Alteration of Piezo1 signaling in type 2 diabetic mice: focus on endothelium and BKCa channel.

Piezo1 mechanosensitive ion channel plays a important role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Ten- to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db-/db-) were used. The second-order mesenteric arteries (~ 150 µm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db-/db- mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db-/db- mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db-/db- mice, while there was no effect in control mice. Co-immunofluorescence staining showed co-localization of Piezo1 and BKCa channel was more pronounced in db-/db- mice than in control mice. These results indicate that the vascular responses induced by Piezo1 activation are different in the mesenteric resistance arteries in type 2 diabetic mice.

Investigating the Cardiovascular Benefits of Dapagliflozin: Vasodilatory Effect on Isolated Rat Coronary Arteries.

Dapagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, is an antidiabetic medication that reduces blood glucose. Although it is well known that dapagliflozin has additional benefits beyond glycemic control, such as reducing blood pressure and lowering the risk of cardiovascular events, no sufficient research data are available on the direct effect of dapagliflozin on cardiovascular function. Thus, in this study, we investigated the direct vascular effect of dapagliflozin on isolated rat coronary arteries. The left descending coronary arteries of 13-week-old male Sprague Dawley rats were cut into segments 2-3 mm long and mounted in a multi-wire myography system to measure isometric tension. Dapagliflozin effectively reduced blood vessel constriction induced by U-46619 (500 nM) in coronary arteries regardless of the endothelium. Treatment with an eNOS inhibitor (L-NNA, 100 µM), sGC inhibitor (ODQ, 5 µM), or COX inhibitor (indomethacin, 3 µM) did not affect the vasodilation induced by dapagliflozin. The application of a Ca2+-activated K+ channel (KCa) blocker (TEA, 2 mM), voltage-dependent K+ channel (KV) blocker (4-AP, 2 mM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (3 µM), and inward-rectifier K+ channel (KIR) blocker (BaCl2, 30 µM) did not affect the dapagliflozin-induced vasodilation either. The treatment with dapagliflozin decreased contractile responses induced by the addition of Ca2+, which suggested that the extracellular Ca2+ influx was inhibited by dapagliflozin. Treatment with dapagliflozin decreased the phosphorylation level of the 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that dapagliflozin has a significant vasodilatory effect on rat coronary arteries. Our findings suggest a novel pharmacologic approach for the treatment of cardiovascular diseases in diabetic patients through the modulation of Ca2+ homeostasis via dapagliflozin administration.

Ezetimibe Induces Vasodilation in Rat Mesenteric Resistance Arteries through Inhibition of Extracellular Ca2+ Influx.

Ezetimibe is a lipid-lowering agent that selectively inhibits cholesterol absorption by binding to the Niemann-Pick C1-like 1 (NPC1L1) protein. Although it is well known that administration of ezetimibe in hypercholesterolemia patients reduces the risk of cardiovascular events through attenuation of atherosclerosis, studies on the direct effect of ezetimibe on vascular function are not sufficient. The aim of the present study was to investigate the vascular effects of ezetimibe in rat mesenteric arteries. In the present study, 12-week-old male Sprague Dawley rats were used. After the rats were sacrificed, the second branches of the mesenteric arteries were isolated and cut into 2-3 mm segments and mounted in a multi-wire myography system to measure isometric tension. Ezetimibe reduced vasoconstriction induced by U46619 (500 nM) in endothelium-intact and endothelium-denuded arteries. Ezetimibe-induced vasodilation was not affected by the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 300 µM) or the non-selective potassium channel blocker, tetraethylammonium (TEA, 10 mM). Moreover, ezetimibe also completely blocked the contraction induced by an increase in external calcium concentration. Ezetimibe significantly reduced vascular contraction induced by L-type Ca2+ channel activator (Bay K 8644, 30 nM). Treatment with ezetimibe decreased the phosphorylation level of 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that ezetimibe has a significant vasodilatory effect in rat mesenteric resistance arteries. These results suggest that ezetimibe may have beneficial cardiovascular effects beyond its cholesterol-lowering properties.

