Figure 6
Figure 6. VSMC adhesion to VWF is because of a Notch-dependent up-regulation of integrin αvβ3. (A-B) The ability of VSMCs to adhere to a VWF-coated substrate was quantified following 18-hour culture on control (IgG) or Jag1-coated plates (to stimulate Notch signaling). Blocking Abs specific to αvβ3 (A, LM609) and the RGD-binding site of VWF (B, anti-152B) prevented Notch-dependent adhesion to VWF, whereas a blocking Ab specific to β1 integrins (A, anti-P4C10) did not. (C-K) Representative images of in vitro fibrin bead angiogenesis assays in which HUVECs (green) were cultured on microcarrier beads and suspended in a fibrin gel with VSMCs (red). (C) A fibrin gel was fixed and immunostained with a fluorescein-labeled Ulex lectin specific to endothelial cells and with Abs to VWF showed that the in vitro endothelial tubes produced a VWF-rich basement membrane. The addition of a blocking Ab to VWF (anti-152B) significantly reduced copatterning, as quantified by pixel overlap in ImageJ, between VSMCs and endothelial tubes (F-G) compared with controls (D-E). Blocking Notch signaling specifically in VSMCs by siRNA knockdown (J-K) significantly reduced copatterning compared with transfection with All Stars negative control siRNA (H-I). Arrows point to regions magnified, shown in panels E, G, I, and K. (L) VSMC-endothelial copatterning was reduced 66% when VWF blocking Ab was added to culture media and 69% when Notch3 was transfected into VSMCs. (M) Notch3 receptor knockdown in VSMCs was confirmed by Western blot in comparison to VSMCs transfected with All Stars negative control siRNA, as was the resultant decrease in Notch signaling as measured by decreased Hey2 expression, a canonical Notch downstream transcription factor. Molecular weight (MW) of N3 precursor: 280 kDa; MW of truncated N3: 120 kDa. Values represent means ± SEM. IgG control (n = 14); α-152b (n = 17); control siRNA (n = 23), Notch3 siRNA (n = 23); where n is the number of beads analyzed; P = 2.5 × 10−13 (α-152b); P = 8.4 × 10−13 (N3 siRNA). Scale bars: (D,F,H,J) 325 μm; and (C,E,G,I,K) 150 μm.

VSMC adhesion to VWF is because of a Notch-dependent up-regulation of integrin αvβ3. (A-B) The ability of VSMCs to adhere to a VWF-coated substrate was quantified following 18-hour culture on control (IgG) or Jag1-coated plates (to stimulate Notch signaling). Blocking Abs specific to αvβ3 (A, LM609) and the RGD-binding site of VWF (B, anti-152B) prevented Notch-dependent adhesion to VWF, whereas a blocking Ab specific to β1 integrins (A, anti-P4C10) did not. (C-K) Representative images of in vitro fibrin bead angiogenesis assays in which HUVECs (green) were cultured on microcarrier beads and suspended in a fibrin gel with VSMCs (red). (C) A fibrin gel was fixed and immunostained with a fluorescein-labeled Ulex lectin specific to endothelial cells and with Abs to VWF showed that the in vitro endothelial tubes produced a VWF-rich basement membrane. The addition of a blocking Ab to VWF (anti-152B) significantly reduced copatterning, as quantified by pixel overlap in ImageJ, between VSMCs and endothelial tubes (F-G) compared with controls (D-E). Blocking Notch signaling specifically in VSMCs by siRNA knockdown (J-K) significantly reduced copatterning compared with transfection with All Stars negative control siRNA (H-I). Arrows point to regions magnified, shown in panels E, G, I, and K. (L) VSMC-endothelial copatterning was reduced 66% when VWF blocking Ab was added to culture media and 69% when Notch3 was transfected into VSMCs. (M) Notch3 receptor knockdown in VSMCs was confirmed by Western blot in comparison to VSMCs transfected with All Stars negative control siRNA, as was the resultant decrease in Notch signaling as measured by decreased Hey2 expression, a canonical Notch downstream transcription factor. Molecular weight (MW) of N3 precursor: 280 kDa; MW of truncated N3: 120 kDa. Values represent means ± SEM. IgG control (n = 14); α-152b (n = 17); control siRNA (n = 23), Notch3 siRNA (n = 23); where n is the number of beads analyzed; P = 2.5 × 10−13 (α-152b); P = 8.4 × 10−13 (N3 siRNA). Scale bars: (D,F,H,J) 325 μm; and (C,E,G,I,K) 150 μm.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal