HIF-1α Overexpression Induces Angiogenesis in Mesenchymal Stem Cells

Abstract

Stem cell therapy continues to be an innovative and promising strategy for heart failure. Stem cell injection alone, however, is hampered by poor cell survival and differentiation. This study was aimed to explore the possibility of improving stem cell therapy through genetic modification of stem cells, in order for them to promote angiogenesis in an auto- and paracrine manner under hypoxic conditions. Hypoxia inducible factor-1α was overexpressed in bone marrow-derived mesenchymal stem cells (MSCs) by stable transduction using a lentiviral vector. Under hypoxic and normoxic conditions, the vascular endothelial growth factor (VEGF) concentration in the cells' supernatant was measured by an enzyme-linked immunosorbent assay. Migration was assayed by wound healing and c-Met expression by flow cytometry. Tube formation was evaluated on a Matrigel basement membrane. The concentration of VEGF was significantly increased in the supernatant of HIF-1α-overexpressing MSCs; this medium was significantly more effective in inducing endothelial cell migration compared to untransduced MSCs. Transduced cells showed increased levels of c-Met expression and were more efficient at tube formation. However, no indication of differentiation toward an endothelial phenotype was observed. This study indicated that genetic modification of MSCs by HIF-1α overexpression has the potential to improve components of the angiogenesis process under a hypoxic condition by paracrine and autocrine mechanisms.

Keywords: HIF-1α; angiogenesis; hypoxia; mesenchymal stem cells.

FIG. 1.

Morphology of mesenchymal stem cells (MSCs) during culture (A) and their expression level of CD31 (1%), CD34 (0.5%), CD105 (92%), and CD73 (80%) (B).

FIG. 2.

Adipogenesis differentiation (A) and osteogenesis differentiation (B) of MSCs; note the vacuoles containing fat and calcium deposition in Oil Red and Alizarin red staining, respectively.

FIG. 3.

Transducted MSCs; note the GFP expression in infected cells. GFP, green fluorescent protein.

FIG. 4.

HIF-1α expression level; lane 1: size marker; lane 2: GAPDH in HIF-MSCs; lane 3: HIF-1α in HIF-MSCs; lane 4: GAPDH in MSCs; lane 5: HIF-1α in MSCs. HIF, hypoxia inducible factor.

FIG. 5.

Tube-formation assay on Matrigel after 24 h hypoxia; (A): MSCs formed aggregations with some sprouting tubes; (B): human umbilical vein endothelial cell (HUVEC) tube formation as endothelial cells; (C): HIF-MSCs, note the organized and interconnected tube formations; (D): most HIF-MSCs expressing GFP contributed in tube formation.

FIG. 6.

Vascular endothelial growth factor concentration in MSC and HIF-MSC media after 24 h hypoxia. Note the significant differences between groups indicated by *p<0.05.

FIG. 7.

Reverse transcriptase-polymerase chain reaction for endothelial markers. (Left) CD34 (152 bp), vWF (196 bp), and Tie2 (171 bp) in HUVECs; (right) HIF-MSCs under hypoxic conditions.

FIG. 8.

Flow cytometry results for c-Met expression; (A): MSCs (1.3%); (B): HIF-MSCs (25.7%).

FIG. 9.

Wound- (region indicated between two red lines, upper panels) healing assay by applying a 24-h conditioned medium of hypoxic MSCs and HIF-MSCs (lower panels) on HUVECs. Note the black arrows showing wound closure only in HIF-MSCs after 20 h, which failed to be closed in MSCs during the same period of culture.