Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.     

Cystic Fibrosis Related Diabetes

Vascular endothelial growth factor (VEGF) plays a pivotal role in the development of diabetic macular edema (DME), the leading cause of vision loss among working-aged individuals. A decade of clinical trials demonstrated that drugs that bind soluble VEGF restore the integrity of the blood-retinal barrier, resolve macular edema, and improve vision in most patients with DME. Four drugs (pegaptanib, ranibizumab, bevacizumab, and aflibercept) effectively treat DME when administered by intravitreal injections. Only ranibizumab has received U.S. Food and Drug Administration (FDA) approval for DME, but bevacizumab is commonly used off-label, and an FDA application for aflibercept is pending. Effective treatment requires repeated injections, although recent data suggest that the treatment burden diminishes after 1 year. Intravitreal therapy is generally safe, although the incidence of systemic thromboembolic events varies among trials.     

Binding and displacement of vascular endothelial growth factor (VEGF) by thrombospondin: Effect on human microvascular endothelial cell proliferation and angiogenesis

Vascular endothelial growth factor (VEGF) is a specific angiogenic factor, and thrombospondin (TSP), is a potent inhibitor of angiogenesis. To better understand the role of TSP as an anti-angiogenic agent, we have identified its specific domains that participate in its anti-angiogenic activity and examined the mechanism of its inhibitory effect on VEGF₁₆₅ induced angiogenesis. Exogenously added TSP inhibited VEGF₁₆₅ induced angiogenesis (proliferation and tube formation of human dermal microvascular endothelial cells {HDMEC} and neovascular outgrowth from human arterial rings). Although both VEGF₁₆₅ and TSP are heparin binding proteins, TSP had a higher affinity for ¹²⁵I-heparin than VEGF₁₆₅ (K _d1 4 nM and K _d2 14 nM for TSP; K _d 91 nM for VEGF₁₆₅). TSP displaced 36% of ¹²⁵I-VEGF₁₆₅ from HDMEC and this was comparable to the 27% reduction in ¹²⁵I-VEGF₁₆₅ binding to HDMEC upon cleavage of cell surface heparan sulfate (HS). About 35% of the mitogenic activity of VEGF₁₆₅ was attributable to its heparin binding region. These results indicate that a proportion of the mitogenic activity of VEGF₁₆₅ is inhibited by TSP via competition for cell surface HS. Further, ¹²⁵I-VEGF₁₆₅ bound directly to TSP in a saturable, concentration dependent manner, and heparin modulated this binding. The mAbs to the heparin binding domain to the type 1 and type 3 repeats of TSP inhibited the binding of VEGF₁₆₅ to TSP, and also blocked the inhibitory effect of TSP on VEGF₁₆₅ induced HDMEC proliferation. We conclude that (i) the anti-angiogenic activity of TSP is localized in its heparin binding domain and type 1 and type 3 repeats (ii) TSP inhibits angiogenesis by at least two separate mechanisms, (a) displacement of VEGF₁₆₅ from endothelial cell HS and (b) direct binding to VEGF₁₆₅. This revised version was published online in June 2006 with corrections to the Cover Date.     

Soluble neuropilin targeted to the skin inhibits vascular permeability

Neuropilin 1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF₁₆₅), an inducer of vascular permeability and angiogenesis. Numerous physiological factors enhance VEGF expression and function but only a few have been shown to be negative regulators. Previously, we have shown that the naturally occurring soluble form of NRP1 (sNRP1) inhibits binding of VEGF₁₆₅ to endothelial cells in vitro and impairs tumor growth in vivo. To investigate the role of sNRP1 in the regulation of vascular development and function, sNRP1 expression was targeted to the skin, where it is not normally expressed, using a keratin 14 (K14) promoter expression construct. K14-sNRP1 transgenic mice displayed normal skin architecture with a subtle abnormal vascular phenotype. While the overall number of skin blood vessels remained unchanged, the lumen size of smooth muscle-associated dermal vessels was reduced. K14-sNRP1 mice had reduced vascular permeability in response to VEGF₁₆₅, but also to VEGF₁₂₁ and platelet activating factor, suggesting that the lack of permeability was not solely due to the sequestration of VEGF. sNRP1 also reversed the increase in inflammation and edema induced by transgenic VEGF overexpression in cutaneous delayed-type hypersensitivity reactions. In summary, sNRP1 appears to primarily regulate vessel permeability while its effect on physiological angiogenesis is less evident in this model.     

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1–4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF₁₁₁a, VEGF₁₁₁b, VEGF₁₂₁a, VEGF₁₂₁b, VEGF₁₅₅a, VEGF₁₅₅b, VEGF₁₆₅a, VEGF₁₆₅b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF₁₆₅b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF₁₁₁b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.     

Intravitreous anti-VEGF for diabetic retinopathy: hopes and fears for a new therapeutic strategy

Vascular endothelial growth factor (VEGF) plays a key role in the development of both proliferative diabetic retinopathy (PDR) and diabetic macular oedema (DMO). In recent years, anti-VEGF agents have emerged as new approaches to the treatment of these devastating diabetic complications. Although Phase III studies in the diabetic population are needed, intravitreal anti-VEGF therapy is currently being used in clinical practice. Intravitreal injection is an effective means of delivering anti-VEGF drugs to the retina. However, this is an invasive procedure associated with potentially serious complications, such as endophthalmitis or retinal detachment, which may be significant for patients requiring serial treatment over many years. In addition, although delivered within the vitreous, anti-VEGF drugs could pass into the systemic circulation, which could potentially result in hypertension, proteinuria, increased cardiovascular events and impaired wound healing. Pegaptanib, ranibizumab and bevacizumab are the currently available anti-VEGF agents. Ranibizumab and bevacizumab block all VEGF isoforms, thus impairing both physiological and pathological neovascularisation. Pegaptanib only blocks the VEGF₁₆₅ isoform, and would therefore seem the best option for avoiding systemic adverse effects in diabetic patients, although this remains to be demonstrated in clinical trials. In this regard, head-to-head studies designed to evaluate not only the efficacy, but also the systemic adverse effects of these drugs in a high-risk population such as diabetic patients are warranted.     

Intramyocardial delivery of VEGF<Subscript>165</Subscript> via a novel biodegradable hydrogel induces angiogenesis and improves cardiac function after rat myocardial infarction

Vascular endothelial growth factor (VEGF), an independent mitogen, has been reported to induce angiogenesis and thus attenuates the damage induced by myocardial infarction (MI). VEGF₁₆₅ is the most abundant and predominant isoform of VEGF. This study investigates whether this effect could be strengthened by local intramyocardial injection of VEGF₁₆₅ along with a novel biodegradable Dex-PCL-HEMA/PNIPAAm hydrogel and ascertains its possible mechanism of action. Rat models of myocardial infarction were induced by coronary artery ligation. Phosphate-buffered saline (PBS group), Dex-PCL-HEMA/PNIPAAm hydrogel (Gel group), phosphate-buffered saline containing VEGF₁₆₅ (VP group), and hydrogel containing VEGF₁₆₅ (VPG group) were injected into a peri-infarcted area of cardiac tissue immediately after myocardial infarction, respectively. The sham group was thoracic but without myocardial infarction. The injection of VEGF₁₆₅ along with a hydrogel induced angiogenesis, reduced collagen content and MI area, inhibited cell apoptosis, increased the level of VEGF₁₆₅ protein and the expression of flk-1 and flt-1, and improved cardiac function compared with the injection of either alone after MI in rats. The results suggest that injection of VEGF₁₆₅ along with a hydrogel acquires more cardioprotective effects than either alone in rat with MI by sustained release of VEGF₁₆₅, then may enhance the feedback between VEGF and its receptors flk-1 and flt-1.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF₁₂₁- and VEGF₁₆₅-overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF₁₈₉-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF₁₂₁ and VEGF₁₆₅, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF₁₈₉, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.     

Anti-vascular endothelial growth factor drug treatment of diabetic macular edema: the evolution continues

Diabetic mellitus is the leading cause of blindness in working aged patients in developing nations. Due to the buildup of abnormal metabolites from several overactive biochemical pathways, chronic hyperglycemia causes oxidative stress in the retina which upregulates vascular endothelial growth factor (VEGF). Together with other growth factors and metabolites, VEGF causes endothelial cell proliferation, vasodilation, recruitment of inflammatory cells, and increased vascular permeability, leading to breakdown of the blood-retinal barrier. This allows trans-cellular exudation into the interstitial space resulting in diabetic macular edema (DME). For over 3 decades the standard treatment for DME has been laser photocoagulation. Though laser reduces the incidence of vision loss by 50%, few eyes with diffuse edema experience improved vision. This has led physicians to use the VEGF-binding drugs pegaptanib, ranibizumab, and aflibercept, each of which has been approved for the treatment of exudative macular degeneration, and bevacizumab which is commonly used off-label for a variety of chorioretinal disorders. Intravitreal administration of each drug frequently causes rapid improvement of DME with sustained improvement in vision through 2 years. Though these drugs significantly outperform laser photocoagulation over treatment periods of 1 year of less, the advantages appear to lessen when trials approach 2 years. Further studies to better determine relative efficacies of anti-VEGF drugs and laser photocoagulation are continuing.     

Anti–neuropilin‐1 peptide inhibition of synoviocyte survival,angiogenesis, and experimental arthritis

Objective

To delineate the role of neuropilin‐1 (NP‐1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation and to determine whether blockade of NP‐1 could suppress synoviocyte survival and angiogenesis.

Methods

VEGF_111–165 peptide, which encompasses the NP‐1 binding domain of VEGF₁₆₅, was generated by cleaving VEGF₁₆₅ with plasmin. The effect of this peptide on the interaction between VEGF₁₆₅ and its receptor was determined by ¹²⁵I‐VEGFR binding assay. Assays to determine synoviocyte apoptosis, adhesion, and migration were performed in the presence of VEGF₁₆₅ and/or the peptide. VEGF₁₆₅‐induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of endothelial cells (ECs). Mice were immunized with type II collagen to induce experimental arthritis.

Results

VEGF_111–165 peptide specifically inhibited the binding of ¹²⁵I‐VEGF₁₆₅ to NP‐1 on rheumatoid synoviocytes and ECs. The peptide eliminated the VEGF₁₆₅‐mediated increase in synoviocyte survival and activation of p‐ERK and Bcl‐2. The peptide also completely inhibited a VEGF₁₆₅‐induced increase in synoviocyte adhesion and migration. In addition, the anti–NP‐1 peptide blocked VEGF₁₆₅‐stimulated proliferation, capillary tube formation, and wounding migration of ECs in vitro. VEGF₁₆₅‐induced neovascularization in a Matrigel plug in mice was also blocked by treatment with the peptide. Finally, subcutaneous injection of anti–NP‐1 peptide suppressed arthritis severity and autoantibody formation in mice with experimental arthritis and inhibited synoviocyte hyperplasia and angiogenesis in arthritic joints.

Conclusion

Anti–NP‐1 peptide suppressed VEGF₁₆₅‐induced increases in synoviocyte survival and angiogenesis, and thereby blocked experimental arthritis. Our findings suggest that anti–NP‐1 peptide could be useful in alleviating chronic arthritis.

     

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF₁₆₅-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of ¹²⁵I-VEGF to 50% at 5 μg/ml and cross-linking of ¹²⁵I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of ¹²⁵I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cγ (PLCγ) and DNA synthesis were augmented by heparin. However, basal PLCγ tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin. This revised version was published online in June 2006 with corrections to the Cover Date.     

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165

Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2'fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF(165) aptamer binds specifically to the VEGF(165) isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF(165)-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF(165) for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF(164)-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF(165)-aptamer complex.     

Advances in the treatment of diabetic retinopathy

As the diabetes epidemic in the United States continues to worsen, so too does the prevalence of diabetic retinopathy (DR). DR is divided broadly into nonproliferative and proliferative stages, with or without vision-threatening macular edema. Progression to proliferative DR is associated with vision loss that is often irreparable, and a rapid decline in health-related quality of life. Vascular endothelial growth factor (VEGF)-A is upregulated in the diabetic eye, and has been identified as a key driver of DR pathogenesis. With this perspective, we review the published phase III clinical trial data of anti-VEGF therapies approved for the treatment of DR in the United States. Using the Early Treatment Diabetic Retinopathy Study Diabetic Retinopathy Severity Scale, in which an improvement of ≥2 steps is considered clinically significant, approximately one-third of patients with DR and macular edema experience this level of improvement after 1 year of treatment with either ranibizumab or aflibercept. The rates of clinically significant DR improvement with ranibizumab could be twice that in the subgroup of patients with moderately severe or severe nonproliferative DR and macular edema. These clinical trial data indicate that intraocular inhibition of VEGF is a rational approach for the management of DR.     

Diabetic retinopathy is associated with a switch in splicing from anti- to pro-angiogenic isoforms of vascular endothelial growth factor

Aims/hypothesis Proliferative diabetic retinopathy results from excess blood vessel growth into the vitreous fluid of the eye. Retinal angiogenesis is regulated by expression of vascular endothelial growth factor (VEGF), and many studies have shown that VEGF is critically involved in proliferative diabetic retinopathy. VEGF is alternatively spliced to form the angiogenic (VEGF_xxx) and potentially anti-angiogenic (VEGF_xxxb) family of isoforms. The VEGF_xxxb family is found in normal tissues, but down-regulated in renal and prostate cancer. Previous studies on endogenous expression of VEGF in the eye have not distinguished between the two families of isoforms.Methods We measured VEGF_xxxb isoform expression in normal human eye tissue (lens, sclera, retina and iris) and vitreous fluid using enzyme-linked immunosorbent assay and Western blotting with a VEGF_xxxb-specific antibody.Results VEGF_xxxb protein was expressed in lens, sclera, retina, iris and vitreous fluid. Multiple isoforms were seen, including VEGF₁₆₅b, VEGF₁₂₁b, VEGF₁₄₅b, VEGF₁₈₃b and VEGF₁₈₉b. In non-diabetic patients, 64±7% of the VEGF in the vitreous was VEGF_xxxb (n=18), whereas in diabetic patients only 12.5±3.6% of total VEGF was VEGF_xxxb.Conclusions/interpretation Since VEGF_xxxb inhibits VEGF_xxx-induced angiogenesis in a one-to-one stoichiometric manner, these results show that in the eye of diabetic patients VEGF splicing was switched from an anti-angiogenic to a pro-angiogenic environment. This occurred through changes to the ratio of VEGF_xxx : VEGF_xxxb. Alterations to splicing, and through that to the balance of VEGF isoforms, could therefore be a potential therapeutic strategy for diabetic retinopathy.Konopatskaya and Perrin are joint first authors.     

Vascular Endothelial Growth Factor (VEGF) in Crohn's Disease Increased Production by Peripheral Blood Mononuclear Cells and Decreased VEGF165 Labeling of Peripheral CD14+ Monocytes

Recently, increased serum levels of vascularendothelial growth factor (VEGF) have been shown inpatients with inflammatory bowel disease. The origins ofthe circulating VEGF are still not described. Monocytes play an important role in the inflammatoryprocess. VEGF binding to monocytes mediates monocyterecruitment and activation. The present studyinvestigates the VEGF production of peripheral bloodmononuclear cells and the ability of peripheral monocytesto bind VEGF₁₆₅ in patients with Crohn'sdisease. Nineteen patients with Crohn's disease and 10healthy volunteers were studied. VEGF₁₆₅labeling of CD14⁺ monocytes was measured using two-color flow cytometry.Density of VEGF labeling was expressed as the meanfluorescence intensity (MFI). Furthermore, VEGF levelswere determined in culture supernatants of unstimulated peripheral blood mononuclear cells. VEGF inculture supernatants was measured using a solid-phaseenzyme-linked immunosorbent assay. There was asignificantly decreased VEGF₁₆₅ labeling ofmonocytes of patients with active Crohn's disease (MFI:369.9 ± 121.6, N = 7, P < 0.002) compared topatients with inactive disease (MFI: 457.7 ±74.5, N = 6) and healthy controls (MFI: 542.9 ±96.2, N = 10). Unstimulated peripheral blood mononuclear cellsof patients with active Crohn's disease producedsignificantly higher amounts of VEGF (1142.6 ±483.9 pg/ml, N = 12, P < 0.001) compared withperipheral blood mononuclear cells of healthy volunteers(113.4 ± 101.8 pg/ml, N = 10). VEGF production byperipheral blood mononuclear cells of patients withactive disease was significantly increased compared to patients with quiescent disease (261.6± 254.8 pg/ml, N = 7, P < 0.001). Inconclusion, our data describe peripheral bloodmononuclear cells as one of the origins of the elevatedVEGF serum levels in patients with active Crohn's disease.Furthermore, a decrease in VEGF₁₆₅ bindingsites on peripheral monocytes of patients with activeCrohn's disease has been shown. The study underlines theimportant role of VEGF in Crohn's disease.     

Semaphorin 3A is expressed in human osteoarthritic cartilage and antagonizes vascular endothelial growth factor 165–promoted chondrocyte migration: An implication for chondrocyte cloning

Objective

Vascular endothelial growth factor 165 (VEGF₁₆₅) and its receptors, including neuropilin 1 (NRP‐1), are overexpressed in human osteoarthritic (OA) articular cartilage, although their functional roles in the cartilage are not fully understood. An axon‐guidance molecule, semaphorin 3A (Sema3A), which binds to NRP‐1, acts as an antagonist of VEGF signaling in endothelial cells. The aim of this study was to examine the expression of Sema3A and the functions of the VEGF₁₆₅/Sema3A/NRP‐1 axis in OA cartilage.

Methods

The expression of Sema3A in OA and normal cartilage samples was examined by real‐time polymerase chain reaction and immunohistochemical analyses. Functional analyses of VEGF₁₆₅ and Sema3A were carried out using OA chondrocytes in culture. The migration activity of chondrocytes was examined in a monolayer wound assay. The effects of Sema3A on VEGF₁₆₅‐induced up‐regulation of matrix metalloproteinases (MMPs) and intracellular signaling were also studied in cultured chondrocytes.

Results

Sema3A expression was significantly elevated in OA cartilage as compared to normal cartilage. Sema3A immunoreactivity directly correlated with the Mankin score and with chondrocyte cloning. VEGF₁₆₅ promoted the migration of chondrocytes, and this activity was suppressed by VEGF receptor 2 tyrosine kinase inhibitors. Sema3A antagonized the chondrocyte migration promoted by VEGF₁₆₅, and the activity was blocked by a selective inhibitor of, or small interfering RNA for, Sema3A. VEGF₁₆₅‐induced overexpression of MMPs and phosphorylation of ERK and focal adhesion kinase in chondrocytes were inhibited by Sema3A.

Conclusion

Our findings provide the first evidence that Sema3A is overexpressed, with a direct correlation with cloning, in OA cartilage and that it suppresses the VEGF₁₆₅‐promoted migration of chondrocytes. Our findings also suggest that Sema3A plays a role in chondrocyte cloning through inhibition of cell migration in OA cartilage.

     

Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-induced angiogenesis and represents a potential target for anticancer therapy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF₁₂₁ and VEGF₁₆₅ isoforms. Three of them completely neutralized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 μg/ml. The most potent one, with a dissociation constant (K _d) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A complete inhibition of the angiogenic response was obtained by daily intraperitoneal injections of 10 μg mAb/mouse. Angiogenesis induced by basic fibroblast growth factor was not inhibited by the mAb. Epitope mapping of the mAb, performed by competitive enzyme-linked immunosorbent assay and Western blot analysis, showed that it did not bind to the reduced and denatured monomer of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop β5 to β6 of VEGF, resulted in the complete loss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeric form, encompassing one of the loop regions involved in KDR receptor binding. The mAb with its strong neutralizing properties represents a useful agent for effective blocking of VEGF-mediated tumor neovascularization. Received: 11 November 1998 / Accepted: 16 December 1998     

Intravitreal anti-VEGF drugs as adjuvant therapy in diabetic retinopathy surgery

The use of intravitreal anti vascular endothelial growth factor (anti-VEGF) drugs such as pegaptanib, ranibizumab and bevacizumab has been widely reported to treat complications such as macular edema and rubeosis. During the past few years they have also been used as an adjuvant therapy to reduce intraocular bleeding during vitrectomy in eyes with proliferative diabetic retinopathy as well as to reduce the occurrence of vitreous haemorrhages in vitrectomized eyes and facilitate glaucoma surgery. In this paper we review the use of anti VEGF drugs in the surgical management of diabetic retinopathy related complications.     

Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan sulphate

Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF₁₆₅) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF₁₆₅. Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF₁₆₅ against thermal and enzyme degradation in vitro, and isolated VEGF₁₆₅ from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n?=?8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.