Gunay Yetik-Anacak

Heat shock protein 90 inhibitors attenuate LPS-induced endothelial hyperpermeability

Endothelial hyperperme ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60(Src), phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and beta-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and beta-catenin expression, and inhibited activation of pp60(Src) and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of beta-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.

Gas what: NO is not the only answer to sexual function

The ability to get and keep an erection is important to men for several reasons and the inability is known as erectile dysfunction (ED). ED has started to be accepted as an early indicator of systemic endothelial dysfunction and subsequently of cardiovascular diseases. The role of NO in endothelial relaxation and erectile function is well accepted. The discovery of NO as a small signalling gasotransmitter led to the investigation of the role of other endogenously derived gases, carbon monoxide (CO) and hydrogen sulphide (H2S) in physiological and pathophysiological conditions. The role of NO and CO in sexual function and dysfunction has been investigated more extensively and, recently, the involvement of H2S in erectile function has also been confirmed. In this review, we focus on the role of these three sister gasotransmitters in the physiology, pharmacology and pathophysiology of sexual function in man, specifically erectile function. We have also reviewed the role of soluble guanylyl cyclase/cGMP pathway as a common target of these gasotransmitters. Several studies have proposed alternative therapies targeting different mechanisms in addition to PDE‐5 inhibition for ED treatment, since some patients do not respond to these drugs. This review highlights complementary and possible coordinated roles for these mediators and treatments targeting these gasotransmitters in erectile function/ED.

Methylglyoxal causes endothelial dysfunction: the role of endothelial nitric oxide synthase and AMP-activated protein kinase α.

Abstract Background: Methylglyoxal is a major precursor in the formation of advanced glycation end products and is associated with the pathogenesis of diabetes-related vascular complications. The aim of this study was to evaluate whether methylglyoxal induces endothelial dysfunction and to determine the contributors involved in this process. Methods: Rat thoracic aortic rings were treated for 24 h with 100 μM methylglyoxal by using an organ culture method. A cumulative dose-response curve to acetylcholine was obtained to determine endothelium-dependent relaxation. The protein levels of endothelial nitric oxide synthase (eNOS) and its phosphorylated form at the serine 1177 site [p-eNOS (Ser1177)], heat shock protein 90 (Hsp90), AMP-activated protein kinase α (AMPKα) and its phosphorylated form at the threonine 172 site [p-AMPKα (Thr172)] were evaluated. Superoxide production was determined by lucigenin-chemiluminescence. Results: Treatment with 100 μM methylglyoxal for 24 h decreased acetylcholine-induced vascular relaxation. The levels of eNOS and p-eNOS (Ser1177) were reduced while no effect on Hsp90 was observed. Levels of p-AMPKα (Thr172) were significantly decreased without any change in total AMPKα protein levels. Superoxide level was not affected by methylglyoxal treatment. Conclusions: In rat aortic rings, methylglyoxal determines a reduction in endothelium-dependent relaxation. This effect seems to be mediated via a reduction in p-eNOS (Ser1177) and p-AMPKα (Thr172).

Angiotensin, Bradykinin and the Endothelium

Angiotensins and kinins are endogenous peptides with diverse biological actions; as such, they represent current and future targets of therapeutic intervention. The field of angiotensin biology has changed significantly over the last 50 years. Our original understanding of the crucial role of angiotensin II in the regulation of vascular tone and electrolyte homeostasis has been expanded to include the discovery of new angiotensins, their important role in cardiovascular inflammation and the development of clinically useful synthesis inhibitors and receptor antagonists. While less applied progress has been achieved in the kinin field, there are continuous discoveries in bradykinin physiology and in the complexity of kinin interactions with other proteins. The present review focuses on mechanisms and interactions of angiotensins and kinins that deal specifically with vascular endothelium.

