Pharmacological modulation of vascular ageing: A review from VascAgeNet.

Vascular ageing, characterized by structural and functional changes in blood vessels of which arterial stiffness and endothelial dysfunction are key components, is associated with increased risk of cardiovascular and other age-related diseases. As the global population continues to age, understanding the underlying mechanisms and developing effective therapeutic interventions to mitigate vascular ageing becomes crucial for improving cardiovascular health outcomes. Therefore, this review provides an overview of the current knowledge on pharmacological modulation of vascular ageing, highlighting key strategies and promising therapeutic targets. Several molecular pathways have been identified as central players in vascular ageing, including oxidative stress and inflammation, the renin-angiotensin-aldosterone system, cellular senescence, macroautophagy, extracellular matrix remodelling, calcification, and gasotransmitter-related signalling. Pharmacological and dietary interventions targeting these pathways have shown potential in ameliorating age-related vascular changes. Nevertheless, the development and application of drugs targeting vascular ageing is complicated by various inherent challenges and limitations, such as certain preclinical methodological considerations, interactions with exercise training and sex/gender-related differences, which should be taken into account. Overall, pharmacological modulation of endothelial dysfunction and arterial stiffness as hallmarks of vascular ageing, holds great promise for improving cardiovascular health in the ageing population. Nonetheless, further research is needed to fully elucidate the underlying mechanisms and optimize the efficacy and safety of these interventions for clinical translation.

The Effects of Novel Triazolopyrimidine Derivatives on H2S Production in Lung and Vascular Tonus in Aorta.

INTRODUCTION: Hydrogen sulfide (H2S), known as a third gasotransmitter, is a signaling molecule that plays a regulatory role in physiological and pathophysiological processes. Decreased H2S levels were reported in inflammatory respiratory diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. H2S donors or drugs that increase H2S have emerged as novel treatments for inflammatory respiratory diseases. We previously showed that resveratrol (RVT) causes vascular relaxation and antioxidant effects by inducing H2S production. In the current study, we synthesized a new molecule Cpd2, as an RVT analog. We examined the effect of Cpd2 and its precursor chalcone compound (Cpd1) on H2S formation under both healthy and oxidative stress conditions in the lung, as well as vascular relaxation in the aorta. METHODS: Cpd2 synthesized from Cpd1 with microwaved in basic conditions. H2S formation was measured by H2S biosensor in the mice lungs under both healthy and pyrogallol-induced oxidative stress conditions in the presence/absence of H2S synthesis inhibitor aminooxyacetic acid (AOAA). The effect of compounds on vascular tonus is investigated in mice aorta by DMT myograph. RESULTS: RVT and Cpd2 significantly increased l-cysteine (l-cys) induced-H2S formation in the lung homogenates of healthy mice, but Cpd1 did not. Superoxide anion generator pyrogallol caused a decrease in H2S levels in mice lungs and Cpd2 restored it. Inhibition of Cpd2-induced H2S formation by AOAA confirmed that Cpd2 increases endogenous H2S formation in both healthy and oxidative stress conditions. Furthermore, we found that both Cpd1 and Cpd2 (10-8-10-4 M) caused vascular relaxation in mice aorta. DISCUSSION AND CONCLUSION: We found that Cpd2, a newly synthesized RVT analog, is an H2S-inducing molecule and vasorelaxant similar to RVT. Since H2S has antioxidant and anti-inflammatory effects, Cpd2 has a potential for the treatment of respiratory diseases where oxidative stress and decreased H2S levels are present.

Vascular ageing: moving from bench towards bedside.

Prevention of cardiovascular disease (CVD) remains one of the largest public health challenges of our time. Identifying individuals at increased cardiovascular risk at an asymptomatic, sub-clinical stage is of paramount importance for minimizing disease progression as well as the substantial health and economic burden associated with overt CVD. Vascular ageing (VA) involves the deterioration in vascular structure and function over time and ultimately leads to damage in the heart, brain, kidney, and other organs. Vascular ageing encompasses the cumulative effect of all cardiovascular risk factors on the arterial wall over the life course and thus may help identify those at elevated cardiovascular risk, early in disease development. Although the concept of VA is gaining interest clinically, it is seldom measured in routine clinical practice due to lack of consensus on how to characterize VA as physiological vs. pathological and various practical issues. In this state-of-the-art review and as a network of scientists, clinicians, engineers, and industry partners with expertise in VA, we address six questions related to VA in an attempt to increase knowledge among the broader medical community and move the routine measurement of VA a little closer from bench towards bedside.

Relaxation mechanisms of chloroform root extracts of Prangos heyniae and Prangos uechtritzii on mouse corpus cavernosum.

Erectile dysfunction (ED) is the inability to achieve/maintain an erection. Because of the side effects, interactions, or ineffectiveness of currently used drugs, novel drug discovery studies are ongoing. The roots of Turkish endemic plants Prangos uechtritzii and Prangos heyniae are traditionally used as aphrodisiacs in Anatolia and contain coumarin-like relaxant compounds. This study aims to reveal the relaxant effect mechanisms of chloroform root extracts of P. heyniae (Ph-CE) and P. uechtritzii (Pu-CE). Isolated organ bath experiments were performed on Swiss albino mouse corpus cavernosum by DMT strip myograph. Relaxant responses to extract (10-7 -10-4  g/ml) were obtained in the presence/absence of NO and H2 S synthesis inhibitors nitro-l-arginine methyl ester (l-NAME, 100 µM) and aminooxyacetic acid (AOAA, 10 mM) respectively. Sodium nitroprusside (SNP, 10-9 to 10-4  M) and Na2 S (10-6 to 3 × 10-3  M)-induced relaxations and CaCl2 (10-6 to 10-4  M), KCl (10-2.1 to 10-0.9  M) and phenylephrine (3 × 10-8 to 3 × 10-5  M)-induced contractions were taken in the presence/absence of the extracts (10-4  g/ml). Relaxations induced by Ph-CE but not by Pu-CE were inhibited in the presence of l-NAME and AOAA. Ph-CE increased Na2 S- and SNP-induced relaxations. Ph-CE and Pu-CE decreased the contractions of KCl, phenylephrine, and CaCl2 . It was concluded that NO and H2 S synthesis/downstream mechanisms play roles in relaxations of Ph-CE but not in Pu-CE-induced relaxations. Inhibition of calcium influx appears to be involved in the relaxant effect of Ph-CE and Pu-CE. Since the extracts act directly by relaxing smooth muscle or through H2 S as well as NO, they may be a potential therapeutic agent in diseases such as ED where the bioavailability of NO is impaired.

H2S releasing sodium sulfide protects against pulmonary hypertension by improving vascular responses in monocrotaline-induced pulmonary hypertension.

Pulmonary arterial hypertension is caused by complex structural and functional changes in the endothelial and smooth muscle cells of pulmonary arteries. Hydrogen sulfide (H2S), a gasotransmitter, can potentially treat pulmonary hypertension by relaxing the pulmonary arteries and decreasing bronchial pressure. Although the role of H2S in systemic circulation has been examined, the H2S levels in pulmonary arteries, the role of H2S in endothelium-dependent vasorelaxation and the L-cysteine/H2S pathway in monocrotaline-induced pulmonary arterial hypertension have not been investigated. The rats were divided into control, monocrotaline, monocrotaline + Na2S, and Na2S groups. The right ventricular pressure and hypertrophy were evaluated. KCl, acetylcholine, and L-cysteine responses were obtained in the main pulmonary arteries by wire myograph. H2S levels were measured in pulmonary arteries and lungs by methylene blue assay. Right ventricular pressure and hypertrophy were increased by monocrotaline and ameliorated by Na2S. The KCl-induced contractions and relaxing responses to acetylcholine and L-cysteine in pulmonary arteries and H2S production in the lungs and pulmonary arteries were significantly attenuated in the monocrotaline group and augmented in the monocrotaline + Na2S group. These findings suggest that H2S levels were reduced, and L-cysteine-induced and endothelium-dependent relaxations were impaired in the pulmonary arteries in monocrotaline-induced pulmonary arterial hypertension. The H2S donor, Na2S, prevented endothelial dysfunction and increased pulmonary artery pressure and hypertrophy. Also, Na2S enhanced the L-cysteine-mediated responses and restored the diminished H2S levels in pulmonary arteries and the lungs. The treatments targeting H2S might be beneficial for promoting vascular alterations, i.e. endothelial dysfunction and impaired H2S-mediated relaxation in pulmonary arterial hypertension.

