Comparative analysis of antiproliferative and vasodilator effects of drugs for pulmonary hypertension: Extensive in vitro study in rats and human.

An effective pulmonary hypertension (PH) treatment should combine antiproliferative and vasodilator effects. We characterized a wide-range of drugs comparing their anti-proliferative vs vasodilator effects in human and rat pulmonary artery smooth muscle cells (PASMC). Key findings: 1) Approved PH drugs (PDE5 inhibitors, sGC stimulators and PGI2 agonists) are preferential vasodilators. 2) cGMP stimulators were more effective in cells derived from hypertensive rats. 3) Nifedipine acted equally as vasodilator and antiproliferative. 4) quercetin and imatinib were potent dual vasodilator/antiproliferative drugs. 5) Tacrolimus and levosimendan lacked antiproliferative effects. 6) Forskolin, pinacidil and hydroxyfasudil were more effective as antiproliferative in human cells.

Corrigendum: Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

[This corrects the article DOI: 10.3389/fphar.2023.1021535.].

Survivin inhibition with YM155 ameliorates experimental pulmonary arterial hypertension.

Background: Imbalance between cell proliferation and apoptosis underlies the development of pulmonary arterial hypertension (PAH). Current vasodilator treatment of PAH does not target the uncontrolled proliferative process in pulmonary arteries. Proteins involved in the apoptosis pathway may play a role in PAH and their inhibition might represent a potential therapeutic target. Survivin is a member of the apoptosis inhibitor protein family involved in cell proliferation. Objectives: This study aimed to explore the potential role of survivin in the pathogenesis of PAH and the effects of its inhibition. Methods: In SU5416/hypoxia-induced PAH mice we assessed the expression of survivin by immunohistochemistry, western-blot analysis, and RT-PCR; the expression of proliferation-related genes (Bcl2 and Mki67); and the effects of the survivin inhibitor YM155. In explanted lungs from patients with PAH we assessed the expression of survivin, BCL2 and MKI67. Results: SU5416/hypoxia mice showed increased expression of survivin in pulmonary arteries and lung tissue extract, and upregulation of survivin, Bcl2 and Mki67 genes. Treatment with YM155 reduced right ventricle (RV) systolic pressure, RV thickness, pulmonary vascular remodeling, and the expression of survivin, Bcl2, and Mki67 to values similar to those in control animals. Lungs of patients with PAH also showed increased expression of survivin in pulmonary arteries and lung extract, and also that of BCL2 and MKI67 genes, compared with control lungs. Conclusion: We conclude that survivin might be involved in the pathogenesis of PAH and that its inhibition with YM155 might represent a novel therapeutic approach that warrants further evaluation.

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-ß signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFß-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1ß expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH.

Biomodulation of Physicochemical Parameters, Aromas, and Sensory Profile of Craft Beers by Using Non-Saccharomyces Yeasts.

Beer is an alcoholic beverage produced by the metabolism of yeasts and made from water, malt, and hops. In recent years, the interest in craft beers has increased considerably due to the demand for new beverages and the consumer's willingness to pay higher prices. This article explores the sensorial changes produced in craft beers by using different Saccharomyces and non-Saccharomyces yeasts with several instrumental and sensory analyses performed. After a primary fermentation process with Saccharomyces cerevisiae or Lachancea thermotolerans, it was observed that green beer brewed with L. thermotolerans had a lower pH (3.41) due to the significant production of l-lactic acid (3.98 g/L) compared to that brewed with S. cerevisiae. Following, the bottle conditioning was carried out with a culture of S. cerevisiae, L. thermotolerans, Hanseniaspora vineae, or Schizosaccharomyces pombe. Of note is the increased production of aromatic esters, including 2-phenylethyl acetate in the H. vineae conditioning, which is associated with a high aromatic quality, as well as ethyl lactate in all samples, whose main fermentation was carried out with L. thermotolerans. Although this research is at an early stage, future complementary studies may shed more light on this topic.