Hydrogen sulfide compensates nitric oxide deficiency in murine corpus cavernosum

Erectile dysfunction (ED) is considered as a marker for cardiovascular diseases. Nitric oxide (NO) deficiency is the major cause of erectile dysfunction (ED). The role of hydrogen sulfide (H2S) in erection has recently been recognized and is receiving attention as a pharmacological target. Several studies have focused on the effect of H2S on NO-dependent relaxation, but the role of NO on H2S in penile tissue has not been studied yet. Unlike NO, H2S is mainly synthesized from smooth muscle cells rather than endothelial cells. We hypothesized that H2S may compensate for the decreased NO bioavailability and may be beneficial in severe ED where endothelial dysfunction is present. Thus we studied the effect of NO deficiency on H2S formation and vasorelaxation induced by l-cysteine, which is the substrate of the H2S producing enzymes in mice corpus cavernosum (MCC). NO deficiency induced by Nω-Nitro-l-arginine (L-NNA) was confirmed by the inhibition of acetylcholine-induced relaxation. l-cysteine, the substrate for the endogenous H2S production, caused a concentration-dependent relaxation that was reduced by CBS/CSE inhibitor aminooxyacetic acid (AOAA) in MCC strips. L-NNA caused a significant increase in l-cysteine-induced relaxation, and this effect was reversed by AOAA. On the contrary, no change in relaxation to NaHS (exogenous H2S donor) in MCC was observed. L-NNA increased H2S formation stimulated by l-cysteine in wild type MCC but not in CSE-/- mice. In parallel, the expression of both cysthationine γ lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3-MST) was increased, whereas cysthationine-β synthase (CBS) was decreased in eNOS-/- MCC. We conclude that H2S plays a compensatory role in the absence of NO by enhancing the relaxation induced by endogenous H2S through CSE and 3-MPST in MCC, without altering downstream mechanisms. We suggest that H2S-targeting drugs may provide the maintenance of compensatory treatment in ED patients. This may be more relevant in ED with severe endothelial dysfunction, as H2S is mainly derived from smooth muscle.

Resveratrol Stimulates Hydrogen Sulfide (H2S) Formation to Relax Murine Corpus Cavernosum

INTRODUCTION Resveratrol (RVT) found in red wine protects against erectile dysfunction and relaxes penile tissue (corpus cavernosum) via a nitric oxide (NO) independent pathway. However, the mechanism remains to be elucidated. Hydrogen sulfide (H2 S) is a potent vasodilator and neuromodulator generated in corpus cavernosum. AIMS We investigated whether RVT caused the relaxation of mice corpus cavernosum (MCC) through H2 S. METHODS H2 S formation is measured by methylene blue assay and vascular reactivity experiments have been performed by DMT strip myograph in CD1 MCC strips. MAIN OUTCOME MEASURES Endothelial NO synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1 mM) or H2 S inhibitor aminooxyacetic acid (AOAA, 2 mM) which inhibits both cystathionine-β-synthase (CBS) and cystathionine-gamma-lyase (CSE) enzyme or combination of AOAA with PAG (CSE inhibitor) has been used in the presence/absence of RVT (0.1 mM, 30 min) to elucidate the role of NO or H2 S pathways on the effects of RVT in MCC. Concentration-dependent relaxations to RVT, L-cysteine, sodium hydrogen sulfide (NaHS) and acetylcholine (ACh) were studied. RESULTS Exposure of murine corpus cavernosum to RVT increased both basal and L-cysteine-stimulated H2 S formation. Both of these effects were reversed by AOAA but not by L-NNA. RVT caused concentration-dependent relaxation of MCC and that RVT-induced relaxation was significantly inhibited by AOAA or AOAA + PAG but not by L-NNA. L-cysteine caused concentration-dependent relaxations, which are inhibited by AOAA or AOAA + PAG significantly. Incubation of MCC with RVT significantly increased L-cysteine-induced relaxation, and this effect was inhibited by AOAA + PAG. However, RVT did not alter the effect of exogenous H2 S (NaHS) or ACh-induced relaxations. CONCLUSIONS These results demonstrate that RVT-induced relaxation is at least partly dependent on H2 S formation and acts independent of eNOS pathway. In phosphodiesterase 5 inhibitor (PDE-5i) nonresponder population, combination therapy with RVT may reverse erectile dysfunction via stimulating endogenous H2 S formation.

