Contraction Of Pulmonary Arteries Research Articles

Contractions Induced in Human Pulmonary Arteries by a H2S Donor, GYY 4137, Are Inhibited by Low-Frequency (20 kHz) Ultrasound.

The present study aimed to investigate the effect of a H2S donor, GYY 4137, on human pulmonary arteries and whether low-frequency ultrasound (20 kHz, 4 W/cm2) inhibits GYY 4137 contractions. Functional studies were conducted on human and rat pulmonary arteries mounted on microvascular myographs. We placed an ultrasonic gadget in the tissue organ bath to insonate the arteries with low-frequency ultrasound. To measure the effect of the low-frequency ultrasound on the entrance of extracellular Ca2+, the preparations were placed in a Ca2+-free solution, and the thromboxane agonist, U46619, and extracellular calcium were added in the presence of insonation. In isolated human pulmonary arteries, GYY 4137 induced contractions, which were most pronounced in the arteries contracted with the thromboxane analogue, U46619. The transient GYY4137 contractions were reversed by low-frequency ultrasound, a blocker of KV7 channels, XE-991 (10 µM), and glibenclamide (1 μM), a blocker of ATP-sensitive channels. Low-frequency ultrasound also inhibited the contractions induced by the smooth muscle entrance of increasing extracellular calcium concentrations. The present findings show that GYY 4137 can cause a transient contraction of pulmonary arteries in human arteries. GYY 4137 alone does not cause significant vascular contraction in rat lung arteries, but it contracts rat lung arteries precontracted with U46619. The transient contractions induced by GYY 4137 can be inhibited by low-frequency ultrasound, probably by counteracting the influx of external Ca2+. The effect of low-frequency ultrasound counteracts contraction in pulmonary arteries; therefore, a possibility could be to develop a larger device allowing treatment of patients with pulmonary hypertension.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension

RationalePulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. MethodsTo test this hypothesis, we exposed global (Gpr75−/−) and endothelial cell (EC) GPR75 knockout (EC-Gpr75−/−) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. ResultsChronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75−/− or EC-Gpr75−/− mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75−/−, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. ConclusionThese results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves.

Pulmonary hypertension due to HFpEF in rats

Heart failure with preserved ejection fraction (HFpEF) is frequently complicated by pulmonary hypertension (PH), which underlying mechanism remains largely unknown. When present, it is associated with a worse outcome and there are no therapeutic options for PH due to HFpEF. In this context, we sought to characterize the pulmonary circulation in an experimental model of HFpEF in rats, replicating the characteristics of HFpEF observed in patients. Obesity-prone (OP) and -resistant Sprague-Dawley rats were respectively fed with a high-fat diet or a standard rat chow for 12 months ( n = 10 rats in each group) and evaluated by echocardiography, left (LV) and right ventricular (RV) catheterization, histological and pulmonary artery vasoactive analyses. In OP rats fed with high-fat diet during 12 months, HFpEF was confirmed with preserved LV ejection fraction, LV diastolic dysfunction (assessed by increased LV end-diastolic pressure) associated to concentric LV hypertrophy and fibrosis. In rats with HFpEF, RV systolic pressure was increased (31.6 ± 1.0 versus 26.3 ± 0.5 mmHg, P < 0.001), with no significant changes in pulmonary artery muscularization and inflammatory infiltration. Echocardiography showed an increase in pulmonary artery (PA) diameter and PA-to-aorta diameter ratio associated with an increased RV/LV ratio. Circulating levels of sST2 were increased in rats with HFpEF, while NT-proBNP levels decreased. Ex vivo pulmonary artery contraction to phenylephrine and endothelin-1 was similar in both groups of rats. Our model shows that rats develop HFpEF associated to typical risk factors and present with PH. However, at this early stage of HFpEF, neither pronounced pulmonary structural changes nor RV functional and structural changes were observed, suggesting that other mechanisms are likely involved in the development of pulmonary vascular disease in HFpEF.