Restoration of Vitamin D Levels Improves Endothelial Function and Increases TASK-Like K+ Currents in Pulmonary Arterial Hypertension Associated with Vitamin D Deficiency.

Background: Vitamin D (vitD) deficiency is highly prevalent in patients with pulmonary arterial hypertension (PAH). Moreover, PAH-patients with lower levels of vitD have worse prognosis. We hypothesize that recovering optimal levels of vitD in an animal model of PAH previously depleted of vitD improves the hemodynamics, the endothelial dysfunction and the ionic remodeling. Methods: Male Wistar rats were fed a vitD-free diet for five weeks and then received a single dose of Su5416 (20 mg/Kg) and were exposed to vitD-free diet and chronic hypoxia (10% O2) for three weeks to induce PAH. Following this, vitD deficient rats with PAH were housed in room air and randomly divided into two groups: (a) continued on vitD-free diet or (b) received an oral dose of 100,000 IU/Kg of vitD plus standard diet for three weeks. Hemodynamics, pulmonary vascular remodeling, pulmonary arterial contractility, and K+ currents were analyzed. Results: Recovering optimal levels of vitD improved endothelial function, measured by an increase in the endothelium-dependent vasodilator response to acetylcholine. It also increased the activity of TASK-1 potassium channels. However, vitD supplementation did not reduce pulmonary pressure and did not ameliorate pulmonary vascular remodeling and right ventricle hypertrophy. Conclusions: Altogether, these data suggest that in animals with PAH and severe deficit of vitD, restoring vitD levels to an optimal range partially improves some pathophysiological features of PAH.

Oxygen-sensitivity and Pulmonary Selectivity of Vasodilators as Potential Drugs for Pulmonary Hypertension.

Current approved therapies for pulmonary hypertension (PH) aim to restore the balance between endothelial mediators in the pulmonary circulation. These drugs may exert vasodilator effects on poorly oxygenated vessels. This may lead to the derivation of blood perfusion towards low ventilated alveoli, i.e., producing ventilation-perfusion mismatch, with detrimental effects on gas exchange. The aim of this study is to analyze the oxygen-sensitivity in vitro of 25 drugs currently used or potentially useful for PH. Additionally, the study analyses the effectiveness of these vasodilators in the pulmonary vs the systemic vessels. Vasodilator responses were recorded in pulmonary arteries (PA) and mesenteric arteries (MA) from rats and in human PA in a wire myograph under different oxygen concentrations. None of the studied drugs showed oxygen selectivity, being equally or more effective as vasodilators under conditions of low oxygen as compared to high oxygen levels. The drugs studied showed low pulmonary selectivity, being equally or more effective as vasodilators in systemic than in PA. A similar behavior was observed for the members within each drug family. In conclusion, none of the drugs showed optimal vasodilator profile, which may limit their therapeutic efficacy in PH.

Hepatic Encephalopathy-Associated Cerebral Vasculopathy in Acute-on-Chronic Liver Failure: Alterations on Endothelial Factor Release and Influence on Cerebrovascular Function.

The acute-on-chronic liver failure (ACLF) is a syndrome characterized by liver decompensation, hepatic encephalopathy (HE) and high mortality. We aimed to determine the mechanisms implicated in the development of HE-associated cerebral vasculopathy in a microsurgical liver cholestasis (MHC) model of ACLF. Microsurgical liver cholestasis was induced by ligating and extracting the common bile duct and four bile ducts. Sham-operated and MHC rats were maintained for eight postoperative weeks Bradykinin-induced vasodilation was greater in middle cerebral arteries from MHC rats. Both Nω-Nitro-L-arginine methyl ester and indomethacin diminished bradykinin-induced vasodilation largely in arteries from MHC rats. Nitrite and prostaglandin (PG) F1α releases were increased, whereas thromboxane (TX) B2 was not modified in arteries from MHC. Expressions of endothelial nitric oxide synthase (eNOS), inducible NOS, and cyclooxygenase (COX) 2 were augmented, and neuronal NOS (nNOS), COX-1, PGI2 synthase, and TXA2S were unmodified. Phosphorylation was augmented for eNOS and unmodified for nNOS. Altogether, these endothelial alterations might collaborate to increase brain blood flow in HE.

