Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.169664
© 2008 American Heart Association, Inc.
Molecular Medicine |
H-Ras Regulates Angiogenesis and Vascular Permeability by Activation of Distinct Downstream Effectors
From the Moores Cancer Center (D.S., D.C.), University of California, San Diego, La Jolla; and Cell Biolabs Inc (J.L.), San Diego, Calif.
Correspondence to David Cheresh, PhD, University of California San Diego, Moores Cancer Center, 3855 Health Sciences Dr, La Jolla, CA 92093. E-mail dcheresh{at}ucsd.edu
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Abstract |
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Angiogenesis and vascular permeability occur following endothelium activation by vascular endothelial growth factor (VEGF). Downstream mechanisms that define these vascular responses remain unknown. H-Ras activation has been associated with the angiogenic response. However, active H-Ras initiates a wide spectrum of other biological responses through multiple downstream effectors. To identify vascular signaling by H-Ras and the immediate effectors we activated the extracellular signal regulated kinase/mitogen-activated protein kinase or phosphatidylinositol 3-kinase (PI3K) pathways in chicken and mouse endothelial tissues by ectopic expression of the Ras effector mutants H-RasV12S35 or H-RasV12C40, respectively. Constitutive activation of the extracellular signal-regulate kinase/mitogen-activated protein kinase pathway by H-RasV12S35 was sufficient to induce angiogenesis and not vascular permeability, whereas activation of the PI3K pathway by H-RasV12C40 was required for both angiogenesis and vascular permeability. Pharmacological inhibition of PI3K (
/β) suppressed both Ras- or VEGF-mediated vascular response in vivo and survival of primary human endothelial cells in vitro. However, inhibition of PI3K (
/
) suppressed Ras- or VEGF-mediated vascular permeability in vivo, with no effect on survival of primary endothelial cells. This was supported by genetic studies because PI3K p110 knockout mice showed impaired vascular permeability response to VEGF or H-RasV12C40 treatment yet produced a wild-type angiogenic response to H-RasV12S35. We conclude that downstream of VEGF, H-Ras serves as a cellular switch that controls neovascularization and vascular permeability by activation of distinct effectors.
Key Words: angiogenesis • endothelial cells • Ras • VEGF • vascular permeability
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Introduction |
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Angiogenesis and vascular permeability occur in response to vascular endothelial growth factor (VEGF) activation of endothelial cells.1 Described first as a vascular permeability factor by Dvorak et al,2 VEGF is the only angiogenic growth factor that induces both vascular phenotypes. Other growth factors like basic fibroblast growth factor or platelet-derived growth factor are only angiogenic.3,4 Genetic deficiency of VEGF results in embryonic lethality because of failed development of vasculature.5 Angiogenesis is also associated with pathological processes such as tumor growth and metastasis, proliferative retinopathies, age-related macular degeneration, and rheumatoid arthritis.6,7
The GTPase Ras, which is proximally positioned upstream of a number of important signal transduction networks, becomes activated in the proangiogenic response to VEGF in adult tissues,8,9 and in developmental angiogenesis because mice deficient in p120-ras GAP or NF-1, which facilitate Ras inactivation, fail to form organized vascular networks,10,11 deletion of Sos1 (a positive regulator of Ras activation) leads to cardiovascular, yolk sac defects and embryonic lethality,12 and disruption of the Ras effector B-Raf results in vascular defects in mice and midgestational death.13 However, the coordinated circuitry and effectors that participate in angiogenic signaling downstream of Ras to manifest distinct vascular responses to specific growth factors have not been established. Additionally, Ras contribution to vascular permeability has not been documented to date.
