Synergistic effect of angiotensin II on vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells

Abstract

Introduction: Increased levels of angiotensin II (Ang II) and activity of Ang II receptor type 1 (AT1R) elicit detrimental effects in cardiovascular disease. However, the role of Ang II receptor type 2 (AT2R) remains poorly defined. Mesenchymal stem cells (MSCs) replenish and repair endothelial cells in the cardiovascular system. Herein, we investigated a novel role of angiotensin signaling in enhancing vascular endothelial growth factor (VEGF)-A-mediated differentiation of MSCs into endothelial cells (ECs).

Methods: Bone marrow was aspirated from the femurs of Yucatan microswine. MSCs were extracted via ficoll density centrifugation technique and were strongly immunopositive for MSC markers, CD44, CD90, and CD105, but negative for hematopoietic markers, CD14 and CD45. Subsequently, naïve MSCs were differentiated for 10 days in varying concentrations and combinations of VEGF-A, Ang II, and AT1R or AT2R antagonists. Markers specific to ECs were determined by FACS analysis.

Results: AT1R and AT2R expression and cellular localization was demonstrated in MSCs stimulated with VEGF-A and Ang II via quantitative RT-PCR and immunofluorescence, respectively. Differentiation of naïve MSCs in media containing Ang II (2 ng/ml) plus low-dose VEGF-A (2 ng/ml) produced a significantly higher percentage of cells that were positive for expression of EC markers (for example, platelet endothelial cell adhesion molecule, vascular endothelial Cadherin and von Willebrand factor) compared to VEGF-A alone. Ang II alone failed to induce EC marker expression. MSCs differentiated with the combination of Ang II and VEGF-A were capable of forming capillary tubes using an in vitro angiogenesis assay. Induction of EC marker expression was greatly attenuated by co-treatment of Ang II/VEGF-A with the AT2R antagonist PD123319, but not the AT1R antagonist telmisartan.

Conclusions: We report the presence of functional AT2R receptor on porcine bone marrow-derived MSCs, where it positively regulates EC differentiation. These findings have significant implications toward therapeutic approaches based on activation of AT2R, which could be a means to stimulate regeneration of damaged endothelium and prevent vascular thrombosis.

Figure 1

Characterization of bone marrow-derived mesenchymal stem cells. Flow cytometry data revealed that mesenchymal stem cells (MSCs) at passages 3 to 5 stained negatively for CD14 (monocyte marker) and CD45 (hematopoietic marker), but expressed surface markers that are indicative of MSC lineage, including CD44 (hyaluronic acid receptor), CD90 (Thy-1), and CD105 (Endoglin). Isolated MSCs exhibited stem-like properties.

Figure 2

Effect of VEGF-A and angiotensin II treatment on the expression of angiotensin receptors. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured in vascular endothelial growth factor (VEGF-A; 2 ng/ml), angiotensin II (Ang II; 2 ng/ml), or combined VEGF-A/Ang II for 24 hours. Expression of angiotensin receptor AT1R and AT2R mRNA was analyzed via quantitative RT-PCR, and human umbilical vein endothelial cells (HUVECs) were used as positive control (A). Immunostaining demonstrating co-localization of AT1R and AT2R on the cell membrane of BM-MSCs treated with VEGF-A (2 ng/ml) plus Ang II (2 ng/ml). AT1R stained in red co-localized with AT2R labeled in green to produce a yellow merge (B). One image representative of three independent experiments is shown. Data are representative of three independent experiments performed from three different cultures from swine bone marrow. *P <0.05 vs. naïve MSCs. ^# P <0.05 vs. Ang II-treated MSCs, n = 3. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Immunopositivity of endothelial cells differentiated from mesenchymal stem cells in the presence of VEGF-A for PECAM, vWF, and VE-cadherin. Representative grids for von Willebrand factor (vWF) are shown. Low-dose vascular endothelial growth factor (VEGF-A) induced a small increase in immunopositivity for platelet endothelial cell adhesion molecule-1 (PECAM-1), vWF, and vascular endothelial cadherin (VE-cadherin) (A, I). Higher concentrations of VEGF-A (25 to 50 ng/ml) induced a marked increase in expression of endothelial cell (EC) markers (B, C, I). Angiotensin II (Ang II; 2 to 50 ng/ml) had no effect on the expression of EC markers (D, E, F, I). Human umbilical vein endothelial cells (HUVECs) were used as an independent positive control showing ≈ 99% immunopositivity to vWF (G, I). Naïve MSCs were negative for EC marker expression (H, I). Graphical representation of the fluorescence-activated cell sorting (FACS) data (I). Note that HUVECs were excluded from the statistical analyses. *P <0.05 vs. naïve MSCs. ^# P <0.05 vs. VEGF-A (2 ng/ml). ^α P <0.05 VEGF-A (50 ng/ml) vs. VEGF-A (25 ng/ml)-treated MSCs, n = 3. MSC, mesenchymal stem cell.

