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. 2021 Mar 4;24(4):102272. doi: 10.1016/j.isci.2021.102272

Vitamin D3 induces mesenchymal-to-endothelial transition and promotes a proangiogenic niche through IGF-1 signaling

Lei Chen 1, Anweshan Samanta 2, Lin Zhao 3, Nathaniel R Dudley 1, Tanner Buehler 1, Robert J Vincent 1, Jeryl Hauptman 3, Magdy Girgis 3, Buddhadeb Dawn 3,4,∗
PMCID: PMC8005757  PMID: 33817577

Summary

Although vitamin D3 (VitD3) prevents angiogenesis in cancer, VitD3 deficiency is associated with greater incidence of cardiovascular events in patients. We examined the influence of VitD3 on the angiogenic potential of mesenchymal stem cells (MSCs). VitD3 treatment increased the expression of proangiogenic molecules in MSCs, which exhibited an endothelial cell-like phenotype and promoted vascularization in vitro and in vivo. VitD3 activated the IGF-1 promoter and boosted IGF-1 receptor (IGF-1R) signaling, which was essential for the mesenchymal-to-endothelial transition (MEndoT) of MSCs. VitD3-treated MSCs created a proangiogenic microenvironment for co-cultured arterial endothelial cells, as well as aortic rings. The induction of MEndoT and angiogenesis promotion by VitD3-stimulated MSCs was attenuated by IGF-1R inhibitor picropodophyllin. We conclude that VitD3 promotes MEndoT in MSCs, and VitD3-treated MSCs augment vascularization by producing a proangiogenic niche through continued IGF-1 secretion. These results suggest a potential therapeutic role of VitD3 toward enhancing MSC-induced angiogenesis.

Subject areas: Biological Sciences, Physiology, Molecular Biology, Cell Biology

Graphical abstract

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Highlights

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    Vitamin D3 (VitD3) treatment induces IGF-1 in mesenchymal stem cells (MSCs)

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    VitD3 promotes mesenchymal-to-endothelial transition in MSCs via IGF-1 signaling

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    Continued IGF-1 secretion by VitD3-treated MSCs creates a proangiogenic niche

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    VitD3 may enhance MSC-induced angiogenesis through dual mechanisms

Biological Sciences; Physiology; Molecular Biology; Cell Biology

Introduction

Bone marrow mesenchymal stem cells (MSCs) have been shown to promote angiogenesis after an ischemic injury (Nagaya et al., 2004). Under specific culture conditions, a small fraction of MSCs express endothelial characteristics (Prockop, 1997). MSCs also participate in angiogenesis through paracrine factors, including vascular endothelial growth factors (VEGFs) and basic fibroblast growth factor (Kinnaird et al., 2004b). However, the results of clinical trials for therapeutic tissue repair with MSCs have produced suboptimal results.

Although vitamin D3 (VitD3) deficiency has been linked to manifold adverse health consequences (Holick, 2006), the underlying pathophysiology and molecular mechanisms remain largely unclear. The effects of VitD3 on angiogenesis remain particularly perplexing. VitD3 has also been shown to suppress endothelial cell sprouting and proliferation in vitro and reduce vascularity of tumors in vivo (Mantell et al., 2000). However, a role of VitD3 in the promotion of vascular regeneration through the SDF-1/CXCR4 axis and recruitment of angiogenic myeloid cells has been reported (Wong et al., 2014). The ability of VitD3 to enhance proliferation and migration of endothelial cells has also been documented (Molinari et al., 2013). In addition, epidemiological data from VitD3-deficient patients show an increased incidence of cardiovascular events (Wang et al., 2008), suggesting its possible proangiogenic influence.

Given the therapeutic potential of MSCs in cardiac repair (Golpanian et al., 2016), we sought to determine whether VitD3 treatment could induce a proangiogenic phenotype in MSCs and promote vascularization. We further investigated the impact of VitD3 treatment on MSC niche. Although a positive association between serum 25(OH)D levels and IGF-1 concentration in humans has been reported (Hypponen et al., 2008), the potential molecular basis remains unknown. Since IGF-1 is known to promote angiogenesis (van Beijnum et al., 2017) and has been reported to increase levels of VEGF and its receptors in certain cells (Menu et al., 2004; Rabinovsky and Draghia-Akli, 2004), we examined whether VitD3 treatment would induce mesenchymal-to-endothelial transition (MEndoT) through IGF-1 upregulation. Our results indicate that VitD3 induces MEndoT in MSCs and augment angiogenesis in a dose-dependent manner. Perhaps more importantly, our findings also show that VitD3-treated MSCs are able to create a proangiogenic niche through increased IGF-1 secretion.

Results

VitD3 induces proangiogenic genes and proteins in MSCs

The role of VitD3 in angiogenesis remains poorly understood. To examine the angiogenic potential of VitD3, we first examined whether exposure to VitD3 would increase the expression of molecules known to promote angiogenesis. MSCs were harvested from murine bone marrow by adhesion and expanded in culture. To analyze the expression profile of these genes, total RNA was extracted from MSCs after 48 h of treatment with VitD3 (0.1 nM and 10 nM) or dimethyl sulfoxide (DMSO, control). Quantitative polymerase chain reaction (qPCR) data in Figure 1A show that VitD3 treatment upregulated the expression of proangiogenic genes, including VEGF, FLK1, endothelin-1, VCAM-1, ICAM-1, VWF, and VE-cadherin in a dose-dependent fashion. The levels of 2 key proangiogenic molecules, VEGF and its receptor FLK1, increased 12.4- and 3.9-fold, respectively, with 10 nM of VitD3 (Figure 1A). Increased VEGF and FLK1 levels by Western blot analysis confirmed the above mRNA findings at the protein level (Figures 1B and 1C). Interestingly, the expression of genes associated with cellular pluripotency such as Oct-4 and SSEA1 was also upregulated by VitD3 treatment (Figure 1A).

Figure 1.

Figure 1

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VitD3 induces proangiogenic molecules in MSCs

(A) qPCR data show increased mRNA levels of proangiogenic as well as pluripotency-related genes in MSCs following exposure to VitD3. Data represent mean ± standard error of the mean (SEM), n = 3.

(B) Representative Western immunoblots show VEGF, FLK1, and β-actin (loading control) protein levels in MSCs following VitD3 stimulation.

(C) Densitometric quantitation of VEGF and FLK1 protein levels.

Data represent mean ± SEM. One-way analysis of variance (ANOVA) with Bonferroni post hoc test, ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n = 3.

VitD3 induces mesenchymal-to-endothelial transition in MSCs

Since the ability of VitD3 to induce endothelial transition in MSCs remains unknown, we examined whether VitD3 could induce this phenotypic shift in MSCs. Treatment of MSCs with VitD3 for 48 h increased VE-cadherin and CD34 protein levels in a dose-dependent manner (Figures 2A and 2B). These results were further validated by staining of MSCs with fluorophore-conjugated anti-VE-cadherin and anti-CD34 antibodies. As shown in Figure 2C, compared with control MSCs, VitD3-treated MSCs exhibited increased fluorescent signals indicating greater expression of VE-cadherin and CD34. As VE-cadherin is essential for cohesion and organization of the intercellular junctions of endothelial cells, it is a reliable indicator of an endothelial phenotype (Breier et al., 1996). These observations therefore suggest that a significant fraction of VitD3-treated MSCs acquire an endothelial-like cellular phenotype.

Figure 2.

Figure 2

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VitD3 induces MEndoT in MSCs

(A) Representative Western immunoblots show VE-cadherin, CD34, and β-actin (loading control) expression in MSCs following VitD3 stimulation.

(B) Densitometric quantitation of VE-cadherin and CD34 protein levels. Data represent mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Bonferroni post hoc test, ns = not significant, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n = 3.

(C) Increased expression of CD34 (FITC, green) and VE-cadherin (TRITC, red) in MSCs following VitD3 treatment. Nuclei are identified in blue (DAPI). Scale bar, 20 μm.

(D) Flow cytometric analysis shows a dose-dependent increase in CD34 expression and minimal expression of CD31 in VitD3-stimulated MSCs. The right part shows the quantitative data. Data represent mean ± SEM. One-way ANOVA with Bonferroni post hoc test, ∗∗∗∗p < 0.0001, n = 3.

To quantify the extent of MEndoT, control and VitD3-treated MSCs were stained with fluorescent conjugated antibodies against CD34 and CD31, two known markers of endothelial lineage (Middleton et al., 2005). Flow cytometric analysis identified a dose-dependent increase in CD34+ MSCs by 1.5- and 3.5-folds in 0.1 nM and 10 nM VitD3-treated MSC groups, respectively, compared with control MSCs (Figure 2D). Interestingly, CD31 expression did not increase significantly following VitD3 treatment, indicating early commitment to endothelial lineage.

VitD3-treated MSCs induce angiogenesis in vitro and in vivo

To determine the significance of the above molecular changes, we examined whether MEndoT in VitD3-treated MSCs would translate into angiogenesis. MSCs were treated with VitD3 for 48 h, and angiogenesis assays were performed in vitro on Matrigel. Compared with controls, VitD3 treatment resulted in greater tube formation by MSCs (Figure 3A), which quantitatively resulted in 2.68- and 11.76-fold greater branch lengths in 0.1 nM and 10 nM VitD3-treated MSCs groups (Figure 3B), respectively.

Figure 3.

Figure 3

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VitD3-treated MSCs promote angiogenesis in vitro and in vivo

(A) MSCs treated with either vehicle (control) or VitD3 produced a tube-like network in a Matrigel assay. Scale bar, 1 mm.

(B) Quantitative assessment of relative branch length. Data represent mean ± standard error of the mean (SEM), Welch's analysis of variance (ANOVA) with Dunnett's T3 post hoc test, ∗p < 0.05, ∗∗p < 0.01, n = 4.

(C) Explanted Matrigel plugs with vehicle- (control) or VitD3-treated MSCs show evidence of perfusion with blood. n = 6 per group.

(D) Isolectin B4 positivity (green) identifies capillary endothelium within Matrigel plugs at 3 weeks after implantation. Scale bar, 10 μm.

(E) Quantitative estimates of capillary densities in Matrigel plugs. Data represent mean ± SEM. One-way ANOVA with Bonferroni post hoc test, ns = not significant, ∗∗p < 0.01, n = 4 per group.

Next, we performed a xenograft assay to examine whether MSCs were capable of promoting angiogenesis in vivo. Control Matrigel plugs or those mixed with VitD3-treated MSCs were transplanted subcutaneously in 8-week-old C57BL/6 male mice. Three weeks later, explanted Matrigel plugs carrying VitD3-treated MSCs macroscopically displayed a pink color indicating perfusion with blood in vivo as compared with a pale color observed in control MSC plugs (Figure 3C). The 10 nM VitD3-treated group showed a significantly darker change in coloration. This indicated the presence of larger amount of blood, which suggested a higher density of functional capillaries, an early stage marker of neovascularization produced by endothelial cells (Akhtar et al., 2002). Sections from Matrigel plugs were stained with isolectin B4 (Figure 3D) and capillary densities quantified using microscopy. The capillary densities were 1.41- and 2.46-folds greater in the 0.1 nM and 10 nM VitD3-treated MSC groups, respectively (Figure 3E). Together, these data indicate that MSCs treated with VitD3 are able to induce greater angiogenesis both in vitro and in vivo.

VitD3 induces and activates the IGF-1 proangiogenic signaling pathway

To elucidate the mechanistic basis of the above observations, we investigated the molecular events that lead to MEndoT of VitD3-treated MSCs. Earlier, we observed that the expression of IGF-1 in MSCs was significantly enhanced following VitD3 treatment (Figure 1A). To further validate this finding, protein expression of IGF-1 was assessed by Western blot analysis following 48 h of VitD3 treatment. We found significant upregulation of IGF-1 by VitD3 in a dose-dependent manner (Figures 4A and 4B). In light of these results, we examined the activation of IGF-1 receptor (IGF-1R) via phosphorylation. The phospho-IGF-1R levels increased by 1.28-, 1.60-, and 1.79-folds in 0.1 nM, 1 nM, and 10 nM VitD3-treated MSC groups, respectively. In addition, we found concomitant increases in the activation of a key downstream factor for angiogenesis, phospho-ERK1/2 (Dunn et al., 2001) (Figures 4A and 4B). To further interrogate the role of IGF-1 signaling in VitD3-treated MSCs, we analyzed IGF-1R signaling using a specific inhibitor, picropodophyllin (PPP). The VitD3-induced increase in phospho-IGF-1R, phospho-ERK1/2, VEGF, and FLK1 was reduced by co-treatment with PPP, and this suppression was also dose dependent (Figures 4C and 4D).

Figure 4.

Figure 4

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VitD3 enhances proangiogenic IGF-1 signaling in MSCs

(A and B) Representative Western immunoblots (A) and quantitative densitometric data (B) show increased protein expression of key components of IGF-1 signaling in VitD3-treated MSCs in a dose-dependent manner. Data represent mean ± standard error of the mean (SEM). Welch's analysis of variance (ANOVA) with Dunnett's T3 post hoc test (IGF-1 and p-IGF-1R) and one-way ANOVA with Bonferroni post hoc test (p-ERK1/2), ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n = 3.

(C and D) Representative Western immunoblots (C) and quantitative densitometric data (D) show reversal of VitD3-induced expression of proangiogenic molecules following co-treatment with PPP, an IGF-1R antagonist, in a dose-dependent manner. Data represent mean ± SEM. Welch's ANOVA with Dunnett's T3 post hoc test (p-IGF-1R and p-ERK1/2) and one-way ANOVA with Bonferroni post hoc test (VEGF and FLK1), ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n = 3.

(E) qPCR data show reversal of VitD3-induced increased expression of proangiogenic genes following co-treatment with PPP, an IGF-1R antagonist. Data represent mean ± SEM, n = 3.

Although IGF-1 signaling has been shown to promote angiogenesis, its role in MSC-induced angiogenesis remains unknown. To address this, qPCR was performed to examine the relative levels of mRNA transcripts encoding proangiogenic factors following treatment with VitD3 with or without PPP. VitD3 treatment resulted in marked increase in mRNA expression of several proangiogenic and pluripotency-related molecules. PPP co-treatment reversed these changes with suppression of VitD3-induced augmentation of mRNA expression of endothelin-1, VCAM-1, ICAM-1, VE-cadherin, VWF, PDGFRβ, α-SMA, CD34, FLK1, and VEGF (Figure 4E), indicating that VitD3-induced activation of IGF-1R signaling is responsible for the induction of a proangiogenic phenotype in MSCs.

To further substantiate these findings at the mRNA level, we performed Western blot analysis using protein samples from cells subjected to analogous treatments. The results confirmed similar effects of PPP co-treatment on protein levels of VE-cadherin and CD34 (Figure 5A). Finally, MSCs treated with VitD3 with/out PPP for 48 h were stained with anti-VE-cadherin and anti-CD34 antibodies followed by fluorescent imaging. Consistently, the VitD3-induced increase in fluoresence signals of VE-cadherin and CD34 was markedly reduced in MSCs co-treated with PPP (Figure 5B).

Figure 5.

Figure 5

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IGF-1R antagonist inhibits both MEndoT and the proangiogenic phenotype of VitD3-treated MSCs

(A) Representative Western immunoblots (upper panel) and quantitative densitometric data (lower panel) show dose-dependent reversal of VitD3-induced increase in VE-cadherin and CD34 levels in VitD3-treated MSCs with PPP co-treatment. Data represent mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Bonferroni post hoc test (VE-cadherin) and Welch's ANOVA with Dunnett's T3 post hoc test (CD34), ns = not significant, ∗p < 0.05, ∗∗∗∗p < 0.0001, n = 3.

