RESUMEN
Cutaneous scarring is a major source of morbidity and current therapies to mitigate scar formation remain ineffective. Although wound fibrosis and inflammation are highly linked, only recently have mechanical forces been implicated in these pathways. Our group has developed a topical polymer device that significantly reduces post-injury scar formation via the manipulation of mechanical forces. Here we extend these studies to examine the genomewide transcriptional effects of mechanomodulation during scar formation using a validated large animal model, the red Duroc pig. We demonstrate that mechanical loading of incisional wounds upregulates expression of genes associated with inflammatory and fibrotic pathways, and that device-mediated offloading of these wounds reverses these effects. Validation studies are needed to clarify the clinical significance of these findings.
Asunto(s)
Regulación hacia Abajo/genética , Inflamación/genética , Transducción de Señal/genética , Transcripción Genética , Cicatrización de Heridas/genética , Heridas y Lesiones/genética , Animales , Modelos Animales de Enfermedad , Redes Reguladoras de Genes , Inflamación/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Sus scrofa , Heridas y Lesiones/patologíaRESUMEN
Despite significant advances in reconstructive surgery, the repair of massive lumbosacral defects poses significant challenges. When the extent of soft tissue loss, tumor resection, and/or radiation therapy preclude the use of traditional local options, such as gluteal advancement flaps or pedicled thigh flaps, then distant flaps are required. We report a case of a 64-year-old male who presented with a large sacral Marjolin's ulcer secondary to recurrent pilonidal cysts and ulcerations. The patient underwent wide local composite resection, which resulted in a wound measuring 450 cm(2) with exposed rectum and sacrum. The massive defect was successfully covered with a free transverse rectus abdominis myocutaneous flap, providing a well-vascularized skin paddle and obviating the need for a latissimus flap with skin graft. The free-TRAM flap proved to be a very robust flap in this situation and would be one of our flaps of choice for similar defects.
Asunto(s)
Carcinoma de Células Escamosas/cirugía , Colgajos Tisulares Libres , Seno Pilonidal/complicaciones , Procedimientos de Cirugía Plástica/métodos , Recto del Abdomen/trasplante , Neoplasias Cutáneas/cirugía , Nalgas/irrigación sanguínea , Carcinoma de Células Escamosas/etiología , Humanos , Región Lumbosacra , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/etiologíaRESUMEN
OBJECTIVE: To test the hypothesis that the mechanical environment of cutaneous wounds can control scar formation. BACKGROUND: Mechanical forces have been recognized to modulate myriad biologic processes, but the role of physical force in scar formation remains unclear. Furthermore, the therapeutic benefits of offloading cutaneous wounds with a device have not been rigorously tested. METHODS: A mechanomodulating polymer device was utilized to manipulate the mechanical environment of closed cutaneous wounds in red Duroc swine. After 8 weeks, wounds subjected to different mechanical stress states underwent immunohistochemical analysis for fibrotic markers. In a phase I clinical study, 9 human patients undergoing elective abdominal surgery were treated postoperatively with a stress-shielding polymer on one side whereas the other side was treated as standard of care. Professional photographs were taken between 8 and 12 months postsurgery and evaluated using a visual analog scale by lay and professional panels. This study is registered with ClinicalTrials.gov, number NCT00766727. RESULTS: Stress shielding of swine incisions reduced histologic scar area by 6- and 9-fold compared to control and elevated stress states, respectively (P < 0.01 for both) and dramatically decreased the histologic expression of profibrotic markers. Closure of high-tension wounds induced human-like scar formation in the red Duroc, a phenotype effectively mitigated with stress shielding of wounds. In the study on humans, stress shielding of abdominal incisions significantly improved scar appearance (P = 0.004) compared with within-patient controls. CONCLUSIONS: These results indicate that mechanical manipulation of the wound environment with a dynamic stress-shielding polymer device can significantly reduce scar formation.
