VEGFR1 Blockade Research Articles

Sunitinib-induced endocapillary proliferative glomerulonephritis with IgA2 deposit in addition to thrombotic microangiopathy: a case report

BackgroundSunitinib, a multi-targeted tyrosine kinase inhibitor, is used as a second-line therapy for gastrointestinal stromal tumors (GIST) resistant to imatinib. However, its impact on the vascular endothelial growth factor (VEGF) pathway can lead to significant toxicities, including hypertension and thrombotic microangiopathy (TMA).Case presentationThis case report describes a unique instance of a patient with metastatic GIST who developed endocapillary proliferative glomerulonephritis (EPGN) with IgA2 deposits and TMA following sunitinib treatment. The patient presented with severe hypertension, nephrotic syndrome, and acute kidney injury. Renal biopsy confirmed the diagnosis, revealing IgA2 deposits, which are not commonly associated with TMA. Discontinuation of sunitinib led to a rapid improvement in renal function and proteinuria. The potential mechanisms underlying sunitinib-induced glomerular injury may involve the blockade of VEGFR-1, affecting immune cell recruitment and function, and the disruption of the nitric oxide and endothelin systems, leading to endothelial damage and immune dysregulation. Management of these toxicities requires a personalized approach, with options ranging from symptomatic relief to drug discontinuation. The use of endothelin receptor antagonists and other therapeutic alternatives for GIST management is discussed.ConclusionsThis case highlights the complex interplay between the therapeutic effects of sunitinib and its potential renal and cardiovascular toxicities, emphasizing the need for close monitoring and effective management strategies to optimize patient outcomes.

VEGFR1 blockade reduces Mycobacterium tuberculosis-induced pathology in immunocompromised mice

Abstract Mycobacterium tuberculosis (Mtb) is a major public health concern that caused 1.6 million deaths in 2021. New treatments are needed, especially in immunocompromised individuals, such as patients with HIV coinfection, who are severely impacted by the disease. We have recently reported that blocking VEGFR1, a receptor that is present on monocytes and contributes to their recruitment to the sites of infection, limits Mtb-induced pathology in immunocompetent mice of both Mtb-resistant (C57BL/6J) and Mtb-supersusceptible (C3HeB/FeJ) strains (Harding et al., 2019). These results offer a way to reduce lung pathology without impeding host defense. Here, we extend this finding by showing that VEGFR1 blockade has similar effects in immunocompromised (RAG1KO) mice as well, measured by a reduction in the area of inflammation and without a change in bacterial burden. Following treatment with the VEGFR1 blocker SU5416, we have found an elevated ratio and an increase in the absolute number of neutrophil granulocytes in the Mtb-infected lungs in both immunosufficient and immunocompromised mice. Surprisingly however, this did not result in exacerbated pathology as the majority of the recruited neutrophils remained in the lung vasculature. Our results indicate that further evaluation of VEGFR1 blockers as an adjunctive treatment to antitubercular drug therapy for immunocompromised populations could be worthwhile. Supported by grants from NIH (R01 HL128778 and R01 NS123449)

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

Binding of the placental growth factor to VEGF receptor type 1 modulates human T cell functions.

The immunosuppressive properties of vascular endothelial growth factors (VEGFs) suggest a new role of angiogenic factors in T cell modulation in cancer and pregnancy. Most of VEGF effects on T cells are mediated through the VEGF receptor type 2 (VEGFR-2). This study aims to investigate the role of placental growth factor (PlGF) as a selective VEGFR-1 ligand in the modulation of human T cells functions. For this, PBMCs from healthy donors were stimulated with anti-CD3 mAbs (a-CD3) or Concanavalin A (ConA) in the absence or presence of PlGF and assessed for T cell proliferation, IL-10 production, programmed cell death, and the expression of inhibitory receptors (PD-1, CTLA-4, TIM-3) using radiometric (3 H-thymidine incorporation) and FACS analysis. We showed that most T cells in freshly isolated PBMCs lacked VEGFR-1. However, activation with a-CD3 or ConA strongly increased the percentages of VEGFR-1 expressing CD4+ and CD8+ T cells. PlGF in a wide dose range suppressed PBMC cell proliferation, inhibiting both CD4+ and CD8+ T cells. Blockade of VEGFR-1, but not VEGFR-2 with neutralizing Abs completely abolished the suppressive effect of PlGF. Furthermore, we found that treatment with PlGF up-regulated IL-10 production in CD4+ and CD8+ T cells, promoted CD8+ T cells apoptosis and enhanced the expression of inhibitory receptors (PD-1 and TIM-3) on activated T cells. Our in vitro findings suggest the involvement of PlGF/VEGFR-1 signaling in the modulation of T cell responses in a-CD3-stimulated PBMCs.

