Intracellular Vascular Endothelial Growth Factor Research Articles

Gatekeeping role of Nf2/Merlin in vascular tip EC induction through suppression of VEGFR2 internalization.

In sprouting angiogenesis, the precise mechanisms underlying how intracellular vascular endothelial growth factor receptor 2 (VEGFR2) signaling is higher in one endothelial cell (EC) compared with its neighbor and acquires the tip EC phenotype under a similar external cue are elusive. Here, we show that Merlin, encoded by the neurofibromatosis type 2 (NF2) gene, suppresses VEGFR2 internalization depending on VE-cadherin density and inhibits tip EC induction. Accordingly, endothelial Nf2 depletion promotes tip EC induction with excessive filopodia by enhancing VEGFR2 internalization in both the growing and matured vessels. Mechanistically, Merlin binds to the VEGFR2–VE-cadherin complex at cell-cell junctions and reduces VEGFR2 internalization–induced downstream signaling during tip EC induction. As a consequence, nonfunctional excessive sprouting occurs during tumor angiogenesis in EC-specific Nf2-deleted mice, leading to delayed tumor growth. Together, Nf2/Merlin is a crucial molecular gatekeeper for tip EC induction, capillary integrity, and proper tumor angiogenesis by suppressing VEGFR2 internalization.

Expression and possible role of Smad3 in postnecrotizing enterocolitis stricture.

To investigate the expression of Smad3 (mothers against decapentaplegic homolog 3) protein in postnecrotizing enterocolitis stricture and its possible mechanism of action. We used immunohistochemistry to detect the expression characteristics of Smad3 and nuclear factor kappa B (NF-κB) proteins in human postnecrotizing enterocolitis stricture. We cultured IEC-6 (crypt epithelial cells of rat small intestine) in vitro and inhibited the expression of Smad3 using siRNA technique. Quantitative PCR, western blotting, and ELISA were used to detect the changes in transforming growth factor-β1 (TGF-β1), NF-κB, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and zonula occludens-1 (ZO-1) messenger RNA (mRNA) and protein expressions in IEC-6 cells. CCK8 kit and Transwell cellular migration were used to detect cell proliferation and migration. Changes in epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) in IEC-6 cells were detected by immunofluorescence technique. The results showed that Smad3 protein and NF-κB protein were overexpressed in narrow intestinal tissues and that Smad3 protein expression was positively correlated with NF-κB protein expression. After inhibiting the expression of Smad3 in IEC-6 cells, the mRNA expressions of NF-κB, TGF-β1, ZO-1, and VEGF decreased, whereas the mRNA expression of TNF-α did not significantly change. TGF-β1, NF-κB, and TNF-α protein expressions in IEC-6 cells decreased, whereas ZO-1 and intracellular VEGF protein expressions increased. IEC-6 cell proliferation and migration capacity decreased. There was no significant change in protein expression levels of EMT markers E-cadherin and vimentin and also extracellular VEGF protein expression. We suspect that the high expression of Smad3 protein in postnecrotizing enterocolitis stricture may promote the occurrence and development of secondary intestinal stenosis. The mechanism may be related to the regulation of TGF-β1, NF-κB, TNF-α, ZO-1, and VEGF mRNA and protein expression. This may also be related to the ability of Smad3 to promote epithelial cell proliferation and migration.

Hydroquinone increases risk for retinal pigment epithelial (RPE) cell degeneration

AbstractPurposeCigarette smoke increases the risk for age‐related macular degeneration (AMD) e.g. through oxidative stress and mitochondrial damage in human retinal pigment epithelial (RPE) cells. Vascular endothelial growth factor (VEGF) promotes choroidal neovascularization in wet AMD, for which anti‐VEGF injections are used as therapy. Since VEGF is important for the RPE cell functionality and integrity, too low VEGF levels increase the risk for RPE cell degeneration. Pigment epithelium‐derived factor (PEDF) improves mitochondrial activity and reduces neovascularization. The aim of the present study was to examine the effects of the cigarette smoke component hydroquinone on the VEGF and PEDF levels in RPE cells.MethodsIL‐1α‐primed ARPE‐19 cells were exposed to hydroquinone (10 μM, 50 μM, 200 μM; 18 h, 1 h or 15 min). Enzyme‐linked immunosorbent assay (ELISA) was used to detect the levels of intracellular VEGF as well as secreted VEGF and PEDF. The 2′,7′‐dichlorofluorescin diacetate (DCFDA) probe was used to detect ROS production. Lactate dehydrogenase (LDH) and intracellular ATP were measured using commercial kits, and total protein concentrations using a protocol based on the Bradford method.ResultsHydroquinone reduced the levels of both intra‐ and extracellular VEGF. In addition, the release of PEDF as well as the ratio between VEGF and PEDF were reduced at all used concentrations. ROS production increased at 50 μM hydroquinone, and the cell membrane was compromised at 200 μM hydroquinone after 15 min exposure. ATP levels were reduced from the 50 μM hydroquinone concentration upwards and total protein levels at all concentrations, already without cytotoxicity.ConclusionsAccording to our present data, hydroquinone favors anti‐angiogenic conditions but predisposes to the RPE cell degeneration, impaired mitochondrial function, and reduced protein synthesis in human RPE cells.

Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer

To examine copper transporter 1 (CTR1) expression in pancreatic carcinoma cells, orthotopic xenograft pancreatic tumor model and clinical samples, and verify the effect of copper chelating agent ammonium tetrathiomolybdate (TM) regulate the expression of CTR1 in pancreatic carcinoma cells and the inhibition of pancreatic carcinoma. The expressions of copper transporter CTR1 and antioxidant protein 1 (ATOX1) in 22 clinical pancreatic ductal carcinoma and paracancer tissues 0.5-1 cm away from the tumor were measured by immunohistochemistry (IHC). PANC-1 cells were used to construct 5 orthotopic xenograft pancreatic tumor of nude mice models. Pancreatic cancer tissues and corresponding normal pancreatic tissues were collected, and the expressions of CTR1 and ATOX1 were detected by IHC and compared with clinical tissues. The proliferation of pancreatic carcinoma cells PANC-1 treated with 10, 30, 50, 100 μmol/L TM for 24 h, 48 h, 72 h was measured by CCK8 assay. The migration abilities of PANC-1 cells treated with 50 μmol/L TM for 24 h, 48 h were detected by scratch test. The expressions of CTR1, vascular endothelial growth factor (VEGF) and CyclinD1 proteins in PANC-1 cells treated with 10, 30, 50, 100 μmol/L TM for 48 h were measured by Western blot. Then the subcutaneous tumor-bearing model of nude mice were established with PANC-1 cells, and the growth of tumor was observed after oral administration of 0.3 mg/d and 1.0 mg/d of TM, respectively. The immunohistochemical results indicated that 19 of the 22 clinical pancreatic ductal cancer tissues of carcinoma patients had high expression of CTR1, and the same high expression of CTR1 was found in the orthotopic transplanted tumor tissues of PANC-1 nude mice. The proliferation inhibition of PANC-1 cells increased with the concentration of TM increased and the treatment time prolonged. The expressions of intracellular CTR1, VEGF and CyclinD1 all decreased with the concentration of TM increased. The cell migration ability decreased after the PANC-1 cells treated with TM. The tumor growth of PANC-1 tumor-bearing nude mice was inhibited after different doses of TM were delivered. The reduction in tumor volume and weight was more pronounced in the high-dose TM group (P<0.05). The expression of CTR1 is abnormally elevated in pancreatic carcinoma, and treatment with copper chelating agent for this target may help to inhibit pancreatic carcinoma.

A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor;ER, endoplasmic reticulum.

Catabolic degradation of endothelial VEGFA via autophagy

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

MiR-20a ameliorates diabetic angiopathy in streptozotocin-induced diabetic rats by regulating intracellular antioxidant enzymes and VEGF.

We evaluated the beneficial effect of miR-20a mimic against diabetic angiopathy (DA) in rats by regulating intracellular antioxidant enzymes and vascular endothelial growth factor (VEGF). Diabetes was induced by intraperitoneal administration of streptozotocin (STZ; 65 mg/kg). Rats were then treated with miR-20a mimic (250 nmol/kg orally) for 8 weeks after STZ administration. The effect of miR-20a mimic against DA in rats was evaluated by estimating serum glucose concentration, lipid profile, Lp-a, kidney function test, inflammatory mediators, and markers of endothelial cell function. Markers of oxidative stress in the aortic tissue were estimated in rats treated with miR-20a mimic. Western blot assay, RT-PCR, and histopathology of kidney and myocardial tissues were also performed. Serum levels of blood glucose and markers of renal function were significantly lower, and the lipid profile improved in the miR-20a mimic group compared to the DA group. Treatment with miR-20a mimic ameliorated the altered markers of endothelial function and oxidative stress, as well as mediators of inflammation, in the DA rats. Protein expressions of ERK1/2, JNK, and p38 MAPK, as well as mRNA expressions of TLR-4 and NF-κB, in aortic tissues were lower in the miR-20a mimic group than in the DA group. The miR-20a mimic group had fewer histopathological changes in kidney and myocardial tissues than the DA group. MiR-20a mimic can protect against DA in rats by regulating vascular endothelial function and oxidative stress.

Abstract 112: Vegf-receptor Inhibitor Impedes Macrophage Function At The End Target Muscle After Nerve Injury

PURPOSE: Recovery in peripheral nerve injuries is limited by the critical time point beyond which muscle denervation is irreversible. A major component of functional recovery is muscle reinnervation, which depends on terminal Schwann cells (tSCs) at the neuromuscular junction (NMJ). The hallmark feature of the tSC response to injury is cytoplasmic process elongation that guides axon growth. In response to injury, macrophages secrete Vascular Endothelial Growth Factor (VEGF) which may induce angiogenesis to support tSC process extension. We hypothesize that VEGF inhibition impedes tSC function at the NMJ, which in turn will impede reinnervation of the end target muscle. METHODS: Sciatic nerve transection with immediate repair was performed in C57BL/6 mice. To evaluate whether VEGF inhibition impedes end target muscle reinnervation, Cabozantinib, a VEGF Receptor-2 (VEGFR-2) inhibitor, was administered to mice via oral gavage for seven days following nerve injury to suppress VEGF secretion. For the control group, saline was given for the same duration. Mice were sacrificed on 14, 21, 28 and 42 days after injury (n = 3 per time point), and the leg muscles were harvested. Immunohistochemical stains for NF200 (axon marker), S100 (Schwann cell marker), VEGF, CD68 (macrophage marker), and bungarotoxin (BTX, motor endplate marker) were used on extensor digitorum longus and tibialis anterior muscle sections. Functional innervation was determined by two methods: 1) immunohistochemical staining with NF-200 for axonal reinnervation into motor endplates marked by BTX in whole muscle mounts and 2) electrophysiological stimulation of the sciatic nerve to test tibialis anterior CMAP amplitude. RESULTS (PRELIMINARY): After nerve injury, extracellular and intracellular VEGF expression increased significantly at postoperative day (POD) 3 compared to non-injured muscle, and then peaked at POD 12. At two weeks (POD14) after injury, motor end plates were 41.2% fully innervated and 37.1% partially innervated in the control group, while the Cabozantinib group had only 7.5% fully innervated and 4.3% partially innervated motor end plates. VEGF expression at POD 14 was greatly different between the two groups (11.48 percent pixels vs 3.49 percent pixels, p <0.001). By POD 21, however, 97% of the motor end plates in the Control group and 93.2% in the Cabozantinib group had reached full innervation. Axonal sprouting between motor end plates was more prevalent in the control group (46.9% vs 18.5%). VEGF expression decreased in both groups by POD 21 and POD 28, while CD68 positive cells co-stained with VEGF remained significantly higher in the control group at POD 28. Electrophysiological stimulation at 10 Hz showed significant differences at POD 21 (t test, mean difference 2.151 mV, p = 0.047) but no differences at POD 42 (t test -0.822, p = 421). CONCLUSIONS: Drug induced VEGF inhibition had a marked but temporary effect on the functional recovery of denervated muscle after nerve injury. Although the differences between the two experimental groups became nonsignificant over time, the effects seen at the neuromuscular junction of the target muscles highlighted the unrecognized role of VEGF in macrophages on the functional recovery after nerve injury.

A multi-responsive biomimetic nano-complex platform for enhanced gene delivery.

RNA interference (RNAi) is widely regarded as a promising technology for disease treatment, yet one major obstacle for its clinical application is the lack of enhanced siRNA delivery vehicles to circumvent complex extra- and intracellular barriers. By integrating unique peculiarities of thioglycolic acid conjugated chitosan nanoparticles (TCS NPs), biomimetic transfersomes (T) and amphiphilic hyaluronic acid (HA-GMS), a novel nano-complex was prepared, where vascular endothelial growth factor (VEGF) siRNA loaded TCS NPs were cloaked by transfersomes with HA-GMS assembled on the surface (HT-TCS-siRNA NPs). The nano-complex provided superior siRNA protection and desirable stability at pH 7.4 and 6.5 (mimicking tumor tissue) and exerted proton sponge effects at acidic pH 5.0 (mimicking endo/lysosomes). The TCS NPs were stable at pH 5.0 but disintegrated in the presence of 10 mM glutathione (GSH) at pH 7.4 (mimicking tumor cytosol), which was favorable to release siRNA to the cytoplasm. In vitro cell uptake and gene silencing assays exhibited enhanced intracellular siRNA accumulation and VEGF silencing efficacy of HT-TCS-siRNA NPs in HeLa cells. The enhanced gene delivery capacity of the multi-responsive biomimetic nano-complex gives them potential for application in cancer therapy.

Intracrine VEGF signalling mediates colorectal cancer cell migration and invasion

Background:Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key regulators of angiogenesis, affecting endothelial cell survival and function. However, the effect of VEGF-VEGFR signalling on tumour cell function is not well understood. Our previous studies in colorectal cancer (CRC) cells have demonstrated an intracrine VEGF/VEGFR1 signalling mechanism that mediates CRC cell survival and chemo-sensitivity. Since extracellular VEGF signalling regulates migration of endothelial cells and various tumour cells, we attempted to determine whether intracrine VEGF signalling affects CRC cell motility.Methods:Migration and invasion of CRC cells, with and without VEGF or VEGFR1 depletion, were assayed using transwell migration chambers. Changes in cell morphology, epithelial-mesenchymal transition (EMT) markers, and markers of cell motility were assessed by immunostaining and western blot.Results:Depletion of intracellular VEGF and VEGFR1 in multiple CRC cell lines led to strong inhibition of migration and invasion of CRC cells. Except for Twist, there were no significant differences in markers of EMT between control and VEGF/VEGFR1-depleted CRC cells. However, VEGF/VEGFR1-depleted CRC cells demonstrated a significant reduction in levels of phosphorylated focal adhesion kinase and its upstream regulators pcMET and pEGFR.Conclusions:Inhibition of intracrine VEGF signalling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

Remarkable expression of vascular endothelial growth factor in bone marrow plasma cells of patients with POEMS syndrome

Vascular endothelial growth factor (VEGF) is pathognomonically elevated in patients with POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes) syndrome. However, its source of overproduction is unclear. As clinical improvement is almost always associated with VEGF reduction after anti-plasma cell therapy, its increase at diagnosis has been attributed to the underlying monoclonal gammopathy, although direct evidence is still lacking. In the current study, we systemically measured VEGF levels in POEMS patients, before and after treatment. Bone marrow plasma cells showed remarkable VEGF expression, in both mRNA and protein levels, which decreased gradually in response to therapy. Of note, statistically linear correlations were observed between serum and bone marrow plasma cell VEGF levels (mRNA vs. serum, rho 0.343, p=0.003; protein vs. serum, rho 0.644, p<0.0001), supporting bone marrow plasma cells as the main source of circulating VEGF. Intriguingly, immunophenotyping revealed that bone marrow plasma cells were polyclonal in most patients at diagnosis. A clear monoclonal population, coexistent with polytypic cells, was only detectable in 11 cases (18%), in which comparable intracellular VEGF expression was observed between these two plasma cell populations (p=0.594), while monoclonal cells showed higher intracellular interleukin-6 expression (p=0.006). These patients had more serum monoclonal protein, less post-therapeutic complete remission, and inferior overall (p=0.027) and progression-free survival (p=0.002). Collectively, bone marrow plasma cells, mainly polyclonal population, are the major source of VEGF overproduction in POEMS patients.

