Percentage Of Medial Wall Thickness Research Articles

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

Exosomal miR-211 contributes to pulmonary hypertension via attenuating CaMK1/PPAR-γaxis

AimExsomes play a significant role in increasing pathophysiological processes by delivering their content. Recently, a variety of studies have showed exosomal microRNAs (miRNAs) are involved in pulmonary hypertension (PH) notably. In this study, we found that exosomal miR-211 was overexpressed in hypoxia-induced PH rats but its intrinsic regulation was unclear. Therefore, our aim was to reveal the underlying mechanism which overexpressed exosomal miR-211 targeted in the development of PH. Methods18 male SD rats were randomly divided into normoxia and hypoxia group, housed in normal or hypoxic chamber for 3 weeks respectively. Then, mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance(PVR), right ventricular hypertrophy index(RV/(LV + S)), the percentage of medial wall area (WA%) and the percentage of medial wall thickness (WT%) were measured. Expression of miR-211 in exosomes was detected by qRT-PCR. Expression of Ca2+/calmodulin-dependent kinase1(CaMK1)and peroxisome proliferator-activated receptors-γ(PPAR-γ)in lung tissue were detected by Western blot(WB); After miR-211 overexpressed exosomes were injected to rats through caudal vein, mPAP, PVR, RV/(LV + S), WA% and WT% were also measured. Sequentially, hypoxia rats were injected with lentivirus riched in miR-211 inhibitor via tail vein, and PH-related indicators were measured. In vitro, after miR-211 was positively or negatively regulated in pulmonary arterial smooth muscle cell (PASMC) by plasmid transfection, proliferation of PASMC was detected by CCK8, as well as the expression of CaMK1 and PPAR- γ. Further, the relationship between CaMK1 and miR-211 was verified by Dual-Luciferase assay. And the regulatory relationship of CaMK1/PPAR- γ aixs was demonstrated in PASMC. ResultsEvident increases of mPAP, PVR, RVHI, WT% and WA% were observed with hypoxia administration. And the concentration of plasma exosomes in hypoxia rats was increased and positively correlated with the above indexes. miR-211 in exosomes of PH was upregulated while the expression of CaMK1 and PPAR-γ decreased in lung tissues. Further, injection of exosomes overexpressed with miR-211 demonstrated that exosomal miR-211 aggravated PH while inhibition of miR-211 attenuated PH in rats. In vitro, overexpression of miR-211 promoted the proliferation of PASMC and inhibited expression of CaMK1 and PPAR-γ in PASMC. And Dual-luciferase assay demonstrated that CaMK1 was a downstream gene of miR-211. Plasmid transfection experiments indicated that CaMK1 can promote PPAR-γ expression. ConclusionExosomal miR-211 promoted PH via inhibiting CaMK1/PPAR-γ axis, promoting PASMC proliferation in rats.

Inhibition of Siah2 ubiquitin ligase ameliorates monocrotaline-induced pulmonary arterial remodeling through inactivation of YAP

AimsIt has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models. Main methodsIntraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit. Key findingsThe protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats. SignificanceSiah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH.

Efficacy of stem cell therapy for pulmonary arterial hypertension: a systematic review and meta-analysis of preclinical studies

BackgroundDespite significant progress in drug treatment, the prognosis of patients with advanced pulmonary arterial hypertension (PAH) remains extremely poor. Many preclinical studies have reported the efficacy of stem cell (SC) therapy for PAH; however, this approach remains controversial. The aim of this systematic review and meta-analysis is to assess the potential efficacy of SC therapy for PAH.MethodsThe Medline, EMBASE, Cochrane Library, and Web of Science databases were searched from inception to August 12, 2018. Preclinical studies that evaluated the use of SC therapy for PAH were included. The primary outcome was pulmonary haemodynamics, as assessed by measurement of the right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), and/or mean right ventricle pressure (mRVP). The secondary outcomes included the weight ratio of the right ventricle to the left ventricle plus septum (RV/LV+S), the right ventricle to body weight ratio (RV/BW), the percentage of pulmonary arteriole area index (WA), and/or the percentage of medial wall thickness of the pulmonary arteriole (WT). The quality of outcomes was evaluated using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) bias risk tool. The inverse-variance method with random-effects modelling was used to calculate pooled weighted mean differences (WMDs) and 95% CIs. Statistical analysis was performed with STATA 14.0.ResultsTwenty-eight eligible articles (722 animals) were included. SC therapy reduced the pooled WMDs (95% CIs) of RVSP, mPAP, mRVP, RV/LV+S, RV/BW, WA, and WT for animals with PAH, with values of − 14.12 (− 14.63, − 13.61), − 11.86 (− 12.35, − 11.36), − 17.33 (− 18.10, − 16.56), − 0.10 (− 0.10, − 0.09), 0.23 (0.21, 0.24), − 13.66 (− 15.71, − 11.62), and − 7.96 (− 7.99, − 7.93), respectively.ConclusionsSC therapy is effective for PAH in preclinical studies. These results may help to standardise preclinical animal studies and provide a theoretical basis for clinical trial design in the future.Systematic review registrationPROSPERO (http://www.crd.york.ac.uk/PROSPERO).

Grape seed proanthocyanidin inhibits monocrotaline-induced pulmonary arterial hypertension via attenuating inflammation: in vivo and in vitro studies

Inflammation in pulmonary arterioles initiates and maintains pathological processes in pulmonary arterial hypertension (PAH), and inhibition of it attenuates PAH development. Grape seed proanthocyanidin (GSP) is believed to be effective in protecting vascular system via inhibiting inflammation, while its effect on pulmonary circulation remains inconclusive. In this study, we made observations in monocrotaline (MCT)-induced PAH rats and found decreases in mean pulmonary arterial pressure, pulmonary vessel resistance, right ventricular hypertrophy index, percentage of medial wall thickness, percentage of medial wall area, and lung weight of wet and dry tissue ratio after GSP administration in vivo. At the cellular and molecular levels, we also found several effects of GSP on MCT-induced PAH: (a) endothelial nitric oxide synthase expression in lung tissue and plasma NO level were increased; (b) Ca2+ level in pulmonary arterial smooth muscle cell (PASMC) was decreased; (c) transcription of inflammatory factors such as myeloperoxidase, interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α) was down-regulated in lung tissue; (d) nuclear factor-κB pathway was inhibited as IκBα was less phosphorylated; (e) TNFα-induced PASMC overproliferation could be inhibited. These results indicated a possible mechanism of GSP reversing pulmonary vascular remodeling and vascular contraction by inhibiting inflammation, and it may be useful for preventing PAH development.

Paclitaxel alleviates monocrotaline-induced pulmonary arterial hypertension via inhibition of FoxO1-mediated autophagy.

It has been demonstrated that activation of autophagy is involved in the development of pulmonary arterial hypertension (PAH). Recent studies have shown that cytosolic forkhead box protein O1 (FoxO1) activates autophagy in cancer cells. Paclitaxel has been found to potentially reverse PAH progression. However, the role of FoxO1 and the effects of paclitaxel on autophagy in the pathogenesis of PAH remain unknown. PAH was generated by intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricular systolic pressure (RVSP), the right ventricle hypertrophy index (RV/LV+S), and the percentage of medial wall thickness (%MT) were used to detect the development of PAH. Hematoxylin and eosin staining was performed to measure pulmonary vascular remodeling. The protein level, phosphorylation, and nucleus translocation of FoxO1 and the levels of LC3A, LC3B, and Beclin-1 were examined by immunoblotting. The results showed that in spite of reduced expression of FoxO1, elevated phosphorylation of FoxO1 caused most of FoxO1 accumulating in cytosolic fraction in MCT-PAH rats. Autophagy was also activated in the MCT-PAH group. In cultured rat pulmonary arterial smooth muscle cells (PASMCs), knockdown of FoxO1 markedly blocked autophagy activation, indicating that elevation of cytosolic FoxO1 stimulates autophagy activation. Treatment of PAH rats with paclitaxel reduced FoxO1 phosphorylation and increased FoxO1 nuclear accumulation, despite increased FoxO1 expression, therefore suppressed autophagy, finally reduced elevated RVSP, RV/LV+S, and %MT in MCT-induced PAH. Taken together, paclitaxel inhibits pulmonary vascular remodeling by FoxO1-mediated autophagy suppression, suggesting that paclitaxel might be a novel therapeutic agent for the prevention and treatment of PAH.

Resveratrol inhibits monocrotaline-induced pulmonary arterial remodeling by suppression of SphK1-mediated NF-κB activation

AimsThis study aims to explore the molecular mechanisms underlying sphingosine kinase 1 (SphK1) inducing pulmonary vascular remodeling and resveratrol suppressing pulmonary arterial hypertension (PAH). Material and methodsmonocrotaline (MCT) was used to induce PAH in rats. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI) and histological analyses including hematoxylin and eosin staining, the percentage of medial wall thickness (%MT), α-SMA staining and Ki67 staining were performed to evaluate the development of PAH. Protein levels of SphK1, nuclear factor-kappaB (NF-κB)-p65 and cyclin D1 were determined using immunoblotting. Sphingosine-1-phosphate (S1P) concentration was measured using enzyme-linked immunosorbent assay. Key findingsSphK1 protein level, S1P production, NF-κB activation and cyclin D1 expression were significantly increased in MCT-induced PAH rats. Inhibition of SphK1 by PF543 suppressed S1P synthesis and NF-κB activation and down-regulated cyclin D1 expression in PAH rats. Suppression of NF-κB by pyrrolidine dithiocarbamate (PDTC) also reduced cyclin D1 expression in PAH model. Treatment of PAH rats with either PF543 or PDTC dramatically decreased RVSP, RVHI and %MT and reduced pulmonary arterial smooth muscle cells proliferation and pulmonary vessel muscularization. In addition, resveratrol effectively inhibited the development of PAH by suppression of SphK1/S1P-mediated NF-κB activation and subsequent cyclin D1 expression. SignificanceSphK1/S1P signaling induces the development of PAH by activation of NF-κB and subsequent up-regulation of cyclin D1 expression. Resveratrol inhibits the MCT-induced PAH by targeting on SphK1 and reverses the downstream changes of SphK1, indicating that resveratrol might be a therapeutic agent for the prevention of PAH.

Activation of AMPK prevents monocrotaline-induced pulmonary arterial hypertension by suppression of NF-κB-mediated autophagy activation

AimsIt has been shown that activation of autophagy is involved in the development of pulmonary arterial hypertension (PAH). Meanwhile, activation of nuclear factor-kappaB (NF-κB) has been found to induce autophagy in several types of human diseases including cancer and cardiac diseases. However, it is still unknown whether NF-κB mediates autophagy activation in PAH, and whether activation of adenosine monophosphate-activated protein kinase (AMPK) benefits PAH by modulation of NF-κB and autophagy. Main methodsRat models of PAH were established by intraperitoneally injection of monocrotaline (MCT). The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and percentage of medial wall thickness (%MT) were performed to evaluate the development of PAH. The translocation of NF-κB p65 from cytosol to nucleus, the protein levels of LC3A, LC3B, and RND3 were determined by immunoblotting. Metformin was used to activate AMPK. Key findingsNF-κB and autophagy were significantly activated in MCT-induced PAH rats, this was accompanied with the reduction of RND3. Pharmacological inhibition of NF-κB suppressed MCT-induced activation of autophagy and down-regulation of RND3 expression and reduced RVSP, RVHI, and %MT in MCT-induced PAH rats. In addition, activation of AMPK by metformin suppressed NF-κB-mediated autophagy activation and down-regulation of RND3 and therefore reduced RVSP, RVHI, and %MT in MCT-induced PAH. SignificanceNF-κB-induced autophagy activation and consequent down-regulation of RND3 contributes to the development of PAH in MCT-treated rats. Activation of AMPK prevents the development of PAH by targeting on NF-κB to suppress autophagy and vascular remodeling.

Effects of FHL1 and P21 on hypoxia-induced pulmonary vascular remodeling in neonatal rats.

Numerous studies have demonstrated that altered expression levels of four and a half LIM domains 1 (FHL1) and P21 are necessary for hypoxia-induced pulmonary vascular remodeling in both adult rats and human patients with idiopathic pulmonary arterial hypertension. However, whether FHL1 and P21 are present in the pulmonary artery and whether these proteins affect pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH) in neonatal rats remain unknown. The present study investigated the effects of altered FHL1 and P21 expression on pulmonary vascular remodeling in neonatal rats with HPH. A total of 32 newborn Sprague-Dawley rats were exposed to hypoxia or room air for 7 or 14 days (n=8/subgroup). Parameters including the percentage of medial wall thickness (WT%), the percentage of medial wall area (WA%), right ventricular (RV) mean pressure, RV hypertrophy index (RVHI) and RV systolic pressure (RVSP) were measured to evaluate the development of HPH. Additionally, the expressions of FHL1 and P21 in the pulmonary artery smooth muscle cells (PASMCs) were measured by reverse transcription-quantitative polymerase chain reaction, western blot analysis and immunohistochemical staining. WA%, WT%, RV mean pressure, RVHI and RVSP were significantly increased in the HPH model group when compared with the control group (P<0.01). The protein expression levels of FHL1 were significantly increased in the HPH group (P<0.05), while the mRNA and protein expression levels of P21 were significantly reduced (P<0.05). Pearson correlation analysis indicated that the protein expressions of FHL1 and P21 were correlated with WA% and WT% (all P<0.001), and that the protein expression of P21 was negatively correlated with that of FHL1 (P<0.01). The results indicated that the expressions of FHL1 and P21 were altered in the PASMCs of newborn rats with HPH. Furthermore, FHL1 and P21 may serve important roles in pulmonary vascular remodeling.

Inhibition of ubiquitin proteasome function prevents monocrotaline-induced pulmonary arterial remodeling

AimsPrevious study has indicated that inhibition of proteasome function ameliorates the development of pulmonary arterial hypertension (PAH), while its underlying mechanisms are still unclear. This study was performed to address these issues. Material and methodsMale Sprague-Dawley (SD) rats were divided into five groups: control group, PAH group, vehicle treated PAH group, MG-132 treated PAH group and bortezomib treated PAH group. PAH model was established by a single intraperitoneal injection of monocrotaline (MCT). MG-132 and bortezomib were administered to inhibit proteasome function. The right ventricular systolic pressure (RVSP), the right ventricle hypertrophy index (RVHI) and the percentage of medial wall thickness (%MT) were used to evaluate the development of PAH. Hematoxylin and eosin staining was performed to measure vascular remodeling. Immunoblotting was used to determine Akt phosphorylation, expression of PTEN and NEDD4, and the level of ubiquitinated-PTEN protein. Key findingsMCT increased RVSP, RVHI and %MT in rats, while these changes were suppressed by treatment of PAH rats with MG-132 or bortezomib. In PAH model, expression of PTEN was decreased and phosphorylation of Akt was increased, these were accompanied by an elevation of NEDD4 protein level. Treatment of PAH model with MG-132 or bortezomib increased PTEN expression and accumulation of ubiquitinated-PTEN protein and decreased Akt phosphorylation, while didn't change NEDD4 expression. SignificanceInhibition of proteasome function ameliorates pulmonary arterial remodeling by suppressing UPS-mediated PTEN degradation and subsequent inhibition of PI3K/Akt pathway, indicating that UPS might be a novel target for prevention of PAH.

Altered expression of PPAR‑γ and TRPC in neonatal rats with persistent pulmonary hypertension.

Persistent pulmonary hypertension of the newborn (PPHN) is a life-threatening disease that is commonly observed in the neonatal intensive care unit. PPHN is pathologically characterized by pulmonary vascular remodeling and, in particular, pulmonary artery smooth muscle cell (PASMC) proliferation. Decreased expression levels of peroxisome proliferator-activated receptor γ (PPAR-γ), which is a member of the nuclear receptor hormone superfamily, in combination with elevated expressions of transient receptor potential cation channel, subfamily C, member 1 (TRPC1) and TRPC6 contributes to the PASMC proliferation and excessive pulmonary vascular remodeling in adult pulmonary hypertension (PH). Whether PPAR-γ, TRPC1 and TRPC6 affect the development of vascular remodeling in PPHN model rats remains unknown. The aim of the present study was to investigate the roles of PPAR-γ, TRPC1 and TRP6 on the pathogenesis of PPHN in rats. The rat model of PPHN was established by exposure to hypoxic conditions and indomethacin treatment. Lung tissues, hearts and blood from PPHN model and Control rats were collected and examined. Parameters, including the percentage of medial wall thickness (WT %), the percentage of medial wall area (WA %), right ventricular hypertrophy (RVH) and the plasma concentration of B-type natriuretic peptide (BNP) were used to estimate the development of PPHN. The expression levels of PPAR-γ, TRPC1 and TRPC6 in lung tissues were detected by immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction. Significant increases were observed in the WT %, WA %, RVH and plasma BNP in the PPHN group compare with the Control group (P<0.01). In addition, the mRNA and protein expression levels of PPAR-γ were markedly downregulated (P<0.05 vs. Control). In the PPHN group, the protein expression levels of TRPC1 and TRPC6 were higher compared to the control group; however, there was no difference in the mRNA expression levels (P>0.05). In conclusion, the present study successfully established a PPHN rat model, and the altered expressions of PPAR-γ, TRPC1 and TRPC6 in the pulmonary artery located in the lungs of newborn rats with PPHN suggested that these proteins may be important mediators of PPHN.

Pulmonary artery denervation improves pulmonary arterial hypertension induced right ventricular dysfunction by modulating the local renin-angiotensin-aldosterone system.

BackgroundPulmonary arterial hypertension (PAH) is commonly accompanied with the activation of the renin-angiotensin-aldosterone system (RAAS). Renal sympathetic denervation (RSD) reduces PAH partly through the inhibition of RAAS. Analogically, we hypothesized that pulmonary artery denervation (PADN) could reverse PAH and PAH-induced right ventricular (RV) dysfunction by downregulating the local RAAS activity.MethodsTwenty-five beagle dogs were randomized into two groups: control group (intra-atrial injection of N-dimethylacetamide, 3 mg/kg, n = 6) and test group (intra-atrial injection of dehydrogenized-monocrotaline, 3 mg/kg, n = 19). Eight weeks later, dogs in the test group with mean pulmonary arterial pressure (mPAP) ≥25 mmHg (n = 16) were reassigned into the sham (n = 8) and PADN groups (n = 8) by chance. After another 6 weeks, the hemodynamics, pulmonary tissue morphology and the local RAAS expression in lung and right heart tissue were measured.ResultsPADN reduced the mPAP (25.94 ± 3.67 mmHg vs 33.72 ± 5.76 mmHg, P < 0.05) and the percentage of medial wall thickness (%MWT) (31.0 ± 2.6 % vs 37.9 ± 2.8 %, P < 0.05) compared with the sham group. PADN attenuated RV dysfunction, marked with reduced atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ratio of right ventricular to left ventricular plus septum weight [RV/(LV + S)]. Moreover, the local RAAS expression was activated in PAH dogs while inhibited after PADN.ConclusionsPADN improves hemodynamics and relieves RV dysfunction in dogs with PAH, which can be associated with the downregulating RAAS activity in local tissue.

Involvement of CapG in proliferation and apoptosis of pulmonary arterial smooth muscle cells and in hypoxia-induced pulmonary hypertension rat model

ABSTRACTPurpose: Actin-binding protein capping protein gelsolin-like (CapG) was preferentially expressed in human pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, and reduced CapG expression was accompanied by impaired migration ability in vitro. We intended to investigate the effects of CapG on rat PASMCs and hypoxia-induced pulmonary hypertension (HPH) rat model. Materials and Methods: We investigated the effect of RNA interference-medicated down-regulation of CapG expression in rat PASMCs as well as in HPH rat model. The proliferation, apoptosis and cell cycle of PASMCs were evaluated. The HPH rat model was established by intratracheal instillation of lentiviral vector and subsequent hypoxia exposure for four weeks. Right ventricular systolic pressure, right ventricular hypertrophy and the percentage of medial wall thickness were measured to evaluate the development of HPH. Results: Knock-down CapG in PASMCs resulted in decreased proliferation, increased apoptosis and induced cell cycle inhibition. Down-regulation of CapG expression locally could attenuate pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy in HPH rat model. Conclusions: Our study indicated that CapG participated in the pathogenesis of pulmonary vascular remodeling in HPH rats, which was probably mediated by promoting the proliferation and inhibiting the apoptosis of PASMCs. We proposed CapG modulating protective effects of pulmonary hypertension.

The potential of asiaticoside for TGF-β1/Smad signaling inhibition in prevention and progression of hypoxia-induced pulmonary hypertension.

Asiaticoside (AS) is a saponin monomer extracted from the medicinal plant Centella asiatica, which has a variety of biological effects. We intended to investigate the effects of asiaticoside on a hypoxia-induced pulmonary hypertension (HPH) rat model and examine the possible effects of asiaticoside on TGF-β1/Smad signaling in vivo and in vitro. The rat HPH model was established by hypoxic exposure and asiaticoside was administered for four weeks. Parameters including the mean pulmonary artery pressure (mPAP), the right ventricular hypertrophy (RVH) and the percentage of medial wall thickness were used to evaluate the development of HPH. TGF-β1, TGF-β receptor, Smad2/3 and phospho-Smad2/3 expressions were detected and the proliferation, migration and apoptosis of pulmonary arterial smooth muscle cells (PASMCs) adjusted by asiaticoside under the hypoxic condition were evaluated. Our data indicate that asiaticoside attenuated pulmonary hypertension, pulmonary vascular remodeling and RV hypertrophy in HPH rats, which was probably mediated by restraining the hypoxia-induced overactive TGF-β1/Smad2/3 signaling and inhibiting the proliferation by inducing apoptosis of the PASMCs. Given the preventative potential in animal models and in vitro, we propose asiaticoside as a promising protective treatment in HPH.

Activation of peroxisome proliferator-activated receptor γ ameliorates monocrotaline-induced pulmonary arterial hypertension in rats.

Activation of peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation of pulmonary artery smooth muscle cells (PASMCs) and vascular remodeling in rats and humans, and therefore improves the development of pulmonary arterial hypertension (PAH). However, molecular mechanisms underlying these effects have not been completely understood. In the present study, the effects of PPARγ activation in monocrotaline (MCT)-induced pulmonary artery remodeling in rats were investigated. Eighteen Sprague-Dawley (SD) rats were randomly assigned into three groups (n=6): Control (Con), PAH and PAH treated with rosiglitazone (MCT + Rosi). The right ventricular systolic pressure (RVSP), the ratio of the right to left ventricle plus septum weight [RV/(LV + S)], the percentage of medial wall thickness (%MT) and wall area (%WA) were used to evaluate the development of PAH. Tissue morphology was measured using hematoxylin and eosin staining. The protein levels of the phosphatase and tensin homologue deleted on chromosome ten (PTEN), Akt (ser473) phosphorylation (p-Akt) and total Akt in intrapulmonary arteries were determined by western blot analysis. MCT treatment significantly increased the RVSP, which was reduced by rosiglitazone treatment. The ratio of RV/(LV + S), %MT and %WA induced by MCT were similarly inhibited, which was associated with the increase of PTEN expression and the inhibition of Akt phosphorylation levels by rosiglitazone. In conclusion, activation of PPARγ ameliorates the proliferation of PASMCs and vascular remodeling by regulating the PTEN/PI3K/Akt pathway, suggesting that the activation of PPARγ has potential benefits for PAH.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

Activation of PPAR-γ ameliorates pulmonary arterial hypertension via inducing heme oxygenase-1 and p21WAF1: An in vivo study in rats

AimsOur previous study has indicated that activation of PPAR-γ inhibits the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) in vitro through inducing the expression of heme oxygenase-1 (HO-1), which in turn up-regulates the p21WAF1 expression. In the present study, we intended to determine whether similar mechanisms have been involved in activation of PPAR-γ inhibition of development of rat PAH model. Material and methodsRat pulmonary arterial hypertension (PAH) model was established by subcutaneous injection of monocrotaline (MCT). Rosiglitazone was administered to activate PPAR-γ. Zinc protoporphyria IX (ZnPP-IX), was used to confirm the role of HO-1 in mediating PPAR-γ function. Parameters including the right ventricle systolic pressure (RVSP), the right ventricular hypertrophy (RVH) and the percentage of medial wall thickness were used to evaluate the development of PAH. Immunoblotting was used to determine the expression of HO-1 and p21WAF1. Key findingsRosiglitazone significantly decreased the RVSP and inhibited the RVH in MCT-induced rat PAH model, and partially inhibited the pulmonary vascular remodeling. These effects were coupled with the sequential increase of HO-1 and p21WAF1 expressions by rosiglitazone. SignificanceActivation of PPAR-γ benefits PAH by inhibiting proliferation of PASMCs and reducing pulmonary vascular remodeling. The present study suggests that enhancing PPAR-γ activity might have potential value in clinical treatment of PAH.

Effect of antenatal tetrandrine administration on endothelin-1 and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia

Purpose The aim of this study was to evaluate the effect of the traditional Chinese medicine tetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) and epidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatic hernia (CDH). Methods A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternally administered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH (n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered by cesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining were performed, and image analysis was performed after lung processing. Results Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in 68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group was significantly lower than those of the Dex and EGF groups ( P < .05). The lungs of fetuses with CDH showed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast to improved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statistical differences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage of medial wall thickness, and vascular volume) were found ( P < .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tet groups ( P < .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groups was stronger than that in the control group ( P < .05). There was no difference in lung EGF and ET-1 immunoreactivity between CDH+Dex and CDH+Tet groups ( P > .05). Conclusion Antenatal treatment with Tet may improve lung growth and vascular remodeling, and its mechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternally may be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping to avoid the side effects of Dex.

Pulmonary arteriole muscularization in lambs with diaphragmatic hernia after combined tracheal occlusion/glucocorticoid therapy

A morphometric study was performed to examine the effects of prenatal glucocorticoids, which were administered 48 hours before birth, on muscularization of small pulmonary arterioles (<60 microm diameter) in lambs with diaphragmatic hernia (DH) after fetal tracheal occlusion (TO). DH was created in 23 fetal sheep at 65 days gestation. TO was performed in 16 of 24 fetuses between 110 and 140 days of gestation; 9 of the fetuses were exposed prenatally to betamethasone (0.5 mg/kg body weight) 48 hours before delivery. Six sham-operated animals served as controls. Sections of paraffin that were embedded in lung tissues were stained with Elastin-Van Gieson, and the percentage of medial wall thickness (MWT) was determined. The percentage of MWT in DH lambs (29.6% +/- 1.9%) was increased compared with sham animals (18.1% +/- 1.3%) and was not different from that of DH/TO animals (30.3% +/- 1.7%). In DH/TO + glucocorticoid lambs, the percentage of MWT (24.6% +/- 1.2%) was significantly lower than in the DH/TO group but was higher than the sham group. In fetuses who underwent prolonged TO therapy for severe DH, prenatal glucocorticoid treatment decreased medial hypertrophy of pulmonary arterioles by approximately 19%. We speculate that such structural changes may have contributed to improve gas exchange that was observed in this model.

Inhibition of rho kinase attenuates high flow induced pulmonary hypertension in rats

The RhoA/Rho kinase pathway may participate in the pathogenesis of hypoxia and monocrotaline induced pulmonary hypertension. This study tested whether RhoA/Rho kinase pathway is involved in the pathogenesis of high flow induced pulmonary hypertension in rats. Male Wistar rats (4 weeks) were randomly divided into 4 shunt groups, 4 treated groups and 4 control groups. Shunt and treated groups underwent left common carotid artery/external jugular vein shunt operation. Control groups underwent sham operation. Treated groups received fasudil treatment and the others received same dose of saline. At weeks 1, 2, 4 and 8 of the study, right ventricular systolic pressure was measured and blood gases were analysed to calculate Qp/Qs. The weight ratio of right ventricle to left ventricle plus septum and the mean percentage of medial wall thickness in moderate sized pulmonary arteries were obtained. RhoA activity in pulmonary arteries was detected using Rho activity assay reagent. Rho kinase activity was quantified by the extent of MYPT1 phosphorylation with Western blot. Proliferating cells were evaluated using proliferating cell nuclear antigen immunohistological staining. Carotid artery/jugular vein shunt resulted in high pulmonary blood flow, both an acute and a chronic elevation of right ventricular systolic pressure, significant medial wall thickening characterized by smooth muscle cells proliferation, right ventricular hypertrophy and increased activation of RhoA and Rho kinase. Fasudil treatment lowered pulmonary artery systolic pressure, suppressed pulmonary artery smooth muscle cells proliferation, attenuated pulmonary artery medial wall thickening and inhibited right ventricular hypertrophy together with significant suppression of Rho kinase activity but not Rho activity. Activated RhoA/Rho kinase pathway is associated with both the acute pulmonary vasoconstriction and the chronic pulmonary artery remodelling of high flow induced pulmonary hypertension. Fasudil treatment could improve pulmonary hypertension by inhibiting Rho kinase activity.