RESUMEN
BACKGROUND: Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. METHODS: Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. RESULTS: The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6ß1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: Thus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.
Asunto(s)
Membrana Corioalantoides/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Proteínas Supresoras de Tumor/farmacología , Gelatina de Wharton/metabolismo , Animales , Bioensayo , Movimiento Celular , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/citología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Células Madre Mesenquimatosas/citología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Receptores de Netrina , Netrina-1 , Cultivo Primario de Células , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Gelatina de Wharton/citologíaRESUMEN
Disorders in skin wound healing are a major health problem that requires the development of innovative treatments. The use of biomaterials as an alternative of skin replacement has become relevant, but its use is still limited due to poor vascularization inside the scaffolds, resulting in insufficient oxygen and growth factors at the wound site. In this study, we have developed a cell-based wound therapy consisting of the application of collagen-based dermal scaffolds containing mesenchymal stem cells from Wharton's jelly (WJ-MSC) in an immunocompetent mouse model of angiogenesis. From our comparative study on the secretion profile between WJ-MSC and adipose tissue-derived MSC, we found a stronger expression of several well-characterized growth factors, such as VEGF-A, angiopoietin-1 and aFGF, which are directly linked to angiogenesis, in the culture supernatant of WJ-MSC, both on monolayer and 3D culture conditions. WJ-MSC proved to be angiogenic both in vitro and in vivo, through tubule formation and CAM assays, respectively. Moreover, WJ-MSC consistently improved the healing response in vivo in a mouse model of human-like dermal repair, by triggering angiogenesis and further providing a suitable matrix for wound repair, without altering the inflammatory response in the animals. Since these cells can be easily isolated, cultured with high expansion rates and cryopreserved, they represent an attractive stem cell source for their use in allogeneic cell transplant and tissue engineering.