Role of Rho‐associated kinase (ROCK) in the different speed of relaxation between pulmonary arteries and mesentery arteries of rats

Pulmonary arteries (PA) are exposed to hemodynamic environment and contractile stimuli different from those of systemic arteries. Previous studies reported that in situapplication of sodium nitroprusside (SNP, NO donor) lowered the PA pressure, which was less prominent than the response of systemic arteries. Here, we investigated the relaxation speed and the in vitro effects of NO‐dependent signaling modulators to compare between isolated PA and mesenteric arteries (MA) of rats. In the dual‐wire myography study, PA showed significantly slower relaxation after the response to 80 mM KCl (80K) or to pharmacological agonists (U46619 and PGF2a). Surprisingly, addition of 30 μM SNP could not prevent the 80K contraction in PA while significantly attenuated the contraction of MA. The pretreatment with soluble guanylate cyclase (sGC) inhibitor (ODQ, 10 μM) markedly further slowed the relaxation of PA after 80K or agonist‐induced contraction. Perplexingly, immunoblot assay revealed higher expression of sGC‐a with similar level of sGC‐b1 in PA than MA. The expression of RhoA‐kinase (ROCK) was higher while myosin light chain phosphatase (MYPT) appeared lower in PA than MA. The treatment with ROCK inhibitor (Y27632, 10 μM) could abolish the different of relaxation response between PA and MA. Taken together, we speculate that despite the higher expression and the functional importance of NO/sGC pathway in PA, the higher level of ROCK and relatively low MYPT in PA are responsible for the characteristic slow relaxation after removal of stimuli.

T18/S19 diphosphorylation of myosin regulatory Light Chain is Responsible for the impaired relaxation of pulmonary arteries in pulmonary hypertensive rats

Pulmonary arteries (PA) are exposed to hemodynamic environment and contractile stimuli different from those of systemic arteries. Previous studies reported that in situapplication of sodium nitroprusside (SNP, NO donor) lowered the PA pressure, which was less prominent than the response of systemic arteries. Here, we investigated the relaxation speed and the in vitro effects of NO‐dependent signaling modulators to compare between isolated PA and mesenteric arteries (MA) of rats. In the dual‐wire myography study, PA showed significantly slower relaxation after the response to 80 mM KCl (80K) or to pharmacological agonists (U46619 and PGF2a). Surprisingly, addition of 30 mM SNP could not prevent the 80K contraction in PA while significantly attenuated the contraction of MA. The pretreatment with soluble guanylate cyclase (sGC) inhibitor (ODQ, 10 mM) markedly further slowed the relaxation of PA after 80K or agonist‐induced contraction. Perplexingly, immunoblot assay revealed higher expression of sGC‐a with similar level of sGC‐b1 in PA than MA. The expression of RhoA‐kinase (ROCK) was higher while myosin light chain phosphatase (MYPT) appeared lower in PA than MA. The treatment with ROCK inhibitor (Y27632, 10 mM) could abolish the different of relaxation response between PA and MA. Taken together, we speculate that despite the higher expression and the functional importance of NO/sGC pathway in PA, the higher level of ROCK and relatively low MYPT in PA are responsible for the characteristic slow relaxation after removal of stimuli.

CRISPR-mediated Bmpr2 point mutation exacerbates late pulmonary vasculopathy and reduces survival in rats with experimental pulmonary hypertension

BackgroundPatients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (Bmpr2) mutations present earlier with severe hemodynamic compromise and have poorer survival outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with Bmpr2 mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant Bmpr2 mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats.MethodsA monoallelic single nucleotide insertion in exon 1 of Bmpr2 (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed.ResultsThe +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/[LV + S]) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type.ConclusionsThe present study demonstrates that the Bmpr2 mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.

Beneficial effects of a preventive treatment with anethole trithione on pulmonary hypertension

