Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Pathological Angiogenesis Requires Syndecan-4 for Efficient VEGFA-Induced VE-Cadherin Internalization.

[Figure: see text].

Defining an Upstream VEGF (Vascular Endothelial Growth Factor) Priming Signature for Downstream Factor-Induced Endothelial Cell-Pericyte Tube Network Coassembly.

OBJECTIVE: In this work, we have sought to define growth factor requirements and the signaling basis for different stages of human vascular morphogenesis and maturation. Approach and Results: Using a serum-free model of endothelial cell (EC) tube morphogenesis in 3-dimensional collagen matrices that depends on a 5 growth factor combination, SCF (stem cell factor), IL (interleukin)-3, SDF (stromal-derived factor)-1α, FGF (fibroblast growth factor)-2, and insulin (factors), we demonstrate that VEGF (vascular endothelial growth factor) pretreatment of ECs for 8 hours (ie, VEGF priming) leads to marked increases in the EC response to the factors which includes; EC tip cells, EC tubulogenesis, pericyte recruitment and proliferation, and basement membrane deposition. VEGF priming requires VEGFR2, and the effect of VEGFR2 is selective to the priming response and does not affect factor-dependent tubulogenesis in the absence of priming. Key molecule and signaling requirements for VEGF priming include RhoA, Rock1 (Rho-kinase), PKCα (protein kinase C α), and PKD2 (protein kinase D2). siRNA suppression or pharmacological blockade of these molecules and signaling pathways interfere with the ability of VEGF to act as an upstream primer of downstream factor-dependent EC tube formation as well as pericyte recruitment. VEGF priming was also associated with the formation of actin stress fibers, activation of focal adhesion components, upregulation of the EC factor receptors, c-Kit, IL-3Rα, and CXCR4 (C-X-C chemokine receptor type 4), and upregulation of EC-derived PDGF (platelet-derived growth factor)-BB, PDGF-DD, and HB-EGF (heparin-binding epidermal growth factor) which collectively affect pericyte recruitment and proliferation. CONCLUSIONS: Overall, this study defines a signaling signature for a separable upstream VEGF priming step, which can activate ECs to respond to downstream factors that are necessary to form branching tube networks with associated mural cells.

Upregulation of MUC5AC by VEGF in human primary bronchial epithelial cells: implications for asthma.

BACKGROUND: Airway mucus hypersecretion is an important pathophysiological feature in asthma. Mucins are glycoproteins that are mainly responsible for the viscoelastic property of mucus, and MUC5AC is a major mucin glycoprotein that is overproduced in asthma. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Therefore, we sought to investigate the effect of VEGF on MUC5AC expression and study the underlying mechanisms. METHODS: In order to elucidate the precise mechanism underlying the effect of VEGF on MUC5AC expression, we tested the effects of VEGF on RhoA activation and the association of caveolin-1 and VEGFR2 in Primary Bronchial Epithelial Cells. RESULTS: VEGF up-regulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and activated RhoA. Additionally, VEGF-induced MUC5AC expression and RhoA activation were enhanced by disrupting caveolae with cholesterol depletion and reversed by cholesterol repletion, and inhibited by a selective VEGF receptor 2 (VEGFR2) inhibitor SU1498. Furthermore, phospho-VEGFR2 expression was decreased via overexpression of caveolin-1. VEGF treatment reduced the association of caveolin-1 and VEGFR2. CONCLUSION: Collectively, our findings suggest that VEGF up-regulates MUC5AC expression and RhoA activation by interaction with VEGFR2, and this phenomenon was related with the association of caveolin-1 and VEGFR2. Further studies on these mechanisms are needed to facilitate the development of treatments for asthma.

A cyclic peptide reproducing the α1 helix of VEGF-B binds to VEGFR-1 and VEGFR-2 and inhibits angiogenesis and tumor growth.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are pivotal regulators of angiogenesis. The VEGF-VEGFR system is therefore an important target of anti-angiogenesis therapy. Based on the X-ray structure of VEGF-B/VEGFR-1 D2, we designed a cyclic peptide (known as VGB1) reproducing the α1 helix and its adjacent region to interfere with signaling through VEGFR-1. Unexpectedly, VGB1 bound VEGFR-2 in addition to VEGFR-1, leading to inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells and 4T1 murine mammary carcinoma cells, which express VGEFR-1 and VEGFR-2, and U87 glioblastoma cells that mostly express VEGFR-2. VGB1 inhibited different aspects of angiogenesis, including proliferation, migration and tube formation of endothelial cells stimulated by VEGF-A through suppression of extracellular signal-regulated kinase 1/2 and AKT (Protein Kinase B) phosphorylation. In a murine 4T1 mammary carcinoma model, VGB1 caused regression of tumors without causing weight loss in association with impaired cell proliferation (decreased Ki67 expression) and angiogenesis (decreased CD31 and CD34 expression), and apoptosis induction (increased TUNEL staining and p53 expression, and decreased Bcl-2 expression). According to far-UV circular dichroism (CD) and molecular dynamic simulation data, VGB1 can adopt a helical structure. These results, for the first time, demonstrate that α1 helix region of VEGF-B recognizes both VEGFR-1 and VEGFR-2.

Effect of melatonin on EGF- and VEGF-induced monolayer permeability of HUVECs.

Melatonin is a natural hormone involved in the regulation of circadian rhythm, immunity, and cardiovascular function. In the present study, we focused on the mechanism of melatonin in the regulation of vascular permeability. We found that melatonin could inhibit both VEGF- and EGF-induced monolayer permeability of human umbilical vein endothelial cells (HUVECs) and change the tyrosine phosphorylation of vascular-endothelial (VE-)cadherin, which was related to endothelial barrier function. In addition, phospho-AKT (Ser473) and phospho-ERK(1/2) played significant roles in the regulation of VE-cadherin phosphorylation. Both the phosphatidylinositol 3-kinase/AKT inhibitor LY49002 and MEK/ERK inhibitor U0126 could inhibit the permeability of HUVECs, but with different effects on tyrosine phosphorylation of VE-cadherin. Melatonin can influence the two growth factor-induced phosphorylation of AKT (Ser473) but not ERK(1/2). Our results show that melatonin can inhibit growth factor-induced monolayer permeability of HUVECs by influencing the phosphorylation of AKT and VE-cadherin. Melatonin can be a potential treatment for diseases associated with abnormal vascular permeability. NEW & NOTEWORTHY We found that melatonin could inhibit both EGF- and VEGF-induced monolayer permeability of human umbilical vein endothelial cells, which is related to phosphorylation of vascular-endothelial cadherin. Blockade of phosphatidylinositol 3-kinase/AKT and MEK/ERK pathways could inhibit the permeability of human umbilical vein endothelial cells, and phosphorylation of AKT (Ser473) might be a critical event in the changing of monolayer permeability and likely has cross-talk with the MEK/ERK pathway.

Anti-PD1 'SHR-1210' aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement.

Monoclonal anti-programmed cell death 1 (PD1) antibodies are successful cancer therapeutics, but it is not well understood why individual antibodies should have idiosyncratic side-effects. As the humanized antibody SHR-1210 causes capillary hemangioma in patients, a unique toxicity amongst anti-PD1 antibodies, we performed human receptor proteome screening to identify nonspecific interactions that might drive angiogenesis. This screen identified that SHR-1210 mediated aberrant, but highly selective, low affinity binding to human receptors such as vascular endothelial growth factor receptor 2 (VEGFR2), frizzled class receptor 5 and UL16 binding protein 2 (ULBP2). SHR-1210 was found to be a potent agonist of human VEGFR2, which may thereby drive hemangioma development via vascular endothelial cell activation. The v-domains of SHR-1210's progenitor murine monoclonal antibody 'Mab005' also exhibited off-target binding and agonism of VEGFR2, proving that the polyspecificity was mediated by the original mouse complementarity-determining regions (CDRs), and had survived the humanization process. Molecular remodelling of SHR-1210 by combinatorial CDR mutagenesis led to deimmunization, normalization of binding affinity to human and cynomolgus PD1, and increased potency in PD1/PD-L1 blockade. Importantly, CDR optimization also ablated all off-target binding, rendering the resulting antibodies fully PD1-specific. As the majority of changes to the paratope were found in the light chain CDRs, the germlining of this domain drove the ablation of off-target binding. The combination of receptor proteome screening and optimization of the antibody binding interface therefore succeeded in generating novel, higher-potency, specificity-enhanced therapeutic IgGs from a single, clinically sub-optimal progenitor. This study showed that highly-specific off-target binding events might be an under-appreciated phenomenon in therapeutic antibody development, but that these unwanted properties can be fully ameliorated by paratope refinement.

Thioredoxin-interacting protein deficiency ameliorates diabetic retinal angiogenesis.

Diabetic retinopathy is the leading cause of blindness among working-aged adults around the world. Hyperglycemia and intraocular vascular endothelial growth factor (VEGF) over-accumulation are essential for the progression of diabetic retinopathy, which eventually results in proliferative diabetic retinopathy, characterized by pathologic angiogenesis and impaired vision. Thioredoxin-interacting protein (TXNIP) was highly induced in retinal endothelial cells under diabetic conditions. However, the role of TXNIP in diabetes-associated retinal angiogenesis remains elusive. Here, we investigated whether the absence of TXNIP alters diabetes-associated retinal angiogenesis. Exposure of human retinal microvascular endothelial cells (HRMECs) to moderately high glucose (MHG) promoted cell migration and tube formation, but not proliferation. Knockdown of TXNIP suppressed moderately high glucose (MHG)-induced reactive oxygen species (ROS) generation, migration, tube formation and activation of Akt/mTOR pathway in HRMECs. Moreover, gene silencing of TXNIP inhibited VEGF-induced angiogenic response by blocking VEGFR2 and downstream signal pathway Akt/mTOR activation in HRMECs. Furthermore, TXNIP knockout inhibited VEGF or VEGF and MHG-induced retinal angiogenesis ex vivo compared with wild-type mice. In conclusion, our study demonstrated that TXNIP deficiency inhibited VEGF or/and MHG-induced angiogenic response in HRMECs and mice retinas and suggested TXNIP may be a potential therapy target for treating proliferative diabetic retinopathy.

Vascular endothelial growth factor influences migration and focal adhesions, but not proliferation or viability, of human neural stem/progenitor cells derived from olfactory epithelium.

In humans, new neurons are continuously added in the olfactory epithelium even in the adulthood. The resident neural stem/progenitor cells (hNS/PCs-OE) in the olfactory epithelium are influenced by several growth factors and neurotrophins. Among these modulators the vascular endothelial growth factor (VEGF) has attracted attention due its implicated in cell proliferation, survival and migration of other type of neural/stem progenitor cells. Interestingly, VEGFr2 receptor expression in olfactory epithelium has been described in amphibians but not in humans. Here we show that VEGFr is expressed in the hNS/PCs-OE. We also investigated the effect of VEGF on the hNS/PCs-OE proliferation, viability and migration in vitro. Additionally, pharmacological approaches showed that VEGF (0.5 ng/ml)-stimulated migration of hNS/PCs-OE was blocked with the compound DMH4, which prevents the activation of VEGFr2. Similar effects were found with the inhibitors for Rac (EHT1864) and p38MAPK (SB203850) proteins, respectively. These observations occurred with changes in focal adhesion contacts. However, no effects of VEGF on proliferation or viability were found in hNS/PCs-OE. Our results suggest that hNS/PCs-OE respond to VEGF involving VEGFr2, Rac and p38MAPK.

Cardiac myocyte p38α kinase regulates angiogenesis via myocyte-endothelial cell cross-talk during stress-induced remodeling in the heart.

Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.

Endothelial SCUBE2 Interacts With VEGFR2 and Regulates VEGF-Induced Angiogenesis.

OBJECTIVE: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, exerts its proangiogenic action by binding to VEGFR2 (VEGF receptor 2), the activity of which is further modulated by VEGFR2 coreceptors such as neuropilins. However, whether VEGFR2 is regulated by additional coreceptors is not clear. To investigate whether SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), a peripheral membrane protein expressed in vascular endothelial cells (ECs) known to bind other signaling receptors, functions as a VEGFR2 coreceptor and to verify the role of SCUBE2 in the VEGF-induced angiogenesis. APPROACH AND RESULTS: SCUBE2 lentiviral overexpression in human ECs increased and short hairpin RNA knockdown inhibited VEGF-induced EC growth and capillary-like network formation on Matrigel. Like VEGF, endothelial SCUBE2 was upregulated by hypoxia-inducible factor-1α at both mRNA and protein levels. EC-specific Scube2 knockout mice were not defective in vascular development but showed impaired VEGF-induced neovascularization in implanted Matrigel plugs and recovery of blood flow after hind-limb ischemia. Coimmunoprecipitation and ligand-binding assays showed that SCUBE2 forms a complex with VEGF and VEGFR2, thus acting as a coreceptor to facilitate VEGF binding and augment VEGFR2 signal activity. SCUBE2 knockdown or genetic knockout suppressed and its overexpression promoted the VEGF-induced activation of downstream proangiogenic and proliferating signals, including VEGFR2 phosphorylation and mitogen-activated protein kinase or AKT activation. CONCLUSIONS: Endothelial SCUBE2 may be a novel coreceptor for VEGFR2 and potentiate VEGF-induced signaling in adult angiogenesis.

VEGF and the diabetic kidney: More than too much of a good thing.

Over a decade and a half has passed since the publication of early reports hinting at a pathogenetic role for vascular endothelial growth factor ("VEGF") in the development of diabetic kidney disease. In diabetic rats, renal mRNA levels of the VEGF-A isoform were upregulated and administration of a VEGF-A neutralizing antibody attenuated albuminuria: VEGF was "bad" in diabetic nephropathy. Since that time, our understanding of the complexity of the renal VEGF system has advanced. Unlike its experimental counterpart, human diabetic nephropathy is associated with diminished VEGF-A levels and experience in the oncological setting has taught us that VEGF blocking therapy can cause adverse renal effects in patients. Correspondingly, investigational studies in cultured cells and rodent models have demonstrated that the biological effects of the VEGF system are dependent not only on the amount of VEGF, but also the type of VEGF, its sites of action and the prevailing milieu. Here we reflect back on the discoveries that have been made since those initial reports that shone the spotlight on the importance of the VEGF system in the diabetic kidney and we consider that the role of VEGF in diabetic nephropathy extends well beyond being "too much of a good thing".

Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling.

Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant.

Molecular controls of arterial morphogenesis.

Formation of arterial vasculature, here termed arteriogenesis, is a central process in embryonic vascular development as well as in adult tissues. Although the process of capillary formation, angiogenesis, is relatively well understood, much remains to be learned about arteriogenesis. Recent discoveries point to the key role played by vascular endothelial growth factor receptor 2 in control of this process and to newly identified control circuits that dramatically influence its activity. The latter can present particularly attractive targets for a new class of therapeutic agents capable of activation of this signaling cascade in a ligand-independent manner, thereby promoting arteriogenesis in diseased tissues.

Total saponins from Albizia julibrissin inhibit vascular endothelial growth factor-mediated angiogenesis in vitro and in vivo.

