1. Activation of Ras in the Vascular Endothelium Induces Brain Vascular Malformations and Hemorrhagic Stroke 
Qing-fen Li, Brandee Decker-Rockefeller, Anshika Bajaj, Kevin Pumiglia 
Cell Reports 
Volume 24, Issue 11, Pages (September 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 2869-2882DOI: (10.1016/j.celrep.2018.08.025)
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3. Figure 1 Generation of Endothelial-Specific, Inducible HRasV12 Mice
(A) Breeding strategy for iEC-HRasV12 double-transgenic mouse (Cdh5:tTAx tetOp:HrasV12).
(B) Western blots of lung endothelial lysates from control (tTA) and iEC-HRasV12 mice (heterozygous genotype [h]) showing the levels of total Ras (Pan-Ras) and changes in the HRAS isoform. n = 4. Error bars represent mean ± SEM. N.S., no statistically significant differences.
(C) Purified endothelial cells from control (tTA) and iEC-HRasV12 mice were used to analyze Ras activation. OD490 is indicative of the level of activated Ras. Quantification data are mean ± SEM. n = 4. ∗∗∗p ≤
(D) Lysates from control and iEC-HRasV12 (h genotype) lung endothelial cells were analyzed for activation of signaling. Total ERK2 protein levels were used as loading control for all three phospho-proteins. Quantification data represented as mean ± SEM. n = 4. ∗p ≤ 0.05.
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4. Figure 2
Activation of Ras in the Endothelium Results in Rapid Lethality with Associated Cerebrovascular Malformations
(A) Control mice and iEC-HRasV12 mice of various genotypes (heterozygous [h], intermediate [I], and homozygous [H]) had doxycycline removed from their diet. Mice were monitored and survival plotted as a Kaplan-Meier curve. p ≤ n = 15.
(B) Gross dissection images of brains from control or iEC-HRasV12 mice. White boxes indicate areas imaged at higher power in the lower panel. Asterisk shows examples of micro-hemorrhage, while arrowheads indicate areas of enlarged, dilated vessels.
(C) H&E staining of cerebellum (top) and immunohistochemistry (bottom) using anti-collagen IV in the cerebrum show vascular malformation in iEC-HRasV12 brain. Green arrows indicate hemorrhagic spots, and black arrows highlight the presence of intense vascular staining, showing an increase in both diameter and number of vessels.
(D) Immunofluorescence co-staining was used to evaluate vascular structures (collagen IV) and the presence of proliferating cells (Ki-67), blood-brain barrier disruption (MECA-32 antibody), and inflammatory cells (CD11b). White arrows highlight staining associated with abnormally dilated vessels in the iEC-HRasV12 mice cerebrum (inset is a higher magnification view of highlighted area).
(E) Markers were quantified by measuring total target molecules signal intensity in a field and normalizing to the signal intensity of collagen IV (or CD31) in the same field. Data (mean ± SEM) were derived from ten randomly selected fields from four mice per genotype. ∗∗∗p <
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5. Figure 3
Induced Activation of HRAS in Endothelial Cells Results in Abnormal Vascular Morphogenesis
(A) Quantification of primary mouse brain endothelial cells grown in culture from three individual experiments and represented as mean ± SEM. ∗∗∗p <
(B) Representative phase-contrast images (top) and immunofluorescence staining show beads of Slik-HRASV12 HUVECs ± doxycycline (100 ng/mL) in a 3D fibrin gel angiogenesis assay at day 7. Middle: CD31 (green); bottom: CD31 (red), Ki-67 (green).
(C) The lengths of VLSs (vascular-like structures) and area of endothelial cells were quantified, and the number of VLSs around the beads was counted manually. Data (mean ± SEM) are derived from 11 randomly selected beads from three individual experiments. ∗∗∗p <
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6. Figure 4
PI-3′ Kinase Inhibition Reverses Abnormal Vascular Morphogenesis Mediated by HRASV12 In Vitro but Not In Vivo
(A) Representative phase-contrast images show beads of control and HRASV12-expressing human endothelial cells ± BYL719 (5 μM) or PD (2 μM) in in vitro 3D fibrin bead assay at day 7.
(B) The number of VLSs, lengths of VLSs, and area of the endothelial cell per beads were quantified, and data (mean ± SEM) are derived from 12 randomly selected beads from three individual experiments. ∗∗p < 0.01.
(C) Representative bead angiogenesis assays for control and PIK3CAH1047R-expressing HUVECs. Images correspond to day 12 and were stained with CD31 (green) and DAPI (blue).
