be mediated by decreased activity of plasma membrane Ca2+ ATPase and could be a mechanism contributing to elevated peripheral resistance and raised blood pressure in preeclampsia.