Anti-angiogenic effect of water extract from the fruiting body of Agrocybe aegerita

The water extract of the mushroom fruiting body of Agrocybe aegerita (AaE) had a total phenolic content of 13.67 ± 0.21 GAE (μM)/mg extract and 5 dominant phenolic acids including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid and sinapic acid were identified which accounted for 2.5% dry weight of the extract. AaE inhibited the vascular endothelial growth factor (VEGF)-induced proliferation in HUVECs shown by the MTT assay. Down-regulation of intracellular reactive oxygen species (ROS) level and VEGF secretion were also observed in Caco-2 cells treated with AaE. Inhibition of other angiogenic cascades in HUVECs by AaE (at 25 μg/mL) included a decrease in the migration of endothelial cells (ECs) by 46.14 ± 8.53% as evidenced by the wound healing assay and transwell insert assay; as well as a complete blocking of the VEGF-induced formation of capillary network on matrigel matrix. Taken together, these results demonstrated the presence of anti-angiogenic properties of AaE in vitro and among other compounds present in AaE phenolics might contribute to the effect. Therefore, Aa could be considered as potential functional food with antitumor effect modulated by inhibition of angiogenesis.

Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib

Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC.

A Paracrine Circuit Regulates Vascular Endothelial Growth Factor Production By Bone Marrow Plasma Cells in Patients with POEMS Syndrome

Background: POEMS syndrome is a rare paraneoplastic disorder, characterized by polyneuropathy, organomegaly, osteosclerosis and extravascular volume overload, due to an underlying plasma cell dyscrasia. Vascular endothelial growth factor (VEGF), a potent angiogenic cytokine that can increase vascular permeability, plays a critical role in its pathogenesis. Despite extensive VEGF studies concerning its role in disease diagnosis, monitoring and response assessment, little is known about its cellular source and mechanisms governing the production of VEGF in POEMS patients.Methods and Materials: Patients with POEMS syndrome, 62 newly diagnosed and 46 of them after lenalidomide-dexamethasone (LenDex) treatment, admitted to Peking Union Medical College Hospital between February 2014 and April 2015, were included in the current study. Clinical information, serum and bone marrow samples were collected with the Institutional approval. Serum levels of VEGF were measured by ELISA. VEGF levels in bone marrow plasma cells were quantified via real-time quantitative PCR and multiparameter flow cytometry, respectively. In addition, the phenotype, clonality and intracellular interleukin-6 (IL-6) expression of bone marrow plasma cells were also analyzed by flow cytometry. Furthermore, immunohistochemical staining was performed to study the plasma cell distribution, clonality and expression of VEGF, IL-6 and hypoxia-inducible factor-1α (HIF-1α) in bone marrow biopsies. Statistical analyses were conducted using SPSS 22, and a p < 0.05 was considered as significant.Results: Serum levels of VEGF were dramatically elevated in patients with newly diagnosed POEMS syndrome (median 5958 pg/mL), significantly higher than both disease and healthy controls (p < 0.001), and can be used as a diagnostic marker (area under curve 0.988, p < 0.001). A cut-off value of 2000 pg/mL had a specificity of 97.7% with a sensitivity of 91.9% in support of the diagnosis. After treatment, VEGF levels decreased gradually (6-cycle after LenDex, median 1184 pg/mL, p < 0.001; 12-cycle after LenDex, median 832 pg/mL, p < 0.001). Bone marrow plasma cells showed remarkable VEGF expression, validated in both mRNA and protein levels, which also decreased in response to LenDex therapy. More importantly, a statistically linear correlation was observed between serum and bone marrow plasma cell VEGF levels (newly diagnosed patients, rho = 0.33, p = 0.01; post-therapeutic patients, rho = 0.53, p < 0.001), supporting that bone marrow plasma cells were the source of circulating VEGF. Intriguingly, immunophenotying revealed that bone marrow plasma cells were polyclonal in most newly diagnosed cases. Monoclonal population, co-existing with polyclonal plasma cells, was observed only in 11 patients (18%). Further analyses showed that the relative amounts of these two populations were similar (41% vs. 59%) and they also had comparable intracellular VEGF expression (mean fluorescent intensity, 2009 vs. 2367, p = 0.594). However, monoclonal plasma cells had significantly higher intracellular IL-6 expression (mean fluorescent intensity, 1635 vs. 865, p = 0.006). In 46 newly diagnosed cases with bone marrow biopsies available, immunohistochemical staining was performed, and plasma cells were typically distributed in a scattered manner, with focal aggregates observed in 21 cases (46%). Intracellular κ and λ light chain staining demonstrated that the background scattered plasma cells were polyclonal, while both monoclonal and polyclonal fractions were observed in the focal area, which showed similar nuclear and intracellular staining of HIF-1α and VEGF, respectively. In contrast, IL-6 was mainly expressed in λ-restricted plasma cells.Conclusion: Bone marrow plasma cells are the potential source of VEGF production in patients with POEMS syndrome. Monoclonal plasma cells, aggregates focally in the bone marrow, may secret IL-6 to stimulate polyclonal plasma cells proliferation, and consequent VEGF production in a paracrine circuit. DisclosuresOff Label Use: Lenalidomide for POEMS syndrome.

Abstract 16: Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells

Abstract Background: Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal associated with the growth and metastasis of tumors. In addition, VEGF has also been implicated in the suppression of immune cell function. The purpose of this study was to characterize VEGF secretion, receptor expression and effect of soluble VEGF neutralization in human lymph node cells both at rest and during activation. Methods: Human lymph nodes derived from patients with melanoma (IRB CASE 3610) containing T cells, B cells, monocyte/macrophages and dendritic cells were cultured with anti-CD3/CD28 beads and IL-2 in order to induce an inflammatory response in vitro. Culture supernatant was assayed for the presence of soluble VEGF on days 4, 7, 11 and 14 of culture. Multiparameter FACS analysis was performed to assess the cell surface expression of VEGF receptor subtypes 1 and 2 over the 14-day culture period. In addition, intracellular expression of cytokines within effector T cells was assessed at the end of the culture. Finally, a parallel culture containing a VEGF neutralizing antibody was performed to assess the effect of VEGF neutralization on effector T cell phenotype. High-throughput T cell receptor CDR3 sequencing was used to determine the molecular sequence of the variable region of the T cell receptors (ImmunoSEQ, Seattle, WA). Results: VEGF was measureable in the culture supernatant on days 4 and beyond. The proportion of cells expressing intracellular VEGF peaked at day 4 and subsequently decreased. CD11b+(monocyte/macrophage), CD11c+ (dendritic cells) and CD19+ (B) cells had the highest proportion of cells which expressed intracellular VEGF. VEGF R1 and R2 were not constitutively expressed on resting immune cells, but receptor expression was upregulated upon activation of the T cells in vitro. Day 14 T cells demonstrated intracellular IFN-γ, IL-2 and TNF-α. The addition of VEGF-blocking antibody in the culture of MDLN cells effectively neutralized the amount of soluble VEGF detected in the culture supernatant at all days. The addition of the VEGF-blocking antibody also increased the proportion of T cells expressing intracellular IFN-γ as compared T cells in the non-VEGF blocked culture and resulted in distinct molecularly defined T cell clones. Conclusion: These results confirm that soluble VEGF is produced by immune cells during activation, even in the absence of tumor. VEGF receptor expression on lymph node immune cell subsets is constitutive, but low, and then upregulated during in vitro activation of T cells, presumably due to secondary cytokine production. Neutralization of soluble VEGF resulted in qualitative differences in effector cytokine expression and T cell receptor sequences. Whether the effect on these T cells is direct via VEGF interaction with the cognate receptor or from an indirect effect on antigen-presenting cells is currently under investigation. Citation Format: Madeleine P. Strohl, Hallie Graor, Mei Zhang, Anthony Visioni, John Ammori, Isabelle Rivers-McCue, Julian Kim. Immunomodulatory effects of VEGF on human lymph node antigen-presenting and lymphoid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 16. doi:10.1158/1538-7445.AM2014-16

Effects of the vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor SU5416 on in vitro cultures of Plasmodium falciparum.

BackgroundVascular endothelial growth factor (VEGF) is taken up by parasitized red blood cells during malaria and stimulates intra-erythrocytic growth of Plasmodium falciparum in vitro. The cause and consequence of this uptake is not understood.MethodsPlasmodium falciparum was cultured in vitro. Parasite growth and intracellular VEGF levels were assessed using flow cytometry. Intracellular VEGF was visualized by fluorescence immunocytochemistry. Phosphorylated tyrosine was measured by western blotting. In vivo assessment of intra-erythrocytic VEGF was performed in Plasmodium berghei ANKA-infected C57BL/6 mice.ResultsVEGF accumulated intracellularly in infected red blood cells, particularly in schizonts. In vitro growth of P. falciparum was unchanged when co-cultured with the anti-VEGF antibody bevacizumab or with an anti-VEGF receptor-1 peptide. In contrast, the VEGF receptor-2 inhibitor, SU5416, dose-dependently inhibited growth. None of the treatments reduced intracellular VEGF levels. Thus, the anti-parasitic effect of SU5416 seemed independent of VEGF uptake. SU5416 reduced phosphorylated tyrosine in parasitized red blood cells. Similarly, the broad-spectrum tyrosine kinase inhibitor genistein dose-dependently inhibited P. falciparum growth and reduced tyrosine phosphorylation. Neither bevacizumab nor anti-VEGF receptor-1 peptide affected tyrosine kinase activity. Finally, in vivo uptake of VEGF in P. berghei ANKA was demonstrated, analogous to the in vitro uptake in P. falciparum, making it a possible model for the effects of VEGF signalling in vivo during malaria.ConclusionsInhibition of VEGFR-2 signalling reduces intra-erythrocytic growth of P. falciparum, likely due to tyrosine kinase inhibition. Internalisation of VEGF in P. falciparum-infected red blood cells does not rely on VEGF receptors. The function of in vivo uptake of VEGF can be studied in rodent malaria models.

In vitro evaluation of optimized liposomes for delivery of small interfering RNA

One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7 ± 2.0 nm and zeta potentials of 4.03 ± 0.69 mV, which had no significant change when stored at 4 °C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent.

Synergistic inhibition of breast cancer by co-delivery of VEGF siRNA and paclitaxel via vapreotide-modified core–shell nanoparticles

A somatostatin analog, vapreotide (VAP), can be used as a ligand for targeting drug delivery based on its high affinity to somatostatin receptors (SSTRs), which is overexpressed in many tumor cells. RNA interference plays an important role on downregulation of vascular endothelial growth factor (VEGF), which is important for tumor growth, progression and metastasis. To improve tumor therapy efficacy, the vapreotide-modified core–shell type nanoparticles co-encapsulating VEGF targeted siRNA (siVEGF) and paclitaxel (PTX), termed as VAP-PLPC/siRNA NPs, were developed in this study. When targeted via somatostatin receptors to tumor cells, the VAP-PLPC/siRNA NPs could simultaneously delivery siVEGF and PTX into cells and achieve a synergistic inhibition of tumor growth. Interestingly, in vitro cell uptake and gene silencing experiments demonstrated that the targeted VAP-PLPC/siRNA NPs exhibited significant higher intracellular siRNA accumulation and VEGF downregulation in human breast cancer MCF-7 cells, compared to those of the non-targeted PEG-PLPC/siRNA NPs. More importantly, in vivo results further demonstrated that the targeted VAP-PLPC/siRNA NPs had significant stronger drug distribution in tumor tissues and tumor growth inhibition efficacy via receptor-mediated targeting delivery, accompany with an obvious inhibition of neovascularization induced by siVEGF silencing. These results suggested that the co-delivery of siRNA and paclitaxel via vapreotide-modified core–shell nanoparticles would be a promising approach for tumor targeted therapy.

Pancreatic duct cells as a source of VEGF in mice

Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Therapeutic Approaches to the Regulation of Wound Angiogenesis

Re-establishment of a functional vascular network is a critical component of successful wound repair. One of the most potent pro-angiogenic agents is vascular endothelial growth factor (VEGF), which, from a basic science and pre-clinical perspective, seems ideal for the therapeutic stimulation of blood vessel growth in non-healing wounds. Current strategies to improve the dysfunctional angiogenesis that occurs in non-healing wounds are inadequate with regard to the nature and magnitude of the clinical problem. However, VEGF therapy has so far been unsuccessful in promoting healing in the clinic. More effective means of delivery to the wound, which take into account the biochemical and spatio-temporal aspects of angiogenesis, may be necessary to realize VEGF's therapeutic potential. Reviewed approaches for the regulation of wound angiogenesis include: targeting regulators of intracellular VEGF signaling, making use of collagen-binding VEGF fusion proteins for increased retention in the wound, and implantation of heterogeneous scaffold systems for spatial control of angiogenesis with simultaneous use of VEGF and its inhibitor. To maximize efficacy of therapeutic VEGF, it may be necessary to also target its intracellular inhibitory mechanisms. Immobilizing VEGF to the wound matrix may increase its bioavailability and therapeutic efficacy. Gaining spatial control of angiogenesis opens up possibilities for advanced directed therapy. The reviewed studies present innovative approaches to in vivo directed modulation of angiogenesis utilizing VEGF biology which can, if taken further and validated in human subjects, have significant impact on clinical wound care in the future.