Xiufeng Yu

The MicroRNA-328 Regulates Hypoxic Pulmonary Hypertension by Targeting at Insulin Growth Factor 1 Receptor and L-Type Calcium Channel-α1C

Chronic hypoxia is the most common cause of secondary pulmonary hypertension, for which the mechanisms are still unclear. Recent studies implicated an important role for microRNAs (miRNAs) in hypoxia-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be implicated in the etiology of hypoxic pulmonary hypertension. Here we show that miRNA-328, a posttranscriptional regulator, was drastically downregulated in the pulmonary artery (PA) after a hypoxic assault. PA rings, Western blot, quantitative real-time PCR, in situ hybridization, and luciferase assay were used to investigate the role of miRNA-328 in hypoxic pulmonary hypertension. We found that hypoxia produced a significant inhibition of miRNA-328 expression, which was involved in PA vasoconstriction and remodeling. Overexpressing miRNA-328 in the transgenic mice remarkably decreased the right ventricular systolic pressure and PA wall thickness under both normoxia and hypoxia. MiRNA-328 inhibited L-type calcium channel-&agr;1C expression through a miRNA-328 binding site within the 3′ untranslational region of L-type calcium channel-&agr;1C. The L-type calcium channel-&agr;1C inhibition attenuated the PA response to KCl. Furthermore, miRNA-328 suppressed the insulin growth factor 1 receptor, ultimately leading to apoptosis of pulmonary arterial smooth muscle cells. The posttranscriptional repression of L-type calcium channel-&agr;1C and insulin growth factor 1 receptor was further confirmed by luciferase reporter assay. These results showed that miRNA-328, an important protecting factor, plays a significant role in PA constriction and remodeling by regulating multiple gene targets in hypoxic pulmonary hypertension.

Modulation of Pulmonary Vascular Remodeling in Hypoxia: Role of 15-LOX-2/15-HETE-MAPKs Pathway

Background: We have previously shown that 15-hydroxyeicosatetraenoic acid (15-HETE) plays a critical role in pulmonary hypertension (PH)-associated vascular remodeling. However, the signaling mechanisms remain unclear. The purpose of this study was to investigate the role of 15-lipoxygenase-2 (15-LO-2)/15-HETE-mitogen-activated protein kinases (MAPKs) pathway in hypoxia-induced pulmonary vascular remodeling and the underlying mechanisms. Methods: The arterial wall thickness was measured by hematoxylin and eosin (HE) staining in distal pulmonary arteries isolated from normal and PAH patient-derived lungs. The protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated p38 mitogen-activated protein kinases (p-p38MAPK) were measured by Western blot in the lungs of PAH patients and hypoxia-induced rats. The apoptosis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) was determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Flow cytometry. The cell proliferation and cell cycle in PASMCs following hypoxia were analyzed by bromodeoxyuridine incorporation and flow cytometry, respectively. Results: Our results showed that the levels of p-ERK and p-p38MAPK were both drastically elevated in lungs from human patients and hypoxic rats. The HE staining revealed that the medial wall thickness was higher in patients with PAH than normal humans. In cultured PASMCs, Hypoxia stimulated the cell proliferation, the cell cycle progression, and subsequently promoted cell differentiation and cell migration leading to the suppressed cell apoptosis. Furthermore, MAPKs- induced cell proliferation and anti-apoptosis in PASMCs is 15-LO-2/15HETE activation-dependent. Conclusion: Our study indicates that hypoxia-induced pulmonary vascular remodeling is associated with increased levels of 15-LO-2 and 15-HETE. 15-LO-2/15-HETE stimulates the cell proliferation and anti-apoptosis in PASMCs through phosphorylation of ERK and p38MAPK, which subsequently contributing to hypoxia-induced pulmonary vascular remodeling.

Key Role of ROS in the Process of 15-Lipoxygenase/15-Hydroxyeicosatetraenoiccid-Induced Pulmonary Vascular Remodeling in Hypoxia Pulmonary Hypertension.