Asunto(s)
Cicatriz/fisiopatología , Modelos Animales de Enfermedad , Cicatrización de Heridas/fisiología , Pared Abdominal/patología , Pared Abdominal/fisiopatología , Pared Abdominal/cirugía , Adulto , Animales , Vendajes , Fenómenos Biomecánicos/fisiología , Cicatriz/patología , Cicatriz Hipertrófica/patología , Cicatriz Hipertrófica/fisiopatología , Cicatriz Hipertrófica/prevención & control , Fuerza Compresiva/fisiología , Femenino , Fibrosis , Humanos , Persona de Mediana Edad , Procedimientos de Cirugía Plástica , Factores de Riesgo , Elastómeros de Silicona , Piel/patología , Piel/fisiopatología , Dehiscencia de la Herida Operatoria/patología , Dehiscencia de la Herida Operatoria/fisiopatología , Porcinos , Adulto JovenRESUMEN
Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt-mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide-3-kinase/Akt inhibitor). We also demonstrated that high-frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α-smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.
Asunto(s)
Cicatriz Hipertrófica/enzimología , Cicatriz Hipertrófica/etiología , Fibroblastos/fisiología , Mecanotransducción Celular/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Androstadienos/farmacología , Animales , Movimiento Celular , Supervivencia Celular , Cicatriz Hipertrófica/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Estrés Mecánico , WortmaninaRESUMEN
BACKGROUND: The success of antimicrobial therapy has been impaired by the emergence of resistant bacterial strains. Antimicrobial peptides are ubiquitous proteins that are part of the innate immune system and are successful against such antibiotic-resistant microorganisms. The authors have previously demonstrated the feasibility of protein delivery via microvascular free flap gene therapy and here they examine this approach for recalcitrant infections. METHODS: The authors investigated the production of the human cathelicidin antimicrobial peptide-LL37, delivered by ex vivo transduction of the rodent superficial inferior epigastric free flap with Ad/CMV-LL37. The vascular permeabilizing agent vascular endothelial growth factor (VEGF) was co-administered during ex vivo transduction with adenoviral vectors in an attempt to augment transduction efficiency. A rodent model of chronic wound/foreign body infection seeded with bioluminescent Staphylococcus aureus was used to assess the biological efficacy of delivering therapeutic antimicrobial genes using this technology. RESULTS: The authors were successful in demonstrating significant LL37 expression, which persisted for 14 days after ex vivo transduction with Ad/CMV-LL37. Transduction efficiency was significantly improved with the co-administration of 5 micrograms of VEGF during transduction without significantly increasing systemic dissemination of adenovirus or systemic toxicity. They were able to demonstrate in the rodent model of chronic wound/foreign body infections a significant reduction in bacterial loads from infected catheters following transduction with Ad/CMV-LL37 and increased bacterial clearance. CONCLUSION: This study demonstrates for the first time that microbicidal gene therapy via microvascular free flaps is able to clear chronic infections such as occurs with osteomyelitis resulting from trauma or an infected foreign body [corrected]
Asunto(s)
Antibacterianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Portadores de Fármacos , Ingeniería Genética , Colgajos Quirúrgicos , Infección de Heridas/tratamiento farmacológico , Animales , Antibacterianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/biosíntesis , Catelicidinas , Enfermedad Crónica , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
BACKGROUND: Distraction osteogenesis is a valuable clinical tool; however the molecular mechanisms governing successful distraction remain unknown. We have used a uniaxial in vitro strain device to simulate the uniaxial mechanical environment of the interfragmentary distraction gap. MATERIALS AND METHODS: Using the Flexcell system, normal human osteoblasts were subjected to different levels of cyclical uniaxial mechanical strain. Cellular morphology, proliferation, migration, and the expression of angiogenic (vascular endothelial growth factor [VEGF] and fibroblast growth factor-2 [FGF-2]) and osteogenic (osteonectin, osteopontin, and osteocalcin) proteins and extracellular matrix molecules (collagen IalphaII) were analyzed in response to uniaxial cyclic strain. RESULTS: Osteoblasts exposed to strain assumed a fusiform spindle-shaped morphology aligning parallel to the axis of uniaxial strain and osteoblasts exposed to strain or conditioned media had a 3-fold increase in proliferation. Osteoblast migration was maximal (5-fold) in response to 9% strain. Angiogenic cytokine, VEGF, and FGF-2, increased 32-fold and 2.6-fold (P < 0.05), respectively. Osteoblasts expressed greater amounts of osteonectin, osteopontin, and osteocalcin (2.1-fold, 1.8-fold, 1.5-fold respectively, P < 0.01) at lower levels of strain (3%). Bone morphogenic protein-2 production increased maximally at 9% strain (1.6-fold, P < 0.01). Collagen I expression increased 13-, 66-, and 153-fold in response to 3, 6, and 9% strain, respectively. CONCLUSIONS: Uniaxial cyclic strain using the Flexcell device under appropriate strain parameters provides a novel in vitro model that induces osteoblast cellular and molecular expression patterns that simulate patterns observed in the in vivo distraction gap.
Asunto(s)
Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis por Distracción , División Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Forma de la Célula/fisiología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Técnicas In Vitro , Osteocalcina/genética , Osteocalcina/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Estrés Mecánico , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism.
Asunto(s)
Apoptosis/fisiología , Cicatriz Hipertrófica , Estrés Mecánico , Animales , Fenómenos Biomecánicos , Proliferación Celular , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Cicatriz Hipertrófica/fisiopatología , Modelos Animales de Enfermedad , Elasticidad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Piel/anatomía & histología , Piel/metabolismo , Piel/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cicatrización de HeridasRESUMEN
BACKGROUND: Long-term survival of a skin graft is dependent on eventual revascularization. The authors' aim in the present study was to determine whether skin graft vascularization occurs by (1) simple reconnection of vessels, (2) ingrowth of recipient vasculature, (3) outgrowth of donor-derived vessels, and/or (4) recruitment of bone marrow-derived endothelial progenitor cells. METHODS: Full-thickness skin grafts (1 x 1 cm) were transferred between wild-type FVB/N mice (n = 20) and transgenic tie2/lacZ mice (n = 20), where lacZ expression is controlled by the endothelial specific tie2 promoter, allowing differentiation of recipient and donor endothelial cells. The contribution of endothelial progenitor cells to skin graft neovascularization was determined using a bone marrow transplant model where tie2/lacZ bone marrow was transplanted into wild-type mice (n = 20). RESULTS: Vascular regression in the graft was observed at the periphery starting on day 3 and moving centrally through day 21, sparing graft vessels in the absolute center of the graft. At the same time, vascular ingrowth occurred from the wound bed to replace the regressing vessels. Furthermore, bone marrow-derived endothelial progenitor cells contributed to these new vessels starting as early as day 7. Surprisingly, the contribution of bone marrow-derived vessels to the overall process was approximately 15 to 20 percent of new endothelial cells. CONCLUSIONS: Replacement of the donor graft vasculature by endothelial and endothelial progenitor cells from the recipient along preexisting channels is the predominant mechanism for skin graft revascularization. This mechanism is likely similar for all nonvascularized free grafts and suggests novel strategies for optimizing the vascularization of tissue constructs engineered in vitro.
Asunto(s)
Células Endoteliales/fisiología , Endotelio Vascular/fisiología , Neovascularización Fisiológica/fisiología , Trasplante de Piel/fisiología , Células Madre/fisiología , Animales , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Ratones , Ratones Transgénicos , Receptor TIE-2/fisiología , Cicatrización de Heridas/fisiología , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: Ischemia is a limiting factor during distraction osteogenesis. The authors sought to determine the extent of ischemia in the distraction zone and whether endothelial progenitor cells home to the distraction zone and participate in local vasculogenesis. METHODS: Laser Doppler imaging was used to assess the extent of blood flow in the distraction zone in gradually distracted, immediately distracted, and osteotomized rat mandibles during activation and consolidation. Animals (n = 50; 25 rats with unilateral gradual distraction and contralateral osteotomy as an internal control, and 25 rats with unilateral immediate distraction) were examined on postoperative days 4, 6, and 8 of activation, and after 1 and 2 weeks of consolidation. Endothelial progenitor cells isolated from human peripheral blood were labeled with fluorescent DiI dye, and 0.5 x 10 cells were injected intra-arterially under direct vision into each carotid artery at the start of activation in nude rats (n = 18) that then underwent the distraction protocol outlined above. RESULTS: Doppler flow analysis demonstrated relative ischemia during the activation period in the distraction osteogenesis group and increased blood flow in the osteotomized control group as compared with flow in a normal hemimandible [normal, 1 (standardized); distraction osteogenesis, 0.58 +/- 0.05; control, 2.58 +/- 0.21; p < 0.05 for both results]. We observed a significantly increased endothelial progenitor cell population at the generate site versus controls at midactivation and at 1 and 2 weeks of consolidation [25 +/- 1.9 versus 1 +/- 0.3 DiI-positive cells per high-power field (p < 0.05), 124 +/- 21 versus 8 +/- 4 DiI-positive cells per high-power field (p < 0.05), and 106 +/- 18 versus 9 +/- 3 DiI-positive cells per high-power field (p < 0.05), respectively]. CONCLUSIONS: These data suggest that the distraction zone becomes relatively ischemic during activation and that endothelial progenitor cells home to the ischemic generate site during the activation phase and remain during the consolidation phase. Selective expansion of these stem cells may be useful in overcoming ischemic limitations of distraction osteogenesis. Moreover, their homing capability may be used to effect site-specific transgene delivery to the generate.
Asunto(s)
Endotelio Vascular/citología , Mandíbula/irrigación sanguínea , Mandíbula/cirugía , Osteogénesis por Distracción , Osteogénesis/fisiología , Células Madre/fisiología , Animales , Isquemia/diagnóstico , Isquemia/fisiopatología , Flujometría por Láser-Doppler , Osteogénesis por Distracción/métodos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect.
Asunto(s)
Sustancias de Crecimiento/administración & dosificación , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Inhibidores de la Angiogénesis/farmacología , Animales , Endostatinas/farmacología , Tejido de Granulación/irrigación sanguínea , Tejido de Granulación/efectos de los fármacos , Ratones , Piel/fisiopatología , Cicatrización de Heridas/fisiologíaRESUMEN
Fibroblasts represent a highly mechanoresponsive cell type known to play key roles in normal and pathologic processes such as wound healing, joint contracture, and hypertrophic scarring. In this study, we used a novel fibroblast-populated collagen lattice (FPCL) isometric tension model, allowing us to apply graded biaxial loads to dermal fibroblasts in a 3-dimensional matrix. Cell morphology demonstrated dose-dependent transition from round cells lacking stress fibers in nonloaded lattices to a broad, elongated morphology with prominent actin stress fibers in 800-mg-loaded lattices. Using quantitative real-time RT-PCR, a dose dependent induction of both collagen-1 and collagen-3 mRNA up to 2.8- and 3-fold, respectively, as well as a 2.5-fold induction of MMP-1 (collagenase) over unloaded FPCLs was observed. Quantitative expression of the proapoptotic gene Bax was down-regulated over 4-fold in mechanically strained FPCLs. These results suggest that mechanical strain up-regulates matrix remodeling genes and down-regulates normal cellular apoptosis, resulting in more cells, each of which produces more matrix. This "double burden" may underlie the pathophysiology of hypertrophic scars and other fibrotic processes in vivo.
Asunto(s)
Cicatriz Hipertrófica/genética , Colágeno , Fibroblastos , Expresión Génica , Adolescente , Adulto , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés MecánicoRESUMEN
Pulsed electromagnetic fields (PEMF) have been shown to be clinically beneficial, but their mechanism of action remains unclear. The present study examined the impact of PEMF on angiogenesis, a process critical for successful healing of various tissues. PEMF increased the degree of endothelial cell tubulization (sevenfold) and proliferation (threefold) in vitro. Media from PEMF cultures had a similar stimulatory effect, but heat denaturation ablated this activity. In addition, conditioned media was able to induce proliferative and chemotactic changes in both human umbilical vein endothelial cells and fibroblasts, but had no effect on osteoblasts. Angiogenic protein screening demonstrated a fivefold increase in fibroblast growth factor beta-2 (FGF-2), as well as smaller increases in other angiogenic growth factors (angiopoietin-2, thrombopoietin, and epidermal growth factor). Northern blot analysis demonstrated an increase in FGF-2 transcription, and FGF-2 neutralizing antibody inhibited the effects of PEMF. In vivo, PEMF exposure increased angiogenesis more than twofold. We conclude that PEMF augments angiogenesis primarily by stimulating endothelial release of FGF-2, inducing paracrine and autocrine changes in the surrounding tissue. These findings suggest a potential role for PEMF in therapeutic angiogenesis.
Asunto(s)
Campos Electromagnéticos , Células Endoteliales/efectos de la radiación , Factor 2 de Crecimiento de Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Neovascularización Fisiológica/efectos de la radiación , Osteoblastos/efectos de la radiación , Animales , División Celular/efectos de la radiación , Movimiento Celular/efectos de la radiación , Colágeno , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Replicación del ADN/efectos de la radiación , Combinación de Medicamentos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Laminina , Ratones , Ratones Transgénicos , Neovascularización Fisiológica/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Comunicación Paracrina , Prostaglandinas/biosíntesis , Proteoglicanos , Ratas , Método Simple Ciego , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Distraction osteogenesis (DO) requires a long consolidation period and has a low but real failure rate. Bone morphogenic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects, but their effects on mandibular DO are unknown. We investigated the effect of local delivery of adenovirus containing the gene for BMP-2 (Adbmp-2) on mandibular DO in a rat model. Rats (n = 54) were distracted to 3 mm over 6 days. At the start of consolidation (POD 10), Adbmp-2 or adenovirus containing the lacZgene (AdlacZ) was injected directly into the distraction zone. After 1, 2, and 4 weeks of consolidation, mandibles were evaluated for amount of bone deposition. Adbmp-2-treated specimens demonstrated an increased amount of new bone formation by radiographic, histologic, and histomorphometric analysis. This study demonstrates that local, adenovirally-mediated delivery of BMP-2 can increase bone deposition during DO, potentially shortening consolidation and enhancing DO in poorly healing mandibles, such as occurs postirradiation.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Terapia Genética/métodos , Mandíbula/cirugía , Osteogénesis por Distracción , Factor de Crecimiento Transformador beta , Adenoviridae , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Immunoblotting , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Diminished production of vascular endothelial growth factor (VEGF) and decreased angiogenesis are thought to contribute to impaired tissue repair in diabetic patients. We examined whether recombinant human VEGF(165) protein would reverse the impaired wound healing phenotype in genetically diabetic mice. Paired full-thickness skin wounds on the dorsum of db/db mice received 20 microg of VEGF every other day for five doses to one wound and vehicle (phosphate-buffered saline) to the other. We demonstrate significantly accelerated repair in VEGF-treated wounds with an average time to resurfacing of 12 days versus 25 days in untreated mice. VEGF-treated wounds were characterized by an early leaky, malformed vasculature followed by abundant granulation tissue deposition. The VEGF-treated wounds demonstrated increased epithelialization, increased matrix deposition, and enhanced cellular proliferation, as