Brian P. Eliceiri

Selective Requirement for Src Kinases during VEGF-Induced Angiogenesis and Vascular Permeability

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.

The role of αv integrins during angiogenesis: insights into potential mechanisms of action and clinical development

Angiogenesis depends on specific molecular interactions between vascular cells and components of the extracellular matrix (ECM). This Perspective focuses on the functional role of integrins in angiogenesis and neovascularization. Specifically, we discuss the mechanism by which antagonists of αv integrins disrupt angiogenesis in vivo and how they may impact patients with cancer and inflammatory disease. Role of ECM and integrins during angiogenesis and vasculogenesis. Angiogenesis depends not only on growth factors and their receptors but is also influenced by receptors for ECM proteins. In general, cell adhesion to the ECM is mediated by integrins, heterodimeric transmembrane proteins that comprise a diverse family of over 15 α and 8 β subunits. Integrin subunits can heterodimerize in over 20 combinations. Different integrin combinations may recognize a single ECM ligand, while others bind several different ECM proteins. Integrin-mediated adhesion leads to intracellular signaling events that regulate cell survival, proliferation, and migration (1). These signals include elevation in intracellular pH and calcium, inositol lipid synthesis, and the tyrosine phosphorylation of a wide range of nonreceptor tyrosine kinases such as focal adhesion kinase and Src kinases, as well as adaptor proteins such as Shc, p130 CAS, and Crk II. These signaling events trigger a number of downstream signals, including activation of the Ras/mitogen-activated protein (MAP) kinase pathway (1).

A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth

Tumor cells are elusive targets for immunotherapy due to their heterogeneity and genetic instability. Here we describe a novel, oral DNA vaccine that targets stable, proliferating endothelial cells in the tumor vasculature rather than tumor cells. Targeting occurs through upregulated vascular-endothelial growth factor receptor 2 (FLK-1) of proliferating endothelial cells in the tumor vasculature. This vaccine effectively protected mice from lethal challenges with melanoma, colon carcinoma and lung carcinoma cells and reduced growth of established metastases in a therapeutic setting. CTL-mediated killing of endothelial cells indicated breaking of peripheral immune tolerance against this self antigen, resulting in markedly reduced dissemination of spontaneous and experimental pulmonary metastases. Angiogenesis in the tumor vasculature was suppressed without impairment of fertility, neuromuscular performance or hematopoiesis, albeit with a slight delay in wound healing. Our strategy circumvents problems in targeting of genetically unstable tumor cells. This approach may provide a new strategy for the rational design of cancer therapies.

Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke

Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.

Src-mediated coupling of focal adhesion kinase to integrin αvβ5 in vascular endothelial growth factor signaling

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin αvβ5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with β1 and β3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/αvβ5 signaling complex. Moreover, formation of this FAK/αvβ5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin β5, but not integrin β3, have a reduced VP response to VEGF. This FAK/αvβ5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin αvβ5 into a FAK-containing signaling complex during growth factor–mediated biological responses.

Adhesion events in angiogenesis

Recent work from several laboratories indicates that the coordination of endothelial cell adhesion events with growth factor receptor inputs regulates endothelial cell responses during angiogenesis. Analyses of the signaling pathways downstream of integrins, cadherins and growth-factor receptors are providing an insight into the molecular basis of known anti-angiogenic strategies, as well as into the design of novel therapies.

Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure

Big mitogen-activated protein kinase 1 (BMK1), also known as ERK5, is a member of the MAPK family. Genetic ablation of BMK1 in mice leads to embryonic lethality, precluding the exploration of pathophysiological roles of BMK1 in adult mice. We generated a BMK1 conditional mutation in mice in which disruption of the BMK1 gene is under the control of the inducible Mx1-Cre transgene. Ablation of BMK1 in adult mice led to lethality within 2-4 weeks after the induction of Cre recombinase. Physiological analysis showed that the blood vessels became abnormally leaky after deletion of the BMK1 gene. Histological analysis revealed that, after BMK1 ablation, hemorrhages occurred in multiple organs in which endothelial cells lining the blood vessels became round, irregularly aligned, and, eventually, apoptotic. In vitro removal of BMK1 protein also led to the death of endothelial cells partially due to the deregulation of transcriptional factor MEF2C, which is a direct substrate of BMK1. Additionally, endothelial-specific BMK1-KO leads to cardiovascular defects identical to that of global BMK1-KO mutants, whereas, surprisingly, mice lacking BMK1 in cardiomyocytes developed to term without any apparent defects. Taken together, the data provide direct genetic evidence that the BMK1 pathway is critical for endothelial function and for maintaining blood vessel integrity.

Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1

The ECM protein Del-1 is one of several novel ECM proteins that accumulate around angiogenic blood vessels in embryonic and tumor tissue and promote angiogenesis in the absence of exogenous growth factors. Del-1 expressed in mouse or rabbit ischemic hind-limb muscle by gene transfer rapidly promotes new blood vessel formation and restores muscle function. This angiogenic ECM protein initiates angiogenesis by binding to integrin alphavbeta5 on resting endothelium, thereby resulting in expression of the transcription factor Hox D3 and integrin alphavbeta3. Hox D3 converts resting endothelium to angiogenic endothelium by inducing expression of proangiogenic molecules such as integrin alphavbeta3. These findings provide evidence for an angiogenic switch that can be initiated in the absence of exogenous growth factors and indicate that the angiogenic matrix protein Del-1 may be a useful tool for the therapy of ischemic disease.

Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells

The enteric nervous system may have an important role in modulating gastrointestinal barrier response to disease through activation of enteric glia cells. In vitro studies have shown that enteric glia activation improves intestinal epithelial barrier function by altering the expression of tight junction proteins. We hypothesized that severe injury would increase expression of glial fibrillary acidic protein (GFAP), a marker of enteric glial activation. We also sought to define the effects of vagal nerve stimulation on enteric glia activation and intestinal barrier function using a model of systemic injury and local gut mucosal involvement. Mice with 30% total body surface area steam burn were used as model of severe injury. Vagal nerve stimulation was performed to assess the role of parasympathetic signaling on enteric glia activation. In vivo intestinal permeability was measured to assess barrier function. Intestine was collected to investigate changes in histology; GFAP expression was assessed by quantitative PCR, by confocal microscopy, and in GFAP-luciferase transgenic mice. Stimulation of the vagus nerve prevented injury-induced intestinal barrier injury. Intestinal GFAP expression increased at early time points following burn and returned to baseline by 24 h after injury. Vagal nerve stimulation prior to injury increased GFAP expression to a greater degree than burn alone. Gastrointestinal bioluminescence was imaged in GFAP-luciferase transgenic animals following either severe burn or vagal stimulation and confirmed the increased expression of intestinal GFAP. Injection of S-nitrosoglutathione, a signaling molecule released by activated enteric glia cells, following burn exerts protective effects similar to vagal nerve stimulation. Intestinal expression of GFAP increases following severe burn injury. Stimulation of the vagus nerve increases enteric glia activation, which is associated with improved intestinal barrier function. The vagus nerve may mediate the signaling that occurs from the central nervous system to the enteric nervous system following gastrointestinal injury.

Magnetic targeting of nanoparticles across the intact blood–brain barrier

Delivery of therapeutic or diagnostic agents across an intact blood-brain barrier (BBB) remains a major challenge. Here we demonstrate in a mouse model that magnetic nanoparticles (MNPs) can cross the normal BBB when subjected to an external magnetic field. Following a systemic administration, an applied external magnetic field mediates the ability of MNPs to permeate the BBB and accumulate in a perivascular zone of the brain parenchyma. Direct tracking and localization inside endothelial cells and in the perivascular extracellular matrix in vivo was established using fluorescent MNPs. These MNPs were inert and associated with low toxicity, using a non-invasive reporter for astrogliosis, biochemical and histological studies. Atomic force microscopy demonstrated that MNPs were internalized by endothelial cells, suggesting that trans-cellular trafficking may be a mechanism for the MNP crossing of the BBB observed. The silica-coated magnetic nanocapsules (SiMNCs) allow on-demand drug release via remote radio frequency (RF) magnetic field. Together, these results establish an effective strategy for regulating the biodistribution of MNPs in the brain through the application of an external magnetic field.