Pulmonary hypertension (PH) is a multifactorial disease characterized by pulmonary arteries (PA) and right ventricular (RV) alterations. Mitochondria dysfunction and associated reactive oxygen species production were shown to contribute to these phenomena. OP2 Drugs start-up demonstrated that anethole trithione (ATT), the active ingredient of its principal lead OP2113, specifically inhibits mitochondrial ROS production from complex I. In this context, this study aims to assess the effects of ATT on PH set-up. By using osmotic mini-pump, influence of a preventive treatment with ATT or its vehicle was tested on PH development in male rats exposed to three weeks of chronic hypoxia (CH - 10 % O2). PA and cardiac PH hallmarks (remodelling, fibrosis and hemodynamic parameters) were assessed. Contraction and intracellular calcium signal of isolated cardiomyocytes from RV were simultaneously measured by IonOptix system. We quantified cardiac mitochondrial mass by measuring VDAC1 expression by western blotting. The effect of preventive or acute treatment with ATT on PA reactivity was measured by isometric tension. In rats with PH, preventive treatment with ATT reduced PA remodelling and RV systolic pressure, whereas RV hypertrophy and fibrosis seemed not to be affected. However, this preventive treatment abolished the decrease of both speed of cardiomyocytes contraction and VDAC1 expression induced by CH. Preventive and acute treatment with ATT reversed PA hyperreactivity to 5-HT in rats with PH. Acute ATT treatment triggered a sustained relaxation of preconstricted PA in both control and PH conditions. L-NAME (eNOS inhibitor) and CHAPS (non-denaturing detergent used to remove endothelium) did not affect the acute effect of ATT on PA contraction and relaxation whereas glibenclamide (KATP channel inhibitor) attenuated ATT induced relaxation. Y27632 (a ROCK inhibitor) and ATT induced additive reduction of 5-HT contraction. Altogether, these results highlighted the benefit of an ATT preventive treatment on PA reactivity and remodelling as well as RV cardiac function suggesting a potential therapeutic interest of ATT in PH set-up. Whereas ATT effect on reactivity seemed to be endothelium and ROCK-independent, KATP channel was partly involved in ATT-induced relaxation.

Right ventricular overloading is attenuated in monocrotaline-induced pulmonary hypertension model rats with a disrupted Gpr143 gene, the gene that encodes the 3,4-l-dihydroxyphenyalanine (l-DOPA) receptor

Pulmonary hypertension (PH) is a severe and progressive disease that causes elevated right ventricular systolic pressure, right ventricular hypertrophy and ultimately right heart failure. However, the underlying pathophysiologic mechanisms are poorly understood. We previously showed that 3,4-l-dihydroxylphenyalanine (DOPA) sensitizes vasomotor response to sympathetic tone via coupling between the adrenergic receptor alpha1 (ADRA1) and a G protein-coupled receptor 143 (GPR143), a DOPA receptor. We investigated whether DOPA similarly enhances ADRA1-mediated contraction in pulmonary arteries isolated from rats, and whether GPR143 is involved in the PH pathogenesis. Pretreating the isolated pulmonary arteries with DOPA 1 μM enhanced vasoconstriction in response to phenylephrine, an ADRA1 agonist, but not to U-46619, a thromboxane A2 agonist or endothelin-1. We generated Gpr143 gene-deficient (Gpr143-/y) rats, and confirmed that DOPA did not augment phenylephrine-induced contractile response in Gpr143-/y rat pulmonary arteries. We utilized a rat model of monocrotaline (MCT)-induced PH. In the MCT model, the right ventricular systolic pressure was attenuated in the Gpr143-/y rats than in WT rats. Phenylephrine-induced cell migration and proliferation were also suppressed in Gpr143-/y pulmonary artery smooth muscle cells than in WT cells. Our result suggests that GPR143 is involved in the PH pathogenesis in the rat models of PH.

Cannabinoids—A New Perspective in Adjuvant Therapy for Pulmonary Hypertension

Currently, no treatment can completely cure pulmonary hypertension (PH), which can lead to right ventricular failure and, consequently, death. Therefore, searching for new therapies remains important. Increased resistance in pulmonary circulation is mainly caused by the excessive contraction and proliferation of small pulmonary arteries. Cannabinoids, a group of lipophilic compounds that all interact with cannabinoid receptors, exert a pulmonary vasodilatory effect through several different mechanisms, including mechanisms that depend on vascular endothelium and/or receptor-based mechanisms, and may also have anti-proliferative and anti-inflammatory properties. The vasodilatory effect is important in regulating pulmonary resistance, which can improve patients’ quality of life. Moreover, experimental studies on the effects of cannabidiol (plant-derived, non-psychoactive cannabinoid) in animal PH models have shown that cannabidiol reduces right ventricular systolic pressure and excessive remodelling and decreases pulmonary vascular hypertrophy and pulmonary vascular resistance. Due to the potentially beneficial effects of cannabinoids on pulmonary circulation and PH, in this work, we review whether cannabinoids can be used as an adjunctive therapy for PH. However, clinical trials are still needed to recommend the use of cannabinoids in the treatment of PH.

Prenatal hypoxia induced ETBR activation and abnormal ROS signalling in pulmonary artery cells of rat offspring

Pulmonary arterial hypertension is a progressive disorder characterized by remodeling and increased small pulmonary arteries resistance. Endothelin-1 (ET-1) was related to PAH and ET-1 receptors were up-regulated selectively in the lung when exposed to toxic factor hypoxia. However, the role of ET-1 signaling in the pathogenesis of prenatal hypoxia-induced pulmonary abnormalities remains to be elucidated. Pregnant rats were divided into prenatal hypoxia (10.5 % O2 from gestational day 4–21) and control group. Their three-month-old offspring male rats were tested for vascular functions and molecular analysis, DNA methylation was assessed for cellular hypoxia. Functional testing showed that ET-1-mediated vasoconstriction was enhanced, and the expressions of endothelin A receptor/B receptor (ETAR/ETBR), inositol 1,4,5-trisphosphate receptor, type 1, and the sensitivity of calcium channels were increased in the small pulmonary arteries following prenatal hypoxia. q-PCR and DHE staining showed that the expressions of NADPH oxidase 1/4 (Nox1/4) were up-regulated, along with the increased production of superoxide anion. Furthermore, superoxide anion promoted ET-1-mediated pulmonary artery contraction. In the pulmonary artery smooth muscle cell experiments, q-PCR, Western Blot, CCK8 and DHE staining showed that the expressions of ETBR, Nox1/4, and superoxide anion were increased by hypoxia, along with promoted cell proliferation. 2,2,6,6-Tetramethyl-1-piperidinyloxy reversed hypoxia-induced cell proliferation. ETBR antagonist BQ788 inhibited hypoxia-increased expressions of Nox1/4, superoxide anion production, and proliferation of cells. Moreover, methylation analysis indicated that hypoxia decreased the methylation levels of the ETBR promoter in the pulmonary artery smooth muscle cells. The results indicated that prenatal toxic factor hypoxia resulted in abnormal ETBR activation, which enhanced ET-1-mediated vasoconstriction of pulmonary arteries and pulmonary artery smooth muscle cell proliferation through ETBR/Nox1/4-derived ROS pathway.

ATT has positive effects on pulmonary hypertension hallmarks

Pulmonary hypertension (PH) is a multifactorial disease characterized by pulmonary arteries (PA) hyperreactivity and remodelling involving an imbalance of contractile and proliferative agents such as 5-hydroxytryptamine (5-HT). Mitochondria and associated reactive oxygen species (ROS) production were shown to contribute to these phenomena. OP2 Drugs recently demonstrated that Anethole Trithione (ATT), the active ingredient of its principal lead OP2113, specifically inhibits mitochondrial ROS (mtROS) production from complex I in its reverse mode. In this context, this study aims to assess the effects of ATT on PH hallmarks. Male rats were either exposed to normoxia or chronic hypoxia (10% O2) during 3 weeks to induce PH (CH-PH). The effect of ATT on PA reactivity was determined ex vivo by isometric tension measurements of arterial rings. PA contraction was induced by 5-HT, prostaglandin F2α (PGF2α), phenylephrine (Phe) or endothelin-1 (ET-1) whereas relaxation was induced by carbachol (Cch) on preconstricted vessels with PGF2α. The effect of ATT on calcium responses induced by 5-HT was assessed in rat PA smooth muscle cells using confocal imaging. Proliferative responses induced by 5-HT were evaluated by ELISA BrdU on human PASMC in presence or absence of ATT. In rats with CH-PH, ATT reduced contractile hyperreactivity induced by 5-HT and PGF2α. In control rats, ATT treatment also decreased contraction to 5-HT and Phe, but not contraction to ET-1 and PGF2α as well as relaxation to Cch. Interestingly, acute ATT exposure did not modify basal cytosolic calcium signal in control rat PASMC. However, the number of 5-HT responding cells was reduced in presence of ATT whereas neither maximal peak of cytosolic calcium responses nor the global calcium variations were affected. Finally, 5-HT-induced proliferation was reduced by ATT in control human PASMC. In conclusion, ATT improves vascular hyperreactivity of PA from rats with CH-PH whereas in control condition ATT altered 5-HT-induced contraction in rat PA, calcium signalling to 5-HT in rat PASMC and 5-HT-induced human PASMC proliferation. Although mechanisms underlying ATT influence on PH hallmarks need to be further elucidated, these Results suggest that OP2113 may be used to develop new PH therapies.

Carbonic anhydrase inhibition improves pulmonary artery reactivity and nitric oxide-mediated relaxation in sugen-hypoxia model of pulmonary hypertension.

Pulmonary hypertension (PH) is a serious disease with pulmonary arterial fibrotic remodeling and limited responsiveness to vasodilators. Our data suggest that mild acidosis induced by carbonic anhydrase inhibition could ameliorate PH, but the vascular mechanisms are unclear. We tested the hypothesis that carbonic anhydrase inhibition ameliorates PH by improving pulmonary vascular reactivity and relaxation mechanisms. Male Sprague-Dawley rats were either control normoxic (Nx), or injected with Sugen 5416 (20 mg/kg, sc) and subjected to hypoxia (9% O2) (Su+Hx), or Su+Hx treated with acetazolamide (ACTZ, 100 mg/kg/day, in drinking water). After measuring the hemodynamics, right ventricular hypertrophy was assessed by Fulton's Index; vascular function was measured in pulmonary artery, aorta, and mesenteric arteries; and pulmonary arteriolar remodeling was assessed in lung sections. Right ventricular systolic pressure and Fulton's Index were increased in Su+Hx and reduced in Su+Hx + ACTZ rats. Pulmonary artery contraction to KCl and phenylephrine were reduced in Su+Hx and improved in Su+Hx + ACTZ. Acetylcholine (ACh)-induced relaxation and nitrate/nitrite production were reduced in pulmonary artery of Su+Hx and improved in Su+Hx + ACTZ. ACh relaxation was blocked by nitric oxide (NO) synthase and guanylate cyclase inhibitors, supporting a role of NO-cGMP. Sodium nitroprusside (SNP)-induced relaxation was reduced in pulmonary artery of Su+Hx, and ACTZ enhanced relaxation to SNP. Contraction/relaxation were not different in aorta or mesenteric arteries of all groups. Pulmonary arterioles showed wall thickening in Su+Hx that was ameliorated in Su+Hx + ACTZ. Thus, amelioration of pulmonary hemodynamics during carbonic anhydrase inhibition involves improved pulmonary artery reactivity and NO-mediated relaxation and may enhance responsiveness to vasodilator therapies in PH.

Downregulation of Soluble Guanylate Cyclase and Protein Kinase G With Upregulated ROCK2 in the Pulmonary Artery Leads to Thromboxane A2 Sensitization in Monocrotaline-Induced Pulmonary Hypertensive Rats.

Thromboxane A2 (TXA2) promotes various physiological responses including pulmonary artery (PA) contraction, and pathophysiological implications have been suggested in cardiovascular diseases including pulmonary hypertension. Here, we investigated the role of TXA2 receptor (TP)-mediated signaling in the pathophysiology of pulmonary arterial hypertension (PAH). The sensitivity of PA to the contractile agonist could be set by relaxing signals such as the nitric oxide (NO), soluble guanylate cyclase (sGC), and cGMP-dependent kinase (PKG) pathways. Changes in the TP agonist (U46619)-induced PA contraction and its modulation by NO/cGMP signaling were analyzed in a monocrotaline-induced PAH rat model (PAH-MCT). In the myograph study, PA from PAH-MCT showed higher responsiveness to U46619, that is decreased EC50. Immunoblot analysis revealed a lower expression of eNOS, sGC, and PKG, while there was a higher expression of RhoA-dependent kinase 2 (ROCK2) in the PA from PAH-MCT than in the control. In PAH-MCT, the higher sensitivity to U46619 was reversed by 8-Br-cGMP, a membrane-permeable cGMP analog, but not by the NO donor, sodium nitroprusside (SNP 30 μM). In contrast, in the control PA, inhibition of sGC by its inhibitor (1H− [1,2,4] oxadiazolo [4,3−a] quinoxalin-1-one (ODQ), 10 μM) lowered the threshold of U46619-induced contraction. In the presence of ODQ, SNP treatment had no effect whereas the addition of 8-Br-cGMP lowered the sensitivity to U46619. The inhibition of ROCK by Y-27632 attenuated the sensitivity to U46619 in both control and PAH-MCT. The study suggests that the attenuation of NO/cGMP signaling and the upregulation of ROCK2 increase the sensitivity to TXA2 in the PAH animal, which might have pathophysiological implications in patients with PAH.

