Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH. METHODS: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx). RESULTS: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a. CONCLUSIONS: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

Exacerbated inflammatory signaling underlies aberrant response to BMP9 in pulmonary arterial hypertension lung endothelial cells.

Imbalanced transforming growth factor beta (TGFß) and bone morphogenetic protein (BMP) signaling are postulated to favor a pathological pulmonary endothelial cell (EC) phenotype in pulmonary arterial hypertension (PAH). BMP9 is shown to reinstate BMP receptor type-II (BMPR2) levels and thereby mitigate hemodynamic and vascular abnormalities in several animal models of pulmonary hypertension (PH). Yet, responses of the pulmonary endothelium of PAH patients to BMP9 are unknown. Therefore, we treated primary PAH patient-derived and healthy pulmonary ECs with BMP9 and observed that stimulation induces transient transcriptional signaling associated with the process of endothelial-to-mesenchymal transition (EndMT). However, solely PAH pulmonary ECs showed signs of a mesenchymal trans-differentiation characterized by a loss of VE-cadherin, induction of transgelin (SM22α), and reorganization of the cytoskeleton. In the PAH cells, a prolonged EndMT signaling was found accompanied by sustained elevation of pro-inflammatory, pro-hypoxic, and pro-apoptotic signaling. Herein we identified interleukin-6 (IL6)-dependent signaling to be the central mediator required for the BMP9-induced phenotypic change in PAH pulmonary ECs. Furthermore, we were able to target the BMP9-induced EndMT process by an IL6 capturing antibody that normalized autocrine IL6 levels, prevented mesenchymal transformation, and maintained a functional EC phenotype in PAH pulmonary ECs. In conclusion, our results show that the BMP9-induced aberrant EndMT in PAH pulmonary ECs is dependent on exacerbated pro-inflammatory signaling mediated through IL6.

Bone Morphogenetic Protein 9 Is a Mechanistic Biomarker of Portopulmonary Hypertension.

RATIONALE: BMP9 (bone morphogenetic protein 9) is a circulating endothelial quiescence factor with protective effects in pulmonary arterial hypertension (PAH). Loss-of-function mutations in BMP9, its receptors, and downstream effectors have been reported in heritable PAH. OBJECTIVES: To determine how an acquired deficiency of BMP9 signaling might contribute to PAH. METHODS: Plasma levels of BMP9 and antagonist soluble endoglin were measured in group 1 PAH, group 2 and 3 pulmonary hypertension (PH), and in patients with severe liver disease without PAH. MEASUREMENTS AND MAIN RESULTS: BMP9 levels were markedly lower in portopulmonary hypertension (PoPH) versus healthy control subjects, or other etiologies of PAH or PH; distinguished PoPH from patients with liver disease without PAH; and were an independent predictor of transplant-free survival. BMP9 levels were decreased in mice with PH associated with CCl4-induced portal hypertension and liver cirrhosis, but were normal in other rodent models of PH. Administration of ALK1-Fc, a BMP9 ligand trap consisting of the activin receptor-like kinase-1 extracellular domain, exacerbated PH and pulmonary vascular remodeling in mice treated with hypoxia versus hypoxia alone. CONCLUSIONS: BMP9 is a sensitive and specific biomarker of PoPH, predicting transplant-free survival and the presence of PAH in liver disease. In rodent models, acquired deficiency of BMP9 signaling can predispose to or exacerbate PH, providing a possible mechanistic link between PoPH and heritable PAH. These findings describe a novel experimental model of severe PH that provides insight into the synergy between pulmonary vascular injury and diminished BMP9 signaling in the pathogenesis of PAH.

Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

A Selective Transforming Growth Factor-ß Ligand Trap Attenuates Pulmonary Hypertension.

RATIONALE: Transforming growth factor-ß (TGF-ß) ligands signal via type I and type II serine-threonine kinase receptors to regulate broad transcriptional programs. Excessive TGF-ß-mediated signaling is implicated in the pathogenesis of pulmonary arterial hypertension, based in part on the ability of broad inhibition of activin-like kinase (ALK) receptors 4/5/7 recognizing TGF-ß, activin, growth and differentiation factor, and nodal ligands to attenuate experimental pulmonary hypertension (PH). These broad inhibition strategies do not delineate the specific contribution of TGF-ß versus a multitude of other ligands, and their translation is limited by cardiovascular and systemic toxicity. OBJECTIVES: We tested the impact of a soluble TGF-ß type II receptor extracellular domain expressed as an immunoglobulin-Fc fusion protein (TGFBRII-Fc), serving as a selective TGF-ß1/3 ligand trap, in several experimental PH models. METHODS: Signaling studies used cultured human pulmonary artery smooth muscle cells. PH was studied in monocrotaline-treated Sprague-Dawley rats, SU5416/hypoxia-treated Sprague-Dawley rats, and SU5416/hypoxia-treated C57BL/6 mice. PH, cardiac function, vascular remodeling, and valve structure were assessed by ultrasound, invasive hemodynamic measurements, and histomorphometry. MEASUREMENTS AND MAIN RESULTS: TGFBRII-Fc is an inhibitor of TGF-ß1 and TGF-ß3, but not TGF-ß2, signaling. In vivo treatment with TGFBRII-Fc attenuated Smad2 phosphorylation, normalized expression of plasminogen activator inhibitor-1, and mitigated PH and pulmonary vascular remodeling in monocrotaline-treated rats, SU5416/hypoxia-treated rats, and SU5416/hypoxia-treated mice. Administration of TGFBRII-Fc to monocrotaline-treated or SU5416/hypoxia-treated rats with established PH improved right ventricular systolic pressures, right ventricular function, and survival. No cardiac structural or valvular abnormalities were observed after treatment with TGFBRII-Fc. CONCLUSIONS: Our findings are consistent with a pathogenetic role of TGF-ß1/3, demonstrating the efficacy and tolerability of selective TGF-ß ligand blockade for improving hemodynamics, remodeling, and survival in multiple experimental PH models.

Chronic black tea extract consumption improves endothelial function in ovariectomized rats.

PURPOSE: Menopause escalates the risk of cardiovascular diseases in women. There is an unmet need for better treatment strategy for estrogen-deficiency-related cardiovascular complications. Here we investigated the impact of chronic black tea extract (BT) consumption on cardiovascular function and lipid metabolism using a rat model of estrogen deficiency. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) and treated with BT (15 mg/kg/day, 4 weeks; active ingredients: theaflavins) or estrogen (E2) treatment for 4 weeks. Serum was collected for measuring cholesterol, triacylglycerol and estradiol levels. Changes in vascular reactivity were examined. The protein levels of NADPH oxidases were assessed by Western blotting. Reactive oxygen species (ROS) level was measured using dihydroethidium fluorescence imaging. The concentrations of cGMP were measured using ELISA kit. RESULTS: Aortic rings from control, BT-treated and E2-treated OVX rats exhibited a greater increase in Phe-induced contraction after inhibition of NO synthase compared with those from OVX rats. ACh-induced endothelium-dependent relaxations were augmented in aortae and renal arteries in BT/E2-treated OVX rats than in OVX rats. BT/E2 treatment improved flow-mediated dilatation in small mesenteric resistance arteries of OVX rats. BT/E2 treatment restored the eNOS phosphorylation level and reversed the up-regulation of NADPH oxidases and ROS overproduction in OVX rat aortae. ACh-stimulated cGMP production was significantly elevated in the aortae from BT- and E2-treated rats compared with those from OVX rats. BT/E2 treatment reduced circulating levels of total cholesterol. CONCLUSIONS: The present study reveals the novel benefits of chronic BT consumption to reverse endothelial dysfunction and favorably modifying cholesterol profile in a rat model of estrogen deficiency and provides insights into developing BT as beneficial dietary supplements for postmenopausal women.

Chronic cranberry juice consumption restores cholesterol profiles and improves endothelial function in ovariectomized rats.

