Rat Pulmonary Artery Research Articles

Vasodilatory action of trans-4-methoxy-β-nitrostyrene in rat isolated pulmonary artery.

Trans-4-methoxy-β-nitrostyrene (T4MN) induced more potent vasorelaxant effects in resistance arteries from hypertensive rats than its parent drug, β-nitrostyrene 1-nitro-2-phenylethene (NPe). To better understand the influence of insertion of the electron-releasing methoxy group in the aromatic ring of NPe, we investigated vasorelaxant effects of T4MN in isolated pulmonary artery and compared them with those of NPe in view of the potential interest of T4MN in pulmonary arterial hypertension. T4MN and NPe both caused concentration-dependent vasorelaxation in pulmonary artery rings pre-contracted with either phenylephrine (1µmol/L) or KCl (60mmol/L), an effect unaffected by endothelium removal. In endothelium-intact preparations pre-contracted with phenylephrine, the vasorelaxant effect of T4MN was more potent than that of NPe. However, unlike NPe, this effect was significantly reduced following pretreatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10µmol/L, a guanylate cyclase inhibitor) or tetraethylammonium (5mmol/L, a potassium channel blocker). T4MN abolished the CaCl2 -induced contractions in pulmonary artery preparations stimulated with phenylephrine (PHE) under Ca2+ -free conditions in the presence of verapamil, to preferentially activate receptor-operated calcium channels. From these findings, we propose that T4MN evokes endothelium-independent vasorelaxant effects in isolated rat pulmonary artery, partially by inhibiting Ca2+ influx through L-type Ca2+ channels, as well as by activating soluble guanylate cyclase and potassium channels. The present results suggest the therapeutic potential of T4MN in treating pulmonary arterial hypertension.

Azelnidipine treatment reduces the expression of Cav1.2 protein

AimsAzelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. Main methodsHEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavβ2c. Key findingsThe surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavβ2c subunits that function as a chaperone of Cav1.2. SignificanceThis study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

Pirfenidone Is a Vasodilator: Involvement of KV7 Channels in the Effect on Endothelium-Dependent Vasodilatation in Type-2 Diabetic Mice.

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16–18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

The preventive and therapeutic effects of AAV1-KLF4-shRNA in cigarette smoke-induced pulmonary hypertension.

We found previously that KLF4 expression was up‐regulated in cultured rat and human pulmonary artery smooth muscle cells (PASMCs) exposed to cigarette smoke (CS) extract and in pulmonary artery from rats with pulmonary hypertension induced by CS. Here, we aim to investigate whether CS‐induced pulmonary hypertension (PH) is prevented and ameliorated by targeted pulmonary vascular gene knockdown of KLF4 via adeno‐associated virus 1 (AAV1)‐KLF4‐shRNA in vivo in rat model. The preventive and therapeutic effects were observed according to the different time‐point of AAV1‐KLF4‐shRNA intratracheal administration. We tested haemodynamic measurements of systemic and pulmonary circulations and observed the degree of pulmonary vascular remodelling. In the preventive experiment, KLF4 expression and some pulmonary circulation hemodynamic measurements such as right ventricular systolic pressure (RVSP), mean right ventricular pressure (mRVP), peak RV pressure rate of rise (dP/dt max) and right ventricle (RV) contractility index were increased significantly in the CS‐induced PH model. While in the prevention group (AAV1‐KLF4‐shRNA group), RVSP, mRVP, dP/dt max and RV contractility index which are associated with systolic function of right ventricle decreased and the degree of pulmonary vascular remodelling relieved. In the therapeutic experiment, we observed a similar trend. Our findings emphasize the feasibility of sustained pulmonary vascular KLF4 gene knockdown using intratracheal delivery of AAV1 in an animal model of cigarette smoke‐induced PH and determined gene transfer of KLF4‐shRNA could prevent and ameliorate the progression of PH.

