A mechanosensitive transcriptional mechanism that controls angiogenesis

Abstract

Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.

Fig. 1. TFII-I and GATA2 control VEGFR2 expression via p190RhoGAP

a) VEGFR2 and TFII-I mRNA levels in cells treated with TFII-I siRNA or lentiviral vectors (virus) relative to control cells (*, p < 0.05; unpaired Student's t-test is used throughout). b) Immunoblots showing VEGFR2, TFII-I, p190RhoGAP, and GAPDH protein levels in cells treated with TFII-I or p190RhoGAP siRNAs, or both. c) Immunofluorescence micrographs showing GATA2 distribution and nuclear DAPI staining in control and p190RhoGAP knockdown cells cultured with 0.3% serum (bar, 5μm). d) VEGFR2 mRNA level in cells transfected with p190RhoGAP siRNA alone or with GATA2 siRNA (*, p<0.01). VEGFR2 promoter activities (e) and mRNA levels (f) in cells transfected with GATA2 or TFII-I siRNA alone or in combination (*, p<0.01; **, p<0.05). g) ChIP analysis showing VEGFR2 promoter co-immunoprecipitating with GATA2 or TFII-I antibodies in cells transfected with TFII-I or GATA2 siRNA (Ct, control; n=3; error bars = s.e.m. throughout).

Fig. 2. Matrix elasticity controls VEGFR2 expression via TFII-I and GATA2

a) Immunofluorescence micrographs showing GATA2 and TFII-I distribution in HMVE cells cultured on the fibronectin-coated gels of different stiffness (Young's moduli of 150 and 4000 Pa) in EBM2 with 0.3% serum (bar, 5μm). b) VEGFR2 mRNA level in HMVE cells cultured on rigid fibronectin-coated glass or the gels of different elasticity (150, 1000 and 4000 Pa; normalized to that in cells on the softest gels; *, p<0.01, **, p<0.05). c) VEGFR2 mRNA levels in HMVE cells on soft gels (150 Pa) transfected with TFII-I or GATA2 siRNA alone, or in combination (normalized to cells on the stiffest gels; *, p<0.01). d) VEGFR2 mRNA levels in HMVE cells on soft gels transfected with p190RhoGAP or GATA2 siRNA alone, or in combination (normalized to cells on stiffest gels; *, p<0.01). Error bars represent s.e.m. of 3 replica experiments.

Fig. 3. Antagonism between GATA2 and TFII-I controls capillary cell migration and tube formation in vitro

a) The motility of HMVE cells transfected with human siRNAs or transduced with lentiviral vectors encoding GATA2 or TFII-I, alone or in combination, was quantitated using the Transwell migration assay (*, p< 0.01). Where indicated, VEGF (10 ng/ml) was added to the lower chamber and SU5416 was added in both chambers. Error bars represent s.e.m. of 3 replica experiments. b) Micrographs showing in vitro tube formation induced by VEGF (10 ng/ml) in HMVE cells transfected with siRNAs or transduced with lentiviral vectors encoding GATA2 or TFII-I, alone or in combination.

Fig. 4. Matrix elasticity controls vessel formation via TFII-I and GATA2 in vivo

Light (H&E-stained) (a,b top), immunofluorescence micrographs (a,b bottom) and vessel densities per high power field (c,d) showing cell infiltration and VEGFR2 expression in Matrigel with different elasticity implanted in mice for 7 days without (a,c) or with TFII-I or Gata2 siRNAs (b,d) (bar, 25 μm). The number of VEGFR2-positive blood vessels was normalized to that in the 700Pa gels in (c) and to that in gels treated with control siRNA in (d) (n=6, mean ± S.E.M., *, p< 0.01).

Fig. 5. TFII-I, GATA2, and p190RhoGAP regulate retinal vessel formation in vivo

a) Confocal fluorescence micrographs showing projected images of vessels stained with Alexa 594-isolectin in the retina of a control mouse eye versus eyes transfected with TFII-I, Gata2, or p190RhoGAP siRNAs (bar = 0.1 mm). b) Confocal fluorescence micrographs showing projected images of vessels stained with Alexa 594-isolectin in the retina of a control mouse eye versus eyes overexpressed TFII-I or GATA2, alone or both in combination (bar = 0.1 mm).