Evaluation of the combination of endothelin receptor antagonists (ERA) and phosphodiesterase-5 inhibitors for the treatment of pulmonary arterial hypertension (PAH) in pathologic human pulmonary arteries in an ex-vivo organ bath model

PurposeMedical combination therapy of pulmonary arterial hypertension (PAH) may alleviate the drawbacks of monotherapy by avoiding drug tolerance and by increasing effectiveness, as shown by the combination of ambrisentan and tadalafil (AMBITION trial). The present ex-vivo study evaluated the combination of the endothelin receptor antagonists (ERA) macitentan and bosentan with the phosphodiesterase-5 (PDE-5) inhibitor vardenafil in pulmonary arteries from patients suffering from terminal lung disease as a model of PAH. MethodsSegments of the pulmonary vessels were excised from resected lungs of patients requiring lung transplantation (LTX). Contraction of pulmonary arteries (PA) was elicited by consecutive dose-response curves of endothelin-1 (ET-1) followed by norepinephrine (NE) to allow inhibition by different pathways. Forces were measured isometrically in an organ bath in the presence and absence of ERA and PDE-5 inhibitors and their combination. ResultsPA of 38 patients were examined between October 2016 and November 2019. Bosentan (1E-7 M) and macitentan (1E-8 M, 3E-8 M, 1E-7 M) inhibited ET-1 induced contractions, whereas vardenafil (1E-6 M, 3E-6 M, 1E-5 M) inhibited only the NE induced part of the contractions. Vardenafil enhanced bosentan-induced inhibition of vasoconstriction in a dose-dependent fashion. Combination effects exceeded single bosentan at 3E-6 M and 1E-5 M vardenafil, and they exceeded single vardenafil at the lower vardenafil concentrations. Macitentan showed a more pronounced inhibition than bosentan regardless of the lower concentrations. Accordingly, combination effects with vardenafil resembled those of macitentan alone. ConclusionsMacitentan and bosentan were potent antagonists of vasoconstriction in PA of LTX patients. The benefit of drug combinations was demonstrated at selected concentrations only owing to a narrow therapeutic range of vardenafil in this ex-vivo model. These results suggest the utility of drug combinations other than the established pair of ambrisentan and tadalafil in PAH treatment but also make a case for a further assessment of vasodilator properties of drugs complementing ERA.

Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction.

The calcium‐activated chloride channel (CaCC) TMEM16A enables chloride secretion across several transporting epithelia, including in the airways. Additional roles for TMEM16A have been proposed, which include regulating mucus production and secretion and stimulating smooth muscle contraction. The aim of the present study was to test whether the pharmacological regulation of TMEM16A channel function, could affect any of these proposed biological roles in the airways. In vitro, neither a potent and selective TMEM16A potentiator (ETX001) nor the potent TMEM16A inhibitor (Ani9) influenced either baseline mucin release or goblet cell numbers in well‐differentiated primary human bronchial epithelial (HBE) cells. In vivo, a TMEM16A potentiator was without effect on goblet cell emptying in an IL‐13 stimulated goblet cell metaplasia model. Using freshly isolated human bronchi and pulmonary arteries, neither ETX001 or Ani9 had any effect on the contractile or relaxant responses of the tissues. In vivo, ETX001 also failed to influence either lung or cardiovascular function when delivered directly into the airways of telemetered rats. Together, these studies do not support a role for TMEM16A in the regulation of goblet cell numbers or baseline mucin release, or on the regulation of airway or pulmonary artery smooth muscle contraction.

Signaling Pathways Involved in Enhanced 5‐HT‐Induced Contraction of Pulmonary Artery Segments From Guinea‐Pigs With Hyperthyroidism

We have previously reported that hyperthyroidism in rats resulted in enhanced 5‐HT‐induced contractions of pulmonary artery segments by a mechanism involving activation of calcium‐activated chloride channels. We have investigated other possible pathways that might be involved in this response in this study. Pulmonary artery ring segments were set up in Krebs’ solution for isometric tension recording. 5‐HT‐induced contraction was significantly enhanced in main pulmonary artery segments from guinea‐pigs with hyperthyroidism compared with a similar preparation from control guinea pigs. Nifedipine (10−6M) and tempol (3×10−3M) significantly reduced 5‐HT‐induced contraction in artery segments from thyroxine‐treated while having no effect in artery segments from control guinea pigs. Y‐27632 (10−5M) and SB 202190 (10−5M) but not PD 98059 (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated guinea pigs. However, the reduction was greater in artery segments from thyroxine‐treated guinea pigs. Genistein (10−5M) significantly reduced 5‐HT‐induced contractions in artery segments from control or thyroxine‐treated guinea pigs. Genistein was equipotent in both cases. These results would suggest that hyperthyroidism‐induced increased 5‐HT‐induced contraction of the guinea pig pulmonary artery is mediated by multiple signaling pathways including increased influx of extracellular calcium through L‐type dihydropyridine channels, tyrosine kinase pathway, Rho‐kinase pathway of calcium sensitization, increased generation of reactive oxygen species and p38 MAPK pathway.

Role of protein kinase D1 in pulmonary hypertension in rats

Protein kinase D (PKD) is known to be implicated in a variety of physiological functions and pathological conditions including cardiovascular diseases, such as cardiac hypertrophy and ischemia reperfusion injury. PKD has been thought to be a new target of the diseases for treatments. Previously, we showed that PKD played a role in pulmonary arterial contraction through MYPT1 phosphorylation and actin polymerization and that inhibition of PKD activity decreased right cardiac ventricular pressure in monocrotaline‐induced pulmonary hypertension (PH) model,. In this study, we further investigated a precise role of PKD in PH and in human pulmonary smooth muscle cells (HPSMCs).Material and methodsEight‐week‐old male rats were given either 60 mg/kg MCT or saline 4 weeks before experiments. Isometric tension was measured using pulmonary arterial ring samples to examine the effect of PKD inhibitor, CRT0066101 on norepinephrine (NE)‐induced contraction and phosphorylation of PKD, myosin phosphatase target subunit 1 (MYPT1), regulatory myosin light chain (RLC) and other PKD substrate proteins. Expression of PKD and phosphorylated‐PKD in rats lungs were also examined with immunohistochemistry using anti‐phospho‐PKD antibodies. We also investigated precise roles of PKD isoforms using HPSMCs with RNA interference.ResultsIn pulmonary arterial rings, basal expression of PKD1 and phosphorylated PKD were increased in pulmonary arteries in PH rats. NE increased phosphorylation of PKD, MYPT1 and RLC in ring samples during contraction, which were attenuated by pretreatment of CRT0066101. In immunohistochemistry, increased expression of PKD1 was observed in pulmonary arteries in PH rats (Fig 1). In HASMCs, increased phosphorylation of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down.ConclusionsPKD1 may play role in contraction of pulmonary arteries and at least partially via MYPT1 phosphorylation. Increased PKD1 could be involved in PH development in rats.Support or Funding InformationGrant‐in‐Aid for Scientific Research (C) 17K11058Immunohistochemistry with anti‐PKD antibody in lung tissue from PH rats.Figure 1

Cytochrome P450 Epoxygenase-Dependent Activation of TRPV4 Channel Participates in Enhanced Serotonin-Induced Pulmonary Vasoconstriction in Chronic Hypoxic Pulmonary Hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic trpv4−/− mice. These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs, in part via the CYP-EET-TRPV4 pathway. Our results further support the notion that manipulation of TRPV4 function may offer a novel therapeutic strategy for the treatment of hypoxia-related pulmonary hypertension.