Long-lasting enfuvirtide carrier pentasaccharide conjugates with potent anti-human immunodeficiency virus type 1 activity.

Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG(12)-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.

PAR-1 activation on human late endothelial progenitor cells enhances angiogenesis in vitro with upregulation of the SDF-1/CXCR4 system.

OBJECTIVE: The importance of PAR-1 in blood vessel development has been demonstrated in knockout mice. As endothelial progenitor cells (EPCs) are involved in postnatal vasculogenesis, we examined whether they express PAR-1 and whether stimulation by the peptide SFLLRN modulates their angiogenic properties. METHODS AND RESULTS: EPC expanded from human CD34+ cord blood cells expressed PAR-1. PAR-1 activation induced EPC proliferation in a concentration-dependent manner far more potently than that of human umbilical vein endothelial cells. PAR-1 activation also enhanced actin reorganization, promoting both spontaneous migration in a Boyden chamber assay and migration toward SDF-1 and VEGF. As shown by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), EPC stimulation by SFLLRN significantly enhanced the mRNA expression of SDF-1 and its receptor CXCR-4. PAR-1 activation also increased CXCR4 expression on EPC and induced SDF-1 secretion, leading to autocrine stimulation. PAR-1 stimulation by SFLLRN also increased the formation of capillary-like structures by EPC in Matrigel, and this effect was abrogated by anti-CXCR-4, anti-SDF-1, and MEK inhibitor pretreatment. CONCLUSIONS: Human EPCs express functional PAR-1. PAR-1 activation promotes cell proliferation and CXCR4-dependent migration and differentiation, leading to a proangiogenic effect.

Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Efficacy of dendrimer-mediated angiostatin and TIMP-2 gene delivery on inhibition of tumor growth and angiogenesis: in vitro and in vivo studies.

Gene transfer is an attractive approach to fight cancer by targeting cancer cells or their vasculature. Our study reports the inhibition of tumor growth and angiogenesis by a nonviral method using dendrimers associated with 36-mer anionic oligomers (ON36) for delivering angiostatin (Kringle 1-3) and tissue inhibitor of metalloproteinase (TIMP)-2 genes. The optimal concentrations of dendrimers and ON36 for an efficient green fluorescent protein (GFP) plasmid delivery in endothelial cells (HMEC-1) and cancer cells (MDA-MB-435) were first chosen. Then the efficacy of transfection was determined by testing angiostatin and TIMP-2 secretion by Western blot and the biologic effects were evaluated. Angiostatin gene transfer markedly reduced in vitro (i) HMEC-1 but not MDA-MB-435 proliferation; (ii) HMEC-1 and MDA-MB-435 wound healing reparation; and (iii) capillary tube formation. TIMP-2 gene transfer did not affect cell proliferation but strongly inhibited (i) wound healing of HMEC-1 and MDA-MB-435 cells; and (ii) capillary tube formation. Supernatants of transfected-MDA-MB-435 cells also inhibited the formation of angiogenic networks on Matrigel, indicating a paracrine effect. In vivo, intratumoral angiostatin or TIMP-2 gene delivery using dendrimers associated with ON36 effectively inhibited tumor growth by 71% and 84%, respectively. Combined gene transfer resulted in 96% inhibition of tumor growth. Tumor-associated vascularization was also greatly reduced. These findings provide a basis for the further development of nonviral delivery of genes to fight cancer.

Insights in the molecular mechanisms of the anti-angiogenic effect of an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce the risk of coronary event by cholesterollowering dependent and independent mechanisms. We have already described that the inhibitory effect of cerivastatin on angiogenesis contribute to the cholesterol-independent beneficial effect and was due to the inhibition of the cell signaling cascade RhoA/FAK/Akt. In this study, new insights in the molecular mechanism of action were provided. It indicates an inhibition of exposure of alpha V beta 3 integrin on cell membrane and a modification of gene expression. The inhibition of angiogenesis could be related to 1) an increase in genes involved in the inhibition of cell proliferation (p19(INK4), p21(Waf/Cip1),Wnt-5a), the inhibition of cell migration (Rho-GDI 1, alpha E-catenin) and 2) a downregulation of genes involved in angiogenesis (PAI-1, Vitronectin, HoxD3, Notch4) or in cell invasion (Semaphorin E). In addition, DNA repair protein genes (MLH1, XRCC1) were increased. This study may indicate new biological interest of genes involved in angiogenesis control.

Differential STAT3, STAT5, and NF-kappaB activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules.

Different forms of interleukin-15 (IL-15) have been identified and shown to elicit different transduction pathways whose impact on hematopoiesis is poorly understood. We demonstrated herein that hematopoietic CD34+ cells constitutively produced endogenous secreted IL-15 (ES-IL-15) that activated different transcription factors and controlled the expression of several functional proteins, depending on the progenitor source. Thus, nuclear factor-kappa B (NF-kappa B) was activated in bone marrow (BM) and cord blood (CB) progenitors, whereas signal transducer and activator of transcription 3 (STAT3) and STAT5 activation was restricted to peripheral granulocyte-colony-stimulating factor (G-CSF)-mobilized and BM progenitors, respectively. ES-IL-15 acts through autocrine/paracrine loops controlled by high-affinity receptors involving IL-15 receptor alpha (IL-15Ralpha). Furthermore, ES-IL-15 was found to differentially control the expression of several functional molecules important for hematopoietic differentiation. Indeed, in BM precursors, neutralizing anti-IL-15 monoclonal antibody (mAb) inhibits the expression of the gamma c chain and of the chemokine stromal derived factor-1 (SDF-1) but had no effect on vascular cell adhesion molecule 1 (VCAM-1) and beta1 integrin adhesion molecule expression. Conversely, in CB progenitors, anti-IL-15 mAb inhibited VCAM-1 and beta1 integrin expression without affecting gammac chain expression and, most important, up-regulated SDF-1 expression. In conclusion, unprimed human hematopoietic CD34+ cells secrete cell-unbound IL-15, which activates through autocrine/paracrine loop distinct signaling pathways, depending on the progenitor source, thereby influencing the expression of several molecules important in the control of hematopoiesis.