RESUMEN
Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell-based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection-free allograft survival with a single perioperative ex vivo infusion of bone marrow-derived mesenchymal stem cells through the allograft vasculature, in the absence of long-term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.
Asunto(s)
Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Miembro Posterior/trasplante , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Trasplante de Piel/efectos adversos , Alotrasplante Compuesto Vascularizado/métodos , Animales , Rechazo de Injerto/etiología , Tolerancia Inmunológica/inmunología , Terapia de Inmunosupresión , Ratas , Ratas Endogámicas Lew , Tolerancia al Trasplante/inmunologíaRESUMEN
Radiation therapy is an effective treatment strategy for many types of cancer but is limited by its side effects on normal tissues, particularly the skin, where persistent and progressive fibrotic changes occur and can impair wound healing. In this study, we attempted to mitigate the effects of irradiation on skin using a novel transcutaneous topical delivery system to locally inhibit p53 up-regulated modulator of apoptosis (PUMA) gene expression with small interfering RNA (siRNA). In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated. Prior to irradiation, PUMA and nonsense siRNA were applied via a novel hydrogel formulation to dorsal skin and reapplied weekly. Skin was harvested at multiple time points to evaluate dermal siRNA penetration, mRNA expression, protein expression, dermal thickness, subcutaneous fat, stiffness, vascular hypertrophy, SCAR index, and reactive oxygen species (ROS) generation. Murine skin treated with topical PUMA siRNA via optimized hydrogel formulation demonstrated effective PUMA inhibition in irradiated tissue at 3-4 days. Tissue stiffness, dermal thickness, vascular hypertrophy, SCAR index, ROS levels, and mRNA levels of MnSOD and TGF-ß were all significantly reduced with siPUMA treatment compared to nonsense controls. Subcutaneous fat area was significantly increased, and levels of SMAD3 and Phospho-SMAD3 expression were unchanged. These results show that PUMA expression can be effectively silenced in vivo using a novel hydrogel lipoplex topical delivery system. Moreover, cutaneous PUMA inhibition mitigates radiation induced changes in tissue character, restoring a near-normal phenotype independent of SMAD3 signaling.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/administración & dosificación , Proteínas Reguladoras de la Apoptosis/farmacología , Traumatismos Experimentales por Radiación/prevención & control , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Cicatrización de Heridas/efectos de los fármacos , Administración Cutánea , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Geles , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BLRESUMEN
Diabetic patients exhibit dysfunction of the normal wound healing process, leading to local ischemia by vascular occlusive disease as well as sustained increases in the proinflammatory cytokines and overproduction of reactive oxygen species (ROS). Of the many sources of ROS, the enzyme xanthine oxidase (XO) has been linked to overproduction of ROS in diabetic environment, and studies have shown that treatment with XO inhibitors decreases XO overactivity and XO-generated ROS. This study evaluates the role of XO in the diabetic wound and the impact of specifically inhibiting its activity on wound healing. Treatment of diabetic wounds with siXDH (xanthine dehydrogenase siRNA) decreased XDH mRNA expression by 51.6%, XO activity by 35.9%, ROS levels by 78.1%, pathologic wound burden by 31.5%, and accelerated wound healing by 7 days (23.3%). Polymerase chain reaction analysis showed that increased XO activity in wild-type wound may be due to XDH to XO conversion and/or XO phosphorylation, but not to gene transcription, whereas increased XO activity in diabetic wounds may also be from gene transcription. These results suggest that XO may be responsible for large proportion of elevated oxidative stress in the diabetic wound environment and that normalizing the metabolic activity of XO using targeted delivery of siXDH may decrease overproduction of ROS and accelerate wound healing in diabetic patients.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Células 3T3 NIH/metabolismo , Estrés Oxidativo/efectos de los fármacos , Purinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Xantina Oxidasa/antagonistas & inhibidores , Animales , Línea Celular , Células Cultivadas , Expresión Génica , Homeostasis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , ARN Mensajero , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Molecular profiling of human biopsies and surgical specimens is frequently complicated by their inherent biological heterogeneity and by the need to conserve tissue for clinical diagnosis. We have developed a set of novel 'tissue print' and 'print-phoresis' technologies to facilitate tissue and tumor-marker profiling under these circumstances. Tissue printing transfers cells and extracellular matrix components from a tissue surface onto nitrocellulose membranes, generating a two-dimensional anatomical image on which molecular markers can be visualized by specific protein and RNA- and DNA-detection techniques. Print-phoresis is a complementary new electrophoresis method in which thin strips from the print are subjected to polyacrylamide gel electrophoresis, providing a straightforward interface between the tissue-print image and gel-based proteomic techniques. Here we have utilized these technologies to identify and characterize markers of tumor invasion of the prostate capsule, an event generally not apparent to the naked eye that may result in tumor at the surgical margins ('positive margins'). We have also shown that tissue-print technologies can provide a general platform for the generation of marker maps that can be superimposed directly onto histopathological and radiological images, permitting molecular identification and classification of individual malignant lesions.
