Endothelial cell culture and lentivirus transduction

Primary cultures of human umbilical vein ECs were obtained from Lonza (#CC-2519) and cultured in complete growth media EGM-2 (Lonza, #CC-3156 and #CC-4176). These cells were transduced at passage 3 with a constitutively active form of R-Ras (R-Ras38V) or insertless control (mock) using pLenti6 lentivirus expression vector (ThermoFisher, K4955-10) to examine the effect of R-Ras signaling in vitro12. R-Ras knockdown was carried out by lentivirus transduction of shRNA to target the RRAS sequence (5′-GGAAATACCAGGAACAAGA-3′). The negative control shRNA, which does not target any known sequence of the human, mouse, rat, or zebrafish origin, was obtained from COSMO BIO co., Ltd. (Tokyo, Japan) and cloned into pSIH-H1-puro vector11. The complementary DNA (cDNA) for an R-Ras mutant (R-Ras38V D64A), which is constitutively active but incapable of activating PI3K36, was constructed by PCR. A GSK-3β mutant (GSK-3β S9A) cDNA was obtained from Addgene (plasmid #49492)50. These cDNAs were subcloned into pLenti6 vector. In vitro transduction was carried out at one multiplicity of infection per cell.

To determine the role of Akt isoforms in mediating the R-Ras effect, EC were first transduced with R-Ras38V, and 48 h later, either Akt isoform was silenced by transfecting 20 nM of Akt1 short interefering RNA (siRNA) (Sigma, SIHK0094), Akt2 siRNA (Sigma, SIHK0097), or non-targeting siRNA controls (Sigma, SIC001, SIC002) using N-TER™ siRNA nanoparticle transfection system (Sigma, N2788). Likewise, Raptor or Rictor siRNA knockdown or GSK-3β S9A lentivirus transduction was carried out at 48 h after the R-Ras38V transduction.

In vitro endothelial sprouting assay

The effect of R-Ras on endothelial morphogenesis was studied in a fibrin gel 3-D culture of ECs51. The R-Ras mutant or shRNA-transduced ECs were coated on cytodex 3 microcarrier beads (GE Healthcare Life Sciences) at 48 h after transduction. EC-coated beads were then resuspended in a 2 mg/ml fibrinogen solution (Sigma-Aldrich) in PBS at a density of 250 beads per ml. 0.5 ml of the EC-coated bead suspension in fibrinogen solution was added to each well of a four-well chamber slide (Nunc® Lab-Tek® II) containing 0.625 U of thrombin (Sigma-Aldrich). After gels have solidified, 0.8 ml of EGM-2 media containing 2 × 105 pericytes (human brain microvascular pericytes, ScienCell, Inc.) as a feeder layer was added onto the gel in each well. The 3-D culture was maintained in the CO 2 incubator at 37 °C for 5–7 days to observe EC sprouting and lumen formation. For counting the number of sprouts, the fibrin gel 3-D culture was fixed and stained with UEA lectin (Vectorlabs, FL-1061). Sprouts were counted at the growing tip of branches.

For Akt silencing in 3-D culture, ECs were transfected with siRNA for either Akt isoform at 48 h after R-Ras38V transduction, and subsequently coated onto microcarrier beads 24 h later. GSK-3β S9A mutant expression in 3-D culture was carried out in a similar time frame after R-Ras38V transduction.

Confocal 3-D reconstruction of endothelial sprouts

Confocal z-stacks of endothelial sprouts in 3-D fibrin culture were captured using Nikon A1R confocal microscope. The images of 20 μm in depth and total 40 steps of z-stacks were taken using the same settings of acquisition for the mock and R-Ras38V-transduced EC sprouts. Three-dimensional reconstruction of the confocal image was generated using Volocity® software (PerkinElmer). Snapshots were taken at 1024 × 1024, 300 dpi at selected angles.

Detergent fractionation of tubulin

To fractionate soluble and insoluble tubulin, cell lysis buffer (0.5% Triton, 85 mM PIPES [pH 6.9], 1 mM EGTA, 1 mM MgCl 2 , 2 M glycerol) was added to cells52. The cell lysates were kept at 4 °C for 3 min, and centrifuged for 20 min at 13 000 r.p.m. at 4 °C to yield a supernatant containing soluble (cytosolic) tubulin and a pellet containing insoluble (cytoskeletal) tubulin52. α-Tubulin in each fraction was determined by western blot. As a control for microtubule stabilization, cells were treated with paclitaxel 5 ng/ml for 30 min in culture prior to cell lysis.

Microtubule disruption by nocodazole

ECs were culture in the fibrin gel 3-D culture system for 5 days to generate endothelial sprouts with lumens. A microtubule disrupting agent, nocodazole (Sigma, M1404), was added at 10 μM into the media, and the 3-D culture was incubated for additional 1.5 h. Bright-field microscope images were obtained before and after nocodazole treatment.

Quantification of lumen size

Bright-field images of endothelial sprouts in 3-D fibrin gel culture were digitally captured with ×20 magnification. The area size of each hallow structure in the endothelial sprouts (lumen) was determined by morphometry analysis. Sprouts from 10 EC-coated beads in two different culture cells were analyzed for each group. Small vacuoles of <5 µm in length were not included in the analysis.

Immunofluorescence

ECs cultured on glass-bottom chamber slides or EC sprouts in 3-D culture were fixed with 2% paraformaldehyde/PBS for 10 min or 30 min, respectively, permeabilized in 0.1% Triton X-100/PBS, and blocked in 10% goat serum/PBS before immunostaining. Cells were stained with α-tubulin (Invitrogen, YOL1/34, diluted 1:500), acetylated-α-tubulin (Lys40) (Cell Signaling Technologies, #5335, diluted 1:250), delta 2-tubulin (Novusbio, NB100-57397, diluted 1:100), detyrosinated α-tubulin (Biolegend, 909503, diluted 1:500), EB1 (Santa Cruz, sc-47704, diluted 1:200), or PODXL (ThermoFisher Scientific, PA5-28116, diluted 1:200) antibody or phalloidin (Invitrogen, A12379, diluted 1:50). ECs and EC sprouts were analyzed by epi-fluorescence (Nikon Eclipse 90i) and confocal (Nikon A1R) microscopy. For quantitative analyses of post-translationally modified and total α-tubulins, the positive area for each immunostaining was determined in each cell by Volocity® software. Total of 15 cells from three different experiments were examined.

Western blotting analyses

Cell lysate was prepared from subconfluent monolayer culture of ECs at 72 h post transduction of R-Ras mutants or control vector. For silencing of an Akt isoform, Raptor, or Rictor, ECs were transfected with siRNA at 48 h after R-Ras38V transduction, and cultured for additional 48 h before cell lysis. The study with GSK-3β S9A transduction was carried out in a similar time frame after R-Ras38V transduction. For PI3K inhibition, ECs were incubated with LY294002 (25 µM) or DMSO (control) for 1 h before cell lysis.

The following antibodies were used for immunoblotting: rabbit anti-phospho-Akt Ser473 (1:1000, Cell Signaling 4060), rabbit anti-Akt (1:1000, Cell Signaling 9272), anti-phospho-Akt1 Ser473 (1:1000, Cell Signaling 9018), rabbit anti-Akt1 (1:1000, Cell Signaling 2938), rabbit anti-phospho-Akt2 Ser474 (1:1000, Cell Signaling 8599), rabbit anti-Akt2 (1:1000, Cell Signaling 3063), rabbit anti-phospho-GSK-3β Ser9 (1:1000, Cell Signaling 9322), rabbit anti-GSK-3β (1:1000, Cell Signaling 12456), rabbit anti-acetylated-α-tubulin Lys40 (1:1000, Cell Signaling 5335), rabbit anti-α-tubulin (1:1000, Cell Signaling 2125), and rabbit anti-GAPDH (1:10,000, Cell Signaling 2118). Full scans of the western blots shown in Figs. 2–4 are provided in Supplementary Figs. 18 and 19.

Immunoprecipitation of phospho-Akt

Cultured ECs were lysed in Triton lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 2 mM CaCl 2 , 150 mM NaCl) containing proteinase inhibitor and phosphatase inhibitor cocktails on ice for 10 min. Cell lysate was incubated with anti-phospho-Akt Ser473 (1:200 dilution) overnight at 4 °C, followed by incubation with 120 µl of protein A magnetic beads (CST #8687) for 2 h at 4 °C. Beads were washed and SDS-PAGE was performed, which was followed by anti-Akt1 or Akt2 western blotting for the detection of phosphorylated Akt isoforms. Lysate of 50 μg (5%) was used to indicate the protein input for immunoprecipitation.

R-Ras activity assay

Pull-down assays were performed to determine R-Ras activity following the manufacturer’s instructions (Cell Signaling, #8821). Briefly, ECs were cultured in low-serum media (2% horse serum without growth factor supplement) for overnight and stimulated with or without 500 ng/ml of recombinant human angiopoietin-1 (R&D Systems) for the indicated times. Cell lysates were prepared and incubated with glutathione resin and GST-Raf1-RBD to pull-down active R-Ras. Western blot analysis of total cell lysate (Input) and the eluted samples (pull-down) were performed using anti-R-Ras antibody (1:2000, Anaspec) and GAPDH antibody (1:10,000, Cell Signaling 2118).

Animals

All animal experiments performed here were approved by the Institutional Animal Care and Use Committee at Sanford Burnham Prebys Medical Discovery Institute. The R-Ras knockout mouse line was generated by Lexicon Genetics. The inactivation of Rras in these mice is caused by an insertion of the gene-trap vector VICTR20 between exons 4 and 5 of Rras on chromosome12. These mice have been backcrossed to wild-type mice with the C57BL/6 genetic background >10 times.

Hindlimb ischemia model

Femoral artery ligation was performed to induce unilateral hindlimb ischemia in 8-week-old male mice53,54. Briefly, left femoral arteries were ligated at two sites at proximal as well as distal to the joint point between profunda and epigastric arteries using polyester 6/0 USP sutures. The contralateral side (right hindlimb) was examined as a normal tissue control.

Assessment of muscle hypoxia and necrosis

The level of hypoxia in the GC muscle was analyzed 2 h after intraperitoneal injection of 60 mg/kg pimonidazole into mice at 7 days post ischemia induction. The hypoxic tissues were stained in paraffin sections using Hypoxyprobe kit (Hypoxyprobe, Inc.). Sections were counter stained with hematoxylin to visualize the total muscle area. Hypoxic area was quantified using HALO™ image analysis platform (Indica Labs). The thresholds were set empirically for identifying the area with strong, medium, or weak staining of hypoxic tissues. The area of each level of staining was presented as percent of such area in the total muscle tissue area examined.

For the analysis of muscle degeneration, 5 µm-thick cross-sections were obtained from mid-portion of GC muscles and stained with hematoxylin and eosin. In addition, triphenyltetrazolium chloride (TTC) staining was used to assess the muscle viability55. Mice were killed at 14 days after femoral artery ligation, and the GC muscles were collected and cut into 2-mm slices. These slices were incubated for 30 min in 2% TTC (Sigma) solution and fixed for 30 min in 10% (vol/vol) buffered formaldehyde. Viable tissues are stained in red and infarct areas are unstained (pale yellow/white).

Immunostaining of GC muscles

Ischemic and contralateral GC muscles were collected from mouse hindlimbs, fixed in 2% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 5 μm thickness. After deparaffinization, rehydration, and antigen retrieval (DAKO), sections were blocked and incubated overnight with primary antibodies for CD31 (BD Biosciences) and R-Ras (AbCam). Sections were then washed in PBS with 0.1% Triton X-100 and incubated with Alexa Flour-conjugated secondary antibodies for 1 h at room temperature for fluorescence microscopy. For immunohistochemistry of R-Ras, sections were stained with anti-R-Ras polyclonal antibodies AR43 (gift from Dr. J. Reed) and peroxidase-conjugated secondary antibody (Vector) followed by staining with AEC kit (Life Technologies). For immunofluorescence analyses of frozen sections, GC muscles were snap-frozen in liquid nitrogen. Muscle cross-sections of 10 or 50 μm-thick longitudinal sections were washed in PBS and incubated overnight at 4 °C with primary antibodies diluted in the antibody dilution solution. Sections were then washed in PBS with 0.1% Triton X-100 and incubated with Alexa Flour-conjugated secondary antibodies for 1 h at room temperature. To assess muscle recovery, immunofluorescence was performed with anti-dystrophin antibody (1:200, EMD Millipore) in cryosections of GC muscles, and images were digitally captured (Eclipse 90i, Nikon). The number of dystrophin+ muscle fibers and total muscle fibers in the field (×20 magnification) were manually counted. The muscle recovery was assessed by the dystrophin+ fibers/total fibers (%) ratio determined in non-necrotic area.

