Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle

VEGFR surface localization plays a critical role in converting extracellular VEGF signaling towards angiogenic outcomes, and the quantitative characterization of these parameters is critical for advancing computational models; however the levels of these receptors on blood vessels is currently unknown. Therefore our aim is to quantitatively determine the VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles. We contextualize this VEGFR quantification through comparison to VEGFR-levels on cells in vitro. Using quantitative fluorescence we measure and compare the levels of VEGFR1 and VEGFR2 on endothelial cells isolated from C57BL/6 and BALB/c gastrocnemius and tibialis anterior hindlimb muscles. Fluorescence measurements are calibrated using beads with known numbers of phycoerythrin molecules. The data show a 2-fold higher VEGFR1 surface localization relative to VEGFR2 with 2,000–3,700 VEGFR1/endothelial cell and 1,300–2,000 VEGFR2/endothelial cell. We determine that endothelial cells from the highly glycolytic muscle, tibialis anterior, contain 30% higher number of VEGFR1 surface receptors than gastrocnemius; BALB/c mice display ∼17% higher number of VEGFR1 than C57BL/6. When we compare these results to mouse fibroblasts in vitro, we observe high levels of VEGFR1 (35,800/cell) and very low levels of VEGFR2 (700/cell), while in human endothelial cells in vitro, we observe that the balance of VEGFRs is inverted, with higher levels VEGFR2 (5,800/cell) and lower levels of VEGFR1 (1,800/cell). Our studies also reveal significant cell-to-cell heterogeneity in receptor expression, and the quantification of these dissimilarities ex vivo for the first time provides insight into the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) signaling.     

Quantitative Characterization of Cellular Membrane-Receptor Heterogeneity through Statistical and Computational Modeling

Cell population heterogeneity can affect cellular response and is a major factor in drug resistance. However, there are few techniques available to represent and explore how heterogeneity is linked to population response. Recent high-throughput genomic, proteomic, and cellomic approaches offer opportunities for profiling heterogeneity on several scales. We have recently examined heterogeneity in vascular endothelial growth factor receptor (VEGFR) membrane localization in endothelial cells. We and others processed the heterogeneous data through ensemble averaging and integrated the data into computational models of anti-angiogenic drug effects in breast cancer. Here we show that additional modeling insight can be gained when cellular heterogeneity is considered. We present comprehensive statistical and computational methods for analyzing cellomic data sets and integrating them into deterministic models. We present a novel method for optimizing the fit of statistical distributions to heterogeneous data sets to preserve important data and exclude outliers. We compare methods of representing heterogeneous data and show methodology can affect model predictions up to 3.9-fold. We find that VEGF levels, a target for tuning angiogenesis, are more sensitive to VEGFR1 cell surface levels than VEGFR2; updating VEGFR1 levels in the tumor model gave a 64% change in free VEGF levels in the blood compartment, whereas updating VEGFR2 levels gave a 17% change. Furthermore, we find that subpopulations of tumor cells and tumor endothelial cells (tEC) expressing high levels of VEGFR (>35,000 VEGFR/cell) negate anti-VEGF treatments. We show that lowering the VEGFR membrane insertion rate for these subpopulations recovers the anti-angiogenic effect of anti-VEGF treatment, revealing new treatment targets for specific tumor cell subpopulations. This novel method of characterizing heterogeneous distributions shows for the first time how different representations of the same data set lead to different predictions of drug efficacy.     

Activation of alternative pathways of angiogenesis and involvement of stem cells following anti-angiogenesis treatment in glioma

Malignant gliomas are hypervascular tumors that are highly resistant to all the currently available multimodal treatments. Therefore, anti-angiogenic therapies targeting VEGF or VEGF receptors (VEGFRs) were designed and thought to be an effective tool for controlling the growth of malignant gliomas. However, recent results of early clinical trials using humanized monoclonal antibodies against VEGF (Bevacizumab), as well as small-molecule tyrosine kinase inhibitors that target different VEGF receptors (VEGFRs) (Vatalanib, Vandetanib, Sunitinib, Sorafenib, etc) alone or in combination with other therapeutic agents demonstrated differing outcomes, with the majority of reports indicating that glioma developed resistance to the employed anti-angiogenic treatments. It has been noted that continued anti-angiogenic therapy targeting only the VEGF-VEGFR system might affect pro-angiogenic factors other than VEGF, such as basic fibroblast growth factor (bFGF), stromal derived factor 1 (SDF-1) and Tie-2. These factors may in turn stimulate angiogenesis by mobilizing bone marrow derived precursor cells, such as endothelial progenitor cells (EPCs), which are known to promote angiogenesis and vasculogenesis. In this short review, the current antiangiogenic treatments, possible mechanisms of activation of alternative pathways of angiogenesis, and possible involvement of bone marrow derived progenitor cells in the failure of anti-angiogenic treatments are discussed.     

