Diabetic Wound Healing and Activation of Nrf2 by Herbal Medicine.

Nrf2 defense is a very important cellular mechanism to control oxidative stress, which is implicated in wound healing. Nrf2 can induce many cytoprotective genes, including HO-1, NQO1 and G6PD. Among many natural products that have been reported as Nrf2 activators, sulforaphane and curcumin have been studied more widely than any others, and both are in clinical trials for non-cancerous disorders. Recently, we reported 4-ethyl catechol and 4-vinyl catechol as Nrf2 co-factors that can induce Nrf2 as potently as sulforaphane and curcumin. These new Nrf2 co-factors were identified in hot aqueous extract of an herbal medicine Barleria lupulina, and fermented Noni (Morinda citrifolia) juice, which are used traditionally for diabetic wound healing.

Anti-inflammatory activity of Barleria lupulina: Identification of active compounds that activate the Nrf2 cell defense pathway, organize cortical actin, reduce stress fibers, and improve cell junctions in microvascular endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hot aqueous extracts of the plant Barleria lupulina (BL) are used for treating inflammatory conditions and diabetic vascular complications. AIM OF THE STUDY: The goal was to identify active compounds in hot aqueous extracts of BL (HAE-BL) that are consistent with a role in reducing inflammation and reducing the vascular pathology associated with diabetes. In particular, we examined activation of the Nrf2 cell defense pathway because our initial findings indicated that HAE-BL activates Nrf2, and because Nrf2 is known to suppress inflammation. Activation of Nrf2 by HAE-BL has not been described previously. MATERIALS AND METHODS: Human endothelial cells, real-time PCR, western blotting, cytoskeletal analyses, and assay-guided fractionation with HPLC were used to identify specific compounds in HAE-BL that activate the Nrf2 cell defense pathway and reduce markers of inflammation in vitro. RESULTS: HAE-BL potently activated the Nrf2 cell defense pathway in endothelial cells consistent with its traditional use and reported success in reducing inflammation. Assay guided fractionation with HPLC identified three alkyl catechols: 4-ethylcatechol, 4-vinylcatechol, and 4-methylcatechol, that are each potent Nrf2 activators. In addition to activating Nrf2, HAE-BL and akyl catechols each profoundly improved organization of the endothelial cell actin cytoskeleton, reduced actin stress fibers, organized cell-cell junctions, and induced expression of mRNA encoding claudin-5 that is important for formation of endothelial tight junctions and reducing vascular leak. CONCLUSIONS: HAE-BL contains important alkyl catechols that potently activate the Nrf2 cell defense pathway, improve organization of the endothelial cell cytoskeleton, and organize tight cell junctions. All of these properties are consistent with a role in reducing inflammation and reducing vascular leak. Because activation of the Nrf2 cell defense pathway also prevents cancers, neuro-degeneration, age-related macular degeneration, and also reduces the severity of chronic obstructive pulmonary disorder and multiple sclerosis, HAE-BL warrants additional consideration for these other serious disorders.

Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet.

The Nrf2 (NFE2L2) cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both in vitro and in vivo. Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The absence of alkyl catechols from the modern Western diet suggests serious negative consequences for Nrf2 cell defense, resulting in reduced protection against multiple chronic diseases associated with oxidative stress.

Calpain inhibition improves collateral-dependent perfusion in a hypercholesterolemic swine model of chronic myocardial ischemia.

PURPOSE: Calpain overexpression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia. METHODS: Yorkshire swine fed a high cholesterol diet for 4 weeks underwent surgical placement of an ameroid constrictor to their left circumflex coronary artery. Three weeks later, animals received no drug, high cholesterol control group (n = 8); low-dose calpain inhibition (0.12 mg/kg; n = 9); or high-dose calpain inhibition (0.25 mg/kg; n = 8). The heart was harvested after 5 weeks. RESULTS: Myocardial perfusion in ischemic myocardium significantly improved with high-dose calpain inhibition at rest and with demand pacing (P = .016 and .011). Endothelium-dependent microvessel relaxation was significantly improved with low-dose calpain inhibition (P = .001). There was a significant increase in capillary density, with low-dose calpain inhibition and high-dose calpain inhibition (P = .01 and .01), and arteriolar density with low-dose calpain inhibition (P = .001). Calpain inhibition significantly increased several proangiogenic proteins, including vascular endothelial growth factor (P = .02), vascular endothelial growth factor receptor 1 (P = .003), vascular endothelial growth factor receptor 2 (P = .003), and talin, a microvascular structural protein (P = .0002). There was a slight increase in proteins implicated in endothelial-dependent (nitric oxide mediated) relaxation, including extracellular signal-regulated kinase, phosphorylated extracellular signal-regulated kinase, and inducible nitric oxide synthase with calpain inhibition. CONCLUSIONS: In the setting of hypercholesterolemia, calpain inhibition improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. Calpain inhibition also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation.

Angiogenesis.

Extracellular matrix (ECM) is essential for all stages of angiogenesis. In the adult, angiogenesis begins with endothelial cell (EC) activation, degradation of vascular basement membrane, and vascular sprouting within interstitial matrix. During this sprouting phase, ECM binding to integrins provides critical signaling support for EC proliferation, survival, and migration. ECM also signals the EC cytoskeleton to initiate blood vessel morphogenesis. Dynamic remodeling of ECM, particularly by membrane-type matrix metalloproteases (MT-MMPs), coordinates formation of vascular tubes with lumens and provides guidance tunnels for pericytes that assist ECs in the assembly of vascular basement membrane. ECM also provides a binding scaffold for a variety of cytokines that exert essential signaling functions during angiogenesis. In the embryo, ECM is equally critical for angiogenesis and vessel stabilization, although there are likely important distinctions from the adult because of differences in composition and abundance of specific ECM components.

Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion.

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to "anti-angiogenesis" strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.

Active Rac1 improves pathologic VEGF neovessel architecture and reduces vascular leak: mechanistic similarities with angiopoietin-1.

Architecturally defective, leaky blood vessels typify pathologic angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). Such neovascular defects aggravate disease pathology and seriously compromise the therapeutic utility of VEGF. Endothelial cell (EC) transduction with active L61Rac1 strongly improved VEGF-driven angiogenesis in vivo as measured by increased neovascular density, enhanced lumen formation, and reduced vessel leakiness. Conversely, transduction with dominant-negative N17Rac1 strongly inhibited neovascularization. In vitro, active L61Rac1 promoted organization of cortical actin filaments and vascular cords and improved EC-EC junctions, indicating that improved cytoskeletal dynamics are important to the mechanism by which active L61Rac1 rectifies VEGF-driven angiogenesis. SEW2871, a sphingosine 1-phosphate receptor-1 agonist that activates Rac1 in ECs, improved cord formation and EC-EC junctions in vitro similarly to active L61Rac. Moreover, SEW2871 administration in vivo markedly improved VEGF neovessel architecture and reduced neovascular leak. Angiopoietin-1, a cytokine that "normalizes" VEGF neovessels in vivo, activated Rac1 and improved cord formation and EC-EC junctions in vitro comparably to active L61Rac1, and a specific Rac1 inhibitor blocked these effects. These studies distinguish augmentation of Rac1 activity as a means to rectify the pathologic angioarchitecture and dysfunctionality of VEGF neovessels, and they identify a rational pharmacologic strategy for improving VEGF angiogenesis.

Cdc42-mediated inhibition of GSK-3ß improves angio-architecture and lumen formation during VEGF-driven pathological angiogenesis.

Vascular endothelial growth factor-A (VEGF) typically induces abnormal angiogenesis in the adult, thereby aggravating disease pathology and limiting utility of VEGF for therapeutic angiogenesis. To identify strategies for rectifying defects in pathological VEGF neovessels, we investigated consequences of modulating the Rho GTPase Cdc42. In a mouse skin model of VEGF-driven pathological angiogenesis, transduction with active Cdc42 (L28Cdc42) markedly improved VEGF neovessels, as measured by increased lumen formation, enlarged vessel diameter, and enhanced perfusion of macromolecular tracers. Conversely, transduction with dominant negative Cdc42 (N17Cdc42) impaired endothelial cell (EC) assembly into lumenized blood vessels and reduced neovessel diameter and tracer perfusion. In vitro, active Cdc42 improved coordination between actin filaments and microtubules and enhanced formation of vascular cords, suggesting that active Cdc42 rectifies defects in angiogenesis by improving cytoskeletal dynamics and capillary morphogenesis. Analyses of Cdc42 signaling in microvascular ECs indicated that active Cdc42 also inhibits glycogen synthase kinase-3ß (GSK-3ß), a multi-functional serine/threonine protein kinase. Pharmacological inhibition of GSK-3ß improved vascular cord formation in vitro and promoted proper neovessel formation in vivo comparably to active Cdc42, thus linking GSK-3ß inhibition to the mechanism by which active Cdc42 rectifies pathological neovascularization. These studies identify activation of Cdc42 and inhibition of GSK-3ß as novel strategies for correcting abnormalities associated with VEGF-driven angiogenesis, and they suggest new approaches for achieving improved therapeutic neovascularization with VEGF.

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

BACKGROUND: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks. CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.

Moderate GSK-3ß inhibition improves neovascular architecture, reduces vascular leakage, and reduces retinal hypoxia in a model of ischemic retinopathy.

In ischemic retinopathies, unrelieved hypoxia induces the formation of architecturally abnormal, leaky blood vessels that damage retina and ultimately can cause blindness. Because these newly formed blood vessels are functionally defective, they fail to alleviate underlying hypoxia, resulting in more pathological neovascularization and more damage to retina. With an established model of ischemic retinopathy, we investigated inhibition of glycogen synthase kinase-3ß (GSK-3ß) as a means for improving the architecture and functionality of pathological blood vessels in retina. In vitro, hypoxia increased GSK-3ß activity in retinal endothelial cells, reduced ß-catenin, and correspondingly impaired integrity of cell/cell junctions. Conversely, GSK-3ß inhibitors restored ß-catenin, improved cell/cell junctions, and enhanced the formation of capillary cords in three-dimensional collagen matrix. In vivo, GSK-3ß inhibitors, at appropriately moderate doses, strongly reduced abnormal vascular tufts, reduced abnormal vascular leakage, and improved vascular coverage and perfusion during the proliferative phase of ischemia-driven retinal neovascularization. Most importantly, these improvements in neovasculature were accompanied by marked reduction in retinal hypoxia, relative to controls. Thus, GSK-3ß inhibitors offer a promising strategy for alleviating retinal hypoxia by correcting key vascular defects typically associated with ischemia-driven neovascularization.

Vascular endothelial growth factor: much more than an angiogenesis factor.

Vascular endothelial growth factor (VEGF) is best known as a cytokine essential for embryonic vasculogenesis and for the angiogenesis associated with various pathologies including cancer. However, VEGF also serves other functions that are less widely recognized. An early study (Berse et al., 1992) revealed widespread expression of VEGF transcripts in adult tissues devoid of ongoing neovascularization, thereby predicting additional VEGF functions distinct from angiogenesis. More recent studies have confirmed that VEGF does indeed serve multiple additional functions, including normal maintenance of endothelial and neural cell compartments. These findings have important implications for the use of VEGF antagonists and VEGF receptor antagonists in patients for which inhibition of pathological angiogenesis is the therapeutic goal.

Extracellular matrix mediates a molecular balance between vascular morphogenesis and regression.

PURPOSE OF REVIEW: We discuss very recent studies that address the critical role of extracellular matrix in controlling the balance between vascular morphogenesis and regression. Much of this work suggests that a balance mechanism exists for controlling the extent of tissue vascularization involving downstream signaling events regulating endothelial cell behaviors in relation to their interactions with extracellular matrix molecules. RECENT FINDINGS: Endothelial gene expression changes and signaling lead to events that not only stimulate vascular morphogenesis but also suppress mechanisms mediated through pro-regression factors such as Rho kinase. At the same time, vascular networks are susceptible to regression mediated by factors such as matrix metalloproteinase-1, matrix metalloproteinase-10, thrombospondin-1, extracellular matrix matricryptic fragments and angiopoietin-2. Pericyte recruitment to such vascular tubes can prevent regression events by delivering molecules such as tissue inhibitor of metalloproteinase-3 and angiopoietin-1 that promote vascular stabilization by decreasing tube susceptibility to these regression stimuli. SUMMARY: Extracellular matrix-derived signals lead to critical morphologic changes mediated through cytoskeletal rearrangements that control the shape, function and signaling events in endothelial cell-lined vessels regulating tube formation, remodeling, stabilization and regression. These signals control both vascular morphogenic and regression events, and thus a molecular balance exists to control the extent and function of vascular tube networks within tissues.

Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity.

Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).

VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype.

Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype.

Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization.

The extracellular matrix (ECM) is critical for all aspects of vascular biology. In concert with supporting cells, endothelial cells (ECs) assemble a laminin-rich basement membrane matrix that provides structural and organizational stability. During the onset of angiogenesis, this basement membrane matrix is degraded by proteinases, among which membrane-type matrix metalloproteinases (MT-MMPs) are particularly significant. As angiogenesis proceeds, ECM serves essential functions in supporting key signaling events involved in regulating EC migration, invasion, proliferation, and survival. Moreover, the provisional ECM serves as a pliable scaffold wherein mechanical guidance forces are established among distal ECs, thereby providing organizational cues in the absence of cell-cell contact. Finally, through specific integrin-dependent signal transduction pathways, ECM controls the EC cytoskeleton to orchestrate the complex process of vascular morphogenesis by which proliferating ECs organize into multicellular tubes with functional lumens. Thus, the composition of ECM and therefore the regulation of ECM degradation and remodeling serves pivotally in the control of lumen and tube formation and, finally, neovessel stability and maturation.

In vivo and in vitro models of Mammalian angiogenesis.

Angiogenesis is a complex process involving the organization of proliferating endothelial cells into new blood vessels. Both in vivo models and in vitro models are important for investigating angiogenesis and for defining the involvement of specific molecules. This chapter describes a basic mouse model of vascular endothelial growth factor-driven angiogenesis in mouse skin together with a modified version of this model in which retrovirus-packaging cells are included as a means to efficiently achieve retroviral transduction in vivo. With this approach, the contributions of specific proteins to angiogenesis can be defined. In addition, we describe a model of capillary morphogenesis in vitro that uses microvascular endothelial cells transduced with retrovirus in culture. This in vitro model provides a complementary strategy for investigating the importance of specific molecules for angiogenesis.

VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.

Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.

Interstitial collagen I stimulates microvascular endothelial cells to form solid cords that imitate precapillary structures found during angiogenesis. Time-lapse microscopy identified cell retraction and disruption of cell-cell contacts as early critical steps in collagen I-induced capillary morphogenesis. These early stages paralleled collagen I activation of Src kinase and GTPase Rho through beta1 integrins. The Src inhibitor PP2, dominant-negative Src, and Rho inhibitor exoenzyme C3 transferase each inhibited collagen I induction of actin stress fibers that mediate cell retraction and each inhibited capillary morphogenesis. Collagen I also disrupted VE-cadherin from intercellular junctions through a Src-dependent mechanism; both the Src inhibitor PP2 and dominant-negative Src preserved VE-cadherin localization to regions of cell-cell contact. An active Src mutant disrupted VE-cadherin and cell-cell contacts similarly to collagen I. In sharp contrast, laminin-1 did not induce capillary morphogenesis, and laminin-1 did not induce activation of Src or Rho. Rather, laminin-1 induced persistent activation of the GTPase Rac. Thus, these studies identify activation of Src and Rho as key mechanisms by which collagen I provokes capillary morphogenesis of microvascular endothelial cells, and they define marked differences between the functions of collagen I and laminin-1 in regulating endothelial cell morphogenesis.

Rho activity critically and selectively regulates endothelial cell organization during angiogenesis.

The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants. In this animal model, dominant negative N19RhoA selectively impaired assembly of ECs into new blood vessels; and, in contrast, active V14RhoA stimulated ECs to form blood vessels with functional lumens. In vitro, dominant negative N19RhoA reduced EC actin stress fibers and prevented ECs from contracting and reorganizing into precapillary cords within collagen gels. In contrast, active V14RhoA promoted EC stress fiber formation, contractility, and organization into cords. Neither N19RhoA nor V14RhoA significantly affected EC proliferation or migration in vitro; and, similarly, neither mutant significantly affected EC density during angiogenesis in vivo. Thus, these studies identify a critical and selective role for Rho activity in regulating EC assembly into new blood vessels, and they identify both negative and positive manipulation of Rho activity, respectively, as strategies for suppressing or promoting the organizational stages of neovascularization.

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.