Pathogenesis Of Hypoxia-induced Pulmonary Hypertension Research Articles

Hub Genes and Immune Cell Infiltration in Hypoxia-Induced Pulmonary Hypertension: Bioinformatics Analysis and In Vivo Validation.

Hypoxia-induced pulmonary hypertension (HPH) represents a severe pulmonary disorder with high morbidity and mortality, which necessitates identifying the critical molecular mechanisms underlying HPH pathogenesis. The mRNA expression microarray GSE15197 (containing 8 pulmonary tissues from HPH and 13 normal controls) was downloaded from Gene Expression Omnibus (GEO). Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed by RStudio software. The Protein-Protein Interaction (PPI) network was visualized and established using Cytoscape, and the cytoHubba app from Cytoscape was used to pick out the hub modules. The infiltration of immune cells in HPH was analyzed using the CIBERSORTx. To confirm the potential hub genes, real-time quantitative reverse transcription PCR (qRT-PCR) was conducted using lung tissues of rat HPH models and controls. A total of 852 upregulated and 547 downregulated genes were identified. The top terms in biological processes were apoptosis, proliferation, and regulation of the MAPK cascade, including ERK1/2. Cytoplasm, cytosol, and membrane were enriched in cellular component groups. Molecular functions mainly focus on protein binding, protein serine/threonine kinase activity and identical protein binding. KEGG analysis identified pathways in cancer, regulation of actin cytoskeleton and rap1 signaling pathway. There was significantly different immune cell infiltration between HPH and normal control samples. High proportions of the memory subsets of B cells and CD4 cells, Macrophages M2 subtype, and resting Dendritic cells were found in HPH samples, while high proportions of naive CD4 cells and resting mast cells were found in normal control samples. The qRT-PCR results showed that among the ten identified hub modules, FBXL3, FBXL13 and XCL1 mRNA levels were upregulated, while NEDD4L, NPFFR2 and EDN3 were downregulated in HPH rats compared with control rats. Our study revealed the key genes and the involvement of immune cell infiltration in HPH, thus providing new insight into the pathogenesis of HPH and potential treatment targets for patients with HPH.

Important Role of Endogenous Nerve Growth Factor Receptor in the Pathogenesis of Hypoxia-Induced Pulmonary Hypertension in Mice.

Pulmonary arterial hypertension (PAH) remains a disease with poor prognosis; thus, a new mechanism for PAH treatment is necessary. Circulating nerve growth factor receptor (Ngfr)-positive cells in peripheral blood mononuclear cells are associated with disease severity and the prognosis of PAH patients; however, the role of Ngfr in PAH is unknown. In this study, we evaluated the function of Ngfr using Ngfr gene-deletion (Ngfr-/-) mice. To elucidate the role of Ngfr in pulmonary hypertension (PH), we used Ngfr-/- mice that were exposed to chronic hypoxic conditions (10% O2) for 3 weeks. The development of hypoxia-induced PH was accelerated in Ngfr-/- mice compared to littermate controls. In contrast, the reconstitution of bone marrow (BM) in Ngfr-/- mice transplanted with wild-type BM cells improved PH. Notably, the exacerbation of PH in Ngfr-/- mice was accompanied by the upregulation of pulmonary vascular remodeling-related genes in lung tissue. In a hypoxia-induced PH model, Ngfr gene deletion resulted in PH exacerbation. This suggests that Ngfr may be a key molecule involved in the pathogenesis of PAH.

