Chronic wounds are associated with inhibition of Smad3 phosphorylation

Abstract

A chronic wound is a heavy burden to the public health system and society. About 1.51- 2.21% of people are affected in the world. An individual chronic wound cost was 9,060 € on average per year, including medical insurance and personal payment. Chronic wounds usually lead to limited mobility, limb pain, and serious stress on emotion. So far, it is still unclear how chronic wounds develop, especially on a molecular level. our purpose was to explore the mechanisms behind the development of chronic wounds and to identify treatment options. To understand the underlying mechanisms, patients with acute wounds (<30 days) and chronic wounds (≥30 days) from the BG clinic in Tübingen were investigated. Considering the important role in wound healing process, TGF-β1-3 were investigated in the wound tissue and the blood of patients. TGF-β1 gene expression was higher in chronic than acute wounds tissue, and active TGF-β1 levels were also higher in the serum of patients with chronic wounds. This result suggested that the TGF-β signaling pathway might be impaired. Hence, phosphorylation of Smad3 was TGF-β signaling. Low levels of phospho-Smad3 in traumatic chronic wounds were checked by Western blot analysis, and acute wound tissue had a significant increase of phospho-Smad3 in the nucleus when compared to the chronic wounds group in the immunohistochemistry staining. Besides the samples from patients with acute and chronic wounds, scab model was developed to investigate the wound healing process. This in vitro model was produced with HaCaT cells and fresh EDTA venous blood and could be kept for more than 2weeks. Different growth factors, cytokines and chemokines, including TGF-B1-3, were identified in this model during the first 7 days, and target genes of TGF-β were also discovered. To further clarify the role of TGF-β1-3, exogeneous TGF-β1-3 or inhibitor of TGF-β (Alk5i) were added to the scabs. it was verified that exogenous TGF-β1 maintained the stability of the scab and promoted the production of Co/1A1, FN1, andCTGF.Smad7, inhibitor of TGF-β, was also increased with exogeneous TGF-β1stimulation. As a versatile growth factor, exogenous TGF-β1 produced more junctions and mesh area in the tube formation assay and promoted the production of angiogenic factors, like VEGFA, Angiopoietin-1, Angiopoietin-2, VEGFR3, IL-2, IL-4, IL-10Endostatin, and GM-CSF.TGF-B1 promoted angiogenesis in the scab model. In this study, high levels of TGF-β1 were detected in wound tissue and serum of patients with chronic wounds, and low levels of phospho-Smad3 confirmed the impaired TGF-β signaling pathway in chronic wounds and explained why increasedTGF-B1 in chronic wounds couldn't improve the wound healing. Besides the data from the clinic, a new in vitro wound model was also established. Similar to real wounds, it could be maintained for 14 days and changed with timeTGF-B1-3 and its target gene expressions were detectable in this model. Among these three TGF-β isoforms, TGF-β1 showed the most obvious effect on the scab stability and angiogenesis ability. This in vitro wound model can be used to track inhibitors and test treatment targets.