Vascular Endothelial Growth Factor Trap Research Articles

Pre-Clinical Studies of a Novel Bispecific Fusion Protein Targeting C3b and VEGF in Neovascular and Nonexudative AMD Models.

KNP-301 is a bi-specific fragment crystallizable region (Fc) fusion protein, which inhibits both C3b and vascular endothelial growth factor (VEGF) simultaneously for patients with late-stage age-related macular degeneration (AMD). The present study evaluated in vitro potency, in vivo efficacy, intravitreal pharmacokinetics (IVT PK), and injectability of KNP-301. C3b and VEGF binding of KNP-301 were assessed by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), and cellular bioassays. A laser-induced choroidal neovascularization (CNV) model and a sodium iodate-induced nonexudative AMD model were used to test the in vivo efficacy of mouse surrogate of KNP-301. Utilizing fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT) scans, the reduction in disease lesions were analyzed in a CNV mouse model. In the nonexudative AMD mouse model, outer nuclear layer (ONL) was assessed by immunofluorescence staining. Lastly, intravitreal pharmacokinetic study was conducted with New Zealand white rabbits via IVT administration of KNP-301 and injectability of KNP-301 was examined by a viscosity test at high concentrations. KNP-301 bound C3b selectively, which resulted in a blockade of the alternative pathway, not the classical pathway. KNP-301 also acted as a VEGF trap, impeding VEGF-mediate signaling. Our dual-blockade strategy was effective in both neovascular and nonexudative AMD models. Moreover, KNP-301 had an advantage of potentially less frequent dosing due to the long half-life in the intravitreal chamber. Our viscosity assessment confirmed that KNP-301 meets the criteria of the IVT injection. Unlike current therapies, KNP-301 is expected to cover patients with late-stage AMD of both neovascular and nonexudative AMD, and its long-term PK profile at the intravitreal chamber would allow convenience in the dosing interval of patients.

The use of aflibercept (VEGF trap) in mitigating sulfur mustard-induced corneal neovascularization in a rabbit model.

Sulfur mustard (SM)-induced ocular injury is characterized by an acute inflammatory response that may become chronic or enter a latent phase with delayed pathology. This study aimed to evaluate the efficacy of ziv-aflibercept and aflibercept in preventing and ameliorating corneal neovascularization (NV), respectively, following chemical eye exposure to SM vapor in a rabbit model. Chemical SM ocular insult was induced in the right eye of rabbits. A single application of ziv-aflibercept was administered 2h or 9 days post-exposure. A single subconjunctival aflibercept treatment in an ocular formulation was administered 4 weeks after SM vapor exposure and subsequent to an initial 1-week treatment with 0.1 % dexamethasone. Clinical monitoring was performed 5-12 weeks post-exposure, and digital corneal pictures were taken to assess the extent of NV. The rabbits were euthanized and the corneas were processed for histological assessment. Treatment with ziv-aflibercept 2h and 9 days post-exposure moderately reduced insult severity and partially delayed or prevented corneal NV. Aflibercept application 4 weeks post-exposure significantly reduced the extent of NV for 8 weeks. The substantial decrease in existing corneal NV in this group was confirmed by histology. These results reveal the powerful anti-angiogenic efficacy of the VEGF-trap for ameliorating existing NV as opposed to preventing NV development, revealing the ability of this treatment to mitigate corneal NV.

Structural Characterization of RC28-E, a Recombinant Fusion Protein With Dual Targets on VEGF and FGF2

Vascular endothelial growth factor (VEGF) and fibroblast growthfactor (FGF) play important roles in angiogenesis-related diseases. RC28-E is a soluble fusion protein composed of the human VEGF receptor 1 (VEGFR1) extracellular domain 2 (ECD 2), VEGFR2 ECD 3, FGFR1 ECDs 2 and 3, and the Fc regions of human immunoglobulin G1. By targeting both VEGF and FGF2, RC28-E may represent a useful antiangiogenetic agent, but structural and functional characterizations of this fusion protein are needed. Liquid chromatography–tandem mass spectrometry, size exclusion high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing, and bio-layer interferometry were used to characterize the properties of RC28-E. The purity of RC28-E was confirmed to be 98% or greater. The glycosylation modification of RC28-E was found to be very complicated, with 11 potential N-linked glycosylation points and 23 types of N-glycans, causing high heterogeneity of the protein. The primary modifications of the amino acid sequence of RC28-E protein included C-terminal K truncation, N-deamidation, and M-oxidation modification. Notably, RC28-E demonstrated a higher affinity for both VEGF and FGF2 than VEGF trap or FGF trap for their respective targets.