(D) Representative images show cerebral vasculature (collagen IV; green) on control and iEC-HRasV12 (intermediate genotype breeding TA X iEC-HRasV12) ± 25 mg/kg BYL719 for 12 days after removal doxycycline from the diet. The white arrows highlight abnormal vasculature.
(E) The diameters of abnormal vessels were quantified. Data (mean ± SEM) are derived from six mice per genotype. ∗∗∗p <
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7. Figure 5
HRAS Inhibition of TGF-β Signaling Contributes to Abnormal Vascular Morphogenesis
(A) BrdU incorporation of HRASV12 HUVECs and control cells to the inhibitory effects of TGF-β1. Data (mean ± SEM) are derived from three individual experiments, normalized to untreated control. ∗∗∗p <
(B) Western blots of lysates from doxycycline-induced HRASV12 HUVECs ± TGF-β1 (0.1 ng/mL) or BMP9 (0.1 ng/mL) showing analysis of pSMAD2/3, pSMAD1/5, and total SMAD2/3, SMAD1/5 (loading controls) in HUVECs.
(C) Quantification of western blots as described in (B). Data represent mean ± SEM. n = 4. ∗∗p < 0.01.
(D) Representative bright-field images (top) and immunofluorescence with anti-CD31 (green)/anti-Ki-67 (red) beads from bead angiogenesis assay of HRASV12 HUVECs ± 0.2 ng/mL TGF-β1, captured on day 7. Arrowheads indicate sheets; arrows point to the tubular structure.
(E) Quantification of the number and lengths of VLSs and area of endothelial cells around each bead are represented as mean ± SEM. n = 8. ∗∗p < 0.01 and ∗∗∗p < 0.001.
(F) Representative bead angiogenesis assays for HUVECs ± 0.5 μΜ SB Images correspond to d12 and were stained with CD31 (green), Ki-67 (red), and DAPI (blue).
(G) The number and lengths of VLSs, and the area of endothelial cells around each bead, were quantified and represented as mean ± SEM. n = 8. ∗∗∗p <
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8. Figure 6
HRas Alters Tip or Stalk Cell Specification and Gene Expression
(A) Diagram of hypothesis that activation of HRAS disrupts balance of tip or stalk cell gene expression, disrupting normal patterning programs and lumenogenesis and sustaining proliferation.
(B and C) Representative bead angiogenesis assays for control and HRASV12 HUVECs at different time points (B). Green arrowhead highlight protrusions. (C) Quantification of protrusion number per bead represented as mean ± SEM (n = 8). One-way ANOVA found that HRASV12 was different from control (as well as the previous day) on day 1 (D1) and D3, but not D7.
(D) Heatmap showing changes in the average arbitrary gene copy (n = 6) of tip or stalk genes in control and HRASV12 HUVECs at various points as measured from RNA isolated from co-culture bead angiogenesis assays.
(E and F) Quantification of angiogenesis gene expression in HRASV12 expressing HUVECs ± LY (5 μM) (E) or ± TGF-β1 (0.2 ng/mL) (F). RNA was isolated from bead angiogenesis assays on day 6. Values are normalized to control, as represented by dotted line. Data (mean ± SEM) are derived from four individual experiments. HRASV12 versus HRASV12 + LY or TGF-β1 (∗p < 0.05 and ∗∗p < 0.01).
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9. Figure 7
HRASV12 Alters Tip or Stalk Gene Expression and Function In Vivo
(A) Analysis of gene expression levels in control and iEC-HRasV12 mouse brain endothelial cells by qRT-PCR. Data (mean ± SEM) are derived from four individual experiments, two or three mice per group. ∗p < 0.05 and ∗∗p < 0.01.
(B and C) Representative images of IB4 staining of whole-mounted retinas of control and iEC-HRasV12 mice at post-natal day 5 (B). White arrow highlights area of vascular fusion, and red asterisks highlight tip cells.
(C) Quantification data are represented as mean ± SEM. n = 12. ∗∗∗p <
(D and E) Immunofluorescence images (D) show PDGFRβ (red) and CD31 (green) in control and iEC-HRasV12 mouse retina in the emerging tips and stalks.
(E) The fluorescence intensity of PDGFRβ and CD31 in the same field was quantified and represented as mean ± SEM of measurements made on six independent fields from at least two separate mice. ∗∗p < 0.05.
Cell Reports  , DOI: ( /j.celrep )
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