Comparative evaluation of relaxant effects of three prangos species on mouse corpus cavernosum: Chemical characterization and the relaxant mechanisms of action of P. pabularia and (+)-oxypeucedanin.

ETHNOPHARMACOLOGICAL RELEVANCE: Erectile dysfunction (ED) is the most common form of sexual dysfunction which has been the topic of great interest through the history by all cultures. It is now among the most treated health problems in men of all ages that develop under the influence of lifestyle factors and some diseases. Plants are extensively used to cure sexual dysfunction for centuries. Roots of Prangos sp. have been used to improve sexual performance in Anatolian traditional medicine and are rich of coumarin, furanocoumarin and their derivatives. Scientific research is necessary to support and validate the ethno-traditional uses of these plants. AIM OF THE STUDY: The aim of this study is to investigate the effects of the root extracts of P. pabularia, P. uechtritzii and P. heyniae on erectile function and to isolate and identify the chemical compounds of the most active extract and reveal possible pharmacological mechanism of the major compound of the extract with the strongest relaxant effect in mouse corpus cavernosum (MCC). MATERIALS AND METHODS: The roots of plants were extracted with chloroform, n-hexane and methanol. The compounds were isolated from the extract by column chromatography and structures were identified by NMR and MS. The relaxant effects of extracts (10-7-10-4 g/mL), (+)-oxypeucedanin (10-7-10-4 M) and Na2S (10-7-3 × 10-3 M) were tested in MCC strips by DMT myograph. To investigate the mechanism, the synthesis inhibitors of aminooxyacetic acid (AOAA, 10-2 M) and nitro-L-arginine methyl ester (L-NAME, 10-4 M) were used, respectively. H2S formation was evaluated basal and L-cysteine (L-cyst)-stimulated conditions by H2S microsensor. RESULTS: All extracts relaxed MCC in a concentration dependent manner. The maximum relaxing effects were achieved with chloroform extracts. Chloroform extract of P. pabularia (Pp-CE) was more potent than the others. Pp-CE-induced relaxations were significantly decreased by AOAA and L-NAME. (+)-Oxypeucedanin, the major compound of Pp-CE, induced relaxant responses and this effect was inhibited by AOAA, but not L-NAME. The relaxation of (+)-oxypeucedanin was found to be similar in view of Emax to positive control H2S donor Na2S. (+)-Oxypeucedanin increased L-cyst-stimulated H2S formation. Augmentation of H2S synthesis with (+)-oxypeucedanin was inhibited by AOAA. CONCLUSIONS: Pp-CE has the strongest effect on relaxation of MCC and this result supports the traditional aphrodisiac use of P. pabularia root extract in Anatolia. The pharmacological mechanisms of Pp-CE to relax MCC involve NO and H2S formation. (+)-Oxypeucedanin could be responsible for the H2S-mediated relaxations of Pp-CE in MCC.

The role of resistin on metabolic syndrome-induced erectile dysfunction and the possible therapeutic effect of Boldine.

