Mouse Vascular Endothelial Growth Factor Research Articles

MP03-03 TRANSCRIPTOME ANALYSIS IN LUMBOSACRAL DORSAL ROOT GANGLIA REVEALS KEY MOLECULAR CHANGES IN ANIMAL MODELS OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME (UCPPS)

You have accessJournal of UrologyCME1 Apr 2023MP03-03 TRANSCRIPTOME ANALYSIS IN LUMBOSACRAL DORSAL ROOT GANGLIA REVEALS KEY MOLECULAR CHANGES IN ANIMAL MODELS OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME (UCPPS) Sathish Kumar Yesupatham and Alison Xiaoqiao Xie Sathish Kumar YesupathamSathish Kumar Yesupatham More articles by this author and Alison Xiaoqiao XieAlison Xiaoqiao Xie More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with UCPPS experience chronic pelvic pain and lower urinary tract symptoms (LUTS), such as urinary frequency, urgency, and nocturia. UCPPS symptoms are often associated with nociceptive sensitization in the peripheral and central nervous system, which underlies symptoms of bladder overactivity, pelvic pain, and centralized pain. Understanding the molecular mechanism underlying the pathogenesis of nociceptive sensitization is necessary for finding better drug targets and neuromodulation protocols to treat UCPPS symptoms. However, despite transcriptomic analysis has been conducted on the L4 & L5 dorsal root ganglia (DRGs) in injury-induced pain models, genomic analysis focusing on bladder sensory pathways have not been performed. The current study aims to characterize transcriptome changes in lumbosacral DRGs receiving bladder and pelvic sensory input in a mouse model of UCPPS. METHODS: Male and female C57BL6/J mice were used in this study. UCPPS symptoms was induced by intravesical instillations of recombinant mouse vascular endothelial growth factor (VEGFA), an animal mode of UCPPS we previously established. As a read-out of visceral hypersensitivity, Von Frey assay was performed and compared between VEGF-instilled experimental group and saline-instilled control group. Mice were sacrificed after VEGF-induced visceral hypersensitivity was observed, and their L1, L2, L6, S1 and S2 DRGs was harvested for bulk-RNA sequencing. RESULTS: Samples were sequenced at a depth of 40 million reads per sample. Data analysis was performed using Basepair Tech platform. Ingenuity pathway analysis (Qiagen) enabled to visualize the biological network changes due to UCPPS. Spliced Transcripts Alignment to a Reference (mm10) revealed changes in the Nociceptor signaling. Principal component analysis score confirms the global gene expression changes in UCPPS-like experimental group. Upstream analysis on VEGF signaling revealed activation/ inhibition of transcriptional factors like ATF3, FOSB, and BCL2. CONCLUSIONS: Our data is the first characterization on key signaling pathway changes in lumbosacral DRGs during the pathogenesis of UCPPS symptoms in animal models. VEGF and its receptors have been recognized as a urine biomarker for UCPPS in patients. Ongoing transcriptome analysis such as Ingenuity pathway analysis and Gene set enrichment analysis will further reveal the etiology of pain centric UCPPS. Source of Funding: NIDDK 25B0965/5R01DK129260-02 (Xie) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e22 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sathish Kumar Yesupatham More articles by this author Alison Xiaoqiao Xie More articles by this author Expand All Advertisement PDF downloadLoading ...

Assessment of mouse VEGF neutralization by ranibizumab and aflibercept.

To assess the interaction between ranibizumab, aflibercept, and mouse vascular endothelial growth factor (VEGF), both in vivo and in vitro. In vivo, the effect of intravitreal injection of ranibizumab and aflibercept on oxygen induced retinopathy (OIR) and the effect of multiple intraperitoneal injections of ranibizumab and aflibercept on neonatal mice were assessed. In vitro, the interaction of mouse VEGF-A with aflibercept or ranibizumab as the primary antibody was analyzed by Western blot. In both experiments using intravitreal injections in OIR mice and multiple intraperitoneal injections in neonatal mice, anti-VEGF effects were observed with aflibercept, but not with ranibizumab. Western blot analysis showed immunoreactive bands for mouse VEGF-A in the aflibercept-probed blot, but not in the ranibizumab-probed blot. Aflibercept but not ranibizumab interacts with mouse VEGF, both in vivo and in vitro. When conducting experiments using anti-VEGF drugs in mice, aflibercept is suitable, but ranibizumab is not.

In Vitro and In Vivo Studies of a Heminecrolysin Toxin-VEGF Fusion Protein as a Novel Therapeutic for Solid Tumor Targeting.

Angiogenesis, the formation of new vessels, is a critical step in the malignancy progression of solid tumors. Many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Pharmaceutically, Vascular Endothelial Growth Factor (VEGF) can deliver coupled toxins to the tumor vessels through VEGF Receptors. In the current study, we designed, expressed, and assessed the in vitro and in vivo toxicities of a novel immunotoxin consisting of mouse VEGF and heminecrolysin toxin (mVEGF-HNc). The fusion protein was expressed in E. coli and purified via Ni+2 affinity chromatography. The biological activity of immunotoxin was evaluated on NIH/3T3 cells and TC1-tumorized mouse model. The mVEGF-NHc showed significant cytotoxicity on the cells as VEGFR-expressing cells. Moreover, the size of the tumor in the mVEGF-HNc-treated group started to reduce after six injections, while it continued to grow in the PBS-received mice. Efficacious targeting of solid tumor cells via mVEGF-HNc suggests its prospective therapeutic potential for cancer therapy.

Abstract LB057: Bevacizumab leaves a photoacoustic fingerprint in breast cancer mouse models

Abstract Background: Photoacoustic tomography (PAT) provides a direct readout of tumor haemoglobin (Hb) concentration and oxygenation. This readout could be used to provide an early indication of response to anti-angiogenic drugs, thus optimizing the management of these therapies. Experimental Procedures: Two cohorts of nude Balb/c were inoculated with either estrogen-dependent MCF-7 (n=7) or estrogen-independent MDA-MB-231 (n=19) cells. Mice were randomly split into Control (Ctrl) and Bevacizumab (Bev,10 mg/Kg, IP, weekly) groups. PAT was performed weekly, starting just before enrolment. Oxy- and deoxy-Hb were quantified in the tumour and reference areas. Total Hb (THb) and O2 saturation (SO2) were calculated. Blood samples and tissues were collected after imaging. Hypoxia (Hypoxyprobe and CA-IX) and vascular density/maturity (CD31 and ASMA) were analyzed by immunohistochemistry. Circulating levels of human and mouse vascular endothelial growth factor (hVEGF and mVEGF) and Hb were measured. Summary of New Data: Reflecting clinical observations, the MCF-7 Bev group and most of the mice from the MDA-MB-231 Bev group (8/11) showed no improvement in survival with Bev treatment. All resistant tumours (Bev MCF-7 and Bev MDA-MB-231 non-responders (Bev-NR)) showed an increase in hVEGF production (Ctrl vs Bev; 73.0513.06 vs. 497103.9 for MCF-7; 247.984.81 vs. 330.4 85.97 for Bev NR). MDA-MB-231 responders (Bev-R) showed a significant decrease in hVEGF (247.984.81 vs. 80.53 26.13). These results indicate that VEGF was only successfully sequestered in a small subset of animals (3/11) and that VEGF overload might be a resistance mechanism. At the final time point, PAT showed no difference between Ctrl and Bev-NR in THb for either group (THbMDA-MB-231 7.2±1.3 vs. 5.5±1.2). THb was found significantly increased in the Bev-R group by PAT (13.9±3.3 p-valuevs.Crtl=0.0339; p-valuevs.Bev=0.0072) and biochemical measurements. SO2 showed a slight but significant decrease between Ctrl and Bev-NR (0.58±0.03 vs. 0.48±0.01). A dramatic decrease was seen in the Bev-R group (0.31±0.07) and significantly different from Ctrl and Bev-NR. SO2 was sensitive to early changes, SO2 values increased in Ctrl and Bev-NR 48h after enrolment (p-values paired t-test pCtrl= 0.0134; pNR=0.0268) while Bev-R shows a trend (p=0.0649). Analysis of SO2MSOT over time shows a significant (p<0.0001) change in slope at 3 weeks after enrolment in the Bev-R group compared to either of the Ctrl or Bev-NR groups. Our results indicate that PAT SO2 can differentiate responders at very early stages of the treatment. Histologically, we observed fewer blood vessels but better structured in Bev-NR than in Bev-R, which showed higher vascular density and also higher levels of hypoxia makers. Statement of the Conclusions: Our results postulates PAT as a low-cost, label-free and non-invasive candidate to monitor response to Bevacizumab over time. Our results also indicate that tumours with functional vasculature may be resistant to Bevacizumab treatment. Citation Format: Isabel Quiros-Gonzalez, Michal R. Tomaszewski, Monika A. Golinska, Emma Brown, Laura Ansel-Bollepalli, Lina Hacker, Dominique-Laurent Couturier, Rosa M. Sainz, Sarah E. Bohndiek. Bevacizumab leaves a photoacoustic fingerprint in breast cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB057.