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo. nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro. AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 108 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

Safety and efficacy review of aflibercept for the treatment of metastatic colorectal cancer

ABSTRACT Introduction Anti-angiogenic drugs are an efficacious class of therapy in the treatment of patients with metastatic colorectal cancer (mCRC). Aflibercept, a vascular endothelial growth factor (VEGF) trap which binds the angiogenic factors VEGF-A, VEGF-B, and placental growth factor (PIGF) is approved in combination with FOLFIRI chemotherapy following progression after an oxaliplatin-containing regimen. Areas covered This report provides a review of the practice-changing clinical studies which have established the use of anti-angiogenic therapy as second-line therapy in mCRC including aflibercept with FOLFIRI (5FU, leucovorin, irinotecan). This review also evaluates aflibercept with other chemotherapy regimens as well as efficacy and safety data from real-world studies. Expert opinion Aflibercept in combination with FOLFIRI chemotherapy is an established safe and efficacious regimen for the treatment of mCRC as second-line chemotherapy. Although several toxicities have been described, the majority are either low grade or manageable by drug cessation and supportive therapies. For optimal outcomes, patient selection and close observation of toxicities is essential.

The Eye in Internal Medicine: Diabetic retinopathy

Diabetic retinopathy has seen tremendous progress in diagnostic tools and treatment in recent 15 years. Sight threatening stages like proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) can be treated much more effectively now. The recognition of vascular endothelial growth factor (VEGF) as a driver of proliferation and macular edema has led to the development of VEGF inhibiting drugs such as antibodies (Bevacizumab), fragments of an antibody (Ranibizumab), or a so called VEGF trap (Aflibercept). Laser treatment is no longer a gold standard. Today laser therapy is part of a combined treatment strategy. DME can be monitored quantitatively by ocular coherence tomography (OCT) measurements. For non-responders to VEGF inhibiting drugs, we have secondline corticosteroidal implants. However, screening examinations and early diagnosis of DME and PDR remain crucial. The same accounts for the collaboration of opthalmologists, general practioners, and diabetologists. Good control of diabetes and blood pressure is still important.

The vascular endothelial growth factor trap aflibercept induces vascular dysfunction and hypertension via attenuation of eNOS/NO signaling in mice.

Aflibercept, as a soluble decoy vascular endothelial growth factor receptor,Which has been used as afirst-line monotherapy for cancers.Aflibercept often causes cardiovascular toxicities including hypertension, but the mechanisms underlyingaflibercept-induced hypertension remain unknown. In this study we investigated the effect of short-term and long-term administrationof aflibercept on blood pressure (BP), vascular function, NO bioavailability, oxidative stress and endothelin 1 (ET-1) in mice and cultured endothelial cells. We showed that injection of a single-dose of aflibercept (18.2, 36.4 mg/kg, iv) rapidly and dose-dependently elevated BP in mice. Aflibercept treatment markedly impaired endothelial-dependent relaxation (EDR) andresulted in NADPH oxidases 1 (NOX1)- and NADPH oxidases4 (NOX4)-mediated generation of ROS, decreased theactivation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) concurrently with a reduction in nitric oxide (NO) production and elevation of ET-1levelsin mouse aortas;theseeffects were greatly attenuated by supplementation ofL-arginine (L-arg, 0.5 or 1.0 g/kg, bid, ig) before aflibercept injection. Similar results were observed in L-arg-pretreated cultured endothelial cells, showing markedly decreased ROS accumulation and AKT/eNOS/NO signaling impairment induced by aflibercept. In order to assess the effects of long-term aflibercept on hypertension and to evaluate the beneficial effects ofL-arg supplementation, we administered these two drugs to WT mice for up to 14 days(at an interval of two days). Long-term administration of aflibercept resulted in a sustained increase in BP and a severely impaired EDR, which are associated with NOX1/NOX4-mediated production of ROS, increase in ET-1, inhibition of AKT/eNOS/NO signaling and a decreased expression of cationic amino acid transporter (CAT-1). The effects causedby long-term administration were greatly attenuated by L-arg supplementation in a dose-dependent manner. We conclude that aflibercept leads to vascular dysfunction and hypertension by inhibiting CAT-1/AKT/eNOS/NO signaling, increasing ET-1, and activating NOX1/NOX4-mediated oxidative stress, which can be suppressed by supplementation ofL-arg. Therefore, L-arg could be a potential therapeutic agent for aflibercept-induced hypertension.

