Skeletal muscle VEGF gradients in peripheral arterial disease: simulations of rest and exercise

VEGF is a key promoter of angiogenesis and a major target of proangiogenic therapy for peripheral arterial disease (PAD). Greater understanding of VEGF angiogenic signaling and guidance by gradients for new capillaries will aid in developing new proangiogenic therapies and improving existing treatments. However, in vivo measurements of VEGF concentration gradients at the cell scale are currently impossible. We have developed a computational model to quantify VEGF distribution in extensor digitorum longus skeletal muscle using measurements of VEGF, VEGF receptor (VEGFR), and neuropilin-1 (NRP1) expression in an experimental model of rat PAD. VEGF is secreted by myocytes, diffuses through and interacts with extracellular matrix and basement membranes, and binds VEGFRs and NRP1 on endothelial cell surfaces of blood vessels. We simulate the effects of increased NRP1 expression and of therapeutic exercise training on VEGF gradients, receptor signaling, and angiogenesis. Our study predicts that angiogenic therapy for PAD may be achieved not only through VEGF upregulation but also through modulation of VEGFRs and NRP1. We predict that expression of 10(4) NRP1/cell can increase VEGF binding to receptors by 1.7-fold (vs. no NRP1); in nonexercise-trained muscle with PAD, angiogenesis is hindered due to limited VEGF upregulation, signaling, and gradients; in exercise-trained muscle, VEGF signaling is enhanced by upregulation of VEGFRs and NRP1, and VEGF signaling is strongest within the first week of exercise therapy; and hypoxia-induced VEGF release is important to direct angiogenesis towards unperfused tissue.     

Interactions between NRP1 and VEGFR2 molecules in the plasma membrane

Here we use a quantitative FRET approach, specifically developed to probe membrane protein interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A (VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the plasma membrane. Our results underscore the complex nature of the interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the need for new methodologies that capture this complexity, and the need for precise physiological measurements of local receptor surface densities in the membrane of cells. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Neuropilin signalling in vessels,neurons and tumours

The neuropilins NRP1 and NRP2 are transmembrane proteins that regulate many different aspects of vascular and neural development. Even though they were originally identified as adhesion molecules, they are most commonly studied as co-receptors for secreted signalling molecules of the class 3 semaphorin (SEMA) and vascular endothelial growth factor (VEGF) families. During nervous system development, both classes of ligands control soma migration, axon patterning and synaptogenesis in the central nervous system, and they additionally help to guide the neural crest cell precursors of neurons and glia in the peripheral nervous system. Both classes of neuropilin ligands also control endothelial cell behaviour, with NRP1 acting as a VEGF-A isoform receptor in blood vascular endothelium and as a semaphorin receptor in lymphatic valve endothelium, and NRP2 promoting lymphatic vessel growth induced by VEGF-C. Here we provide an overview of neuropilin function in neurons and neural crest cells, discuss current knowledge of neuropilin signalling in the vasculature and conclude with a summary of neuropilin roles in cancer.     

Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.     

Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.     

VEGF receptor signalling - in control of vascular function

Vascular endothelial growth-factor receptors (VEGFRs) regulate the cardiovascular system. VEGFR1 is required for the recruitment of haematopoietic precursors and migration of monocytes and macrophages, whereas VEGFR2 and VEGFR3 are essential for the functions of vascular endothelial and lymphendothelial cells, respectively. Recent insights have shed light onto VEGFR signal transduction and the interplay between different VEGFRs and VEGF co-receptors in development, adult physiology and disease.     

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.     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

VEGF受体功能研究进展

血管内皮生长因子受体（VEGFR）调控心血管系统的发育。VEGFR1对于造血祖细胞的招募及单核巨噬细胞的迁移是必需的;VEGFR2、VEGFR3在调控血管及淋巴管内皮细胞的功能时发挥重要作用,而现在很多研究都聚焦于阻断VEGFR信号通路以达到阻断肿瘤血管生长的目的。     

Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF(165). Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF(165) binding to NRP1, surprisingly reduces VEGF(121)-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF(121) binds directly to NRP1; however, unlike VEGF(165), VEGF(121) is not sufficient to bridge the NRP1.VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF(165), but not VEGF(121) binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.     

Molecular MRI assessment of vascular endothelial growth factor receptor‐2 in rat C6 gliomas

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide‐based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti‐VEGFR2 monoclonal antibody, shown by varied increases in T₁ signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin‐Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.     

Angiotensin II directly impairs adipogenic differentiation of human preadipose cells

microRNAs (miRNAs) are small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. Reduced expression of miR-338 has been reported in several types of cancers; however, the role of miR-338 in oral squamous cell carcinoma (OSCC) has not been elucidated. In this study, we demonstrated that miR-338 was dramatically downregulated in OSCC tissues and cell lines. Overexpression of miR-338 significantly inhibited proliferation, colony formation, migration, and invasion of OSCC cells. In addition, neuropilin1 (NRP1) was identified as a target of miR-338 in OSCC cells and inversely correlated with miR-338 in OSCC tissues. Furthermore, restoration of NRP1 attenuated the tumor-suppressive effects of miR-338. Taken together, miR-338 might inhibit growth and metastasis of OSCC cells by targeting NRP1.     

Epidermal Expression of Neuropilin 1 Protects Murine keratinocytes from UVB-induced apoptosis

Background

Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear.

Methods and Results

To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (−/−) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher.

Conclusion

NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.     

Mechanisms Controlling the Effects of Bevacizumab (Avastin) on the Inhibition of Early but Not Late Formed Corneal Neovascularization

Purpose

To evaluate the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab on the inhibition of corneal neovascularization (NV).

Methods

Corneal NV was induced by closed eye contact lens wear followed by a silk suture tarsorrhaphy in rabbits. Weekly subconjunctival injections of bevacizumab (5.0 mg) for 1 month were started immediately (early treatment group) or 1 month after induction of corneal NV with continuous induction (late treatment group). The severity of corneal NV was evaluated. Immunostaining was used to evaluate the intracorneal diffusion of bevacizumab, and the existence of pericytes and smooth muscle cells around the NV. The expression of AM-3K, an anti-macrophage antibody, vascular endothelial growth factor (VEGF) with its receptors (VEGFR1 and VEGFR2), and vascular endothelial apoptosis were also evaluated. Western blot analysis was performed to quantify the expression level of VEGF, VEGFR1 and VEGFR2 on corneal epithelium and stroma in different groups.

Results

Early treatment with bevacizumab inhibited corneal NV more significantly than late treatment. Intracorneal diffusion of bevacizumab was not different among different groups. Immunostaining showed pericytes and smooth muscle cells around newly formed vessels as early as 2 weeks after induction. Immunostaining and Western blot analysis showed that VEGF, VEGFR1, and VEGFR2 on corneal stroma increased significantly in no treatment groups and late treatment groups, but not in early treatment group. Bevacizumab significantly inhibited macrophage infiltration in the early but not late treatment group. Sporadic vascular endothelial apoptosis was found at 4 weeks in the late but not early treatment group.

Conclusions

Early but not late injection of bevacizumab inhibited corneal NV. Late injection of bevacizumab did not alter macrophage infiltration, and can''t inhibit the expression of VEGF, VEGFR1, and VEGFR2 on corneal vessels. The inhibition of corneal NV in early treatment group does not occur via vascular endothelial apoptosis.