Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner.

Ilexgenin A is a natural triterpenoid with beneficial effects on lipid disorders. This study aimed to investigate the effects of ilexgenin A on endothelial homeostasis and its mechanisms. Palmitate (PA) stimulation induced endoplasmic reticulum stress (ER stress) and subsequent thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in endothelial cells, leading to endothelial dysfunction. Ilexgenin A enhanced LKB1-dependent AMPK activity and improved ER stress by suppression of ROS-associated TXNIP induction. However, these effects were blocked by knockdown of AMPKα, indicating AMPK is essential for its action in suppression of ER stress. Meanwhile, ilexgenin A inhibited NLRP3 inflammasome activation by down-regulation of NLRP3 and cleaved caspase-1 induction, and thereby reduced IL-1ß secretion. It also inhibited inflammation and apoptosis exposed to PA insult. Consistent with these results in endothelial cells, ilexgenin A attenuated ER stress and restored the loss of eNOS activity in vascular endothelium, and thereby improved endothelium-dependent vasodilation in rat aorta. A further analysis in high-fat fed mice showed that oral administration of ilexgenin A blocked ER stress/NLRP3 activation with reduced ROS generation and increased NO production in vascular endothelium, well confirming the beneficial effect of ilexgenin A on endothelial homeostasis in vivo. Taken together, these results show ER stress-associated TXNIP/NLRP3 inflammasome activation was responsible for endothelial dysfunction and ilexgenin A ameliorated endothelial dysfunction by suppressing ER-stress and TXNIP/NLRP3 inflammasome activation with a regulation of AMPK. This finding suggests that the application of ilexgenin A is useful in the management of cardiovascular diseases in obesity.

Pharmacological activation of AMPK ameliorates perivascular adipose/endothelial dysfunction in a manner interdependent on AMPK and SIRT1.

Adipose and endothelial dysfunction is tightly associated with cardiovascular diseases in obesity and insulin resistance. Because perivascular adipose tissue (PVAT) surrounds vessels directly and influences vessel functions through paracrine effect, and AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) show similarities in modulation of metabolic pathway, we hypothesized that activation of AMPK and SIRT1 in PVAT might regulate the endothelial function in pathological settings. Thus, in this study, we focused on the regulation of AMPK and SIRT1 activities implicated in adipocytokine expression and endothelial homeostasis under inflammatory conditions by using salicylate, metformin, AICA riboside (AICAR) and resveratrol as AMPK activating agents. We prepared conditioned medium (CM) by stimulating PVAT with palmitic acid (PA) and observed the effects of AMPK activating agents on adipocytokine expression and vessel vasodilation in rats. Moreover, we explored the effects of resveratrol and metformin in fructose-fed rats. We observed that PA stimulation induced inflammation and dysregulation of adipocytokine expression accompanied with reduced AMPK activity and SIRT1 abundance in PVAT. AMPK activating agents inhibited NF-κB p65 phosphorylation and suppressed gene expression of pro-inflammatory adipocytokines, and upregulated adiponectin and PPARγ expression in PVAT in an AMPK/SIRT1-interdependent manner. Meanwhile, CM stimulation impaired endothelium-dependent vasodilation in response to acetylcholine (ACh). Pretreatment of CM with AMPK-activating agents enhanced eNOS phosphorylation in the aorta and restored the loss of endothelium-dependent vasodilation, whereas this action was abolished by co-treatment with AMPK inhibitor compound C or SIRT1 inhibitor nicotinamide. Long-term fructose-feeding in rats induced dysregulation of adipocytokine expression in PVAT and the loss of endothelium-dependent vasodilation, whereas these alterations were reversed by oral administration of resveratrol and metformin. Altogether, pharmacological activation of AMPK beneficially regulated adipocytokine expression in PVAT and thus ameliorated endothelial dysfunction against inflammatory insult in an AMPK/SIRT1-interdependent manner.

Effects of 3-methyladenine, an autophagy inhibitor, on the elevated blood pressure and arterial dysfunction of angiotensin II-induced hypertensive mice.

Autophagy is an intracellular degradation system that disassembles cytoplasmic components through autophagosomes fused with lysosomes. Recently, it has been reported that autophagy is associated with cardiovascular diseases, including pulmonary hypertension, atherosclerosis, and myocardial ischemia. However, the involvement of autophagy in hypertension is not well understood. In the present study, we hypothesized that excessive autophagy contributes to the dysfunction of mesenteric arteries in angiotensin II (Ang II)-induced hypertensive mice. Treatment of an autophagy inhibitor, 3-methyladenine (3-MA), reduced the elevated blood pressure and wall thickness, and improved endothelium-dependent relaxation in mesenteric arteries of Ang II-treated mice. The expression levels of autophagy markers, beclin1 and LC3 II, were significantly increased by Ang II infusion, which was reduced by treatment of 3-MA. Furthermore, treatment of 3-MA induced vasodilation in the mesenteric resistance arteries pre-contracted with U46619 or phenylephrine, which was dependent on endothelium. Interestingly, nitric oxide production and phosphorylated endothelial nitric oxide synthase (p-eNOS) at S1177 in the mesenteric arteries of Ang II-treated mice were increased by treatment with 3-MA. In HUVECs, p-eNOS was reduced by Ang II, which was increased by treatment of 3-MA. 3-MA had direct vasodilatory effect on the pre-contracted mesenteric arteries. In cultured vascular smooth muscle cells (VSMCs), Ang II induced increase in beclin1 and LC3 II and decrease in p62, which was reversed by treatment of 3-MA. These results suggest that autophagy inhibition exerts beneficial effects on the dysfunction of mesenteric arteries in hypertension.

Onvansertib, a polo-like kinase 1 inhibitor, inhibits prostate stromal cell growth and prostate smooth muscle contraction, which is additive to inhibition by α1-blockers.

Prostate smooth muscle contraction and prostate enlargement contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Recent evidence demonstrated that inhibitors for polo-like kinases (PLKs) inhibit smooth muscle contraction of human prostate tissues. However, their additive effects to α1-blockers, and effects on prostate growth are unknown. Here, we examined effects of a novel and highly selective PLK1 inhibitor, onvansertib on prostate smooth muscle contraction alone and in combination with α1-blockers, and on proliferation and viability of prostate stromal cells (WPMY-1). Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Proliferation and viability were assessed by plate colony, EdU, and CCK-8 assay. Electric field stimulation (EFS)-induced contractions of human prostate tissues were inhibited to 34% by 100 nM and 1 µM onvansertib at 32 Hz, and to 48% and 47% by the α1-blockers tamsulosin and silodosin. Combination of onvansertib with tamsulosin or silodosin further reduced EFS-induced contractions in comparison to α1-blockers alone (59% and 61% respectively), and to onvansertib alone (68% for both). Noradrenaline-, phenylephrine-, methoxamine-, endothelin-1-, and ATP-induced contractions were inhibited by onvansertib (100 nM) to similar extent. Viability and proliferation of WPMY-1 cells were reduced in a concentration- and time-dependent manner (24-72 h, 10-100 nM). Onvansertib inhibits neurogenic, adrenergic, and endothelin-1- and ATP-induced contractions of human prostate smooth muscle, and proliferation of stromal cells. Contractions are reduced not more than 50% by α1-blockers. Combination of α1-blockers with onvansertib provides additive inhibition of prostate contractions.

Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2/3, CMPD101.

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α1-adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of ß2-adrenoceptors or ß2-adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions.

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Quercetin and pioglitazone synergistically reverse endothelial dysfunction in isolated aorta from fructose-streptozotocin (F-STZ)-induced diabetic rats.

Pioglitazone is an anti-diabetic drug with potential to cause adverse effects following prolonged use. This study, therefore, investigated the effects of combination treatment of a subliminal concentration of pioglitazone and quercetin, a potent antioxidant, on vascular reactivity of aorta isolated from fructose-streptozotocin (F-STZ)-induced diabetic rats. Relaxation to acetylcholine and sodium nitroprusside, and contraction to phenylephrine were tested in organ bath chambers following pre-incubation with vehicle (DMSO; 0.05%), quercetin (10-7 M), pioglitazone (10-7 M), or their combination (P+Q; 10-7 M each drug). Subliminal concentration of quercetin or pioglitazone did not alter the acetylcholine- induced relaxation nor the phenylephrine-induced contraction in both normal rat and diabetic F-STZ induced tissues. However, P+Q combination synergistically improved the impaired acetylcholine-induced relaxation and decreased the elevated phenylephrine-induced contraction in aortic rings from diabetic, but not in the normal rats. Neither mono nor combination treatment altered sodium nitroprusside-induced relaxation. The combination also synergistically decreased superoxide anion and increased nitric oxide production compared to the individual treatments in aorta from diabetic rats. Overall, these data demonstrated a synergistic effect, in which, a combination (P+Q; 10-7 M each drug) caused a significantly greater effect than 10-6 M of either agent in improving endothelial function of isolated diabetic aorta. In conclusion, a combination of subliminal concentrations of pioglitazone and quercetin is able to decrease oxidative stress and provide synergistic vascular protection in type 2 diabetes mellitus and thus the possibility of using quercetin as a supplement to pioglitazone in the treatment of diabetes with the goal of reducing pioglitazone toxicity.