Role of ATP and Cl<sup>–</sup> transporters in regulation of contractile activity of pulmonary artery smooth muscles in hyposmotic conditions

Objective . The role of Cl – -transport in ATP-dependent regulation of contractile activity of rat pulmonary artery (PA) smooth muscle cells was studied. Design and methods . The study was performed on endotheliumdenuded ring segments of the PA of male Wistar rats. Mechanical tension was measured using organ bath technique. Contractions of the PA segments were induced by high-potassium solution (30 mM KCl), hyposmotic solution (40 mM NaCl), as well as restoration of the medium osmolarity (120 mM NaCl) after incubation in hyposmotic solution. Inhibitor of Na + , K + , 2Cl – cotransport (NKCC) bumetanide (10 μM, 30 minutes preincubation), nonselective Cl – -channel blocker SITS (100 μM) and Ca2 + -activated Cl – channels blocker niflumic acid (NA, 10 μM) were used to modulate the Cl – -transport. Results . ATP (10–500 μM) did not affect vascular tone of PA segments incubated in Krebs solution (120 mM NaCl), while 500 and 1000 μM ATP led to the development of transient contractions, the amplitude of which decreased in the presence of bumetanide, SITS and NA. Incubation of PA segments in a hyposmotic medium (40 mM NaCl) caused the development of transient contraction. Subsequent recovery of the medium osmolarity (120 mM NaCl) induced another transient contraction — isosmotic striction. ATP (500 μM) eliminated the relaxation phase of hyposmotic striction, and completely suppressed the development of isosmotic striction. Bumetanide did not affect the action of ATP during isosmotic striction, but restored the relaxation phase of hyposmotic striction. SITS and NA eliminated the effect of ATP on hypo- and isosmotic striction. Conclusions . The constrictive effect of ATP on the smooth muscle cells of the PA is associated with the activation of mechanisms of transmembrane Cl – -redistribution, in which NKCC and Ca2 + -activated Cl – channels are involved.

Activation of yes-associated protein mediates sphingosine-1-phosphate-induced proliferation and migration of pulmonary artery smooth muscle cells and its potential mechanisms.

The aims of the present study were to examine the molecular mechanisms underlying sphingosine-1-phosphate (S1P)-induced rat pulmonary artery smooth muscle cells (PASMCs) proliferation/migration and to determine the effect of yes-associated protein (YAP) activation on S1P-induced PASMCs proliferation/migration and its potential mechanisms. S1P induced YAP dephosphorylation and nuclear translocation, upregulated microRNA-130a/b (miR-130a/b) expression, reduced bone morphogenetic protein receptor 2 (BMPR2), and inhibitor of DNA binding 1(Id1) expression, and promoted PASMCs proliferation and migration. Pretreatment of cells with Rho-associated protein kinase (ROCK) inhibitor Y27632 suppressed S1P-induced YAP activation, miR-130a/b upregulation, BMPR2/Id1 downregulation, and PASMCs proliferation/migration. Knockdown of YAP using small interfering RNA also suppressed S1P-induced alterations of miR-130a/b, BMPR2, Id1, and PASMCs behavior. In addition, luciferase reporter assay indicated that miR-130a/b directly regulated BMPR2 expression in PASMCs. Inhibition of miR-130a/b functions by anti-miRNA oligonucleotides attenuated S1P-induced BMPR2/Id1 downregulation and the proliferation and migration of PASMCs. Taken together, our study indicates that S1P induces activation of YAP through ROCK signaling and subsequently increases miR-130a/b expression, which, in turn, downregulates BMPR2 and Id1 leading to PASMCs proliferation and migration.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01−100 μM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 μM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 μM), the DP1 receptor antagonist BW A868C (1 μM) also inhibited relaxation reaching significance above 10 μM. In contrast, the EP3 receptor antagonist L798106 (1 μM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 μM) blocked transforming growth factor β1 (TGF-β1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 μM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-β1 in the presence of TRE.

PDGF/MEK/ERK axis represses Ca2+ clearance via decreasing the abundance of plasma membrane Ca2+ pump PMCA4 in pulmonary arterial smooth muscle cells.

Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by vascular remodeling and vasoconstriction, which is associated with increased intracellular calcium ion concentration ([Ca2+]i). Platelet-derived growth factor-BB (PDGF-BB) is the most potent mitogen for pulmonary arterial smooth muscle cells (PASMCs) and is involved in vascular remodeling during PAH development. PDGF signaling has been proved to participate in maintaining Ca2+ homeostasis of PASMCs; however, the mechanism needs to be further elucidated. Here, we illuminate that the expression of plasma membrane calcium-transporting ATPase 4 (PMCA4) was downregulated in PASMCs after PDGF-BB stimulation, which could be abolished by restraining the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK). Functionally, suppression of PMCA4 attenuated the [Ca2+]i clearance in PASMCs after Ca2+ entry, promoting cell proliferation and elevating cell locomotion through mediating formation of focal adhesion. Additionally, the expression of PMCA4 was decreased in the pulmonary artery of monocrotaline (MCT)- or hypoxia-induced PAH rats. Moreover, knockdown of PMCA4 could increase the right ventricular systolic pressure (RVSP) and wall thickness (WT) of pulmonary artery in rats raised under normal conditions. Taken together, our findings demonstrate the importance of the PDGF/MEK/ERK/PMCA4 axis in intracellular Ca2+ homeostasis in PASMCs, indicating a functional role of PMCA4 in pulmonary arterial remodeling and PAH development.

Transplantation of viable mitochondria improves right ventricular performance and pulmonary artery remodeling in rats with pulmonary arterial hypertension

ObjectiveBecause mitochondrial dysfunction is a key factor in the progression of pulmonary hypertension, this study tested the hypothesis that transplantation of exogenous viable mitochondria can reverse pulmonary artery remodeling and restore right ventricular performance in pulmonary hypertension. MethodsPulmonary hypertension was induced by parenteral injection of monocrotaline (60 mg/kg) and creation of a left-to-right shunt aortocaval fistula in rats. Three weeks after creation of fistula, the animals were randomly assigned to receive intravenous delivery of placebo solution or allogeneic mitochondria once weekly for 3 consecutive weeks. Mitochondria (100 μg) were isolated from the freshly harvested soleus muscles of naïve rats. Transthoracic echocardiography was performed at 3 weeks after mitochondrial delivery. ResultsEx vivo heart-lung block images acquired by an IVIS Spectrum (PerkinElmer, Waltham, Mass) imaging system confirmed the enhancement of MitoTracker (Invitrogen, Carlsbad, Calif) fluorescence in the pulmonary arteries. Mitochondria transplantation significantly increased lung tissue adenosine triphosphate concentrations and improved right ventricular performance, as evidenced by a reduction in serum levels of B-type natriuretic peptide and ventricular diameter. Right ventricular mass and wall thickness were restored in the mitochondrial group. In the pulmonary arteries of rats that received mitochondrial treatment, vascular smooth muscle cells expressed higher levels of α-smooth muscle actin and smooth muscle myosin heavy chain II, indicating the maintenance of the nonproliferative, contractile phenotype. The hyper-reactivity of isolated pulmonary arteries to α-adrenergic stimulation was also attenuated after mitochondrial transplantation. ConclusionsTransplantation of viable mitochondria can restore the contractile phenotype and vasoreactivity of the pulmonary artery, thereby reducing the afterload and right ventricular remodeling in rats with established pulmonary hypertension. The improvement in overall right ventricular performance suggests that mitochondrial transplantation can be a revolutionary clinical therapeutic option for the management of pulmonary hypertension.

Chemerin Added to Endothelin-1 Promotes Rat Pulmonary Artery Smooth Muscle Cell Proliferation and Migration

BackgroundWhile chemerin has been shown to increase proliferation and migration of systemic vascular smooth muscle cells (SMCs) contributing therefore to the development of hypertension, this remains to be clarified for the pulmonary circulation.MethodsExpression of chemerin and its three receptors (CMKRL1, CCRL2, GPR1) was examined by immunohistochemistry and RTq-PCR in lungs, pulmonary artery, and thoracic aorta from Wistar rats. Primary cultured rat pulmonary artery and thoracic aorta SMCs treated with recombinant chemerin (tested from 5.10–9 to 10–7 mol/L) were assessed for proliferation and migration (both with 10–7 mol/L endothelin-1), as well as for staurosporine-induced apoptosis.ResultsIn pulmonary artery and thoracic aorta, CMKLR1 expression was detected in both endothelial cells and SMCs. In primary cultured pulmonary artery SMCs, chemerin and its three receptors were expressed, and CMKLR1 expression was higher than those of CCRL2 and GPR1. Chemerin added to endothelin-1 increased pulmonary artery SMC proliferation, while chemerin or endothelin-1 alone did not. This effect was less pronounced in thoracic aorta SMCs. Chemerin induced pulmonary artery and thoracic aorta SMC migration, which was exacerbated by endothelin-1 and more pronounced in thoracic aorta SMCs. Chemerin concentration-dependently reduced staurosporine-induced apoptosis in both pulmonary artery and thoracic aorta SMCs. In pulmonary artery SMCs, endothelin-1 treatment increased the expression of CMKLR1, CCRL2, and GPR1, while these expressions were not altered in thoracic aorta SMCs.ConclusionChemerin/CMKRL1 signaling, in conjunction with a key mediator in the pathogenesis of pulmonary hypertensive diseases, endothelin-1, stimulated proliferation and migration, and increased resistance to apoptosis in rat primary cultured pulmonary artery SMCs. Our results suggest that this signaling could play a role in pulmonary artery remodeling observed in pulmonary hypertension.

Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia.

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

Apoptosis signal-regulating kinase 1 inhibition in in vivo and in vitro models of pulmonary hypertension.

Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O2) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast.

A20 attenuates hypoxia-induced pulmonary arterial hypertension by inhibiting NF-κB activation and pulmonary artery smooth muscle cell proliferation

PAH is a progressive disease characterized by uncontrolled proliferation of PASMCs. Zinc finger protein A20 is a negative feedback regulatory protein of NF-κB activity. The aim of this study was to evaluate zinc finger protein A20 can alleviate PAH in hypoxia exposed mice. C57BL/6 mice received a tail vein injection of adenovirus-mediated ad-A20 and ad-A20 shRNA were exposed to hypoxia. PASMCs isolated from rat pulmonary arteries were cultured in hypoxia, and were transfection of A20 adenovirus. Pulmonary hemodynamic parameters were measured by right heart catheterization. Pulmonary vascular morphological changes were analyzed by HE and α-SMA staining. The expression changes of A20, NF-κB and its downstream protein were detected. The expression of phospho-p65 was increased with the prolongation of hypoxia time. The expression of A20 in lung tissue of chronic hypoxia group decreased with the prolongation of hypoxia time. Adenovirus-mediated A20 (ad-A20) overexpression significantly attenuated the abnormally increased RVSP, RV/(LV + S) ratio, WT%, WA%, α-SMA and the pulmonary vessel muscularization. Ad-A20 treatment markedly attenuated the degradation of phospho-p65 and inhibited the induction of phospho-IκBα induced by hypoxia treatment. Furthermore, silencing A20 abolished the protection by anti-inflammatory activity and the inhibitory effect on cell proliferation. We showed that Zinc finger protein A20 can block NF-κB signaling pathway, alleviates the hypoxia-induced abnormal elevation of pulmonary arterial pressure, hyperproliferation of PASMCs and the pulmonary vascular remodeling.

Impact of cigarette smoking on nitric oxide-sensitive and nitric oxide-insensitive soluble guanylate cyclase-mediated vascular tone regulation.

Cigarette smoking induces vascular endothelial dysfunction characterized by impaired nitric oxide (NO) bioavailability. There are two types of soluble guanylate cyclase (sGC), which is a cellular target of NO: NO-sensitive reduced form (the heme moiety with a ferrous iron) and NO-insensitive oxidized (the heme moiety with a ferric iron)/heme-free form. This study investigated the influence of cigarette smoking on NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation in organ chamber experiments with isolated rat and human arteries. The rats were subcutaneously administered phosphate-buffered saline (PBS), nicotine-free cigarette smoke extract (N(-)-CSE) or nicotine-containing cigarette smoke extract (N(+)-CSE) for 4 weeks. Plasma thiobarbituric acid reactive substance (TBARS) levels were higher in the N(+)-CSE group than those in the N(-)-CSE group, and TBARS levels for these groups were higher than those for the PBS group. In the aorta and the pulmonary artery in rats administered N(-)-CSE or N(+)-CSE, acetylcholine-induced relaxation was significantly impaired compared with that in rats administered PBS; there was no significant difference in the relaxation between the N(-)-CSE and N(+)-CSE groups. However, sodium nitroprusside (NO-sensitive sGC stimulant)- and BAY 60-2770 (NO-insensitive sGC stimulant)-induced relaxations were not different among the three groups, regardless of the vessel type. In addition, in the human gastroepiploic artery, the relaxant responses to these sGC-targeting drugs were identical between nonsmokers and smokers. These findings suggest that NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation functions normally even in blood vessels damaged by cigarette smoking.

Molecular cloning and characterization of a cathepsin L-like cysteine protease of Angiostrongylus cantonensis

Angiostrongylus cantonensis is a parasitic nematode dwelling in the heart and pulmonary arteries of rats, which can cause angiostrongyliasis in human by accidental infections, manifested as eosinophilic meningitis or meningoencephalitis. Cysteine proteases are the major class of endopeptidases that are expressed at a high level in A. cantonensis, which suggests it may play key roles in pathogenesis of the disease. In this study, the biological properties of the cathepsin L-like peptidase (Ac-cathL) of A. cantonensis were investigated. The Ac-cathL gene was identified from the fourth stage cDNA library of A. cantonensis, and then cloned and characterized by bioinformatics analysis and heterologous expression. The open reading frame (ORF) of Ac-cathL (1068 bp) encodes a protein of 355 amino acids with an estimated molecular weight of 58.0 kDa. Sequence analysis and multiple sequence alignment demonstrated that Ac-cathL resembles members of cathepsin L family of other parasites and mammals. Stage-dependent mRNA expression analysis showed that Ac-cathL transcripts were expressed in all stages of A. cantonensis, with the highest expression in female stage. The recombinant Ac-cathL (rAc-cathL) expressed in Escherichia coli exhibited protease activity in acidic pH as demonstrated by gelatin zymography, as well as hydrolytic activity against natural substrates, including BSA, human IgG and human fibrinogen. Immunolocalization revealed that Ac-cathL is localized in tegument of the 18 days post infection stage and uterus of the female adult stage. Therefore, these results implied that the Ac-cathL plays important roles in host tissue migration, nutrition uptake and immune evasion.

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.

Potential role of cartilage oligomeric matrix protein in the modulation of pulmonary arterial smooth muscle superoxide by hypoxia.

Changes in reactive oxygen species and extracellular matrix seem to participate in pulmonary hypertension development. Because we recently reported evidence for chronic hypoxia decreasing expression of cartilage oligomeric matrix protein (COMP) and evidence for this controlling loss of pulmonary arterial smooth muscle bone morphogenetic protein receptor-2 (BMPR2) and contractile phenotype proteins, we examined if changes in superoxide metabolism could be an important factor in a bovine pulmonary artery (BPA), organoid cultured under hypoxia for 48 h model. Hypoxia (3% O2) caused a depletion of COMP in BPA, but not in bovine coronary arteries. Knockdown of COMP by small-interfering RNA (siRNA) increased BPA levels of mitochondrial and extra-mitochondrial superoxide detected by MitoSOX and dihydroethidium (DHE) HPLC products. COMP siRNA-treated BPA showed reduced levels of SOD2 and SOD3 and increased levels of NADPH oxidases NOX2 and NOX4. Hypoxia increased BPA levels of MitoSOX-detected superoxide and caused changes in NOX2 and SOD2 expression similar to COMP siRNA, and exogenous COMP (0.5 μM) prevented the effects of hypoxia. In the presence of COMP, BMPR2 siRNA-treated BPA showed increases in superoxide detected by MitoSOX and depletion of SOD2. Superoxide scavengers (0.5 μM TEMPO or mitoTEMPO) maintained the expression of contractile phenotype proteins calponin and SM22α decreased by 48 h hypoxia (1% O2). Adenoviral delivery of BMPR2 to rat pulmonary artery smooth muscle cells prevented the depletion of calponin and SM22α by COMP siRNA. Thus, COMP regulation of BMPR2 appears to have an important role in controlling hypoxia-elicited changes in BPA superoxide and its potential regulation of contractile phenotype proteins.

Evaluation of Antispasmodic Effect of Arcapillin on Smooth Muscles of Rats

Background: Arcapillin, 2',4',5-trihydroxy-5',6,7–trimethoxyflavone, is a flavone aglycone isolated from several Artemisia species, though, it was first identified from A. capillaris. The Artemisia species are used in folk medicine as a remedy for gastrointestinal and liver illnesses, hypertension, fever and inflammation. Studies indicated a potential role of arcapillin to relieve symptoms of liver disorders; however, there is no report yet in the literature of its effect on smooth muscles. Objective: Our study aims to evaluate the effect of arcapillin, isolated from A. monosperma, on the contractile activity of rat smooth muscles. Materials and Methods: Increased concentrations of arcapillin were tested on isolated rat ileum, pulmonary artery, trachea, and urinary bladder. The muscle contraction was recorded upon addition of arcapillin in eight cumulative concentrations of half log units in the range of [10-7 M -3×10-4 M]. Depending on the organ-containing muscles, the preparations were treated with arcapillin either at basal tonus or after pre-stimulated via a contractile agent; 10-3 M O-acetylcholine on ileum and 10-5 M L-phenylephrine on pulmonary artery rings. Control tissues were treated with sodium hydroxide in an equivalent concentration to that used to dissolve the flavone. Results: Arcapillin caused a dose-dependent relaxation on ileum preparation and pulmonary artery. The inhibition of the contractile activity of ileum was reversible within 60 seconds after washing off the flavone. The urinary bladder showed a slight increase in contraction at the highest concentrations starting at [10-4 M] of arcapillin. There was no observed effect on the contraction of tracheal smooth muscles by all tested concentrations of arcapillin. Conclusion: The antispasmodic activity of arcapillin may contribute to the pharmaceutical importance of A. monosperma in particularly to treat gastrointestinal disorders.

Downregulation of miR‑98 contributes to hypoxic pulmonary hypertension by targeting ALK1.

Chronic hypoxia is one of the most common causes of secondary pulmonary hypertension, the mechanisms of which remain unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit the translation or accelerate the degradation of mRNA. Previous studies have demonstrated that deregulated miRNA expression contributes to various cellular processes including cell apoptosis and proliferation, which are mediated by hypoxia. In the present study, the expression of miR-98 was identified to be decreased in the lung tissue of a hypoxic pulmonary hypertension (HPH) rat model and pulmonary artery (PA) smooth muscle cells (PASMCs), which was induced by hypoxia. By transfecting miR-98 mimics into PASMCs, the high expression of miR-98 inhibited cell proliferation, but upregulated hypoxia-induced PASMCs apoptosis. However, these effects of miR-98 mimics on PASMCs were reversed by ALK1 (activin receptor-like kinase-1) overexpression. ALK1 was identified as a candidate target of miR-98. In addition, overexpressing miR-98 markedly decreased the pulmonary artery wall thickness and the right ventricular systolic pressure in rats induced by hypoxia. These results provided clear evidence that miR-98 was a direct regulator of ALK1, and that the downregulation of miR-98 contributed to the pathogenesis of HPH. These results provide a novel potential therapeutic strategy for the treatment of HPH.

INCREASED EXPRESSION OF THE NERVE GROWTH FACTOR (NGF) IN PULMONARY ARTERY IS ACCOMPANIED BY CHANGES OF PARTICULAR MICRORNAS LEVELS IN MONOCROTALINE-INDUCED PULMONARY HYPERTENSION

Objective: NGF is a member of the neurotrophin family, whose role has been described in the development and persistence of pulmonary hypertension (PH). Let-7 family is a group of microRNAs that targets NGF and downregulates its expression. On the other hand, microRNA miR-21, has been shown to support NGF signalling, and its expression is increased after NGF stimulation in neuronal cells. Furthermore, miR-21 was reported to contribute to proliferation and migration of lung vascular smooth muscle cells and to decrease BMPR-II expression. In this study, we examined the role of these microRNAs in regulation of NGF expression in pulmonary artery and lungs and, consequently, in the development of experimental monocrotaline-induced PH. Design and method: Male Wistar rats were injected either with monocrotaline (s.c. 60 mg/kg, MCT group) or with vehicle (CON group). Four weeks after MCT treatment, vital functions and right ventricular pressure of rats were measured. Expression of let-7 family, miR-21 and NGF was determined by qRT-PCR in rat pulmonary artery and whole lungs. Results: After MCT administration, we observed decreased oxygen saturation, increased right ventricular pressure and right ventricular hypertrophy (P < 0.05 vs CON). NGF gene expression was significantly increased in pulmonary artery (+106%, P < 0.05 vs CON). In the let-7 microRNA family, expression of two members, namely let-7 g and let-7i was significantly decreased (−31% and −34% respectively, P < 0.05 vs CON), whereas expression of miR-21 was significantly increased (+37%, P < 0.05 vs CON). In lung tissue, we did not note any change in NGF gene expression, together with no significant changes in expression of let-7 family. However, there was a significant increase in miR-21 expression (+92%, P < 0.05 vs CON). Conclusions: The decreased expression of let-7 g and let-7i microRNAs is in line with the increase in NGF expression in pulmonary artery. Additionally, increased miR-21 expression could further enhance NGF signalling. Therefore, we conclude that the let-7 – NGF – miR-21 axis could be a significant contributor to pulmonary vascular pathophysiology in MCT-induced PH.