CKLF1, transcriptionally activated by FOXC1, promotes hypoxia/reoxygenation­induced oxidative stress and inflammation in H9c2 cells by NLRP3 inflammasome activation.

Myocardial ischemia/reperfusion (I/R) injury is a clinical challenge in the treatment of ischemic heart disease. The present study aimed to establish a hypoxia/reoxygenation (H/R)-induced H9c2 cell model to explore the role and mechanism of chemokine-like factor 1 (CKLF1) in myocardial I/R injury. First, CKLF1 expression was measured in H/R-induced H9c2 cells by reverse transcription-quantitative PCR and western blotting. Subsequently, after CKLF1 silencing, cell viability and apoptosis were evaluated by Cell Counting Kit-8 assay and flow cytometry. In addition, 2,7-dichlorodihydrofluorescein diacetate staining was used to assess the levels of cellular reactive oxygen species. Additionally, the levels of superoxide dismutase, glutathione peroxidase and malondialdehyde, and the contents of inflammatory factors IL-6, IL-1ß and TNF-α were detected using corresponding commercially available kits. Western blotting was used to examine the expression levels of proteins involved in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. The JASPAR database predicted that forkhead box protein C1 (FOXC1) would bind to the CKLF1 promoter region, and dual luciferase and chromatin immunoprecipitation assays were performed to verify it. Subsequently, FOXC1 overexpression and CKLF1 silencing were used to clarify the regulatory mechanism of FOXC1 on CKLF1 in H/R-induced H9c2 cells. The results revealed that CKLF1 expression was markedly enhanced in H/R-stimulated H9c2 cells. CKLF1 knockdown enhanced the viability and inhibited the apoptosis of H9c2 cells exposed to H/R. Moreover, the oxidative stress and inflammation induced by H/R were alleviated following CKLF1 silencing. CKLF1 knockdown also inhibited NLRP3 inflammasome activation. Furthermore, FOXC1 bound to the CKLF1 promoter region to upregulate CKLF1 expression, and FOXC1 overexpression alleviated the effects of CKLF1 knockdown on H9c2 cell damage induced by H/R via activation of the NLRP3 inflammasome. In conclusion, CKLF1 transcriptionally activated by FOXC1 may promote H/R-induced oxidative stress and inflammation in H9c2 cells via NLRP3 inflammasome activation.

Transcription factor JDP2 activates PDE4B to participate in hypoxia/reoxygenation-induced H9c2 cell injury.

Myocardial ischemia/reperfusion (I/R) injury is a clinical challenge in the treatment of acute myocardial infarction (AMI). Phosphodiesterase 4B (PDE4B) expression is upregulated in AMI tissues. Thus, the present study aimed to investigate the role of PDE4B in myocardial I/R injury. H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro myocardial I/R model. PDE4B expression was detected via reverse transcription-quantitative PCR (RT-qPCR) and western blotting before and after transfection with PDE4B interference plasmids in H/R-stimulated H9c2 cells. Cell viability and cytotoxicity were assessed using the Cell Counting Kit-8 and lactate dehydrogenase assays, respectively. Furthermore, oxidative stress was assessed using malondialdehyde, superoxide dismutase and glutathione/glutathione oxidized ratio detection kits. Cell apoptosis was detected via a TUNEL assay and western blotting. c-Jun dimerization protein 2 (JDP2) expression was also detected via RT-qPCR and western blotting. The dual luciferase reporter and chromatin immunoprecipitation assays were performed to verify the interaction between JDP2 and PDE4B. Following co-transfection with PDE4B interference plasmid and JDP2 overexpression plasmid, cell viability, cytotoxicity, oxidative stress and cell apoptosis were assessed. The results demonstrated that PDE4B knockdown reversed H/R-induced loss of viability and cytotoxicity of H9c2 cells. H/R-induced oxidative stress and cardiomyocyte apoptosis were also alleviated by PDE4B knockdown. In addition, the transcription factor JDP2 was expressed at high levels in H/R-stimulated H9c2 cells, and JDP2 overexpression upregulated PDE4B expression. Notably, JDP2 overexpression partly reversed the ameliorative effect of PDE4B knockdown on H/R-induced H9c2 injury. Taken together, the results of the present study suggested that JDP2-activated PDE4B contributed to H/R-induced H9c2 cell injury.

VEGF treatment promotes bone marrow-derived CXCR4+ mesenchymal stromal stem cell differentiation into vessel endothelial cells.

Stem/progenitor cells serve an important role in the process of blood vessel repair. However, the mechanism of vascular repair mediated by C-X-C chemokine receptor type 4-positive (CXCR4+) bone marrow-derived mesenchymal stem cells (BMSCs) following myocardial infarction remains unclear. The aim of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on vessel endothelial differentiation from BMSCs. CXCR4+ BMSCs were isolated from the femoral bone marrow of 2-month-old mice and the cells were treated with VEGF. Expression of endothelial cell markers and the functional properties were assessed by reverse transcription-quantitative polymerase chain reaction, flow cytometry and vascular formation analyses. The results indicated that the CXCR4+ BMSCs from femoral bone marrow cells expressed putative cell surface markers of mesenchymal stem cells. Treatment with VEGF induced platelet/endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) expression at the transcriptional and translational levels, compared with untreated controls. Moreover, VEGF treatment induced CXCR4+ BMSCs to form hollow tube-like structures on Matrigel, suggesting that the differentiated endothelial cells had the functional properties of blood vessels. The results demonstrate that the CXCR4+ BMSCs were able to differentiate into vessel endothelial cells following VEGF treatment. For cell transplantation in vascular disease, it may be concluded that CXCR4+ BMSCs are a novel source of endothelial progenitor cells with high potential for application in vascular repair.

In silico Analysis of TCR Vß7 of Two Patients with Type 1 Diabetes Mellitus.

OBJECTIVE: To compare the sequences and crystal structures of variable region of beta chain 7 (Vß7) of T cell receptor (TCR) of two patients with type 1 diabetes mellitus (T1DM). PATIENTS AND METHODS: The skewness of TCR Vß7 of two T1DM patients were detected with real-time florescence quantitative polymerase chain reaction (FQ-PCR) and deoxyribonucleic acid (DNA) melting curve analysis technique followed by being sequenced, the crystal structures of them were simulated according to CPH models 2.0 Server, IMGT database, and RasMol 2 software. RESULTS: The whole sequences of TCR Vß7 of T1DM patient-1 were "CASRTAGQYEQYFGPGTR", that of patient-2 were "CASRTAGQYEQFFGPGTR"; the only difference between them lied on the 12(th) amino acid. The crystal structures of Vß7 of the two patients simulated with backbone model were rather similar, while that with sphere model were obviously different. CONCLUSION: Although the TCR Vß7 of the T1DM patients share the similar gene sequences, their crystal structures simulated with sphere model are different, and the mechanism needs further study.