Immunostaining of whole-mounted muscle bundles

GC muscles were fixed in 2% paraformaldehyde for 1 h at 4 °C and washed three times for 10 min each with PBST (0.2% Triton X-100 in PBS). A small bundle of muscle was carefully dissected by fine forceps from the center portion of the GC muscle. The muscle tissues were then blocked with 1% BSA/PBST (5% normal goat serum) for 1 h at room temperature. The tissues were incubated with rocking overnight at 4 °C with anti-CD31 antibody (1:100 dilution, BD Biosciences, 550274). Next day, tissues were washed overnight in PBS at 4 °C, followed by anti-rat IgG-Alexa 647 conjugate (1:500) at 4 °C for overnight. Immunofluorescence images were acquired using Nikon A1R laser scanning confocal microscope or Nikon 90i fluorescence microscopy.

BrdU incorporation assay

BrdU (50 mg/kg) was injected i.p. at 13 days after femoral artery ligation, and the GC muscles were harvested 24 h later. The muscle tissues were snap-frozen in liquid nitrogen. For staining BrdU-labeled cells, frozen sections were incubated with 5 μg/ml proteinase K in PBST for 10 min at room temperature. Sections were then fixed with 1% formaldehyde for 10 min and rinsed in DNase I buffer (50 mM Tris-HCl, 10 mM MgCl 2 , pH 7.5) for 20 min. The sections were incubated with RNase-free DNase I (0.1 U/μl) in DNase I buffer for 2 h. DNase I was heat-inactivated by incubation at 70 °C for 10 min in preheated 50 mM Tris-HCl, pH 7.5. After blocking nonspecific binding with blocking buffer (10% normal goat serum, 0.2% BSA, 0.3% PBST), the sections were incubated overnight at room temperature with mouse anti-BrdU monoclonal antibody (1:500, ThermoFisher Scientific, MA3-071) and rat anti-mouse CD31 antibody (1:200, BD Biosciences, 550274), followed by incubation with fluorescently labeled secondary antibodies. BrdU and CD31 staining in GC muscles was analyzed by fluorescent microscopy. The percentage of mitotic ECs was determined by: number of BrdU+ nuclei within CD31+ area/total number of nuclei within CD31+ area × 100.

Vascular area and density

To assess reparative angiogenesis, a subset of animals (five animals per group) was killed and GC muscles collected at 7 or 14 days after arterial ligation. Cryosections of the GC muscles were incubated with the anti-CD31 antibody (1:200, BD Biosciences, 550274) overnight at 4 °C followed by Alexa Fluor 555 anti-rat immunoglobulin G (1:500; ThermoFisher) for 1 h and analyzed by fluorescence microscopy. The number of CD31+ objects per field (vessel density) and CD31+ area per field (vascular area) were determined in five random fields for each slide using Volocity® image software (PerkinElmer). All data are presented as mean ± SEM.

Transmission electron microscopy

GC muscles were collected at 14 days after arterial ligation and cut into small pieces (1–3 mm3), fixed in Karnovsky’s fixative (Electron Microscopy Sciences) for 4 h, and washed with PBS two times. Thin sections of GC muscles were prepared and stained with 2% uranyl acetate for 30 s and applied to a continuous carbon grid. The sections were photographed using FEI Morgagni Transmission Electron Microscope system (FEI Company).

Laser Doppler imaging

Peripheral blood flow in the mouse foot was analyzed by laser Doppler imaging (PeriScan PIM 3 System, PERIMED). Post-surgical scans were performed immediately after the arterial ligation to confirm the interruption of blood flow in lower limb. Mice were scanned at days 1, 3, 5, 7, and 14. Tissue perfusion was quantified in region of interest defined in the ischemic limb relative to the contralateral, non-ligated side and was displayed as color-coded images. Flow images were then analyzed with blood perfusion imaging software (PIMSoft) and reported as the ratio of flow in the ischemic to non-ischemic hindlimb. Seventeen mice were studied for each group.

Lectin perfusion

To histologically visualize the perfusion of newly formed intramuscular capillaries and microvessels, mice were intravenously injected with biotin-labeled tomato lectin (Vector) at 14 days post ischemia induction. Mice were killed 5 min later and GC muscles were isolated. A small piece (100 μm thickness) from the center portion of the GC muscle was carefully dissected and fixed in 2% paraformaldehyde. Small bundles of GC muscles were doubly stained in whole-mount for CD31 and biotinylated lectin to visualize all vessels (perfused and non-perfused) and perfused vessels, respectively. The images of 10 random areas in each specimen were captured, five mice per group. The perfusion efficiency was calculated as a ratio of perfused vessel area (CD31/lectin double positive area) to total vascular area (CD31 positive area) by Volocity® image software (PerkinElmer).

Endothelial cell-targeted in vivo gene transfer

A lentiviral expression vector, pLenti6/Cdh5-R-Ras38V, was generated for EC-specific expression of R-Ras38V in mouse tissues. This construct was generated by replacing CMV promoter in the original pLenti6 vector with the mouse VE-cadherin (Cdh5) promoter sequence35. A similar expression vector was constructed for R-Ras38V D64A mutant expression. A lentiviral vector with Cdh5 promoter without cDNA insert was also generated as a control.

For R-Ras KO rescue experiments, hindlimb ischemia was induced in the R-Ras KO mice, and 3 days later, mice were randomly assigned into two groups. The lentivirus harboring the pLenti6/Cdh5-R-Ras38V expression vector or control vector was intramuscularly injected into the GC muscle at three injection sites, 10 µl each, 1.2 × 107 TU total using a Hamilton syringe. At day 14, mice were intravenously perfused with biotinylated tomato lectin through the tail vein 5 min before resecting the GC muscles for analysis. Five mice per group were examined. To confirm the induction of R-Ras38V expression, total tissue RNA was isolated from the GC muscle using RNeasy Mini Kit (Qiagen, 74134). cDNA was synthesized using a high capacity cDNA reverse transcription Kit (Applied Biosystems, 4368814). Reverse transcription PCR (RT-PCR) was carried out using a primer set for human RRAS: 5′-GACCCCACTATTGAGGACTCC-3′ and 5′-CTACAGGAGGACGCAGGG-3′. A primer set for mouse Ppia (Integrated DNA Technologies) was used to standardize RT-PCR.

Akt inhibitor treatment

A set of mice that received pLenti6/Cdh5-R-Ras38V lentivirus injection into GC muscles were treated with an Akt inhibitor MK-2206. MK-2206 (Apexbio Technology LLC) was dissolved in 30% captisol® (Ligand Pharmaceuticals, Inc.) and administered p.o. at 120 mg/kg by gavage every 2 days37 starting from 1 day after the lentivirus injection (day 4 post femoral artery ligation) for total five times. At day 14, 100 µl of 1 mg/ml tomato lectin was injected i.v. for vessel perfusion analysis, and mice were killed to collect GC muscles. A control group received vehicle alone via gavage. Three animals were examined per group.

Endothelial cell isolation from GC muscle

GC muscles were resected at 14 days post ischemia induction and dissociated into single cell suspension by enzymatic digestion with collagenase II (500 U/ml, Sigma #6885), collagenase D (1.5 U/ml, Roche #11088882001), and dispase II (2.4 U/ml, Roche, #04942078001). Endothelial cells were sorted from the cell suspension using anti-mouse CD31 MACS magnetic microbeads and MS column (Miltenyi Biotec). Endothelial cell purity was assessed by VE-Cadherin expression and LDL uptake assay (Biomedical Technologies Inc., BT-902). The isolated cells were lysed and used for western blot analyses to determine R-Ras expression, Akt and GSK-3β phosphorylation, and tubulin acetylation.

Adenovirus-mediated VEGF gene therapy

Adenovirus carrying mouse VEGF-A 164 (Cell Biolabs Inc.) was injected at 3 × 108 p.f.u. (1 × 108 p.f.u./10 μl × 3 injections) into the calf (GC) muscles of wild-type mice immediately after femoral artery ligation. The GC muscles were collected for immunofluorescence analyses 11 days later. Frozen sections of 50 μm were used for lectin perfusion assay and PODXL staining. Frozen sections of 10 μm were used to detect R-Ras expression.

Statistical analysis

Statistical analyses were performed using the two-tailed Student’s t test to compare two experimental groups and the one-way analysis of variance with Tukey’s multiple comparison test for multiple experimental groups. Calculations were performed using GraphPad Prism v7.00 software. P < 0.05 was considered significant. Error bars represent the SEM. Data on the graphs are presented as mean ± SEM.

Data availability

All data relevant to this study are presented in the manuscript’s main figures or the Supplementary files and available from the authors upon request.