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(Circulation. 2003;107:1532.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Small GTP-Binding Protein Rac Is an Essential Mediator of Vascular Endothelial Growth Factor-Induced Endothelial Fenestrations and Vascular Permeability

Anna Eriksson, PhD^*; Renhai Cao, PhD^*; Joy Roy, MD, PhD; Katerina Tritsaris, PhD; Claes Wahlestedt, MD, PhD; Steen Dissing, PhD; Johan Thyberg, MD, PhD; Yihai Cao, MD, PhD

From the Microbiology and Tumor Biology Center (A.E., R.C., Y.C.), the Center for Genomics and Bioinformatics (C.W.), and the Department of Cell and Molecular Biology (J.T.), Karolinska Institute, Stockholm, Sweden; the Department of Surgical Sciences (J.R.), Karolinska Hospital, Stockholm, Sweden; and the Department of Medical Physiology (K.T., S.D.), The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

Correspondence to Dr Yihai Cao, Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail yihai.cao{at}mtc.ki.se

	Abstract

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Background— Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces both angiogenesis and vascular permeability. Although its angiogenic activity has been well characterized, the signaling pathways of VEGF-induced permeability remain poorly understood.

Methods and Results— Using the mouse corneal micropocket assay, Miles assay, and a combination of cytochemical, electron microscopic, and biochemical assays, we demonstrate that VEGF-induced vascular leakage partly can be separated from its angiogenic activity. VEGF but not FGF-2 induced capillaries with a highly fenestrated endothelium, a feature linked with increased vascular permeability. A cell-permeable Rac antagonist (TAT-RacN17) converted VEGF-induced, leaky vascular plexuses into well-defined vascular networks. In addition, this Rac mutant blocked formation of VEGF-induced endothelial fenestrations and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase C (PLC), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling.

Conclusions— Our findings indicate that the small GTP-binding protein Rac is a key component in mediation of VEGF-induced vascular permeability but less so in neovascularization. This may have conceptual implications for applying Rac antagonists in treatment and prevention of VEGF-induced vascular leakage and edema in connection with ischemic disorders.

Key Words: vasculature • receptors • endothelium • ischemia • proteins

	Introduction

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Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is the prototype of a family of growth factors, including at least four other structurally related proteins, placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D.^1–4 The VEGF family members are important regulators of angiogenesis and vascular permeability. Their functions are mediated by activation of three structurally homologous tyrosine kinase receptors, VEGFR-1, VEGFR-2, and VEGFR-3.⁵ VEGF and PlGF-2 also bind to a nontyrosine kinase receptor, neuropilin-1,^6,7 the biological signals of which are not known. Despite their structural similarities, the members of the VEGF family have distinct yet overlapping functions in regulation of vasculogenesis and angiogenesis. For example, VEGF induces vasculogenesis, angiogenesis, and vascular permeability; PlGF and VEGF-B do not stimulate angiogenesis or vascular permeability per se but modulate VEGF-induced angiogenesis and vascular permeability in positive and negative manners.^8,9 VEGF-C and VEGF-D induce both angiogenesis and lymphangiogenesis.^5,10,11 Accumulating evidence indicates that in response to VEGF, VEGFR-2 mediates signals for blood vessel growth and vascular permeability, and VEGFR-3 transduces signals for lymphatic vessel growth.^12,13 The function of VEGFR-1 is poorly understood.

VEGF was initially characterized as a vascular permeability factor secreted by tumor cells.¹⁴ Since the tumor vasculature is disorganized, fenestrated, dilated, and hemorrhagic, it has been suggested that VEGF is responsible for induction of vascular leakage in tumors.^1,3,14,15 Persistent expression of VEGF, VEGFR-1, and VEGFR-2 is also noted in tissues with fenestrated endothelium, such as the choroid plexus and kidney glomeruli.¹⁶ It has therefore been speculated that continuous production of VEGF is involved in induction and maintenance of physiological and pathological endothelial fenestrations. Although these studies provide compelling evidence that VEGF is involved in formation of vascular fenestrations, both tumors and healthy tissues may express other factors involved in this process. Thus, pure in vivo and in vitro systems are required to characterize the molecular mechanisms by which VEGF induces fenestrations. In the present study, the mouse corneal angiogenesis assay, Miles assay, and endothelial cell cultures were used to explore this topic. Our results demonstrate that the GTP-binding protein Rac is a basic component in the VEGF-induced signal transduction system leading to formation of endothelial cell fenestrations and increased vascular permeability.

	Methods

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Reagents, Cells, and Animals
Recombinant human VEGF₁₆₅ was prepared as described.¹⁷ Recombinant human FGF-2 was obtained from Pharmacia and Upjohn (Stockholm, Sweden). Stable PAE cell lines expressing VEGFR-2 (PAE/VEGFR-2 cells) were provided by Dr Lena Claesson-Welsh (Uppsala University, Uppsala, Sweden). The GST-Pak-CD (PAK-CRIB domain) construct was a gift from Dr John Collard (Netherlands Cancer Institute, Amsterdam, Netherlands). Plasmids encoding TAT-RacN17 and TAT-ß-galactosidase were kindly provided by Dr Stephen Dowdy (Washington University, St Louis, Mo). Male 5- to 6-week-old C57BL/6 mice were acclimated and caged in groups of four. Female 6-week-old BALB/c mice were individually caged. Animals were anesthetized with Hypnorm:Dormicum:H₂O (1:1:2) IP and killed with CO₂. All animal studies were reviewed and approved by the animal care and use committee of the Stockholm Animal Board.

Mouse Corneal Angiogenesis Assay
The mouse corneal angiogenesis assay was performed as described.^18,19 Corneal micropockets were created with a modified von Graefe cataract knife in eyes of male 5- to 6-week-old C57BL/6 mice. A micropellet (0.35x0.35 mm) of sucrose and aluminum sulfate (Bukh Meditec) coated with hydron polymer type NCC containing 160 ng VEGF or 80 ng FGF-2 was implanted into each pocket. In Rac-neutralizing experiments, 80 ng VEGF with or without 240 ng TAT-RacN17 was used. The pellet was positioned 1.0 to 1.2 mm from the corneal limbus. The eyes were examined by a slit-lamp biomicroscope on day 5 or 6 after pellet implantation. Vessel length and clock hours of circumferential neovascularization were measured.

Electron Microscopy
Six days after implantation of VEGF, FGF-2, or VEGF/TAT-RacN17 pellets into corneas, the animals were killed and the eyes were removed and immersed in 3% glutaraldehyde in 100 mmol/L sodium cacodylate-HCl buffer (pH 7.3) with 50 mmol/L sucrose. Cornea parts containing new blood vessels were dissected out, cut into small pieces, and put in fresh fixative. After rinsing in buffer, the specimens were postfixed in 1.5% osmium tetroxide in 100 mmol/L cacodylate buffer (pH 7.3) with 0.7% potassium ferrocyanate 2 hours at 4°C, dehydrated in graded ethanol, stained with 2% uranyl acetate in ethanol, and embedded in low-viscosity epoxy resin. Thin sections were cut perpendicular to the surface of the cornea with diamond knifes on a Leica Ultracut, picked up on grids, stained with lead citrate, and examined in a Philips CM120 electron microscope at 80 kV. For quantitative evaluation, segments of individual capillaries with a wall thickness <0.5 µm were photographed at a final magnification of x50 000 (one cornea from 3 to 4 animals per group). The number of endothelial fenestrations (per unit cell section length), the number of caveolae (both on the luminal and abluminal sides), and the minimal thickness of the endothelium within these segments were determined. In addition, the number of ferritin particles in a 1.0-µm-wide zone outside the endothelial cells were counted under a magnifier.

Purification of TAT-RacN17
Recombinant TAT-RacN17 protein was purified with a denaturing His-tag affinity purification protocol as described.²⁰ Briefly, Escherichia coli cells expressing TAT-RacN17 were induced with 0.1 mmol/L IPTG for 4 hours. Cell pellets were collected, washed with PBS, and resuspended in buffer Z (8 mol/L urea, 100 mmol/L NaCl, 20 mmol/L HEPES) followed by sonication on ice. The clarified sonicates were loaded on a TALONresin column equilibrated with buffer Z containing 10 mmol/L imidazole. After extensive washing, the His-tagged bound proteins were eluted sequentially with buffer Z containing 0.1 and 1 mol/L imidazole. The pooled eluates were desalted against a PD10 column equilibrated with PBS/10% glycerol. The protein concentration and purity was determined on a denaturing NuPAGE gel using bovine albumin as standard. Purified protein fractions were dialyzed against water and lyophilized before use. TAT-ß-galactosidase was purified by a similar protocol by using PBS with protease inhibitors instead of buffer Z. Both TAT-RacN17 and TAT-ß-galactosidase were tagged with hemagglutinin (HA).

Permeability Assay
Six-week-old female BALB/c white mice were shaved. Four days later, they were anesthetized, and 150 µL 1% Evans blue solution was injected in the tail vein. After 5 minutes, 100 ng TAT-RacN17 protein in a volume of 20 µL PBS was given by intradermal injection into the mid dorsum. After 1 hour, 50 ng VEGF in 20 µL PBS was injected in the same and an adjacent location. The extravasation of Evans blue was recorded with a digital camera for up to 4 hours. As control, 50 ng BSA or 100 ng TAT-RacN17 was injected into adjacent locations on the same animal.

Immunohistochemistry
The growth factor–implanted mouse eyes were enucleated at day 5 after implantation, frozen on dry ice, and stored at -80°C before use. Frozen sections of 10 µm were cut with a cryomicrotome. Sections were air-dried for 10 minutes, fixed with acetone, and blocked with 30% nonimmune goat serum. Endogenous biotin was blocked using an avidin-biotin reagent (Vector Laboratories). The slides were sequentially stained with mouse anti-HA (Sigma), FITC-conjugated anti-mouse IgG, biotinylated rat anti-mouse CD31 (1:100, Pharmingen), and streptavidin-conjugated Cy3 for 30 to 60 minutes at 20°C. After washing in PBS, slides were mounted in 90% glycerol and examined under a Nikon fluorescence microscope. Images were collected with a digital imaging system and further analyzed with the use of the Adobe Photoshop 6.0 program.

Detection of GTP-Rac
PAE/VEGFR-2 cells (5x10⁶) were starved in serum-free Ham’s F12 medium for 19 hours, pretreated with or without 100 nmol/L wortmannin for 90 minutes, and stimulated with 50 ng/mL VEGF for 10 minutes. Cells were lysed in a GST-Fish buffer (50 mmol/L Tris pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mmol/L NaCl, 10 mmol/L MgCl₂, 10 µg/µL aprotinin/leupeptin, 1 mmol/L PMSF). After centrifugation of cell lysates, equal amounts of supernatants were used for binding to GST-PAK-CD-Sepharose as described.²¹ Briefly, supernatants of bacterial lysates (1.2 mL) containing GST-PAK were incubated in end-to-end rotation with 300 µL of 50% GSH-Sepharose in PBS for 1 hour at 4°C. The bound Sepharose beads were then washed 3 times with bacterial lysis buffer and resuspended in 0.5 mL GST-Fish buffer. GST-PAK Sepharose in 100 µL was incubated with cell lysates for 1 hour. After extensive washing, bound material was released with LDS loading buffer and analyzed by Western blotting (ECL) with the use of a mouse anti-human Rac1 monoclonal antibody (1:1000, BD Science).

Signal Transduction Assays
PAE/VEGFR-2 cells were grown to 90% confluence in 60-mm dishes, washed, and incubated for 30 minutes in serum-free medium (RPMI 1640). Cellular activity was stopped by adding 500 µL LDS lysis buffer containing 1.2 µg/mL aprotinin, pepstatin, and leupeptin and 1.25 mmol/L NaF, PMSF, and sodium orthovanadate. Samples were mixed for 30 seconds and centrifuged for 10 minutes at 14 000 rpm. DNA was removed, and equal amounts of protein samples were separated by SDS-PAGE with the use of a 10% BIS-Tris gel. Proteins were transferred to nitrocellulose membranes, and nonspecific sites were blocked with 5% bovine albumin in PBS/0.1% Tween. To visualize equal amounts of protein loading in each lane, membranes were stained with ponceau dye. Membranes were probed overnight at 4°C with antibodies solubilized in PBS/5% BSA/0.1% Tween for detecting P-Akt (Ser473) and P-eNOS (Ser1177) (Cell Signaling Technology), P-Erk 1/2 (Tyr204), and P-KDR (Oncogene Research Products). This was followed by incubation for 1 hour in PBS/1% BSA/0.1% Tween with peroxidase-conjugated rabbit immunoglobulin diluted 1:1000 for P-Akt, P-eNOS, and P-PLC and 1:40000 for P-VEGFR-2 and with peroxidase-conjugated mouse immunoglobulin diluted 1:5000 for P-Erk1/2. Protein bands were visualized by enhanced chemiluminiscence.