Vasodilatory Effect of Alpinia officinarum Extract in Rat Mesenteric Arteries.

BACKGROUND: Alpinia officinarum (A. officinarum) is known to exhibit a beneficial effect for anti-inflammatory, anti-oxidant, and anti-hyperlipidemic effects. However, no sufficient research data are available on the cardiovascular effect of A. officinarum. Thus, in this study, we investigate whether A. officinarum extract has direct effects on vascular reactivity. METHODS: To examine whether A. officinarum extract affects vascular functionality, we measured isometric tension in rat mesenteric resistance arteries using a wire myograph. After arteries were pre-contracted with high-K+ (70 mM), phenylephrine (5 µM), or U46619 (1 µM), A. officinarum extract was treated. RESULTS: A. officinarum extract induced vasodilation in a concentration-dependent manner, and this effect was endothelium independent. To further investigate the mechanism, we incubated arteries in a Ca2+-free and high-K+ solution, followed by the cumulative addition of CaCl2 (0.01-2.5 mM) with or without A. officinarum extract (30 µg/mL). Pre-treatment of A. officinarum extract reduced the contractile responses induced by cumulative administration of Ca2+, which suggests that extracellular Ca2+ influx was inhibited by the treatment of A. officinarum extract. These results were associated with a reduction in phosphorylated MLC20 in VSMCs treated with A. officinarum extract. Furthermore, eucalyptol, an active compound of A. officinarum extract, had a similar effect as A. officinarum extract, which causes vasodilation in mesenteric resistance arteries. CONCLUSION: A. officinarum extract and its active compound eucalyptol induce concentration-dependent vasodilation in mesenteric resistance arteries. These results suggest that administration of A. officinarum extract could exert beneficial effects to treat high blood pressure.

Vasorelaxant Effect of Trachelospermi caulis Extract on Rat Mesenteric Resistance Arteries.

BACKGROUND: Trachelospermi caulis (T. caulis) has been used as a traditional herbal medicine in Asian countries. Although it is well known that T. caulis has beneficial effects, no sufficient research data are available on the cardiovascular effect of T. caulis. We investigated whether T. caulis extract has vascular effects in rat resistance arteries in this study. METHODS: To examine whether T. caulis extract affects vascular reactivity, we measured isometric tension of rat mesenteric resistance arteries using a multi-wire myograph system. T. caulis extract was administered after arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM). Vanillin, a single active component of T. caulis, was used to treat mesenteric arteries. RESULTS: T. caulis extract caused vascular relaxation in a concentration-dependent manner, which was endothelium-independent. To further identify the mechanism, we incubated the arteries in Ca2+-free solution containing high K+, followed by a cumulative administration of CaCl2 (0.01-2.0 mM) with or without T. caulis extract (250 µg/mL). The treatment of T. caulis extract decreased contractile responses induced by the addition of Ca2+, which suggested that the extracellular Ca2+ influx was inhibited by the T. caulis extract. Moreover, an active compound of T. caulis extract, vanillin, also induced vasodilation in mesenteric resistance arteries. CONCLUSION: T. caulis extract and its active compound, vanillin, concentration-dependently induced vascular relaxation in mesenteric resistance arteries. These results suggest that the administration of T. caulis extract could help decrease blood pressure.

Vanillin Induces Relaxation in Rat Mesenteric Resistance Arteries by Inhibiting Extracellular Ca2+ Influx.

Vanillin is a phenolic aldehyde, which is found in plant species of the Vanilla genus. Although recent studies have suggested that vanillin has various beneficial properties, the effect of vanillin on blood vessels has not been studied well. In the present study, we investigated whether vanillin has vascular effects in rat mesenteric resistance arteries. To examine the vascular effect of vanillin, we measured the isometric tension of arteries using a multi-wire myograph system. After the arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM), vanillin was administered. Vanillin induced concentration-dependent vasodilation. Endothelial denudation or treatment of eNOS inhibitor (L-NNA, 300 µM) did not affect the vasodilation induced by vanillin. Treatment of K+ channel inhibitor (TEA, 10 mM) or sGC inhibitor (ODQ, 10 µM) or COX-2 inhibitor (indomethacin, 10 µM) did not affect the vanillin-induced vasodilation either. The treatment of vanillin decreased the contractile responses induced by Ca2+ addition. Furthermore, vanillin significantly reduced vascular contraction induced by BAY K 8644 (30 nM). Vanillin induced concentration-dependent vascular relaxation in rat mesenteric resistance arteries, which was endothelium-independent. Inhibition of extracellular Ca2+ influx was involved in vanillin-induced vasodilation. Treatment of vanillin reduced phopsho-MLC20 in vascular smooth muscle cells. These results suggest the possibility of vanillin as a potent vasodilatory molecule.

MicroRNAs and obesity-induced endothelial dysfunction: key paradigms in molecular therapy.

The endothelium plays a pivotal role in maintaining vascular health. Obesity is a global epidemic that has seen dramatic increases in both adult and pediatric populations. Obesity perturbs the integrity of normal endothelium, leading to endothelial dysfunction which predisposes the patient to cardiovascular diseases. MicroRNAs (miRNAs) are short, single-stranded, non-coding RNA molecules that play important roles in a variety of cellular processes such as differentiation, proliferation, apoptosis, and stress response; their alteration contributes to the development of many pathologies including obesity. Mediators of obesity-induced endothelial dysfunction include altered endothelial nitric oxide synthase (eNOS), Sirtuin 1 (SIRT1), oxidative stress, autophagy machinery and endoplasmic reticulum (ER) stress. All of these factors have been shown to be either directly or indirectly caused by gene regulatory mechanisms of miRNAs. In this review, we aim to provide a comprehensive description of the therapeutic potential of miRNAs to treat obesity-induced endothelial dysfunction. This may lead to the identification of new targets for interventions that may prevent or delay the development of obesity-related cardiovascular disease.

Stimulation of autophagy improves vascular function in the mesenteric arteries of type 2 diabetic mice.

NEW FINDINGS: What is the central question of this study? What is the role of autophagy in vascular dysfunction in type 2 diabetes? What is the main finding and its importance? Autophagy is decreased in the mesenteric arteries of type 2 diabetic mice, and stimulation of autophagy using rapamycin and trehalose improves vascular function, which is associated with normalization of myogenic response and endothelium-dependent relaxation. ABSTRACT: Vascular dysfunction is a major complication in type 2 diabetes (T2D). It has been suggested that dysregulation of autophagy is associated with various cardiovascular diseases. However, the relationship between autophagy and vascular dysfunction in T2D remains unclear. Thus, we examined whether reduced autophagy is involved in vascular dysfunction and whether stimulation of autophagy could improve vascular function in diabetes. Ten- to twelve-week-old male type 2 diabetic (db- /db- ) mice and their control (db- /db+ ) mice were treated with rapamycin or trehalose. Mesenteric arteries (MAs) were mounted for arteriography and their diameter was measured. Western blot analysis and immunofluorescence staining were assessed. Myogenic response (MR) was significantly increased, whereas endothelium-dependent relaxation (EDR) was significantly attenuated in the MAs of diabetic mice. These results were associated with increased expression of LC3II, p62 and beclin-1 in diabetic mice. Treatment with autophagy stimulators significantly reduced the potentiation of MR and improved EDR in the diabetic mice. Furthermore, autophagy stimulation normalized expression of LC3II, p62 and beclin-1 in the diabetic mice. In addition, phosphorylation level of endothelial nitric oxide synthase was decreased in diabetic mice and was restored by rapamycin and trehalose. T2D impairs vascular function by dysregulated autophagy. Therefore, autophagy could be a potential target for overcoming diabetic microvascular complications.

Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries.

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).

Involvement of inhibitor kappa B kinase 2 (IKK2) in the regulation of vascular tone.

Inhibitor kappa B kinase 2 (IKK2) plays an essential role in the activation of nuclear factor kappa B (NF-kB). Recently, it has been suggested that IKK2 acts as a myosin light chain kinase (MLCK) and contributes to vasoconstriction in mouse aorta. However, the underlying mechanisms are still unknown. Therefore, we investigated whether IKK2 acts as a MLCK or regulates the activity of myosin light chain phosphatase (MLCP). Pressure myograph was used to measure vascular tone in rat mesenteric arteries. Immunofluorescence staining was performed to identify phosphorylation levels of MLC (ser19), MYPT1 (thr853 and thr696) and CPI-17 (thr38). SC-514 (IKK2 inhibitor, 50 µM) induced relaxation in the mesenteric arteries pre-contracted with 70 mM high K+ solution or U-46619 (thromboxane analog, 5 µM). The relaxation induced by SC-514 was increased in the arteries pre-contracted with U-46619 compared to arteries pre-contracted with 70 mM high K+ solution. U-46619-induced contraction was decreased by treatment of SC-514 in the presence of MLCK inhibitor, ML-7 (10 µM). In the absence of intracellular Ca2+, U-46619 still induced contraction, which was decreased by treatment of SC-514. Furthermore, phosphorylation levels of MLC (ser19) and MYPT1 (thr853) were decreased by treatment of SC-514. IKK2 is involved in the vascular contraction through regulation of MLCP activity by phosphorylating MYPT1 at thr853 in rat mesenteric arteries. These findings suggest IKK2 could be a new pharmacological target for specific therapies of various vascular diseases.

Inhibition of endoplasmic reticulum stress improves coronary artery function in type 2 diabetic mice.

NEW FINDINGS: What is the central question of this study? Endoplasmic reticulum (ER) stress has been reported to be involved in type 2 diabetes; however, the role of exacerbated ER stress in vascular dysfunction in type 2 diabetes remains unknown. What is the main finding and its importance? The main findings of this study are that ER stress is increased in the coronary arteries in type 2 diabetes, and inhibition of ER stress using taurine-conjugated ursodeoxycholic acid improves vascular function, which is associated with normalization of the myogenic response and endothelium-dependent relaxation. Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributory factor in cardiovascular diseases, the relationship between ER stress and vascular dysfunction in type 2 diabetes remains unclear. Thus, in the present study, we examined whether ER stress contributes to coronary artery dysfunction and whether inhibition of ER stress ameliorates vascular function in type 2 diabetes. Type 2 diabetic mice and their control counterparts were treated with an ER stress inhibitor (taurine-conjugated ursodeoxycholic acid, 150 mg kg(-1)  day(-1) , by i.p. injection) for 2 weeks or not treated. The myogenic response and endothelium-dependent relaxation were measured in pressurized coronary arteries. In type 2 diabetic mice, blood glucose and body weight were elevated compared with control mice. The myogenic response was potentiated and endothelium-dependent relaxation impaired in coronary arteries from the type 2 diabetic mice. Interestingly, treatment with the ER stress inhibitor normalized the myogenic responses and endothelium-dependent relaxation. These data were associated with an increase in ER stress marker expression or phosphorylation (IRE1-XBP-1 and PERK-eIF2α) in type 2 diabetic mice, which were reduced by treatment with the ER stress inhibitor. Inhibition of ER stress normalizes the myogenic response and improves vascular function in type 2 diabetes. Therefore, ER stress could be a potential target for cardiovascular diseases in diabetes mellitus.

Nuclear factor kappa B inhibition improves conductance artery function in type 2 diabetic mice.

BACKGROUND: We previously reported that enhanced nuclear factor kappa B (NFκB) activity is responsible for resistance arteries dysfunction in type 2 diabetic mice. METHODS: In this study, we aimed to determine whether augmented NFκB activity also impairs conductance artery (thoracic aorta) function in type 2 diabetic mice. We treated type 2 diabetic (db(-) /db(-) ) and control (db(-) /db(+) ) mice with two NFκB inhibitors (dehydroxymethylepoxyquinomicin, 6 mg/kg, twice a week and IKK-NBD peptide, 500 µg/kg/day) for 4 weeks. RESULTS: As expected, the NFκB inhibition did not affect blood glucose level and body weight. Thoracic aorta vascular endothelium-dependent relaxation (EDR), determined by the wire myograph, was impaired in diabetic mice compared with control and was significantly improved after NFκB inhibition. Interestingly, thoracic EDR was also rescued in db(-) /db(-p50NFκB-/-) and db(-) /db(-PARP-1-/-) double knockout mice compared with db(-) /db(-) mice. Similarly, the acute in vitro down regulation of NFκB-p65 using p65 shRNA lentiviral particles in arteries from db(-) /db(-) mice also improved thoracic aorta EDR. Western blot analysis showed that the p65NFκB phosphorylation, cleaved PARP-1 and COX-2 expression were increased in thoracic aorta from diabetic mice, which were restored after NFκB inhibition and in db(-) /db(-p-50NFκB-/-) and db(-) /db(-PARP-1-/-) mice. CONCLUSIONS: The present results indicate that in male type 2 diabetic mice, the augmented NFκB activity also impairs conductance artery function through PARP-1 and COX-2-dependent mechanisms.

Circumferential submucosal incision prior to endoscopic mucosal resection provides comparable clinical outcomes to submucosal dissection for well-differentiated neuroendocrine tumors of the rectum.

BACKGROUND: Small rectal neuroendocrine tumors (NETs) can be treated with endoscopic resection. Endoscopic submucosal dissection (ESD) has been accepted as a reliable technique, but it is difficult. We evaluated the feasibility and efficacy of precut and endoscopic mucosal resection (CSI-EMR) for rectal NETs compared to ESD. METHODS: Patients with rectal NETs were enrolled consecutively. ESD or CSI-EMR was performed at operator's discretion. Histological and clinical outcomes were measured and compared between the two treatment modalities. RESULTS: Thirty-three patients were enrolled in the study. Seventeen NETs were treated by the ESD method and 16 were treated by CSI-EMR. Both groups had similar mean tumor diameters (ESD 7.53 ± 1.94 vs. CSI-EMR 6.63 ± 1.99 mm; p = 0.197). En bloc resection was achieved in 100 % of ESD group and 87.5 % of CSI-EMR group. Lateral margin involvement occurred in one patient in ESD group and two in CSI-EMR group. The histologically complete resection rate was 88.2 % (15 of 17) in the ESD group and 81.2 % (13 of 16) in CSI-EMR group (p = 0.592). One case of perforation occurred in both groups. Delayed bleeding did not occur. None of the measured outcomes were different between the two groups. Operating time was significant shorter in CSI-EMR group than in ESD group (9.69 vs. 20.12 min, respectively; p value = 0.004). CONCLUSIONS: CSI-EMR results in reliable clinical outcomes for small rectal NETs comparable to those of ESD. CSI-EMR is technically feasible and more time saving.

Enhanced p22phox expression impairs vascular function through p38 and ERK1/2 MAP kinase-dependent mechanisms in type 2 diabetic mice.

Type 2 diabetes is associated with vascular complication. We hypothesized that increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p22(phox) expression impairs vascular endothelium-dependent relaxation (EDR) in type 2 diabetes. Type 2 diabetic (db(-)/db(-)) and control (db(-)/db(+)) mice were treated with reactive oxygen species (ROS) scavenger, polyethylene glycol superoxide dismutase (1,000 U/kg daily ip), or small interfering RNA p22(phox) (p22(phox)-lentivirus-small interfering RNA, 100 µg iv, 2 times/wk) for 1 mo. EDR was impaired in microvascular bed (coronary arteriole and femoral and mesenteric resistance arteries) from diabetic mice compared with control. Interestingly, ROS scavenger and p22(phox) downregulation did not affect blood glucose level or body weight but significantly improved EDR. Mitogen-activated protein kinases (ERK1/2 and p38) phosphorylation and NADPH oxidase activity were increased in arteries from diabetic mice and were reduced after ROS scavenger or p22(phox) downregulation in db(-)/db(-) mice. The present study showed that enhanced p22(phox) expression causes vascular dysfunction through ERK1/2 and p38-mitogen-activated protein kinase-dependent mechanisms in male type 2 diabetic mice. Therefore, p22(phox) could be an important target to improve vascular function in diabetes.

Vasodilator responses to acetylcholine are not mediated by the activation of soluble guanylate cyclase or TRPV4 channels in the rat.

The effects of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), an inhibitor of the activation of soluble guanylate cyclase (sGC) on responses to NO donors acetylcholine (ACh) and bradykinin (BK) were investigated in the pulmonary and systemic vascular beds of the rat. In these studies the administration of ODQ in a dose of 5 mg/kg iv attenuated vasodilator responses to five different NO donors without inhibiting responses to ACh and BK in the systemic and pulmonary vascular beds of the rat. Vasodilator responses to ACh were not inhibited by l-NAME or the transient receptor vanilloid type 4 (TRPV4) antagonist GSK-2193874, which attenuated vasodilator responses to the TRPV4 agonist GSK-1016790A. ODQ did not inhibit vasodilator responses to agents reported to act in an NO-independent manner or to vasoconstrictor agents, and ODQ did not increase blood methemoglobin levels, suggesting that off target effects were minimal. These results show that ODQ in a dose that inhibited NO donor-mediated responses did not alter vasodilator responses to ACh in the pulmonary and systemic vascular beds and did not alter systemic vasodilator responses to BK. The present results indicate that decreases in pulmonary and systemic arterial pressures in response to ACh are not mediated by the activation of sGC or TRPV4 channels and that ODQ can be used to study the role of the activation of sGC in mediating vasodilator responses in the rat.

Serine-threonine kinase with-no-lysine 4 (WNK4) controls blood pressure via transient receptor potential canonical 3 (TRPC3) in the vasculature.

Mutations in the serine-threonine kinase with-no-lysine 4 (WNK4) cause pseudohypoaldosteronism type 2 (PHAII), a Mendelian form of human hypertension. WNK4 regulates diverse ion transporters in the kidney, and dysregulation of renal transporters is considered the main cause of the WNK4 mutation-associated hypertension. Another determinant of hypertension is vascular tone that is regulated by Ca(2+)-dependent blood vessel constriction. However, the role of WNK4 in vasoconstriction as part of its function to regulate blood pressure is not known. Here, we report that WNK4 is a unique modulator of blood pressure by restricting Ca(2+) influx via the transient receptor potential canonical 3 (TRPC3) channel in the vasculature. Loss of WNK4 markedly augmented TRPC3-mediated Ca(2+) influx in vascular smooth muscle cells (VSMCs) in response to α-adrenoreceptor stimulation, which is the pathological hallmark of hypertension in resistance arteries. Notably, WNK4 depletion induced hypertrophic cell growth in VSMCs and increased vasoconstriction in small mesenteric arteries via TRPC3-mediated Ca(2+) influx. In addition, WNK4 mutants harboring the Q562E PHAII-causing or the D318A kinase-inactive mutation failed to mediate TRPC3 inhibition. These results define a previously undescribed function of WNK4 and reveal a unique therapeutic target to control blood pressure in WNK4-related hypertension.

Chronic inhibition of epidermal growth factor receptor tyrosine kinase and extracellular signal-regulated kinases 1 and 2 (ERK1/2) augments vascular response to limb ischemia in type 2 diabetic mice.

Type 2 diabetes is a key risk factor for ischemia-dependent pathology; therefore, a significant medical need exists to develop novel therapies that increase the formation of new vessels. We explored the therapeutic potential of epidermal growth factor receptor tyrosine kinase (EGFRtk) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibition in impaired ischemia-induced neovascularization in type 2 diabetes. Unilateral femoral artery ligation was performed in diabetic (db(-)/db(-)) and their control (db(-)/db(+)) mice for 4 weeks, followed by treatments with EGFRtk and ERK1/2 inhibitors (AG1478, 10 mg/kg/day and U0126, 400 µg/kg/day, respectively) for 3 weeks. Neovascularization, blood flow recovery, vascular and capillary density, and endothelial nitric oxide synthase activity were significantly impaired and were associated with enhanced EGFRtk and ERK1/2 activity in db(-)/db(-) mice. EGFRtk and ERK1/2 inhibitors did not have any effect in control mice, while in db(-)/db(-) mice there was a significant increase in neovascularization, blood flow recovery, vascular and capillary density, endothelial nitric oxide synthase activity, and were associated with a decrease in EGFRtk and ERK1/2 activity. Our data demonstrated that the inhibition of EGFRtk and ERK1/2 restored ischemia-induced neovascularization and blood flow recovery in type 2 diabetic mice. Thus, EGFRtk and ERK1/2 could be possible targets to protect from ischemia-induced vascular pathology in type 2 diabetes.

Chronic inhibition of endoplasmic reticulum stress and inflammation prevents ischaemia-induced vascular pathology in type II diabetic mice.

Endoplasmic reticulum (ER) stress and inflammation are important mechanisms that underlie many of the serious consequences of type II diabetes. However, the role of ER stress and inflammation in impaired ischaemia-induced neovascularization in type II diabetes is unknown. We studied ischaemia-induced neovascularization in the hind-limb of 4-week-old db - /db- mice and their controls treated with or without the ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) and interleukin-1 receptor antagonist (anakinra, 0.5 µg/mouse per day) for 4 weeks. Blood pressure was similar in all groups of mice. Blood glucose, insulin levels, and body weight were reduced in db - /db- mice treated with TUDCA. Increased cholesterol and reduced adiponectin in db - /db- mice were restored by TUDCA and anakinra treatment. ER stress and inflammation in the ischaemic hind-limb in db - /db- mice were attenuated by TUDCA and anakinra treatment. Ischaemia-induced neovascularization and blood flow recovery were significantly reduced in db - /db- mice compared to control. Interestingly, neovascularization and blood flow recovery were restored in db - /db- mice treated with TUDCA or anakinra compared to non-treated db - /db- mice. TUDCA and anakinra enhanced eNOS-cGMP, VEGFR2, and reduced ERK1/2 MAP-kinase signalling, while endothelial progenitor cell number was similar in all groups of mice. Our findings demonstrate that the inhibition of ER stress and inflammation prevents impaired ischaemia-induced neovascularization in type II diabetic mice. Thus, ER stress and inflammation could be potential targets for a novel therapeutic approach to prevent impaired ischaemia-induced vascular pathology in type II diabetes.

Sodium nitrite therapy rescues ischemia-induced neovascularization and blood flow recovery in hypertension.

Arterial hypertension is a major risk factor that can lead to complication of peripheral vascular disease due, in part, to endothelial dysfunction. Because sodium nitrite (SN) can be converted to nitric oxide (NO), which counteracts endothelial dysfunction, we explored the effect of nitrite on neovascularization following hind limb ischemia in different models of hypertension (HT). Chronic delivery of angiotensin II (Ang II, 400 ng/kg/min) or N(omega)-nitro-L-arginine-methyl-ester (L-NAME, 0.1 g/L) was used for a 2-week period to induce hypertension. Mice were subjected to femoral artery ligation-induced ischemia in the hind limb followed by treatment with SN (50 mg/L) for 2 weeks. SN significantly reduced systolic arterial blood pressure in mice receiving Ang II and L-NAME but had no effect in sham animals. After 2 weeks, blood flow and microangiography showed 60 % ± 1.0 recovery in sham compared with 40 % ± 1.3 in HT mice. Importantly, sham and HT mice treated with SN showed a 100 % blood flow recovery associated with normalization in capillary density. The inhibition of xanthine-oxido-reductase (allopurinol) or VEGFR (SU-5416) prevented the neovascularization in HT mice treated with SN. Cyclic GMP (cGMP) content in the hind limb was significantly increased in mice treated with SN compared with non-treated mice. Nitrite/nitrate content was only increased in the sham group treated with SN. Immunoprecipitation and Western blot analysis revealed an increase in eNOS/Akt/VEGFR phosphorylation in skeletal muscle from mice treated with SN compared with non-treated mice. Our findings indicate that SN therapy rescues the neovascularization and blood flow recovery in the ischemic hind limb of sham and HT mice likely through the Akt/NO/cGMP and VEGFR pathways.

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.