Objective-The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue-derived cells (ADSC) on angiogenic cell therapy. Method and Results-ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and ␣-smooth muscle actin-positive cells in ischemic tissue. Conclusion-These
We have developed a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. Efficiency of gene transfer was greatly enhanced by protamine sulfate and centrifugation. Fluorescein isothiocyanate-labeled oligodeoxynucleotides (FITC-ODN) were also delivered to cells at > 95% efficiency. When HVJ envelope vector was injected into organs directly, reporter gene expression was observed in organs including liver, brain, skin, uterus, tumor masses, lung, and eye. When HVJ envelope vector containing luciferase gene was injected into mouse tail vein, luciferase gene expression was detected primarily in spleen. FITC-ODN were also delivered to spleen cells by intravenous injection of HVJ envelope. These results suggest that HVJ envelope vector will be useful for both ex vivo and in vivo gene therapy experiments.
Purpose: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppresses the growth of human cancer cells as effectively as replicationcompetent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E.Experimental Design: The molecules responsible for HVJ-E-induced cancer cell death were elucidated in prostate cancer cell lines, and the effect of HVJ-E on orthotopic prostate cancers was evaluated in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice.Results: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells but not the viability of the noncancerous prostate epithelium. Knockdown experiments using siRNAs showed that the cancer cell-selective killing induced by HVJ-E was mediated by retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were upregulated by HVJ-E in the castration-resistant prostate cancer cell line PC3 but not in the noncancerous prostate epithelial cell line PNT2. TRAIL siRNA but not Noxa siRNA significantly inhibited HVJ-E-induced cell death in PC3 cells. However, Noxa siRNA effectively suppressed HVJ-E-induced cell death in DU145 cells, another castration-resistant prostate cancer cell line, in which Noxa but not TRAIL was upregulated by HVJ-E. Furthermore, the orthotopic prostate cancers were dramatically eradicated in immunodeficient mice injected with HVJ-E.Conclusion: The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy.
Objective-We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson's disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-B (NF-B) inactivation in ECs and vascular smooth muscle cells (VSMCs). Methods and Results-From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-␣-induced NF-B activity in vascular cells and increased inhibitor of kappa B-␣ (IB-␣), possibly through the attenuation of IB-␣ ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-B activity in VSMCs. Conclusions-These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-B activity. (Arterioscler Thromb Vasc
Background-Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. Methods and Results-Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs)were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c-fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF-and vascular endothelial growth factor 165-injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. Conclusions-These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.
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