VEGFs Gene On Treatment Of Secondary Lymohedema And On Bone Marrow Stromal Cells Differentiation In Vitro

Lymphedema is a problem of tissue fluid imbalance often due to defects in lymphatic uptake and/or transport.Primary lymphedema results from developmental defects of the lymphatic system with fewer vessels leading to insufficiencies in transport,whereas the more common secondary lymphedema arises as a consequence of surgical,malignant,inflammatory,or traumatic disruption of the lymphatics. Although secondary lymphedema is a common clinical condition,treatment for this disabling condition remains limited and largely ineffective.The rebuild of circulatory structures is a good choice to treat lymphedema,but the way how to rebuild lymphatic vessels effectively is still under study.Recent molecular studies have begun to elucidate the basis for lymphangiogenesis which has been shown to be stimulated by various cytokines, including those in the vascular endothelial growth factor(VEGF) family:VEGF, VEGF-B,VEGF-C,VEGF-D,Orfvirus-encoded VEGF-E,and the placenta growth factor.These ligands bind to VEGF receptors(VEGFR)-1,VEGFR-2,and VEGFR-3 with partially overlapping receptor specificities.In traditionally,the prototype VEGF binds VEGF receptor(VEGFR)-1 and VEGFR-2 and is angiogenic,whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3,is either angiogenic or lymphangiogenic in different assays,and the affinity of VEGF-C toward their receptors is regulated by proteolytic processing.The affinity of themature,proteolytically processed form of VEGF-C is 40 times higher for VEGFR-3 than for VEGFR-2.Furthermore,in adults VEGFR-3 is largely absent from blood vessel endothelia and remains predominantly expressed in the lymphatic endothelium.Targeted gene inactivation and transgene approaches in mice have also revealed critical roles for VEGF-C in lymphatic vessel function Adenoviral expression of VEGF-C can induce lymphangiogenesis in the skin of normal mice.In order to resolve the questions about lymphangiogenic specificity of VEGF family in lymphedema,the scientists in Japan and Poland found that Embryonic Stem Cells treating with VEGF-A and VEGF-C individually promote lymphatic endothelial cell formation in vitro and enhance formation of lymphatic vessel structures OP9 culture condition or 3-dimensional collagen matrix.It is powerful new in vitro tool to dissect the molecular mechanisms that treat lymphedema and control lymphatic vessel formation.Bone marrow stromal cells(MSCs),also known as mesenchymal stem cells or colony-forming units(CFU) fibroblastic,are nonhematopoietic multipotent stem-like cells that adhere to culture dishes.They are capable of clonal expansion in culture, MSCs share characteristics with other multipotent stem cells such as neural stem cells, hematopoietic stem cells because they possess the ability to self-renew and can differentiate not only into osteoblasts,chondrocytes,neurons,and skeletal muscle cells but also into vascular endothelial cells and cardiomyocytes,it has been became a kinds of ideal cell in tissue and cell repair for its merits as followed:1.Bone marrow is an altenrative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation,which obviates immunologicin compatibilities is possible.2.Of the various p rogenitorc eUsth ate xistw ithinb onem arrow,m esenchymal stem cells(MSC) are particularly attractive for clinical use because they are easily isolated,can be expanded in culture,and can be genetically manipulated using currently available molecular techniques.3.MSCs are adult stem cells,has no ethics questions. Objectives:1.In order to resolve the questions about lymphangiogenic specificity of VEGF-C in lymphedema,We investigated the efficacy of local transfer of naked plasmid DNA encoding human VEGF-C(pcDNA3.1-VEGF-C) on a rat model of hindlimb lymphedema by following changes in lymphedema volume using water displacement volumetry,magnetic resonance imaging (MRI) and B ultrasound techniques.We additionally examined lymphangiogenesis using histological and immunohistochemical examinations.2.Although many researches have demonstrated that bone marrow-derived mesenchymal stern cells(MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes,chondroeytes,and adipocytes in vivo and in vitro,little information is available regarding its potential to differentiate into lymphatic endothelial cell.Thus,we investigated the differentiation of MSCs into cells of the lymphatic endothelial lineage,and investigated its bionomics by culture bone marrow stem cells on matrige with the presence of endothelial growth supplements,Methods:1,We produced a surgical model of secondary lymphedema in the rat hindlimb and treated with local intradermal VEGF-C(pcDNA3.1-VEGF-C) transfection to investigate the efficacy of gene transfer.Magnetic resonance imaging(MRI),B ultrasound,and water displacement volumetry demonstrated a change of lymphedema in therapy group as compared to controls.Histological and immunofluorescent studies demonstrated the numbers of newly formed lymphatic vessels in therapy group.Our results indicate that VEGF-C gene therapy has produced new lymphatic vessels.2,Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described,and flow cytometry to detect the surface markers: CD29,CD90,CD34 and CD45.3,Purified MSCs were then plated onto dishes at cell density of 1 to 1.5×10~4 cells/cm~2 in the presence of VEGF(20 ng/mL) and / or VEGF-C(50 ng/mL) cultured in differentiation medium for 4 or 7 days to media containing 2% FBS.Thenexamined CD34,CD31,Prox-1 and LYVE-1 by immunocytochemistry,RT-PCR,Western Blot..4,Prepares collagen gel on the ice box and then put into37℃incubator. Inoculate the cells with good growth condition on the surface of the collagen gel.Then observe the growth of cultured cells by inverted phase contrast microscope.Results:1,The rat model got typical secondary lymphedema after operation, and one week after therapy,magnetic resonance imaging(MRI)(P<0.05),B ultrasound(P<0.05),and water displacement volumetry(P<0.05) demonstrated a reduction of lymphedema in therapy group as compared to controls.Histological and immunofluorescent studies demonstrated numerous newly formed lymphatic vessels in therapy group.2,Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described,and examination demonstrated be positive for CD29 and CD90 surface markers and negative for CD34 and CD45 by f low cytometry.5,Purified MSCs were then plated onto dishes in the presence of VEGF-A and /or VEGF-C cultured in differentiation medium for 10 days to media containing 2%FBS.The immunocytochemistry,RT-PCR,Western Blot demonstrated that compared to control group,the groups of VEGF-A, VEGF-C and VEGF-A +VEGF-C express Prox-1 and LYVE-1 but not CD34 or CD31.6,Lymphatic endothelial cells induced from MSCs can not grow with tubiform structure in collagen gel.Conclusions:1,Our results indicate that VEGF-C gene therapy has produced new lymphatic vessels which may have improved functional lymphatic drainage to reduce lymphedema volume in our model.2,In differentiation medium,MSCs treating with VEGF-A,VEGF-C and VEGF-A + VEGF-C individually promote lymphatic endothelial cell formation in vitro,and express the marks of Prox-1 and LYVE-1,but cannot grow with tubiform structure in collagen gelBring New Ideas:1,We investigated the efficacy of local transfer of naked plasmid DNA encoding human VEGF-C(pcDNA3.1-VEGF-C) on a rat model of hindlimb lymphedema by following changes in lymphedema volume using water displacement volumetry,magnetic resonance imaging(MRI) and B ultrasound techniques.2,Induced marrow stromal stem cells to lymphatic endotheliai cell with with VEGF-A,VEGF-C and VEGF-A + VEGF-C individually and detected by Immunocytochemistry,PT-PCR,Western Blot et al.