A systems biology perspective on sVEGFR1: its biological function,pathogenic role and therapeutic use

Angiogenesis is the growth of new capillaries from pre-existent microvasculature. A wide range of pathological conditions, from atherosclerosis to cancer, can be attributed to either excessive or deficient angiogenesis. Central to the physiological regulation of angiogenesis is the vascular endothelial growth factor (VEGF) system – its ligands and receptors (VEGFRs) are thus prime molecular targets of pro-angiogenic and anti-angiogenic therapies. Of growing interest as a prognostic marker and therapeutic target in angiogenesis-dependent diseases is soluble VEGF receptor-1 (sVEGFR1, also known as sFlt-1) – a truncated version of the cell membrane-spanning VEGFR1. For instance, it is known that sVEGFR1 is involved in the endothelial dysfunction characterizing the pregnancy disorder of pre-eclampsia, and sVEGFR1’s therapeutic potential as an anti-angiogenic agent is being evaluated in pre-clinical models of cancer. This mini review begins with an examination of the protein domain structure and biomolecular interactions of sVEGFR1 in relation to the full-length VEGFR1. A synopsis of known and inferred physiological and pathological roles of sVEGFR1 is then given, with emphasis on the utility of computational systems biology models in deciphering the molecular mechanisms by which sVEGFR1’s purported biological functions occur. Finally, we present the need for a systems biology perspective in interpreting circulating VEGF and sVEGFR1 concentrations as surrogate markers of angiogenic status in angiogenesis-dependent diseases.     

Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-Cgamma-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.     

Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.     

Glucocorticoid effects on angiogenesis are associated with mTOR pathway activity

Glucocorticoids (GC) often are administered during pregnancy, but despite their widespread use in clinical practice, it remains uncertain how GC exposure affects pro-angiogenic factors and their receptors. We investigated the effects of GC on vascular endothelial growth factor (VEGF), placental growth factor (PIGF), vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2) protein and mRNA expressions and investigated the possible association of GC with the Akt/mTOR pathway. We incubated human umbilical vein endothelial cells (HUVECs) with a synthetic GC, triamcinolone acetonide (TA). TA administration caused decreased cellular and soluble VEGF and VEGFR1 protein expressions and increased soluble VEGFR2 expression. VEGF, VEGFR1 and VEGFR2 mRNA expressions were altered in a time and dose dependent manner. PIGF protein expression was unaffected by TA treatment, but PIGF mRNA expression decreased in a dose dependent manner after incubation for 48 and 72 h. Phospho-mTOR and phospho-Akt expressions were unaffected. Phospho-p70S6K and phospho-4EBP1 protein expressions and the vascular network forming capacity of HUVECs decreased in a dose dependent manner. We found that GC exert detrimental effects on angiogenesis by altering cellular and soluble angiogenic protein and mRNA levels, and vascular network forming capacities by the Akt/mTOR pathway.     

Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130^Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.     

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway—alone or in combination—would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.     

Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model

p21-activated kinase 1 (Pak1)—a key node protein kinase regulating various cellular process including angiogenesis—has been recognised to be a therapeutic target for multitude of diseases, and hence, various small molecule inhibitors targeting its activity have been tested. However, the direct toxic and anti-angiogenic effects of these pharmacologic agents have not been examined. In this study, we evaluate the translational efficacy of Pak1 inhibitor IPA-3 using zebrafish toxicity model system to stratify its anti-angiogenic potential and off-target effects to streamline the compound for further therapeutic usage. The morphometric analysis has shown explicit delay in hatching, tail bending, pericardial sac oedema and abnormal angiogenesis. We provide novel evidence that Pak1 inhibitor could act as anti-angiogenic agents by impeding the development of sub-intestinal vessel (SIV) and intersegmental vessels (ISVs) by suppressing the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), neurophilin 1 (NRP1) and its downstream genes matrix metalloproteinase (MMP)-2 and MMP-9. Knockdown studies using 2-O-methylated oligoribonucleotides targeting Pak1 also revealed similar phenotypes with inhibition of angiogenesis accompanied with deregulation of major angiogenic factor and cardiac-specific genes. Taken together, our findings indicate that Pak1 signalling facilitates enhanced angiogenesis and also advocated the design and use of small molecule inhibitors of Pak1 as potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting anomalous angiogenesis.     

