Karolina Kublickiene

Effects in Postmenopausal Women of Estradiol and Medroxyprogesterone Alone and Combined on Resistance Artery Function and Endothelial Morphology and Movement

CONTEXT Endothelial dysfunction in resistance arteries after menopause is important for the development of high blood pressure and cardiovascular disease. OBJECTIVES Our objectives were to study the effects of different hormone replacement therapies on the function and morphology of isolated resistance arteries, and to look for their mechanistic basis. DESIGN AND SETTING This was a randomized, placebo-controlled double-blind study in a University hospital, along with laboratory based studies. PATIENTS AND INTERVENTIONS We isolated resistance arteries in sc biopsies from 55 postmenopausal women before and after 3-month therapy with estradiol (E2), medroxyprogesterone acetate (MPA), E2 plus MPA, or placebo. In addition, we studied isolated human endothelial cells. MAIN OUTCOME MEASURES AND RESULTS Artery flow-mediated dilatation was augmented after treatment with E2 or E2 plus MPA, whereas MPA or placebo had no effect. Pressure-induced myogenic tone was reduced by E2 plus MPA, whereas it was unchanged in the other groups. Scanning microscopy showed that E2 improved endothelial cell morphology and decreased signs of endothelial apoptosis, but the addition of MPA impaired these events. E2, MPA, or the combination all increased the expression and phosphorylation of the actin-binding protein, moesin and of the focal adhesion complex controller, focal adhesion kinase, and induced the rearrangement of cytoskeletal actin and vinculin fibers. All treatments promoted endothelial cell horizontal migration, with E2 inducing the strongest effect. CONCLUSIONS This study suggests that hormone replacement therapy with estrogens or in combination with MPA may benefit the function of resistance arteries and may preserve the morphological integrity of endothelial cells by regulatory actions on the cytoskeleton.

The oestrogen receptor β contributes to sex related differences in endothelial function of murine small arteries via EDHF

Sex related differences in cardiovascular function have been reported in oestrogen receptor β knockout (ERβKO) mice. In this study we examined the role of endothelium‐derived hyperpolarizing factor (EDHF) in differences in small artery endothelial function between ERβKO and wild‐type (WT) mice. Small femoral arteries were isolated from ERβKO and WT mice and mounted on a wire myograph. Concentration–response curves to ACh were compared before and after incubation with inhibitors of nitric oxide (NO) and prostacyclin (PGI2) synthesis. Comparison of the expression of the principal vascular connexins (Cx37, 40 and 43), implicated in EDHF‐mediated dilatation were undertaken by immunohistochemistry. Vascular ultrastructure was studied by transmission and scanning electron microscopy. ACh‐induced relaxation of arteries (< 200 μm internal diameter) was greater in WT females versus males and was attributable to a greater EDHF component of relaxation. This sex difference was absent in ERβKO mice. Arteries from ERβKO males (but not females) were more sensitive to ACh compared to WT. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF‐mediated responses was confirmed in male arteries. The absence of ERβ had no influence on expression of main Cx subtypes within vascular wall or on ultrastructure and morphology of the endothelium. The data suggest that in WT male mice, ERβ reduces EDHF‐mediated relaxation through gap junction communication.

Diverse Mechanisms of Endothelium-Derived Hyperpolarizing Factor-Mediated Dilatation in Small Myometrial Arteries in Normal Human Pregnancy and Preeclampsia

This ex vivo study focuses on the mechanisms of endothelium-dependent dilatation in the uterine circulation of normal pregnancy (n = 12) and in women with preeclampsia (n = 12). Arteries (internal diameter, ∼250 μm) isolated by myometrial biopsy from women undergoing planned cesarean delivery or delivery as a result of the deterioration of preeclampsia were studied using a wire myograph. Bradykinin-induced dilatation was assessed in the presence and/or absence of pharmacological inhibitors to determine the contribution of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF), as well as that of EDHF-mediated pathways such as myoendothelial gap junctions (MEGJs) and products of arachidonic acid, H2O2 and cytochrome P450 2C9 (CYP2C9). Transmission electron microscopy was used to visualize morphological prerequisites for MEGJs. In normal pregnancy, EDHF through MEGJs appeared to be a predominant mediator conferring endothelium-dependent relaxation in small myometrial arteries. In preeclampsia, bradykinin-induced relaxation was reduced via compromised EDHF-type responses, in which the contribution of MEGJs became negligible. The attenuated role of MEGJs to endothelium-dependent relaxation was partly compensated through the contribution of H2O2 or other endothelium-derived relaxing factors. CYP2C9 products of arachidonic acid had no effect on EDHF-type relaxation in arteries of women with normal pregnancy or with preeclampsia. We suggest that EDHF-type responses via MEGJs are primarily targeted in small myometrial arteries in women with preeclampsia. This could significantly contribute to the impaired uteroplacental blood flow in this disorder.

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.

Circulating levels of autoantibodies to oxidized low-density lipoprotein and C-reactive protein levels correlate with endothelial function in resistance arteries in men with coronary heart disease.

The association between C-reactive protein (CRP) and future cardiovascular risk has been of particular interest during recent years. Oxidized low-density lipoprotein (ox-LDL) is another marker linking the immune system with the atherogenic process. The aim of this study was to examine whether ox-LDL and CRP were associated with endothelial function in peripheral resistance arteries. Twenty-five men with a previous hospital-diagnosed myocardial infarction were enrolled in the study. The exclusion criterion was a history of diabetes mellitus. IgG and IgM autoantibodies to malonyldialdehyde low-density lipoprotein (MDA-LDL) and high-sensitivity CRP (hs-CRP) were measured. Flow-mediated dilatation was measured in isolated resistance arteries from subcutaneous fat biopsies. Endothelial function test reflecting the maximum vessel dilatation in male subjects was inversely related to MDALDL IgG autoantibody levels (r = −0.6 and P = 0.003). Comparison of hs-CRP levels and of maximum vessel dilatation in males revealed also an inverse relation (r= −0.4 and P = 0.04). In conclusion, a clear correlation exists between flow-mediated dilatation in subcutaneous resistance arteries and plasma levels of MDA-LDL IgG autoantibody and CRP in male patients with coronary heart disease.

The role of estrogen receptor subtypes for vascular maintenance.

Protective role of estrogens (E2) against cardiovascular disease has been appreciated for many years until the equivocal results of cardiovascular outcomes in clinical trials on hormone replacement therapy were reported. Although new ongoing trials aim to resolve these discrepancies, it is obvious that cardiovascular effects of E2 are complex and diverse. To understand further the cardiovascular effects of E2, the detailed knowledge on the specific role of both classical estrogen receptor (ER) subtypes and G protein-coupled receptor-30 in the vasculature are of importance. In this article, we review the current knowledge about the pattern of ERα and ERβ expression in human vasculature, the genomic and non-genomic cardiovascular effects of E2versus subtype selective ERα and ERβ stimulation on isolated arteries and in different knockout animal models. The results indicate that although ERα and ERβ are expressed in the endothelium and media of human arteries, there is no definite evidence for predominant expression of one over another, the pattern depends on vascular bed, sex and diseased condition. Data from the experiments on isolated arteries and in ER knockout animal models may indicate that activation of specific ER subtypes could provide additional cardiovascular protective effects. However, a clear role for each ERs have to be finalised with focus on mechanisms and by exploring the potential of ERs-selective agonists for clinical utility.

Beneficial vasoactive endothelial effects of fluvastatin: focus on prostacyclin and nitric oxide

Statins are believed to exert beneficial effects against cardiovascular disease beyond correction of dyslipidemia. There are however still very sparse data on how individual statins interact with the production of vasoactive eicosanoids and nitric oxide (NO) in human vascular endothelial cells. Here we have determined how fluvastatin affects the mRNA expression of genes associated with vascular reactivity as well as the formation of two major vasodilators, prostacyclin (PGI2) and NO, in human endothelial cells. Also, the influence of fluvastatin on arterial resistance was assessed in isolated small arteries. We show that the promoter activity of prostacyclin synthase (PTGIS), the mRNA expression of PTGIS and endothelial nitric oxide synthase (eNOS), and the production of PGI2 and NO are significantly induced by fluvastatin. Also, strong rapid dilatation ex vivo was observed, with the equal contribution of PGI2 and NO. Our findings in cell culture experiments and in isolated human arteries indicate that fluvastatin-evoked endothelium-derived vasodilator production may confer protection of the endothelial cells via both acute and long-term effects of fluvastatin treatment. If these effects take place in vivo, we suggest a protective pleiotropic role of fluvastatin on the cardiovascular system, particularly at the level of the vascular endothelium, to ameliorate the process of atherogenesis and in the acute manner to reduce vascular tone.

Expression of heat shock proteins and nitrotyrosine in small arteries from patients with coronary heart disease

Heat shock proteins (HSPs) have been suggested to play an important role in the pathogenesis of cardiovascular disease; however, their levels in resistance arteries and their role as useful markers for endothelial dysfunction are not well known. In this paper we studied the levels of HSP90, HSP70, HSP60, HSP27, and of the oxidative stress marker nitrotyrosine (NT) in isolated small subcutaneous arteries from female and male patients with coronary heart disease (CHD) and compared them with healthy controls. HSPs and NT levels were analyzed by immunohistochemistry (IHC) with streptavidin-biotin complex and 3,3′-diaminobenzidine (DAB) staining. The results were assessed with a semi-quantitative method. The study showed lower levels of HSP90 in arteries from both male and female patients when compared to the healthy controls, while levels of HSP70 were lower only in male patients versus controls. The levels of HSP60 and HSP27 did not show any significant difference in either the male or the female groups. NT levels were higher in the arteries from female patients as compared to controls. In conclusion, the present study strengthens the concept that HSPs may play an important role in the pathogenesis of CHD, and that at least two of them, HSP70 and HSP90, may have useful applications as markers of vascular dysfunction in resistance arteries.

Altered responsiveness of small uterine arteries in women with idiopathic menorrhagia

OBJECTIVE This study was undertaken to study vascular reactivity of small myometrial arteries in women with idiopathic menorrhagia. STUDY DESIGN Small myometrial arteries were isolated from 6 patients with idiopathic menorrhagia and 4 controls. The contractile responses to thromboxane mimetic (U46619) and endothelin-1 were assessed before and after incubation with N(w)-nitro-L arginine methyl ester alone or in combination with indomethacin (Indo). Endothelium-dependent dilation to bradykinin and basal tension were compared before and after incubation with N(w)-nitro-L arginine methyl ester alone, or with N(w)-nitro-L arginine methyl ester in combination with indomethacin. RESULTS Constriction to endothelin-1 was enhanced in idiopathic menorrhagia arteries (P < .05). Idiopathic menorrhagia arteries demonstrated enhanced basal tension after incubation with N(w)-nitro-L arginine methyl ester, which was further exaggerated by indomethacin. NOS inhibition had no effect on basal tension in controls, but basal tension was enhanced after inhibition of cyclooxygenase-derived products (P < .05). Bradykinin-mediated dilation was significantly increased in idiopathic menorrhagia (P < .05). CONCLUSION The presence of functional alterations in small myometrial arteries could contribute to idiopathic menorrhagia.

Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue

Abstract Objective: Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue. Design: Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction. Results: In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction. Conclusion: ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.