Therapeutic approaches to control tissue repair and fibrosis: Extracellular matrix as a game changer.

Organ fibrosis is characterized by the accumulation of disorganized and stiff extracellular matrix (ECM) and represents the final stage of several life-threatening diseases. The progressive replacement of normal tissue by fibrotic ECM impedes organ functionality to the point of failure. Fibrosis affects millions of people worldwide with no effective cure for various reasons: (a) Due to the lack of clinical biomarkers and non-invasive detection methods fibrosis is often diagnosed too late, when organs are already destroyed beyond repair. (b) Fibrosis can be understood as dysregulated tissue repair that evolved robust programs to be able to respond to various injury scenarios. The redundant nature of these programs often evades linear therapeutic strategies. (c) Fibrosis perpetuates itself by establishing conditions that activate normal into fibrogenic cells which, in turn, create a pro-fibrotic environment. ECM takes center stage in the process of fibrosis as a defining feature and thus potential diagnostic biomarker. The ECM is also a main promoter of the disease process by providing lasting physicochemical pro-fibrotic cues to residing and recruiting cells. Effective anti-fibrotic therapies will need to take the lasting (mis-) instructive character of scar ECM into account. To restore organ functionality, it will be important to (re)turn fibrotic scar into functional ECM, for instance by dissolving fibrotic ECM and delivering cells with regenerative potential.

The fibronectin ED-A domain enhances recruitment of latent TGF-ß-binding protein-1 to the fibroblast matrix.

Dysregulated secretion and extracellular activation of TGF-ß1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-ß-binding protein-1 (LTBP-1) and thus stores TGF-ß1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-ß1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-ß1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.

Healthy human second-trimester fetal skin is deficient in leukocytes and associated homing chemokines.

The lack of immune cells in mid-gestational fetal skin is often mentioned as a key factor underlying scarless healing. However, the scarless healing ability is conserved until long after the immune system in the fetus is fully developed. Therefore, we studied human second-trimester fetal skin and compared the numbers of immune cells and chemokine levels from fetal skin with adult skin. By using immunohistochemistry, we show that healthy fetal skin contains significant lower numbers of CD68(+) -macrophages, Tryptase(+) -mast cells, Langerin(+) -Langerhans cells, CD1a(+) -dendritic cells, and CD3(+) -T cells compared to adult skin. Staining with an early lineage leukocyte marker, i.e., CD45, verified that the number of CD45(+) -immune cells was indeed significantly lower in fetal skin but that sufficient numbers of immune cells were present in the fetal lymph node. No differences in the vascular network were observed between fetal and adult skin. Moreover, significant lower levels of lymphocyte chemokines CCL17, CCL21, and CCL27 were observed in fetal skin. However, levels of inflammatory interleukins such as IL-6, IL-8, and IL-10 were undetectable and levels of CCL2 were similar in healthy fetal and adult skin. In conclusion, this study shows that second-trimester fetal skin contains low levels of immune cells and leukocyte chemokines compared to adult skin. This immune cell deficiency includes CD45(+) leukocytes, despite the abundant presence of these cells in the lymph node. The immune deficiency in healthy second-trimester fetal skin may result in reduced inflammation during wound healing, and could underlie the scarless healing capacities of the fetal skin.

Altered TGF-ß signaling in fetal fibroblasts: what is known about the underlying mechanisms?

Scarless wound healing is a unique and intrinsic capacity of the fetal skin that is not fully understood. Further insight into the underlying mechanisms of fetal wound healing may lead to new therapeutic approaches promoting adult scarless wound healing. Differences between fetal and adult wound healing are found in the extracellular matrix, the inflammatory reaction and the levels of growth factors present in the wound. This review focuses specifically on transforming growth factor ß (TGF-ß), as this growth factor is prominently involved in wound healing and fibroblast-to-myofibroblast differentiation. Although fetal fibroblasts do respond to TGF-ß, they lack a proliferative and a contractile response and display short-lived myofibroblast differentiation, autocrine response, and collagen up-regulation in comparison with adult fibroblasts. Curiously, prolonged TGF-ß activation is associated with fibrosis, and therefore, this short-lived response in fetal fibroblasts might contribute to scarless healing. This review gives an overview of the current knowledge on TGF-ß signaling and the intracellular TGF-ß signaling pathway in fetal fibroblasts. Furthermore, this review also describes the various components that regulate the cellular TGF-ß response and hypothesizes about the possible roles these components might play in the altered response of fetal fibroblasts to TGF-ß.

Tissue engineered tubular construct for urinary diversion in a preclinical porcine model.

PURPOSE: The ileal conduit has been considered the gold standard urinary diversion for patients with bladder cancer and pediatric patients. Complications are mainly related to the use of gastrointestinal tissue. Tissue engineering may be the technical platform on which to develop alternatives to gastrointestinal tissue. We developed a collagen-polymer conduit and evaluated its applicability for urinary diversion in pigs. MATERIALS AND METHODS: Tubular constructs 12 cm long and 15 mm in diameter were prepared from bovine type I collagen and Vypro® II synthetic polymer mesh. Characterized tubes were sterilized, seeded with and without primary porcine bladder urothelial cells, and implanted as an incontinent urostomy using the right ureter in 10 female Landrace pigs. At 1 month the newly formed tissue structure was functionally and microscopically evaluated by loopogram and immunohistochemistry, respectively. RESULTS: The survival rate was 80% with 1 related and 1 unrelated death. By 1 month the collagen was resorbed and a retroperitoneal tunnel had formed that withstood 40 cm H(2)O water pressure. In 5 cases the tunnel functioned as a urostomy. Histological analysis revealed a moderate immune response, neovascularization and urothelial cells in the construct lumen. The polymer mesh provoked fibroblast deposition and tissue contraction. No major differences were observed between cellular and acellular constructs. CONCLUSIONS: After implanting the tubular constructs a retroperitoneal tunnel was formed that functioned as a urinary conduit in most cases. Improved large tubular scaffolds may generate alternatives to gastrointestinal tissue for urinary diversion.