Uncovered Contribution of Kv7 Channels to Pulmonary Vascular Tone in Pulmonary Arterial Hypertension.

K+ channels play a fundamental role regulating membrane potential of pulmonary artery (PA) smooth muscle cells and their impairment is a common feature in pulmonary arterial hypertension (PAH). K+ voltage-gated channel subfamily Q (KCNQ1-5) or Kv7 channels and their regulatory subunits subfamily E (KCNE) regulatory subunits are known to regulate vascular tone, but whether Kv7 channel function is impaired in PAH and how this can affect the rationale for targeting Kv7 channels in PAH remains unknown. Here, we have studied the role of Kv7/KCNE subunits in rat PA and their possible alteration in PAH. Using the patch-clamp technique, we found that the total K+ current is reduced in PA smooth muscle cells from pulmonary hypertension animals (SU5416 plus hypoxia) and Kv7 currents made a higher contribution to the net K+ current. Likewise, enhanced vascular responses to Kv7 channel modulators were found in pulmonary hypertension rats. Accordingly, KCNE4 subunit was highly upregulated in lungs from pulmonary hypertension animals and patients. Additionally, Kv7 channel activity was enhanced in the presence of Kv1.5 and TASK-1 channel inhibitors and this was associated with an increased KCNE4 membrane abundance. Compared with systemic arteries, PA showed a poor response to Kv7 channel modulators which was associated with reduced expression and membrane abundance of Kv7.4 and KCNE4. Our data indicate that Kv7 channel function is preserved and KCNE4 is upregulated in PAH. Therefore, compared with other downregulated channels, the contribution of Kv7 channels is increased in PAH resulting in an enhanced sensitivity to Kv7 channel modulators. This study provides insight into the potential usefulness of targeting Kv7 channels in PAH.

Vitamin D deficiency downregulates TASK-1 channels and induces pulmonary vascular dysfunction.

Vitamin D (VitD) receptor regulates the expression of several genes involved in signaling pathways affected in pulmonary hypertension (PH). VitD deficiency is highly prevalent in PH, and low levels are associated with poor prognosis. We investigated if VitD deficiency may predispose to or exacerbate PH. Male Wistar rats were fed with a standard or a VitD-free diet for 5 wk. Next, rats were further divided into controls or PH, which was induced by a single dose of Su-5416 (20 mg/kg) and exposure to hypoxia (10% O2) for 2 wk. VitD deficiency had no effect on pulmonary pressure in normoxic rats, indicating that, by itself, it does not trigger PH. However, it induced several moderate but significant changes characteristic of PH in the pulmonary arteries, such as increased muscularization, endothelial dysfunction, increased survivin, and reduced bone morphogenetic protein (Bmp) 4, Bmp6, DNA damage-inducible transcript 4, and K+ two-pore domain channel subfamily K member 3 (Kcnk3) expression. Myocytes isolated from pulmonary arteries from VitD-deficient rats had a reduced whole voltage-dependent potassium current density and acid-sensitive (TASK-like) potassium currents. In rats with PH induced by Su-5416 plus hypoxia, VitD-free diet induced a modest increase in pulmonary pressure, worsened endothelial function, increased the hyperreactivity to serotonin, arterial muscularization, decreased total and TASK-1 potassium currents, and further depolarized the pulmonary artery smooth muscle cell membrane. In human pulmonary artery smooth muscle cells from controls and patients with PH, the active form of VitD calcitriol significantly increased KCNK3 mRNA expression. Altogether, these data strongly suggest that the deficit in VitD induces pulmonary vascular dysfunction.

Effect of Coffee Cascara Dietary Fiber on the Physicochemical, Nutritional and Sensory Properties of a Gluten-Free Bread Formulation.