assessed by uptake of 5-bromodeoxyuridine. Analysis of gene expression by real-time reverse transcriptase-polymerase chain reaction demonstrates a significant up-regulation of platelet-derived growth factor-B and fibroblast growth factor-2 in VEGF-treated wounds, which corresponds with the increased granulation tissue in these wounds. These experiments also demonstrated an increase in the rate of repair of the contralateral phosphate-buffered saline-treated wound when compared to wounds in diabetic mice never exposed to VEGF (18 days versus 25 days), suggesting that topical VEGF had a systemic effect. We observed increased numbers of circulating VEGFR2(+)/CD11b(-) cells in the VEGF-treated mice by fluorescence-activated cell sorting analysis, which likely represent an endothelial precursor population. In diabetic mice with bone marrow replaced by that of tie2/lacZ mice we demonstrate that the local recruitment of bone marrow-derived endothelial lineage lacZ+ cells was augmented by topical VEGF. We conclude that topical VEGF is able to improve wound healing by locally up-regulating growth factors important for tissue repair and by systemically mobilizing bone marrow-derived cells, including a population that contributes to blood vessel formation, and recruiting these cells to the local wound environment where they are able to accelerate repair. Thus, VEGF therapy may be useful in the treatment of diabetic complications characterized by impaired neovascularization.
Asunto(s)
Diabetes Mellitus Tipo 2/fisiopatología , Proteínas Recombinantes/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Femenino , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/genética , Movilización de Célula Madre Hematopoyética , Humanos , Ratones , Ratones Mutantes , Ratones Transgénicos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/patología , beta-Galactosidasa/genéticaRESUMEN
The impact of inhibitors of tumor angiogenesis (endostatin, angiostatin) on the neovascularization required for the healing of transferred tissue has not been examined. We investigated the effect of endostatin on the functional neovascularization of random pattern flaps. C57BL6 mice were pretreated with endostatin beginning 3 days prior to surgery (n = 10), and daily injections continued throughout the study. Dorsal random cutaneous flaps were raised in both treatment and control (saline-treated) groups. The remaining cranial attachment was divided on day 9. Oxygen tension (PO2) was measured using a microprobe on days 1, 3, 5 and 16. Flaps were harvested and the vasculature was stained with CD31 on day 16. We found that endostatin significantly decreased flap survival. Mice that were treated with endostatin had fewer CD31+ blood vessels, worse flap perfusion at all time points, and lower oxygen tensions throughout the length of the flap. These findings have potential implications for the patients undergoing antiangiogenesis therapy who require surgical reconstruction.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Endostatinas/administración & dosificación , Isquemia , Neovascularización Fisiológica/efectos de los fármacos , Colgajos Quirúrgicos/irrigación sanguínea , Animales , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Piel/irrigación sanguíneaRESUMEN
UNLABELLED: Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS: This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.
Asunto(s)
Antígenos CD34/biosíntesis , Glicoproteínas/biosíntesis , Hemangioma/metabolismo , Células Madre/metabolismo , Antígeno AC133 , Antígenos CD , Membrana Celular/metabolismo , Células Cultivadas , Preescolar , Citometría de Flujo , Transportador de Glucosa de Tipo 1 , Hemangioma/patología , Humanos , Lactante , Laminina/biosíntesis , Leucocitos Mononucleares/inmunología , Microscopía Fluorescente , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/biosíntesis , Células Neoplásicas Circulantes , Péptidos , Receptores de IgG/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The success of an arterial reconstruction is dependent on multiple factors, including patient selection, quality of the vessels, type of vascular conduit employed, and the surgical technique adopted. Transit time flow measurement is a technique that is now the standard of care for immediate graft patency verification following coronary artery bypass graft surgery. This study demonstrates that transit time flow measurement can be successfully used to evaluate renal transplant anastomoses: accurate intraoperative measurements provide a rapid objective assessment of renal graft patency.