PURPOSE: Postmenopausal women experience higher risks for cardiovascular diseases than age-matched men and pre-menopausal women. There is a need for better treatment strategy for estrogen-deficient-related cardiovascular complications. We and others have recently reported that activated renin-angiotensin system and the associated oxidative stress impaired endothelium-dependent relaxation in ovariectomized rat, while angiotensin receptor blocker rescues endothelial dysfunction. Dietary supplements and lifestyle modifications provide an alternative way to improve cardiovascular health. The present study tests the hypothesis that chronic cranberry juice consumption improves cholesterol profiles and vascular functions in estrogen-deficient animal model. The effect of cranberry consumption on expression and activity of renin-angiotensin system in the vasculature will be determined. METHODS: Ovariectomized rats were treated daily with commercial cranberry juice at 7 mg/kg for 8 weeks, a dosage comparable to recent clinical studies. Serum was collected for measuring cholesterol levels while aorta was isolated for isometric force assay and expression studies. RESULTS: Cranberry juice consumption reduced circulating levels of total cholesterol, triacylglycerols, HDL, nHDL, and nHDL/HDL ratio. Meanwhile, cranberry juice consumption improved endothelium-dependent relaxation in aorta of ovariectomized rats by restoring p-eNOS level (endothelial nitric oxide synthase phosphorylated at ser-1177), reversing the up-regulated levels of renin-angiotensin system markers (angiotensin-converting enzyme, angiotensin II, and angiotensin II type 1 receptor), and normalizing the elevated NAD(P)H oxidase expression and oxidative stress. CONCLUSIONS: Our data demonstrate the novel cardiovascular benefits of cranberry juice consumption in improving both vascular functions and cholesterol profiles, providing insight into developing cranberry products into useful dietary supplements for postmenopausal women.

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidative stress-dependent p38MAPK and JNK pathway.

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.

Sphingosine kinase 2 mediates cerebral preconditioning and protects the mouse brain against ischemic injury.

BACKGROUND AND PURPOSE: Cerebral preconditioning provides insights into endogenous mechanisms that protect the brain from ischemic injury. Hypoxia and the anesthetic isoflurane are powerful preconditioning agents. Recent data show that sphingosine 1-phosphate receptor stimulation improves outcome in rodent models of stroke. Endogenous sphingosine 1-phosphate levels are controlled by the expression and activity of sphingosine kinases (SPK). We hypothesize that SPK upregulation mediates preconditioning induced by isoflurane and hypoxia and reduces ischemic injury. METHODS: Male wild-type C57BL/J, SPK1(-/-) and SPK2(-/-) mice were exposed to isoflurane or hypoxia preconditioning before transient middle cerebral artery occlusion. Infarct volume and neurological outcome were measured 24 hours later. SPK inhibitors (SKI-II and ABC294640) were used to test the involvement of SPK2. Expressions of SPK1, SPK2, and hypoxia-inducible factor 1α were determined. Primary cultures of mouse cortical neurons were exposed to isoflurane before glutamate- or hydrogen peroxide-induced cell death. RESULTS: Isoflurane preconditioning and hypoxia preconditioning significantly reduced infarct volume and improved neurological outcome in wild-type and SPK1(-/-) mice but not in SPK2(-/-) mice. Pretreatment with SKI-II or ABC294640 abolished the isoflurane preconditioning-induced tolerance. Western blot showed a rapid and sustained increase in SPK2 level, whereas SPK1 level was similar between preconditioned mice and controls. Hypoxia-inducible factor 1α was upregulated in wild-type isoflurane-preconditioned mice but not in SPK2(-/-). Isoflurane preconditioning protected primary neurons against cell death, which was abolished in ABC294640-treated cells. CONCLUSIONS: Applying genetic and pharmacological approaches, we demonstrate that neuronal SPK2 isoform plays an important role in cerebral preconditioning.

Fingolimod provides long-term protection in rodent models of cerebral ischemia.

OBJECTIVE: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. METHODS: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. RESULTS: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha. INTERPRETATION: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Raloxifene improves vascular reactivity in pressurized septal coronary arteries of ovariectomized hamsters fed cholesterol diet.

Although vascular effects of selective estrogen receptor modulators (SERMs) have been extensively examined in conduit arteries, whether SERMs could favorably modulate myogenic response in resistance arteries is unknown. The impact of raloxifene therapy and cholesterol diet on myogenic constriction during estrogen deficiency is unresolved. This study investigated changes in vascular reactivity and myogenic responses in female ovariectomized (Ovx) hamsters fed high-cholesterol diet (HCD) with and without chronic treatment of raloxifene. Functional studies were performed on hamster septal coronary arteries cannulated in a pressure myograph. Acetylcholine (ACh)-induced dilatation was reduced in arteries from cholesterol-fed Ovx hamsters, but not in those from cholesterol-fed hamsters, while pressure-induced myogenic constriction was unaffected. Chronic treatment with raloxifene restored ACh-induced dilatation in cholesterol-fed Ovx hamsters. U46619-induced constriction was increased in arteries from cholesterol-fed Ovx hamsters but not from cholesterol-fed control hamsters, which was normalized by chronic raloxifene treatment. The pressure-diameter relationship is presented as normalized diameter versus intraluminal pressure, while the effect of ACh or U46619 is expressed as percentage of tone at 80 mm Hg. Two-way analysis of variance (ANOVA) followed by Bonferroni post-tests were used for statistical evaluation among different treatment groups. P<0.05 was taken as statistically significant. The present results show that chronic treatment with raloxifene could benefit myogenically active coronary arteries by (i) restoring ACh-induced dilatation and (ii) reducing U46619-induced constriction without affecting pressure-induced myogenic responses in cholesterol-fed hamsters during estrogen deficiency. If such benefit can be observed in humans, raloxifene and other SERMs may be useful to preserve endothelial function and curtail vascular hypersensitivity in resistance coronary arteries in post-menopausal women with hypercholesterolemia or hyperlipidemia, a lipid condition implicated in the pathogenesis of myocardial infarction.

Cyclooxygenase-2-derived prostaglandin F2alpha mediates endothelium-dependent contractions in the aortae of hamsters with increased impact during aging.

Hypertension and vascular dysfunction result in the increased release of endothelium-derived contracting factors (EDCFs), whose identity is poorly defined. We tested the hypothesis that endothelial cyclooxygenase (COX)-2 can generate EDCFs and identified the possible EDCF candidate. Changes in isometric tension of aortae of young and aged hamsters were recorded on myograph. Real-time changes in intracellular calcium concentrations ([Ca(2+)](i)) in native aortic endothelial cells were measured by imaging. Endothelium-dependent contractions were triggered by acetylcholine (ACh) after inhibition of nitric oxide production and they were abolished by COX-2 but not COX-1 inhibitors or by thromboxane-prostanoid receptor antagonists. 2-Aminoethoxydiphenyl borate (cation channel blocker) eliminated endothelium-dependent contractions and ACh-stimulated rises in endothelial cell [Ca(2+)](i). RT-PCR and Western blotting showed COX-2 expression mainly in the endothelium. Enzyme immunoassay and high-performance liquid chromatography-coupled mass spectrometry showed release of prostaglandin (PG)F(2alpha) and prostacyclin (PGI(2)) increased by ACh; only PGF(2alpha) caused contraction at relevant concentrations. COX-2 expression, ACh-stimulated contractions, and vascular sensitivity to PGF(2alpha) were augmented in aortae from aged hamsters. Human renal arteries also showed thromboxane-prostanoid receptor-mediated ACh- or PGF(2alpha)-induced contractions and COX-2-dependent release of PGF(2alpha). The present study demonstrates that PGF(2alpha), derived from COX-2, which is localized primarily in the endothelium, is the most likely EDCF underlying endothelium-dependent, thromboxane-prostanoid receptor-mediated contractions to ACh in hamster aortae. These contractions involved increases in endothelial cell [Ca(2+)](i). The results support a critical role of COX-2 in endothelium-dependent contractions in this species with an increased importance during aging and, possibly, a similar relevance in humans.

Therapeutically relevant concentrations of raloxifene dilate pressurized rat resistance arteries via calcium-dependent endothelial nitric oxide synthase activation.