Efficacy of Aflibercept Treatment and Its Effect on the Retinal Perfusion in the Oxygen-Induced Retinopathy Mouse Model of Retinopathy of Prematurity

Introduction: Bevacizumab and ranibizumab, which are anti-vascular endothelial growth factor (VEGF) medications, are used frequently in the treatment for retinopathy of prematurity (ROP) in infants. Aflibercept, or VEGF Trap, has been used anecdotally, but translation and clinical studies are lacking. Objective: This study investigates the efficacy of aflibercept at reducing areas of non-perfused retina and studies its effect on normal angiogenesis in the oxygen-induced retinopathy mouse model of ROP. Methods: C57BL/6 J mice were assigned to room air control (n = 21 eyes) or hyperoxia with 75% oxygen (n = 84 eyes). The hyperoxic mice were assigned to 1 of 3 groups: 0 ng (n = 14 eyes), 100 ng (n = 35 eyes), or 1,000 ng (n = 35 eyes) of intravitreal aflibercept administered on postnatal day 14. Eyes were enucleated at PN17 and PN25 postinjection. Retinas were stained with anti-collagen IV antibody and photographed with microscopy. Areas of perfused and non-perfused retina were quantified using ImageJ software. Statistical comparisons were made using ANOVA with Tukey post hoc comparisons. Results: At PN17, there was no significant difference in the area of non-perfused retina between the hyperoxic control and the 100 and 1,000 ng aflibercept groups. At PN25, the 100 ng (p < 0.05) and 1,000 ng (p = 0.008) treatment groups displayed less non-perfusion compared to hyperoxic controls. At the 1,000 ng dose, there was increased non-perfusion compared to the 100 ng dose (p = 0.02). There was reduced non-perfusion by PN25 compared to PN17 for the 100 ng group (p < 0.05), with no difference in the 1,000 ng group. Conclusions: The study shows that the area of non-perfused retina decreases effectively with aflibercept at PN25 with 100 ng dosage. With the 1,000 ng dosage, there is an inhibition of the physiologic angiogenesis with a higher area of non-perfused retina.

A Biopolymer‐Fused Form of VEGF‐B for Treatment of Preeclampsia

Preeclampsia is a complex syndrome hallmarked by the development of elevated blood pressure and proteinuria during the second and third trimester of pregnancy. There is an increase in a number of anti‐angiogenic factors in these women. These factors are believed to drive endothelial dysfunction and contribute to the elevation in blood pressure and other maternal symptoms. Vascular Endothelial Growth Factor (VEGF) is a family of growth factors that play a role in angiogenesis, endothelial cell health and maintenance. The VEGF family of proteins have varying affinities for the two major VEGF receptors, Flt‐1 and Flk‐1. Flk‐1 signaling is the major driver of VEGF‐induced angiogenesis and increased vascular permeability, whereas Flt‐1 is less angiogenic and may serve to modulate bioavailable VEGF. Furthermore, the soluble extracellular domain of Flt‐1 (sFlt‐1) is increased in early‐onset preeclampsia and serves as a VEGF trap. Previous studies have shown that administration of the isoform VEGF‐A in the Reduced Uterine Perfusion Pressure (RUPP) rat model significantly reduced blood pressure. While effective at reducing placental‐ischemia induced hypertension, VEGF‐A at high doses can produce unwanted side effects including unopposed angiogenesis and increased vascular permeability. These undesired effects of VEGF‐A are believed to be mediated by the Flk‐1 receptor. VEGF‐B, another isoform of VEGF, has a high affinity for Flt‐1 and s‐Flt1, but does not bind Flk‐1. We hypothesize that VEGF‐B will liberate endogenous VEGF‐A by successfully antagonizing circulating sFlt‐1, without inducing unwanted angiogenesis and increased vascular permeability. The purpose of this study was to determine the in‐vitro activity, pharmacokinetics, and biodistribution of a biopolymer delivered form of VEGF‐B compared to our previously published biopolymer‐delivered VEGF‐A. Both VEGF‐B and VEGF‐A were conjugated to an elastin like polypeptide (ELP) in order to aid in purification and delivery of the proteins. Angiogenic potential of ELP‐VEGF‐B and ELP‐VEGF‐A was evaluated using a tube formation assay in human umbilical vein endothelial cells (HUVECs). Pharmacokinetics, biodistribution, and placental transfer of fluorophore‐labeled ELP‐VEGF‐B were determined after intravenous (IV) or subcutaneous (SC) administration into pregnant rats. Tubule formation analysis indicated that ELP‐VEGF‐B had less angiogenic potential than ELP‐VEGF‐A. IV administration of ELPVEGF‐B cleared from the plasma in a bi‐phasic manner indicative of a two‐compartment pharmacokinetic model. Whereas, SC administration of ELP‐VEGF‐B showed accumulation of protein in the plasma over the first 24h, followed by a slow clearance. ELP‐VEGF‐B accumulated predominantly in the maternal liver, with lower but significant accumulation in the kidneys, placenta, and brain. Importantly, the biopolymer‐fused ELP‐VEGF‐B did not cross the placental barrier into the fetal circulation. In conclusion, our data suggest that ELP‐VEGF‐B has a similar pharmacokinetic profile to that of VEGF‐A, but does not induce as robust of an angiogenic response and may prove to be a safer therapeutic agent.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of diffusion-weighted magnetic resonance imaging in the evaluation of the response to AAV2-VEGF-Trap neoadjuvant treatment in a triple-negative breast cancer animal model.

ObjectiveEvaluation of the efficacy of adeno‐associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap (AAV2‐VEGF‐Trap) alone or combination with paclitaxel in a mouse model of triple‐negative breast cancer (TNBC) using diffusion‐weighted magnetic resonance imaging (DW‐MRI) and in vivo fluorescence imaging.Materials and MethodsXenografted TNBC tumors were established by subcutaneous injection of MDA‐MB‐231 cells into nude mice. Tumors were treated with AAV2‐VEGF‐Trap, paclitaxel, AAV2‐VEGF‐Trap combined with paclitaxel and control. A 7.0‐Tesla magnetic resonance (MR) was used to obtain the apparent diffusion coefficient (ADC) values and ΔADC values. In vivo fluorescence imaging coupled with the optical imaging probe AngioSense680 EX was acquired to obtain average luminous intensity values. Immunohistochemical staining of tumor Ki‐67 and vascular endothelial cell marker antigen (CD31) were used to evaluate the effects on tumor proliferation and angiogenesis.ResultsThe combination of AAV2‐VEGF‐Trap with paclitaxel exhibited greater tumor growth inhibition compared with the other groups. The ADC values in the paclitaxel group and the AAV2‐VEGF‐Trap in combination with paclitaxel group were significant greater compared with the control group, and the ΔADC values of all treatment groups were significantly increased compared with the control group on the 14th day after administration. Decreased microvessel density and luminous intensity in the treatment groups that contain AAV2‐VEGF‐Trap were observed. Reduced proliferation activity was noted in groups that contained paclitaxel.ConclusionAAV2‐VEGF‐Trap inhibits TNBC growth though inhibiting tumor neovascularization with a single intravenous injection, and AAV2‐VEGF‐Trap exhibits a synergistic effect when used in combination with paclitaxel for TNBC neoadjuvant therapy. In vivo fluorescence imaging can detect the anti‐angiogenesis effect of AAV2‐VEGF‐Trap early and noninvasively. DW‐MRI can longitudinally monitor the neoadjuvant efficacy of TNBC.

