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1.
Proc Natl Acad Sci U S A ; 108(52): 20976-81, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22171002

RESUMO

Neovascularization is a critical determinant of wound-healing outcomes for deep burn injuries. We hypothesize that dextran-based hydrogels can serve as instructive scaffolds to promote neovascularization and skin regeneration in third-degree burn wounds. Dextran hydrogels are soft and pliable, offering opportunities to improve the management of burn wound treatment. We first developed a procedure to treat burn wounds on mice with dextran hydrogels. In this procedure, we followed clinical practice of wound excision to remove full-thickness burned skin, and then covered the wound with the dextran hydrogel and a dressing layer. Our procedure allows the hydrogel to remain intact and securely in place during the entire healing period, thus offering opportunities to simplify the management of burn wound treatment. A 3-week comparative study indicated that dextran hydrogel promoted dermal regeneration with complete skin appendages. The hydrogel scaffold facilitated early inflammatory cell infiltration that led to its rapid degradation, promoting the infiltration of angiogenic cells into the healing wounds. Endothelial cells homed into the hydrogel scaffolds to enable neovascularization by day 7, resulting in an increased blood flow significantly greater than treated and untreated controls. By day 21, burn wounds treated with hydrogel developed a mature epithelial structure with hair follicles and sebaceous glands. After 5 weeks of treatment, the hydrogel scaffolds promoted new hair growth and epidermal morphology and thickness similar to normal mouse skin. Collectively, our evidence shows that customized dextran-based hydrogel alone, with no additional growth factors, cytokines, or cells, promoted remarkable neovascularization and skin regeneration and may lead to novel treatments for dermal wounds.


Assuntos
Queimaduras/terapia , Dextranos/uso terapêutico , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapêutico , Neovascularização Fisiológica/fisiologia , Fenômenos Fisiológicos da Pele , Alicerces Teciduais , Cicatrização/fisiologia , Análise de Variância , Animais , Camundongos , Microscopia Eletrônica de Varredura
2.
Wound Repair Regen ; 19(2): 205-13, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21362088

RESUMO

The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF's downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products vascular endothelial growth factor and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and vascular endothelial growth factor all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.


Assuntos
Queimaduras/metabolismo , Quimiocinas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Queimaduras/patologia , Queimaduras/fisiopatologia , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Movimento Celular , Quimiocina CXCL12/metabolismo , Citoproteção , Imuno-Histoquímica , Queratinas/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Análise em Microsséries , Neovascularização Fisiológica , Neutrófilos/patologia , Nitroimidazóis , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia
3.
Wound Repair Regen ; 18(2): 193-201, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20163569

RESUMO

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that controls vascular responses to hypoxia and ischemia. In this study, mice that were heterozygous (HET) for a null allele at the locus encoding the HIF-1alpha subunit (HET mice) and their wild-type (WT) littermates were subjected to a thermal injury involving 10% of the body surface area. HIF-1alpha protein levels were increased in burn wounds of WT but not of HET mice on day 2. The serum levels of stromal-derived factor 1alpha, which binds to CXCR4, were increased on day 2 in WT but not in HET mice. Circulating angiogenic cells were also increased on day 2 in WT but not in HET mice and included CXCR4(+)Sca1(+) cells. Laser Doppler perfusion imaging demonstrated increased blood flow in burn wounds of WT but not HET mice on day 7. Immunohistochemistry on day 7 revealed a reduced number of CD31(+) vessels at the healing margin of burn wounds in HET as compared with WT mice. Vessel maturation was also impaired in wounds of HET mice as determined by the number of alpha-smooth muscle actin-positive vessels on day 21. The remaining wound area on day 14 was significantly increased in HET mice compared with WT littermates. The percentage of healed wounds on day 14 was significantly decreased in HET mice. These data delineate a signaling pathway by which HIF-1 promotes angiogenesis during burn wound healing.


