Bianca Barreira

HIF-2α-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction.

Aims Hypoxic conditions stimulate pulmonary vasoconstriction and vascular remodelling, both pathognomonic changes in pulmonary arterial hypertension (PAH). The secreted protein thrombospondin-1 (TSP1) is involved in the maintenance of lung homeostasis. New work identified a role for TSP1 in promoting PAH. Nonetheless, it is largely unknown how hypoxia regulates TSP1 in the lung and whether this contributes to pathological events during PAH. Methods and results In cell and animal experiments, we found that hypoxia induces TSP1 in lungs, pulmonary artery smooth muscle cells and endothelial cells, and pulmonary fibroblasts. Using a murine model of constitutive hypoxia, gene silencing, and luciferase reporter experiments, we found that hypoxia-mediated induction of pulmonary TSP1 is a hypoxia-inducible factor (HIF)-2α-dependent process. Additionally, hypoxic tsp1−/− pulmonary fibroblasts and pulmonary artery smooth muscle cell displayed decreased migration compared with wild-type (WT) cells. Furthermore, hypoxia-mediated induction of TSP1 destabilized endothelial cell–cell interactions. This provides genetic evidence that TSP1 contributes to vascular remodelling during PAH. Expanding cell data to whole tissues, we found that, under hypoxia, pulmonary arteries (PAs) from WT mice had significantly decreased sensitivity to acetylcholine (Ach)-stimulated endothelial-dependent vasodilation. In contrast, hypoxic tsp1−/− PAs retained sensitivity to Ach, mediated in part by TSP1 regulation of pulmonary Kv channels. Translating these preclinical studies, we find in the lungs from individuals with end-stage PAH, both TSP1 and HIF-2α protein expression increased in the pulmonary vasculature compared with non-PAH controls. Conclusions These findings demonstrate that HIF-2α is clearly implicated in the TSP1 pulmonary regulation and provide new insights on its contribution to PAH-driven vascular remodelling and vasoconstriction.

Kv7 channels critically determine coronary artery reactivity: left–right differences and down-regulation by hyperglycaemia

AIMS Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. METHODS AND RESULTS We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. CONCLUSION Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.

The Flavonoid Quercetin Reverses Pulmonary Hypertension in Rats

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.

Ceramide Mediates Acute Oxygen Sensing in Vascular Tissues

AIMS A variety of vessels, such as resistance pulmonary arteries (PA) and fetoplacental arteries and the ductus arteriosus (DA) are specialized in sensing and responding to changes in oxygen tension. Despite opposite stimuli, normoxic DA contraction and hypoxic fetoplacental and PA vasoconstriction share some mechanistic features. Activation of neutral sphingomyelinase (nSMase) and subsequent ceramide production has been involved in hypoxic pulmonary vasoconstriction (HPV). Herein we aimed to study the possible role of nSMase-derived ceramide as a common factor in the acute oxygen-sensing function of specialized vascular tissues. RESULTS The nSMase inhibitor GW4869 and an anticeramide antibody reduced the hypoxic vasoconstriction in chicken PA and chorioallantoic arteries (CA) and the normoxic contraction of chicken DA. Incubation with interference RNA targeted to SMPD3 also inhibited HPV. Moreover, ceramide and reactive oxygen species production were increased by hypoxia in PA and by normoxia in DA. Either bacterial sphingomyelinase or ceramide mimicked the contractile responses of hypoxia in PA and CA and those of normoxia in the DA. Furthermore, ceramide inhibited voltage-gated potassium currents present in smooth muscle cells from PA and DA. Finally, the role of nSMase in acute oxygen sensing was also observed in human PA and DA. INNOVATION These data provide evidence for the proposal that nSMase-derived ceramide is a critical player in acute oxygen-sensing in specialized vascular tissues. CONCLUSION Our results indicate that an increase in ceramide generation is involved in the vasoconstrictor responses induced by two opposite stimuli, such as hypoxia (in PA and CA) and normoxia (in DA).

Different patterns of pulmonary vascular disease induced by type 1 diabetes and moderate hypoxia in rats

Although type 1 and type 2 diabetes are strongly associated with systemic cardiovascular morbidity, the relationship with pulmonary vascular disease had been almost disregarded until recent epidemiological data revealed that diabetes might be a risk factor for pulmonary hypertension. Recent experimental studies suggest that diabetes induces changes in lung function insufficient to elevate pulmonary pressure. The aim of this study was to assess the effects of diabetes on the sensitivity to other risk factors for pulmonary hypertension. We therefore analysed the effects of the combination of diabetes with exposure to moderate hypoxia on classical markers of pulmonary hypertension. Control (saline‐treated) and diabetic (70 mg kg−1 streptozotocin‐treated) male Wistar–Kyoto rats were followed for 4 weeks and exposed to normoxia or moderate normobaric hypoxia (14%) for another 2 weeks. Hypoxia, but not diabetes, strongly reduced voltage‐gated potassium currents, whereas diabetes, but not hypoxia, induced pulmonary artery endothelial dysfunction. Both factors independently induced pulmonary vascular remodelling and downregulated the lung bone morphogenetic protein receptor type 2. However, diabetes, but not hypoxia, induced pulmonary infiltration of macrophages, which was markedly increased when both factors were combined. Diabetes plus hypoxia induced a modest increase in diastolic and mean pulmonary artery pressure and right ventricular weight, while each of the two factors alone had no significant effect. The pattern of changes in markers of pulmonary hypertension was different for moderate hypoxia and diabetes, with no synergic effect except for macrophage recruitment, and the combination of both factors was required to induce a moderate elevation in pulmonary arterial pressure.