BACKGROUND: Resistin is known as a potential mediator of obesity-associated insulin resistance. The high resistin level disrupts nitric oxide (NO)-mediated relaxation which is also important in erectile function. An antioxidant alkaloid, Boldine, is known as anti-diabetic and protects endothelial functions. OBJECTIVES: We aimed to investigate resistin expression in penile tissue in the presence of insulin resistance (IR) and the effect of Boldine treatment on erectile functions in the metabolic syndrome (MetS) rat model. MATERIALS AND METHODS: Wistar rats were randomly divided into three groups: Control, MetS, and boldine treated MetS group. MetS parameters were assessed by serum triglycerides (TG), uric acid (UA), glucose, insulin levels, HOMA index, and waist circumference (WC)/tibia length (TL) ratio. To evaluate erectile functions, intracavernous pressure (ICP)/mean arterial pressure (MAP) ratio was performed during cavernous nerve stimulation. Protein expressions of resistin, endothelial nitric oxide synthase (eNOS), p(S1177) eNOS, and insulin receptor-ß were evaluated by Western blotting. RESULTS: TG, glucose, insulin levels, weight, WC/TL ratio, HOMA index and resistin expression in penile tissue were significantly increased and ICP/MAP values, and p (S1177) eNOS expression in penile tissue were decreased in MetS group. Boldine treatment enhanced ICP/MAP values, insulin receptor-ß and p(S1177) eNOS expressions compared with the MetS group. DISCUSSION AND CONCLUSION: MetS caused a deterioration in erectile function accompanied by an increase in resistin expression and a reduction in eNOS enzyme activation in the rat penile tissues. Boldine treatment resulted in an improvement in erectile function, independent of resistin expression.

The anti-angiogenic potential of (±) gossypol in comparison to suramin.

Cotton, a staple fiber that grows around the seeds of the cotton plants (Gossypium), is produced throughout the world, and its by products, such as cotton fibers, cotton-seed oil, and cottonseed proteins, have a variety of applications. Cotton-seed contains gossypol, a natural phenol compound. (±)-Gossypol is a yellowish polyphenol that is derived from different parts of the cotton plant and contains potent anticancer properties. Tumor growth and metastasis are mainly related to angiogenesis; therefore, anti-angiogenic therapy targets the new blood vessels that provide oxygen and nutrients to actively proliferating tumor cells. The aim of the present study was to evaluate the anti-angiogenic potential of (±)-gossypol in vitro. (±)-Gossypol has anti-proliferative effects on cancer cell lines; however, its anti-angiogenic effects on normal cells have not been studied. Anti-proliferative activities of gossypol assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, anti-angiogenic activities using tube formation assay, and cell migration inhibition capability using a wound-healing assay on human umbilical vein endothelial cells (HUVECs) were revealed. (±)-Gossypol displayed the following potent anti-angiogenic activities in vitro: it inhibited the cell viability of HUVECs, it inhibited the migration of HUVECs, and disrupted endothelial tube formation in a dose-dependent manner. In addition, the anti-angiogenic effects of (±)-gossypol were investigated in ovo in a model using a chick chorioallantoic membrane (CAM). Decreases in capillary density were assessed and scored. (±)-Gossypol showed dose-dependent anti-angiogenic effects on CAM. These findings suggest that (±)-gossypol can be used as a new anti-angiogenic agent.

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

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 NFκB 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 NFκB signaling leading the repression of NFκB transcriptional activation and suppressed cell proliferation. The results suggested that these molecules might have significant potential for chemoprevention of prostate carcinogenesis induced by inflammatory NFκB signaling pathway.

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.

Hydrogen sulfide: A novel mechanism for the vascular protection by resveratrol under oxidative stress in mouse aorta.

Reactive oxygen species (ROS) decreases bioavailability of nitric oxide (NO) and impairs NO-dependent relaxations. Like NO, hydrogen sulfide (H2S) is an antioxidant and vasodilator; however, the effect of ROS on H2S-induced relaxations is unknown. Here we investigated whether ROS altered the effect of H2S on vascular tone in mouse aorta and determined whether resveratrol (RVT) protects it via H2S. Pyrogallol induced ROS formation. It also decreased H2S formation and relaxation induced by l-cysteine and in mouse aorta. Pyrogallol did not alter sodium hydrogensulfide (NaHS)-induced relaxation suggesting that the pyrogallol effect on l-cysteine relaxations was due to endogenous H2S formation. RVT inhibited ROS formation, enhanced l-cysteine-induced relaxations and increased H2S level in aortas exposed to pyrogallol suggesting that RVT protects against "H2S-dysfunctions" by inducing H2S formation. Indeed, H2S synthesis inhibitor AOAA inhibited the protective effects of RVT. RVT had no effect on Ach-induced relaxation that is NO dependent and the stimulatory effect of RVT on H2S-dependent relaxation was also independent of NO. These results demonstrate that oxidative stress impairs endogenous H2S-induced relaxations and RVT offers protection by inducing H2S suggesting that targeting endogenous H2S pathway may prevent vascular dysfunctions associated by oxidative stress.

