Cell Vascular Endothelial Growth Factor Production Research Articles

Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

IL-17 Promotes Angiogenic Factors IL-6, IL-8, and Vegf Production via Stat1 in Lung Adenocarcinoma.

Inflammation and angiogenesis are two hallmarks of carcinoma. The proinflammatory cytokine interleukin-17 (IL-17) facilitates angiogenesis in lung cancer; however, the underlying mechanism is not fully understood. In this study, tumour microvessel density (MVD) was positively associated with IL-17, interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial cell growth factor (VEGF) expression in human lung adenocarcinoma tissues, and it was increased in tumour tissues of A549-IL-17 cell-bearing nude mice. Importantly, positive correlations were also detected between IL-17 expression and IL-6, IL-8 and VEGF expression in human lung adenocarcinoma tissues. Furthermore, IL-6, IL-8 and VEGF production, as well as STAT1 phosphorylation, were increased in tumour tissues of A549-IL-17 cell-bearing nude mice in vivo and in A549 and H292 cells following IL-17 stimulation in vitro. In addition, STAT1 knockdown using an inhibitor and siRNA attenuated the IL-17-mediated increases in IL-6, IL-8 and VEGF expression in A549 and H292 cells. In conclusion, IL-17 may promote the production of the angiogenic inducers IL-6, IL-8 and VEGF via STAT1 signalling in lung adenocarcinoma.

TNFSF15 suppresses VEGF production in endothelial cells by stimulating miR-29b expression via activation of JNK-GATA3 signals.

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in promoting neovascularization. VEGF gene expression in vascular endothelial cells in normal tissues is maintained at low levels but becomes highly up-regulated in a variety of disease settings including cancers. Tumor necrosis factor superfamily 15 (TNFSF15; VEGI; TL1A) is an anti-angiogenic cytokine prominently produced by endothelial cells in a normal vasculature. We report here that VEGF production in mouse endothelial cell line bEnd.3 can be inhibited by TNFSF15 via microRNA-29b (miR-29b) that targets the 3'-UTR of VEGF transcript. Blocking TNFSF15 activity by using either siRNA against the TNFSF15 receptor known as death domain-containing receptor-3 (DR3; TNFRSF25), or a neutralizing antibody 4-3H against TNFSF15, led to inhibition of miR-29b expression and reinvigoration of VEGF production. In addition, we found that TNFSF15 activated the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. Treatment of the cells either with SP600125, an inhibitor of JNK, or with JNK siRNA, led to eradication of TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. These findings suggest that VEGF gene expression can be suppressed by TNFSF15-stimulated activation of the JNK-GATA3 signaling pathway which gives rise to up-regulation of miR-29b.

Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3.

What are the roles of the microRNA miR-210-an miRNA that is up-regulated in endometriotic cyst stromal cells (ECSCs)-in the pathogenesis of endometriosis? Up-regulated miR-210 expression in ECSCs is involved in their proliferation, resistance to apoptosis and angiogenesis through signal transducer and activator of transcription (STAT) 3. In the pathogenesis of endometriosis, a number of roles for microRNAs (miRNAs) are becoming apparent. ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometriotic tissues (patients aged 24-40 years undergoing salpingo-oophorectomy or evisceration for the treatment of ovarian endometriotic cysts, n = 10) and the eutopic endometrial tissues without endometriosis (premenopausal patients aged 35-45 years undergoing hysterectomies for subserousal leiomyoma, n = 13), respectively. We used a global gene expression microarray technique to identify downstream targets of miR-210, and we assessed the functions of miR-210 in the pathogenesis of endometriosis by using the miR-210-transfected NESCs. Gene expression microarray analysis revealed that one of the key target molecules of miR-210 is STAT3. In the NESCs, in comparison to the control, miR-210 transfection resulted in the induction of cell proliferation (P < 0.0005), the production of vascular endothelial cell growth factor (VEGF) (P < 0.0005) and the inhibition of apoptosis (P < 0.05) through STAT3 activation [increased levels of mRNA (P < 0.0005), and protein (P < 0.005)]. In the ECSCs, inhibitors of STAT3 inhibited the cell proliferation and VEGF production (P < 0.05), and induced the apoptosis of these cells (P < 0.05). The roles of aberrant miR-210 expression were investigated only in the stromal component of ectopic and eutopic endometrium. Control endometrial tissues were obtained from premenopausal patients who had subserosal leiomyoma and NESC gene expression patterns may be altered in these women. Furthermore, the effects of STAT3 inhibitors were evaluated only in ECSCs and not in NESCs. The present findings indicate that miR-210 induces NESCs to differentiate into the endometriotic phenotype and we speculate that up-regulated miR-210 expression in ECSCs is involved in the creation of the endometriosis-specific cellular dysfunctions through epigenetic mechanisms. The data indicate that STAT3 inhibitors may be promising candidates for the treatment of endometriosis. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K.N., no. 25861500 to Y.K. and no. 23592407 to H.N.). There are no conflicts of interest to declare.

Stanniocalcin, a Potential Ovarian Angiogenesis Regulator, Does Not Affect Endothelial Cell Apoptosis

The ovarian follicle represents an outstanding model for the investigation of the angiogenic process. In fact, follicular development is associated with an extensive neovascularization, and physiological angiogenesis is necessary for folliculogenesis. However, although the major factors triggering the angiogenic events have been thoroughly investigated and are now relatively well defined, information about the molecular events involved in the modulation and/or arrest of neovascularization is still scarce. Therefore, this research focused on the potential involvement of stanniocalcin (STC), a protein whose biological function is still unclear, in the control mechanisms of follicular angiogenesis. We evaluated the effect of 5 and 50 ng/mL STC on the production of the main proangiogenic factor, vascular endothelial growth factor (VEGF), by swine granulosa cells. Moreover, STC's effect on cell viability and the modulation of caspase-3 and -7 activities in swine aortic endothelial cells were also examined. Granulosa cell VEGF production was significantly (P < 0.001) inhibited by STC. Endothelial cell viability was significantly (P < 0.001) increased, whereas caspase activities were reduced (P < 0.01) by STC. These data indicate that STC is not angiosuppressive for endothelial cells, although it could potentially modulate local angiogenesis acting at the granulosa cell level.

Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production

Modulating the paracrine effects of bone marrow mesenchymal stem cells (BMSCs) may be important for the treatment of ischemic myocardial tissue. In this regard, endogenous estrogen may enhance BMSC vascular endothelial growth factor (VEGF) production. However, little information exists regarding the effect of testosterone on stem cell function. We hypothesized that 1) endogenous or exogenous estrogen will enhance stem cell production of VEGF and 2) endogenous or exogenous testosterone will inhibit BMSC VEGF production. BMSCs were collected from adult male, female, castrated male, and ovariectomized female rats. One hundred thousand cells were incubated with testosterone (1, 10, or 100 nM) or estrogen (0.15, 1.5, or 15 nM) for 48 h. Cell supernatants were collected, and VEGF was measured by ELISA. BMSCs harvested from castrated males, normal females, and ovariectomized females produced more VEGF compared with normal males. Castration was associated with the highest level (1,018 +/- 98.26 pg/ml) of VEGF production by BMSCs, which was significantly more than that produced by BMSCs harvested from normal male and normal female animals. Exogenous testosterone significantly reduced VEGF production in BMSCs harvested from ovariectomized females in a dose-dependent manner. Exogenous estrogen did not alter BMSC VEGF production. These findings suggest that testosterone may work on BMSCs to decrease protective growth factor production and that effective removal of testosterone's deleterious effects via castration may prove to be beneficial in terms of protective factor production. By manipulating the mechanisms that BMSCs use to produce growth factors, we may be able to engineer stem cells to produce maximum growth factors during therapeutic use.

Lenalidomide Displays Direct Anti-Non-Hodgkin's Lymphoma (NHL) Cell Activity in Association with Enhanced SPARC Expression but Independent of Its Ability to Strongly Inhibit NHL Cell VEGF Production In Vitro.

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin's lymphoma (NHL). We have previously shown that lenalidomide can directly inhibit the proliferation of NHL cells and synergize with dexamethasone in inducing cell cycle arrest at the G1/S transition leading to caspase activation and apoptosis. In this study we have studied the potential for lenalidomide mediated growth inhibition, apoptosis and inhibition of angiogenic factors in NHL. We have assessed proliferation, expression of p21 and SPARC tumor suppressor genes and the production of the angiogenic factor vascular endothelial growth factor (VEGF) in six established NHL cells lines in vitro. The effect of lenalidomide on NHL cells was determined after 1–3 days using 3H-thymidine incorporation, microbead array and real time PCR. We found that lenalidomide displays anti-proliferative effects on Namalwa, Jeko-1 and Rec-1 cells when used alone and in combination with dexamethasone. However, several NHL cell lines, such as Granta-519, DB, and JVM-2, appear to be resistant. We also found that lenalidomide strongly inhibited VEGF production (but not mRNA levels) by the three sensitive lines (but not the three resistant lines) at much lower concentrations than required for inhibitory effects on proliferation. Excess recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on Rec-1 proliferation suggesting that there is no connection between these two effects. Lenalidomide strongly increased the expression of tumor suppressor genes, such as p21cip/kip and SPARC (secreted protein acidic and rich in cysteine). In particular, elevation of SPARC mRNA correlated with the anti-proliferative effect of lenalidomide and was not seen in the resistant cells, whereas elevated p21 was observed in both sensitive and resistant (DB) cells. The effect of SPARC gene silencing on lenalidomide-induced effects is being assessed in siRNA studies. In conclusion, lenalidomide demonstrates anti-proliferative activity against certain sensitive NHL cells, particularly in combination with dexamethasone. Potent anti-VEGF activity of lenalidomide appears to be independent of proliferation and supports its anti-angiogenic potential. Lenalidomide-induced up-regulation of SPARC mRNA appears to correlate with sensitivity and may have biomarker potential in NHL.

Evaluation of VEGF‐mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast‐mediated crosstalk to endothelial cells

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.

Immunomodulatory Role of Vascular Endothelial Growth Factor and Angiopoietin-1 in Airway Remodeling

The blood vessels formed in asthmatic airways are involved in inflammatory and airway remodeling processes in chronic asthma. Vascular endothelial cell growth factor (VEGF) and angiopoietin-1 (Ang-1) are primary angiogenic growth factors, involved in the formation of such blood vessels. VEGF has been reported to contribute to non-specific airway hyper-responsiveness, have chemotactic effects on eosinophils, and enhance airway smooth muscle cell proliferation. Furthermore, Th2 cells have receptors for VEGF, and Th2-associated cytokines increase VEGF production. There are reports that elevated levels of VEGF correlates with the severity of asthma. Ang-1 has been shown to induce pro-inflammatory effects such as eosinophil chemotaxis via tie-2 receptors. Reports indicate ang-1 contribution to increased secretion of matrix metalloproteinase-2 (MMP-2) and decreased secretion of tissue inhibitors of metalloproteinase-2 (TIMP-2). However, Ang-1 has also been shown to exhibit several anti-inflammatory properties such as suppressing expression of adhesion molecules, blocking vascular permeability and eosinophil chemotaxis induced by VEGF. These findings support the notion that apart from their roles in blood vessels formation, these angiogenic growth factors are directly involved in the pathogenesis of chronic asthma. This paper reviews individual and combined roles of VEGF and Ang-1. The potential therapeutic applications involving these factors are also discussed.

Hypoxia-inducible factor 1alpha and antiangiogenic activity of farnesyltransferase inhibitor SCH66336 in human aerodigestive tract cancer.

The farnesyltransferase inhibitor SCH66336, in combination with other receptor tyrosine kinase inhibitors, inhibits the growth of non-small-cell lung cancer (NSCLC) cells. We examined whether SCH66336 inhibits angiogenesis of aerodigestive tract cancer cells. Antiangiogenic activities of SCH66336 against NSCLC, head and neck squamous cell carcinoma (HNSCC), and endothelial cells were examined with cell proliferation, capillary tube formation, and chick aorta (under hypoxic, normoxic, insulin-like growth factor I (IGF)-stimulated, and unstimulated conditions); reverse transcription-polymerase chain reaction; and western blot analyses. The specific roles of the ubiquitin-mediated proteasome machinery, mitogen-activated protein kinase (MAPK) and Akt pathways, and heat shock protein 90 (Hsp90) in the SCH66336-mediated degradation of hypoxia-inducible factor 1alpha (HIF-1alpha) were assessed with ubiquitin inhibitors and adenoviral vectors that express constitutively active MAP kinase kinase (MEK)1, constitutively active Akt, or Hsp90. SCH66336 showed antiangiogenic activities and decreased the expression of vascular endothelial cell growth factor (VEGF) and HIF-1alpha in hypoxic, IGF-stimulated, and unstimulated aerodigestive tract cancer and endothelial cells. SCH66336 reduced the half-life of the HIF-1alpha protein, and ubiquitin inhibitors protected the hypoxia- or IGF-stimulated HIF-1alpha protein from SCH66336-mediated degradation. SCH66336 inhibited the interaction between HIF-1alpha and Hsp90. The overexpression of Hsp90, but not constitutive Akt or constitutive MEK, restored HIF-1alpha expression in IGF-stimulated or hypoxic cells but not in unstimulated cells. SCH66336 appears to inhibit angiogenic activities of NSCLC and HNSCC cells by decreasing hypoxia- or IGF-stimulated HIF-1alpha expression and to inhibit VEGF production by inhibiting the interaction between HIF-1alpha and Hsp90, resulting in the proteasomal degradation of HIF-1alpha.

Anti-angiogenesis effects of meisoindigo on chronic myelogenous leukemia in vitro

Meisoindigo, an active compound of a Chinese anti-leukemia medicine, has been effectively used in the treatment of chronic myelogenous leukemia (CML). Increasing evidences have demonstrated that angiogenesis is an important pathobiologic feature of CML. The anti-angiogenesis effect of meisoindigo on CML is unknown. In this study, we determined the effects of meisoindigo on the apoptosis, adherence and differentiation of endothelial cells as well as the secretion of vascular endothelial growth factor (VEGF) by CML cells. We found that VEGF level, measured by enzyme-linked immunosobent assay (ELISA), was higher in bone marrow plasma from CML patients compared with the healthy controls (334.83 ± 23.09 ng/L versus 102.36 ± 38.76 ng/L, P < 0.01). CML cell VEGF production was decreased after CML cells were treated with 10 μmol/L meisoindigo compared with the controls (212.10 ± 46.13 ng/L versus 293.75 ± 64.79 ng/L, P < 0.05). Ten micromole per liter of meisoindigo could induce time-dependent apoptosis of ECV304 cells determined by annexin-V. Treatment of ECV304 cells with meisoindigo for 48 h reduced the number of adherent cells and the expression of VCAM-1 compared with the control cells (43.78 ± 9.09% versus 73.51 ± 3.21%, P < 0.05). Meisoindigo also inhibited tubule formation of HUVECs in an in vitro Matrigel after HUVECs were incubated with meisoindigo for 6 h. Our findings suggest that meisoindigo could inhibit angiogeneic process through decreasing the VEGF secretion in leukemic cells and also through inhibiting the proliferation, adhesion and differentiation of endothelial cells, causing the interruption of a reciprocal stimulatory loop between leukemic and endothelial cells. This effect may contribute to the anti-leukemic effect of this drug.

Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes

Development of the retinal vasculature is controlled by a hierarchy of interactions among retinal neurons, astrocytes and blood vessels. Retinal neurons release platelet-derived growth factor (PDGFA) to stimulate proliferation of astrocytes, which in turn stimulate blood vessel growth by secreting vascular endothelial cell growth factor (VEGF). Presumably, there must be counteractive mechanisms for limiting astrocyte proliferation and VEGF production to prevent runaway angiogenesis. Here, we present evidence that the developing vessels provide feedback signals that trigger astrocyte differentiation--marked by cessation of cell division, upregulation of glial fibrillary acidic protein (GFAP) and downregulation of VEGF. We prevented retinal vessel development by raising newborn mice in a high-oxygen atmosphere, which leads, paradoxically, to retinal hypoxia (confirmed by using the oxygen-sensing reagent EF5). The forced absence of vessels caused prolonged astrocyte proliferation and inhibited astrocyte differentiation in vivo. We could reproduce these effects by culturing retinal astrocytes in a low oxygen atmosphere, raising the possibility that blood-borne oxygen itself might induce astrocyte differentiation and indirectly prevent further elaboration of the vascular network.

CXCL12 and Vascular Endothelial Growth Factor Synergistically Induce Neoangiogenesis in Human Ovarian Cancers

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.

Regulation of the pro-angiogenic microenvironment by carboxyamido-triazole.

Anti-angiogenic agents regulate tumor growth by inhibiting endothelial cell proliferation and invasion. Carboxyamido-triazole (CAI), an inhibitor of non-voltage-operated calcium entry and calcium influx-mediated pathways, has angiogenesis and invasion inhibitory activity. We hypothesized that CAI may express its anti-angiogenic effects through negative regulation of pro-angiogenic cytokine production and/or function. In vivo, orally administered CAI prevented A2058 human melanoma xenograft growth and concomitantly resulted in a marked reduction in circulating vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In vitro, A2058 cell secretion of VEGF was inhibited by CAI treatment under limiting micronutrient conditions that approximate the tumor microenvironment, media restriction, and acidification to pH 6.8 (P=0.0003 and P=0.0006, respectively). VEGF and HIF-1alpha message and protein were also reduced by CAI treatment. Oral CAI treatment reduced vascular ingrowth in vivo into VEGF-containing Matrigel plugs. Commensurate with those findings, human umbilical vein endothelial cell (HUVEC) migration towards VEGF was reduced below background by exposure to CAI in the migration chamber (P<0.0001). An 88% reduction in circulating IL-8 concentration was measured in CAI-treated animals. However, IL-8 protein secretion and gene expression were increased by CAI treatment in culture (P< or =0.01), where CAI caused a dose-dependent acidification of the culture milieu (P< or =0.005). This paradox suggests that IL-8 production in vitro may be more sensitive to ambient pH than cytosolic calcium. These observations suggest that CAI inhibition of tumor cell VEGF production and endothelial cell response to VEGF results in disruption of signaling between the tumor and its microenvironment, causing a net anti-angiogenic effect.

A nitric oxide synthase inhibitor, N(G)-nitro-l-arginine-methyl-ester, exerts potent antiangiogenic effects on plasmacytoma in a newly established multiple myeloma severe combined immunodeficient mouse model.

Angiogenesis is one of critical factors in sustaining the growth, invasion and metastasis of certain solid tumours and haematological malignancies such as multiple myeloma (MM). In the present study, we examined the anticancer potential of an inhibitor of nitric oxide synthase (NOS), NG-nitro-l-arginine methyl ester (L-NAME), in a novel severe combined immunodeficient mouse model (KHM mouse) that harbours the highly sanguineous plasmacytoma cell line KHM-4, derived from a patient with highly chemoresistant MM. KHM mice were intraperitoneally administered with either L-NAME, doxorubicin, melphalan, or paclitaxel. A significant reduction in tumour sizes was noted in L-NAME-administered KHM mice while no significant reduction was observed in melphalan- or doxorubicin-administered mice. A profound decrease in angiogenesis was observed in tumour tissues from L-NAME- and paclitaxel-administered KHM mice. A marked decrease in human vascular endothelial cell growth factor (VEGF) levels was identified in tumour tissues from L-NAME-administered KHM mice, strongly suggesting that L-NAME suppressed VEGF production by tumour cells through its inhibition of NOS in tumour cells, resulting in reduced neovasculization in mice leading to the regression of tumour sizes. The present data represent the first observations that certain NOS inhibitors potentially serve as experimental agents for the treatment of chemoresistant MM and plasmacytoma.

Capsaicin regulates vascular endothelial cell growth factor expression by modulation of hypoxia inducing factor-1alpha in human malignant melanoma cells.

Capsaicin (8-Methyl-N-Vanillyl-6-nonenamide), a known natural dietary chemopreventive agent, inhibits malignant melanoma cell proliferation. In the present study, we examined the effect of capsaicin on constitutive and induced vascular endothelial cell growth factor (VEGF) expression in malignant melanoma cells. Capsaicin treatment resulted in enhanced VEGF protein secretion in malignant melanoma cells independent of IL-1beta and TNF-alpha. The observed up-regulation of VEGF production by capsaicin was concentration- and duration-dependent and was inversely associated with inhibition of melanoma cell proliferation. We observed an increase in hypoxia-inducible factor (HIF)-1alpha and VEGF mRNA expression in capsaicin-treated melanoma cells. Further, an electrophoretic mobility shift assay (EMSA) from nuclear extracts from melanoma cells showed a decrease in constitutive activation of NF-kappaB and enhanced HIF-1alpha binding activity to hypoxia response element (HRE) in melanoma cells treated with different concentrations of capsaicin. These data suggest that inhibition of cellular proliferation by capsaicin follows enhanced VEGF production by remaining melanoma cells by enhancing HIF-1alpha expression and binding to HRE.

Osteopontin induces angiogenesis of murine neuroblastoma cells in mice.

Angiogenesis is an essential process for tumor growth and is regulated by tumor-derived angiogenic cytokines. Osteopontin (OPN) is one of the cytokines produced by various tumor cells and is suggested to be involved in angiogenesis by upregulating endothelial cell migration in cooperation with vascular endothelial cell growth factor (VEGF). To provide evidence of OPN involvement in a causal role in tumor angiogenesis, we generated a stable transfectant from murine neuroblastoma C1300 cells to constitutively secrete high levels of murine OPN. The OPN mRNA expression and protein secretion were confirmed by RT-PCR and ELISA, respectively. The biological activity of secreted OPN was determined with migration assay by using human umbilical vein endothelial cells (HUVEC). Transfection with OPN gene did not increase VEGF production and did not affect gene expression of other angiogenic factors confirmed by complementary DNA macroarray system. To demonstrate the effect of OPN on tumor-induced angiogenesis in vivo, millipore chambers containing OPN-transfected or control cells were implanted to the dorsal air sac of mice. The OPN-transfected cells significantly induced neovascularization in comparison to the control cells in mice. Conclusively, these results provide direct evidence of OPN involvement in the role of tumor angiogenesis.

Evidence of a leading role for VEGF in Bartonella henselae-induced endothelial cell proliferations.

Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis (BA) and bacillary peliosis (BP). The pathomechanisms of these tumorous proliferations are unknown. Our results suggest a novel bacterial two-step pathogenicity strategy, in which the pathogen triggers growth factor production for subsequent proliferation of its own host cells. In fact, B. henselae induces host cell production of the angiogenic factor vascular endothelial growth factor (VEGF), leading to proliferation of endothelial cells. The presence of B. henselae pili was associated with host cell VEGF production, as a Pil- mutant of B. henselae was unable to induce VEGF production. In turn, VEGF-stimulated endothelial cells promoted the growth of B. henselae. Immunohistochemistry for VEGF in specimens from patients with BA or BP revealed increased VEGF expression in vivo. These findings suggest a novel bacteria-dependent mechanism of tumour growth.

Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats

Background : The aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats. Methods : We investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro. Results : The expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h. Conclusions : These data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.

Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis

Arsenic trioxide (As2O3) has recently been used successfully in the treatment of acute promyelocytic leukemia and has been shown to induce partial differentiation and apoptosis of leukemic cells in vitro. However, the mechanism by which As2O3 exerts its antileukemic effect remains uncertain. Emerging data suggest that the endothelium and angiogenesis play a seminal role in the proliferation of liquid tumors, such as leukemia. We have shown that activated endothelial cells release cytokines that may stimulate leukemic cell growth. Leukemic cells, in turn, can release endothelial growth factors, such as vascular endothelial growth factor (VEGF). On the basis of these observations, we hypothesized that As2O3 may interrupt a reciprocal loop between leukemic cells and the endothelium by direct action on both cell types. We have shown that treatment of proliferating layers of human umbilical vein endothelial cells (HUVECs) with a variety of concentrations of As2O3results in a reproducible dose- and time-dependent sequence of events marked by change to an activated morphology, up-regulation of endothelial cell adhesion markers, and apoptosis. Also, treatment with As2O3 caused inhibition of VEGF production in the leukemic cell line HEL. Finally, incubation of HUVECs with As2O3 prevented capillary tubule and branch formation in an in vitro endothelial cell–differentiation assay. In conclusion, we believe that As2O3 interrupts a reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of both cell types and by inhibiting leukemic cell VEGF production.