Tongxinluo Inhibits Cyclooxygenase-2, Inducible Nitric Oxide Synthase, Hypoxia-inducible Factor-2α/Vascular Endothelial Growth Factor to Antagonize Injury in Hypoxia-stimulated Cardiac Microvascular Endothelial Cells

Background： Endothelial dysflinction is considered as the initiating process and pathological basis of cardiovascnlar disease. Cyclooxygenase-2 （COX-2） and prostacyclin synthase （PGIS）, inducible nitric oxide synthase （iNOS） and endothelial NOS （eNOS） are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia （Hx）, Hx-inducible factor （HIF）-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor （VEGF）, restllting in abnormal proliferation. Whether and how Tongxinluo （TXL） modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells （HCM ECs） have not been clarified. Methods： HCMEC were treated with CoCl2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α. and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations, In addition, the effector molecular of inflammation prostaglandin E2 （PGE2） and the oxidative marker nitrotyrosine （NT） was adopted to reflect HCMEC in.jury. Results： Hx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PG E, and NT contents, attenuating the inflammatory and oxidative injury. Conclusions： TXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and H IF-2α/VEGF to antagonize Hx-induced HCMEC injury.

Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells

Background： Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease.Although tongxinluo （TXL） has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-1 1 in hypoxia-stimulated human cardiac microvascular endothelial cells （HCMECs） have not been reported.Methods： In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL.First, the messenger RNA （mRNA） expression of claudin-5, claudin-9, and claudin-l 1 was confirmed.Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment.Furthermore, the distribution and content histone H3K9 acetylation （H3K9ac） in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.Results： We found that hypoxia-suppressed claudin-9 gene expression in HCMECs （F=7.244;P =0.011） and the hypoxia-suppressed claudin-9 could be reversed by TXL （F=61.911;P =0.000）, which was verified by its protein content changes （F=29.142;P =0.000）.Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury.Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter （F=37.766;P =0.000）, activating claudin-9 transcription.Conclusions： The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.