High Glucose Levels Promote Switch to Synthetic Vascular Smooth Muscle Cells via Lactate/GPR81

Figure 1. Contractile vSMCs and synthetic vSMCs respond to high glucose concentration differently. (a) Glucose uptake in smooth muscle cells (SMCs). Contractile vSMCs (vcSMCs), synthetic vSMCs (vsSMCs), and human arterial SMCs (haSMCs) (Fisher Scientific, USA) were cultured in basic DMEM with 25 mM glucose for 20 min; then, intracellular glucose concentrations were determined (Abcam, Cambridge, MA, USA) and normalized to measurements in haSMC. (b) ATP production in SMCs. haSMCs and vSMCs were cultured in 25 mM glucose; then, intracellular ATP levels were measured at 24 h, normalized to the level of haSMCs, and presented as a percentage. (c) Cell viability of SMC. Contractile vSMCs and synthetic vSMCs show different viability in high-glucose medium. (d,e) Dynamic change in lactate in glucose culture. The vSMCs derived from hiPSCs were cultured in (d) 5.5 mM (normal glucose, 1 g/L, NG) and (e) 25 mM glucose (high glucose, 4.5 g/L, HG). Lactate was measured at indicated time points with lactate assay kit (Biovision, Milpitas, CA, USA). Contractile vSMCs and synthetic vSMCs produce lactate differently depending on glucose concentration. (f,g) Comparison of lactate production in medium of…

Figure 2. vSMCs utilize glucose differently. There are distinct metabolic differences between contractile vSMCs and synthetic vSMCs in proteomics and metabolomics analyses. Metabolic pathway map summarizing only the significant results from proteomics and [13C]-labeled glucose metabolomics analyses. Cell sample treatment and handling were described in the method section. The bar graphs represent the detected levels of proteins or [13C]-labeled metabolites in synthetic vSMCs (red bar) and contractile vSMCs (blue bar). All data were obtained from independent experiments. Data present 30 min 13C pulse samples. (a) Glycolysis pathway (left panel) and lactate production (right panel) map summarizing the results from proteomics and [13C]-labeled glucose metabolomics analyses. Glycolysis activity in synthetic vSMCs was lower than contractile vSMCs but lactate production was higher in synthetic vSMCs. (b) The TCA cycle pathway map summarizing the results from proteomics and [13C]-labeled glucose metabolomics analyses. The higher enzymes and metabolites indicate that the TCA cycle activity in synthetic vSMCs was higher than in contractile vSMCs. (c) The expression of glucose transporter 1 (GLUT1)…

Figure 3. High glucose concentration promotes SMC phenotype switch. (a) The contractile vSMCs were cultured in 5.5 mM (NG, normal glucose concentration) and 25 mM glucose (HG, high glucose concentration) for 4 days, and their morphologies were evaluated via light microscopy; bar = 100 μm. D0: day 0; D4: day 4. (b) vSMC proliferation was measured with MTT assay (Millipore, St Louis, MO, USA) at days 0 and 4 after vSMCs were cultured in NG and HG for 4 days. (c) Ki67 immunofluorescence stain. vSMCs were fixed, stained with ki67 antibody and DAPI, and visualized with Leica microscope cells. (d) vSMC migration was measured with migration kit (Millipore, St Louis, MO, USA) at day 4 after vSMCs were cultured in NG and HG for 4 days. (e) The expression of markers for the contractile (myosin heavy chain 11 [MYH11], transgelin (TAGN)) and synthetic (collagen I [Col 1] and vimentin [VMT]) vSMC phenotype was evaluated via quantitative RT-PCR on day 4 for vSMCs. (f) Protein levels of MYH11, TAGN, VMT, and Col I were evaluated with Western blot and ELISA (collagen I, R&D scientific, Minneapolis, MN, USA) on day 4 for vSMCs. (g) Immunocytometry. Contractile vSMCs were cultured in NG and HG media for 4…

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…

Figure 5. GPR81 regulates expression of genes involved in lactate metabolism. Contractile vSMCs were cultured in fresh DMEM containing NG (5.5 mM glucose), HG (25 mM glucose), or lactate (10 mM) for 4 days and the cells were used to analyze MCT1, MCT4, PGC-1α, and CD147 mRNA levels with RT-PCR (a) and protein levels with Western blot (b). MCTs, PGC-1α, and CD147 are higher in HG or lactate culture than in NG culture. Relative mRNA expression via RT-PCR (c) and protein by Western blot (d). Knocking down GPR81 inhibited the increase in RNA levels of MCT1, MCT4, PGC-1α, and CD147 with RT-PCR and Protein levels of MCT1, MCT4, PGC-1α, and CD147 with Western blot analysis (e). All data are shown as mean ± S.E. * p < 0.05, ** p < 0.01, *** p < 0.001 for all panels; n ≥ 3 independent experiments for each experimental condition or group.

Figure 6. GPR81 in diabetic patients and high-lipid, high-glucose diet TallyHo mice. Tissues from diabetic patients were used to analyze GPR81 expression in vSMCs and surrounding cells with immunohistochemistry (IHC). Serial 5 µm sections of human lung (a–d) were used for IHC. Diabetic lung tissues had higher blood vessel counts (SMA staining in brown, quantified with Image J Program) and GPR81 expression (red) than control tissues. (a) Control nondiabetic lung tissue stained with SMA; (b) GPR81 stain on serial section close to section (a); (c) diabetic lung tissue stained with SMA; (d) GPR81 stain on serial section close to section (c). (e) The blood vessels were counted in the above IHC slides, and the result showed that there are more blood vessels and GPR81-positive blood vessels in diabetic tissues (n = 5) than in control tissues (n = 5). A total of 4 fields (200X) from each case was counted. (f) Lung tissue lactate assay. Human lung tissue from control and diabetic patients were used to measure lactate. Diabetic lung demonstrated higher LDHa activity (left) and lactate levels (right) than the control sample. (n = 5). TallyHo mice were also used to observe if the high-glucose diet…