Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice.

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 +/- 6.9 microm in diabetic mice and 34.4 +/- 3.9 microm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 +/- 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.

Induction of angiogenesis in omentum with vascular endothelial growth factor: influence on the viability of encapsulated rat pancreatic islets during transplantation.

Transplantation of pancreatic islets is proposed as a treatment for type 1 diabetes, but insufficient blood supply can cause the loss of viable grafted islets. In the present study, we investigated the influence of vascular endothelial growth factor (VEGF) on the angiogenesis of omentum during encapsulated islet allotransplantation and consequently on islet survival. Fifty rat islets, cultured for 24 h, were encapsulated in the presence or absence of human VEGF and implanted in the peritoneal cavity of rats (n = 6). After 7, 14 and 28 days of implantation, encapsulation devices with surrounding omentum were removed. Histological analysis of this tissue was performed. Cellular adhesion at the membrane surface was characterized by a phagocytosis test. The morphological aspect of the islets was analyzed and their functionality was evaluated by measuring insulin secretion. At each step of the study, there was a two-fold increase in the number of vessels in the presence of VEGF. In addition, VEGF increased the vessel diameter and the surface area of the angiogenic pedicle. Moreover, the presence of VEGF significantly decreased the distance between the devices and vessels (16.2 +/- 5.6 vs. 51.6 +/- 10.1 microm, p < 0.001). Membrane surface analysis showed a decrease in macrophage adhesion in the presence of VEGF. Furthermore, islet structure and functionality was preserved in the presence of VEGF. Stimulation of angiogenesis of omentum induced by VEGF is associated with preservation of islet viability. Local delivery of VEGF proved to be a relevant approach to ameliorate the outcome of islet transplantation.

Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders.

Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.

[In vitro and in vivo inhibition of HIV1 replication by retroviral transfer of interferon alpha, beta, or gamma genes: application to gene therapy of AIDS]. / Inhibition in vitro et in vivo de la réplication du VIH1 par transfert rétroviral des gènes d'interféron alpha, beta ou gamma: application à la thérapie génique du sida.

Somatic gene therapy is defined as the transfer of a heterologous gene into an organism for the purpose of correcting a genetic defect or providing a new therapeutic function to the target cell and thus inducing a cure or improving associated symptoms. While encouraging results have been generated by recent clinical evaluation of combination of anti-viral drugs, Aids still constitute an obvious candidate among the infectious diseases which might be treated by gene therapy. We have therefore chosen to develop and evaluate a gene therapy strategy based on the transfer into human target cells of HIV1-inducible interferon (IFN) alpha, beta or gamma genes. In a preliminary study, myeloïd U937 cell lines transfected with expression vectors containing the IFN alpha, beta or gamma genes under the control of the long terminal repeat (LTR) sequences of HIV1 were shown to be strongly resistant against an in vitro and in vivo (in HIV1 challenged SCID mice model) HIV1 infection. This cellular resistance was correlated with a strong induction of transgenic IFN synthesis and for IFN gamma, with a defect of HIV particles maturation. Secondly, construction and production of high titer retroviral vectors containing Tat-inducible IFN genes allowed efficient transduction of lymphoïd cell lines and human primary lymphocytes. These transduced cells were shown to be highly resistant against laboratory and primary HIV isolates. Taken together, our in vitro and in vivo results suggest that HIV1 inducible IFN gene therapy can be beneficial to HIV-infected individuals provided the fact that methods are developed that allow the efficient transduction of human hematopoïetic stem cells.

Experimental gene therapy: the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice.

OBJECTIVES: To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer on interferon (IFN)-alpha, -beta and -gamma genes to human cells. DESIGN: Human U937 promonocytic cells were stably transfected with Tat-inducible IFN expression vectors conferring an antiviral state against infection with HIV. METHODS: Transfected cells were either infected by HIV-1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo. RESULTS: U937 cell lines stably carrying IFN transgenes under the positive control of the HIV-1 Tat protein were highly resistant to HIV-1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV-1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN-U937 cells permitted full HIV-2 replication. Transfected cells injected into SCID mice and challenged against HIV-1 were strongly resistant to infection when cells were transduced with IFN-alpha of IFN-beta genes. However, IFN-gamma-transfected cells permitted HIV-1 infection in vivo despite the induction of a high level of IFN-gamma secretion. The quantity of proviral DNA was 10(5)-fold lower in IFN-alpha- or IFN-beta-transfected U937 cells collected from these SCID mice than that in non-transfected cells. CONCLUSIONS: Our results substantiated the validity of a strategy, bases on the transfer of HIV-1-inducible IFN-alpha or IFN-beta genes, to confer antiviral resistance to human cells.

HIV Tat protein potentiates in vitro granulomonocytic progenitor cell growth.

The direct involvement of hematopoietic progenitor cells in AIDS which results in dysregulated hematopoiesis, probably due to regulatory factors yet to be determined, has been reported by us and others. In this study we demonstrate that the HIV-2 Tat gene product is released in supernatant of HIV-2-infected long-term bone marrow cultures (LTBMC). These Tat-containing supernatants specifically enhanced growth of CFU-GM in agar culture system and this promoting activity was specifically neutralized with anti-Tat antibodies. The use of different recombinant Tat proteins confirmed that Tat has been responsible for enhanced in vitro growth of CFU-GM after HIV-2 infection of LTBMC. Moreover, limiting dilution analysis showed that Tat acts directly on the CD34+ cell population in which it increases the frequency of IL3-responding cells.

Involvement of HIV nef protein in abnormal hematopoiesis in AIDS: in vitro study on bone marrow progenitor cells.

In previous studies (1-4), we described disrupted hematopoiesis in long-term human bone marrow cultures infected with HIV. Conditioned medium (CM) from HIV-1 non-productively infected liquid cultures inhibits the proliferation of granulo-monocytic progenitor cells (CFU-GM) in clonogenic assays. A growth-inhibitory soluble factor was therefore suspected and we report here that this factor has been identified as the nef gene product. These nef-containing supernatants are specifically neutralized by anti-nef antibodies and recombinant nef has the same growth inhibitory properties. We used a nef-deficient virus to confirm that nef protein is responsible for the in vitro growth inhibition of CFU-GM.

Effects of thymus humoral factor gamma-2 (THF gamma 2) on lymphohaematopoietic progenitor cells: an in vitro study.

Thymus humoral factor gamma-2 (THF gamma 2), an immunomodulatory factor, was previously shown to exert a regulatory effect upon human haematopoietic development. In the present study, we were primarily interested in determining whether THF gamma 2 has an immunorestorative effect after HIV infection. Although no inhibition of viral production by T-cluster-forming cells and their mature progeny was observed, this thymus-derived compound prevented the dramatic decrease in CD4+ cells observed in cultures of immature T cells from normal bone marrow infected with HIV 1.

Severe in vitro inhibition of erythropoiesis and transient stimulation of granulopoiesis after bone-marrow infection with eight different HIV-2 isolates.

OBJECTIVE: To correlate severe anaemia and frequent neutropenia in HIV-2-infected patients with an inhibitory effect on bone-marrow progenitors common to several HIV-2 isolates. DESIGN: The effects of eight HIV-2 isolates on early (BFU-E) and late (CFU-E) erythroid progenitors and on granulomonocytic (CFU-GM) progenitors, produced in long-term bone-marrow cultures (LTBMC), were studied. METHODS: Absolute numbers of BFU-E, CFU-E and CFU-GM per culture flask were calculated weekly for each HIV-2-infected LTBMC using semi-solid clonogenic assays, and compared with those obtained in mock-infected LTBMC. Levels of significance for comparisons were determined by an analysis of variance (ANOVA). RESULTS: Pooled data from 24 series of LTBMC (three series for each HIV-2 isolate) revealed 80 and 100% inhibition of BFU-E and CFU-E on days 6 and 12 of LTBMC, respectively, while transient stimulation of CFU-GM was observed between days 14 and 20 of LTBMC, followed by total inhibition on day 30. CONCLUSIONS: These results confirm a direct inhibitory effect of HIV-2 on in vitro haematopoiesis. The similar pattern of erythroid progenitor inhibition obtained from seven out of eight isolates suggests that the inhibitory effect on erythropoiesis is a feature common to a large number of HIV-2 isolates, which correlates with clinical findings.

The effects of HIV on hematopoiesis.

Hematologic abnormalities are commonly associated with infection by the human immunodeficiency virus (HIV) that is the etiologic agent of the acquired immunodeficiency syndrome (AIDS). Bone marrow examinations reveal frequent dysplasia, dyserythropoïesis, lymphocytic infiltration and plasmocytosis. These marrow morphologic characteristics are strongly associated with peripheral blood cytopenia of one or more lineage: anemia, granulocytopenia or thrombocytopenia are frequently described. The pathophysiology of these observed deficiencies is still unclear, although several mechanisms have been postulated as possible explanations for the hematological features of AIDS patients. The hematologic manifestations observed in patients with AIDS or AIDS-related complex (ARC) and the postulated pathogenetic mechanisms are discussed in this review.

Modulation of normal human erythropoietic progenitor cells in long-term liquid cultures after HIV-1 infection.

Long-term bone marrow cultures (LTBMC) have been infected by two isolates of human immunodeficiency virus type 1 (HIV-1) (HIV-1 LAV and HIV-1 NDK) at multiplicities of infection ranging from 10(2) to 2.10(6) tissue culture infectious units (TCIU) per 10(6) bone marrow mononuclear cells (BMMNC). These infected cells are nonproducer cells and the viruses can be rescued by coculture with peripheral blood lymphocytes, cord blood lymphocytes, or BMMNC and not by the CEM cell line. HIV-1 clearly is not cytopathic for these cells. Following production and growth of erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E) for at least 6 weeks after infection with HIV-1 NDK, colony assays displayed a 50% inhibition of BFU-E production during 3 weeks of LTBMC. This was followed by a stimulation phase. On the contrary, HIV-1 LAV induces a 150% stimulation of BFU-E production, followed by 50% inhibition. Production of CFU-E was inhibited by 80-100% with the two isolates of HIV-1 after four weeks of LTBMC. Stimulatory and inhibitory activities were recovered from supernatants of infected LTBMC and lymphoid CEM cell lines, suggesting that HIV-1 induces release of a humoral factor responsible for disruption of hemopoietic progenitor cell production in vitro and consequently for hematologic abnormalities in AIDS patients.

Transient stimulation of granulopoiesis and drastic inhibition of erythropoiesis in HIV-2-infected long-term liquid bone marrow cultures.

Impaired hematopoiesis is commonly associated with human immunodeficiency virus HIV-1 and HIV-2-related AIDS. HIV-1 infection of hematopoietic progenitors has been studied, whereas HIV-2 infection of these cells is less well documented. In this work, we studied myeloid and erythroid progenitor production and differentiation in long-term bone marrow (LTBM) cultures after HIV-2 infection. A nonadherent fraction from these cultures containing the hematopoietic progenitors is nonproductively infected with HIV-2, whereas stroma cells replicate the virus only weakly. HIV-2 can be rescued from nonadherent T-depleted bone marrow cells, and its replication in stroma cells is amplified by cocultivation with HIV permissive cells. Colony assays performed weekly during the 6 weeks of LTBM cultures revealed a 100% inhibition of erythroid colony-forming unit (CFU)-E and erythroid burst-forming unit (BFU)-E production after 12 days of culture, whereas granulomonocytic colony forming units (CFU-GM) production was stimulated until day 20 and then disappeared on day 30. Stimulatory and inhibitory activities were recovered from supernatants of infected LTBM cultures and an infected lymphoid CEM cell line, suggesting that release of viral factor(s) may be responsible for HIV-2-induced impairment of hematopoietic progenitor production in vitro. Based on these results, an indirect effect of HIV-2 infection on the commitment of myeloid and erythroid progenitors, resulting in a dysregulated hematopoiesis, is postulated.