(B) The inhibition of VitD3-induced angiogenic phenotype in MSCs by IGF-1R antagonist PPP was further confirmed with fluorescent detection of VE-cadherin (red) and CD34 (green) in MSCs treated with VitD3 with and without PPP. Scale bar, 20 μm.

(C and D) Representative images (C) and quantitative assessment of branch length (D) show robust augmentation of capillary-like tube formation by MSCs on Matrigel following VitD3 treatment and inhibition of the same by co-treatment with IGF-1R antagonist PPP. Scale bar, 100 μm.

Data represent mean ± SEM. Welch's ANOVA with Dunnett's T3 post hoc test, ns = not significant, ∗p < 0.05, n = 4.

VEGF and FLK1 (Figures 4C and 4D) are two of the most important proangiogenic signaling molecules. Thus, suppression of VEGF and FLK1 expression by PPP indicated a negative impact of PPP on angiogenesis by MSCs. To examine this, VitD3-treated MSCs with or without PPP co-treatment were subjected to an in vitro angiogenesis assay on Matrigel. As shown in Figure 5C, the branch formation competence of MSCs was greatly reduced by PPP. Compared with untreated MSCs (control), branch length increased by 11.76-fold in the VitD3-treated MSC group, which was reduced to a mere 3.45-fold increase with PPP co-treatment (Figure 5D). The suppression of both angiogenesis-related gene expression and branch formation by IGF-1R inhibitor PPP indicates that IGF-1, through the expression of VEGF-FLK1, is a major factor contributing to the angiogenesis-promoting effects of VitD3 in MSCs.

IGF-1 identified as the target gene for VitD3

The functions of VitD3 are predominantly activated through binding to its specific receptor, the vitamin D receptor (VDR) (Brumbaugh and Haussler, 1973). To determine whether the VitD3-VDR complex is a transcriptional activator of IGF-1, chromatin immunoprecipitation (ChIP) assay was performed for IGF-1 in response to VitD3 treatment of MSCs. The results of the ChIP assay indicated that VDR directly binds to the promoter region of IGF-1 and that this binding was more robust in the presence of increasing doses of VitD3 (Figure 6A). Compared with untreated MSC controls, the VDR binding activities were 7.28- and 43.76-folds greater in MSCs treated with 0.1 nM and 10 nM of VitD3, respectively (Figure 6B).

Figure 6.

Figure 6

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VitD3/VDR directly binds to the IGF-1 promoter and increases activity

(A and B) The binding of VDR following VitD3 stimulation in MSCs was analyzed by ChIP assay (A) and the relative densities of DNA bands were quantified (B). Data represent mean ± standard error of the mean (SEM). n = 4.

(C) The potential VDR response elements within the promoter region of IGF-1 (top) and the corresponding mutant VDR response elements (bottom, with mutated nucleotides underlined).

(D) Luciferase reporter constructs containing either wild-type or mutant promoter sequences were transfected into MSCs for VitD3 stimulation, and the relative levels of luciferase were analyzed following quantification with the internal control.

Data represent mean ± SEM. One-way or Welch's analysis of variance (ANOVA) with Bonferroni or Dunnett's T3 post hoc tests, respectively, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n = 3.

Furthermore, the analysis of murine IGF-1 genomic sequence identified two potential VDR response elements in the promoter region of IGF-1. Next, to evaluate whether these response elements within the IGF-1 promoter confer VitD3 and VDR-dependent transcriptional activation, luciferase assay was performed. A DNA fragment of the IGF-1 promoter was subcloned into the firefly luciferase reporter vector, pGL3-Basic (renamed IGF-1-WT). In addition, the VDR binding elements within the IGF-1 promoter region were mutated and subcloned into the pGL3-Basic vector and renamed IGF-1-mut1 and IGF-1-mut2 (Figure 6C). As shown in Figure 6D, the induction of the luciferase signal was enhanced by 1.43- and 4.32-folds in MSCs treated with 0.1 nM and 10 nM of VitD3, respectively, compared with controls, suggesting that VitD3, through its activation of the VDR, can activate IGF-1 expression. The respective reporter signals of the IGF-1-mutants induced by VitD3 were considerably less when compared with the wild-type IGF-1 promoter vector (IGF-1-WT) during stimulation with the same concentrations of VitD3 (Figure 6D), confirming the role of VDR binding to IGF-1 promoter in response to VitD3 stimulation.

