Development of angiogenic cell and gene therapy by transplantation of umbilical cord blood with vascular endothelial growth factor gene.

Endothelial progenitor cells (EPCs) are present in the mononuclear cells (MNCs) of umbilical cord blood and peripheral blood. To establish the efficiency of angiogenic cell and gene therapies, we transfected the human vascular endothelial growth factor (hVEGF) gene into cord blood MNCs to enhance endothelialization. MNCs from cord blood and peripheral blood were isolated and transfected with pCR3 expressing hVEGF165 or GFP by the Hemagglutinating Virus of Japan (HVJ)-envelope and the cells were cultured in endothelium basal medium-2. The number of attached cells from cord blood was higher than that from peripheral blood. Attached cells expressed Flk-1, VE-cadherin, PECAM-1, CD34, and Tie-2. The increase in the number of attached cells was transient with the transfection of vascular endothelial growth factor (VEGF) gene early in the experimental period. Flt-1 mRNA was not expressed early in the culture period, but was expressed at 2 weeks after separation. VEGF gene transfer into MNCs at 12 days after separation, i.e., when Flt-1 mRNA was expressed continuously, increased the number of attached cells. We evaluated the effects of the transplantation of cord blood MNCs expressing the hVEGF gene on regional blood flow in an ischemic area in a rat model of chronic hindlimb ischemia. Blood flow was significantly improved in nude rats that received transplanted control MNCs. Transplantation of cord blood MNCs transfected with the hVEGF gene yielded greater improvements in blood flow. These results indicate that the hVEGF gene enhances endothelialization of EPCs, and that the transplantation of cord blood MNCs transfected with the VEGF gene may be feasible for the treatment of ischemic diseases as a type of angiogenic cell and gene therapy.

[1]  A. Quyyumi,et al.  Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. , 2003, The New England journal of medicine.

[2]  Stefanie Dimmeler,et al.  Transdifferentiation of Blood-Derived Human Adult Endothelial Progenitor Cells Into Functionally Active Cardiomyocytes , 2003, Circulation.

[3]  M. Sands,et al.  VEGF increases engraftment of bone marrow-derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[4]  K. Shimada,et al.  Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial , 2002, The Lancet.

[5]  R. Nagai,et al.  Role of Fas/Fas ligand interaction in ischemia-induced collateral vessel growth. , 2002, Hypertension research : official journal of the Japanese Society of Hypertension.

[6]  E. Scott,et al.  Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion , 2002, Nature.

[7]  J. Isner,et al.  Endothelial Progenitor Cell Vascular Endothelial Growth Factor Gene Transfer for Vascular Regeneration , 2002, Circulation.

[8]  Y. Kaneda,et al.  Safety evaluation of hemagglutinating virus of Japan--artificial viral envelope liposomes in nonhuman primates. , 2001, Human gene therapy.

[9]  T. Murohara,et al.  Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. , 2000, The Journal of clinical investigation.

[10]  J. Isner,et al.  Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Isner,et al.  Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. , 1999, Circulation research.

[12]  J. Isner,et al.  VEGF contributes to postnatal neovascularization by mobilizing bone marrow‐derived endothelial progenitor cells , 1999, The EMBO journal.

[13]  J. Isner,et al.  Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. , 1999, The Journal of clinical investigation.

[14]  Takayuki Asahara,et al.  Isolation of Putative Progenitor Endothelial Cells for Angiogenesis , 1997, Science.

[15]  B. Keyt,et al.  Identification of Vascular Endothelial Growth Factor Determinants for Binding KDR and FLT-1 Receptors , 1996, The Journal of Biological Chemistry.

[16]  W. Risau.  Differentiation of endothelium , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  D. Donner,et al.  Vascular Endothelial Cell Growth Factor Promotes Tyrosine Phosphorylation of Mediators of Signal Transduction That Contain SH2 Domains , 1995, The Journal of Biological Chemistry.

[18]  H. Federoff,et al.  Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice. , 1995, Circulation research.

[19]  C B Harley,et al.  Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Lansdorp,et al.  Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. , 1994, Blood.

[21]  W. Risau,et al.  Induction of vasculogenesis and hematopoiesis in vitro. , 1992, Development.

[22]  D. Orlic.  Stem cell repair in ischemic heart disease: An experimental model , 2002, International journal of hematology.

[23]  K. Rajewsky,et al.  Perspectives Series: Molecular Medicine in Genetically Engineered Animals , 1996 .

[24]  H. Mocharla,et al.  Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping. , 1990, Gene.