TRB3 mediates vascular remodeling by activating the MAPK signaling pathway in hypoxic pulmonary hypertension

BackgroundHypoxic pulmonary hypertension (PH) is a refractory pulmonary vascular remodeling disease, and the efficiency of current PH treatment strategies is unsatisfactory. Tribbles homolog 3 (TRB3), a member of the pseudokinase family, is upregulated in diverse types of cellular stresses and functions as either a pro-proliferative or pro-apoptotic factor depending on the specific microenvironment. The regulatory mechanisms of TRB3 in hypoxic PH are poorly understood.MethodsWe performed studies using TRB3-specific silencing and overexpressing lentiviral vectors to investigate the potential roles of TRB3 on hypoxic pulmonary artery smooth muscle cells (PASMCs). Adeno-associated virus type 1(AVV1) vectors encoding short-hairpin RNAs against rat TRB3 were used to assess the role of TRB3 on hypoxic PH. TRB3 protein expression in PH patients was explored in clinical samples by western blot analysis.ResultsThe results of whole-rat genome oligo microarrays showed that the expression of TRB3 and endoplasmic reticulum stress (ERS)-related genes was upregulated in hypoxic PASMCs. TRB3 protein expression was significantly upregulated by hypoxia and thapsigargin. In addition, 4-PBA and 4μ8C, both inhibitors of ERS, decreased the expression of TRB3. TRB3 knockdown promoted apoptosis and damaged the proliferative and migratory abilities of hypoxic PASMCs as well as inhibited activation of the MAPK signaling pathway. TRB3 overexpression stimulated the proliferation and migration of PASMCs but decreased the apoptosis of PASMCs, which was partly reversed by specific inhibitors of ERK, JNK and p38 MAPK. The Co-IP results revealed that TRB3 directly interacts with ERK, JNK, and p38 MAPK. Knockdown of TRB3 in rat lung tissue reduced the right ventricular systolic pressure and decreased pulmonary medial wall thickness in hypoxic PH model rats. Further, the expression of TRB3 in lung tissues was higher in patients with PH compared with those who have normal pulmonary artery pressure.ConclusionsTRB3 was upregulated in hypoxic PASMCs and was affected by ERS. TRB3 plays a key role in the pathogenesis of hypoxia-induced PH by binding and activating the ERK, JNK, and p38 MAPK pathways. Thus, TRB3 might be a promising target for the treatment of hypoxic PH.

Dysregulated lncRNA TUG1 in different pulmonary artery cells under hypoxia.

BackgroundAt present, the role of lncRNAs in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH) is not fully understood. This study aimed to explore differences in the hypoxia-induced expression of lncRNAs and their potential role in multiple pulmonary artery cells.MethodsLncRNA expression in pulmonary artery smooth muscle cells (PASMCs), pulmonary microvascular endothelial cells (PMECs), and pericytes (PCs) was analyzed by high-throughput sequencing and compared between normoxic and hypoxic cells. Bioinformatics analysis was conducted to predict their functions.ResultsPASMCs, PMECs, and PCs displayed 275 (140 upregulated), 251 (162 upregulated), and 290 (176 upregulated) different lncRNAs, respectively. Among these, lncRNA TUG1 levels increased in PASMCs and PCs but decreased in PMECs. Bioinformatics analysis indicated that lncRNA TUG1 might target miR-145-5p, thereby affecting SOX4 and BMF expression, and could also regulate miR-129-5p levels to affect CYP1B1 and VCP expression. It could also regulate miR-138-5p levels to affect KCNK3 and RHOC expression.ConclusionsHypoxia exposure of vascular cells resulted in differential expression of lncRNAs, especially lncRNA TUG1, which showed significant abnormal expression in all three types of vascular cells under hypoxia. Our results suggested that abnormal expression of lncRNA TUG1 might be involved in the regulation of pulmonary vascular cell function under hypoxia.

Tribbles Homolog 3-Mediated Vascular Insulin Resistance Contributes to Hypoxic Pulmonary Hypertension in Intermittent Hypoxia Rat Model.