VitD3-treated MSCs promote a proangiogenic niche

MSCs have the ability to generate a specific microenvironment through the secretion of various factors (Kinnaird et al., 2004a, 2004b). Our results show the ability of VitD3 treatment to induce IGF-1 expression in MSCs (Figures 1A and 4A). As a secreted protein, IGF-1 has the potential to produce a niche within the surrounding cells by binding to its cognate receptor. To evaluate this niche-forming ability of IGF-1, MSCs were subjected to either 0.1 nM or 10 nM of VitD3 for 3 days and then cultured in fresh media (without VitD3) for an additional 5 days. The IGF-1 concentrations in the culture media were quantified by enzyme-linked immunosorbent assay (ELISA) on days 1, 2, 3, 4, 5, 6, and 8. The IGF-1 concentration in culture media increased significantly by day 3 with VitD3 stimulation. Compared with the 0.07 ng/mL of IGF-1 in control MSC media, IGF-1 levels in 0.1 nM and 10 nM VitD3-treated MSC groups were 0.44 ng/mL and 0.50 ng/mL, respectively. After switching to VitD3-free culture media, the IGF-1 concentration decreased slightly on day 4 but then continued to rise subsequently despite the removal of VitD3 stimulation. The IGF-1 levels in media containing non-treated MSCs (control) were low (Figure 7A). The IGF-1 levels in media containing MSCs previously treated with 10 nM of VitD3 were greater compared with levels in 0.1 nM of VitD3-treated MSCs or control MSCs (Figure 7A).

Figure 7.

Figure 7

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VitD3-treated MSCs produce an IGF-1 microenvironment for angiogenesis

(A) IGF-1 levels increase in the culture medium in a dose-dependent manner following exposure of MSCs to VitD3. IGF-1 levels continue to increase even after cessation of VitD3 stimulation. Data represent mean ± standard error of the mean (SEM). n = 3.

(B) Cartoon showing the experimental set up with VitD3-treated MSCs in contactless co-culture.

(C and D) Representative images (C) and quantitative data (D) show significant augmentation of bead branching with VitD3-treated MSCs and reversal of the same with VitD3 and PPP co-stimulated MSCs. Scale bar, 100 μm. Data represent mean ± SEM. One-way analysis of variance (ANOVA) with Bonferroni post hoc test (protrusion length) and Welch's ANOVA with Dunnett's T3 post hoc test (protrusions per bead), ns = not significant, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n = 8-10 per group.

(E and F) Representative images (E) and quantitative assessment of neovascularization surface area (F) from the aortic ring assay show enhanced sprouting induced by VitD3-treated MSCs. This effect was neutralized when MSCs were treated with PPP. Scale bar, 100 μm. Data represent mean ± SEM. Welch's ANOVA with Dunnett's T3 post hoc test, ns = not significant, ∗∗p < 0.01, n = 5 per group.