Figure 4

Synergistic effect of angiotensin II and low-dose VEGF-A to promote mesenchymal stem cell differentiation into endothelial cells. Co-culture of naïve bone marrow-derived mesenchymal stem cells (BM-MSC) with vascular endothelial growth factor (VEGF-A; 2 ng/ml) and angiotensin II (Ang II; 2 ng/ml) resulted in significantly increased expression of platelet endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF), and vascular endothelial cadherin (VE-cadherin) (A). Increasing the dose of Ang II in combination with the low dose of VEGF-A did not result in further increase in EC marker expression (B, C, D). Note that human umbilical vein endothelial cells were excluded from the statistical analyses. *P <0.05 vs. naïve MSCs. ^# P <0.05 vs. VEGF-A (2 ng/ml)-treated MSCs, n = 3.

Figure 5

Cell morphology of differentiated mesenchymal stem cells into endothelial cells. Images taken by inverted microscope after 10 days of stimulation. Control mesenchymal stem cells (MSCs) cultured in growth media retained fibroblastoid morphology (A). MSCs differentiated in high-dose vascular endothelial growth factor (VEGF-A) changed into the cobblestone shape typical of endothelial cells (B); however, low-dose VEGF-A produced less of an effect (C). Combination treatment with low-dose VEGF-A and low-dose angiotensin II (Ang II) also induced cobblestone morphology (D), but Ang II alone had no effect (E). Positive control (human umbilical vein endothelial cells (HUVECs)) demonstrated rigid cobblestone morphology (F). Capillary formation by differentiated MSCs into endothelial cells (ECs) (G, H, I, J, K, L). After 10 days of stimulation, cells were seeded onto ECMatrix gel. Naïve MSCs cultured in control media and incubated on the matrix did not form elliptical structure (G). High-dose VEGF-A was capable of forming characteristic capillary structures associated with EC regeneration (H). MSCs differentiated in low-dose VEGF-A started forming elliptical structures. However, individual cells did not connect with each other (I). Combination treatment with low dose VEGF-A and Ang II resulted in complex mesh-like formation in addition to closed polygonal structure (J). Ang II alone did not induce any mesh-like structure or connections (K). The effect of the combined treatment was more striking compared with HUVEC cells forming capillaries (L). One image representative of three independent experiments performed from three different swine bone marrow samples is shown.

Figure 6

Effect of antagonists for AT1R and AT2R on immunopositivity for endothelial cell markers. Mesenchymal stem cells (MSCs) were pretreated with either angiotensin receptor AT1R or AT2R inhibitor and then cultured with angiotensin II (Ang II) and vascular endothelial growth factor (VEGF-A). PD123319, an AT2R inhibitor, significantly reduced the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF) and vascular endothelial cadherin (VE-cadherin) (B), whereas there was a lesser effect of the AT1R inhibitor telmisartan (A). Bar diagram showing data from six individual experiments (C). Note that human umbilical vein endothelial cells were excluded from the statistical analyses. *P <0.05 vs. VEGF-A (2 ng/ml) plus Ang II (2 ng/ml)-treated MSCs. ^# P <0.05 telmisartan vs. PD123319-treated MSCs, n = 3.

Figure 7

Silencing of endogenous VEGF-A lacks an effect on mesenchymal stem cell differentiation into endothelial cells. Differentiating mesenchymal stem cells (MSCs) were transfected with small interfering RNA (siRNA) directed against vascular endothelial growth factor (VEGF-A) and then examined for endothelial cell (EC) marker expression. Treatment of MSCs with the cocktail of VEGF-A (2 ng/ml) plus angiotensin II (Ang II; 2 ng/ml) for 24 hours induced a synergistic increase in VEGF transcript as compared with VEGF-A alone or Ang II alone (A). VEGF-A mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (A). Standardization experiments demonstrated that 50 nM siRNA directed against VEGF completely abrogated VEGF protein expression (B). Fluorescence-activated cell sorting analysis revealed that there was no significant difference in EC marker expression between differentiating MSCs that received VEGF-A siRNA or scrambled control siRNA (C, D, E). Note that human umbilical vein endothelial cells (HUVECs) were excluded from the statistical analyses. *P <0.05 vs. naïve MSCs. ^# P <0.05 vs. VEGF-A (2 ng/ml)-treated MSCs or Ang II (2 ng/ml)-treated MSCs alone, n = 3.

Figure 8

Summary of synergistic effect of angiotensin II on VEGF-A-mediated differentiation of BM-MSCs into endothelial cells. Ang II, angiotensin II; ATR, angiotensin receptor; BM, MSC, bone-marrow-derived mesenchymal stem cells; PECAM-1, platelet endothelial cell adhesion molecule-1; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.