The role of eNOS on the compensatory regulation of vascular tonus by H2S in mouse carotid arteries

The gasotransmitter nitric oxide (NO) has an important role in vascular function and a decrease in its bioavailability is accepted as a main pathological mechanism for cardiovascular diseases. However, other gasotransmitters such as hydrogen sulfide (H2S) are also generated by the endothelium and can also affect vascular tone and a crosstalk may exist between H2S and NO. We therefore investigated the consequences of deficiency, replacement or overexpression of endothelial nitric oxide synthase (eNOS) on H2S-induced vascular responses in murine carotid arteries. In pre-contracted carotid arteries from wild-type (WT) mice, l-cysteine elicited relaxation that was inhibited by the H2S synthesis inhibitor amino-oxyacetic acid (AOAA). Genetic deletion of eNOS increased l-cysteine-induced relaxation compared to WT, but the replacement of eNOS by adenoviral transfection or H2S synthesis inhibition by AOAA reversed it. Furthermore, eNOS deletion did not alter NaHS-induced relaxation in carotid arteries while eNOS overexpression/replacement increased NaHS-induced relaxation responses in carotid arteries from WT or eNOS-/-. We suggest that, endogenously produced H2S can compensate for impaired vasodilatory responses in the absence of NO to maintain vascular patency; while, eNOS abundance can limit endogenous H2S-induced vascular responses in mice carotid arteries. Our result suggests that endogenous vs. exogenous H2S-induced relaxation are reciprocally regulated by NO in mice carotid arteries.

Cycloartane-type sapogenol derivatives inhibit NFκB activation as chemopreventive strategy for inflammation-induced prostate carcinogenesis

HIGHLIGHTS9,19‐cyclolanostane derivatives starting from cycloastragenol were synthesized.Five analogs were selected to be screened for their anti‐inflammatory properties.The compounds inhibited NF&kgr;B activation.They were shown to be promising anti‐inflammatory agents.The compounds are noteworthy candidates for prostate cancer chemoprevention. ABSTRACT Chronic inflammation is associated to 25% of cancer cases according to epidemiological data. Therefore, inhibition of inflammation‐induced carcinogenesis can be an efficient therapeutic approach for cancer chemoprevention in drug development studies. It is also determined that anti‐inflammatory drugs reduce cancer incidence. Cell culture‐based in vitro screening methods are used as a fast and efficient method to investigate the biological activities of the biomolecules. In addition, saponins are molecules that are isolated from natural sources and are known to have potential for tumor inhibition. Studies on the preparation of analogues of cycloartane‐type sapogenols (9,19‐cyclolanostanes) have so far been limited. Therefore we have decided to direct our efforts toward the exploration of new anti‐tumor agents prepared from cycloastragenol and its production artifact astragenol. The semi‐synthetic derivatives were prepared mainly by oxidation, condensation, alkylation, acylation, and elimination reactions. After preliminary studies, five sapogenol analogues, two of which were new compounds (2 and 3), were selected and screened for their inhibitory activity on cell viability and NF&kgr;B signaling pathway activity in LNCaP prostate cancer cells. We found that the astragenol derivatives 1 and 2 as well as cycloastragenol derivatives 3, 4, and 5 exhibited strong inhibitory activity on NF&kgr;B signaling leading the repression of NF&kgr;B transcriptional activation and suppressed cell proliferation. The results suggested that these molecules might have significant potential for chemoprevention of prostate carcinogenesis induced by inflammatory NF&kgr;B signaling pathway.

Apoptotic Effect of STAMP2 Transfection to PC3 Cells and Caspase Inductionwith Hydrogen Peroxide Incubation at Lncap Cells- Role of P53

STAMP genes STAMP1/STEAP2 and STAMP2/STEAP4 are expressed in androgen receptor positive-prostate cancer cell line LNCaP, and this regulation was reported, previously. The androgen induction was done at LNCaP cells. STAMP2/STEAP4 was silenced for 1, 2 and 5 days. Using real-time RT-PCR, an apoptosis panel including proapoptotic and/or apoptotic genes was investigated for gene expression alterations in STAMP expressing LNCaP cells in comparison to P53-null PC3 cells in which STAMP2 gene was transfected. In addition, promoter region of the STAMP2 gene was analyzed and putative p53 and androgen receptor (AR) response elements were identified. Thus, p53 gene was silenced using siRNA approach and was induced by hydrogen peroxide. Taken together, prostate-specific STAMP2 gene and its regulation with the p53- and caspase-related pathway was characterized. These results will shed light on understanding the control of proliferation in neoplastic transformation in prostate cancer.