To examine the paracrine functionality of this niche, MSCs were treated with 10 nM of VitD3 for 3 days and then changed to fresh medium before co-culture experiments with endothelial cells (Figure 7B). Primary mouse pulmonary artery endothelial cells (MAECs) were incubated with Cytodex-3 beads, transplanted into a fibrinogen pad, and maintained in culture. After 3 to 5 days of co-culture in VitD3-free EGM-2MV medium, MAEC beads were co-cultured with MSCs without contact (Figure 7B). Compared with vehicle control, co-culturing with untreated MSCs (MSC-control) did not enhance branching (Figures 7C and 7D). However, MAECs co-cultured with VitD3-treated MSCs exhibited significantly more branching compared with MAECs co-cultured with control MSCs. The mean branch length was approximately 2.24-folds greater and mean protrusions per bead was approximately 4-folds higher in the group co-cultured with VitD3-treated MSCs compared with respective parameters in the MSC-control group (Figure 7D). However, the increase in branch length and the number of protrusions from MAEC beads co-cultured with VitD3-treated MSCs were significantly suppressed by the addition of 1 μM of PPP in the co-culture medium (Figures 7C and 7D). To further substantiate the niche-enhancing attributes of VitD3-treated MSCs, we also performed similar co-culture assays with murine aortic rings. Pieces of thoracic aorta harvested from 8-week-old C57BL/6 male mice were cut into 1-mm rings, embedded in Matrigel, and co-cultured with vehicle, control MSCs, or VitD3-treated MSCs as above. After 5 days, greater micro-vessel outgrowths surrounding the aortic rings were noted in co-cultures with VitD3-treated MSCs as compared with the MSC-control group (Figures 7E and 7F). This enhancement in outgrowths was suppressed by the addition of PPP to co-culture (Figures 7E and 7F). Together, these results identify IGF-1 as the primary factor secreted by VitD3-treated MSCs that produces a niche conducive for angiogenesis.

Discussion

The role of VitD3 in angiogenesis remains controversial. Our findings establish a proangiogenic influence of VitD3 on MSCs, which have been used in clinical trials of cardiac repair with mixed results. The salient findings include the following: (i) treatment with VitD3 induces proangiogenic genes, increases the expression of endothelial markers, and promotes MEndoT in MSCs;( ii) VitD3-treated MSCs induce angiogenesis both in vitro and in vivo in an IGF-1-dependent manner; (iii) VitD3/VDR complex binds to the IGF-1 promoter to induce IGF-1 expression; and (iv) VitD3 promotes the formation of a proangiogenic niche by MSCs through enhanced IGF-1 secretion. These results from both in vitro and in vivo studies underscore a beneficial role of VitD3 in vascular health through angiogenesis. This is particularly appealing because MSCs have therapeutic potential for diverse diseases, including cardiovascular ischemic diseases (Afzal et al., 2015; Golpanian et al., 2016). The current data bid well for successful clinical translation of VitD3-treated MSCs for vasculogenesis in patients with ischemic conditions.

Although sparse evidence suggests that VitD3 can influence cellular fate and inhibit epithelial to mesenchymal transition of cancer cells (Upadhyay et al., 2013), its role in endothelial transformation of mesenchymal cells remains unknown. The commitment to endothelial cell lineage is important because it increases the capacity for angiogenesis and capillary formation (Ribatti and Crivellato, 2012). The current results show upregulation of endothelial genes in VitD3-treated MSCs, which is corroborated by increased expression of both VE-cadherin and CD34, distinctive cell surface markers of endothelial lineage (Breier et al., 1996; Middleton et al., 2005). Together, these results establish the induction of MEndoT in VitD3-treated MSCs. The upregulation of other endothelial-related genes including VWF, ICAM-1, and VCAM-1 further establishes the endothelial nature of MSCs treated with VitD3.

The current results show an increased expression of IGF-1 in MSCs in response to VitD3 treatment. In addition, the increase in phospho-IGF-1R in VitD3-treated MSCs indicated the activation of IGF-1 signaling in these cells. In previous studies, IGF-1 has been shown to stimulate migration and tube-forming activities of endothelial cells, supporting a proangiogenic role (Nakao-Hayashi et al., 1992). The current data also show that the inhibition of IGF-1 signaling by PPP attenuated the induction of proangiogenic genes and blocked the acquisition of an endothelial phenotype by VitD3-treated MSCs, indicating a causal role of IGF-1 signaling toward VitD3-induced MEndoT. Concomitant exposure to PPP reduced the ability of VitD3-treated MSCs to form tubes on Matrigel, consistent with reduced capacity for angiogenesis. The mechanistic basis of IGF-1 induction by VitD3 was further confirmed by the ChIP assay that showed increased binding of the VitD3 response region on the IGF-1 promoter with increasing doses of VitD3. Collectively, these findings identify an important role of IGF-1 as the molecular link between VitD3 treatment and induction of MEndoT in MSCs.

