Figure 4.

GPR81 expression in vSMC varies with glucose concentration and is required for vSMC ECM production and migration.Contractile vSMC were cultured in normal glucose concentration (NG, 5.5 mM), in high glucose concentration (HG, 25 mM) or in lactate (10 mM) media for 4 days; then, the cells were used to evaluate GPR81 in (a) RT-PCR (b), Western blot. GPR81 was expressed higher in contractile vSMC cells cultured in HG and lactate. (c) Knocking down GPR81 with GPR81shRNA shows the GPR81 expression was decreased in both NG and HG conditions, a finding confirmed with RT-PCR and Western Blot. (d). Contractile vSMC with scramble shRNA have higher proliferation in HG than NG culture, but inhibited proliferation in vSMC with GPR81 shRNA in HG culture. (e) Contractile vSMC with scramble shRNA have higher proliferation marker ki67 in HG than NG culture but inhibited proliferation in vSMC with GPR81 shRNA in HG culture with RT-PCR and Western blot. (f) Contractile vSMC with scramble shRNA have higher migration in HG had higher proliferation than NG culture but inhibited migration in vSMCs with GPR81 shRNA in HG culture. (g) Contractile vSMC with scramble shRNA have higher MMP2 in HG than NG culture but inhibited in vSMC with GPR81 shRNA in HG culture with RT-PCR and Western blot. The expression levels of synthetic vSMC marker vimentin (VMT), contractile markers transgelin (TAGN) and myosin heavy chain 11(MYH11) and ECM protein collagen I in same set cell lysate as d and f were evaluated via quantitative RT-PCR (h), Western blot (i) and collagen I levels were determined via ELISA (j). All data are shown as mean ± S.E. ** p < 0.01, *** p < 0.001 for all panels; n ≥ 3 independent experiments for each experimental condition or group.