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity.

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.

In vivo solid tumor targeting with recombinant VEGF-diphtheria immunotoxin.

Objective(s):A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. Materials and Methods:In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Results:Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Conclusion:Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.

Novel Dual-Targeting Antibody Fragment IDB0062 Overcomes Anti-Vascular Endothelial Growth Factor Drug Limitations in Age-Related Macular Degeneration.

PurposeRepeated administration of anti–vascular endothelial growth factor drugs to treat age-related macular degeneration leads to resistance. To overcome this drawback, we developed the novel recombinant dual-targeting antibody fragment IDB0062, which is comprised of the anti–vascular endothelial growth factor A Fab and neuropilin 1-targeting peptide, and we assessed its properties.MethodsWe compared the in vitro activity of IDB0062 and conventional drugs using cell proliferation, wound healing, and Transwell assays. The in vivo efficacy of IDB0062 was determined using mouse choroidal neovascularization and oxygen-induced retinopathy models. To evaluate the ocular distribution of IDB0062, we intravitreally administered IDB0062 and ranibizumab to cynomolgus monkeys and measured the retinal drug levels.ResultsIDB0062 effectively inhibited not only vascular endothelial growth factor A in vitro but also placenta growth factor 2, vascular endothelial growth factor B, and platelet-derived growth factor BB, which induce vascular endothelial growth factor A–independent angiogenesis. In addition, IDB0062 showed non-inferior efficacy compared with aflibercept in vivo despite the low selectivity for mouse vascular endothelial growth factor A. In the monkey intravitreal pharmacokinetic study, IDB0062 improved drug distribution in the retina compared with ranibizumab, confirming the accelerated onset of pharmacological action when IDB0062 is injected in the vitreous humor.ConclusionsThrough neuropilin 1 binding, IDB0062 can improve the efficacy and accelerate the onset of pharmacological action in the posterior segment, which is targeted for macular degeneration, thereby improving drug responsiveness in drug-resistant patients.Translational RelevanceConsidering its novel mechanism of action, IDB0062 may help in controlling resistance to conventional anti–vascular endothelial growth factor drugs in clinical settings.

PD01-11 SENSORY GLIAL GQ-GPCR SIGNALING ALLEVIATES VISCERAL PAIN AND IMPROVES MICTURITION FUNCTION IN AN ANIMAL MODEL OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis (PD01)1 Sep 2021PD01-11 SENSORY GLIAL GQ-GPCR SIGNALING ALLEVIATES VISCERAL PAIN AND IMPROVES MICTURITION FUNCTION IN AN ANIMAL MODEL OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME Alison Xie, Randall Meacham, and Anna Malykhina Alison XieAlison Xie More articles by this author , Randall MeachamRandall Meacham More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001965.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with urological chronic pelvic pain syndrome (UCPPS) experience chronic pelvic pain (CPP) and lower urinary tract symptoms (LUTS). Afferent hypersensitivity in bladder-projecting sensory neurons plays an important role in the generation and maintenance of UCPPS symptoms, especially visceral pain and urinary frequency. We found that Gq-GPCR activation in satellite glial cells (SGCs) in the sensory ganglia decreases afferent sensitivity in lumbar nociceptive pathways, and could be a promising and highly innovative approach to alleviate the symptoms of bladder overactivity and pain. In this study, we aimed to test if activation of lumbosacral sensory glial Gq-GPCR signaling alleviates visceral pain, and improves LUTS in UCPPS mouse models. METHODS: A mouse model of UCPPS was induced by intravesical instillation of recombinant mouse vascular endothelial growth factor (VEGFA). VEGF levels were associated with CPP in UCPPS patients, and in animal models of bladder overactivity. In mice, VEGFA induces sensory nerve remodeling in the urinary bladder, visceral mechanical hypersensitivity. Pharmacogenetic activation of spinal and sensory ganglionic glia was achieved by expressing Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gq-DREADD) in GFAP+ glia via Gfap-hM3Dq transgenic mice or targeted adeno-associated viral vector (AAV) delivery. Gq-DREADD agonist, clozapine N-oxide (CNO), was used to selectively activate Gq-GPCR signaling in GFAP+ glia and to decreases sensory neuronal excitability in vivo in awake animals. Afferent sensitivity and bladder function were evaluated by clinically-relevant visceral nociceptive assays and awake cystometry. RESULTS: Lumbosacral glial Gq-GPCR activation significantly attenuated VEGFA-induced visceral hypersensitivity, increased micturition efficiency, and shortened intermicturition intervals. The glia-mediated neuromodulatory effects were long-lasting and dose-dependent, suggesting broader implication of neuromodulation in treating pain and hypersensitivity in pelvic diseases. CONCLUSIONS: Our data demonstrated that sensory glial-mediated neuromodulation effectively alleviated visceral pain and regulated micturition in animal model of UCPPS by inhibiting afferent excitability in the lumbosacral spinal cord and respective sensory ganglia. These results suggest a strong therapeutic potential of targeting GFAP-expressing glia for the treatment of chronic pelvic pain and LUTS in UCPPS patients. Source of Funding: Colorado Pilot Program Mentored Award, CCTSI, PI: Alison Xiaoqiao XieR01 DK121506-01, PI: Anna Malykhina © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e35-e35 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xie More articles by this author Randall Meacham More articles by this author Anna Malykhina More articles by this author Expand All Advertisement Loading ...

A pilot study investigating a novel particle-based growth factor delivery system for preimplantation embryo culture.

Can vascular endothelial growth factor (VEGF)-loaded silica supraparticles (V-SPs) be used as a novel mode of delivering VEGF to the developing preimplantation embryo in vitro? Supplementation of embryo culture media with V-SPs promoted embryonic development in a manner equivalent to media supplemented with free VEGF. VEGF is a maternally derived growth factor that promotes preimplantation embryonic development in vitro. However, its use in clinical media has limitations due to its low stability in solution. This study was a laboratory-based analysis utilising a mouse model. V-SPs were prepared in vitro and supplemented to embryonic culture media. The bioactivity of V-SPs was determined by analysis of blastocyst developmental outcomes (blastocyst development rate and total cell number). SPs were loaded with fluorescently labelled VEGF and release kinetics were characterised. Bioactivity of unlabelled VEGF released from V-SPs was determined by analysis of embryo developmental outcomes (blastocyst developmental rate and total cell number) following individual mouse embryo culture in 20 µl of G1/G2 media at 5% oxygen, supplemented with 10 ng/ml recombinant mouse VEGF in solution or with V-SPs. The bioactivity of freeze-dried V-SPs was also assessed to determine the efficacy of cryostorage. VEGF release kinetics were characterised by an initial burst of VEGF from loaded spheres followed by a consistent lower level of VEGF release over 48 h. VEGF released from V-SPs resulted in significant increases in total blastocyst cell number relative to the control (P < 0.001), replicating the effects of medium freely supplemented with fresh VEGF (P < 0.001). Similarly, freeze dried V-SPs exerted comparable effects on embryonic development (P < 0.05). N/A. In this proof of principle study, the effects of V-SPs on embryonic development were only analysed in a mouse model. These findings suggest that SPs represent a novel method by which a targeted dose of therapeutic agents (e.g. bioactive VEGF) can be delivered to the developing in vitro embryo to promote embryonic development, an approach that negates the breakdown of VEGF associated with storage in solution. As such, V-SPs may be an alternative and effective method of delivering bioactive VEGF to the developing in vitro embryo; however, the potential use of V-SPs in clinical IVF requires further investigation. This work was funded by the University of Melbourne. The authors have no conflict of interest to declare.

Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of ∼ 5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-γ), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

Exosomes derived from human mesenchymal stem cells preserve mouse islet survival and insulin secretion function.

Islet cell death and loss of function after isolation and before transplantation is considered a key barrier to successful islet transplantation outcomes. Mesenchymal stem cells (MSCs) have been used to protect isolated islets owing to their paracrine potential partially through the secretion of vascular endothelial growth factor (VEGF). The paracrine functions of MSCs are also mediated, at least in part, by the release of extracellular vesicles including exosomes. In the present study, we examined (i) the effect of exosomes from human MSCs on the survival and function of isolated mouse islets and (ii) whether exosomes contain VEGF and the potential impact of exosomal VEGF on the survival of mouse islets. Isolated mouse islets were cultured for three days with MSC-derived exosomes (MSC-Exo), MSCs, or MSC-conditioned media without exosomes (MSC-CM-without-Exo). We investigated the effects of the exosomes, MSCs, and conditioned media on islet viability, apoptosis and function. Besides the expression of apoptotic and pro-survival genes, the production of human and mouse VEGF proteins was evaluated. The MSCs and MSC-Exo, but not the MSC-CM-without-Exo, significantly decreased the percentage of apoptotic cells and increased islet viability following the downregulation of pro-apoptotic genes and the upregulation of pro-survival factors, as well as the promotion of insulin secretion. Human VEGF was observed in the isolated exosomes, and the gene expression and protein production of mouse VEGF significantly increased in islets cultured with MSC-Exo. MSC-derived exosomes are as efficient as parent MSCs for mitigating cell death and improving islet survival and function. This cytoprotective effect was probably mediated by VEGF transfer, suggesting a pivotal strategy for ameliorating islet transplantation outcomes.

Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development

ABSTRACTPurpose/Aim: Vascular endothelial growth factor (VEGF) dysregulation is implicated in the pathogenesis of retinopathy of prematurity (ROP). Identifying the factors that contribute to VEGF regulation during normal retinal vascularization is the key to ROP prevention. Currently, physiologic hypoxia is thought to be responsible for retinal VEGF regulation in utero. However, a potential hormonal contribution to VEGF regulation during eye development has not been fully investigated. The placental hormone, human chorionic gonadotropin and the pituitary hormone, and luteinizing hormone (LH) induce VEGF expression in several tissue types. Both of these gonadotropins activate the same LH receptor (LHR) in the human body; LHRs are expressed in the retina. In this study, we aimed to show that LHR signaling participates in VEGF regulation in the developing eye.Methods: When offspring from breeding pairs of LHR knockout mice (lhrkos) reached 21 days old, eyes and serum were extracted from homozygote lhrkos and wildtype (WT) siblings. VEGF levels were measured using Mouse VEGF Quantikine immunoassay kit. Retinas were incubated with isolectin for endothelial cell staining, flat mounted and imaged by confocal microscopy. Retinal vascular density was quantified using Imaris software. Some eyes were sectioned and stained for histopathologic review.Results: Ocular VEGF and retinal vascular volumes were significantly reduced by ~ 15% in lhrko eyes. Serum VEGF was not changed. The lhrko retinas did not display any anomalies.Conclusions: We provide evidence that LHR signaling plays a role in VEGF regulation and vascularization in the developing eye. Given that human preterm infants may have altered LHR-activity, the effect of gonadotropins on eye development should be further studied to identify novel strategies for ROP prevention.

Dimethyloxalylglycine-embedded Poly(ε-caprolactone) Fiber Meshes Promote Odontoblastic Differentiation of Human Dental Pulp–derived Cells

IntroductionThe in vivo effect of prolyl hydroxylase inhibitors on the regeneration of the pulp-dentin complex is unclear. The purpose of this study was to investigate the effect of dimethyloxalylglycine (DMOG)-embedded poly(ε-caprolactone) fiber (PCLF/DMOG) on odontoblastic differentiation of human dental pulp–derived cells (hDPCs) by transplantation of the dentin slice model. MethodsThe hDPCs were seeded onto electrospun PCLF and PCLF/DMOG in dentin slices and then transplanted into nude mice. The surface topography was evaluated for both PCLFs, and DMOG release from the PCLF/DMOG was examined. The effects of the PCLF/DMOG were assessed by histology and quantitative reverse transcription polymerase chain reaction. ResultsThe PCLF/DMOG-treated dentin slices showed higher cellularity with a palisading arrangement of hDPCs and organized collagen fibers. We found that the PCLF/DMOG significantly stimulated the expression of vascular endothelial growth factor, dentin sialoprotein, and bone sialoprotein in the hDPCs (P < .05) and mouse vascular endothelial growth factor A, mouse platelet endothelial cell adhesion molecule 1, and mouse neurofilament light polypeptide in the surrounding host cells (P < .05). ConclusionsThese results show that PCLF/DMOG has potential in pulp-dentin complex regeneration by promoting odontoblastic differentiation of hDPCs and by enhancing host cell recruitment, angiogenesis, and neurogenesis through the released DMOG-mediated cell responses.

In vivo immunotherapy of lung cancer using cross-species reactive vascular endothelial growth factor nanobodies.

Objective(s):Lung cancer is the main leading cause of cancer death worldwide. Angiogenesis is the main step in proliferation and spreading of tumor cells. Targeting vascular endothelial growth factor (VEGF) is an effective approach for inhibition of cancer angiogenesis. Nanobodies (NBs) are a novel class of antibodies derived from the camel. Unique characteristics of Nbs like their small size and good penetration to tumor tissues makes them promising tools in drug development. Development of NBs targeting both human and mouse VEGF is required for understanding their in vivo functions. Therefore, development of cross-species reactive anti-VEGF Nbs for immunotherapy of lung cancer was the main aim of the current study.Materials and Methods:Here we developed NBs from Camelus dromedarius library with high specificity and binding affinity to both human and mouse VEGF. In vitro and In vivo function of developed NB was evaluated on human endothelial cells and lung epithelial tumor cells (TC-1).Results:A nanobody showed the highest affinity to human and mouse VEGF and potently inhibited VEGF in the ELISA experiment. Anti-VEGF NBs significantly inhibited in vitro human endothelial cell migration through blockade of VEGF (P=0.045). Anti-VEGF NBs also significantly inhibited in vivo TC-1 growth in a dose-dependent manner (P=0.001) and resulted in higher survival rate in the nanobody treated groupConclusion:These findings demonstrate the potential of anti-VEGF NBs in tumor growth inhibition and are promising as novel cancer therapeutic candidate.

Production and Characterization of Novel Camel Single Domain Antibody Targeting Mouse Vascular Endothelial Growth Factor.

Camel single domain antibody known as Nanobody™ refers to a novel class of monoclonal antibodies with appropriate pharmacological properties. Nanobody is an antigen-binding site of camel heavy chain antibody also known as VHH. Expression in a microbial system, stability in difficult conditions and extremes of PH, and nanomolar affinity to target an appropriate drug format makes Nanobody a potential for drug discovery. Needs for Nanobody function evaluation in animal models turned our interest to develop anti-mouse vascular endothelial growth factor (mVEGF) Nanobodies using phage display as a potent technique in the isolation of antibodies. Isolation of anti-mVEGF Nanobodies was performed on Camelus dromedarius immune library through four consecutive rounds of biopanning on immobilized mVEGF. Enrichment of the Nanobody library was monitored by polyclonal phage-ELISA, and specific Nanobodies were selected using periplasmic extract-ELISA. Selected Nanobodies were expressed in WK6 Escherichia coli cells and purified using immobilized metal affinity chromatography. Specificity and affinity of selected Nanobodies were evaluated on immobilized mVEGF. Results demonstrated the successful enrichment of the Nanobody library. Two clones named Nb5 and Nb10 were selected through screening procedures according to their signal value in periplasmic extract-ELISA. Selected Nanobodies specifically reacted to mVEGF, but cross-reactivity with other antigens was not observed. Evaluated affinity for the Nanobodies was in nanomolar range. Taken together, according to the results, the selected Nanobodies promise to be a novel tool in research and for further development of diagnostic or therapeutic purposes in pharmaceutical science.

Effect of Human Endothelial Progenitor Cell (EPC)- or Mouse Vascular Endothelial Growth Factor-Derived Vessel Formation on the Survival of Vitrified/Warmed Mouse Ovarian Grafts

The aim of this study was to evaluate the effectiveness of improving angiogenesis at graft sites on the survival of follicles in transplanted ovarian tissue. Matrigel containing 5 × 105 of cord blood-derived endothelial progenitor cells (EPCs) or 200 ng of mouse vascular endothelial growth factor (VEGF) was injected subcutaneously into BALB/c-Nu mice. After 1 week, vitrified/warmed ovaries from female B6D2F1 mice were subcutaneously transplanted into the injection sites. After 1, 2, and 4 weeks posttransplantation, the ovaries were recovered and subjected to histological analysis. Oocytes were collected from the transplanted ovaries, and their fertilization, embryonic development, and delivery were also observed. Vitrified/warmed ovaries transplanted into EPC- or VEGF-treated sites developed more blood vessels and showed better follicle survival than those transplanted into sham-injected sites. Normal embryonic development and consequent live births were obtained using oocytes recovered from cryopreserved/transplanted ovaries. Treatment with EPCs or VEGF could prevent the ischemic damage during the early revascularization stage of ovarian transplantation.

Methodological differences account for inconsistencies in reported free VEGF concentrations in pregnant rats

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/ml) and high (527-1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82-105%) but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

Vascular Endothelial Growth Factor Promotes Fibrosis Resolution and Repair in Mice

Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored. We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays. VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9. In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function.

Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor β (TGF-β) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

Abstract 326: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix Stores and Induces Vascular Permeability in vivo

Introduction The formation of unstable and leaky neovessels underlies the pathogenesis of a large number of chronic inflammatory diseases. Granzyme B (GZMB) is a serine protease that is expressed and released by a variety of immune cells and accumulates in the extracellular matrix (ECM) during chronic inflammation where it cleaves a number of ECM proteins, including fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN in both normal and diseased tissue. We hypothesize that GZMB cleavage of FN will release VEGF from its extracellular stores and promote vascular permeability as a mechanism that contributes to neovessel leakage during chronic inflammation. Methods GZMB-mediated VEGF release from either FN coated wells or endogenously produce endothelial cell (EC) matrix was measured by VEGF ELISA. VEGF-release supernatants were used to treat EC and VEGF receptor 2 (VEGFR2) activation was evaluated by immunoblotting for phosphorylated VEGFR2. Evan’s blue was injected intravenously to CD1 mice followed by ear injection of either mouse GZMB, saline control, GZMB + neutralizing mouse VEGF antibody or GZMB+ IgG control (n=5 for each experimental group). Vascular leakage was evaluated by Evan’s blue dye extraction. Results GZMB effectively releases VEGF from both FN and from EC matrix, while inhibition of GZMB prevented VEGF release. GZMB-mediated VEGF release resulted in significant activation of VEGFR2 in EC monolayer signified by increased VEGFR2 phosphorylation. GZMB ear injection resulted in a significant increase in vascular permeability in vivo. Importantly, co-injection of GZMB and neutralizing mouse VEGF antibody significantly reduced vascular leakage compared to co-injection of GZMB and matching IgG control. Conclusions and Impact GZMB increases VEGF bioavailability by releasing it from the ECM leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.