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

New insights into the control of small artery function in human pregnancy and estrogen receptor beta knockout mice

Background: Available data clearly indicates functional and morphological differences between small and large arteries, and observations from studies on large arteries may not be applicable to understand the physiology of small arteries (~200-300mum) that actively participate in the regulation of peripheral vascular resistance, blood pressure and flow to target organs. These events confer cardiovascular adaptation to normal pregnancy (NP), however they are disturbed in preeclampsia (PE) and in estrogen receptor beta knockout (ERbetaKO) mice at a certain age. Aims: (1) To assess endothelial function and morphology with focus on the role and mechanisms of endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small subcutaneous arteries isolated from pregnant women with and without PE; (2) To estimate the predisposition for sex difference in blood pressure of ERbetaKO mice at the level of small arteries function with a focus on endothelium-dependent dilatation (EDHF) and adrenergic vasoconstriction. Methodology: Small subcutaneous arteries obtained from pregnant women and femoral arteries obtained from age-matched (14-22 weeks old) female and male wild type (WT, ERbeta +/+) and ERbetaKO mice were used in a wire-myography set-up for functional studies. Immunohistochemistry for connexins (Cx) and/or ER subtypes (as appropriate), as well as scanning and transmission electron microscopy techniques were also utilized for evaluation of small arteries morphology with particular focus on prerequisite for gap junction communications. Results and conclusions: (1) The overall endothelium-dependent response in arteries from pregnant women with and without PE was similar. However, EDHF-mediated relaxation was reduced in PE. The results demonstrated heterogeneity in the relative contribution of endothelium-derived factors and in the mechanisms responsible for the EDHF-mediated relaxation in PE. Gap junctions and/or H2O2 and/or cytochrome P450 epoxygenase metabolites of arachidonic acid appeared to be involved in the EDHF-mediated response in PE. In NP women, communication via gap junctions via Cx 43 represented a common pathway responsible for EDHF action. The link between morphological alterations within the vascular wall, and changes in the contribution of gap junctions to EDHFmediated relaxation of small arteries isolated from women with PE was suggested. (2) Endothelium-dependent relaxation in arteries (<200mum of internal diameter) was greater in WT females vs. males, and this was attributed to a greater EDHF component in the relaxation. This difference was absent in ERbetaKO mice. The data suggests that in WT male mice ERbeta reduces EDHFmediated relaxation. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF-mediated responses was indicated in male arteries. However, the absence of ERbeta had no influence on expression of the main Cx subtypes within the vascular wall or on the ultrastructure and morphology of the endothelium. The increased EDHF contribution to endotheliumdependent dilatation in ERbetaKO male mice vs. WT could not explain the hypertension observed in ERbetaKO animals. (3) Femoral arteries from ERbetaKO male mice demonstrated an enhancement of the contractile response to alpha1-adrenoceptor agonist (phenylephrine) that was accompanied by elevated basal tension attributable to endothelial factors. Contractile responses to the mixed adrenoceptor agonist, norepinephrine, were similar in ERbetaKO and WT mice; however the addition of beta-adrenoceptor inhibitor unmasked the enhancement of the underlying alpha1-adrenoceptor responsiveness pertinent to males. beta-Adrenoceptor-mediated dilatation was also enhanced in ERbetaKO vs. WT males. We suggest that ERbeta modifies the adrenergic control of small artery tone in males, but not in females. The alterations in the adrenergic modulation of small artery tone might commence the hypertension in ERbetaKO males. Significance: Heterogeneity in manifestation of functional and morphological signs of endothelial dysfunction at the level of small arteries in PE indicates a complexity and multifactor genesis of this pregnancy-related disorder. The relative importance of ERbeta for the control of small artery function found in males in the rodent model substantiates a gender-related approach for prevention and treatment of cardiovascular disease

Contractile response to NOS/COX inhibitors of arteries from controls (n = 26) <i>vs.</i> ESRD patients with (n = 32).

<p>*, <i>P</i><0.05 ESRD <i>vs.</i> controls.</p

Transmission electron images of arteries from ESRD patients.

<p>The lower magnification pictures (A,B) show an overview of the vascular wall with endothelium (End) and smooth muscle (SM) being separated by the internal elastic lamina (IEL). The areas denoted by the boxes are magnified and show the sites of intercellular contacts that could be considered as prerequisites of myoendothelial gap junctions (C, D). The width of the gap is ∼20 nm (C, arrow), ∼11 nm (D, arrow). Bar: (A) 2 µm; (B) 3 µm; (C) 0.1 µm; (D) 0,2 µm.</p

Endothelial nitric oxide synthase (eNOS) expression in arteries from controls (n = 6) and ESRD patients (n = 10).

<p>Endothelial nitric oxide synthase (eNOS) expression in arteries from controls (n = 6) and ESRD patients (n = 10).</p

Spearman rank correlation between plasma levels of asymmetrical dimethyl L-arginine (ADMA, µmol/L) and artery sensitivity to endothelium-dependent vasodilators (pEC₅₀, A, B) or vasoconstriction in response to NOS/COX inhibition (L-NAME+Indo, C, D) in ESRD patients (A, C) and controls (B, D).

<p>Spearman rank correlation between plasma levels of asymmetrical dimethyl L-arginine (ADMA, µmol/L) and artery sensitivity to endothelium-dependent vasodilators (pEC₅₀, A, B) or vasoconstriction in response to NOS/COX inhibition (L-NAME+Indo, C, D) in ESRD patients (A, C) and controls (B, D).</p

Concentration response curves to acetylcholine (ACh, A) and bradykinin (BK, B).

<p>Responses in physiological salt solution (PSS) and after incubation with <i>N</i>^ω-nitro-l-arginine methyl ester plus indomethacin alone (L-NAME+Indo) or together with 18α-glycyrrhetinic acid (L-NAME+Indo+18α-GA) in arteries from ESRD patients (n = 32 for ACh and n = 22 for BK) and controls (n = 23 for ACh and n = 17 for BK). * ESRD <i>vs.</i> controls, <i>P</i><0.05.</p