Fig 1: CGRP stimulated the proliferation of EPCs in dose- and time-dependent manners. After 24 h, 48 h, and 72 h of incubation with or without a series of concentrations CGRP (10− 8–10− 12 M) in high-glucose DMEM with 10% FBS, cell viability was assessed by the optical density via CCK-8 staining (a). Q-PCR was used to detect two proliferation-related genes, cyclin D1 (b) and cyclin E (c). Flow cytometry analysis of the cell cycle after 72 h of incubation (d). Data represent the means ± SEM of three experiments. * p < 0.05 compared with control
Fig 2: CGRP enhances proangiogenic features of MSCs and indirectly contributes to increased angiogenesis effects of HUVECs. (a) RT-qPCR measurement of proangiogenic gene expression levels in MSCs (n = 6). (b) Representative images of tube formation by HUVECs cultured in MSC-derived conditioned medium (CM) and (d) its quantitative measurement accordingly (n = 6). (c) Representative images of transmembrane migrated HUVECs cocultured with MSCs and (e) its quantitative measurement accordingly (n = 6); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as compared with control and CGRP-BIBN groups.
Fig 3: ERK1/2, p38, and JNK phosphorylation during EPC proliferation as assessed by Western blot analysis. Cells were incubated in complete DMEM with CGRP or CGRP combined with CGRP8–37, PD98059, SB203580, or SP600125 and incubated for 24 h, 48 h, or 72 h. # p < 0.05 compared with the CGRP-only group
Fig 4: Western blotting of CGRP-related proteins. (a) Western blotting and (b)–(e) quantitative analysis indicated that CGRP promoted the protein contents of CALCRL, RAMP-1, pPKA/PKA, and pCREB/CREB. ∗p < 0.05, #p < 0.01, as compared with control and CGRP-BIBN groups (n = 6).
Fig 5: (a) Cell viability assay by CCK-8 assay for MSC proliferation in different concentrations of CGRP for 2 days (n = 6). (b) cAMP levels were released by MSCs cultured in different concentrations of CGRP for 2 days. (c) ALP staining of MSCs in different groups after culture for 4 and 7 days, respectively (scale bar: 250 μm) and (e) ALP activity of MSCs cultured in different groups after culture for 4 and 7 days (n = 6). (d) Representative ARS stained the mineral deposition (red, red arrows) of MSCs after culture for 14 days (scale bar: 250 μm). (f) Quantification of MSCs mineralization (n = 6) from different groups after culture for 14 days. ∗p < 0.05, #p < 0.01.
Supplier Page from Abcam for Rat CGRP peptide