Nader Rahimi

Vascular Endothelial Growth Factor and Hepatocyte Growth Factor Levels Are Differentially Elevated in Patients with Advanced Retinopathy of Prematurity

Although the roles of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in angiogenesis are well described, the putative roles of these factors in retinopathy of prematurity (ROP) remain unknown. We evaluated VEGF and HGF protein levels in subretinal fluid of eyes with ROP, and expression of their corresponding receptors in retrolental membranes associated with stage 5 ROP. We examined subretinal fluid samples from eyes using rhegmatogenous retinal detachment as a control. VEGF and HGF were differentially elevated in eyes with ROP. In Stage 5 ROP (n = 22), the mean VEGF and HGF levels were 14.77 +/- 14.01 ng/ml and 16.56 +/- 9.62 ng/ml, respectively. Interestingly, in patients with active stage 4 ROP, mean VEGF levels were highly elevated (44.16 +/- 18.72 ng/ml), whereas mean HGF levels remained very low (4.77 +/- 2.50 ng/ml). Next, we investigated in vivo expression of VEGF receptor-2 and HGF receptor in retrolental membranes from 16 patients with stage 5 ROP. Both VEGF receptor-2 and HGF receptor proteins were detected mainly in posterior portions of the membrane as well as in vessel walls and along the retinal interface where angiogenesis was active. These findings together suggest that VEGF and HGF play important roles in the pathogenesis of ROP.

IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis

Background Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.

PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

ABSTRACT The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.

Identification of ligand-induced proteolytic cleavage and ectodomain shedding of VEGFR-1/FLT1 in leukemic cancer cells.

Vascular endothelial growth factor receptor-1/fms-related tyrosine kinase 1 (VEGFR-1/FLT1) is expressed as a membrane-bound receptor tyrosine kinase and as an alternatively spliced soluble protein (sVEGFR-1) containing the 1-6 IgG-like domain of its ectodomain. sVEGFR-1 is known as a naturally occurring inhibitor of angiogenesis and as a surrogate marker for cancer progression; it is also linked to pregnancy-induced hypertension called preeclampsia and to avascularity of normal cornea. It remains an open question whether alternative mRNA splicing is the only mechanism by which sVEGFR-1 is generated. In this study, we show that in leukemic cancer cells, PlGF and VEGF-A both induce tyrosine phosphorylation of VEGFR-1 and render it susceptible to ectodomain shedding, resulting in the generation of sVEGFR-1 and an intracellular cytoplasmic fragment. Activation of protein kinase C and tumor necrosis factor-alpha-converting enzyme family metalloproteases are critically required for the occurrence of sVEGFR-1. Following the removal of the ectodomain, the remnant of VEGFR-1 remains attached to the membrane, and the activity of gamma-secretase/presenilin is required for its release from the cell membrane. We propose that sVEGFR-1 produced via ectodomain shedding plays a prominent role in the VEGF receptor system by antagonizing VEGF receptor signaling by acting as a dominant-negative form and/or forming a nonsignaling dimerizing complex with VEGF receptors.

A Single Amino Acid Substitution in the Activation Loop Defines the Decoy Characteristic of VEGFR-1/FLT-1

VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn1050) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp1050) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.

A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCγ1 activation and angiogenesis

Activation of phospholipase Cγ1 (PLCγ1) by vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells in part is responsible for angiogenesis in vivo. The cellular mechanisms exerting negative control over PLCγ1 activation, however, remain unaddressed. Here by using in vitro and in vivo binding assays, we show that the Casitas B-lineage lymphoma (c-Cbl) E3 ubiquitin ligase constitutively associates with PLCγ1 via its C-terminal domain and conditionally interacts with VEGFR-2 via the N-terminal/TKB domain. Site-directed mutagenesis of VEGFR-2 showed that full activation of c-Cbl requires its direct association with phospho-tyrosines 1052 and 1057 of VEGFR-2 via its TKB domain and indirect association with phospho-tyrosine 1173 of VEGFR-2 via PLCγ1. The tertiary complex formation between VEGFR-2, PLCγ1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLCγ1 by a proteolysis-independent mechanism. Further analysis showed that association of c-Cbl with VEGFR-2 does not impact ubiquitylation, down-regulation, or tyrosine phosphorylation of VEGFR-2. Silencing of c-Cbl by siRNA revealed that endogenous c-Cbl plays an inhibitory role in angiogenesis. Our data demonstrate that corecruitment of c-Cbl and PLCγ1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.

