We have analysed whether pulmonary arterial hypertension (PAH) alters the rat faecal microbiota. Wistar rats were injected with the VEGF receptor antagonist SU5416 (20 mg/kg s.c.) and followed for 2 weeks kept in hypoxia (10% O₂, PAH) or injected with vehicle and kept in normoxia (controls). Faecal samples were obtained and microbiome composition was determined by 16S rRNA gene sequencing and bioinformatic analysis. No effect of PAH on the global microbiome was found (α- or β-diversity). However, PAH-exposed rats showed gut dysbiosis as indicated by a taxonomy-based analysis. Specifically, PAH rats had a three-fold increase in Firmicutes-to-Bacteroidetes ratio. Within the Firmicutes phylum, there were no large changes in the relative abundance of the bacterial families in PAH. Among Bacteroidetes, all families were less abundant in PAH. A clear separation was observed between the control and PAH clusters based on short chain fatty acid producing bacterial genera. Moreover, acetate was reduced in the serum of PAH rats. In conclusion, faecal microbiota composition is altered as a result of PAH. This misbalanced bacterial ecosystem might in turn play a pathophysiological role in PAH by altering the immunologic, hormonal and metabolic homeostasis.

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. K_v channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of K_v1.5 and K_v7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA.Experimental Approach: K_v 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 K_v 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 K_v7 channel inhibitors and by the GC inhibitor ODQ but preserved when K_v1.5 channels were inhibited. Additionally, DEA‐NO enhanced K_v7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased K_v currents at all potentials ≥−40 mV and induced membrane hyperpolarization. Both effects were prevented by K_v7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by K_v7 inhibitors.Conclusions and Implications: NO donors and riociguat enhance K_v7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP‐induced PA vasodilation. Our study identifies K_v7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of 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.

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.

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.