Vascularization and VEGF expression in bovine yolk sacs: Impact of reproductive techniques

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Targeting VEGFR1- and VEGFR2-expressing non-tumor cells is essential for esophageal cancer therapy.

Increasing appreciation of tumor heterogeneity and the tumor-host interaction has stimulated interest in developing novel therapies that target both tumor cells and tumor microenvironment. Bone marrow derived cells (BMDCs) constitute important components of the tumor microenvironment. In this study, we aim to investigate the significance of VEGFR1- and VEGFR2-expressing non-tumor cells, including BMDCs, in esophageal cancer (EC) progression and in VEGFR1/VEGFR2-targeted therapies. Here we report that VEGFR1 or VEGFR2 blockade can significantly attenuate VEGF-induced Src and Erk signaling, as well as the proliferation and migration of VEGFR1⁺ and VEGFR2⁺ bone marrow cells and their pro-invasive effect on cancer cells. Importantly, our in vivo data show for the first time that systemic blockade of VEGFR1⁺ or VEGFR2⁺ non-tumor cells with neutralizing antibodies is sufficient to significantly suppress esophageal tumor growth, angiogenesis and metastasis in mice. Moreover, our tissue microarray study of human EC clinical specimens showed the clinicopathological significance of VEGFR1 and VEGFR2 in EC, which suggest that anti-VEGFR1/VEGFR2 therapies may be particularly beneficial for patients with aggressive EC. In conclusion, this study demonstrates the important contributions of VEGFR1⁺ and VEGFR2⁺ non-tumor cells in esophageal cancer progression, and substantiates the validity of these receptors as therapeutic targets for this deadly disease.

Vascular endothelial growth factor-receptor 1 inhibition aggravates diabetic nephropathy through eNOS signaling pathway in db/db mice.

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice.While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling.In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.

Aldosterone Promotes Vascular Remodeling by Direct Effects on Smooth Muscle Cell Mineralocorticoid Receptors

Vascular remodeling occurs after endothelial injury, resulting in smooth muscle cell (SMC) proliferation and vascular fibrosis. We previously demonstrated that the blood pressure-regulating hormone aldosterone enhances vascular remodeling in mice at sites of endothelial injury in a placental growth factor-dependent manner. We now test the hypothesis that SMC mineralocorticoid receptors (MRs) directly mediate the remodeling effects of aldosterone and further explore the mechanism. A wire-induced carotid injury model was performed in wild-type mice and mice with inducible SMC-specific deletion of the MR. Aldosterone did not affect re-endothelialization after injury in wild-type mice. Deletion of SMC-MR prevented the 79% increase in SMC proliferation induced by aldosterone after injury in MR-Intact littermates. Moreover, both injury-induced and aldosterone-enhanced vascular fibrosis were attenuated in SMC-specific MR knockout mice. Further exploration of the mechanism revealed that aldosterone-induced vascular remodeling is prevented by in vivo blockade of the placental growth factor-specific receptor, type 1 vascular endothelial growth factor receptor (VEGFR1), the receptor for placental growth factor. Immunohistochemistry of carotid vessels shows that the induction of VEGFR1 expression in SMC after vascular injury is attenuated by 72% in SMC-specific MR knockout mice. Moreover, aldosterone induction of vascular placental growth factor mRNA expression and protein release are also prevented in vessels lacking SMC-MR. These studies reveal that SMC-MR is necessary for aldosterone-induced vascular remodeling independent of renal effects on blood pressure. SMC-MR contributes to induction of SMC VEGFR1 in the area of vascular injury and to aldosterone-enhanced vascular placental growth factor expression and hence the detrimental effects of aldosterone are prevented by VEGFR1 blockade. This study supports exploring MR antagonists and VEGFR1 blockade to prevent pathological vascular remodeling induced by aldosterone.

Abstract 302: Smooth Muscle Cell Mineralocorticoid Receptors Directly Mediate Aldosterone-induced Remodeling After Vascular Injury Via Type 1 Vegf Receptors

Background and Significance Endothelial damage initiates vascular remodeling with smooth muscle cell (SMC) proliferation and vascular fibrosis. Excessive remodeling, as in restenosis after vascular procedures or atherosclerosis, limits blood flow to distal tissues. In animals, aldosterone (aldo) infusion enhances remodeling which is reversed by aldo antagonists, implicating the mineralocorticoid receptor (MR). We previously showed that MR is expressed in SMC and that aldo induces the type 1 VEGF receptor (VEGFR1) in injured vessels. Hypothesis and Results We hypothesize that aldo promotes pathological vascular remodeling by directly activating MR in SMC and promoting SMC proliferation via VEGFR1 signaling. We show in vivo that aldo does not affect re-endothelialization after injury supporting our focus on SMC. To test this hypothesis we measured aldo-induced remodeling after wire carotid injury in mice with inducible SMC-specific deletion of MR (SMC-MR-KO). The 80% increase in SMC proliferation with aldo in control littermates is completely prevented in SMC-MR-KO mice (p&lt;0.005, n=11). To examine the mechanism, mouse carotid SMC were treated with aldo in vitro producing a dose dependent proliferative response (1,5,10 and100nM aldo, 9%±2, 17%±3, 21%±6, and 25%±7 vs control, respectively, p&lt;0.05, n=8). The increase in proliferation with 10 nM aldo+IgG was completely inhibited by treatment with specific VEGFR1-blocking antibody (Ab, p=0.04, n=8). Immunohistochemistry of carotid vessels reveals that VEGFR1 is induced in SMC after injury and further enhanced by aldo (55%±0.5 increase with injury, 74%± 0.5 with aldo+injury vs uninjured control, p&lt;0.05, n=8).We examined the role of VEGFR1 in vivo and VEGFR1 blockade significantly reduces aldo-induced SMC proliferation after injury (46%±4 decrease vs aldo+IgG, p&lt;0.05, n=9). Tail cuff measurements reveal no difference in blood pressure with any of the treatments. Conclusions These data support that SMC MR directly mediates aldo-induced vascular remodeling independent blood pressure. The mechanism involves VEGFR1 that is induced on SMC in the area of injury. This study supports exploring VEGFR1-blocking drugs to prevent pathological vascular remodeling induced by aldo in humans.

Blockade of VEGFR-1 and VEGFR-2 Enhances Paclitaxel Sensitivity in Gastric Cancer Cells

PurposeHypoxia-inducible factor-1α (HIF-1α) increases transcription of the vascular endothelial growth factor (VEGF) gene. Inhibition of VEGF abolishes VEGF mediated induction of HIF-1α. Recent reports suggested that HIF-1α also mediated the induction of class III β-tubulin (TUBB3) in hypoxia. TUBB3 confers resistance to taxanes. Inhibition of VEGF may decrease the expression of HIF-1α and TUBB3. This study was undertaken to investigate the roles of vascular endothelial growth factor receptor (VEGFR) in gastric cancer cell behavior and to identify methods to overcome paclitaxel resistance in vitro.Materials and MethodsThe protein expression levels of HIF-1α and TUBB3 were measured in human gastric cancer cell lines (AGS) under normoxic and hypoxic conditions. The relationship between TUBB3 and paclitaxel resistance was assessed with small interfering TUBB3 RNA. AGS cells were treated with anti-VEGFR-1, anti-VEGFR-2, placental growth factor (PlGF), bevacizuamb, and paclitaxel.ResultsHypoxia induced paclitaxel resistance was decreased by knockdown of TUBB3. Induction of HIF-1α and TUBB3 in AGS is VEGFR-1 mediated and PlGF dependent. Hypoxia-dependent upregulation of HIF-1α and TUBB3 was reduced in response to paclitaxel treatment. Expressions of HIF-1α and TUBB3 were most decreased when AGS cells were treated with a combination of paclitaxel and anti-VEGFR-1. AGS cell cytotoxicity was most increased in response to paclitaxel, anti-VEGFR-1, and anti-VEGFR-2.ConclusionWe suggest that blockade of VEGFR-1 and VEGFR-2 enhances paclitaxel sensitivity in TUBB3-expressing gastric cancer cells.

Tumor cell-derived placental growth factor sensitizes antiangiogenic and antitumor effects of anti-VEGF drugs

The role of placental growth factor (PlGF) in modulation of tumor angiogenesis and tumor growth remains an enigma. Furthermore, anti-PlGF therapy in tumor angiogenesis and tumor growth remains controversial in preclinical tumor models. Here we show that in both human and mouse tumors, PlGF induced the formation of dilated and normalized vascular networks that were hypersensitive to anti-VEGF and anti-VEGFR-2 therapy, leading to dormancy of a substantial number of avascular tumors. Loss-of-function using plgf shRNA in a human choriocarcinoma significantly accelerated tumor growth rates and acquired resistance to anti-VEGF drugs, whereas gain-of-function of PlGF in a mouse tumor increased anti-VEGF sensitivity. Further, we show that VEGFR-2 and VEGFR-1 blocking antibodies displayed opposing effects on tumor angiogenesis. VEGFR-1 blockade and genetic deletion of the tyrosine kinase domain of VEGFR-1 resulted in enhanced tumor angiogenesis. These findings demonstrate that tumor-derived PlGF negatively modulates tumor angiogenesis and tumor growth and may potentially serve as a predictive marker of anti-VEGF cancer therapy.

Blockade of VEGFR1 and 2 Suppresses Pathological Angiogenesis and Vascular Leakage in the Eye

ObjectiveVEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.Materials and MethodsNeutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [3H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.ResultsVEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.ConclusionOur study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.

Simultaneous blockade of VEGFR‐1 and VEGFR‐2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation

Metastasis continues to be the major cause of morbidity and mortality in malignant melanoma. In our study, we explored whether inhibition of VEGFR-1 or VEGFR-2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR-1 or -2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR-1 and -2 signaling strongly suppressed progression in both B16 tumor models. There was no expression of VEGFR-1 or -2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor-like cells during melanoma metastasis formation, we observed an enhanced proliferation and mobilization of VEGFR-1+ myeloid and VEGFR-2+ endothelial cells with progenitor potential by the induction of melanoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimetastatic effects exerted by combined inhibition of VEGFR-1 and -2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR-1 signaling led to a strong reduction of the CD45-positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma metastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR-2-dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells.

Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoic acid

Vascular endothelial growth factor (VEGF) is a potent signalling molecule that acts through two tyrosine kinase receptors, VEGFR1 and VEGFR2. The upregulation of VEGF and its receptors is important in tumour-associated angiogenesis; however, recent studies suggest that several tumour cells express VEGF receptors and may be influenced by autocrine VEGF signalling. Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma, and is dependent on autocrine signalling for its growth. The alveolar subtype of RMS is often characterized by the presence of a PAX3-FKHR translocation, and when introduced into non-RMS cells, the resultant fusion protein induces expression of VEGFR1. In our study, we examined the expression of VEGF and its receptors in RMS, and autocrine effects of VEGF on cell growth. VEGF and receptor mRNA and protein were found to be expressed in RMS cells. Exogenous VEGF addition resulted in extracellular signal-regulated kinase-1/2 phosphorylation and cell proliferation, and both were reduced by VEGFR1 blockade. Growth was also slowed by VEGFR1 inhibitor alone. Treatment of RMS cells with all-trans-retinoic acid decreased VEGF secretion and slowed cell growth, which was rescued by VEGF. These data suggest that autocrine VEGF signalling likely influences RMS growth and its inhibition may be an effective treatment for RMS.

Fetal Stromal–Dependent Paracrine and Intracrine Vascular Endothelial Growth Factor-A/Vascular Endothelial Growth Factor Receptor-1 Signaling Promotes Proliferation and Motility of Human Primary Myeloma Cells

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.

Vascular Endothelial Growth Factor Receptor-1 Modulates Vascular Endothelial Growth Factor-Mediated Angiogenesis via Nitric Oxide

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.