This study aimed to investigate the role of vascular insulin resistance (VIR) and Tribbles homolog 3 (TRIB3) in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). Rats were subjected to low air pressure and low oxygen intermittently for 4 weeks to induce HPH. The mean right ventricular pressure (mRVP), mean pulmonary arterial pressure (mPAP), and right ventricular index (RVI) were significantly increased in HPH rats. Pulmonary arteries from HPH rats showed VIR with reduced vasodilating effect of insulin. The protein levels of peroxisome proliferator-activated receptor gamma (PPARγ), phosphoinositide 3-kinase (PI3K), phosphorylations of Akt, and endothelial nitric oxide (NO) synthase (eNOS) were decreased, and TRIB3 and phosphorylated extracellular signal-regulated protein kinases (ERK1/2) were increased in pulmonary arteries of HPH rats. Early treatment of pioglitazone (PIO) partially reversed the development of HPH, improved insulin-induced vasodilation, and alleviated the imbalance of the insulin signaling. The overexpression of TRIB3 in rat pulmonary arterial endothelial cells (PAECs) reduced the levels of PPARγ, PI3K, phosphorylated Akt (p-Akt), and phosphorylated eNOS (p-eNOS) and increased p-ERK1/2 and the synthesis of endothelin-1 (ET-1), which were further intensified under hypoxic conditions. Moreover, TRIB3 knockdown caused significant improvement in Akt and eNOS phosphorylations and, otherwise, a reduction of ERK1/2 activation in PAECs after hypoxia. In conclusion, impaired insulin-induced pulmonary vasodilation and the imbalance of insulin-induced signaling mediated by TRIB3 upregulation in the endothelium contribute to the development of HPH. Early PIO treatment improves vascular insulin sensitivity that may help to limit the progression of hypoxic pulmonary hypertension.

C-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

Beraprost Upregulates KV Channel Expression and Function via EP4 Receptor in Pulmonary Artery Smooth Muscle Cells Obtained from Rats with Hypoxia-Induced Pulmonary Hypertension

The reduced expression and function of voltage-dependent potassium (K_V) channels have been involved in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH), leading to pulmonary vasoconstriction and vascular remodeling, while the upregulation of K_V channels is of therapeutic significance for pulmonary hypertension. Beraprost sodium (BPS) has been shown to be effective in patients with pulmonary hypertension. However, the effect of BPS on O₂-sensitive K_V channels in pulmonary artery smooth muscle cells (PASMCs) remains unclear. In the present study, the effect of BPS on rats with HPH was observed, and the influence of BPS on the expression and function of O₂-sensitive K_V channels in PASMCs was investigated. The results revealed that BPS reduced mean pulmonary artery pressure, suppressed right ventricular hypertrophy, and attenuated the remodeling of pulmonary arteries in rats exposed to discontinuous hypoxia for 4 weeks (8 h/day). This was accompanied with the significantly upregulated expression of K_V channel α-subunits (K_V1.2, K_V1.5 and K_V2.1) and O₂-sensitive voltage-gated K⁺ (K_V) channel current (I_K(V)) in small pulmonary arteries in HPH model rats, as well as in hypoxia-induced PASMCs. Furthermore, in vitrostudies have revealed that the upregulation of BPS on O₂-sensitive K_V channels was significantly inhibited after treatment with prostaglandin E₂ receptor subtype EP₄ antagonist GW627368X. Taken together, these results suggest that BPS attenuates the development of HPH through the upregulation of O₂-sensitive K_V channels, which was probably via the EP₄ receptor-related pathway.

Down-regulation of lncRNA MEG3 promotes hypoxia-induced human pulmonary artery smooth muscle cell proliferation and migration via repressing PTEN by sponging miR-21

Hypoxia-induced pulmonary hypertension is a life-threatening disease arising from a progressive increase in pulmonary vascular resistance, irreversible pulmonary vascular remodeling and resulting in right ventricular failure. Recent studies suggested that pulmonary artery smooth muscle cell proliferation and migration played an important role in the pathogenesis of hypoxia-induced pulmonary hypertension. However, the mechanisms of hypoxia-induced pulmonary hypertension are complicated and largely unclear. In this study, we discovered that lncRNA MEG3 was down-regulated in human pulmonary artery smooth muscle cell in hypoxia, and inhibition of MEG3 promoted the cell proliferation and cell migration in both normal and hypoxia condition. Further study demonstrated that MEG3 exerted its function via regulation of miR-21 expression in both normal and hypoxia condition. In addition, we displayed the modulation of PTEN by miR-21 and their role in hypoxia. Ultimately, our study illustrated that MEG3 exerts its role via miR-21/PTEN axis in human pulmonary artery smooth muscle cell under both normal and hypoxia conditions.

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension.

AimsRhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect.Methods and resultsThe analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.ConclusionsOur study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

Smooth Muscle Insulin-Like Growth Factor-1 Mediates Hypoxia-Induced Pulmonary Hypertension in Neonatal Mice.

Insulin-like growth factor (IGF)-1 is a potent mitogen of vascular smooth muscle cells (SMCs), but its role in pulmonary vascular remodeling associated with pulmonary hypertension (PH) is not clear. In an earlier study, we implicated IGF-1 in the pathogenesis of hypoxia-induced PH in neonatal mice. In this study, we hypothesized that hypoxia-induced up-regulation of IGF-1 in vascular smooth muscle is directly responsible for pulmonary vascular remodeling and PH. We studied neonatal and adult mice with smooth muscle-specific deletion of IGF-1 and also used an inhibitor of IGF-1 receptor (IGF-1R), OSI-906, in neonatal mice. We found that, in neonatal mice, SMC-specific deletion of IGF-1 or IGF-1R inhibition with OSI-906 attenuated hypoxia-induced pulmonary vascular remodeling in small arteries, right ventricular hypertrophy, and right ventricular systolic pressure. Pulmonary arterial SMCs from IGF-1-deleted mice or after OSI-906 treatment exhibited reduced proliferative potential. However, in adult mice, smooth muscle-specific deletion of IGF-1 had no effect on hypoxia-induced PH. Our data suggest that vascular smooth muscle-derived IGF-1 plays a critical role in hypoxia-induced PH in neonatal mice but not in adult mice. We speculate that the IGF-1/IGF-1R axis is important in pathogenesis of PH in the developing lung and may be amenable to therapeutic manipulation in this age group.

Megakaryocytic leukemia 1 directs a histone H3 lysine 4 methyltransferase complex to regulate hypoxic pulmonary hypertension.

Enhanced interaction between vascular endothelial cells and circulating leukocytes, as a result of transcriptional activation of cell adhesion molecules (CAM), helps establish a proinflammatory milieu contributing to the pathogenesis of chronic hypoxia-induced pulmonary hypertension. The molecular switch that dictates CAM transactivation is not clearly defined. Our goal was to determine the involvement of the transcriptional modulator megakaryocytic leukemia 1 (MKL1), also known as myocardin-related transcription factor A (MRTF-A), in CAM transactivation and the underlying mechanism. We report here that compared with wild-type littermates, MKL1/MRTF-A knockout mice were more resistant to the development of hypoxia-induced pulmonary hypertension when exposed to low oxygen pressure. Notably, CAM induction in knockout mice was significantly attenuated with a concomitant reduction of leukocyte adhesion. In cultured vascular endothelial cells, overexpression of MKL1/MRTF-A enhanced, whereas depletion of MKL1/MRTF-A dampened, hypoxia-induced CAM transactivation. In response to hypoxia, MKL1/MRTF-A formed a complex with NF-κB on the CAM promoters. Of interest, MKL1/MRTF-A was responsible for recruiting a histone H3 lysine 4 methyltransferase complex to the CAM promoters. Finally, endothelial-specific silencing of ASH2 and WDR5, 2 key components of the histone H3 lysine 4 methyltransferase complex, ameliorated hypoxia-induced pulmonary hypertension in mice. In conclusion, our data suggest that MKL1/MRTF-A, by coordinating key epigenetic alterations on CAM promoters, provides a critical link to hypoxia-induced endothelial malfunction and contributes to the pathogenesis of hypoxia-induced pulmonary hypertension.

