Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)–specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.     

KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling

Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1^+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Inhibitory effects of isoproterenol on PAF-induced endothelial cell permeability and morphological changes

Using a model to study vascular permeability under hydrostatically perfused bovine pulmonary artery endothelial cell (EC) monolayers and a software to automatically analyse cell morphological parameters in a computer image workstation, the effects of isoproterenol (IPN) on platelet-activating factor (PAF)-induced changes in EC monolayer permeability and cell morphological parameters were studied. Albumin has the fortifying effect on endothelial barrier function. After treatment of EC monolayer with 10-8mol/L PAF, trans-monolayer permeability increased, cell surface area decreased, and intercellular space enlarged. As pretreatment with 10-4mol/L IPN, PAF-induced EC permeability increment and morphological changes were blocked. The results suggest that EC contraction and intercellular gap expansion are important mechanisms for PAF-induced high vascular permeability. IPN inhibits the effects of PAF via stabilization of EC morphology and prevention of intercellular gap formation.     

In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A.

VEGF-A is a major angiogenesis and permeability factor. Its cellular effects, which can be used as targets in anti-angiogenesis therapy, have mainly been studied in vitro using endothelial cell cultures. The purpose of the present study was to further characterize these effects in vivo in vascular endothelial cells and pericytes, in an experimental monkey model of VEGF-A-induced iris neovascularization. Two cynomolgus monkeys (Macaca fascicularis) received four injections of 0.5 microg VEGF-A in the vitreous of one eye and PBS in the other eye. After sacrifice at day 9, eyes were enucleated and iris samples were snap-frozen for immunohistochemistry (IHC) and stained with a panel of antibodies recognizing endothelial and pericyte determinants related to angiogenesis and permeability. After VEGF-A treatment, the pre-existing iris vasculature showed increased permeability, hypertrophy, and activation, as demonstrated by increased staining of CD31, PAL-E, tPA, uPA, uPAR, Glut-1, and alphavbeta3 and alphavbeta5 integrins, VEGF receptors VEGFR-1, -2 and -3, and Tie-2 in endothelial cells, and of NG2 proteoglycan, uPA, uPAR, integrins and VEGFR-1 in pericytes. Vascular sprouts at the anterior surface of the iris were positive for the same antigens except for tPA, Glut-1, and Tie-2, which were notably absent. Moreover, in these sprouts VEGFR-2 and VEGFR-3 expression was very high in endothelial cells, whereas many pericytes were present that were positive for PDGFR-beta, VEGFR-1, and NG2 proteoglycan and negative for alpha-SMA. In conclusion, proteins that play a role in angiogenesis are upregulated in both pre-existing and newly formed iris vasculature after treatment with VEGF-A. VEGF-A induces hypertrophy and loss of barrier function in pre-existing vessels, and induces angiogenic sprouting, characterized by marked expression of VEGFR-3 and lack of expression of tPA and Tie-2 in endothelial cells, and lack of alpha-SMA in pericytes. Our in vivo study indicates a role for alpha-SMA-negative pericytes in early stages of angiogenesis. Therefore, our findings shed new light on the temporal and spatial role of several proteins in the angiogenic cascade in vivo.     

Vascular permeability induced by VEGF family members in vivo: role of endogenous PAF and NO synthesis

We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet-activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF-induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF-A(165), VEGF-A(121), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased vascular permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME) abrogated protein extravasation mediated by VEGF-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR-2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF-A(165)-mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF-A(165) proinflammatory activities.     

Phenotypic changes and possible angiogenic roles of pericytes during wound healing in the mouse skin

Pericytes (PCs) are attracting increasing attention as a crucial target for anti-angiogenic therapy. In this study, we sought to determine the functional significance of PCs during angiogenesis by using a skin wound healing model in which different angiogenic stages are identifiable. Angiogenesis was first observed on Day 3 after wounding and increased greatly on Day 5. On Day 5, the leading edge of the regenerating vessels (vascular advancing front; VAF) appeared to be composed of immature vessels, and was further divided into "tip" and "following" regions according to maturational differences. PCs distributed in regenerating vessels showed phenotypic differences according to different regions. PCs that expressed PDGFR-β alone and lacked vascular basement membrane (BM) were predominant in the tip region of the VAF, while PCs that expressed both PDGFR-β and NG2 with their BM coating were numerous in the following regions toward the rear of the VAF. Moreover, PCs in the VAF expressed VEGF-A and associated with most proliferating endothelial cells (ECs). VEGF-A expression of PCs and the proliferating ECs totally disappeared in the region toward the rear of the VAF. We conclude that PCs can differ in their phenotype according to the stage of angiogenesis during wound healing. They may promote angiogenesis at the initial stage but might in turn stabilize the newly formed vessels at the later stage.     

IL-2-activated human killer lymphocytes but not their secreted products mediate increase in albumin flux across cultured endothelial monolayers. Implications for vascular leak syndrome

When cultured with IL-2, human lymphoid cells acquire the ability to lyse various NK-resistant tumor targets. Due to their anti-tumor cytolytic effect, clinical trials with IL-2 alone or IL-2 + IL-2-activated killer (IAK) lymphocytes have been undertaken. However, infusion of therapeutically effective doses of IL-2 is associated with the development of systemic toxicity characterized by exaggerated endothelial permeability, also known as vascular leak syndrome. The present study was designed to examine the effects of IAK cells and their secreted products on vascular endothelial permeability by using an in vitro endothelial permeability model in which the flux of FITC-albumin across endothelial cell (EC) monolayers was measured. When endothelial monolayers were exposed to IAK cells for 2 h, significant increases in the transendothelial permeability to albumin were observed. Exposure of EC to lymphocytes cultured in the absence of IL-2 did not induce significant alteration in the endothelial permeability. In addition, neither culture supernatants of IAK cells nor purified recombinant cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-6, TNF-alpha, GM-CSF, M-CSF, and IFN-gamma, had any effect on endothelial permeability in this model. Prior activation of EC with TNF-alpha did not alter the increased permeability induced by IAK cells or lack of it by nonactivated lymphocytes. Dexamethasone treatment of IAK cells abolished their anti-tumor cytolytic effect but only partially inhibited their ability to induce increased endothelial permeability. Pretreatment of IAK cells with mAb directed at the CD11a/CD18 (LFA-1) adhesion complex, and that of EC with mAb directed at the ICAM-1 molecule, inhibited the IAK cell-induced increase in endothelial permeability. These results demonstrate that direct cell-to-cell contact between IAK cells and EC is necessary and sufficient to cause increased endothelial permeability in this model system, and may therefore be an important factor contributing to the development of the vascular leak syndrome observed clinically.     

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.