OBJECTIVE: Selective estrogen receptor modulators (SERMs) inhibit constriction of mammalian conduit arteries. However, it is unknown whether SERMs at therapeutically achievable concentrations could reduce vascular tone in resistance arteries. The present study aimed to examine roles of Ca(2+) influx in endothelium and endothelial nitric oxide synthase (eNOS) activation in dilatations induced by raloxifene, a second-generation SERM in myogenically active arteries. METHODS AND RESULTS: Small mesenteric arteries from Sprague-Dawley rats were isolated and mounted in a pressure myograph for measurement of changes in vessel diameter. [Ca(2+)](i) images on native endothelial cells of intact arteries were determined by the fluorescence imaging technique, and phosphorylation of eNOS was assayed by Western blotting. Raloxifene (0.3 to 10 nmol/L) produced dilatations on established steady myogenic constriction. Female rat arteries dilated significantly more in response to raloxifene than male arteries. Raloxifene-induced dilatations of female arteries were blunted by N(G)-nitro-l-arginine methyl ester but unaffected by 1400W, charybdotoxin plus apamin, wortmannin, or LY294002. Raloxifene (3 nmol/L) triggered rises in endothelial cell [Ca(2+)](i) and increased eNOS phosphorylation at Ser1177. Both effects were greater in arteries from female rats than in arteries from male rats. Increases in endothelial cell [Ca(2+)](i) and in eNOS phosphorylation were prevented by removal of extracellular Ca(2+) ions. Finally, ICI 182,780 did not affect the raloxifene-stimulated rise in endothelial cell [Ca(2+)](i), eNOS phosphorylation, and vasodilatations. Chronic raloxifene treatment reduced myogenic constriction in arteries from female but not male rats. CONCLUSION: Raloxifene at therapeutically relevant concentrations inhibits myogenic constriction by an NO-dependent mechanism that causally involves the elevated [Ca(2+)](i) in endothelial cells and subsequent eNOS activation. Raloxifene dilates resistance arteries more effectively in female rats, indicating its significant gender-related action on endothelial cells in microcirculation.

ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension.

Human genetics, biomarker, and animal studies implicate loss of function in bone morphogenetic protein (BMP) signaling and maladaptive transforming growth factor-ß (TGFß) signaling as drivers of pulmonary arterial hypertension (PAH). Although sharing common receptors and effectors with BMP/TGFß, the function of activin and growth and differentiation factor (GDF) ligands in PAH are less well defined. Increased expression of GDF8, GDF11, and activin A was detected in lung lesions from humans with PAH and experimental rodent models of pulmonary hypertension (PH). ACTRIIA-Fc, a potent GDF8/11 and activin ligand trap, was used to test the roles of these ligands in animal and cellular models of PH. By blocking GDF8/11- and activin-mediated SMAD2/3 activation in vascular cells, ACTRIIA-Fc attenuated proliferation of pulmonary arterial smooth muscle cells and pulmonary microvascular endothelial cells. In several experimental models of PH, prophylactic administration of ACTRIIA-Fc markedly improved hemodynamics, right ventricular (RV) hypertrophy, RV function, and arteriolar remodeling. When administered after the establishment of hemodynamically severe PH in a vasculoproliferative model, ACTRIIA-Fc was more effective than vasodilator in attenuating PH and arteriolar remodeling. Potent antiremodeling effects of ACTRIIA-Fc were associated with inhibition of SMAD2/3 activation and downstream transcriptional activity, inhibition of proliferation, and enhancement of apoptosis in the vascular wall. ACTRIIA-Fc reveals an unexpectedly prominent role of GDF8, GDF11, and activin as drivers of pulmonary vascular disease and represents a therapeutic strategy for restoring the balance between SMAD1/5/9 and SMAD2/3 signaling in PAH.

Exercise, vascular wall and cardiovascular diseases: an update (part 2).