Therapeutic Inhibition of VEGF Signaling and Associated Nephrotoxicities.

Inhibition of vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling is a common therapeutic strategy in oncology, with new drugs continuously in development. In this review, we consider the experimental and clinical evidence behind the diverse nephrotoxicities associated with the inhibition of this pathway. We also review the renal effects of VEGF inhibition's mediation of key downstream signaling pathways, specifically MAPK/ERK1/2, endothelial nitric oxide synthase, and mammalian target of rapamycin (mTOR). Direct VEGFA inhibition via antibody binding or VEGF trap (a soluble decoy receptor) is associated with renal-specific thrombotic microangiopathy (TMA). Reports also indicate that tyrosine kinase inhibition of the VEGF receptors is preferentially associated with glomerulopathies such as minimal change disease and FSGS. Inhibition of the downstream pathway RAF/MAPK/ERK has largely been associated with tubulointerstitial injury. Inhibition of mTOR is most commonly associated with albuminuria and podocyte injury, but has also been linked to renal-specific TMA. In all, we review the experimentally validated mechanisms by which VEGFA-VEGFR2 inhibitors contribute to nephrotoxicity, as well as the wide range of clinical manifestations that have been reported with their use. We also highlight potential avenues for future research to elucidate mechanisms for minimizing nephrotoxicity while maintaining therapeutic efficacy.

Adeno-associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap: a new treatment option for glioma

ABSTRACTBackground: Adeno-associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap (AAV2-VEGF-Trap) has been reported to inhibit the growth of primary tumor as well as distant metastasis in 4T1 metastatic breast cancer models. The aim of this study was to investigate the inhibiting efficacy of AAV2-VEGF-Trap for glioma.Methods: The intracranial transplanted model of glioma in rats was established. They were treated with AAV2-VEGF-Trap, bevacizumab (BEV), temozolomide (TMZ), TMZ combined with AAV2-VEGF-Trap, TMZ combined with BEV and the control group, respectively. A 7.0 Tesla magnetic resonance (MR) was used to assess the tumor volumes and obtain the apparent diffusion coefficient (ADC) values. Immunohistochemical and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining were used to evaluate the effects on tumor angiogenesis, proliferation and apoptosis.Results: The combination of TMZ with AAV2-VEGF-Trap or BEV showed greater tumor growth inhibition than the other groups, and the ADC values in these two groups were larger than that of the control group. The decreased microvessel density in treatment groups which contain AAV2-VEGF-Trap or BEV was observed. The reduced proliferation activity in groups containing TMZ and increased apoptotic tumor cells in TMZ combined with AAV2-VEGF-Trap group and TMZ combined with BEV group were detected. In addition, there were no differences in antitumor effect, ADC values, Ki-67 and CD31 staining and apoptosis analysis between the two combined therapy groups.Conclusion: AAV2-VEGF-Trap has an obvious anti-angiogenic effect and inhibits the growth of glioma just by a single intravenous injection, which is similar to BEV. Moreover, there is a synergistic antitumor effect between AAV2-VEGF-Trap and TMZ.

Protective effects of a novel drug RC28-E blocking both VEGF and FGF2 on early diabetic rat retina.

To investigate protective effects of a novel recombinant decoy receptor drug RC28-E on retinal damage in early diabetic rats. The streptozotocin (STZ)-induced diabetic rats were randomly divided into 6 groups: diabetes mellitus (DM) group (saline, 3 µL/eye); RC28-E at low (0.33 µg/µL, 3 µL), medium (1 µg/µL, 3 µL), and high (3 µg/µL, 3 µL) dose groups; vascular endothelial growth factor (VEGF) Trap group (1 µg/µL, 3 µL); fibroblast growth factor (FGF) Trap group (1 µg/µL, 3 µL). Normal control group was included. At week 1 and 4 following diabetic induction, the rats were intravitreally injected with the corresponding solutions. At week 6 following the induction, apoptosis in retinal vessels was detected by TUNEL staining. Glial fibrillary acidic protein (GFAP) expression was examined by immunofluorescence. Blood-retinal barrier (BRB) breakdown was assessed by Evans blue assay. Ultrastructural changes in choroidal and retinal vessels were analyzed by transmission electron microscopy (TEM). Content of VEGF and FGF proteins in retina was measured by enzyme linked immunosorbent assay (ELISA). The retinal expression of intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), VEGF and FGF genes was examined by quantitative polymerase chain reaction (qPCR). TUNEL staining showed that the aberrantly increased apoptotic cells death in diabetic retinal vascular network was significantly reduced by treatments of medium and high dose RC28-E, VEGF Trap, and FGF Trap (all P<0.05), the effects of medium and high dose RC28-E or FGF Trap were greater than VEGF Trap (P<0.01). GFAP staining suggested that reactive gliosis was substantially inhibited in all RC28-E and VEGF Trap groups, but the inhibition in FGF Trap group was not as prominent. Evans blue assay demonstrated that only high dose RC28-E could significantly reduce vascular leakage in early diabetic retina (P<0.01). TEM revealed that the ultrastructures in choroidal and retinal vessels were damaged in early diabetic retina, which was ameliorated to differential extents by each drug. The expression of VEGF and FGF2 proteins was significantly upregulated in early diabetic retina, and normalized by RC28-E at all dosages and by the corresponding Traps. The upregulation of ICAM-1 and TNF-α in diabetic retina was substantially suppressed by RC28-E and positive control drugs. Dual blockade of VEGF and FGF2 by RC28-E generates remarkable protective effects, including anti-apoptosis, anti-gliosis, anti-leakage, and improving ultrastructures and proinflammatory microenvironment, in early diabetic retina, thereby supporting further development of RC28-E into a novel and effective drug to diabetic retinopathy (DR).

34 - Combined cell and gene therapy towards the treatment of age-related macular degeneration and diabetic retinopathy

Abnormal and leaky blood vessel growth (neovascularization, NV), caused by an up-regulation of VEGF, is the common cause for vascular leakage, vessel dilatation, tortuosity, haemorrhage, cell death and ultimately blindness in age-related macular degeneration and diabetic retinopathy. Current anti-VEGF treatments (e.g. Avastin) can improve visual function but only temporarily and therefore patients require monthly eye injections. In addition, the anti-VEGF therapies cause side effects such as stroke, gastrointestinal perforations and bleeding. To address these problems, we have generated a novel bi-functional VEGF trap (VEGF Sticky-trap) that is able to trap VEGF as well as bind (“stick”) to the extracellular matrix (ECM). This consequently allows inhibition of neovascularization only at the site of administration without causing systemic side effects as we have demonstrated in a retinopathy mouse model (OIR). To avoid the invasiveness of current therapies we have genetically engineered human embryonic cells that express VEGF Sticky-trap in an inducible manner and further differentiated the cells to retinal pigment epithelium. We have shown that VEGF Sticky-trap expressed by these cells in-vitro is able to bind to ECM and trap soluble VEGF only upon doxycycline induction. In addition, we have demonstrated that these cells incorporate into the subretinal space long-term (up to 10 month), express VEGF Sticky-trap that a) remains bound to eye ECM and b) inhibits laser-induced chorodial NV. Here we have shown that the combination of stem cell and novel anti-VEGF gene therapy can inhibit neovascularization in a controlled and long-term manner; potentially improve vision, while avoiding the risk of side effects.

Angiogenic role of miR-20a in breast cancer.

BackgroundAngiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma.MethodsTube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples.ResultsIn vitro angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (p = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (p<0.001). This association was independent of tumor subtype and VEGFA expression.ConclusionsTransfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.

Efficacy of Lenvatinib, a multitargeted tyrosine kinase inhibitor, on laser-induced CNV mouse model of neovascular AMD

Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ± 0.2939 vs 5.202 ± 0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ± 0.4334 vs 3.122 ± 0.3466 (P = .0064) at day 14th, 1.401 ± 0.6577 vs 2.781 ± 0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ± 0.77 h, the Tmax was 4.60 ± 0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

Germany: Rowa – plastics masterbatch & colour matching

Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ± 0.2939 vs 5.202 ± 0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ± 0.4334 vs 3.122 ± 0.3466 (P = .0064) at day 14th, 1.401 ± 0.6577 vs 2.781 ± 0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ± 0.77 h, the Tmax was 4.60 ± 0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

Early Actions of Anti–Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Antiangiogenic Activity of rTRAIL is Potentiated by Sflt-1, a VEGF Trap

Objective: Anti-angiogenesis and pro-apoptosis are the two processes which are strategically used to target tumors. The soluble form of VEGF receptor 1 (Flt-1) is a modulator of VEGF activity and could be useful as a trap to intrinsically sequester VEGF in tumor cells. In the present study, we have investigated the role of rTRAIL in inhibiting VEGF-mediated angiogenesis and its synergistic potentiation by sFlt-1. Methods: We have expressed human recombinant TRAIL, to study its role in anti-angiogenesis and a 2-domain active variant of sFlt-1 by bacterial expression to be used as a trap for VEGF. 3[H] thymidine incorporation assay was used to confirm the inhibitory activity of sFlt-1 and/or rTRAIL on cell proliferation. The synergistic anti-migratory and anti-angiogenic activity of sFlt-1 and rTRAIL were assessed using endothelial cell wound healing and tube formation assay in-vitro and by rat corneal micropocket assay in-vivo. Results: Inhibition of cell proliferation was evident in sFlt-1 and rTRAIL treated cells in a dose -dependent manner with more than 60% reduction in proliferation rate as compared to the tumor cells treated with sFlt-1 and rTRAIL alone. Furthermore, the anti-metastatic and anti-angiogenic activity of sFlt-1 and rTRAIL in combination was evident in endothelial wound healing and tube formation with a significant reduction in the number of cells migrated into the wounded area and the number of honey comb-like structures with the length of the tubes formed to mimic blood vessel formation in-vitro respectively. The in-vivo corneal micropocket assay confirmed the anti-angiogenic effect of sFlt-1 and rTRAIL in combination. Conclusion: The synergistic role of sFlt-1 and rTRAIL as the potential inhibitors of VEGF- mediated angiogenesis could be therapeutically exploited.

Examination of the foreign body response to biomaterials by nonlinear intravital microscopy.

Implanted biomaterials often fail because they elicit a foreign body response (FBR) and concomitant fibrotic encapsulation. To design clinically relevant interference approaches, it is crucial to first examine the FBR mechanisms. Here, we report the development and validation of infrared-excited nonlinear microscopy to resolve the three-dimensional (3D) organization and fate of 3D-electrospun scaffolds implanted deep into the skin of mice, and the following step-wise FBR process. We observed that immigrating myeloid cells (predominantly macrophages of the M1 type) engaged and became immobilized along the scaffold/tissue interface, before forming multinucleated giant cells. Both macrophages and giant cells locally produced vascular endothelial growth factor (VEGF), which initiated and maintained an immature neovessel network, followed by formation of a dense collagen capsule 2–4 weeks post-implantation. Elimination of the macrophage/giant-cell compartment by clodronate and/or neutralization of VEGF by VEGF Trap significantly diminished giant-cell accumulation, neovascularization and fibrosis. Our findings identify macrophages and giant cells as incendiaries of the fibrotic encapsulation of engrafted biomaterials via VEGF release and neovascularization, and therefore as targets for therapy.