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.     

Vascular endothelial growth factor and hepatocyte regeneration in acetaminophen toxicity

VEGF or VEGF-A is a major regulator of angiogenesis and has been recently shown to be important in organ repair. The potential role of VEGF in acetaminophen (APAP)-induced hepatotoxicity and recovery was investigated in B6C3F1 male mice. Mice were treated with APAP (300 mg/kg ip) and killed at various time points that reflect both the acute and recovery stages of toxicity. VEGF-A protein levels were increased 7-fold at 8 h and followed the development of hepatotoxicity. VEGF receptor 1, 2, and 3 (VEGFR1, VEGFR2, and VEGFR3, respectively) expression increased throughout the time course, with maximal expression at 48, 8, and 72 h, respectively. Treatment with the VEGF receptor inhibitor SU5416 (25 mg/kg ip at 3 h) had no effect on toxicity at 6 or 24 h. In further studies, the role of SU5416 on the late stages of toxicity was examined. Treatment of mice with APAP and SU5416 (25 mg/kg ip at 3 h) resulted in decreased expression of PCNA, a marker of cellular proliferation. Expression of platelet endothelial cell adhesion molecule, a measure of small vessel density, and endothelial nitric oxide synthase (NOS), a downstream target of VEGFR2, were increased at 48 and 72 h following toxic doses of APAP, and treatment with SU5416 decreased their expression. These data indicate that endogenous VEGF is critically important to the process of hepatocyte regeneration in APAP-induced hepatotoxicity in the mouse.     

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3–0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function.In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells.Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Non-redundant roles of the Gab1 and Gab2 scaffolding adapters in VEGF-mediated signalling,migration, and survival of endothelial cells

Gab1 was previously described as a positive modulator of Akt, Src, ERK1/2, endothelial cell migration, and capillary formation in response to vascular endothelial growth factor (VEGF). However, its involvement in endothelial cell survival, as well as the potential contribution of the other family member Gab2 to signalling and biological responses remained unknown. Here, we show that Gab2 is tyrosine phosphorylated in a Grb2-dependent manner downstream of activated VEGF receptor-2 (VEGFR2), and that it associates with signalling proteins including PI3K and SHP2, but apparently not with the receptor. Similarly to Gab1, over-expression of Gab2 induces endothelial cell migration in response to VEGF, whereas its depletion using siRNAs results in its reduction. Importantly, depletion of both Gab1 and Gab2 leads to an even greater inhibition of VEGF-induced cell migration. However, contrary to what has been reported for Gab1, the silencing of Gab2 results in increased Src, Akt and ERK1/2 activation, slightly reduced p38 phosphorylation, and up-regulation of Gab1 protein levels. Accordingly, re-expression of Gab2 in Gab2?/? fibroblasts leads to opposite results, suggesting that the modulation of both Gab2 and Gab1 expression in these conditions might contribute to the impaired signalling observed. Consistent with their opposite roles on Akt, the depletion of Gab1, but not of Gab2, results in reduced FOXO1 phosphorylation and VEGF-mediated endothelial cell survival. Mutation of VEGFR2 Y801 and Y1214, which abrogates the phosphorylation of Gab1, also correlates with inhibition of Akt. Altogether, these results underscore the non-redundant and essential roles of Gab1 and Gab2 in endothelial cells, and suggest major contributions of these proteins during in vivo angiogenesis.     

Structural basis of the junctional anchorage of the cerebral cavernous malformations complex

The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (K_d = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr^1,380-Phe^1,381), thus defining a new mode of FERM domain–membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM–membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.     

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.     

Pancreatic Endoplasmic Reticulum Kinase Activation Promotes Medulloblastoma Cell Migration and Invasion through Induction of Vascular Endothelial Growth Factor A

Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.     

Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

Glycation of extracellular matrix proteins has been demonstrated to contribute to the pathogenesis of vascular complications. However, no previous report has shown the role of glycated fibronectin (FN) in vascular endothelial growth factor (VEGF)‐induced angiogenesis. Thus, this study aimed to investigate the effects of glycated FN on VEGF signalling and to clarify the molecular mechanisms involved. FN was incubated with methylglyoxal (MGO) in vitro to synthesize glycated FN, and human umbilical vein endothelial cells (HUVECs) were seeded onto unmodified and MGO‐glycated FN. Then, VEGF‐induced angiogenesis and VEGF‐induced VEGF receptor‐2 (VEGFR‐2) signalling activation were measured. The results demonstrated that normal FN‐positive bands (260 kD) vanished and advanced glycation end products (AGEs) appeared in MGO‐glycated FN and glycated FN clearly changed to a higher molecular mass. The glycation of FN inhibited VEGF‐induced VEGF receptor‐2 (VEGFR‐2), Akt and ERK1/2 activation and VEGF‐induced cell migration, proliferation and tube formation. The glycation of FN also inhibited the recruitment of c‐Src to VEGFR‐2 by sequestering c‐Src through receptor for AGEs (RAGE) and the anti‐RAGE antibody restored VEGF‐induced VEGFR‐2, Akt and ERK1/2 phosphorylation, endothelial cell migration, proliferation and tube formation. Furthermore, the glycation of FN significantly inhibited VEGF‐induced neovascularization in the Matrigel plugs implanted into subcutaneous tissue of mice. Taken together, these data suggest that the glycation of FN may inhibit VEGF signalling and VEGF‐induced angiogenesis by uncoupling VEGFR‐2‐c‐Src interaction. This may provide a novel mechanism for the impaired angiogenesis in diabetic ischaemic diseases.     

Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function

Parathyroid hormone‐related protein (PTHrP) stimulates osteoblastic function through its N‐ and C‐terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107–139) and the shorter PTHrP (107–111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3‐E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr‐1059 within 5–10 min in both MC3T3‐E1 and rat osteoblastic osteosarcoma UMR‐106 cells. This phosphorylation elicited by osteostatin appears to be VEGF‐independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal‐regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin‐dependent induction of ERK and Akt activation was abrogated by SU6656. Up‐regulation of VEGF and osteoprotegerin gene expression as well as the pro‐survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C‐terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function. J. Cell. Biochem. 114: 1404–1413, 2013. © 2012 Wiley Periodicals, Inc.     

Loss of CCM3 impairs DLL4‐Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations

CCM3, a product of the cerebral cavernous malformation 3 or programmed cell death 10 gene (CCM3/PDCD10), is broadly expressed throughout development in both vertebrates and invertebrates. Increasing evidence indicates a crucial role of CCM3 in vascular development and in regulation of angiogenesis and apoptosis. Furthermore, loss of CCM3 causes inherited (familial) cerebral cavernous malformation (CCM), a common brain vascular anomaly involving aberrant angiogenesis. This study focused on signalling pathways underlying the angiogenic functions of CCM3. Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways. Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3‐silence‐mediated impairment of Notch signalling and reduced the ratio of VEGF‐R2 to VEGF‐R1 expression. Importantly, restoration of DLL4‐Notch signalling entirely rescued the hyper‐angiogenic phenotype induced by CCM3 silence. A concomitant loss of CCM3 and the core components of DLL4‐Notch signalling were also demonstrated in CCM3‐deficient endothelial cells derived from human CCM lesions (CCMEC) and in a CCM3 germline mutation carrier. This study defined DLL4 as a key downstream target of CCM3 in endothelial cells. CCM3/DLL4‐Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.     

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.     

VEGFR1 (Flt1) Regulates Rab4 Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear. Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports αvβ3 integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and αvβ3 integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.     

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.     

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.     

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model

Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework.