Neural Peptide α-CGRP Coregulated Angiogenesis and Osteogenesis via Promoting the Cross-Talk between Mesenchymal Stem Cells and Endothelial Cells

Abstract

Background: The coupled vascularization and bone remodeling are key steps during bone healing, during which the cross-talk between mesenchymal stem cells (MSCs) and endothelial cells plays vital roles. Evidence indicates the well-characterized neuropeptide Calcitonin Gene-Related Peptide-α (CGRP) is proven to play an important role during bone regeneration. However, the regulatory effects of αCGRP on angiogenesis and osteogenesis, as well as underlying cellular and molecular mechanisms, remain unclear.

Aim: The present study was performed to verify the availability of the CGRP for osteogenic capacity in MSCs and explore its potential underlying molecular mechanism. After that, the promoted angiogenic effect of CGRP as well as its underlying mechanisms was studied.

Methods and results: The results showed that CGRP could significantly increase the cyclic adenosine monophosphate (cAMP) level and promote the osteogenesis ability of MSCs via cAMP/PKA signaling pathway. Direct exposure to CGRP increased nitric oxide synthase expression, the release of NO, tube formation, and wound healing of human umbilical vein endothelial cells (HUVEC). The CGRP-treated MSCs were observed with high expression levels of angiogenic factors, such as bFGF and VEGF-α; the conditioned medium derived from CGRP-treated MSCs was also able to promote tube formation and transmembrane migration of HUVECs.

Conclusion: These findings demonstrate the coregulated angiogenesis and osteogenesis effects of CGRP, especially for its regulation effects on the cross-talk between mesenchymal stem cells and endothelial cells.

Figure 1

(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.

Figure 2

Relative osteogenic-specific mRNA expressions of MSCs cultured in different groups for 14 days. The value was normalized to β-actin (n = 6). ^∗p < 0.05, ^#p < 0.01.

Figure 3

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).

Figure 4

CGRP enhances angiogenesis of HUVECs. (a) F-actin staining and (b) iNOS staining of HUVECs; (c) NO release of HUVEC (n = 12); (d) quantitative measurement and (f) representative images of tube formation by HUVECs after 24-hour incubation (n = 6); (e) quantitative evaluation and representative images wound healing closure of HUVECs (n = 6); ∗p < 0.05, ∗∗p < 0.01, and ^∗∗∗p < 0.001, as compared with control and CGRP-BIBN groups.

Figure 5

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.

Scheme 1

Molecular mechanisms mediating coregulated angiogenesis and osteogenesis effects of CGRP.