Role of acid sphingomyelinase and IL-6 as mediators of endotoxin-induced pulmonary vascular dysfunction

Background Pulmonary hypertension (PH) is frequently observed in patients with acute respiratory distress syndrome (ARDS) and it is associated with an increased risk of mortality. Both acid sphingomyelinase (aSMase) activity and interleukin 6 (IL-6) levels are increased in patients with sepsis and correlate with worst outcomes, but their role in pulmonary vascular dysfunction pathogenesis has not yet been elucidated. Therefore, the aim of this study was to determine the potential contribution of aSMase and IL-6 in the pulmonary vascular dysfunction induced by lipopolysaccharide (LPS). Methods Rat or human pulmonary arteries (PAs) or their cultured smooth muscle cells (SMCs) were exposed to LPS, SMase or IL-6 in the absence or presence of a range of pharmacological inhibitors. The effects of aSMase inhibition in vivo with D609 on pulmonary arterial pressure and inflammation were assessed following intratracheal administration of LPS. Results LPS increased ceramide and IL-6 production in rat pulmonary artery smooth muscle cells (PASMCs) and inhibited pulmonary vasoconstriction induced by phenylephrine or hypoxia (HPV), induced endothelial dysfunction and potentiated the contractile responses to serotonin. Exogenous SMase and IL-6 mimicked the effects of LPS on endothelial dysfunction, HPV failure and hyperresponsiveness to serotonin in PA; whereas blockade of aSMase or IL-6 prevented LPS-induced effects. Finally, administration of the aSMase inhibitor D609 limited the development of endotoxin-induced PH and ventilation-perfusion mismatch. The protective effects of D609 were validated in isolated human PAs. Conclusions Our data indicate that aSMase and IL-6 are not simply biomarkers of poor outcomes but pathogenic mediators of pulmonary vascular dysfunction in ARDS secondary to Gram-negative infections.

Activation of PPARβ/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries

PPARβ/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARβ/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARβ/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARβ/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.

Chemical and biological assessment of metal organic frameworks (MOFs) in pulmonary cells and in an acute in vivo model: relevance to pulmonary arterial hypertension therapy

Pulmonary arterial hypertension (PAH) is a progressive and debilitating condition. Despite promoting vasodilation, current drugs have a therapeutic window within which they are limited by systemic side effects. Nanomedicine uses nanoparticles to improve drug delivery and/or reduce side effects. We hypothesize that this approach could be used to deliver PAH drugs avoiding the systemic circulation. Here we report the use of iron metal organic framework (MOF) MIL-89 and PEGylated MIL-89 (MIL-89 PEG) as suitable carriers for PAH drugs. We assessed their effects on viability and inflammatory responses in a wide range of lung cells including endothelial cells grown from blood of donors with/without PAH. Both MOFs conformed to the predicted structures with MIL-89 PEG being more stable at room temperature. At concentrations up to 10 or 30 µg/mL, toxicity was only seen in pulmonary artery smooth muscle cells where both MOFs reduced cell viability and CXCL8 release. In endothelial cells from both control donors and PAH patients, both preparations inhibited the release of CXCL8 and endothelin-1 and in macrophages inhibited inducible nitric oxide synthase activity. Finally, MIL-89 was well-tolerated and accumulated in the rat lungs when given in vivo. Thus, the prototypes MIL-89 and MIL-89 PEG with core capacity suitable to accommodate PAH drugs are relatively non-toxic and may have the added advantage of being anti-inflammatory and reducing the release of endothelin-1. These data are consistent with the idea that these materials may not only be useful as drug carriers in PAH but also offer some therapeutic benefit in their own right.

Effects of Quercetin in a Rat Model of Hemorrhagic Traumatic Shock and Reperfusion

Background: We hypothesized that treatment with quercetin could result in improved hemodynamics, lung inflammatory parameters and mortality in a rat model of hemorrhagic shock. Methods: Rats were anesthetized (80 mg/kg ketamine plus 8 mg/kg xylazine i.p.). The protocol included laparotomy for 15 min (trauma), hemorrhagic shock (blood withdrawal to reduce the mean arterial pressure to 35 mmHg) for 75 min and resuscitation by re-infusion of all the shed blood plus lactate Ringer for 90 min. Intravenous quercetin (50 mg/kg) or vehicle were administered during resuscitation. Results: There was a trend for increased survival 84.6% (11/13) in the treated group vs. the shock group 68.4% (13/19, p > 0.05 Kaplan–Meier). Quercetin fully prevented the development of lung edema. The activity of aSMase was increased in the shock group compared to the sham group and the quercetin prevented this effect. However, other inflammatory markers such as myeloperoxidase activity, interleukin-6 in plasma or bronchoalveolar fluid were similar in the sham and shock groups. We found no bacterial DNA in plasma in these animals. Conclusions: Quercetin partially prevented the changes in blood pressure and lung injury in shock associated to hemorrhage and reperfusion.

miR‐1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries

The expression of miR‐1 is increased in lungs from the Hyp/Su5416 PAH rat model. Pulmonary artery smooth muscle cells from this animal model are more depolarized and show decreased expression and activity of voltage‐dependent potassium channel (Kv)1.5. miR‐1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. Antagomir‐1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition.