Co-profiling reveals distinct patterns of genomic chromatin accessibility and gene expression in pulmonary hypertension caused by chronic hypoxia.

INTRODUCTION: Aberrant gene expression is a key mechanism underlying pulmonary hypertension (PH) development. The alterations of genomic chromatin accessibility and their relationship with the aberrant gene expressions in PH are poorly understood. We used bulk Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) in pulmonary artery smooth muscle cells (PASMCs) of chronic hypoxia-exposed rats mimicking group 3 human PH. METHODS: Adult Sprague Dawley rats were commercially obtained from Hunan SJA (Hunan SJA Laboratory Animal Co., Changsha, China) and randomizedly allocated into four groups exposing to nomobaric hypoxia or normoxia for 1 or 28 days respectively. After the assessment of pulmonary hemodynamics, smooth muscle cells were isolated from intralobular arteries and simultaneously subjected to bulk Assay of ATAC-seq and RNA-seq. RESULTS: Hypoxic exposure for continuous 28-days, but not for 1-day, induced established PH phenotypes in rats. ATAC-seq revealed a major distribution of differential accessibility regions (DARs) annotated to the genome in out-of-promoter regions, following 1-day or 28-days hypoxia. 1188 DAR-associated genes and 378 differentially expressed genes (DEGs) were identified in rats after exposure to 1-day hypoxia, while 238 DAR-associated genes and 452 DEGs for 28-days hypoxia. Most of the DAR-associated genes or DEGs in 1-day did not overlap with that of 28-days hypoxia. A Pearson correlation analysis indicated no significant correlation between ATAC-seq and RNA-seq. CONCLUSIONS: The alterations in genomic chromatin accessibility and genes expression of PASMCs in the initial stage of hypoxia are distinct from the established stage of hypoxia-induced PH. The genomic differential accessibility regions may not be the main mechanisms directly underlying the differentially expressed genes observed either in the initial or established stages of PH. Thus the time-course alterations of gene expression and their possible indirect link with genomic chromatin accessibility warrant more attention in mechanistic study of pulmonary hypertension.

Calcium Sensing Receptor Variants Increase Pulmonary Hypertension Susceptibility.

BACKGROUND: Pulmonary arterial hypertension is an incurable disease, in which the extracellular CaSR (calcium sensing receptor) is mechanistically important. This study was aimed to genetically link the CaSR gene and function to the disease severity. METHODS: Sanger sequencing, Sugen/hypoxia pulmonary arterial hypertension rat model, CaSR mutated rat, transcriptional reporter assay and measurement of CaSR activity were used. RESULTS: Sanger sequencing identified a significant association between the variant rs1042636(A>G), located in CaSR exon 7, and idiopathic pulmonary arterial hypertension (IPAH) formation in patients. The frequency of 2968G homozygotes was higher in patients with IPAH compared with healthy individuals (23.6% versus 17.5%; P=0.001, OR=1.864), and the minor alleles of rs6776158, rs1048213, and rs9883099, located in CaSR promoter, raised the IPAH odds ratio to 2.173. Patients with IPAH carrying heterozygotes or homozygotes genotype of rs1042636 showed markedly higher pulmonary artery pressure and reduced survival compared with individuals carrying the wild-type allele. The minor alleles of rs6776158, rs1048213, and rs9883099 increased CaSR expression in reporter assay. In Sugen/hypoxia pulmonary arterial hypertension rats, the point mutation replicating rs1042636 found in IPAH exacerbated pulmonary arterial hypertension severity by promoting the overexpression and the enhanced activity of CaSR. CONCLUSIONS: Our functional genomic analysis thus indicates that the CaSR minor alleles of rs1042636, rs6776158, rs1048213, and rs9883099 contribute to the development and severity of IPAH. These findings may benefit clinical prognosis and treatment for IPAH.

Comprehensive identification of signaling pathways for idiopathic pulmonary arterial hypertension.

Whole exome sequencing (WES) was used in the research of familial pulmonary arterial hypertension (FPAH). CAV1 and KCNK3 were found as two novel candidate genes of FPAH. However, few pathogenic genes were identified in idiopathic pulmonary arterial hypertension (IPAH). We conducted WES in 20 unrelated IPAH patients who did not carry the known PAH-pathogenic variants among BMPR2, CAV1, KCNK3, SMAD9, ALK1, and ENG. We found a total of 4,950 variants in 3,534 genes, including 4,444 single-nucleotide polymorphisms and 506 insertions/deletions (InDels). Through the comprehensive and multilevel analysis, we disclosed several novel signaling cascades significantly connected to IPAH, including variants related to cadherin signaling pathway, dilated cardiomyopathy, glucose metabolism, immune response, mucin-type O-glycosylation, phospholipase C (PLC)-activating G protein-coupled receptor (GPCR) signaling pathway, vascular contraction and generation, and voltage-dependent Ca2+ channels. We also conducted validation studies in five mutant genes related to PLC-activating GPCR signaling pathway potentially involved in intracellular calcium regulation through Sanger sequencing for mutation accuracy, qRT-PCR for mRNA stability, immunofluorescence for subcellular localization, Western blotting for protein level, Fura-2 imaging for intracellular calcium, and proliferation analysis for cell function. The validation experiments showed that those variants in CCR5 and C3AR1 significantly increased the rise of intracellular calcium and the variant in CCR5 profoundly enhanced proliferative capacity of human pulmonary artery smooth muscle cells. Thus, our study suggests that multiple genetically affected signaling pathways take effect together to cause the formation of IPAH and the development of right heart failure and may further provide new therapy targets or putative clues for the present treatments such as limited therapeutic effectiveness of Ca2+ channel blockers.

