RESUMO
Keloids present a formidable clinical challenge. Surgical excision in conjunction with radiation therapy may decrease the chance of keloid recurrence. Split-thickness skin grafts, however, are more prone to failure in the setting of radiation. In this report, we present a patient with a recurrent auricular keloid who underwent excision and immediate Integra (Integra LifeSciences, Plainsboro, NJ) application, followed by high-dose rate brachytherapy and interval split-thickness skin graft placement. A 23-year-old woman with a history of a recurrent auricular keloid after previous surgical excision, corticosteroid injection, and radiation underwent reexcision of her keloid. Integra was used to cover the resultant exposed auricular perichondrium. The patient then received high-dose rate brachytherapy (1500 cGy) on postoperative days 1 and 2, followed by definitive split-thickness skin graft placement 3 weeks after her initial surgery. The patient recovered from all interventions without complication. There was no evidence of keloid formation 27 months after the interval split-thickness skin graft placement at either the auricular recipient or thigh donor sites. We report the first case of a 2-stage reconstruction of a recurrent auricular keloid (composed of keloid excision and placement of Integra in conjunction with high-dose rate brachytherapy, followed by interval split-thickness skin grafting), resulting in an acceptable cosmetic result without evidence of recurrence at long-term follow-up.
Assuntos
Braquiterapia/métodos , Orelha Externa , Queloide/radioterapia , Queloide/cirurgia , Transplante de Pele/métodos , Feminino , Humanos , Dosagem Radioterapêutica , Recidiva , Coxa da Perna , Adulto JovemRESUMO
Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.
Assuntos
Apoptose/efeitos dos fármacos , Violeta Genciana/farmacologia , Mitocôndrias/metabolismo , Compostos de Amônio Quaternário/farmacologia , Tiorredoxinas/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Fator de Indução de Apoptose/análise , Fator de Indução de Apoptose/metabolismo , Cátions/química , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/análise , Citocromos c/metabolismo , Fibroblastos , Violeta Genciana/química , Violeta Genciana/uso terapêutico , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Oxirredução , Protease La/metabolismo , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/uso terapêutico , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/biossíntese , Compostos de Tritil/química , Compostos de Tritil/farmacologia , Compostos de Tritil/uso terapêutico , Regulação para CimaRESUMO
BACKGROUND: Recent evidence suggests that hydrogen sulfide is capable of mitigating the degree of cellular damage associated with ischemia-reperfusion injury (IRI). METHODS: This study evaluated the potential utility of hydrogen sulfide in preventing IRI in skeletal muscle by using in vitro (cultured myotubes subjected to sequential hypoxia and normoxia) and in vivo (mouse hind limb ischemia, followed by reperfusion) models to determine whether intravenous hydrogen sulfide delivered after the ischemic event had occurred (pharmacologic postconditioning) conferred protection against IRI. Injury score and apoptotic index were determined by analysis of specimens stained with hematoxylin and eosin and terminal deoxynucleotide transferase-mediated deoxy-uridine triphosphate nick-end labeling, respectively. RESULTS: In vitro, hydrogen sulfide reduced the apoptotic index after 1, 3, or 5 hours of hypoxia by as much as 75% (P = .002), 80% (P = .006), and 83% (P < .001), respectively. In vivo, hydrogen sulfide delivered after the onset of hind limb ischemia and before reperfusion resulted in protection against IRI-induced cellular changes, which was validated by significant decreases in the injury score and apoptotic index. The timing of hydrogen sulfide delivery was crucial: when delivered 20 minutes before reperfusion, hydrogen sulfide conferred significant cytoprotection (P < .001), but treatment 1 minute before reperfusion did not provide protection (P = NS). CONCLUSIONS: These findings confirm that hydrogen sulfide limits IRI-induced cellular damage in myotubes and skeletal muscle, even when delivered after the onset of ischemia in this murine model. These data suggest that when given in the appropriate dose and within the proper time frame, hydrogen sulfide may have significant therapeutic applications in multiple clinical scenarios.
Assuntos
Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Sulfetos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular , Estado Terminal , Citoproteção , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Membro Posterior , Injeções Intravenosas , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Mioblastos Esqueléticos/patologia , Traumatismo por Reperfusão/patologia , Índice de Gravidade de Doença , Fatores de TempoRESUMO
BACKGROUND AND AIM: To investigate whether pharmacologic post-conditioning of intestinal tissue with hydrogen sulfide (HS) protects against ischemia reperfusion injury (IRI). METHODS: In vitro, enterocytes were made hypoxic for 1, 2, or 3 h, treated with media containing between 0 and 100 µM HS 20 min prior to the end of the hypoxic period, then returned to normoxia for 3 h. An apoptotic index (AI) was determined for each time point and (HS). In vivo, jejunal ischemia was induced in male Sprague-Dawley rats for 1, 2, or 3 h; 20 min prior to the end of the ischemic period animals were given an intravenous injection of NaHS sufficient to raise the bloodstream concentration to 0, 10 µM, or 100 µM HS. This was followed by jejunal reperfusion for 3 h, histologic processing, and measurement of villus height. RESULTS: In vitro, there was a significant decrease in AI compared with non-HS-treated control at all time points after treatment with 10 µM HS, and at the 2 h time point with 100 µM HS (P < 0.017). In vivo, after 1 h ischemia, qualitative reduction of injury was noted with 10 µM and 100 µM; after 2 h ischemia, reduction was noted with 10 µM but not 100 µM; and after 3 h ischemia, there was no injury reduction. HS treatment resulted in significant quantitative preservation (P < 0.05) of villus height at all time points and doses, except for 3 h ischemia and delivery of 100 µM (P = 0.129). CONCLUSIONS: Hydrogen sulfide provides significant protection to intestinal tissues in vitro and in vivo when delivered after the onset of ischemia.
Assuntos
Sulfeto de Hidrogênio/uso terapêutico , Enteropatias/tratamento farmacológico , Doenças do Jejuno/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sulfeto de Hidrogênio/administração & dosagem , Enteropatias/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Doenças do Jejuno/patologia , Jejuno/irrigação sanguínea , Masculino , Fotomicrografia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Resultado do TratamentoRESUMO
BACKGROUND: This study assessed the feasibility of creating a tissue engineering platform by decellularization of fasciocutaneous tissue. MATERIALS AND METHODS: A fasciocutaneous flap based upon the superficial inferior epigastric artery was harvested from the abdominal wall of 8-wk-old male Sprague-Dawley rats. All cellular components were removed by sequential treatment with sodium azide, DNAse, and sodium deoxycholate. The degree of decellularization was qualitatively assessed by histology and quantitatively assessed by spectrophotometry. Persistence of relevant extracellular matrix proteins was shown following staining with orcein and hematoxylin. The duration of circuit patency was determined by continuous perfusion with a peristaltic perfusion pump. RESULTS: Gross and histologic examination demonstrated removal of cellular constituents with preservation of tissue matrix architecture, including macrochannels and microchannels. This was confirmed by the application of spectrophotometry to DNA isolates, which showed that the decellularized flap retained 4.04 ng/µL DNA, compared with the non-processed control, which retained 37.03 ng/µL DNA, and the acellular control, which was read as having 0.65 ng/µL DNA. The extracellular matrix of vessel walls was shown to remain intact. Peristaltic perfusion of the cannulated pedicle inflow channel with phosphate buffered saline at a rate of 200 µL/min confirmed circuit patency for 6 h. CONCLUSION: Fasciocutaneous flaps harvested with an intact vascular pedicle and associated tissue vascular network can be successfully decellularized and perfused ex vivo. This methodology, which is scalable to human size tissues, provides promise as a technique for the production of customizable engineered tissues.
Assuntos
Derme/cirurgia , Artérias Epigástricas/cirurgia , Retalhos Cirúrgicos/irrigação sanguínea , Engenharia Tecidual/métodos , Alicerces Teciduais , Parede Abdominal/cirurgia , Animais , Fasciotomia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , EspectrofotometriaRESUMO
BACKGROUND: Varicose veins and other vascular abnormalities are common clinical entities. Treatment options include vein stripping, sclerotherapy, and endovenous laser treatment, but all involve some degree of invasive intervention. The purpose of this study was to determine ex vivo the effectiveness of a novel hand-held, battery-operated, high-intensity focused ultrasound (HIFU) device for transcutaneous venous ablation. METHODS: The ultrasound device is 14 x 9 x 4 cm, weighs 650 g, and is powered by 4 lithium ion battery packs. An ex vivo testing platform consisting of two different models comprised of sequentially layered skin-muscle-vein or skin-fat-vein was developed, and specimens were treated with HIFU. The tissues were then disassembled, imaged, and processed for histology. The luminal cross-sectional area of vein that had been treated with HIFU and nontreated controls were measured, and the values presented as median and interquartile range (IQR). The values were compared using a Wilcoxon rank-sum test, and statistical significance was set at P < .05. RESULTS: On gross and histologic examination, veins that had been treated with HIFU showed evidence of coagulation necrosis. The surface of the muscle in direct contact with the vein had a pinpoint area of coagulation, whereas the adjacent fat appeared undisturbed; the skin, fat, and the surface of the muscle in contact with the transducer remained completely unaffected. The cross-sectional area was 3.79 mm(2) (IQR, 3.38-4.22) of the control vein lumen and 0.16 mm(2) (IQR, 0.04-0.39) in those that had been treated with HIFU (P = .0304). CONCLUSION: This inexpensive, portable HIFU device has the potential to allow clinicians to easily perform venous ablation in a manner that is entirely noninvasive and without the expense or inconvenience of large, complicated devices. This device represents a significant step forward in the development of new applications for HIFU technology.
Assuntos
Tecido Adiposo/irrigação sanguínea , Ablação por Ultrassom Focalizado de Alta Intensidade/instrumentação , Pele/irrigação sanguínea , Animais , Coagulação Sanguínea , Fontes de Energia Elétrica , Desenho de Equipamento , Humanos , Teste de Materiais , Ratos , Ratos Sprague-Dawley , Ovinos , Suínos , Veias/patologiaRESUMO
Hemangiomas are the most common type of tumor in infants. As they are endothelial cell-derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T-transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/-, and Ang2-/- mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/- cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/- cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.
Assuntos
Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Hemangioma/tratamento farmacológico , NADPH Oxidases/antagonistas & inibidores , Angiopoietina-2/fisiologia , Animais , Células Endoteliais/metabolismo , Hemangioma/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/fisiologia , Proteínas/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
PURPOSE: Melanoma is a solid tumor that is notoriously resistant to chemotherapy, and its incidence is rapidly increasing. Recently, several signaling pathways have been shown to contribute to melanoma tumorigenesis, including constitutive activation of mitogen-activated protein kinase, Akt, and Stat-3. The activation of multiple pathways may account in part for the difficulty in treatment of melanoma. In a recent screen of compounds, we found that an organopalladium compound, Tris (dibenzylideneacetone) dipalladium (Tris DBA), showed significant antiproliferative activity against melanoma cells. Studies were carried out to determine the mechanism of action of Tris DBA. EXPERIMENTAL DESIGN: Tris DBA was tested on efficacy on proliferation of human and murine melanoma cells. To find the mechanism of action of Tris DBA, we did Western blot and gene array analyses. The ability of Tris DBA to block tumor growth in vivo was assessed. RESULTS: Tris DBA has activity against B16 murine and A375 human melanoma in vivo. Tris DBA inhibits several signaling pathways including activation of mitogen-activated protein kinase, Akt, Stat-3, and S6 kinase activation, suggesting an upstream target. Tris DBA was found to be a potent inhibitor of N-myristoyltransferase-1, which is required for optimal activity of membrane-based signaling molecules. Tris DBA showed potent antitumor activity in vivo against melanoma. CONCLUSION: Tris DBA is thus a novel inhibitor of N-myristoyltransferase-1 with significant antitumor activity and is well tolerated in vivo. Further preclinical evaluation of Tris DBA and related complexes is warranted.
Assuntos
Aciltransferases/antagonistas & inibidores , Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Nus , Transplante de Neoplasias , Compostos Organometálicos/farmacologia , Paládio/farmacologiaRESUMO
BACKGROUND: Vascular malformations are clinical disorders in which endothelial cells fail to remodel and/or undergo programmed cell death, leading to abnormal persistence of blood vessels. The abnormal persistence of vessels makes therapy difficult because these lesions are resistant to interventions that are effective against hemangiomas. Akt1 is a serine-threonine protein kinase, which is a key mediator of resistance to programmed cell death. Our objective was to determine whether sustained activation of Akt1 could lead to vascular malformation in mice. OBSERVATIONS: We examined the effect of constitutive activation of Akt1 in murine endothelial cells (MS1 cells). Overexpression of active AKT1 in MS1 cells led to the development of vascular malformations, characterized by wide endothelial lumens and minimal investment of smooth muscle surrounding the vessels. The histologic features of these vascular malformations is distinct from ras-transformed MS1 cells (angiosarcoma) and suggest that differing signal abnormalities give rise to human vascular malformations vs malignant vascular tumors. CONCLUSIONS: Inhibition of Akt signaling may be useful in the treatment of vascular malformations. Examination of problematic hemangiomas and vascular malformations for the presence of activated Akt or downstream targets of Akt, such as mammalian target of rapamycin (mTOR), may predict response to treatment with Akt inhibitors or rapamycin. This study provides a potential rationale for the systemic and topical use of these inhibitors for vascular malformations and hemangiomas.
Assuntos
Vasos Sanguíneos/anormalidades , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pele/irrigação sanguínea , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Masculino , Camundongos , Camundongos NusRESUMO
Hemangioma of infancy is the most common neoplasm of childhood. While hemangiomas are classic examples of angiogenesis, the angiogenic factors responsible for hemangiomas are not fully understood. Previously, we demonstrated that malignant endothelial tumors arise in the setting of autocrine loops involving vascular endothelial growth factor (VEGF) and its major mitogenic receptor vascular endothelial growth factor receptor 2. Hemangiomas of infancy differ from malignant endothelial tumors in that they usually regress, or can be induced to regress by pharmacologic means, suggesting that angiogenesis in hemangiomas differs fundamentally from that of malignant endothelial tumors. Here, we demonstrate constitutive activation of the endothelial tie-2 receptor in human hemangioma of infancy and, using a murine model of hemangioma, bEnd.3 cells; we show that bEnd.3 hemangiomas produce both angiopoietin-2 (ang-2) and its receptor, tie-2, in vivo. We also demonstrate that inhibition of tie-2 signaling with a soluble tie-2 receptor decreases bEnd.3 hemangioma growth in vivo. The efficacy of tie-2 blockade suggests that either tie-2 activation or ang-2 may be required for in vivo growth. To address this issue, we used tie-2-deficient bEnd.3 hemangioma cells, which, surprisingly, were fully proficient in in vivo growth. Previous studies from our laboratory and others have implicated reactive oxygen-generating nox enzymes in the angiogenic switch, so we examined the effect of nox inhibitors on ang-2 production in vitro and on bEnd.3 tumor growth in vivo. We then inhibited ang-2 production pharmacologically using novel inhibitors of nox enzymes and found that this treatment nearly abolished bEnd.3 hemangioma growth in vivo. Signal-transduction blockade targeting ang-2 production may be useful in the treatment of human hemangiomas in vivo.