BACKGROUND: Visceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo. METHODS: We investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD). RESULTS: Transgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3β and induction of XBP1/IRE1α, but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1. CONCLUSION: These data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

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.

Binding of epsin ubiquitin-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and physiological angiogenesis. Deletion of epsins in vessel endothelium produces uncontrolled tumor angiogenesis and retards tumor growth in animal models. The aim of this study is to test the therapeutic efficacy and targeting specificity of a chemically-synthesized peptide, UPI, which compete for epsin binding sites in VEGFR2 and potentially inhibits Epsin-VEGFR2 interaction in vivo, in an attempt to reproduce an epsin-deficient phenotype in tumor angiogenesis. Our data show that UPI treatment significantly inhibits and shrinks tumor growth in GL261 glioma tumor model. UPI peptide specifically targets VEGFR2 signaling pathway revealed by genetic and biochemical approaches. Furthermore, we demonstrated that UPI peptide treatment caused serious thrombosis in tumor vessels and damages tumor cells after a long-term UPI peptide administration. Besides, we revealed that UPI peptides were unexpectedly targeted cancer cells and induced apoptosis. We conclude that UPI peptide is a potent inhibitor to glioma tumor growth through specific targeting of VEGFR2 signaling in the tumor vasculature and cancer cells, which may offer a potentially novel treatment for cancer patients who are resistant to current anti-VEGF therapies.

As cancer therapeutics continues to improve and progress, the adverse side effects associated with anticancer treatments have also attracted more attention and have become extensively explored. Consequently, the importance of posttreatment follow-ups is becoming increasingly relevant to the discussion. Contemporary treatment methods, such as tyrosine kinase inhibitors, anthracycline chemotherapy, and immunotherapy regimens are effective in treating different modalities of cancers; however, these reagents act through interference with DNA replication or prevent DNA repair, causing endothelial dysfunction, generating reactive oxygen species, or eliciting non-specific immune responses. Therefore, cardiotoxic effects, such as hypertension, heart failure, and left ventricular dysfunction, arise posttreatment. Rising awareness of cardiovascular complications has led to meticulous attention for the evolution of treatment strategies and carefully monitoring between enhanced treatment effectiveness and minimization of adverse toxicity to the cardiovasculature, in which psychological assessments, early detection methods such as biomarkers, magnetic resonance imaging, and various drugs to reverse the damage from cardiotoxic events are more prevalent and their emphasis has increased tremendously. Fully understanding the mechanisms by which the risk factors action for various patients undergoing cancer treatment is also becoming more prevalent in preventing cardiotoxicity down the line.

Epsins, endocytic adaptor proteins required for internalization of ubiquitylated receptors, are generally upregulated in human cancers. It has been characterized that mice deficient of epsins in the endothelium inhibit tumor growth by dysregulating vascular endothelial growth factor receptor-2 (VEGFR2) signaling and non-productive tumor angiogenesis. Binding of the epsin ubiquitin (Ub)-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and degradation, indicative of epsin UIM as a potential therapeutic target. A Computer Assisted Drug Design approach was utilized to create the UIM mimetic peptides for the functional competition of epsin binding sites in ubiquitylated VEGFR2 in vivo. Specifically targeting VEGFR2 in the tumor vasculature, the chemically synthesized chimeric UIM peptide, UPI, causes non-functional tumor angiogenesis, retards tumor growth, and increases survival rates in several tumor models. The authors showed that UPI binds ubiquitylated VEGFR2 to form a supercomplex in an Ub-dependent fashion. Collectively, the UPI targeting strategy offers a potentially novel treatment for cancer patients who are resistant to current anti-angiogenic therapies. In this review, the authors outline the main points of this research specifically as a potential application for glioma tumor therapy.

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.

The kidney's filtration activity is essential for removing toxins and waste products from the body. The vascular endothelial cells of the glomerulus are fenestrated, flattened, and surrounded by podocytes, specialized cells that support glomerular endothelial cells. Mucin-type core 1-derived O-glycans (O-glycans) are highly expressed on both glomerular capillary endothelial cells and their supporting podocytes, but their biological role is unclear. Biosynthesis of core 1-derived O-glycans is catalyzed by the glycosyltransferase core 1 β1,3-galactosyltransferase (C1galt1). Here we report that neonatal or adult mice with inducible deletion of C1galt1 (iC1galt1-/-) exhibit spontaneous proteinuria and rapidly progressing glomerulosclerosis. Ultrastructural analysis of the glomerular filtration barrier components revealed that loss of O-glycans results in altered podocyte foot processes. Further analysis indicated that O-glycan is essential for the normal signaling function of podocalyxin, a podocyte foot process-associated glycoprotein. Our results reveal a new function of O-glycosylation in the integrity of the glomerular filtration barrier.

The retina is a light sensitive tissue that contains specialized photoreceptor cells called rods and cones which process visual signals. These signals are relayed to the brain through interneurons and the fibers of the optic nerve. The retina is susceptible to a variety of degenerative diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP) and other inherited retinal degenerations. In order to reveal the mechanism underlying these diseases and to find methods for the prevention/treatment of retinal degeneration, animal models have been generated to mimic human eye diseases. In this paper, several well-characterized and commonly used animal models are reviewed. Of particular interest are the contributions of these models to our understanding of the mechanisms of retinal degeneration and thereby providing novel treatment options including gene therapy, stem cell therapy, nanomedicine, and CRISPR/Cas9 genome editing. Role of newly-identified adaptor protein epsins from our laboratory is discussed in retinal angiogenesis and VEGFR2 signaling.

It is well established that diabetes mellitus accelerates atherosclerotic vascular disease. Endothelial injury has been proposed to be the initial event in the pathogenesis of atherosclerosis. Endothelium not only acts as a semi-selective barrier but also serves physiological and metabolic functions. Diabetes or high glucose in circulation triggers a series of intracellular responses and organ damage such as endothelial dysfunction and apoptosis. One such response is high glucose-induced chronic endoplasmic reticulum stress in the endothelium. The unfolded protein response is an acute reaction that enables cells to overcome endoplasmic reticulum stress. However, when chronically persistent, endoplasmic reticulum stress response could ultimately lead to endothelial dysfunction and atherosclerosis. Herein, we discuss the scientific advances in understanding endoplasmic reticulum stress-induced endothelial dysfunction, the pathogenesis of diabetes-accelerated atherosclerosis and endoplasmic reticulum stress as a potential target in therapies for diabetic atherosclerosis.

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.