T-type voltage gated calcium channels are involved in endothelium-dependent relaxation of mice pulmonary artery.

In pulmonary arterial endothelial cells, Ca2+ channels and intracellular Ca2+ concentration ([Ca2+]i) control the release of vasorelaxant factors such as nitric oxide and are involved in the regulation of pulmonary arterial blood pressure. The present study was undertaken to investigate the implication of T-type voltage-gated Ca2+ channels (T-VGCCs, Cav3.1 channel) in the endothelium-dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Cav3.1-/- mice. Endothelial [Ca2+]i and NO production were measured, on whole vessels, with the fluo-4 and DAF-fm probes. Acetylcholine (ACh) induced a nitric oxide- and endothelium-dependent relaxation that was significantly reduced in pulmonary arteries from Cav3.1-/- compared to wild type mice as well as in the presence of T-VGCC inhibitors (NNC 55-0396 or mibefradil). ACh also increased endothelial [Ca2+]i and NO production that were both reduced in Cav3.1-/- compared to wild type mice or in the presence of T-VGCC inhibitors. Immunofluorescence labeling revealed the presence of Cav3.1 channels in endothelial cells that co-localized with endothelial nitric oxide synthase in arteries from wild type mice. TRPV4-, beta2 adrenergic- and nitric oxide donors (SNP)-mediated relaxation were not altered in Cav3.1-/- compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Cav3.1 channels activity. The present study thus demonstrates that T-VGCCs, mainly Cav3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca2+]i and ACh-mediated relaxation.

Inhibition of agonist-induced smooth muscle contraction by picotamide in the male human lower urinary tract outflow region.

Male lower urinary tract symptoms (LUTS) due to bladder outlet obstruction are characterized by abnormal smooth muscle contractions in the lower urinary tract. Alpha1-adrenoceptor antagonists may induce smooth muscle relaxation in the outflow region and represent the current gold standard of medical treatment. However, results may be unsatisfactory or inadequate. Apart from α1-adrenoceptor agonists, smooth muscle contraction in the outflow region may be induced by thromboxane A2 (TXA2), endothelins, or muscarinic receptor agonists. Here, we studied effects of the thromboxane A2 receptor (TP receptor) antagonist picotamide on contraction in the human male bladder trigone and prostate. Carbachol, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619, and electric field stimulation (EFS) induced concentration- or frequency-dependent contractions of trigone tissues in an organ bath. Picotamide (300µM) inhibited carbachol-, phenylephrine-, U46619-, and EFS-induced contractions. Endothelins 1-3 induced concentration-dependent contractions of prostate tissues, which were inhibited by picotamide. Analyses using real time polymerase chain reaction and antibodies suggested expression of thromboxane A2 receptors and synthase in trigone smooth muscle cells. Thromboxane B2 (the stable metabolite of thromboxane A2) was detectable by enzyme immune assay in trigone samples, with most values ranging between 50 and 150pg/mg trigone protein. Picotamide inhibits contractions induced by different stimuli in the human lower urinary tract, including cholinergic, adrenergic, thromboxane A2- and endothelin-induced, and neurogenic contractions in different locations of the outflow region. This distinguishes picotamide from current medical treatments for LUTS, and suggests that picotamide may induce urodynamic effects in vivo.

Impaired endothelium-derived hyperpolarization-type relaxation in superior mesenteric arteries isolated from female Otsuka Long-Evans Tokushima Fatty rats.