Hypoxia-Induced Mitogenic Factor Acts as a Nonclassical Ligand of Calcium-Sensing Receptor, Therapeutically Exploitable for Intermittent Hypoxia-Induced Pulmonary Hypertension.

Hypoxia-induced mitogenic factor (HIMF) is an inflammatory cytokine playing important role(s) in the development of hypoxic pulmonary hypertension. The molecular target mediating HIMF-stimulated downstream events remains unclear. The coimmunoprecipitation screen identified extracellular calcium-sensing receptor (CaSR) as the binding partner for HIMF in pulmonary artery smooth muscle cells. The yeast 2-hybrid assay then revealed the binding of HIMF to the intracellular, not the extracellular, domain of extracellular CaSR. The binding of HIMF enhanced the activity of extracellular CaSR and mediated hypoxia-evoked proliferation of pulmonary artery smooth cells and the development of pulmonary vascular remodeling and pulmonary hypertension, all of which was specifically attenuated by a synthesized membrane-permeable peptide flanking the core amino acids of the intracellular binding domain of extracellular CaSR. Thus, HIMF induces pulmonary hypertension as a nonclassical ligand of extracellular CaSR, and the binding motif of extracellular CaSR can be therapeutically exploitable.

Monocrotaline Induces Endothelial Injury and Pulmonary Hypertension by Targeting the Extracellular Calcium-Sensing Receptor.

BACKGROUND: Monocrotaline has been widely used to establish an animal model of pulmonary hypertension. The molecular target underlying monocrotaline-induced pulmonary artery endothelial injury and pulmonary hypertension remains unknown. The extracellular calcium-sensing receptor (CaSR) and particularly its extracellular domain hold the potential structural basis for monocrotaline to bind. This study aimed to reveal whether monocrotaline induces pulmonary hypertension by targeting the CaSR. METHODS AND RESULTS: Nuclear magnetic resonance screening through WaterLOGSY (water ligand-observed gradient spectroscopy) and saturation transfer difference on protein preparation demonstrated the binding of monocrotaline to the CaSR. Immunocytochemical staining showed colocalization of monocrotaline with the CaSR in cultured pulmonary artery endothelial cells. Cellular thermal shift assay further verified the binding of monocrotaline to the CaSR in pulmonary arteries from monocrotaline-injected rats. Monocrotaline enhanced the assembly of CaSR, triggered the mobilization of calcium signaling, and damaged pulmonary artery endothelial cells in a CaSR-dependent manner. Finally, monocrotaline-induced pulmonary hypertension in rats was significantly attenuated or abolished by the inhibitor, the general or lung knockdown or knockout of CaSR. CONCLUSIONS: Monocrotaline aggregates on and activates the CaSR of pulmonary artery endothelial cells to trigger endothelial damage and, ultimately, induces pulmonary hypertension.

Mitochondrial transplantation attenuates hypoxic pulmonary hypertension.

Mitochondria are essential for the onset of hypoxia-induced pulmonary vasoconstriction and pulmonary vascular-remodeling, two major aspects underlying the development of pulmonary hypertension, an incurable disease. However, hypoxia induces relaxation of systemic arteries such as femoral arteries and mitochondrial heterogeneity controls the distinct responses of pulmonary versus femoral artery smooth muscle cells to hypoxia in vitro. The aim of this study was to determine whether mitochondrial heterogeneity can be experimentally exploited in vivo for a potential treatment against pulmonary hypertension. The intact mitochondria were transplanted into Sprague-Dawley rat pulmonary artery smooth muscle cells in vivo via intravenous administration. The immune-florescent staining and ultrastructural examinations on pulmonary arteries confirmed the intracellular distribution of exogenous mitochondria and revealed the possible mitochondrial transfer from pulmonary artery endothelial cells into smooth muscle cells in part through their intercellular space and intercellular junctions. The transplantation of mitochondria derived from femoral artery smooth muscle cells inhibited acute hypoxia-triggered pulmonary vasoconstriction, attenuated chronic hypoxia-induced pulmonary vascular remodeling, and thus prevented the development of pulmonary hypertension or cured the established pulmonary hypertension in rats exposed to chronic hypoxia. Our findings suggest that mitochondrial transplantation possesses potential implications for exploring a novel therapeutic and preventive strategy against pulmonary hypertension.

Estrogen Secreted by Mesenchymal Stem Cells Necessarily Determines Their Feasibility of Therapeutical Application.

Mesenchymal stem cells are therapeutically applicable and involved in the development of some types of diseases including estrogen (E2)-related ones. Little is known about E2 secretion by mesenchymal stem cells and its potential influence on their therapeutical applications. Our in vitro experiments showed that BMSCs cultured from C57BL/6J mice secreted E2 in a time-dependent manner. In vivo study identified a significantly increased E2 level in serum after a single administration of BMSCs, and a sustained elevation of E2 level upon a repetitive administration. Morris water maze test in the ovariectomised (OVX) mouse model revealed BMSCs transplantation ameliorated OVX-induced memory deficits by secreted E2. On the contrary, in endometriosis model, BMSCs transplantation aggravated endometriotic lesions because of E2 secretion. Mechanistically, the aromatase cytochrome P450 appeared to be critical for the biosynthesis and exerted effects of estrogen secretion by BMSCs. Our findings suggested that BMSCs transplantation is on the one hand an attractive option for the therapeutic treatment of diseases associated with E2 deficits in part through E2 secretion, on the other hand a detrimental factor for the E2-exasperated diseases largely via E2 production. It is important and necessary to monitor serum E2 level before and after the initiation of BMSCs therapy.