Ras regulates cell growth, survival, and proliferation in all eukaryotic cells through signaling pathways that respond to peptide growth factors, cytokines, and hormones. These factors activate many downstream effectors through Ras, including Raf, p120 Ras GAP, RalGDS, phosphatidylinositol 3-kinase (PI3K), etc.14,15 Deciphering the contribution of each specific Ras effector is challenging. Ras activity is controlled by the GTP/GDP cycle, with residues corresponding to switch I (30 to 37) and II (59 to 76) regions defining conformational differences between the inactive GDP- and active GTP-Ras. An intact Ras effector domain (residues 32 to 40) is essential for all effectors interactions.16 Mutation of spanning residues 25 to 45 results in differential impairment of effector interactions and provides thus useful elegant tools to isolate contribution of specific effectors to Ras function.16,17 Using such effector mutants, studies have revealed a bifurcation of the signaling pathways downstream of Ras leading to remodeling of the actin cytoskeleton and DNA synthesis.18
Here, we have used 2 such Ras effector mutants to identify selective contributions of Ras effectors of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) or PI3K pathway to vascular phenotypes in vivo. RasV12C40 (G12
V12, T40C40) binds to and selectively activates PI3K, whereas RasV12S35 (G12V12, Y35S35) binds to Raf1 and selectively activates the ERK/MAPK pathway.18 Ectopic expression of these mutations in chick or mouse endothelial tissues show that Ras-induced selective activation of the ERK/MAPK pathway or the PI3K pathway is sufficient to induce differential vascular phenotypes in vivo. Although VEGF-induced angiogenesis is accompanied by a vascular permeability response, VEGF-induced vascular permeability is not required for angiogenesis.19 Thus, angiogenesis and vascular permeability are regulated independently downstream of VEGF. Here, we provide the first evidence that Ras may function as a cellular switch that controls angiogenesis and vascular permeability by activation of distinct downstream effectors.
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Materials and Methods |
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Endothelial Cell Morphogenesis, Permeability, and Survival/Proliferation Assays were performed according to established protocols detailed in the figure legends and in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Angiogenesis Assays
Ten-day fertilized chick embryos (standard pathogen-free grade; SPAFAS, Preston, Conn) were incubated at 37°C, 70% humidity. The chorioallantoic membrane (CAM) was exposed and treated as detailed previously.8,20 Sterile cortisone acetate-treated filter disks were soaked with 20 µL of VEGF (200 ng) in PBS, PBS alone, or adenovirus (108 plaque-forming units [pfu] in PBS) and added directly to the CAM (N=24 for each treatment). Blocking inhibitors (at concentrations indicated in the figure legends) were added in a volume of 10 µL 1 hour before VEGF treatment and daily at 24-hour intervals for 3 days. VEGF concentration and doses of PD98059, PI3K inhibitors, and adenoviruses were based on previously published results.8,20 Five days after treatment CAMs were explanted examined for new vessel branch points (capillary-sized). Quantification and photographs were obtained at x4 magnification through an Olympus SZH10 microscope using a Spot Camera and a Spot Diagnostic Detection System.8,20
Gene delivery of the human RasV12, RasV12S35, or RasV12C40 cDNA in adenovirus vectors in mice was completed by intradermal injection of the vectors in the ears of Nu/Nu, p110+/+, or p110–/– mice (3x108 pfu per injection). Human green fluorescent protein (GFP), RasN17, or VEGF cDNA in same vectors were used as internal controls and injected in the opposite ears as shown. Neovascularization, first observed 36 hours postinjection, was quantified by counting blood vessel branch points 8 days postinfection at x4 magnification through an Olympus SZH10 microscope. Photographs were obtained at the same magnification.
Miles Assay
All animal studies followed current NIH Guidelines for the Use of Laboratory Animals and institutional animal care and use committee-approved protocols. Nu/Nu mice, p110+/+, or p110–/– mice (18 to 21 g) were dosed IP with inhibitors or vehicle, when inhibitors were used. One hour after inhibitor treatment, at time 0 with no treatment, or 5 days postinfection with adenovirus, 100 µL of 1% Evans blue dye (Sigma, St Louis, Mo) was administered intravenously. For VEGF or cytokine treatment, animals were injected intradermally on each flank with 100 µL of either saline, VEGF (400 ng), or pertussis toxin (600 ng). Thirty minutes following dye delivery, mice were perfused, injection sites were photographed, and circular regions including the injection sites (8 mm diameter) were excised. Permeability was quantified by elution of the Evan’s blue in these sections in 400 µL of formamide at 56°C for 24 hours, followed by absorbance measurements at 600 nm.
The expanded Materials and Methods section includes resources and detailed procedures on antibodies and reagents, cell culture, adenovirus preparation, Western blotting, RNA isolation and cDNA synthesis, quantitative real-time RT-PCR, immunohistochemistry and microscopy, and statistics.
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Results |
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Selective Activation of MAPK Pathway by RasV12S35 Is Sufficient to Induce Angiogenesis
Tube formation by endothelial cells is a critical step in angiogenesis.9 An in vitro endothelial cell morphogenesis assay using human umbilical vein endothelial cells (HUVECs) expressing RasV12, RasV12S35, and RasV12C40 was performed. Representative photographs are shown in Figure 1A, top, and the total tube length (millimeters) in 3 separate (x10) fields is shown in Figure 1A, bottom. Compared with VEGF, RasV12 and RasV12S35 induced a significant increase in formation of capillary-like tubular structures that are sustained for up to 120 versus 72 hours for VEGF. This is sustained by constitutive activation of the ERK/PI3K by the selective Ras mutations. In the VEGF treatment, an immediate activation of ERK/PI3K is induced by VEGF, followed by depletion/inactivation of the VEGF from the serum at 37°C with time (Figure IA in the online data supplement). Significantly, HUVECs expressing RasV12 and RasV12S35 induced similar levels of branching morphogenesis, whereas RasV12C40 failed to induce tube formation. Further treatment of HUVECs expressing RasV12S35 or RasV12C40 with VEGF produces little increase in morphogenesis without a synergistic effect (supplemental Figure IB). These findings reveal that Ras-induced activation of the ERK/MAPK pathway in cultured HUVECs is sufficient to induce tube formation in vitro, whereas activation of PI3K is not.
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Activated Ras and Ras effector mutants were transduced into HUVECs using adenoviral vectors. Equivalent expression by the adenoviral constructs carrying RasV12, RasV12S35, or RasV12C40 mutations, and GFP was observed by immunoblotting and immunofluorescence analysis (supplemental Figures II and III). The selective effectors activated by the Ras mutations were identified by monitoring kinase activities (phosphorylation status of specific substrates), as shown in supplemental Figure III. Constitutively active Ras (RasV12) induced ERK/MAPK activation as monitored by increased P-Erk levels (supplemental Figure III) and increased P-Akt, a measure of its capacity to activate PI3K.18 RasV12S35 produced increased P-Erk, but failed to induce phosphorylation of Akt, whereas RasV12C40 produced increased P-Akt compared to controls, yet had no impact on Erk.
The outcome of selective activation of the ERK/MAPK pathway by H-Ras in endothelial tissue in vivo was assessed by ectopic expression of Ras mutations in the chick CAM. Filter disks saturated with AdRasV12, AdRasV12S35, or AdRasV12C40 were placed on the CAM of 10-day-old chick embryos (N=24 for each treatment), and the angiogenic response was assessed 5 days postinfection (Materials and Methods). Representative images of the angiogenic response to treatments are shown in Figure 1B. Lysates of the transduced CAMs were evaluated for Ras expression and ERK and PI3K activity by immunoblotting specific antibodies to Ras, P-Erk, and P-Akt (Ser473) (Figure 1C). A marked angiogenic response associated with activated Erk was detected in the CAMs treated with VEGF or those expressing RasV12 and RasV12S35 compared with controls (Figure 1C and 1D). CAMs expressing RasV12C40 showed no angiogenic response or Erk activation (Figure 1C and 1D) even though phosphorylation of Akt in these tissues is observed (Figure 1C). Ectopic expression of RasN17, a dominant negative Ras (S17N17) disrupted the angiogenic response to VEGF in CAMs (Figure 1D), indicating that Ras activation is required for the angiogenic response downstream of VEGF. Detergent lysates of these CAMs (15 minutes after VEGF treatment) were evaluated for Ras expression and ERK and PI3K activity as above (Figure 1C). Our findings indicate that Ras-induced selective activation of the ERK/MAPK pathway is sufficient for neovascularization both in vitro and in vivo.
Intradermal Expression of RasV12S35 and RasV12C40 in Mice Leads to Angiogenesis or Vascular Permeability
We used a murine model20 to observe the outcome of ectopic expression of Ras mutations on vascular phenotypes. Intradermal injections of the AdRasV12S35 or AdRasV12C40 were performed in the right ear, whereas the internal controls, AdVEGF and AdGFP, were injected in the left ear of each mouse (N=18 per each treatment). VEGF overexpression produced a robust neovascular response and extensive vascular permeability (Figure 2A and 2B).20 Representative images are shown (Figure 2B). Although both RasV12S35 and RasV12 mutations induced new blood vessel growth (Figure 2A and 2B), RasV12 produced a combined angiogenic and vascular permeability response, although the response was not synergistic (fold compared with VEGF and each other) (Figure 2A and 2B). Comparatively, RasV12C40 showed no angiogenic response (Figure 2A and 2B). Consistent with the results in the CAM model, these findings indicate that selective activation of the ERK/MAPK pathway by the RasV12S35 mutant is sufficient to induce an angiogenic phenotype in vivo. Expression of RasN17 before VEGF stimulation disrupted the vascular permeability response to VEGF in a Miles assay (Figure 2C), indicating that active Ras is required for the vascular permeability response downstream of VEGF.
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Significantly, RasV12C40 mutation produced no angiogenic response (Figure 2A and 2B) but induced considerable vascular permeability (Figure 2D and 2E), as measured in a Miles assay (Materials and Methods). RasV12C40 induced 
4.5-fold increase in vascular permeability compared with the GFP controls and only 1.5-fold less response than the corresponding VEGF adenovirus (Figure 2D). Representative images are shown in Figure 2E. In contrast, RasV12S35 mutation produced no vascular permeability relative to RasV12C40 or VEGF (Figure 2D and 2E). RasV12 induced a permeability response comparable with that of VEGF, consistent with its activation of both the ERK/MAPK and PI3K pathway. These results demonstrate that selective activation of the PI3K pathway by RasV12C40 mutant is sufficient to induce vascular permeability.
To associate the vascular effects observed were with gene expression in endothelial cells, we analyzed the adenoviral-transduced murine tissues. Tissue sections were examined for the endothelial marker CD31 and GFP expression driven from the same CMV promoter as the Ras cDNA in the adenoviral vectors (supplemental Figure IV). We observed colocalization of GFP and CD31 (supplemental Figure IVA, arrowheads), consistent with the endothelium expressing the adenoviral genes. To evaluate additionally for Ras-mediated ERK/MAPK and PI3K activation, tissue sections were stained for colocalization of P-ERK and/or P-Akt and CD31 (supplemental Figure IVB). Consistent with the results in vitro (supplemental Figure III), we identify colocalization of increased P-Erk and CD31 in AdRasV12S35-treated sections (supplemental Figure IVB, a) and colocalization of increased P-Akt and CD31 in AdRasV12C40 treatment (supplemental Figure IVB, b) relative to control treatment (supplemental Figure IVB, c). Control AdGFP-treated sections were stained for CD31 (supplemental Figure IVB, c) or treated with secondary antibodies alone before staining for P-Erk and P-Akt (supplemental Figure IVB, d). To determine whether ectopic expression of RasV12, RasV12S35, and RasV12C40 leads to altered VEGF expression, we isolated the total RNA form these tissues and performed reverse transcription followed by real-time quantitative PCR analysis of VEGF-A expression relative to the endogenous gene cyclophilin (CPH) (Materials and Methods). We found no evidence of increased VEGF-A expression with RasV12, RasV12S35, and RasV12C40 overexpression in the mouse ears (supplemental Table I). Additionally, treated tissues did not show altered VEGF levels by Western blotting (data not shown), indicating that VEGF half-life has not been altered by posttranslational stabilization on adenoviral treatment. To exclude other potential paracrine effects induced by the Ras mutations, we additionally evaluated the effects of various autacoid inhibitors and the PI3K/ inhibitor TG100-115 on the vascular permeability induced by RasV12C40 in vitro (expanded Materials and Methods and Figure VA through VF). TG100-115 blocked the transendothelial flux of fluorescein isothiocyanate (FITC) fluorescent beads associated with RasV12C40 treatment (supplemental Figure VB), whereas the NO inhibitor N
-nitro-L-arginine, the serotonin inhibitor 4-chloro-L-phenylalanine, the inhibitor of cyclooxygenase-1 and cyclooxygenase-2 indomethacin, and the histamine inhibitor cyproheptadine hydrochloride had no specific effect on the RasV12C40-induced transendothelial flux of FITC beads (supplemental Figure VC through VF). These findings support our conclusion that the vascular permeability associated with ectopic expression of RasV12C40 in vitro and in vivo is specific to the activation of PI3K pathway.
RasV12C40- or VEGF-Induced Vascular Permeability but Not Angiogenesis Is Blocked by Pharmacological or Genetic Disruption of PI3K /
ERK and PI3K activation have been linked to cell survival and angiogenesis.21 To identify further Ras effectors in endothelial phenotypes, mice transduced with AdRasV12C40, AdRasV12S35 or treated with VEGF were exposed to inhibitors of PI3K or MEK (Figure 3A and 3B), and angiogenesis or vascular permeability were measured as described above. Inhibitors of PI3K and PI3K β22 disrupted both angiogenesis and vascular permeability in vivo regardless of the stimulus (Figure 3A and 3B). In vitro, we observed a dose-dependent inhibition of HUVEC survival by these inhibitors (Figure 4A). These results imply a general survival function for these 2 isoforms in vascular cells as observed previously in other tissues.23 Significantly, an inhibitor of PI3K/,24 TG100-115, disrupted vascular permeability induced by RasV12C40 or VEGF in vivo (Figure 3A) yet had no effect on angiogenesis (Figure 3B) or in vitro survival of HUVECs (Figure 4A). Blockade of MEK completely disrupted angiogenesis (Figure 3B) without influencing vascular permeability induced by RasV12C40 or VEGF (Figure 3A). To further evaluate the role of PI3K in the vascular permeability response, we performed the Miles assay in mice deficient in p110 · p110–/– mice showed impaired vascular permeability induced by RasV12C40 or VEGF (Figure 4C) and a normal/wild-type response to VEGF- or RasV12S35-induced angiogenesis (data not shown). As seen in Figure 2, we found that Ras activation of MEK/ERK is sufficient for angiogenesis. Significantly, Ras activation of / PI3K isoforms is sufficient for vascular permeability, and Ras activation of the and/or β PI3K isoforms is required for survival of endothelial cells and thus indirectly participates to the angiogenic response.
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To correlate these in vivo responses with downstream effectors, we monitored Akt or Erk phosphorylation. VEGF treatment induced Akt and Erk phosphorylation readily detectable in tissue lysates, and pretreatment with TG100-115 partially blocked Akt phosphorylation with no effect on Erk (Figure 4B). Similar Akt inhibition was observed for tissues expressing RasV12C40 and treated with PI3K inhibitors (data not shown). We observed inhibition of both Akt and Erk phosphorylation upon treatment with VEGF and PI3K and -β inhibitors (Figure 4B). We note that Akt activation by phosphorylation at Ser473 recognized here by a pan antibody does not differentiate substrate specificity. Our data, thus, indicate that Ras/ERK/MEK activation is sufficient to induce neovascularization, whereas Ras/PI3K/ activation appears sufficient to disrupt vascular barrier function.
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Discussion |
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Under pathological conditions, such as ischemic injury or cancer, blood vessels undergo proliferation as well as loss of barrier function. Although growth factors and inflammatory mediators can influence the growth and integrity of blood vessels, it is unclear how angiogenesis and vascular permeability are differentiated intracellularly. Many growth factors and cytokines activate Ras, which, in turn, stimulates a wide range of signaling pathways. Here, we asked whether selective Ras mutations distinguish angiogenesis and vascular permeability via activation of downstream effectors. We show that Ras-induced selective activation of MEK/ERK pathway is sufficient to mediate angiogenesis, whereas Ras-induced selective activation of the PI3K
//Akt pathway promotes vascular permeability. This is depicted schematically in Figure 5.
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VEGF stimulation of new blood vessel growth is critical for embryonic development.5 However, the ability of VEGF to promote vascular permeability has profound pathological consequences.25 For example, following stroke or myocardial infarction, VEGF-mediated vascular permeability causes a significant increase in the level of infarcted tissue immediately following injury.24,26 Given that the blood vessel growth–promoting activity of VEGF would benefit ischemic tissues, it is important to understand how vascular permeability and neovascularization are regulated at the molecular levels. Previous studies suggest distinct vascular responses mediated by differential regulation of signaling pathways downstream of VEGF or other growth factors: inhibition of protein kinase C, a VEGF downstream effector, disrupts angiogenesis while enhancing VEGF-induced vascular permeability27; whereas both VEGF and basic fibroblast growth factor are angiogenic, only VEGF mediates vascular permeability3; mice deficient in individual Src family kinases pp60c-src or pp62c-yes show angiogenesis but no vascular permeability in response to VEGF.19,20 Our data provide first evidence that Ras can serve as a cellular switch for angiogenesis or vascular permeability downstream of VEGF by activation of distinct effectors of the MEK/ERK or PI3K///Akt pathway, respectively.
We used both in vitro and in vivo approaches to assess the role of Ras signaling in endothelial cell signaling. We found that ectopic expression of RasV12S35, and RasV12 on basal RasV12 background, promoted branching morphogenesis and tube formation by HUVECs in vitro, whereas expression of RasV12C40 did not. Thus, Ras to Erk signaling appears sufficient for endothelial cell tube formation. In vivo, we observed similarly that only RasV12S35 and RasV12 induced angiogenesis in the embryonic chick CAM or mouse ears. Because RasV12 and RasV12S35 promoted Ras to Erk signaling and RasV12C40 activated PI3K signaling to Akt, the lack of an angiogenic response to RasV12C40 was not due to its incapacity to initiate a signaling response. Rather, selective Ras-derived signals propagate distinct vascular responses. Activation of effectors of the Ras/ERK pathway, Raf or MEK1, has been shown to promote proliferative responses and growth, associated with the angiogenic response observed here.8 Consistently, we observed inhibition of MEK-1 disrupted the angiogenic response induced by RasV12S35 in vivo. We asked whether these vascular phenotypes may be indirect autocrine/paracrine effects mediated by increased VEGF expression in the cells expressing the Ras mutations. We found that ectopic expression of RasV12, RasV12S35, or RasV12C40 does not alter VEGF expression or increase the half-life of VEGF by posttranslational stabilization in vivo, consistent with previous in vitro analyses.13 Comparatively, the vascular phenotypes observed here are clearly distinct as RasV12S35 induces angiogenesis, whereas RasV12C40 induces vascular leak and VEGF is concurrently angiogenic and a vascular permeability factor.
Although the RasV12C40-induced activation of the PI3K pathway is sufficient to induce vascular permeability, not all PI3K isoforms function in this regard. PI3K and -β isoforms have been linked to general cell survival in other tissues,28 whereas PI3K and - have been associated with inflammation.24 Accordingly, inhibitors of PI3K and -β isoforms disrupted both vascular growth and permeability because of their ability to induce dose-dependent inhibition of endothelial cell survival (in vitro). In agreement, p110- and p110β-deficient mice are embryonic lethal due to defects in DNA synthesis and cell survival.28,29 In contrast, inhibition of the PI3K/ isoforms selectively blocked vascular permeability yet had no effect on new blood vessel growth or HUVEC survival in vitro. Moreover, the vascular permeability response to both RasV12C40 and VEGF was significantly diminished in p110–/– mice untreated and treated further with a PI3K inhibitor (data not shown). These findings suggest that PI3K and are be both necessary and sufficient for vascular permeability. Consistent with this proposal, p110- and p110-deficient mice are viable but show reduced inflammatory and immune response, which may be associated, in part, with reduced edema.24 In contrast to the role that PI3K plays in vascular permeability, inhibition of MEK-1 has no effect on vascular permeability (Figure 3A). However, it clearly prevents the angiogenic response induced by RasV12S35 or VEGF (Figure 3B), which is consistent previous studies.4,8,23
Vascular barrier function depends on the integrity of VE-cadherin–mediated cell–cell junctions and the phosphorylation state of VE-cadherin and associated proteins.30,31 Therefore, we probed for phosphorylation of VE-cadherin in the RasV12C40 and VEGF-treated tissues. We observed phosphorylation of VE-cadherin in both treatments (Figure 4D and supplemental Figure VI), consistent with disassembly of the adherens junctions.30 We have also considered the involvement of Src in RasV12C40 mediated vascular permeability and found that a Src inhibitor that blocks VEGF-mediated leak had no effect on RasC40-induced vascular permeability (data not shown). This suggests that Src mediates vascular permeability independently or upstream of Ras. In fact, integrin ligation leading to Src activation may involve Ras downstream in this process.8,20
VEGF-induced vascular permeability is known to precede calcium/solute flux, and tissue perfusion in vasodilatation,32 regulate the female menstrual cycle,33 and fibrin deposition in wound repair.34 VEGF or other growth factor-mediated angiogenesis is observed in early development21 and in the adult associated with implantation and placentation in the ovary and uterus.33 We establish here that differential Ras activation is sufficient to mediate vascular permeability or angiogenesis. When the mechanisms governing these phenotypes become deregulated pathologies occur. As such, mutational activation of Ras is not well tolerated, as seen in the Costello syndrome, the Noonan syndrome, and the cardio-facio-cutaneous syndrome.15,35 Vascular edema and VEGF expression also occur during ischemic injury and cancer.36 Therefore, it is imperative to understand how Ras and its immediate effectors differentially regulate angiogenesis and the vascular barrier function downstream of VEGF.
A number of clinical trials have been aimed at disrupting Ras signal transduction. The fact that Ras controls a broad spectrum of biological responses may explain why this approach has not met success. For example, inhibitors of farnesylation that disrupt H-Ras activation were inefficient because of transfer of farnesyl/geranyl specificity, and combination therapies were found highly toxic.15 Similarly, the first approved VEGF inhibitors in cancer have been found to prolong survival in cancer patients by months. However, VEGF neutralization caused a large increase in the circulating red blood cells,37 which, although acceptable to terminal patients, is of concern to individuals with non–life-threatening conditions, such us blindness or arthritis. Therefore, efforts that have been more focused on therapies targeting the ERK/MAPK and PI3K pathways independently may provide a greater degree of safety and ultimately improve the treatment options for arthritis, blindness, ischemic disease, defective wound repair, endometriosis, and, last, cancer.33,36
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Acknowledgments |
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We thank Drs Dwayne Stupack, Lisette Acevedo, Wolf Wrasidlo, Jeff Lindquist, and Dave Mikolon (Moores Cancer Center) for the excellent suggestions and technical support.
Sources of Funding
This study was funded by NIH grants CA45726 and CA50286. D.S. was supported by a Susan G. Komen Breast Cancer Foundation fellowship.
Disclosures
None.
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Footnotes |
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Original received December 8, 2007; revision received April 21, 2008; accepted April 24, 2008.
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References |
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