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.

Megakaryocytic Leukemia 1 (MKL1) Regulates Hypoxia Induced Pulmonary Hypertension in Rats

Hypoxia induced pulmonary hypertension (HPH) represents a complex pathology that involves active vascular remodeling, loss of vascular tone, enhanced pulmonary inflammation, and increased deposition of extracellular matrix proteins. Megakaryocytic leukemia 1 (MKL1) is a transcriptional regulator known to influence cellular response to stress signals in the vasculature. We report here that in response to chronic hypobaric hypoxia, MKL1 expression was up-regulated in the lungs in rats. Short hairpin RNA (shRNA) mediated depletion of MKL1 significantly ameliorated the elevation of pulmonary arterial pressure in vivo with a marked alleviation of vascular remodeling. MKL1 silencing also restored the expression of NO, a key vasoactive molecule necessary for the maintenance of vascular tone. In addition, hypoxia induced pulmonary inflammation was dampened in the absence of MKL1 as evidenced by normalized levels of pro-inflammatory cytokines and chemokines as well as reduced infiltration of pro-inflammatory immune cells in the lungs. Of note, MKL1 knockdown attenuated fibrogenesis in the lungs as indicated by picrosirius red staining. Finally, we demonstrate that MKL1 mediated transcriptional activation of type I collagen genes in smooth muscle cells under hypoxic conditions. In conclusion, we data highlight a previously unidentified role for MKL1 in the pathogenesis of HPH and as such lay down groundwork for future investigation and drug development.

Brahma-related gene 1 (Brg1) epigenetically regulates CAM activation during hypoxic pulmonary hypertension

Establishment of an inflammatory milieu following elevated leukocyte adhesion to the vascular endothelium, which is mediated by transcriptional activation of cell adhesion molecules (CAMs), contributes to the pathogenesis of chronic hypoxia-induced pulmonary hypertension (HPH). The epigenetic switch that dictates CAM transactivation in response to hypoxia in endothelial cells leading up to HPH is not fully appreciated. We report here that brahma-related gene 1 (Brg1) and brahma (Brm), two catalytic components of the mammalian chromatin remodelling complex, were induced in cultured endothelial cells challenged with hypoxia in vitro as well as in pulmonary arteries in an animal model of HPH. Over-expression of Brg1/Brm enhanced, while the depletion of Brg1/Brm attenuated, CAM transactivation and adhesion of leukocytes. Endothelial-specific deletion of Brg1/Brm ameliorated vascular inflammation and HPH in mice. Chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that hypoxia up-regulated the occupancies of Brg1 and Brm on CAM promoters in a nuclear factor κB (NF-κB) -dependent manner. Finally, Brg1 and Brm activated CAM transcription by altering the chromatin structure surrounding the CAM promoters. Our data suggest that Brg1 provides the crucial epigenetic link to hypoxia-induced CAM induction and leukocyte adhesion that engenders endothelial malfunction and pathogenesis of HPH. As such, targeting Brg1 in endothelial cells may yield promising strategies in the intervention and/or prevention of HPH.

MicroRNA‐98 negatively regulates hypoxia‐induced endothelin‐1 expression in human pulmonary artery endothelial cells

