Genetic ablation of Rac1 in endothelial cells does not impair tumor growth or tumor angiogenesis.

<p><b>A.</b> Western blot analysis of Rac1 flox/flox PDGFB-iCreER OHT-treated primary endothelial cell extracts showed that Rac1 was barely detectable (lane 3) when compared with Rac1 flox/flox OHT-treated (lane 1) or Rac1 flox/flox PDGFB-iCreER vehicle treated (lane 2) endothelial cell extracts (left panel). Western blots of extracts from non-endothelial cells, treated as described above, showed no differences in Rac1 expression (right panel). Hsc-70 provided loading controls. Bar graphs represent densitometric values relative to Hsc-70. N = 2 independent experiments. <b>B.</b> B16F0 tumor volume and angiogenesis (blood vessel density) from tumors grown in OHT-treated Rac1 flox/flox (1, white), or placebo- (2, grey) or OHT- (3, black) treated Rac1 flox/flox PDGFB-iCreER mice. Left bar graph shows mean tumor volume in mm³ (+ s.e.m) for 10-day-old tumors. No significant differences in tumor size were observed between groups. N = 6–7 animals per group. Right bar graph shows mean number of PECAM-1 positive vessels per tumor area per mm² (+ s.e.m.) for 10-day-old tumors. No significant differences in microvessel density were observed between groups. N = 4 animals per group. <b>C.</b> B16F0 tumor volume and angiogenesis (blood vessel density) from tumors grown in OHT-treated Rac1 flox/flox PDGFB-iCreER animals. Tumors were injected at days 5 and 10 (after initial inoculation) with pSico-Con (1, white) or pSico- β3 (2, black) and harvested at day 14. Left bar graph shows mean tumor volume in mm³ (+ s.e.m). <i>P</i><0.05. N = 5 animals per group. Right bar graph shows mean number of endomucin positive vessels per tumor area per mm² (+ s.e.m.). <i>P</i><0.001. N = 5 animals per group.</p

Endothelial-specific Rac1-depletion does not impair tumor growth wild-type mice but does in β3-null mice.

<p><b>A.</b> Murine B16F0 cells (10⁶) were injected subcutaneously into the flanks of wild-type/Tie1-Cre⁺ or β3-null/Tie1-Cre⁺ mice. Lentiviral vector suspensions (10⁶ i.u/ml) of pSico-Con, pSico-Rac1 and pSico-Flk-1 were injected intratumorally on days 5 and 10 after tumor cell injection. Representative macroscopic appearance of 14-day-old B16F0 pSico-Con-, pSico-Rac1- and pSico-Flk-1- treated melanomas in both genotypes. Scale bar: 5 mm. Bar graph shows mean tumor volume per mm³ (+ s.e.m.). Tumor size was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P<0.05) and in pSico-Rac1 treated β3-null/Tie1-Cre⁺ but not in pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 4–6 animals per condition. <b>B.</b> Representative merged images of PECAM-1 (red) and GFP (green) -immunostained sections from pSico-Rac1-treated B16F0 tumors grown in wild-type, wild-type/Tie1-Cre⁺, β3-null and β3-null/Tie1-Cre⁺ mice. PECAM-1-positive staining identified endothelium. GFP-positive staining was observed in tumor cells (concave arrowheads) and in PECAM+ endothelium (arrows) of blood vessels in B16F0 tumors from wild-type and β3-null control mice, indicating successful pSico-Rac1 lentivirus infection <i>in vivo</i>. Loss of GFP detection in most PECAM-1-positive microvessels (small arrowheads), but not in B16F0 tumor cells, was observed in pSico-treated tumors grown in wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice, indicating successful endothelial-specific Cre recombination <i>in vivo</i>. Scale bar: 10 µm. Bar graph shows mean numbers of PECAM-1⁺/GFP⁻ vessels per unit area of tumor section per mm² (+ s.e.m). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice (*P<0.05) and in pSico-Rac1-treated β3-null/Tie1-Cre⁺ mice but not pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 4 animals per condition.</p

VEGF-mediated angiogenesis is dependent on endothelial-Rac1 expression in β3-null mice but not wild-type mice.

<p><b>A.</b> Representative images of 14-day-old VEGF-impregnated sponges in wild-type/Tie1-Cre⁺ and β3-null/Tie1-Cre⁺ mice after treatment with pSico-Con, pSico-Rac1 and pSico-Flk-1. Endomucin-positive staining (blue) identified microvessels. Scale bar: 50 µm. Bar graph represents mean number of microvessels per area of sponge (+ s.e.m.). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P<0.05), and in pSico-Rac1-treated β3-null/Tie1-Cre⁺ mice, but not pSico-Rac1-treated wild-type/Tie1-Cre⁺ mice (<i>n.s.d</i>, no significant differences). N = 6 sponges per group. <b>B.</b> Subcutaneous sponges implanted into wild-type mice were injected with either PBS or VEGF in the presence (Rac1 siRNA) or absence (Con siRNA) of Rac1-specific siRNA. Bar graph shows mean number of laminin-positive vessels per area of sponge per mm² (+ s.e.m). Blood vessel density was increased significantly in VEGF alone-treated (*P<0.01) when compared with PBS controls. Rac1 siRNA-treatment had no significant effect (<i>n.s.d</i>, no significant differences). N = 6–10 sponges per group. <b>C.</b> Semi-quantitative RT-PCR (left) and Western blot (right) analyzes from wild-type and β3-null aortic explants transfected with either Con or Rac1 specific-siRNA. Rac1 siRNA reduced significantly the expression of both Rac1 mRNA (*P<0.01) and protein (*P<0.001) levels. RT-PCR for Αctin and Western blotting for Hsc-70 provided mRNA and protein loading controls, respectively. Bar graphs represent mean Rac1 mRNA and Rac1 protein levels (+ s.e.m.). N = 3 independent experiments. <b>D. </b><i>Ex vivo</i> mouse aortic ring assays. Representative high-power light micrographs of VEGF-mediated microvessel sprouting from wild-type and β3-null mouse aortic rings treated with Con or Rac1-specific siRNA and Con siRNA plus DC101. Bar graphs represent quantification of microvessel numbers from 5-day-old VEGF-stimulated aortic ring cultures transfected with Mock, Con or Rac1 siRNA in the presence or absence of DC101. Sprouting angiogenesis was reduced significantly after DC101 treatment of aortas and in Rac1-specific siRNA transfected β3-null, but not wild-type, aortas. Bar graph represents mean number microvessel sprouts/aortic ring (+ s.e.m.).*P<0.01; <i>n.s.d</i>, no significant differences. N = 3-5 independent experiments.</p

Increased active levels of Rac1 in β3-integrin-deficient endothelial cells.

<p><b>A.</b> Active levels of Rac1 were examined by GST-PAK pull-downs. Western blot analysis of active Rac1 bound to GST-PAK (Rac1-GTP) and total Rac1 from wild-type, β3-integrin null, and β3-null primary lung endothelial cells transduced with human β3-integrin (rescue). Immunoblots were quantified by densitometry, and the levels of GTP-bound Rac1 normalised to total Rac1 levels. Active levels of Rac1 were increased approximately 3 fold in β3-null endothelial cells when compared with wild-type controls (*P<0.01), however, total levels of Rac1 were expressed equally in both genotypes. Furthermore, active levels of Rac1 were reduced to wild-type levels in rescue cells (*P<0.01). Results shown are the means + s.e.m of 3-4 independent experiments. <b>B.</b> Flow-cytometric analysis shows that surface levels of β3-integrin were not detectable in β3-null (dashed line) cells when compared with wild-types (bold line) endothelial cells (left panel). Rescue cells expressed β3-integrin (bold line, right panel). Grey peaks represent isotope IgG controls. Bar graph shows means + s.e.m of relative surface β3-integrin expression in wild-type (white), β3-null (black) and rescue (grey) endothelial cells compared with negative control (*P<0.001, N = 3 independent experiments).</p

Rac1-depletion in endothelial cells does not affect the expression and activity of other Rho-related GTPases.

<p><b>A.</b> Analysis of mRNA expression of Rac isoforms by RT-PCR. RNA was extracted from wild-type and β3-null endothelial cells transfected with Con or Rac1 siRNA. As expected, mRNA expression of Rac1 was significantly reduced in Rac1-depleted cells of both genotypes (*P<0.001). Rac2 and Rac3 isoforms were not detected in either Con- or Rac1-siRNA transfected endothelial cells. Extracts of lung (lu), spleen (sp) and brain (br) act as positive controls for Rac1, Rac2 and Rac3, respectively. Actin mRNA provided the internal control. L: ladder. Bar graphs represent relative mRNA levels of Rac1 after Mock- (white), Con- (grey) and Rac1-siRNA (black) treatment in both genotypes. <b>B.</b> Western blot analyses show that Rac1 expression, as expected, was significantly reduced in Rac1-depleted cells of both genotypes (*P<0.01). In contrast, Cdc42 and RhoA protein expression was not significantly affected in endothelial cells after Rac1-siRNA in both genotypes. In addition, for both wild-type and β3-null endothelial cells active levels of Rac1, Cdc42 and RhoA were examined using GST-PAK pull-down (Rac1 and Cdc42) and G-LISA® (RhoA) assays on Mock (white), Con- (grey) and Rac1-siRNA (black) transfected cells. Bars graphs show mean relative protein (top) and active (bottom) levels (+ s.e.m.) of Rac1, Cdc42, and RhoA in Mock (white), Con- (grey) and Rac1-siRNA (black) treated wild-type and β3-null cells. N = 3 independent experiments.</p

Endothelial Rac1-depletion does not affect the expression of Flk-1.

<p><b>A.</b> Western blot analysis of Flk-1 expression levels in primary wild-type and β3-null endothelial cells transfected with scrambled (Con siRNA) or Rac1 (Rac1 siRNA) siRNAs. Although Flk-1 expression was increased significantly in β3-null endothelial cells when compared with wild-types (*P<0.01), Rac1-depletion did not affect Flk-1 levels (<i>n.s.d</i>) in either genotype. Bar graph represents mean (+ s.e.m.) of Flk-1 expression relative to Hsc-70 loading control (N = 3–4 independent experiments). <b>B.</b> HUVEC were transfected with vector only, a wild-type Rac1 construct (Rac1 WT), or a constitutively active Rac1 construct (Rac1 QL) and seeded on confluent fibroblasts. Tubules were visualized by PECAM1 staining 5 days after seeding. The mean number of branch points (+SEM) is shown in the accompanying bar chart. (n = 12 microscopic fields per condition). Scale bar: 100 µm.</p

Cre-regulated depletion of Rac1.

<p><b>A.</b> Wild-type primary endothelial cells were infected with pSico-Rac1 lentivirus. High-efficiency transduction was achieved as indicated by uniform GFP expression in infected cells (left panel). Cells sorted for GFP positivity and transfected with a Cre-recombinase expression plasmid (pTURBO-Cre) showed a significant loss of GFP (right panel). Scale bar: 10 µm. <b>B.</b> One week after pTURBO-Cre transfection, levels of mRNA, detected by semi-quantitative RT-PCR (upper panels), and protein, detected by Western blotting (lower panels), showed successful Rac1- and Flk-1-depletion in pSico-Rac1 and pSico-Flk-1 infected cells, respectively. Αctin RT-PCR and Western blotting for Hsc-70 were carried out to ensure equal RNA and protein loading, respectively. Bar graphs represent densitometric readouts of mRNA of relative Actin or Hsc-70 protein levels, respectively. *P<0.01. N = 3 independent experiments.</p