Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Neuropilin-1 is upregulated in hepatocellular carcinoma and contributes to tumour growth and vascular remodelling.

BACKGROUND & AIMS: Neuropilin-1 (NRP1) is a transmembrane co-receptor for semaphorins and heparin-binding pro-angiogenic cytokines, principally members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and malignant progression of many cancers. The role of NRP1 in the development of hepatocellular carcinoma (HCC) is not completely understood. METHODS: We used human tissue microarrays and a mouse transgenic model of HCC to establish the spatio-temporal patterns of NRP1 expression in HCC. To evaluate the therapeutic potential of targeting NRP1 in HCC, we treated HCC mice with peptide N, an NRP1 binding recombinant protein and competitive inhibitor of the VEGF-A(165)/NRP1 interaction. RESULTS: We demonstrate that NRP1 is expressed in hepatic endothelial cells of both human healthy biopsies and in HCC samples, but not in normal hepatocytes. We found that increased NRP1 expression in human tumour hepatocytes is significantly associated with primary HCC. Using RT-PCR, Western blot and immunofluorescence analysis we show that NRP1 expression in the liver of transgenic HCC mice is increased with disease progression, in both vascular and tumour compartments. Blocking NRP1 function with peptide N leads to the inhibition of vascular remodelling and tumour liver growth in HCC mice. CONCLUSIONS: Our results indicate a specific role of NRP1 in HCC growth and vascular remodelling and highlight the possibility of therapeutically targeting NRP1 for the treatment of HCC.

Small interfering RNAs induce target-independent inhibition of tumor growth and vasculature remodeling in a mouse model of hepatocellular carcinoma.

RNA interference mediated by small interfering RNAs (siRNAs) has emerged as a potential therapeutic approach to treat various diseases, including cancer. Recent studies with several animal models of posttraumatic revascularization demonstrated that synthetic siRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3 (TLR3)/interferon pathway and suppression of angiogenesis. To analyze the impact of siRNAs on tumor angiogenesis, we injected transgenic mice developing hepatocellular carcinoma (HCC) with either control siRNAs or siRNA targeting neuropilin-1. We found that treatment with these siRNAs led to a comparable reduction in tumor liver volume and to inhibition of tumor vasculature remodeling. We further determined that TLR3, which recognizes double-stranded siRNA, was up-regulated in mouse HCC. Treatment of HCC mice with polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 agonist, led to both a reduction of tumor liver enlargement and a decrease in hepatic arterial blood flow, indicating that TLR3 is functional and may mediate both anti-angiogenic and anti-tumor responses. We also demonstrated that siRNAs increased interferon-γ levels in the liver. In vitro, interferon-γ inhibited proliferation of endothelial cells. In addition, we found that siRNAs inhibited endothelial cell proliferation and morphogenesis in an interferon-γ-independent manner. Our results suggest that synthetic siRNAs inhibit target-independently HCC growth and angiogenesis through the activation of the innate interferon response and by directly inhibiting endothelial cell function.

Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B.

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Coadministration of endothelial and smooth muscle progenitor cells enhances the efficiency of proangiogenic cell-based therapy.

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells.

BACKGROUND INFORMATION: Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre-existing vascular bed. VEGF-A (vascular endothelial growth factor-A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF-A-driven angiogenesis, although the signalling mechanisms underlying this co-operation are not completely understood. RESULTS: We analysed the effects of the combination of VEGF-A and HGF on the activation of VEGFR-2 (VEGF receptor-2) and c-met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR-2 and c-met do not physically associate and do not transphosphorylate each other, suggesting that co-operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF-A(165) and HGF stimulate a similar set of MAPKs (mitogen-activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF-A(165) and HGF. Moreover, the combination of VEGF-A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF-A(165) and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF-A(165) and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular-like patterns in vitro in a Rho- or Rac-dependent manner. CONCLUSIONS: Under angiogenic conditions, combining VEGF-A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.

Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition.

AIMS: We analysed the possible protective role of human endothelial (EPCs) and smooth muscle (SPCs) progenitor cells on atherosclerosis development in apoE(-/-)RAG2(-/-) mice. We determined plasma levels of SPCs in coronary patients. METHODS AND RESULTS: ApoE(-/-)RAG2(-/-) mice received four intravenous injections of saline, 5 x 10(5) SPCs, or 5 x 10(5) EPCs every other week, one (preventive approach) or 12(curative approach) weeks after starting a high fat diet. Derived-SPC levels were quantified from blood mononuclear cells of patients with stable angina (n = 10) and acute coronary syndromes (ACS, n = 9). SPCs reduced atherosclerosis development by 42% (P < 0.001), but had no effect on lesion progression. In the SPC group, collagen and smooth muscle cell content were increased (+80%, P < 0.001, +46%, P < 0.05, respectively), and macrophage content was decreased (-41%, P < 0.05). In the curative approach, macrophage content decreased by 40.5% (P < 0.05) after SPC injection. EPC injection had no effect on atherosclerosis development or progression. Peripheral blood-derived SPC levels were reduced in patients with ACS compared with stable angina patients (P < 0.05). CONCLUSION: We demonstrate that SPCs limit plaque development and promote changes in plaque composition towards a stable phenotype in mice. Our finding in patients suggests that reduced peripheral blood-derived SPC levels might represent a mechanism contributing to plaque destabilization.

Increase in vascular permeability and vasodilation are critical for proangiogenic effects of stem cell therapy.

BACKGROUND: Proangiogenic cell therapy based on administration of bone marrow-derived mononuclear cells (BMCs) or endothelial progenitor cells (EPCs) is now under investigation in humans for the treatment of ischemic diseases. However, mechanisms leading to the beneficial effects of BMCs and EPCs remain unclear. METHODS AND RESULTS: BMC- and CD34+-derived progenitor cells interacted with ischemic femoral arteries through SDF-1 and CXCR4 signaling and released nitric oxide (NO) via an endothelial nitric oxide synthase (eNOS)-dependent pathway. BMC-induced NO production promoted a marked vasodilation and disrupted vascular endothelial-cadherin/beta-catenin complexes, leading to increased vascular permeability. NO-dependent vasodilation and hyperpermeability were critical for BMC infiltration in ischemic tissues and their proangiogenic potential in a model of hindlimb ischemia in mice. CONCLUSIONS: Our results propose a new concept that proangiogenic progenitor cell activity does not rely only on their ability to differentiate into endothelial cells but rather on their capacity to modulate the function of preexisting vessels.

Tetrapeptide AcSDKP induces postischemic neovascularization through monocyte chemoattractant protein-1 signaling.

BACKGROUND: We investigated the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in a model of surgically induced hindlimb ischemia. METHODS AND RESULTS: Hindlimb ischemia was induced by femoral artery ligature and an osmotic minipump was implanted subcutaneously to deliver low (0.12 mg/kg per day) or high (1.2 mg/kg per day) doses of AcSDKP, for 7 or 21 days. Angiography scores, arteriole density, capillary number, and foot perfusion were increased at day 21 in the high-dose AcSDKP-treated mice (by 1.9-, 1.8-, 1.3-, and 1.6-fold, respectively) compared with control animals (P<0.05, P<0.01, P<0.01, respectively). AcSDKP treatment for 24 hours upregulated the monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels by 1.5-fold in cultured endothelial cells (P<0.01). In the ischemic hindlimb model, administration of AcSDKP also enhanced MCP-1 mRNA levels by 90-fold in ischemic leg (P<0.001) and MCP-1 plasma levels by 3-fold (P<0.001 versus untreated ischemic control mice). MCP-1 levels upregulation were associated with a 2.3-fold increase in the number of Mac3-positive cells in ischemic area of AcSDKP-treated mice (P<0.001 versus untreated animals). Interestingly, AcSDKP-induced monocyte/macrophage infiltration and postischemic neovascularization was fully blunted in MCP-1-deficient animals. CONCLUSIONS: AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway.

Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.

Two-dimensional color-coded pulsed Doppler ultrasonography (US) with a 12-MHz linear transducer was used to follow tumor growth and neoangiogenesis development in 12 transgenic mice developing a whole liver hepatocellular carcinoma (HCC) induced by the expression of SV40-T antigen. In this model, male mice developed HCC at various temporal and histologic stages (hyperplastic, four-eight wk; nodular, 12 wk; diffuse carcinoma, 16-20 wk), whereas female mice remained tumor free. Seven age-matched tumor-free mice were used as controls. Liver volume was calculated from B-mode images of the abdomen. Blood flow waveforms were recorded from the hepatic tumor-feeding artery upstream from the tumor vessels, allowing quantitative blood flow velocity measurements. Measurements were performed every four weeks from four to 20 weeks. As early as the hyperplastic stage (eight weeks), liver volume was increased by 2.7-fold, hepatic artery peak-systolic blood flow velocities (BFV) by 1.5-fold, end-diastolic BFV by 1.6-fold and mean BFV by 2.0-fold compared with control values (p < 0.001). Differences increased until 20 weeks and peak-systolic reached 90 +/- 6, end-diastolic 54 +/- 5 and mean BFV 48 +/- 5 cm s(-1). Successive measurements of BFV were reproducible and intraobserver repeatability coefficient values were <3 cm s(-1). In contrast, mesenteric artery BFV, which did not supply tumor region, did not show any significant difference with respect to control values. Thus, an increase in BFV constitutes a functional evaluation of tumor vascularity. In preclinical studies in small animals, measurements of liver volume and blood flow velocities in hepatic tumor-feeding artery provide a useful, reproducible, noninvasive, easy-to-repeat tool to monitor tumor growth and neoangiogenesis in hepatocellular carcinoma in mice.

Interleukin-18/interleukin-18 binding protein signaling modulates ischemia-induced neovascularization in mice hindlimb.

Identification of factors that may stimulate ischemia-induced neovascularization without increasing atherosclerotic plaque progression is of major therapeutic importance. We hypothesized that interleukin-18 binding protein (IL-18BP), a major antiinflammatory protein with plaque-stabilizing activities, may affect the neovascularization in mice ischemic hindlimb. Ischemia was produced by artery femoral occlusion in mice that were subjected to in vivo intramuscular electrotransfer of either an empty plasmid or a murine IL-18BP plasmid. Angiographic score, capillary density (CD31 staining), and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/nonischemic leg ratio by respectively 1.6-, 1.4-, and 1.5-fold in IL-18BP-treated mice compared with controls (P<0.01). This was associated with a significant 2-fold increase in both vascular endothelial growth factor (VEGF) and phospho-Akt protein content in the ischemic hindlimb of IL-18BP-treated mice (P<0.05). Similar results were obtained in IL-18-deficient mice. Because bone marrow-derived endothelial progenitor cells (BM-EPCs) are involved in postnatal vasculogenesis, EPCs were isolated and cultivated from bone marrow mononuclear cells. IL-18BP treatment led to a significant 1.8-fold increase in the percentage of BM-EPCs characterized as cells positive for both AcLDL-Dil and von Willebrand factor (P<0.001). In conclusion, IL-18BP stimulates ischemia-induced neovascularization in association with an activation of VEGF/Akt signaling and an increase in BM-EPCs mobilization and differentiation. Our findings strongly suggest a major antiangiogenic role of endogenous IL-18 in postischemic injury.

Herpes simplex virus type 1 colitis in a patient with common variable immunodeficiency syndrome.

We report on a case of herpes simplex virus (HSV) type 1 colitis in a 69-year-old patient with common variable immunodeficiency syndrome. A treatment with polyvalent immunoglobulins was discontinued in April 2001. In March 2004 she developed chronic diarrhoea related to rectosigmoidal and caecal ulcerations. In November 2004, HSV was recovered in tissue culture from colonic biopsies. Valaciclovir was then started, leading the patient to clinical remission at day 4, and continued for a 6-week course (without any secondary antiviral prophylaxis). Colonic biopsies were negative for HSV by tissue culture and PCR within 3 weeks of antiviral treatment. Intravenous polyvalent immunoglobulin infusions were readministered within the third week of antiviral treatment. She has declared no clinical event since this period. Three months after the antiviral treatment was achieved, a rectosigmoidoscopy showed an ad-integrum macroscopic and histological mucosal healing whereas PCR was negative for HSV in the colonic tissue. As a large proportion of patients with common variable immunodeficiency syndrome present not only as a humoral immunodeficiency but also as a defect in the cellular immunity compartment (with T-cell deficits), HSV, as well as cytomegalovirus, should be investigated in patients with common variable immunodeficiency syndrome presenting colitis.

Transplantation of bone marrow-derived mononuclear cells in ischemic apolipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition.

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC-based therapy in the setting of atherosclerosis. METHODS AND RESULTS: Apolipoprotein E (apoE)-knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (n=10) or 10(6) BM-MNCs from wild-type animals (n=10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (P<0.0001), the 33% increase in capillary density (P=0.01), and the 65% increase in tissue Doppler perfusion score (P=0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 10(6) BM-MNCs into apoE-KO mice with hindlimb ischemia induced a significant 48% to 72% increase in lesion size compared with the other 3 groups (P=0.0025), despite similar total cholesterol levels. Transplantation of 10(5) BM-MNCs produced similar results, whereas transplantation of 10(6) apoE-KO-derived BM-MNCs had neither proangiogenic nor proatherogenic effects. There was no difference in plaque composition between groups. CONCLUSIONS: BM-MNC therapy is unlikely to affect atherosclerotic plaque stability in the short term. However, it may promote further atherosclerotic plaque progression in an ischemic setting.

Ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature.

OBJECTIVES: Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site. METHODS: Mononuclear cells (MNCs) or CD34+ -enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model. RESULTS: The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed alpha-smooth muscle actin (alpha-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature. CONCLUSION: We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.

Strategies to Enhance the Efficiency of Endothelial Progenitor Cell Therapy by Ephrin B2 Pretreatment and Coadministration with Smooth Muscle Progenitor Cells on Vascular Function During the Wound-Healing Process in Irradiated or Nonirradiated Condition.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization. We therefore assessed the effect of a therapeutic strategy based on EPC administration in the healing of radiation-induced damage. To improve cell therapy for clinical use, we used pretreatment with ephrin B2-Fc (Eph-B2-Fc) and/or coadministration with smooth muscle progenitor cells. At day 3, EPCs promoted dermal wound healing in both nonirradiated and irradiated mice by 1.2- and 1.15-fold, respectively, compared with animals injected with phosphate-buffered saline. In addition, EPCs also improved skin-blood perfusion and capillary density in both irradiated and nonirradiated mice compared with PBS-injected animals. We also demonstrated that activation with Eph-B2-Fc increased wound closure by 1.6-fold compared with unstimulated EPCs in nonirradiated mice. Interestingly, the beneficial effect of Eph-B2-Fc was abolished in irradiated animals. In addition, we found that Eph-B2-Fc stimulation did not improve EPC-induced vascular permeability or adhesiveness compared to unstimulated EPCs. We hypothesized that this effect was due to high oxidative stress during irradiation, leading to inhibition of EPCs' beneficial effect on vascular function. In this line, we demonstrated that, in irradiated conditions, N-acetyl-l-cysteine treatment restored the beneficial effect of EPC stimulation with Eph-B2-Fc in the wound healing process. In conclusion, stimulation by Eph-B2-Fc improved the beneficial effect of EPCs in physiological conditions and irradiated conditions only in association with antioxidant treatment. Additionally, cotherapy was beneficial in pathological conditions.

Inhibition of K562 leukemia angiogenesis and growth by expression of antisense vascular endothelial growth factor (VEGF) sequence.

Vascular endothelial growth factor (VEGF), a major angiogenic factor, plays a key role in the growth of solid tumor. Recently, expression of VEGF and its receptors has been found on leukemic cells as well as on endothelial cells. VEGF may fulfill a fundamental role in promoting tumor angiogenesis and proliferation by stimulating both endothelial cells and leukemic cells. To investigate the role of VEGF in the angiogenesis and growth of leukemic cell, we used an antisense strategy to downregulate VEGF expression in K562 cells, a human erythroleukemia cell line. Expression of antisense-VEGF in K562 cells reduced the secretion of VEGF protein and inhibited cell survival. The proliferation and migration of human umbilical vein endothelial cells were decreased in response to the conditioned medium (CM) from K562 cells expressed antisense-VEGF, compared to CM from K562 cells transfected with vector control. Moreover, subcutaneous injection of nude mice with antisense-VEGF K562 cells inhibited tumor growth with a reduction of the density of microvessels and an increased apoptosis in those tumors, compared to vector control K562 cells. These results suggest that the efficient downregulation of the VEGF production in leukemic cells using antisense-VEGF may constitute a novel strategy of treatment in leukemia.

Adenovirus-mediated gene therapy with an antiangiogenic fragment of thrombospondin-1 inhibits human leukemia xenograft growth in nude mice.

Recent investigations support the idea that angiogenesis is involved in the pathophysiology of leukemia. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein showing angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. The aim of the present study was to establish a recombinant adenovirus (ADV.TSP-1(f)) expressing antiangiogenic fragment of TSP-1 (TSP-1(f)), and to determine the feasibility for use of the adenovirally expressed TSP-1(f) in leukemia gene therapy. The results of this investigation showed that TSP-1(f) was expressed efficiently in adenovirus-transduced human myelogenous leukemia K562 cells. Compared to the controls, although there was almost no effect on proliferation of K562 cells in vitro, adenovirus-mediated TSP-1(f) transduction inhibited the growth of K562 xenografts dramatically. Furthermore, the microvessel density (MVD) was much lower in the ADV.TSP-1(f)-treated tumors compared to the controls. These data support the use of in vivo gene delivery approach to produce antiangiogenic fragment of TSP-1 for leukemia therapy.

Suppression of tumor growth by viral vector-mediated gene transfer of N-terminal truncated platelet factor 4.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

[Mechanisms and role of lymphangiogenesis in cancer metastasis]. / Mécanisme de la lymphangiogenèse tumorale, longtemps oubliée, dans la dissémination métastatique.

An important critical point in tumor progression is the acquisition of metastatic potential. The presence of metastases in regional lymph nodes is an indicator of poor survival. The vascular endothelial growth factor (VEGF) family of growth factors and receptors is involved in vasculogenesis and angiogenesis. Among them, VEGF-C and VEGF-D regulate the lymphatic vessels development and growth via their binding to their receptor VEGFR3. The expression of VEGF-C or VEGF-D is demonstrated in various human tumors and can be used as pronostic factors in some of them. With the aid of these molecules and the discovery of specific lymphatic markers, lymphatic endothelial cells can be isolated and lymphatic vessels can be identified within tumors. The role of lymphangiogenesis in promoting the metastatic spread of tumor cells has been studied in animal models.