Endothelin‐1 (ET‐1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of hypoxia‐induced pulmonary hypertension (PH). We previously reported that PPARγ activation attenuates hypoxia‐induced ET‐1 expression and pulmonary vascular cell proliferation. The contribution of posttranscriptional mechanisms to these findings has not been defined. We hypothesized that PPARγ regulates microRNA (miR)‐98 to modulate ET‐1 expression and endothelial proliferation. Human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia (1% O2) or normoxia (21% O2) for 72 h. PPARγ was activated in HPAEC by treatment with rosiglitazone (RSG, 10 μM) during the last 24 h. Hypoxia reduced miR‐98 expression, and RSG attenuated reductions in miR‐98 expression. PPARγ knockdown with siRNA (100 nM) reduced miR‐98 expression and increased expression of NF‐κB‐p50 and ET‐1. Transfection with a miR‐98 mimic (30 nM) decreased ET‐1 mRNA levels and HPAEC proliferation, but did not alter PPARγ expression. These findings suggest that PPARγ activation during hypoxia restores reduced levels of miR‐98 to suppress ET‐1 expression and HPAEC proliferation. PPARγ‐mediated regulation of microRNAs may represent a novel therapeutic target to modulate PH pathogenesis. Funding: Atlanta VAMC Research Service, NIH DK074519/HL102167 (CMH), Emory/Children Healthcare of Atlanta CEB F16788‐00 (BYK).

Abstract 9348: ROCK2 in Vascular Smooth Muscle Cells Plays a Crucial Role for Hypoxia-Induced Pulmonary Hypertension in Mice

Background: Pulmonary hypertension (PH) still remains a fatal disease characterized by progressive increase in pulmonary vascular resistance. We have previously demonstrated that the Rho-kinase (ROCK) pathway in vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of cardiovascular diseases by enhancing their migration and proliferation. Rho-kinase has 2 isoforms, ROCK1 and ROCK2, with different functions; ROCK1 for circulating inflammatory cells and ROCK2 for the vasculature. In this study, we thus examined whether ROCK2 in VSMC is involved in the pathogenesis of PH. Methods and Results: We developed VSMC-specific ROCK2-deficient (ROCK2 +/- ) and VSMC-specific ROCK2-overexpressing (ROCK2-Tg) mice. Both genotypes showed normal growth under physiological conditions. Chronic hypoxia (10% O 2 for 4 weeks) significantly increased ROCK2 expression and ROCK activity in lung tissues from littermates (P<0.05, n=4 each). The extent of the hypoxia-induced PH, as evaluated by the extent of pulmonary vascular remodeling, right ventricular (RV) systolic pressure and RV hypertrophy, was significantly reduced in ROCK2 +/- mice and was significantly enhanced in ROCK2-Tg mice compared with the littermates (all P<0.05). Immunostaining showed that the number of CD45 + inflammatory cells in the lung was significantly reduced in the ROCK2 +/- mice and was increased in the ROCK2-Tg mice compared with the littermates, suggesting the role of vascular ROCK2 in inflammatory cell recruitment. In addition, VSMC proliferation (Ki67 + cells) significantly correlated with the ROCK2 expression after chronic hypoxia, suggesting the role of ROCK2 in VSMC proliferation. In cultured mouse aortic VSMC, ROCK2 expression was decreased in ROCK2 +/- VSMC (-21%) and was increased in ROCK2-Tg VSMC (+100%) compared with littermate VSMC. Hypoxia (1% O 2 for 24 hours) significantly increased ROCK activity in littermate VSMC (P<0.05, n=5 each). Scratch assay showed that migration activity was significantly reduced in the ROCK2 +/- VSMC and was increased in the ROCK2-Tg VSMC compared with littermate VSMC (P<0.01). Conclusions: ROCK2 in VSMC plays a crucial role in the pathogenesis of hypoxia-induced PH through enhanced VSMC migration and proliferation.

Early Macrophage Recruitment and Alternative Activation Are Critical for the Later Development of Hypoxia-Induced Pulmonary Hypertension

Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however, its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and to elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1, an anti-inflammatory enzyme, is protective in HPH. We generated bitransgenic mice that overexpress human heme oxygenase-1 under doxycycline control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of doxycycline resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of found in inflammatory zone-1, arginase-1, and chitinase-3-like-3. A brief 2-day pulse of doxycycline delayed, but did not prevent, the peak of hypoxic inflammation, and could not protect against HPH. In contrast, a 7-day doxycycline treatment sustained high heme oxygenase-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage interleukin-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted the proliferation of pulmonary artery smooth muscle cells, whereas treatment with carbon monoxide, a heme oxygenase-1 enzymatic product, abrogated this effect. Early recruitment and alternative activation of macrophages in hypoxic lungs are critical for the later development of HPH. Heme oxygenase-1 may confer protection from HPH by effectively modifying the macrophage activation state in hypoxia.

Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle‐like cells regulated by myocardin involved in hypoxia‐induced pulmonary vascular remodelling

Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle alpha-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin.

Hypoxia inducible factor-1 alpha correlates the expression of heme oxygenase 1 gene in pulmonary arteries of rat with hypoxia-induced pulmonary hypertension.

To test the hypothesis that hypoxia inducible factor-1 alpha (HIF-1alpha) up-regulated the expression of heme oxygenase-1 (HO-1) gene in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension, 8 male Wistar rats in each of 5 groups were exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index were measured. Lungs were inflation fixed for immunohistochemistry, in situ hybridization; frozen for later measurement of HO-1 enzyme activity. mPAP increased significantly after 7 d of hypoxia [(18.4 +/- 0.4) mmHg, P<0.05], reaching its peak after 14 d of hypoxia, then remained stable. Pulmonary artery remodeling became to develop significantly after 14 d of hypoxia. HIF-1alpha protein in control was poorly positive (0.05 +/- 0.01), but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1alpha protein were markedly up-regulated after 3 d and 7 d of hypoxia (0.20 +/- 0.02; 0.22 +/- 0.02, P<0.05), then declined after 14 d and 21 d of hypoxia. HIF-1alpha mRNA staining was poorly positive in control, hypoxia for 3 and 7 d, but enhanced significantly after 14 d of hypoxia (0.20 +/- 0.02, P<0.05), then remained stable. HO-1 protein increased after 7 d of hypoxia (0.10 +/- 0.01, P<0.05), reaching its peak after 14 d of hypoxia (0.21 +/- 0.02, P<0.05), then remained stable. HO-1 mRNA increased after 3 d of hypoxia, reaching its peak after 7 d of hypoxia (0.17 +/- 0.01, P<0.05), then declined. Linear correlation analysis showed that HIF-1alpha mRNA, HO-1 protein and mPAP were associated with pulmonary remodeling. HIF-1alpha protein (tunica intima) was conversely correlated with HIF-1alpha mRNA (r=0.921,P<0.01), HO-1 protein was conversely correlated with HIF-1alpha protein (tunica intima) (r=0.821, P<0.01). HIF-1alpha and HO-1 were both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rat. Hypoxia inducible factor-1 alpha correlated the expression of heme oxygenase 1 gene in pulmonary arteries of rat with hypoxia-induced pulmonary hypertension.

Exhaled NO is reduced at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets.

Altered nitric oxide (NO) production could contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. To determine whether parameters of lung NO are altered at an early stage of hypoxia-induced pulmonary hypertension, newborn piglets were exposed to room air (control, n = 21) or 10% O(2) (hypoxia, n = 19) for 3-4 days. Some lungs were isolated and perfused for measurement of exhaled NO output and the perfusate accumulation of nitrite and nitrate (NOx-), the stable metabolites of NO. Pulmonary arteries (20-600-microm diameter) and their accompanying airways were dissected from other lungs and incubated for NOx- determination. Abundances of the nitric oxide synthase (NOS) isoforms endothelial NOS and neural NOS were assessed in homogenates of PAs and airways. The perfusate NOx- accumulation was similar, whereas exhaled NO output was lower for isolated lungs of hypoxic, compared with control, piglets. The incubation solution NOx- did not differ between pulmonary arteries (PAs) of the two groups but was lower for airways of hypoxic, compared with control, piglets. Abundances of both eNOS and nNOS proteins were similar for PA homogenates from the two groups of piglets but were increased in airway homogenates of hypoxic compared with controls. The NO pathway is altered in airways, but not in PAs, at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets.