CircItgb5 promotes synthetic phenotype of pulmonary artery smooth muscle cells via interacting with miR-96-5p and Uba1 in monocrotaline-induced pulmonary arterial hypertension

Abstract

Background Pulmonary arterial hypertension (PAH) is a rare but fatal cardiopulmonary disease mainly characterized by pulmonary vascular remodeling. Aberrant expression of circRNAs has been reported to play a crucial role in pulmonary vascular remodeling. The existing literature predominantly centers on studies that examined the sponge mechanism of circRNAs. However, the mechanism of circRNAs in regulating PAH-related protein remains largely unknown. This study aimed to investigate the effect of circItgb5 on pulmonary vascular remodeling and the underlying functional mechanism. Materials and methods High-throughput circRNAs sequencing was used to detect circItgb5 expression in control and PDGF-BB-treated pulmonary arterial smooth muscle cells (PASMCs). Localization of circItgb5 in PASMCs was determined via the fluorescence in situ hybridization assay. Sanger sequencing was applied to analyze the circularization of Itgb5. The identification of proteins interacting with circItgb5 was achieved through a RNA pull-down assay. To assess the impact of circItgb5 on PASMCs proliferation, an EdU assay was employed. Additionally, the cell cycle of PASMCs was examined using a flow cytometry assay. Western blotting was used to detect biomarkers associated with the phenotypic switch of PASMCs. Furthermore, a monocrotaline (MCT)-induced PAH rat model was established to explore the effect of silencing circItgb5 on pulmonary vascular remodeling. Results CircItgb5 was significantly upregulated in PDGF-BB-treated PASMCs and was predominately localized in the cytoplasm of PASMCs. In vivo experiments revealed that the knockdown of circItgb5 attenuated MCT-induced pulmonary vascular remodeling and right ventricular hypertrophy. In vitro experiments revealed that circItgb5 promoted the transition of PASMCs to synthetic phenotype. Mechanistically, circItgb5 sponged miR-96-5p to increase mTOR level and interacted with Uba1 protein to activate the Ube2n/Mdm2/ACE2 pathway. Conclusions CircItgb5 promoted the transition of PASMCs to synthetic phenotype by interacting with miR-96-5p and Uba1 protein. Knockdown of circItgb5 mitigated pulmonary arterial pressure, pulmonary vascular remodeling and right ventricular hypertrophy. Overall, circItgb5 has the potential for application as a therapeutic target for PAH.

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR‐509‐5p/multiple C2 and transmembrane domain containing 2 axis

“…MCT+ sh‐circST6GAL1 or MCT+ sh‐NC mice were injected AAV9‐sh‐circST6GAL1 or AAV9‐sh‐NC 10 days before MCT injection via the tail vein. Right ventricular systolic pressure (RVSP) was determined after MCT treatment in mice as described in previous reports 10,17 . After 30 days, all the mice were anesthetized with isoflurane and killed, the pulmonary artery specimens were collected, and part of the tissues was stored in 4% paraformaldehyde for hematoxylin and eosin staining and immunohistochemistry analysis, and the remaining tissues were frozen in liquid nitrogen for Western blotting.…”

“… 9 Deficiency of circItgb5 is alleviated in PAH rat model. 10 CircRNA FEACR could suppress ferroptosis and ameliorated myocardial ischemia/reperfusion injury. 11 CircST6GAL1 (has_circ_0068481) is originated from ST6GAL1 gene in chr3: 186756529‐186761098; it was found to be elevated in the serum of idiopathic PAH patients and might be a sensitive and specific marker for idiopathic PAH prediction.…”

“…For example, circ_0091822 enhanced ox‐LDL‐induced migration, invasion, and proliferation in vascular SMCs (VSMCs) during atherosclerosis 9 . Deficiency of circItgb5 is alleviated in PAH rat model 10 . CircRNA FEACR could suppress ferroptosis and ameliorated myocardial ischemia/reperfusion injury 11 .…”

Activation of the p62‐Keap1‐Nrf2 pathway improves pulmonary arterial hypertension in MCT‐induced rats by inhibiting autophagy

“…People generally believe that the proliferation of PASMC plays a crucial role in pulmonary vascular remodeling. 32 Effective strategies for the proliferation and migration of PASMC may represent a breakthrough in PAH treatment. 33 Therefore, we chose PASMC for further study.…”

Roles of Integrin in Cardiovascular Diseases: From Basic Research to Clinical Implications

“…Activates the Ubal protein/Ube2n/Mdm2/ACE2 and miR-96-5p/mTOR signaling pathways, thereby promoting PASMC proliferation [123,124] Abbreviations: PASMC: pulmonary arterial smooth muscle cell, PPARγ: peroxisome proliferator-activated receptor γ, OPN: osteopontin, MMP8: matrix metalloproteinase-8, PDGF-BB: platelet-derived growth factor-BB, FAK: focal adhesion kinase, YAP: Yes-associated prtein, TAZ: PDZ-binding motif, Pyk2: proline-rich tyrosine kinase 2, ERK: extracellular regulated protein kinases, NF-kB: nuclear factor kappa-B, OPG: osteoclastogenesis inhibitory factor, Akt: protein kinase B, Ube2n: ubiquitin conjugating enzyme E2 N, Mdm2: murine double minute2, ACE2: angiotensin-converting enzyme 2, mTOR: mammalian target of rapamycin.…”