There is much evidence extolling the virtues of physical activity on cardiovascular disease (CVD). The evidence derives from different population groups where leisure time physical activity reduced the risk of coronary heart disease and cardiovascular mortality in both men and women. Recent meta-analyses have shown that large risk reductions for both ischaemic and haemorrhagic stroke can be achieved by moderate or intense physical activity. There are many data from human and animal studies confirming a beneficial role for exercise in the prevention and treatment of CVD. Physical inactivity and obesity/overweight are not only associated with a number of health-related risk factors, but are considered to be independent risk factors for CVD, type 2 diabetes mellitus and hypertension. Clinical trials confirm that lifestyle interventions (dietary modification and increased physical activity) reduce the risk of progressing from impaired glucose tolerance to type 2 diabetes. Moreover, epidemiological studies indicate that the risk of hypertension increases by being overweight. Modest increases in exercise intensity and frequency have hypotensive effects in sedentary hypertensive patients. Long-term training improves endothelium-dependent dilatation in the aorta and resistance arteries of the heart, whereas short-term training increases endothelial function in coronary conduit arteries. Overall, more scientific evidence will undoubtedly encourage the widespread advocacy of the clinical benefits of exercise therapy in the prevention and treatment of CVD.

Exercise, vascular wall and cardiovascular diseases: an update (Part 1).

Cardiovascular disease (CVD) remains the leading cause of morbidity and premature mortality in both women and men in most industrialized countries, and has for some time also established a prominent role in developing nations. In fact, obesity, diabetes mellitus and hypertension are now commonplace even in children and youths. Regular exercise is rapidly gaining widespread advocacy as a preventative measure in schools, medical circles and in the popular media. There is overwhelming evidence garnered from a number of sources, including epidemiological, prospective cohort and intervention studies, suggesting that CVD is largely a disease associated with physical inactivity. A rapidly advancing body of human and animal data confirms an important beneficial role for exercise in the prevention and treatment of CVD. In Part 1 of this review we discuss the impact of exercise on CVD, and we highlight the effects of exercise on (i) endothelial function by regulation of endothelial genes mediating oxidative metabolism, inflammation, apoptosis, cellular growth and proliferation, increased superoxide dismutase (SOD)-1, down-regulation of p67phox, changes in intracellular calcium level, increased vascular endothelial nitric oxide synthase (eNOS), expression and eNOS Ser-1177 phosphorylation; (ii) vascular smooth muscle function by either an increased affinity of the Ca2+ extrusion mechanism or an augmented Ca2+ buffering system by the superficial sarcoplasmic reticulum to increase Ca2+ sequestration, increase in K+ channel activity and/or expression, and increase in L-type Ca2+ current density; (iii) antioxidant systems by elevation of Mn-SOD, Cu/Zn-SOD and catalase, increases in glutathione peroxidase activity and activation of vascular nicotinamide adenine dinucleotide phosphate [(NAD(P)H] oxidase and p22phox expression; (iv) heat shock protein (HSP) expression by stimulating HSP70 expression in myocardium, skeletal muscle and even in human leucocytes, probably through heat shock transcription factor 1 activity; (v) inflammation by reducing serum inflammatory cytokines such as high-sensitivity C-reactive protein (hCRP), interleukin (IL)-6, IL-18 and tumour necrosis factor-alpha and by regulating Toll-like receptor 4 pathway. Exercise also alters vascular remodelling, which involves two forms of vessel growth including angiogenesis and arteriogenesis. Angiogenesis refers to the formation of new capillary networks. Arteriogenesis refers to the growth of pre-existent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. Another aim of this review is to focus on exercise-related cardiovascular protection against CVD and associated risk factors such as aging, coronary heart disease, hypertension, heart failure, diabetes mellitus and peripheral arterial diseases mediated by vascular remodelling. Lastly, this review examines the benefits of exercise in mitigating pre-eclampsia during pregnancy by mechanisms that include improved blood flow, reduced blood pressure, enhanced placental growth and vascularity, increased activity of antioxidant enzymes, reduced oxidative stress and restored vascular endothelial dysfunction.

Apelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice.

OBJECTIVE: The apelin receptor APJ is a putative receptor protein related to angiotensin (Ang) type 1 receptor. The apelin-APJ system has been implicated in diabetes, but its role in the diabetic vasculature and the mechanisms involved remain unclear. Our aim here was to explore the regulatory role of apelin in the aortic vascular tone in diabetic mice. METHODS: A Multi Myograph system was used to determine the isometric vessel tone in aortic rings from diabetic db/db and control db/m+ mice. The mRNA, phosphorylation, and protein levels of APJ, Akt, and endothelial nitric oxide synthase (eNOS) were analyzed by reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: There is depressed expression of APJ, enhanced contractile response to Ang II, and reduced relaxation response to acetylcholine in aortas from db/db mice. Apelin treatment strikingly reversed the altered aortic vascular responsiveness to Ang II and acetylcholine in db/db mice, both of which were abolished by the eNOS inhibitor NG-nitro-L-arginine methyl ester. Finally, in db/db mice, considerable increases in phosphorylation of Akt on serine 473 and of eNOS on serine 1177 were found in aortas pretreated with apelin. CONCLUSIONS: Apelin treatment modulates the abnormal aortic vascular tone in response to Ang II and acetylcholine by potentiating phosphorylation of Akt and eNOS in diabetic mice, suggesting that the apelin-APJ system might be an important regulator of vascular function in diabetes.

NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension.

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-ß (SMAD-TGF-ß) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-ß ligand trap and, thus, was not contingent on TGF-ß signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-ß-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.

Tamoxifen and estrogen attenuate enhanced vascular reactivity induced by estrogen deficiency in rat carotid arteries.

Recent clinical trials showed that estrogen usage in postmenopausal women did not affect coronary heart disease incidence, in contrast to several laboratory studies showing that estrogen decreased vascular reactivity. We speculated that, in some arteries, estrogen deficiency enhances endothelial function to compensate for the increased vascular smooth muscle reactivity. In this study, we examined the role of endothelium-derived vasoactive factors and the influence of in vivo estrogen and/or tamoxifen treatment on vascular reactivity of estrogen-deficient rats. Common carotid arteries were isolated from sham-operated (control), ovariectomized (Ovx), estrogen- or tamoxifen-treated Ovx rats, and Ovx rats co-treated with estrogen and tamoxifen. U46619 or phenylephrine induced similar contractions in endothelium-intact rings from all groups. Interestingly, removal of endothelium unmasked enhanced contractions in Ovx rats, which was prevented by estrogen, tamoxifen, or estrogen+tamoxifen treatment. Contractions to high K(+) were higher in both endothelium-intact and endothelium-denuded arteries from Ovx rats. Estrogen or tamoxifen treatment normalized high K(+)-induced contraction. A gap junction blocker, 18alpha-glycyrrhetinic acid, revealed enhanced contractions to U46619 in the absence or presence of l-NNA. Western blotting showed enhanced expressions of gap junctional connexin 43 in Ovx group. This study suggests that ovariectomy increases functional expression of gap junction-mediated endothelium-derived hyperpolarizing factor. Also, vascular effects of ovariectomy can be reversed by estrogen, tamoxifen or estrogen+tamoxifen treatment, suggesting that tamoxifen confers estrogenic effects in the vascular system.

The novel peptide apelin regulates intrarenal artery tone in diabetic mice.

Apelin, a newly identified angiotensin (Ang) II homologue, has been implicated in diabetes. We previously reported that apelin exerts an opposing influence on the Ang II signaling. Our aim was to further implore whether apelin could regulate intrarenal artery tone in response to Ang II and Ang IV in diabetes. A Multi Myograph system was used to determine the isometric renal artery tone in diabetic db/db and control db/m+ mice. The phosphorylation, and protein levels of endothelial nitric oxide (NO) synthase (eNOS), and apelin receptor APJ were analyzed by Western blotting. Diminished expression of APJ protein and enhanced contractile responses to Ang II and Ang IV were exhibited in renal arteries from db/db mice. Apelin supplement reversed the abnormal renal vascular responsiveness to Ang II and acetylcholine, but not to Ang IV in db/db mice. Finally, in db/db mice, significant increases in phosphorylation of eNOS on serine 1177 and in NO generation were found in renal arteries pretreated with apelin. Our findings provide novel evidence for the regulatory roles of renal apelin system in vascular functions in diabetes. Apelin treatment may regulate the balance between Ang II and NO and thereby exert beneficial effects on the diabetic vascular pathophysiology.