Novel anti-angiogenic peptides derived from ELR-containing CXC chemokines

Angiogenesis is tightly regulated by numerous endogenous pro- and anti-angiogenic proteins and peptides. Among these are the CXC chemokines, a set of multifunctional peptides. CXC chemokines containing the ELR motif act as pro-angiogenic agents by regulating both endothelial cell proliferation and migration. Here we show that a set of six 22-24-amino acid peptides derived from the pro-angiogenic ELR-containing CXC chemokines exhibit notable anti-proliferative and anti-migratory activity in vitro; we call these peptides chemokinostatins. The ability of the identified peptides to inhibit the basic components of angiogenesis even though they are derived from pro-angiogenic proteins contributes towards the understanding of the diverse role of the CXC chemokine family in angiogenesis.     

Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.     

Receptor Tyrosine Kinase-Mediated Angiogenesis

The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.     

Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogenesis in mice

Vascular endothelial growth factor (VEGF) regulates vasculogenesis and angiogenesis by using two tyrosine kinase receptors, VEGFR1 and VEGFR2. VEGFR1 null mutant mice die on embryonic day 8.5 (E8.5) to E9.0 due to an overgrowth of endothelial cells and vascular disorganization, suggesting that VEGFR1 plays a negative role in angiogenesis. We previously showed that the tyrosine kinase (TK) domain of VEGFR1 is dispensable for embryogenesis, since VEGFR1 TK-deficient mice survived and were basically healthy. However, the molecular basis for this is not yet clearly understood. To test the hypothesis that the specific role of VEGFR1 during early embryogenesis is to recruit its ligand to the cell membrane, we deleted the transmembrane (TM) domain in TK-deficient VEGFR1 mice. Surprisingly, about half of the VEGFR1(TM-TK)-deficient mice succumbed to embryonic lethality due to a poor development of blood vessels, whereas other mice were healthy. In VEGFR1(TM-TK)(-/-) mice with growth arrest, membrane-targeted VEGF was reduced, resulting in the suppression of VEGFR2 phosphorylation. Furthermore, the embryonic lethality in VEGFR1(TM-TK)(-/-) mice was significantly increased to 80 to 90% when the genotype of VEGFR2 was changed from homozygous (+/+) to heterozygous (+/-) in 129/C57BL6 mice. These results strongly suggest that the membrane-fixed ligand-binding region of VEGFR1 traps VEGF for the appropriate regulation of VEGF signaling in vascular endothelial cells during early embryogenesis.     

Mechanistic insight into activation of MAPK signaling by pro-angiogenic factors

Background

Angiogenesis is important in physiological and pathological conditions, as blood vessels provide nutrients and oxygen needed for tissue growth and survival. Therefore, targeting angiogenesis is a prominent strategy in both tissue engineering and cancer treatment. However, not all of the approaches to promote or inhibit angiogenesis lead to successful outcomes. Angiogenesis-based therapies primarily target pro-angiogenic factors such as vascular endothelial growth factor-A (VEGF) or fibroblast growth factor (FGF) in isolation. However, pre-clinical and clinical evidence shows these therapies often have limited effects. To improve therapeutic strategies, including targeting FGF and VEGF in combination, we need a quantitative understanding of the how the promoters combine to stimulate angiogenesis.

Results

In this study, we trained and validated a detailed mathematical model to quantitatively characterize the crosstalk of FGF and VEGF intracellular signaling. This signaling is initiated by FGF binding to the FGF receptor 1 (FGFR1) and heparan sulfate glycosaminoglycans (HSGAGs) or VEGF binding to VEGF receptor 2 (VEGFR2) to promote downstream signaling. The model focuses on FGF- and VEGF-induced mitogen-activated protein kinase (MAPK) signaling and phosphorylation of extracellular regulated kinase (ERK), which promotes cell proliferation. We apply the model to predict the dynamics of phosphorylated ERK (pERK) in response to the stimulation by FGF and VEGF individually and in combination. The model predicts that FGF and VEGF have differential effects on pERK. Additionally, since VEGFR2 upregulation has been observed in pathological conditions, we apply the model to investigate the effects of VEGFR2 density and trafficking parameters. The model predictions show that these parameters significantly influence the response to VEGF stimulation.

Conclusions

The model agrees with experimental data and is a framework to synthesize and quantitatively explain experimental studies. Ultimately, the model provides mechanistic insight into FGF and VEGF interactions needed to identify potential targets for pro- or anti-angiogenic therapies.

     

Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA.

Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.