This study aimed to assess the physicochemical, nutritional and sensory properties of gluten-free breads containing isolated coffee cascara dietary fiber (ICCDF) as a food ingredient. ICCDF was obtained by aqueous extraction. The oil and water holding capacity and the nutritional profile of the novel ingredient were determined. Its safety was certificated by analysis of ochratoxin A, caffeine and gluten. Gluten-free bread formulations were prepared enriching a commercial bakery premix in rice protein (8%) and ICCDF (3% and 4.5%). Nutritional profile of the novel gluten-free breads (dietary fiber, protein, amino acids, lipids, fatty acid profile and resistant starch), as well as bread volume, crumb density, moisture, firmness, elasticity and color intensity were determined. A sensory quantitative descriptive analysis of the breads was conducted using eight trained panelists. New breads showed significantly higher (p < 0.05) content of dietary fiber and protein than the control bread. The addition of ICCDF allowed increasing dough yield, a less crumb firmness and a higher crumb elasticity. The nutrition claims "source of protein and high in dietary fiber" were assigned to the new formulations. In conclusion, a certificated gluten-free bread with improved nutritional and physicochemical properties and good sensorial profile was obtained.

Total, Bioavailable, and Free Vitamin D Levels and Their Prognostic Value in Pulmonary Arterial Hypertension.

Introduction: Epidemiological studies suggest a relationship between vitamin D deficiency and cardiovascular and respiratory diseases. However, whether total, bioavailable, and/or free vitamin D levels have a prognostic role in pulmonary arterial hypertension (PAH) is unknown. We aimed to determine total, bioavailable, and free 25-hydroxy-vitamin D (25(OH)vitD) plasma levels and their prognostic value in PAH patients. Methods: In total, 67 samples of plasma from Spanish patients with idiopathic, heritable, or drug-induced PAH were obtained from the Spanish PH Biobank and compared to a cohort of 100 healthy subjects. Clinical parameters were obtained from the Spanish Registry of PAH (REHAP). Results: Seventy percent of PAH patients had severe vitamin D deficiency (total 25(OH)vitD < 10 ng/mL) and secondary hyperparathyroidism. PAH patients with total 25(OH)vitD plasma above the median of this cohort (7.17 ng/mL) had better functional class and higher 6-min walking distance and TAPSE (tricuspid annular plane systolic excursion). The main outcome measure of survival was significantly increased in these patients (age-adjusted hazard ratio: 5.40 (95% confidence interval: 2.88 to 10.12)). Vitamin D-binding protein (DBP) and albumin plasma levels were downregulated in PAH. Bioavailable 25(OH)vitD was decreased in PAH patients compared to the control cohort. Lower levels of bioavailable 25(OH)vitD (<0.91 ng/mL) were associated with more advanced functional class, lower exercise capacity, and higher risk of mortality. Free 25(OH)vitD did not change in PAH; however, lower free 25(OH)vitD (<1.53 pg/mL) values were also associated with high risk of mortality. Conclusions: Vitamin D deficiency is highly prevalent in PAH, and low levels of total 25(OH)vitD were associated with poor prognosis.

Impact of Nutrition on Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by sustained vasoconstriction, vascular remodeling, inflammation, and in situ thrombosis. Although there have been important advances in the knowledge of the pathophysiology of PAH, it remains a debilitating, limiting, and rapidly progressive disease. Vitamin D and iron deficiency are worldwide health problems of pandemic proportions. Notably, these nutritional alterations are largely more prevalent in PAH patients than in the general population and there are several pieces of evidence suggesting that they may trigger or aggravate disease progression. There are also several case reports associating scurvy, due to severe vitamin C deficiency, with PAH. Flavonoids such as quercetin, isoflavonoids such as genistein, and other dietary polyphenols including resveratrol slow the progression of the disease in animal models of PAH. Finally, the role of the gut microbiota and its interplay with the diet, host immune system, and energy metabolism is emerging in multiple cardiovascular diseases. The alteration of the gut microbiota has also been reported in animal models of PAH. It is thus possible that in the near future interventions targeting the nutritional status and the gut dysbiosis will improve the outcome of these patients.

Impact of Vitamin D Deficit on the Rat Gut Microbiome.

Inadequate immunologic, metabolic and cardiovascular homeostasis has been related to either an alteration of the gut microbiota or to vitamin D deficiency. We analyzed whether vitamin D deficiency alters rat gut microbiota. Male Wistar rats were fed a standard or a vitamin D-free diet for seven weeks. The microbiome composition was determined in fecal samples by 16S rRNA gene sequencing. The vitamin D-free diet produced mild changes on α- diversity but no effect on ß-diversity in the global microbiome. Markers of gut dysbiosis like Firmicutes-to-Bacteroidetes ratio or the short chain fatty acid producing bacterial genera were not significantly affected by vitamin D deficiency. Notably, there was an increase in the relative abundance of the Enterobacteriaceae, with significant rises in its associated genera Escherichia, Candidatus blochmannia and Enterobacter in vitamin D deficient rats. Prevotella and Actinomyces were also increased and Odoribacteraceae and its genus Butyricimonas were decreased in rats with vitamin D-free diet. In conclusion, vitamin D deficit does not induce gut dysbiosis but produces some specific changes in bacterial taxa, which may play a pathophysiological role in the immunologic dysregulation associated with this hypovitaminosis.

miR-1 induces endothelial dysfunction in rat pulmonary arteries.

Endothelial dysfunction plays a central role in the pathophysiology of pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are small single-strand and non-coding RNAs that negatively regulate gene function by binding to the 3'-untranslated region (3'-UTR) of specific mRNAs. microRNA-1 (miR-1) is upregulated in plasma from idiopathic PAH patients and in lungs from an experimental model of PAH. However, the role of miRNA-1 on endothelial dysfunction is unknown. The aim of this study was to analyze the effects of miR-1 on endothelial function in rat pulmonary arteries (PA). Endothelial function was studied in PA from PAH or healthy animals and mounted in a wire myograph. Some PA from control animals were transfected with miR-1 or scramble miR. Superoxide anion production by miR-1 was quantified by dihydroethidium (DHE) fluorescence in rat PA smooth muscle cells (PASMC). Bioinformatic analysis identified superoxide dismutase-1 (SOD1), connexin-43 (Cx43), caveolin 2 (CAV2) and Krüppel-like factor 4 (KLF4) as potential targets of miR-1. The expression of SOD1, Cx43, CAV2, and KLF4 was determined by qRT-PCR and western blot in PASMC. PA incubated with miR-1 presented decreased endothelium-dependent relaxation to acetylcholine. We also found an increase in the production of O2- and decreased expression of SOD1, Cx43, CAV2, and KLF4 in PASMC induced by miR-1, which may contribute to endothelial dysfunction. In conclusion, these data indicate that miR-1 induces endothelial dysfunction, suggesting a pathophysiological role in PAH.

Activation of Kv 7 channels as a novel mechanism for NO/cGMP-induced pulmonary vasodilation.

BACKGROUND AND PURPOSE: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. Kv channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of Kv 1.5 and Kv 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA. EXPERIMENTAL APPROACH: Kv currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph. KEY RESULTS: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on Kv currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by Kv 7 channel inhibitors and by the GC inhibitor ODQ but preserved when Kv 1.5 channels were inhibited. Additionally, DEA-NO enhanced Kv 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased Kv currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by Kv 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by Kv 7 inhibitors. CONCLUSIONS AND IMPLICATIONS: NO donors and riociguat enhance Kv 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies Kv 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension.

Elevated pulmonary arterial pressure in Zucker diabetic fatty rats.

Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5, Kcnk3, Kcnq1, Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension.

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

KEY POINTS: 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. ABSTRACT: Impairment of the voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs are non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region region of specific mRNAs. The present study aimed to analyse the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of the patch clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells. A luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces pulmonary artery smooth muscle cell hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH.