Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis.

RATIONALE: We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE: The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS: Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Endothelial epsins as regulators and potential therapeutic targets of tumor angiogenesis.

VEGF-driven tumor angiogenesis has been validated as a central target in several tumor types deserving of continuous and further considerations to improve the efficacy and selectivity of the current therapeutic paradigms. Epsins, a family of endocytic clathrin adaptors, have been implicated in regulating endothelial cell VEGFR2 signaling, where its inactivation leads to nonproductive leaky neo-angiogenesis and, therefore, impedes tumor development and progression. Targeting endothelial epsins is of special significance due to its lack of affecting other angiogenic-signaling pathways or disrupting normal quiescent vessels, suggesting a selective modulation of tumor angiogenesis. This review highlights seminal findings on the critical role of endothelial epsins in tumor angiogenesis and their underlying molecular events, as well as strategies to prohibit the normal function of endogenous endothelial epsins that capitalize on these newly understood mechanisms.

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes.

Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

Eating the Dead to Keep Atherosclerosis at Bay.

Atherosclerosis is the primary cause of coronary heart disease (CHD), ischemic stroke, and peripheral arterial disease. Despite effective lipid-lowering therapies and prevention programs, atherosclerosis is still the leading cause of mortality in the United States. Moreover, the prevalence of CHD in developing countries worldwide is rapidly increasing at a rate expected to overtake those of cancer and diabetes. Prominent risk factors include the hardening of arteries and high levels of cholesterol, which lead to the initiation and progression of atherosclerosis. However, cell death and efferocytosis are critical components of both atherosclerotic plaque progression and regression, yet, few currently available therapies focus on these processes. Thus, understanding the causes of cell death within the atherosclerotic plaque, the consequences of cell death, and the mechanisms of apoptotic cell clearance may enable the development of new therapies to treat cardiovascular disease. Here, we review how endoplasmic reticulum stress and cholesterol metabolism lead to cell death and inflammation, how dying cells affect plaque progression, and how autophagy and the clearance of dead cells ameliorates the inflammatory environment of the plaque. In addition, we review current research aimed at alleviating these processes and specifically targeting therapeutics to the site of the plaque.

Motif mimetic of epsin perturbs tumor growth and metastasis.

Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium-targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin-interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium-specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.

Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

Clinical presentation and molecular characterization of group B rotaviruses in diarrhoea patients in Bangladesh.

A total of 1106 stool samples collected from diarrhoea patients admitted to Dhaka hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, during January-December 2008 were analysed for the presence of rotavirus-specific RNA by PAGE. The group B-specific RNA migration pattern was detected in 26 patients (2.4%) and group A-specific pattern in 259 patients (23.4%). Clinical data from group A and group B rotavirus-infected patients indicated that episodes did not differ much in the prevalence of diarrhoea, number of stools, outcome or differences in gender. However, abdominal pain was more common in group B rotavirus infections (36 vs 15%, P=0.02) and the virus was responsible for more severe dehydration compared with group A-infected patients (12 vs 3%, P=0.04). Sequence analyses of VP4, VP7 and NSP2 indicated that an Indian-Bangladeshi lineage of the virus, which is different from both the prototype (Chinese) lineage and from the animal group B rotaviruses, has been circulating in Bangladesh. Continuous monitoring of group B rotaviruses both in hospitals and in the community will be helpful to determine the true burden of group B rotaviruses.