Dried stem bark from Albizia julibrissin (AJ) is a highly valued Traditional Chinese Medicine, which has been shown to suppress tumor growth and angiogenesis. Total saponins from AJ (TSAJ) are one of the most bioactive components of AJ extract. The present study evaluated the anti­tumor and anti­angiogenic effects of TSAJ in vitro and in vivo. The anti­angiogenic activity of TSAJ was investigated by measuring the effects on vascular endothelial growth factor (VEGF)­induced proliferation, migration and tube formation of Ea.hy926 endothelial cells in vitro. The expression levels of proteins associated with VEGF­induced angiogenesis were determined by western blotting. Furthermore, in vivo Matrigel™ plug and H22 hepatoma tumor models were used to verify the anti­angiogenic effects of TSAJ. The present study demonstrated that TSAJ significantly inhibited VEGF­mediated endothelial cell proliferation, migration and tube formation of Ea.hy926 cells in vitro. The anti­angiogenic effects of TSAJ were modulated by suppression of phosphorylated­(p­) focal adhesion kinase, p­Akt, and p­extracellular signal­regulated kinase in the VEGF/VEGF receptor 2 (R2) signaling pathway. Furthermore, oral administration of TSAJ significantly inhibited tumor growth and tumor­induced angiogenesis, as well as the formation of functional vessels, in the Matrigel™ plug model. These results suggest that TSAJ may be a potential anti­angiogenic agent that targets the VEGF/VEGFR2 signaling pathway, and inhibits tumor­induced angiogenesis.

Functional binding surface of a ß-hairpin VEGF receptor targeting peptide determined by NMR spectroscopy in living cells.

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells.

Alpha-tocopherol ether-linked acetic acid (α-TEA) has been reported to exhibit both anti-tumor and anti-metastatic activities in cell culture and animal studies. However, it is unclear whether α-TEA possesses anti-angiogenic effects. In this study, we investigated the effect of α-TEA on vascular endothelial growth factor (VEGF)-induced angiogenesis and matrix metalloproteinase (MMP) expression both in vitro and ex vivo. We found that the α-TEA inhibited tube formation, invasion, and migration in human umbilical vein endothelial cells (HUVECs) and that such actions were accompanied by reduced expression of MMP-2. α-TEA also inhibited ex vivo angiogenesis, as indicated by chicken egg chorioallantoic membrane assay. We further showed that α-TEA attenuated protein expression of VEGF receptor-2 (VEGFR-2)-mediated p38 mitogen-activated protein kinase (p38), phosphorylated p38, and focal adhesion kinase (FAK). Moreover, α-TEA (30 µM) significantly up-regulated protein expression of tissue inhibitors of MMP (TIMP)-2 (by 138%) and the metastasis suppressor gene nm23-H1 (by 54%). These results demonstrate that the anti-angiogenic effect of α-TEA both in vitro and ex vivo and its possible mechanistic action appears to involve the inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways and through up-regulation of TIMP-2 and nm23-H1 expression.

Neuropilin-1 enforces extracellular matrix signalling via ABL1 to promote angiogenesis.

Neuropilin-1 (NRP1), together with neuropilin-2, belongs to the neuropilin family. Neuropilins are transmembrane proteins essential for vascular and neural development and act as co-receptors for secreted signalling molecules of the class 3 semaphorin and vascular endothelial growth factor A (VEGF-A) families. NRP1 promotes VEGF-A signal in blood vascular endothelium and semaphorin signal in lymphatic endothelium, by forming complexes with its co-receptors. Mouse mutant studies established that NRP1 expression is essential during development because mice lacking NRP1 expression die embryonically and show severe neuronal and cardiovascular defects. Even though the contribution of NRP1 to vascular development has been mainly ascribed to its function as a VEGF-A receptor, recent evidence suggests that NRP1 contributes to angiogenesis through VEGF-independent mechanisms. In the present paper, we provide an overview of NRP1 functions in the vasculature and discuss current knowledge of NRP1-dependent signalling in the endothelium.

Mechanistic basis for the potent anti-angiogenic activity of semaphorin 3F.

Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.

NGF promotes hemodynamic recovery in a rabbit hindlimb ischemic model through trkA- and VEGFR2-dependent pathways.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 µm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.