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-ß 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 (H2 S) 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-ß-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.

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

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).

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.

Nitric oxide and the endothelium: history and impact on cardiovascular disease.

There are few discoveries with the magnitude of the impact that NO has had on biology during the 25 years since its discovery. There is hardly a disease today not associated with altered NO homeostasis. In fact, despite numerous other endothelial functions, endothelial dysfunction has become synonymous with reduced biological activity of NO. Translating the preclinical discoveries in NO biology to new modalities for disease management has not been as impressive. Beyond the success of drugs for erectile dysfunction, clinical trials of nitric oxide synthase inhibitor have been proven either ineffective or wrought with side effects. NO donors (e.g., nitroglycerine) remain frequently used cardiovascular agents, but were discovered before 1980. Gene therapy studies have yet to become clinically useful. There is no doubt that endothelial- and NO-dysfunction is a hallmark of cardiovascular disease, including diseases which are considered as major current public health concerns: hypertension, obesity, diabetes, malnutrition. In many cases, cardiovascular disease (CVD) can be prevented by identifying and controlling modifiable risk factors. One conceivable approach to the management of multiple risk factors in CVD could be to treat endothelial dysfunction (e.g., by enhancing eNOS expression), since many CVD risk factors are related to endothelial dysfunction. In this regard one goal may include optimizing eNOS function. This can be realized by supplementing co-factors, e.g., BH4, or substrate, L-arginine, by increasing cGMP availability via phosphodiesterase inhibitors or sGC activators or by increasing NO bioavailability via antioxidants. The association of other proteins with the nitric oxide synthase (NOS) isoforms and sGC could also serve as experimental and potentially therapeutic targets to modulate NO bioactivity. There is tremendous promise behind NO itself as well as the numerous other molecules and processes associated with the L-arginine-NO-cGMP pathway. Collaborative efforts among bench scientists, clinical investigators and epidemiologists are the key in realizing this promise.

Effects of hsp90 binding inhibitors on sGC-mediated vascular relaxation.

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations.

Diverse effects of calcium channel blockers in the collar model.

OBJECTIVE: Calcium channel blockers (CCBs) are among the most frequently prescribed cardiovascular drugs. It has been shown that these drugs have antiatherosclerotic effects in both experimental and clinical settings. However, calcium channel blockers have markedly different chemical structures and different effects on the cardiovascular system. We investigated the effect of CD-832, a Ca(+2) channel antagonist, on collar-induced intimal thickening, as well as accompanied reactivity changes in rabbit carotid artery. METHODS AND RESULTS: Rabbits received 5 mg/kg/day CD-832 or vehicle (polyethylene glycol, 0.5 ml/kg/day) intramuscularly for a week before and 2 weeks after the collar application. Histological and isometric force measurements were performed in segments from sham and collared carotid arteries. A three-week treatment with CD-832 did not inhibit the intimal thickening caused by perivascular application of a silicone collar. Potassium chloride (KCl), phenylephrine, 5-hydroxytryptamine (5-HT, serotonin) and histamine induced concentration-dependent contractions in both sham-operated (sham) and collared arteries. Collar-induced attenuations in maximum KCl, histamine, phenylephrine and 5-HT contractions were not affected by CD-832. Collaring caused an increase in pD2 values of 5-HT and a decrease in those of phenylephrine, histamine and acetylcholine. CD-832 did not affect the altered sensitivity to these agonists. CONCLUSIONS: These results demonstrate that, in rabbit carotid artery, CD-832 did not inhibit the collar-induced intimal thickening and did not affect the accompanying changes in vascular reactivity.