	Results

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Induction of Different Vascular Networks by VEGF and FGF-2
We took advantage of the avascular feature of the mouse cornea to avoid interpretation problems caused by preexisting blood vessels. VEGF and FGF-2 were surgically implanted into the corneas of C57BL/6 mice and outgrowth of new blood vessels was examined on day 5. The angiogenic response stimulated by VEGF was intensive, with a high density of capillary sprouts (Figure 1b) that tended to form plexuses by fusion of capillaries at the growing edge (Figure 1b, small arrows). In contrast, new microvessels induced by FGF-2 grew from the limbus toward the pellet and appeared as well-defined vascular networks, occasionally penetrating into the pellet (Figure 1a). Thus, the angiogenic patterns in corneas stimulated by VEGF and FGF-2 were different, suggesting that the structure of the blood vessels could also be different.

View larger version (83K): [in this window] [in a new window]   Figure 1. Corneal vascular networks induced by FGF-2 and VEGF. Pellets containing FGF-2 (a) or VEGF (b) were implanted into mouse corneas; corneal neovascularization was examined on day 5. Large arrows in a and b mark implanted pellets; small arrows in b mark plexuses of fused capillaries at front edge. Blood vessels were stained with anti-CD31 (red) in corneas implanted with FGF-2 (c) and VEGF (d). Electron microscopy shows blood capillaries growing into the cornea after stimulation with FGF-2 (e) and VEGF (f). Arrows mark caveolae; arrowheads mark fenestrations. L indicates capillary lumen; M, collagen matrix; P, perivascular cell. Bars, 0.2 µm. Thin-walled (<0.5 µm) segments of capillaries were photographed at x50 000 and examined for quantification of fenestrations (g), caveolae (h), and ferritin particles (i) present in the pericapillary space (1-µm-wide zone) 1 hour after intravenous injection as described in Methods section. Data represent means of at least 15 capillary segments (±SEM) from 3 to 4 eyes. Statistical significance was calculated by Student’s t test. ***P<0.001.

Histological examination of corneal sections confirmed that FGF-2–induced vascular networks consisted of well-defined single capillaries (Figure 1c), whereas VEGF-induced vessels were disorganized and dilated with interconnected capillary lumens (Figure 1d). Electron microscopy further revealed that VEGF-induced capillaries consisted of a single layer of endothelial cells connected by cell-cell junctions and partly surrounded by pericytes (Figure 1f). No smooth muscle cells were evident, and the endothelium was typically in direct contact with the collagenous matrix of the cornea. Notably, VEGF-induced capillaries demonstrated numerous fenestrations, with a diameter of 50 nm and usually containing a fine diaphragm (Figure 1f, arrowheads). Their frequency varied, but up to 2 to 3 fenestrations were found per micrometer of endothelial cell cross section in the thinnest parts of the capillary walls (Figure 1g). The number of fenestrations in newly formed capillaries correlated with the number of ferritin particles penetrating into the surrounding extracellular space within 1 hour after intravenous injection (Figure 1i). Accordingly, no fenestrations were detected in capillaries generated after stimulation with FGF-2 (Figure 1, e and g) and few ferritin particles leaked extracellularly as compared with VEGF-induced vessels (Figure 1i). Interestingly, no larger gaps between adjacent endothelial cells were observed either in VEGF-induced or FGF-2–induced vessels, and no clear differences were noted in caveolae numbers (Figure 1h). It therefore seems unlikely that intercellular gaps or caveolae were responsible for the variations in extravasation of ferritin between VEGF-induced and FGF-2–induced vessels. These findings agree with the notion that endothelial fenestrations participate in transport not only of water and small solutes but also of macromolecules between blood and tissues.²²

Rac is a Key Mediator of VEGF-Induced Vascular Permeability
The VEGF-induced thin endothelium with frequent fenestrations suggests that endothelial cells undergo morphological changes. The small GTP-binding protein Rac is a critical player in regulation of cell shape. To study the role of Rac in VEGF-induced angiogenesis and vascular permeability in vivo, TAT-RacN17 protein, a cell-permeable dominant-negative mutant of Rac,²⁰ was implanted alone or with VEGF in mouse corneas and corneal neovascularization was monitored at day 5. Vascular plexuses with fused capillaries of the type induced by VEGF (Figure 2a, small arrows) were not seen after treatment with VEGF/TAT-RacN17 but replaced by well-defined microvascular networks (Figure 2b, small arrows). These microvessels appeared as single sprouts of limbal vessels and were similar to those found in FGF-2–induced angiogenesis. There were no signs of capillary fusion in the VEGF/TAT-RacN17–coimplanted corneas. Quantitative analysis revealed that TAT-RacN17 partially blocked VEGF-induced angiogenesis (Figure 2d). As controls, TAT-RacN17 itself did not induce angiogenesis (Figure 2, c and d), and VEGF/TAT-ß-galactosidase–induced vessels were indistinguishable from VEGF-induced vessels (data not shown). In agreement with the altered neovascularization patterns, histological examination of corneal tissues showed that VEGF/TAT-RacN17–induced vessels consisted of single, well-separated capillaries (Figure 2e). To confirm if the TAT-RacN17 protein was taken up by capillaries, corneal sections were double-stained with antibodies against CD31 (Figure 2e, red), a marker of blood vessel endothelial cells, and antibodies against hemagglutinin-tagged TAT-RacN17 (Figure 2f, green). Overlapping of CD31-positive and TAT-RacN17–positive signals in the same sections revealed that virtually all microvessels were TAT-RacN17–positive (Figure 2g, yellow). As a control, TAT–ß-gal was likewise shown to be transduced into the cells of the cornea (Figure 2, h through j).

View larger version (71K): [in this window] [in a new window]   Figure 2. Change of neovascularization patterns by TAT-RacN17. VEGF alone (a), VEGF/TAT-RacN17 (b), or TAT-RacN17 alone (c) was implanted into mouse corneas. Corneal neovascularization was examined on day 5. Large arrows in a, b, and c mark implanted pellets; small arrows mark plexuses of fused capillaries at front edge. Small arrows in b point to single microvessels. d, Measurement of areas of corneal neovascularization. Data represent means of 10 to 12 eyes (±SEM). Corneal sections are stained with anti-CD31 (e and h, red) and anti-HA (f and i, green) from VEGF/TAT-RacN17–implanted (e through g) and VEGF/TAT–ß-galactosidase–implanted (h through j) corneas. Overlapping signals (yellow color in g and j) represent microvessels that have taken up TAT-tagged proteins. Electron microscopy shows thin parts of microvessels growing into the cornea after stimulation with VEGF (k) and VEGF/TAT-RacN17 (l). Arrowheads mark endothelial fenestrations (k). L indicates capillary lumen; M, collagen matrix. Thin-walled (<0.5 µm) segments of capillaries were photographed at x50 000 and examined for quantification of fenestrations (m) and caveolae (n) and measurement of minimal thickness of the endothelium (o) as described in Methods section. Data represent means of at least 15 capillary segments (±SEM) from 3 to 4 eyes. Statistical significance was calculated by Student’s t test. *P<0.05, **P<0.01.

To see if TAT-RacN17 affected VEGF-induced vascular fenestrations, corneal vessels were examined by electron microscopy. Again, VEGF-induced vessels consisted of a thin endothelium with frequent fenestrations (Figure 2k, arrowheads). In contrast, microvessels induced by VEGF/TAT-RacN17 essentially lacked fenestrations (Figure 2l), suggesting that TAT-RacN17 effectively attenuated VEGF-induced vascular fenestrations. Quantitative analysis showed that barely detectable levels of fenestrations were found in VEGF/TAT-RacN17–induced vessels (Figure 2m). In contrast, an increase of endothelial caveolae was found in VEGF/TAT-RacN17–induced vessels as compared with those induced by VEGF alone (Figure 2n), again indicating that caveolae are not the main mediators of VEGF-stimulated vascular leakage. Consistent with this finding, the endothelium induced by VEGF/TAT-RacN17 was significantly thicker than that induced by VEGF alone (Figure 2o).

As demonstrated above, with the use of ferritin as tracer, endothelial fenestrations provide a structural basis for increased vascular permeability. To further study this process, we carried out a modified Miles assay in mice. This assay has been widely used to detect the permeability effect mediated by VEGF/VPF.¹⁴ We previously reported that FGF-2 did not induce vascular permeability in this assay.⁴⁰ Intradermal injections of VEGF but not BSA induced a rapid permeability response as measured by extravasation of Evans blue dye injected intravenously (Figure 3). The VEGF-induced permeability was detectable 3 minutes after injection and maximal after 1 hour. It should be noted that skin capillaries become fenestrated within 10 minutes of VEGF application.²³ The permeability effect was completely prevented when the TAT-RacN17 protein was preinjected in the same location as VEGF. Injection of TAT-RacN17 itself did not induce extravasation of Evans blue dye (Figure 3). These results confirm that Rac is a key mediator of VEGF-induced vascular permeability.

View larger version (64K): [in this window] [in a new window]   Figure 3. Dermal vascular permeability. Extravasation of Evans blue dye after intradermal injections of VEGF, TAT-RacN17 plus VEGF, TAT-RacN17, and BSA (negative control) was recorded with a digital camera system at various time points.

Signaling Pathways of VEGF-Induced Rac Activation
The above results indicate that Rac promotes but is not strictly required for VEGF-induced angiogenesis, whereas it is a necessary part of the signal transduction system mediating the effect of VEGF on formation of endothelial fenestrations and vascular permeability. Earlier studies have demonstrated that VEGF primarily acts through VEGFR-2 to induce angiogenesis and vascular permeability. To further dissect the VEGF-Rac signaling pathways and to understand the underlying mechanisms, we studied the VEGFR-2–transduced signaling pathways in PAE/VEGFR-2 cells. On VEGF stimulation (10 ng/mL), VEGFR-2 became phosphorylated, which led to phosphorylation of PLC, Akt, eNOS, and Erk1/2 in a time-dependent manner (Figure 4). Maximum phosphorylation was observed between 10 and 15 minutes for PLC, Akt, and eNOS, whereas maximum phosphorylation for Erk1/2 was observed after 30 to 60 minutes. To characterize to what extent phosphatidylinositol-3-OH kinase (PI3K) was involved in activating Rac and the other signaling cascades, the specific PI3K inhibitor wortmannin was used. This drug blocked phosphorylation of Akt and eNOS over a time span of 30 minutes, consistent with Akt being an important activator of eNOS in endothelial cells. The functional relation between VEGFR-2 and Rac was examined by using a pull-down assay with GST-PAK bound to glutathione-coupled agarose beads and subsequent blotting with a Rac antibody. This allowed us to specifically detect the activated fraction of Rac. After 10 minutes of VEGF stimulation, an at least 2-fold increase of GTP-Rac was observed, and wortmannin prevented this effect (Figure 4). We found that wortmannin inhibited Rac activation as well as phosphorylation of Akt and eNOS but only to a small extent affected Erk1/2 phosphorylation (Figure 4). We also conducted experiments with TAT-RacN17 and in two experiments found that TAT-RacN17 (15 µg/mL) inhibited the VEGF-induced P-Akt formation by 80% and 90% measured after 20 minutes (data not shown). We did not find an inhibitory effect of TAT-RacN17 on VEGF-induced phosphorylation of Erk1/2. These data, applying both wortmannin and RacN17, are in accordance with the notion that inhibition of Rac and consequently P-Akt and P-eNOS formation will affect vascular permeability to a larger extent than endothelial cell proliferation, a process that is strongly dependent on P-Erk1/2 activity.

View larger version (43K): [in this window] [in a new window]   Figure 4. Activation of Rac and other signaling molecules. In PAE/VEGFR-2 cells, VEGF-induced (10 ng/mL) phosphorylation of VEGFR-2, PLC, Erk1/2, Akt, e-NOS, and activation of Rac were detected by Western blotting. Equal amounts of cell lysates were used in each lane. VEGF-induced autophosphorylation of VEGFR-2 was measured after 10 minutes of exposure to 10 ng/mL VEGF and other phosphoenzymes at times indicated. Formation of GTP-Rac was measured after 10 minutes of incubation. Cells were preincubated for 10 minutes with wortmannin (100 nmol/L) to block PI3K activity.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our findings provide evidence that VEGF-induced angiogenesis can partly be differentiated from its permeability effect. Although these two functions are probably mediated by the same receptor, VEGFR-2, their signaling pathways show a downstream divergence. The need for Rac in VEGF-induced vascular permeability suggests that reorganization of the endothelial cytoskeleton is critical for this process. Accordingly, VEGF-induced capillaries consisted of a thin endothelium with numerous fenestrations through which injected ferritin molecules could pass.²² The fact that a permeable and dominant-negative mutant of Rac (TAT-RacN17) affected angiogenesis less than permeability further suggests that migration and proliferation of endothelial cells, two key steps in the former process, may be less dependent on Rac signaling than formation and maintenance of fenestrations. Notably, Rac was recently reported to promote assembly of capillary tubes in vitro.²⁴ The observation that circulating endothelial progenitors may take part in blood vessel growth²⁵ likewise points to a role of Rac in the angiogenic process. However, this does not exclude that the permeability-stimulating effect of VEGF shows a stronger requirement for Rac than angiogenesis.

Since other angiogenic factors such as FGF-2 and PDGF induce angiogenesis but not increased permeability, vascular leakage is evidently not a prerequisite for blood vessel growth. The signal transduction pathways of VEGF/VEGFR-2 are complex.⁴ Activation of PLC and its downstream targets PKC and Erk1/2 have been identified as angiogenic signals,^26,27 whereas PI3K and Akt increase NO production and promote endothelial survival.²⁸ Our data reveal that PI3K acts upstream of Rac. Recently, it has, however, become evident that a paradoxical relation exists in the activation patterns of small GTPases and PI3K. Thus, Rac/Cdc42 has been localized both upstream and downstream from PI3K. This was explained by a positive feedback loop involving both Rac/Cdc42 and PI3K.²⁹ Our data are consistent with the model (see also Reference 29), according to which PI3K through SH2 domains in its regulatory subunit (p85) binds to phosphotyrosine residues in VEGFR-2 and becomes activated by tyrosine phosphorylation.³⁰ Lipid products of PI3K may then bind to and activate Rac GTPase by stimulating GDP dissociation.³¹ In PI3K downstream signaling, Rac thus defines the vascular fenestration-permeability pathway. Our finding that wortmannin inhibited Rac activation as well as Akt and eNOS phosphorylation but only little affected Erk1/2 phosphorylation is in accordance with the notion that inhibition of Rac and consequently eNOS will affect vascular permeability more than endothelial cell proliferation, a process strongly dependent on Erk1/2 activity.

Compared with other proangiogenic factors, VEGF (-A) has several unique features, including its requirement in endothelial cell differentiation and vasculogenesis during embryonic development and in induction of vascular permeability.^1,16,32,33 In addition, VEGF expression levels can be regulated by multiple other factors and hypoxia.³⁴ Interestingly, VEGF is strongly expressed in nearly all tumors.³⁵ Accumulating evidence has shown that it is a key factor for tumor angiogenesis, and tumor vessels are leaky and tortuous.³⁶ However, it is not known if these features are directly linked to tumor invasion and metastasis. Again, activation of Rac by VEGF could play an important role for malformation of tumor vessels. Despite these effects on blood vessels, VEGF is in clinical trials for treatment of ischemic limbs and myocardial infarction. However, VEGF-based angiogenic therapy has encountered severe side effects, including local edema, increase of atherosclerotic plaque formation, hypertension, and aberrant vascular proliferation.^37–39 These effects on blood vessels are much like those found in tumor vasculature and are involved in vascular permeability. Thus, vascular leakage must be prevented in VEGF-based angiogenic therapy. Rac antagonists could be important agents to inhibit vascular permeability while still allowing VEGF to induce new vessels. It remains to be investigated whether Rac antagonists are general inhibitors of vascular permeability induced by other factors such as histamine and thrombin. Although we have only characterized the role of Rac in mediation of VEGF-induced vascular permeability, we anticipate that other antagonists of small GTP-binding proteins including Rho and Cdc42 may also inhibit VEGF-induced vascular permeability. These data attract attention to the divergence of signaling pathways in VEGF-induced angiogenesis and vascular permeability.

	Acknowledgments

 
This work was supported by the Human Frontier Science Program, the Swedish Cancer Foundation, the Karolinska Institute, Åke Wiberg’s Foundation, the Swedish Research Council, the Swedish Heart Lung Foundation, and the King Gustaf V 80th Birthday Fund, as well as the Danish Health Science Research Council to Drs Tritsaris and Dissing. Dr Y. Cao is supported by the Karolinska Institute and the Swedish Research Council.

	Footnotes

 
*Drs Eriksson and R. Cao contributed equally to this work.

Received September 3, 2002; revision received November 27, 2002; accepted December 3, 2002.

	References

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