Comparative Structure‐Function Analysis of VEGFR‐1 and VEGFR‐2

Abstract: Activation of vascular endothelial growth factor receptor‐1 and ‐2 (VEGFR‐1 and VEGFR‐2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which activation of VEGFRs elicits these cellular events is not fully understood. We recently constructed chimeric receptors containing the extracellular domain of human CSF‐1R/c‐fms fused with the entire transmembrane and cytoplasmic domains of murine VEGFR‐1 and VEGFR‐2. Selective activation of chimeric VEGFR‐2, but not chimeric VEGFR‐1, stimulated endothelial cell growth, migration, and differentiation. Stimulation of cells coexpressing chimeric VEGFR‐1 and VEGFR‐2 suppressed VEGFR‐2‐mediated endothelial cell growth. Site‐directed mutagenesis demonstrated that tyrosines 799 and 1173 are required for VEGFR‐2‐mediated endothelial cell growth and activation of PI3 kinase. Further site‐directed mutagenesis demonstrated that tyrosine 1212, located in the carboxyl tail of VEGFR‐2, is required for the ligand‐dependent autophosphorylation of the receptor and its ability to activate signaling proteins. Collectively, our results suggest that activation of VEGFR‐1 and VEGFR‐2 differentially regulates endothelial cell function and angiogenesis. Second, activation of VEGFR‐2 is associated with many endothelial cell functions, including cell proliferation, migration, and differentiation. Third, activation of PI3 kinase by VEGFR‐2 regulates endothelial cell proliferation.

The Ubiquitin-Proteasome System Meets Angiogenesis

A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system (UPS) plays a central role in fine-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR-2, angiogenic signaling proteins (e.g., the PLCγ1 and PI3 kinase/AKT pathways), and other non-VEGF angiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in angiogenesis-associated diseases. Mol Cancer Ther; 11(3); 538–48. ©2012 AACR.

Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis

IGPR-1 is a novel adhesion molecule that regulates cell–cell interaction. IGPR-1 associates with several SH3-containing proteins, including SPIN90/WISH, and regulates capillary tube formation of primary endothelial cells.

c-Cbl, a Ubiquitin E3 Ligase That Targets Active β-Catenin A NOVEL LAYER OF Wnt SIGNALING REGULATION

Background: Several E3 ligases regulate cytosolic β-catenin during Wnt-off phase. The fate of critical form active β-catenin in Wnt-on phase remains poorly defined. Results: Casitas B-lineage lymphoma (c-Cbl) ubiquitinates cytosolic β-catenin and translocates to the nucleus with Wnt induction to also ubiquitinate active nuclear β-catenin. Conclusion: c-Cbl is a unique E3 ligase targeting active nuclear β-catenin. Significance: This study uncovers a novel layer of Wnt regulation. Regulation of transcriptionally active nuclear β-catenin during the Wnt-on phase is crucial to ensure controlled induction of Wnt target genes. Several ubiquitin E3 ligases are known to regulate cytosolic β-catenin during the Wnt-off phase, but little is known about the fate of active nuclear β-catenin in the Wnt-on phase. We now describe ubiquitination of active β-catenin in the Wnt-on phase by a RING finger ubiquitin E3 ligase, Casitas B-lineage lymphoma (c-Cbl) in endothelial cells. c-Cbl binds preferentially to nuclearly active β-catenin in the Wnt-on phase via the armadillo repeat region. Wild-type c-Cbl suppresses and E3 ligase-deficient c-Cbl-70Z increases Wnt signaling. Wnt induces nuclear translocation of c-Cbl where it ubiquitinates nuclear β-catenin. Deletion of the c-Cbl UBA domain abrogates its dimerization, binding to β-catenin, Wnt-induced c-Cbl nuclear translocation, and ubiquitination of nuclear β-catenin. c-Cbl activity inhibits pro-angiogenic Wnt targets IL-8 and VEGF levels and angiogenesis in a β-catenin-dependent manner. This study defines for the first time c-Cbl as a ubiquitin E3 ligase that targets nuclearly active β-catenin in the Wnt-on phase and uncovers a novel layer of regulation of Wnt signaling.