Interestingly, IGF-1 produced in VitD3-treated MSCs exerted additional biological function beyond the induction of MEndoT. Our data show that IGF-1 continued to be secreted by VitD3-treated MSCs, even after VitD3 was removed from the culture medium. This prolonged secretion of IGF-1 by MSCs enhances an angiogenic niche conducive to the formation of new vasculature. Indeed, in our contactless co-culture system, VitD3-treated MSCs were able to induce angiogenesis in both MAECs and murine aortic rings. These observations suggest that following in vivo transplantation, VitD3-treated MSCs may induce angiogenesis through IGF-1-mediated niche formation in recipient tissues.

The precise role of VitD3 in endothelial cell biology and function may vary depending on the context (Jamali et al., 2018). Although an inhibitory influence of VitD3 on angiogenesis has been reported in the setting of malignancy (Mantell et al., 2000), epidemiological studies have identified an adverse association between VitD3 deficiency and incident cardiovascular events (Wang et al., 2008). In this context, the current findings identify two important mechanisms (Figure 8) through which VitD3 may augment vascularization. First, the generation of angiogenic cells from transplanted MSCs through VitD3-induced MEndoT may play a direct role toward increasing vascularity. Our data show an important role of VitD3-induced IGF-1 in this conversion of MSCs into cells that may directly effect angiogenesis. Second, the protracted secretion of IGF-1 by VitD3-treated MSCs may amplify the vasculogenic effects of donor MSCs by enabling the formation of proangiogenic niches at sites of MSC transplantation, with potential recruitment of host cells and additional vascularization. Collectively, these observations support an important role of VitD3 toward the promotion of angiogenesis, and may explain the increased cardiovascular events in patients with VitD3 deficiency. These findings also support the potential efficacy of VitD3-treated MSCs for therapeutic angiogenesis in ischemic tissues.

Figure 8.

Figure 8

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Schematic representation of dual mechanisms underlying VitD3-induced angiogenesis

VitD3 induces IGF-1 in MSCs, which promotes angiogenesis through MEndoT. In addition, VitD3-treated MSCs continue to secrete IGF-1, thereby creating a proangiogenic niche, which further amplifies the angiogenic effects of VitD3.

Limitations of the study

Although epidemiological evidence has identified an association between VitD3 deficiency and adverse cardiovascular events, it is difficult to reproduce the vascular effects of human VitD3 deficiency in isolation in a mouse model. Therefore, we focused on the elucidation of potential mechanisms through which VitD3 may exert vascular benefits. Although our current results identify how VitD3 signaling may influence angiogenesis, additional studies in a model of ischemia and VitD3-treated MSC transplantation in vivo will be necessary in the future.

Resource availability

Lead contact

Further information and request for resources and reagents should be directed to Prof. Buddhadeb Dawn (buddha.dawn@unlv.edu).

Materials availability

Any unique reagent or material will be available from the lead contact upon request.

Data and code availability

The published article includes all data generated or analyzed during this study. This study did not generate any unique code.

Methods

All methods can be found in the accompanying transparent methods supplemental file.

Acknowledgments

This study was supported in part by NIH grant R01 HL-117730.

Author contributions

Conceptualization, L.C. and B.D.; methodology, L.C., M.G., and R.J.V.; formal analysis, L.C. and L.Z.; investigation, L.C., A.S., and L.Z.; writing – original draft, L.C. and N.R.D.; writing – review and editing, L.Z., L.C., T.B., and B.D.; visualization, L.C., L.Z., T.B., and B.D.; supervision, R.J.V., J.H., and B.D.; funding acquisition, B.D.

Declaration of interests

The authors declare no competing interests related to this research. B.D. is one of the founders of BBBAHMS Regen, Inc.

Published: April 23, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102272.

Supplemental information

Document S1. Transparent methods
mmc1.pdf (795.3KB, pdf)

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods
mmc1.pdf (795.3KB, pdf)

Data Availability Statement

The published article includes all data generated or analyzed during this study. This study did not generate any unique code.

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