We previously reported that 15-lipoxygenase (15-LO) and its metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) were up-regulated in pulmonary arterial cells from both pulmonary artery hypertension patients and hypoxic rats and that these factors mediated the progression of pulmonary hypertension (PH) by affecting the proliferation and apoptosis of pulmonary arterial (PA) cells. However, the underlying mechanisms of the remodeling induced by 15-HETE have remained unclear. As reactive oxygen species (ROS) and 15-LO are both induced by hypoxia, it is possible that ROS are involved in the events of hypoxia-induced 15-LO expression that lead to PH. We employed immunohistochemistry, tube formation assays, bromodeoxyuridine (BrdU) incorporation assays, and cell cycle analyses to explore the role of ROS in the process of 15-HETE-mediated hypoxic pulmonary hypertension (HPH). We found that exogenous 15-HETE facilitated the generation of ROS and that this effect was mainly localized to mitochondria. In particular, the mitochondrial electron transport chain and nicotinamide-adenine dinucleotide phosphate oxidase 4 (Nox4) were responsible for the significant 15-HETE-stimulated increase in ROS production. Moreover, ROS induced by 15-HETE stimulated endothelial cell (EC) migration and promoted pulmonary artery smooth muscle cell (PASMC) proliferation under hypoxia via the p38 MAPK pathway. These results indicated that 15-HETE-regulated ROS mediated hypoxia-induced pulmonary vascular remodeling (PVR) via the p38 MAPK pathway.

The role of PDGF-B/TGF-β1/neprilysin network in regulating endothelial-to-mesenchymal transition in pulmonary artery remodeling.

Endothelial-to-mesenchymal transition (EndoMT) has been recognized as a major reason for the pulmonary artery remodeling (PAR) in pulmonary artery hypertension (PAH). However, the molecular mechanisms and regulatory pathways involved in the EndoMT remain undefined. In the present study, we have confirmed that EndoMT was occurred in pulmonary arteries of rats induced by hypoxia and monocrotaline and in hypoxic pulmonary artery endothelial cells (PAECs). Moreover, hypoxia increased the expression of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) and decreased the expression of neprilysin (NEP), which contributed to the hypoxia-induced EndoMT of PAECs. Furthermore, a reciprocal regulation of PDGF-B and TGF-β1 induced by decreasing NEP promoted the EndoMT of PAECs under hypoxia, which was a novel molecular mechanism to reveal the EndoMT participating in PAR. More importantly, imatinib, a PDGF receptor antagonist, relieved PAR and EndoMT in PAH rats. Thus, our results identify a novel mechanism to reveal the formation of EndoMT in PAH, and imply that imatinib may serve as a new therapeutic approach for treatment of the third cardiovascular disease.

The Critical Role of Dynamin-Related Protein 1 in Hypoxia-Induced Pulmonary Vascular Angiogenesis.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by pulmonary vascular obstruction due in part to excessive pulmonary artery endothelial cells (PAECs) migration and proliferation. The mitochondrial fission protein dynamin‐related protein‐1 (DRP1) has important influence on pulmonary vascular remodeling. However, whether DRP1 participates in the development and progression of pulmonary vascular angiogenesis has not been reported previously. To test the hypothesis that DRP1 promotes the angiogenesis via promoting the proliferation, stimulating migration, and inhibiting the apoptosis of PAECs in mitochondrial Ca2+‐dependent manner, we performed following studies. Using hemodynamic analysis and morphometric assay, we found that DRP1 mediated the elevation of right ventricular systemic pressure (RVSP), right heart hypertrophy, and increase of pulmonary microvessels induced by hypoxia. DRP1 inhibition reversed tube network formation in vitro stimulated by hypoxia. The mitochondrial Ca2+ inhibited by hypoxia was recovered by DRP1 silencing. Moreover, pulmonary vascular angiogenesis promoted by DRP1 was reversed by the specific mitochondrial Ca2+ uniporter inhibitor Ru360. In addition, DRP1 promoted the proliferation and migration of PAECs in mitochondrial Ca2+‐dependent manner. Besides, DRP1 decreased mitochondrial membrane potential, reduced the DNA fragmentation, and inhibited the caspase‐3 activation, which were all aggravated by Ru360. Therefore, these results indicate that the mitochondrial fission machinery promotes migration, facilitates proliferation, and prevents from apoptosis via mitochondrial Ca2+‐dependent pathway in endothelial cells leading to pulmonary angiogenesis. J. Cell. Biochem. 116: 1993–2007, 2015.

15-Lipoxygenase and 15-hydroxyeicosatetraenoic acid regulate intravascular thrombosis in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a disease characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, pulmonary vascular thrombotic lesions, and right heart failure. Recent studies suggest that 15-lipoxygenase (15-LO)/15-hydroxyeicosatetraenoic acid (15-HETE) play an important role in PAH, acting on arterial walls. Here, we show evidence for the action of the 15-LO/15-HETE signaling in the pulmonary vascular thrombotic lesions in the experimental PAH models. Platelet deposition was augmented in rats exposed to hypoxia and Sugen 5416, which were both prevented by nordihydroguaiaretic acid (NDGA), a 15-LO inhibitor. Chronic hypoxic resulted in the platelet deposition specifically in pulmonary vasculature, which was reversed by 15-LO inhibitor. The 15-LO pathway mediated in the endothelial dysfunction induced by hypoxia in vivo. Meanwhile, 15-HETE positively regulated the generation of IL-6 and monocyte chemoattractant protein-1 (MCP-1). The coagulation and platelet activation induced by hypoxia were reversed by 15-LO inhibitor NDGA or the MCP-1 inhibitor synthesis inhibitor bindarit in rats. The 15-LO/15-HETE signaling promoted the coagulation and platelet activation, which was suppressed by MCP-1 inhibition. These results therefore suggest that 15-LO/15-HETE signaling plays a role in platelet activation and pulmonary vascular thrombosis in PAH, involving MCP-1.

Positive feedback-loop of telomerase reverse transcriptase and 15-lipoxygenase-2 promotes pulmonary hypertension.

Objective Pulmonary hypertension (PH) is characterized with pulmonary vasoconstriction and vascular remodeling mediated by 15-lipoxygenase (15-LO)/15-hydroxyeicosatetraenoic acid (15-HETE) according to our previous studies. Meanwhile, telomerase reverse transcriptase (TERT) activity is highly correlated with vascular injury and remodeling, suggesting that TERT may be an essential determinant in the development of PH. The aim of this study was to determine the contribution and molecular mechanisms of TERT in the pathogenesis of PH. Approach and Results We measured the right ventricular systolic pressure (RVSP) and ventricular weight, analyzed morphometric change of the pulmonary vessels in the hypoxia or monocrotaline treated rats. Bromodeoxyuridine incorporation, transwell assay and flow cytometry in pulmonary smooth muscle cells were performed to investigate the roles and relationship of TERT and 15-LO/15-HETE in PH. We revealed that the expression of TERT was increased in pulmonary vasculature of patients with PH and in the monocrotaline or hypoxia rat model of PH. The up-regulation of TERT was associated with experimental elevated RVSP and pulmonary vascular remodeling. Coimmunoprecipitation experiments identified TERT as a novel interacting partner of 15-LO-2. TERT and 15-LO-2 augmented protein expression of each other. In addition, the proliferation, migration and cell-cycle transition from G0/G1 phase to S phase induced by hypoxia were inhibited by TERT knockdown, which were rescued by 15-HETE addition. Conclusions These results demonstrate that TERT regulates pulmonary vascular remodeling. TERT and 15-LO-2 form a positive feedback loop and together promote proliferation and migration of pulmonary artery smooth muscle cells, creating a self-amplifying circuit which propels pulmonary hypertension.

Acetylated cyclophilin A is a major mediator in hypoxia-induced autophagy and pulmonary vascular angiogenesis.

Background: Autophagy is a major intracellular degradation and recycling process that maintains cellular homeostasis, which is involved in structural and functional abnormalities of pulmonary vasculature in hypoxic pulmonary arterial hypertension (HPAH). Cyclophilin A (CyPA) is a secreted, oxidative stress-induced factor. Its role in inducing autophagy and augmenting endothelial cell dysfunction has never been explored. Methods: Lungs from rats exposed to chronic hypoxia were examined for autophagy with electron microscopy, western blotting, and fluorescence microscopy. Results: Activated autophagy was seen in the endothelium of the pulmonary artery from experimental rat models of HPAH and cultured bovine pulmonary arterial endothelial cells under hypoxia. Inhibiting autophagy attenuated the pathological progression of HPAH and repressed endothelial cell migration and angiogenesis. We also showed that CyPA was upregulated and acetylated under hypoxia and led to the abnormal occurrence of autophagy through its interaction with autophagy protein 5 and autophagy protein 7. Moreover, acetylated CyPA was essential for the excessive proliferation, migration, and tube formation networks of pulmonary arterial endothelial cells. Conclusion: Our results indicate the crucial role of acetylated CyPA in the abnormal occurrence of autophagy and subsequent pulmonary vascular angiogenesis.

Inhibition of CXCL12/CXCR4 suppresses pulmonary arterial smooth muscle cell proliferation and cell cycle progression via PI3K/Akt pathway under hypoxia

Abstract Stromal cell–derived factor 1 (CXCL12) and its receptor CXC chemokine receptor 4 (CXCR4) are known to modulate hypoxia-induced pulmonary hypertension (PH) and vascular remodeling by mobilization and recruitment of progenitor cells to the pulmonary vasculature. However, little is known about CXCL12/CXCR4 regulating proliferation and cell cycle progression of pulmonary arterial smooth muscle cells (PASMCs). To determine whether CXCL12/CXCR4 regulates PASMC proliferation and the cell cycle, immunohistochemistry, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed in this study. Our results showed that CXCR4 was induced by hypoxia in pulmonary arteries and PASMCs of rats. Hypoxia-increased cell viability, DNA synthesis and proliferating cell nuclear antigen expression were blocked by administration of CXCR4 antagonist AMD3100, silencing CXCR4 or CXCL12. Furthermore, inhibition of CXCL12/CXCR4 suppressed cell cycle progression, decreased the number of cells in S+G2/M phase and attenuated the expression of proteins that regulate the cell cycle progression at these phases. In addition, PI3K/Akt signaling mediated CXCL12/CXCR4 regulating proliferation and cell cycle progression in PASMCs. Thus, these results indicate that blockade of CXCL12/CXCR4 inhibited PASMC proliferation and cell cycle progression in hypoxia-induced PH via PI3K/Akt signaling pathway.

Inward Rectifier K+ Currents Are Regulated by CaMKII in Endothelial Cells of Primarily Cultured Bovine Pulmonary Arteries.

Endothelium lines the interior surface of vascular walls and regulates vascular tones. The endothelial cells sense and respond to chemical and mechanical stimuli in the circulation, and couple the stimulus signals to vascular smooth muscles, in which inward rectifier K+ currents (Kir) play an important role. Here we applied several complementary strategies to determine the Kir subunit in primarily cultured pulmonary arterial endothelial cells (PAECs) that was regulated by the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). In whole-cell voltage clamp, the Kir currents were sensitive to micromolar concentrations of extracellular Ba2+. In excised inside-out patches, an inward rectifier K+ current was observed with single-channel conductance 32.43 ± 0.45 pS and Popen 0.27 ± 0.04, which were consistent with known unitary conductance of Kir 2.1. RT-PCR and western blot results showed that expression of Kir 2.1 was significantly stronger than that of other subtypes in PAECs. Pharmacological analysis of the Kir currents demonstrated that insensitivity to intracellular ATP, pinacidil, glibenclamide, pH, GDP-β-S and choleratoxin suggested that currents weren’t determined by KATP, Kir2.3, Kir2.4 and Kir3.x. The currents were strongly suppressed by exposure to CaMKII inhibitor W-7 and KN-62. The expression of Kir2.1 was inhibited by knocking down CaMKII. Consistently, vasodilation was suppressed by Ba2+, W-7 and KN-62 in isolated and perfused pulmonary arterial rings. These results suggest that the PAECs express an inward rectifier K+ current that is carried dominantly by Kir2.1, and this K+ channel appears to be targeted by CaMKII-dependent intracellular signaling systems.