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Neoplasias de la Próstata/diagnóstico , Proteínas , Biomarcadores , Humanos , Masculino , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Proteínas/metabolismoRESUMEN
The molecular and cellular level reaches of the metabolic dysregulations that characterize diabetes are yet to be fully discovered. As mechanisms underlying management of reactive oxygen species (ROS) gain interest as crucial factors in cell integrity, questions arise about the role of redox cues in the regulation and maintenance of bone marrow-derived multipotent stromal cells (BMSCs) that contribute to wound healing, particularly in diabetes. Through comparison of BMSCs from wild-type and diabetic mice, with a known redox and metabolic disorder, we found that the cytoprotective nuclear factor erythroid-related factor 2 (Nrf2)/kelch-like erythroid cell-derived protein 1 (Keap1) pathway is dysregulated and functionally insufficient in diabetic BMSCs (dBMSCs). Nrf2 is basally active, but in chronic ROS, we found irregular inhibition of Nrf2 by Keap1, altered metabolism, and limited BMSC multipotency. Forced upregulation of Nrf2-directed transcription, through knockdown of Keap1, restores redox homeostasis. Normalized Nrf2/Keap1 signaling restores multipotent cell properties in dBMSCs through Sox2 expression. These restored BMSCs can resume their role in regenerative tissue repair and promote healing of diabetic wounds. Knowledge of diabetes and hyperglycemia-induced deficits in BMSC regulation, and strategies to reverse them, offers translational promise. Our study establishes Nrf2/Keap1 as a cytoprotective pathway, as well as a metabolic rheostat, that affects cell maintenance and differentiation switches in BMSCs.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Animales , Western Blotting , Diferenciación Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Widespread application of vascularized composite allotransplantation (VCA) is currently limited by the required lifelong systemic immunosuppression and its associated morbidity and mortality. This study evaluated the efficacy of ex vivo (after procurement but before transplantation) engineering of allografts using small interfering RNA to knockdown major histocompatibility complex I (MHC-I) and prolong rejection-free survival. METHODS: Endothelial cells (ECs) were transfected with small interfering RNA targeted against MHC-I (siMHC-I) for all in vitro experiments. MHC-I surface expression and knockdown duration were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. After stimulating Lewis recipient cytotoxic lymphocytes (CTL) with allogeneic controls or siMHC-I-silenced ECs, lymphocyte proliferation, CTL-mediated and natural killer-mediated EC lysis were measured. Using an established VCA rat model, allografts were perfused ex vivo with siMHC-I before transplantation. Allografts were analyzed for MHC-I expression and clinical/histologic evidence of rejection. RESULTS: Treatment with siMHC-I resulted in 80% knockdown of mRNA and 87% reduction in cell surface expression for up to 7 days in vitro (P < 0.05). Treatment of ECs with siMHC-I reduced lymphocyte proliferation and CTL-mediated cytotoxicity (77% and 50%, respectively, P < 0.01), without increasing natural killer-mediated cytotoxicity (P = 0.66). In a rat VCA model, ex vivo perfusion with siMHC-I reduced expression in all tissue compartments by at least 50% (P < 0.05). Knockdown prolonged rejection-free survival by 60% compared with nonsense-treated controls (P < 0.05). CONCLUSIONS: Ex vivo siMHC-I engineering can effectively modify allografts and significantly prolong rejection-free allograft survival. This novel approach may help reduce future systemic immunosuppression requirements in VCA recipients.
RESUMEN
Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.
Asunto(s)
Células de la Médula Ósea/inmunología , Proliferación Celular , Microambiente Celular/inmunología , Inmunomodulación/inmunología , Células Madre Mesenquimatosas/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células de la Médula Ósea/metabolismo , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Hipoxia de la Célula , Células Cultivadas , Microambiente Celular/efectos de los fármacos , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratas Endogámicas Lew , Linfocitos T Reguladores/metabolismoRESUMEN
Chronic hyperglycemia impairs intracellular redox homeostasis and contributes to impaired diabetic tissue regeneration. The Keap1/Nrf2 pathway is a critical regulator of the endogenous antioxidant response system, and its dysfunction has been implicated in numerous pathologies. Here we characterize the effect of chronic hyperglycemia on Nrf2 signaling within a diabetic cutaneous regeneration model. We characterized the effects of chronic hyperglycemia on the Keap1/Nrf2 pathway within models of diabetic cutaneous wound regeneration. We assessed reactive oxygen species (ROS) production and antioxidant gene expression following alterations in the Nrf2 suppressor Keap1 and the subsequent changes in Nrf2 signaling. We also developed a topical small interfering RNA (siRNA)-based therapy to restore redox homeostasis within diabetic wounds. Western blotting demonstrated that chronic hyperglycemia-associated oxidative stress inhibits nuclear translocation of Nrf2 and impairs activation of antioxidant genes, thus contributing to ROS accumulation. Keap1 inhibition increased Nrf2 nuclear translocation, increased antioxidant gene expression, and reduced ROS production to normoglycemic levels, both in vitro and in vivo. Topical siKeap1 therapy resulted in improved regenerative capacity of diabetic wounds and accelerated closure. We report that chronic hyperglycemia weakens the endogenous antioxidant response, and the consequences of this defect are manifested by intracellular redox dysregulation, which can be restored by Keap1 inhibition. Targeted siRNA-based therapy represents a novel, efficacious strategy to reestablish redox homeostasis and accelerate diabetic cutaneous tissue regeneration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Diabetes Mellitus/metabolismo , Hiperglucemia/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regeneración/fisiología , Piel/metabolismo , Cicatrización de Heridas/fisiología , Heridas y Lesiones/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Proteínas del Citoesqueleto/genética , Diabetes Mellitus/genética , Técnica del Anticuerpo Fluorescente , Glutatión , Hiperglucemia/genética , Inmunoprecipitación , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Factor 2 Relacionado con NF-E2/genética , Células 3T3 NIH , Estrés Oxidativo/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Piel/lesionesRESUMEN
BACKGROUND: Fat grafting is limited by unpredictable long-term graft retention. The authors postulate that injury to the donor-derived microvasculature during harvest and subsequent ischemia may account for this clinical variability. They examined the use of the U.S. Food and Drug Administration-approved phosphodiesterase-5 inhibitor sildenafil citrate to protect graft microvasculature and its role in revascularization and survival. METHODS: Inguinal fat of donor Tie2/LacZ mice was infiltrated with sildenafil or saline, harvested, and transplanted onto the dorsa of recipient FVB mice. Additional donor mice were perfused with intraarterial trypsin to inactivate the fat graft microvasculature before harvest and transplantation. Differences in graft revascularization, perfusion, volume of retention, and biochemical changes were assessed. RESULTS: Surviving fat grafts were characterized by exclusively donor-derived vasculature inosculating with the recipient circulation at the graft periphery. Inactivation of donor-derived microvasculature decreased early graft perfusion and led to nearly total graft loss by 8 weeks. Sildenafil attenuated vascular ischemic injury, consistent with reductions in VCAM-1 and SDF1α expression at 48 hours and 4-fold increases in microvasculature survival by 2 weeks over controls. Compared with controls, targeted sildenafil treatment improved early graft perfusion, doubled graft retention at 12 weeks (83 percent versus 39 percent; p < 0.05), ultimately retaining 64 percent of the original graft volume by 24 weeks (compared to 4 percent; p < 0.05) with superior histologic features. CONCLUSIONS: Fat graft vascularization is critically dependent on maintenance of the donor microvasculature. Sildenafil protects the donor microvasculature during transfer and revascularization, increasing long-term volume retention. These data demonstrate a rapidly translatable method of increasing predictability and durability of fat grafting in clinical practice.
Asunto(s)
Tejido Adiposo/trasplante , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Piperazinas/uso terapéutico , Sulfonamidas/uso terapéutico , Trasplantes/irrigación sanguínea , Tejido Adiposo/irrigación sanguínea , Angiopoyetina 1/metabolismo , Animales , Endotelio Vascular/metabolismo , Endotelio Vascular/trasplante , Regulación de la Expresión Génica , Genes Reporteros , Supervivencia de Injerto , Operón Lac , Ratones , Ratones Transgénicos , Microvasos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Piperazinas/administración & dosificación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Purinas/administración & dosificación , Purinas/uso terapéutico , Receptor TIE-2/genética , Citrato de Sildenafil , Sulfonamidas/administración & dosificación , Recolección de Tejidos y Órganos/métodos , Trasplante Autólogo/métodos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Aesthetic procedures are significant sources of revenue for plastic surgeons. With the popularity of nonsurgical aesthetic procedures, many plastic surgeons question how to best tailor their aesthetic practice. METHODS: Revenue generated from surgical and minimally invasive aesthetic procedures performed in the United States between 2000 and 2011 was calculated from the American Society of Plastic Surgeons' annual reports. Regression analysis was performed against six commonly cited economic indicators. RESULTS: In 2011, revenue from minimally invasive procedures increased from $3.0 billion to $5.7 billion (90 percent growth), whereas revenue from surgical procedures decreased from $6.6 billion to $6.0 billion (10 percent decline). Between 2000 and 2011, minimally invasive procedure market share grew from 30 percent to nearly 50 percent. Linear regression analysis revealed significant correlations between surgical procedure revenue and indicators of macroeconomic climate: Dow Jones Industrial Average (R = 0.72; p < 0.01), Standard & Poor's 500 Index (R = 0.64, p < 0.05), and unemployment rate (R = -0.81; p < 0.001). Minimally invasive procedure revenue was significantly correlated with indicators related to microeconomic decision trends: disposable income per capita (R = 0.93; p < 0.001), real gross domestic product per capita (R = 0.88; p < 0.001), and home price index (R = 0.63; p < 0.05). No economic indicator in this study was found to be significantly correlated with both surgical and minimally invasive revenue. CONCLUSION: Despite economic turbulence, minimally invasive procedures are the most rapidly growing source of revenue and are poised to be the dominant source of revenue in the aesthetic market.
Asunto(s)
Técnicas Cosméticas/economía , Procedimientos de Cirugía Plástica/economía , Cirugía Plástica/economía , Antidiscinéticos/economía , Toxinas Botulínicas/economía , Humanos , Renta , Procedimientos Quirúrgicos Mínimamente Invasivos/economía , Desempleo , Estados UnidosRESUMEN
BACKGROUND: Non-healing foot ulcers are the most common cause of non-traumatic amputation and hospitalization amongst diabetics in the developed world. Impaired wound neovascularization perpetuates a cycle of dysfunctional tissue repair and regeneration. Evidence implicates defective mobilization of marrow-derived progenitor cells (PCs) as a fundamental cause of impaired diabetic neovascularization. Currently, there are no FDA-approved therapies to address this defect. Here we report an endogenous PC strategy to improve diabetic wound neovascularization and closure through a combination therapy of AMD3100, which mobilizes marrow-derived PCs by competitively binding to the cell surface CXCR4 receptor, and PDGF-BB, which is a protein known to enhance cell growth, progenitor cell migration and angiogenesis. METHODS AND RESULTS: Wounded mice were assigned to 1 of 5 experimental arms (n = 8/arm): saline treated wild-type, saline treated diabetic, AMD3100 treated diabetic, PDGF-BB treated diabetic, and AMD3100/PDGF-BB treated diabetic. Circulating PC number and wound vascularity were analyzed for each group (n = 8/group). Cellular function was assessed in the presence of AMD3100. Using a validated preclinical model of type II diabetic wound healing, we show that AMD3100 therapy (10 mg/kg; i.p. daily) alone can rescue diabetes-specific defects in PC mobilization, but cannot restore normal wound neovascularization. Through further investigation, we demonstrate an acquired trafficking-defect within AMD3100-treated diabetic PCs that can be rescued by PDGF-BB (2 µg; topical) supplementation within the wound environment. Finally, we determine that combination therapy restores diabetic wound neovascularization and accelerates time to wound closure by 40%. CONCLUSIONS: Combination AMD3100 and PDGF-BB therapy synergistically improves BM PC mobilization and trafficking, resulting in significantly improved diabetic wound closure and neovascularization. The success of this endogenous, cell-based strategy to improve diabetic wound healing using FDA-approved therapies is inherently translatable.
Asunto(s)
Complicaciones de la Diabetes/terapia , Diabetes Mellitus Experimental/complicaciones , Cicatrización de Heridas , Heridas y Lesiones/etiología , Heridas y Lesiones/terapia , Animales , Becaplermina , Bencilaminas , Ciclamas , Modelos Animales de Enfermedad , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/farmacología , Ratones , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas c-sis/administración & dosificación , Proteínas Proto-Oncogénicas c-sis/farmacología , Úlcera Cutánea/etiología , Úlcera Cutánea/terapia , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
BACKGROUND: Radioprotective modalities such as dose fractionation and pharmacologic agents such as amifostine have been used to protect bone and other types of normal tissue from the damaging effects of ionizing radiation without significantly impacting tumor kill. To better understand the cellular mechanism of radioprotection of osseous tissue, the authors sought to determine the effect of dose fractionation and amifostine on isolated osteoblasts. METHODS: Isolated primary rat calvarial osteoblasts were exposed to single or fractionated doses of ionizing radiation both with and without amifostine pretreatment. Endpoints included cell growth (n = 4), vascular endothelial growth factor production as measured by enzyme-linked immunosorbent assay (n = 3), and early osteodifferentiation as measured by a quantitative alkaline phosphatase assay (n = 3). RESULTS: Both dose fractionation and amifostine protect osteoblasts from the growth inhibitory effects of ionizing radiation. Fractionation but not amifostine was protective for hypoxia-induced vascular endothelial growth factor production (used as a surrogate marker of normal osteoblast function). Neither fractionation nor amifostine could prevent the inhibitory effect of ionizing radiation on normal osteoblast osteodifferentiation as measured by alkaline phosphatase production. CONCLUSIONS: Both dose fractionation and amifostine have valid roles as radioprotectants for osteoblasts and can act in an additive fashion. Radioprotection of cell growth and viability does not necessarily correlate with preservation of normal cellular function. Combination protocols involving dose fractionation and amifostine may be effective in radioprotection of osteoblasts and normal osseous tissue.
Asunto(s)
Amifostina/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/efectos de la radiación , Protectores contra Radiación/farmacología , Animales , Animales Recién Nacidos , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Fraccionamiento de la Dosis de Radiación , Osteoblastos/fisiología , Ratas , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
BACKGROUND: Lymphedema is a common debilitating sequela of lymph node dissection. Although numerous clinical studies suggest that factors that lead to fibrosis are associated with the development of lymphedema, this relationship has not been proven. The purpose of these experiments was therefore to evaluate lymphatic regeneration in the setting of variable soft-tissue fibrosis. METHODS: A section of mouse tail skin including the capillary and collecting lymphatics was excised. Experimental animals (n = 20) were treated with topical collagen type I gel and a moist dressing, whereas control animals (n = 20) underwent excision followed by moist dressing alone. Fibrosis, acute lymphedema, lymphatic function, gene expression, lymphatic endothelial cell proliferation, and lymphatic fibrosis were evaluated at various time points. RESULTS: Collagen gel treatment significantly decreased fibrosis, with an attendant decrease in acute lymphedema and improved lymphatic function. Tails treated with collagen gel demonstrated greater numbers of lymphatic vessels, more normal lymphatic architecture, and more proliferating lymphatic endothelial cells. These findings appeared to be independent of vascular endothelial growth factor C expression. Decreased fibrosis was associated with a significant decrease in the expression of extracellular matrix components. Finally, decreased soft-tissue fibrosis was associated with a significant decrease in lymphatic fibrosis as evidenced by the number of lymphatic endothelial cells that coexpressed lymphatic and fibroblast markers. CONCLUSIONS: Soft-tissue fibrosis is associated with impairment in lymphatic regeneration and lymphatic function. These defects occur as a consequence of impaired lymphatic endothelial cell proliferation, abnormal lymphatic microarchitecture, and lymphatic fibrosis. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of normal capillary lymphatics.
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Vasos Linfáticos/fisiopatología , Regeneración/fisiología , Animales , Proliferación Celular , Colágeno Tipo I/administración & dosificación , Células Endoteliales/fisiología , Femenino , Fibrosis , Geles , Inmunohistoquímica , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Linfedema/fisiopatología , Ratones , Ratones Endogámicos , Modelos Animales , Reacción en Cadena de la Polimerasa , Cola (estructura animal) , Factor C de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Hyperbaric oxygen (HBO) therapy is used in the treatment of osteoradionecrosis. Although HBO is thought to improve radiation-induced hypocellularity and bone tissue hypoxia, the precise effects of HBO on bone cells such as osteoblasts have not been described. In this study, our goal was to assess the effect of HBO on irradiated and nonirradiated primary osteoblast cultures and assess for changes in growth, apoptosis, cell cycle profile, differentiation, and gene expression. We found that daily HBO treatments caused a 24% decrease in cell growth after 9 days in culture. Hyperbaric oxygen negatively affects growth by inducing osteoblast apoptosis and cell cycle arrest. Hyperbaric oxygen leads to G1/S cell cycle arrest in unirradiated osteoblasts where as it causes G2/M arrest in cells that were previously irradiated with either 7 or 12 Gy of ionizing radiation. Although radiation was shown to have a dose-dependent inhibitory effect on early osteoblast differentiation as measured by alkaline phosphatase activity, HBO did not have a significant effect on osteoblast differentiation. Microarray analysis revealed that exposure to HBO leads to a differential expression of a variety of gene families including stress response pathways. In summary, although successive daily HBO treatments resulted in growth delay, osteoblast function as measured by the ability to produce alkaline phosphatase was not significantly affected. These data suggest that HBO does not have any positive effects on either normal or radiation-damaged osteoblasts in vitro.
Asunto(s)
Oxigenoterapia Hiperbárica/efectos adversos , Osteoblastos/citología , Osteoblastos/efectos de la radiación , Oxígeno/toxicidad , Fosfatasa Alcalina/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
BACKGROUND: Although it is clear that radiation therapy can cause tissue injury, the degree of injury that is observed clinically can be highly variable. It is possible that variability in the methods by which ionizing radiation is delivered can contribute to some of the observed variability. Thus, the purpose of this study was to assess the effects of various fractionation schedules on the growth and differentiation potential of isolated mesenchymal stem cells in vitro. METHODS: Isolated mesenchymal stem cells (triplicate studies) were exposed to a dose of 12 Gy of ionizing radiation as a single dose, in two doses of 6 Gy, or in six doses of 2 Gy. Cellular proliferation and the potential for differentiation along the bone and fat lineage were assessed. Potential mechanisms for injury and protection were evaluated by analyzing the expression of p21 and manganese superoxide dismutase. RESULTS: Delivery of radiation in multiple doses confers significant radioprotection to mesenchymal stem cell proliferation and potential for differentiation. In contrast, delivery of 12 Gy of radiation as a single dose or as two equal doses of 6 Gy results in marked deficiencies in cellular proliferation and potential for multilineage cellular differentiation. CONCLUSIONS: The authors have demonstrated that even minor alterations in fractionation of radiation dose can result in significant effects on the potential of mesenchymal stem cells to differentiate. These findings imply that at least some of the variability in tissue damage after radiation therapy observed clinically may be attributable to differences in the delivery of ionizing radiation.
Asunto(s)
Células Madre Mesenquimatosas/efectos de la radiación , Células Madre Pluripotentes/efectos de la radiación , Protección Radiológica , Animales , Huesos/citología , Diferenciación Celular/efectos de la radiación , División Celular/efectos de la radiación , Lipogénesis/fisiología , Ratones , Ratones Endogámicos BALB C , Osteogénesis/fisiología , ARN/análisis , Dosis de Radiación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.