Assuntos
Queimaduras/metabolismo , Leucócitos Mononucleares/metabolismo , Neovascularização Fisiológica , Animais , Movimento Celular , Quimiocina CXCL12/sangue , Heterozigoto , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fluxometria por Laser-Doppler , Camundongos , Camundongos Transgênicos , Receptores CXCR4/metabolismo , Fluxo Sanguíneo Regional , Cicatrização
5.
Ann Surg Oncol ; 12(12): 1025-36, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16244806

RESUMO

BACKGROUND: Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates angiogenesis and tumor proliferation. The VEGF signaling pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase 1; sFlk-1), which bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV) amplicons are replication-incompetent viruses used for gene delivery. We attempted to attenuate angiogenesis and inhibit pancreatic tumor growth through HSV amplicon-mediated expression of sFlk-1 under hypoxic control. METHODS: A multimerized hypoxia-responsive enhancer (10 x HRE) was cloned upstream of the sFlk-1 gene (10 x HRE/sFlk-1). A novel HSV amplicon expressing 10 x HRE/sFlk-1 was genetically engineered (HSV10 x HRE/sFlk-1).Human pancreatic adenocarcinoma cells (AsPC1) were transduced with HSV10 x HRE/sFlk-1 and incubated in normoxia (21% oxygen) or hypoxia (1% oxygen). Capillary inhibition was evaluated by human umbilical vein endothelial cell assay. Western blot assessed sFlk-1 expression. AsPC1 flank tumor xenografts (n = 24) were transduced with HSV10 x HRE/sFlk-1. RESULTS: Media from normoxic AsPC1 transduced with HSV10 x HRE/sFlk-1 yielded a 36% reduction in capillary formation versus controls (P < .05), whereas hypoxic AsPC1 yielded a 76% reduction (P < .005). Western blot of AsPC1 transduced with HSV10 x HRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia versus normoxia. AsPC1 flank tumors treated with HSV10 x HRE/sFlk-1 exhibited a 59% reduction in volume versus controls (P < .000001). CONCLUSIONS: HSV amplicon delivery of a hypoxia-inducible soluble VEGF receptor significantly reduces new vessel formation and tumor growth. Tumor hypoxia can thus be used to direct antiangiogenic therapy to pancreatic adenocarcinoma.


Assuntos
Adenocarcinoma/terapia , Inibidores da Angiogênese/administração & dosagem , Fator 1 Induzível por Hipóxia , Neovascularização Fisiológica/efeitos dos fármacos , Neoplasias Pancreáticas/terapia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Adenocarcinoma/patologia , Western Blotting , Hipóxia Celular , Elementos Facilitadores Genéticos , Vetores Genéticos/genética , Humanos , Neoplasias Pancreáticas/patologia , Simplexvirus/genética
6.
J Gastrointest Surg ; 8(7): 812-22; discussion 822-3, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15531234

RESUMO

Tumor hypoxia induces vascular endothelial growth factor (VEGF) expression, which stimulates tumor angiogenesis. The VEGF pathway is inhibited by soluble VEGF receptors (soluble fetal liver kinase-1 [sFlk-1]) that bind VEGF and block its interaction with endothelial cells. Herpes simplex virus (HSV)-derived amplicons are replication-incompetent viruses used for gene delivery. We attempt to attenuate angiogenesis and inhibit hepatoma growth through amplicon-mediated expression of sFlk-1 under hypoxic control. A multimerized hypoxia-responsive enhancer (10xHRE) was cloned upstream of the sFlk-1 gene (10xHRE/sFlk-1). An amplicon expressing 10xHRE/sFlk-1 was genetically engineered (HSV10xHRE/sFlk-1). SK-HEP-1 human hepatoma cells were transduced with HSV10xHRE/sFlk-1 and incubated in normoxia (21% O2) or hypoxia (1% O2). Human umbilical vein endothelial cell assay evaluated capillary inhibition. Western blot assessed sFlk-1 expression. SK-HEP-1 flank tumors (n = 24) in athymic mice were treated with HSV10xHRE/sFlk-1. Media from hypoxic SK-HEP-1 transduced with HSV10xHRE/sFlk-1 yielded an 80% reduction in capillary formation (P < 0.005), whereas normoxic SK-HEP-1 yielded a 25% reduction (P < 0.05). Western blot of SK-HEP-1 transduced with HSV10xHRE/sFlk-1 demonstrated greater sFlk-1 expression in hypoxia vs. normoxia. SK-HEP-1 tumors treated with HSV10xHRE/sFlk-1 yielded a 72% reduction in volume vs. the control group (P < 0.000001). HSV amplicon-mediated delivery of a hypoxia-inducible soluble VEGF receptor substantially reduces new vessel formation and tumor growth in hepatoma.


Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma Hepatocelular/irrigação sanguínea , Neoplasias Hepáticas/irrigação sanguínea , Simplexvirus/genética , Animais , Western Blotting , Hipóxia Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Vetores Genéticos/genética , Sequências Hélice-Alça-Hélice/genética , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Camundongos Nus , Proteínas Nucleares/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Transcrição/genética
7.
Surgery ; 136(3): 579-84, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15349105

RESUMO

BACKGROUND: G207 is an oncolytic herpes virus whose replicative cycle requires cellular ribonucleotide reductase (RR) for viral DNA synthesis. We attempt to enhance viral cytotoxicity in carcinoembryonic antigen (CEA)-producing colorectal cancer (CRC) cells through CEA-driven RR production. METHODS: CEA enzyme-linked immunosorbent assay was performed on LS174T and HCT-8 human CRC cells. The CEA enhancer-promoter (CEA E-P) was functionally assessed by luciferase assay. CEA E-P was cloned upstream of UL39, the gene encoding the large subunit of RR. Cells were transfected with CEA E-P/UL39 and infected with G207 for cytotoxicity assays. LS174T, with or without CEA E-P/UL39, were implanted into athymic mouse flanks (n = 28) and treated with G207. RESULTS: CEA levels were 7-fold higher in LS174T versus HCT-8 ( P <.00001). CEA E-P increased luciferase expression 7.5-fold in LS174T ( P <.01), with no increase in HCT-8. G207 cytotoxicity of'CEA E-P/UL39-transfected LS174T cells increased 69% by day 10 versus nontransfected cells ( P <.001), with no significant increase in HCT-8. Combining CEA E-P/UL39 with G207 in LS174T flank tumors resulted in a 65% decrease in tumor volume versus G207, phosphate-buffered saline, or'CEA E-P/UL39 alone ( P <.0001). CONCLUSIONS: CEA-driven RR production by CEA-secreting CRC cells significantly improves oncolytic viral cytotoxicity and specificity in vitro, and reduces tumor burden in vivo.


Assuntos
Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/terapia , Terapia Genética/métodos , Simplexvirus/genética , Morte Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Ribonucleotídeo Redutases/fisiologia , Replicação Viral/fisiologia , Vírus/genética
8.
Ann Surg ; 240(4): 659-65; discussion 665-6, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15383793

RESUMO

OBJECTIVE: To determine whether alpha-fetoprotein (AFP)-regulated ribonucleotide reductase (RR) production would promote more vigorous and specific viral killing in AFP-expressing hepatocellular carcinoma (HCC). BACKGROUND: AFP is expressed in over 70% of primary HCC but not in normal adult liver. AFP production by HCC can be exploited to target viral killing of tumor cells. G207 is an oncolytic herpes virus lacking UL39, the gene encoding RR. RR is an enzyme required for viral DNA synthesis and cytotoxicity. METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed for AFP levels on Hep3B and PLC5 human HCC cells. An AFP-albumin enhancer-promoter complex (AFP-alb) was constructed in a luciferase vector to assess function. AFP-alb was cloned upstream of UL39 (AFP-alb/UL39) and transfected into HCC cells for G207 cytotoxicity assays. Viral plaque forming assays evaluated G207 replication. Hep3B flank tumors, with and without AFP-alb/UL39 transfection, were established in athymic mice (n = 28) and treated with G207. RESULTS: Hep3B had 5-fold higher AFP levels than PLC5 (P < 0.00001). AFP-alb increased luciferase expression 72-fold in Hep3B (P < 0.001) and 3-fold in PLC5 (P < 0.001). AFP-alb/UL39 transfection increased G207 cytotoxicity 93% in Hep3B (P < 0.0005), with no significant increase in PLC5. Peak viral titers were 46-fold higher in Hep3B transfected with AFP-alb/UL39 versus mock-transfected cells (P < 0.01), with no significant change in PLC5. Flanks tumors transfected with AFP-alb/UL39 and treated with G207 demonstrated a 76% volume reduction versus mock-transfected tumors infected with G207 (P < 0.0001). CONCLUSIONS: AFP-driven RR production by hepatoma cells significantly enhances herpes viral cytotoxicity and specificity in vitro and reduces tumor burden in vivo.


Assuntos
Carcinoma Hepatocelular/terapia , Simplexvirus/genética , alfa-Fetoproteínas/genética , Adulto , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Reporter/genética , Genes Virais/genética , Vetores Genéticos/genética , Humanos , Luciferases/genética , Masculino , Camundongos , Camundongos Nus , Mutação/genética , Transplante de Neoplasias , Regiões Promotoras Genéticas/genética , Ribonucleotídeo Redutases/genética , Transfecção , Replicação Viral
9.
Surgery ; 136(2): 199-204, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15300180

RESUMO

BACKGROUND: Hypoxia is a common tumor condition correlated with therapeutic resistance. Ribonucleotide reductase (RR) is a rate-limiting enzyme for viral replication. We hypothesize that hypoxia-driven transcription of UL39, the gene encoding the large subunit of RR, would enhance herpes oncolytic viral therapy in breast cancer. METHODS: Hypoxia-inducible factor 1alpha (HIF-1alpha) ELISA was performed on MCF7 human breast cancer cells in hypoxia (1% O2) or normoxia (21% O2). A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested in a luciferase assay. 10xHRE was cloned upstream of the UL39 gene (10xHRE-UL39). MCF7 cells were transfected with 10xHRE-UL39, incubated in hypoxia or normoxia, and infected with G207. Cytotoxicity assays and viral titers were performed. RESULTS: HIF-1alpha levels increased 7-fold in hypoxic MCF7 cells (P < .001). 10xHRE increased luciferase gene expression 61-fold in hypoxia versus controls (P < .01). G207 cytotoxicity of 10xHRE-UL39-transfected, hypoxic MCF7 cells increased 74% versus mock-transfected, hypoxic MCF7 cells (P < .001). In normoxia, 10xHRE-UL39 transfection did not significantly improve G207 cytotoxicity. 10xHRE-UL39 transfection improved peak viral titers 69-fold in hypoxia (P < .005), with no significant difference in normoxia. CONCLUSION: Hypoxia-driven RR production significantly enhances G207 cytotoxicity in hypoxic breast cancer cells, which would otherwise be resistant to herpes viral therapy alone.


Assuntos
Neoplasias da Mama/terapia , Hipóxia Celular , Simplexvirus/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Luciferases/metabolismo , Fatores de Transcrição/análise , Replicação Viral
10.
Ann Surg ; 239(6): 892-9; discussion 899-902, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15166969

RESUMO

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.


Assuntos
Neoplasias Colorretais/terapia , Terapia Genética/métodos , Herpesvirus Humano 1/fisiologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/terapia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/patologia , Neoplasias Colorretais/virologia , Vetores Genéticos , Herpesvirus Humano 1/genética , Hipóxia , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
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