Endothelium-derived hyperpolarization (EDH) is an important signaling mechanism of endothelium-dependent vasorelaxation, and little attention has been paid to the EDH-type responses in female metabolic syndrome such as that observed with type-2 diabetes. We previously reported that EDH-type relaxation was impaired in superior mesenteric arteries from male Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type-2 diabetes, however, the response was unclear in female OLETF rat. Thus, the aim of this study was to examine if EDH-type relaxation was altered in superior mesenteric arteries isolated from female OLETF rats compared to age-matched, control female Long-Evans Tokushima Otsuka (LETO) rats at age 50-59 weeks. We investigated concentration-relaxation curves for acetylcholine (at age 50-53 weeks), NS309 (an activator of small- and intermediate-conductance calcium-activated potassium channels) (at age 50-53 weeks), and GSK1016790A (an agonist of transient receptor potential vanilloid type 4, TRPV4) (at age 58 or 59 weeks) in the presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine and the cyclooxygenase inhibitor indomethacin to investigate EDH-type responses in the superior mesenteric artery. Obesity, mild hyperglycemia, hyperinsulinemia, and hyperlipidemia (i.e., increased total cholesterol, triglyceride, and non-esterified fatty acids) were more frequent in OLETF rats than in age-matched LETO rats at age 50-53 weeks. Acetylcholine-, NS309-, and GSK1016790A-induced relaxations in arteries from OLETF rats were all significantly reduced compared to those in LETO rats. These results indicated that EDH-type relaxations were impaired in female OLETF rats. This novel experimental model may provide new insights into vascular dysfunction in metabolic syndrome in females.

Apigenin and naringenin ameliorate PKCßII-associated endothelial dysfunction via regulating ROS/caspase-3 and NO pathway in endothelial cells exposed to high glucose.

Endothelial dysfunction is a key event in the progression of atherosclerosis with diabetes. Increasing cell apoptosis may lead to endothelial dysfunction. Apigenin and naringenin are two kinds of widely used flavones. In the present study, we investigated whether and how apigenin and naringenin reduced endothelial dysfunction induced by high glucose in endothelial cells. We showed that apigenin and naringenin protected against endothelial dysfunction via inhibiting phosphorylation of protein kinase C ßII (PKCßII) expression and downstream reactive oxygen species (ROS) production in endothelial cells exposed to high glucose. Furthermore, we demonstrated that apigenin and naringenin reduced high glucose-increased apoptosis, Bax expression, caspase-3 activity and phosphorylation of NF-κB in endothelial cells. Moreover, apigenin and naringenin effectively restored high glucose-reduced Bcl-2 expression and Akt phosphorylation. Importantly, apigenin and naringenin significantly increased NO production in endothelial cells subjected to high glucose challenge. Consistently, high glucose stimulation impaired acetylcholine (ACh)-mediated vasodilation in the rat aorta, apigenin and naringenin treatment restored the impaired endothelium-dependent vasodilation via dramatically increasing eNOS activity and nitric oxide (NO) level. Taken together, our results manifest that apigenin and naringenin can ameliorate endothelial dysfunction via regulating ROS/caspase-3 and NO pathway.

Apigenin and naringenin regulate glucose and lipid metabolism, and ameliorate vascular dysfunction in type 2 diabetic rats.

Vascular endothelial dysfunction is regarded as the initial step of vascular complications in diabetes mellitus. This study investigated the amelioration of apigenin and naringenin in type 2 diabetic (T2D) rats induced by high-fat diet and streptozotocin and explored the underlying mechanism. Apigenin or naringenin was intragastrically administered at 50 or 100mg/kg once a day for 6 weeks. Biochemical parameters including blood glucose, glycated serum protein, serum lipid, insulin, superoxide dismutase (SOD), malonaldehyde and intercellular adhesion molecule-1 (ICAM-1) were measured. Vascular reactivity in isolated thoracic aortic rings was examined. Pathological features of the thoracic aorta were further observed through optical microscopy and transmission electron microscopy. Lastly, we evaluated their effects on insulin resistance of palmitic acid (PA)-induced endothelial cells. Compared with diabetic control group, apigenin and naringenin significantly decreased the levels of blood glucose, serum lipid, malonaldehyde, ICAM-1 and insulin resistance index, increased SOD activity and improved impaired glucose tolerance. Apigenin and naringenin restored phenylephrine-mediated contractions and acetylcholine or insulin-induced relaxations in aortic tissues. Furthermore, pathological damage in the thoracic aorta of apigenin and naringenin groups was more remissive than diabetic control group. In vitro, apigenin and naringenin inhibited NF-κB activation and ICAM-1 mRNA expression in PA-treated endothelial cells and improved nitric oxide production in the presence of insulin. In conclusion, both apigenin and naringenin can ameliorate glucose and lipid metabolism, as well as endothelial dysfunction in T2D rats at least in part by down-regulating oxidative stress and inflammation. In general, apigenin showed greater potency than naringenin equivalent.