Inflammation and fibrosis in chronic liver diseases including non-alcoholic fatty liver disease and hepatitis C.

At present chronic liver disease (CLD), the third commonest cause of premature death in the United Kingdom is detected late, when interventions are ineffective, resulting in considerable morbidity and mortality. Injury to the liver, the largest solid organ in the body, leads to a cascade of inflammatory events. Chronic inflammation leads to the activation of hepatic stellate cells that undergo trans-differentiation to become myofibroblasts, the main extra-cellular matrix producing cells in the liver; over time increased extra-cellular matrix production results in the formation of liver fibrosis. Although fibrogenesis may be viewed as having evolved as a "wound healing" process that preserves tissue integrity, sustained chronic fibrosis can become pathogenic culminating in CLD, cirrhosis and its associated complications. As the reference standard for detecting liver fibrosis, liver biopsy, is invasive and has an associated morbidity, the diagnostic assessment of CLD by non-invasive testing is attractive. Accordingly, in this review the mechanisms by which liver inflammation and fibrosis develop in chronic liver diseases are explored to identify appropriate and meaningful diagnostic targets for clinical practice. Due to differing disease prevalence and treatment efficacy, disease specific diagnostic targets are required to optimally manage individual CLDs such as non-alcoholic fatty liver disease and chronic hepatitis C infection. To facilitate this, a review of the pathogenesis of both conditions is also conducted. Finally, the evidence for hepatic fibrosis regression and the mechanisms by which this occurs are discussed, including the current use of antifibrotic therapy.

In Atp7b-/- Mice Modeling Wilson's Disease Liver Repopulation With Bone Marrow-Derived Myofibroblasts or Inflammatory Cells and Not Hepatocytes Is Deleterious.

In Wilson's disease, Atp7b mutations impair copper excretion with liver or brain damage. Healthy transplanted hepatocytes repopulate the liver, excrete copper, and reverse hepatic damage in animal models of Wilson's disease. In Fah-/- mice with tyrosinemia and α-1 antitrypsin mutant mice, liver disease is resolved by expansions of healthy hepatocytes derived from transplanted healthy bone marrow stem cells. This potential of stem cells has not been defined for Wilson's disease. In diseased Atp7b-/- mice, we reconstituted bone marrow with donor cells expressing green fluorescent protein reporter from healthy transgenic mice. Mature hepatocytes originating from donor bone marrow were identified by immunostaining for green fluorescence protein and bile canalicular marker, dipeptidylpeptidase-4. Mesenchymal and inflammatory cell markers were used for other cells from donor bone marrow cells. Gene expression, liver tests, and tissues were analyzed for outcomes in Atp7b-/- mice. After bone marrow transplantation in Atp7b-/- mice, donor-derived hepatocytes containing bile canaliculi appeared within weeks. Despite this maturity, donor-derived hepatocytes neither divided nor expanded. The liver of Atp7b-/- mice was not repopulated by donor-derived hepatocytes: Atp7b mRNA remained undetectable; liver tests, copper content, and fibrosis actually worsened. Restriction of proliferation in hepatocytes accompanied oxidative DNA damage. By contrast, donor-derived mesenchymal and inflammatory cells extensively proliferated. These contributed to fibrogenesis through greater expression of inflammatory cytokines. In Wilson's disease, donor bone marrow-derived cells underwent different fates: hepatocytes failed to proliferate; inflammatory cells proliferated to worsen disease outcomes. This will help guide stem cell therapies for conditions with proinflammatory or profibrogenic microenvironments.

Urethral Reconstruction Using Mesothelial Cell-Seeded Autogenous Granulation Tissue Tube: An Experimental Study in Male Rabbits.

Objective. This study was to evaluate the utility of the compound graft for tubularized urethroplasty by seeding mesothelial cells onto autogenous granulation tissue. Methods. Silastic tubes were implanted subcutaneously in 18 male rabbits, of which nine underwent omentum biopsies simultaneously for in vitro expansion of mesothelial cells. The granulation tissue covering the tubes was harvested 2 weeks after operation. Mesothelial cells were seeded onto and cocultured with the tissue for 7 days. A pendulous urethral segment of 1.5 cm was totally excised. Urethroplasty was performed with mesothelial cell-seeded tissue tubes in an end-to-end fashion in nine rabbits and with unseeded grafts in others as controls. Serial urethrograms were performed at 1, 2, and 6 months postoperatively. Meanwhile, the neourethra was harvested and analyzed grossly and histologically. Results. Urethrograms showed cell-seeded grafts maintained wide at each time point, while strictures formation was found in unseeded grafts. Histologically, layers of urothelium surrounded by increasingly organized smooth muscles were observed in seeded grafts. In contrast, myofibroblasts accumulation and extensive scarring occurred in unseeded grafts. Conclusions. Mesothelial cell-seeded granulation tissue tube can be successfully used for tubularized urethroplasty in male rabbits.

The chick chorioallantoic membrane: a model of short-term study of Dupuytren's disease.

Dupuytren's disease is a progressive fibroproliferative disorder that impairs hand function by altering the normal structures of the palmar fascial bands. Nodules composed almost entirely of myofibroblasts and cords are pathognomonic of Dupuytren's disease. The myofibroblasts express alpha-smooth muscle actin that is especially involved in development of the disease. We aimed to evaluate whether the xenograft of Dupuytren's fibromatosis taken from operating room and transplanted on chick chorioallantoic membrane (CAM) survives with its histological and immunohistological features. Fresh samples obtained from eight patients with Dupuytren's disease were minced and immediately inoculated onto 24 CAMs of 8-day-old chick embryos. The implanted CAMs were examined daily by stereomicroscopy and finally the xenografts were examined and characterized in histological sections using a panel of antibodies. The xenografts were incorporated into the CAMs 6-7 days after transplantation, continued to grow and stimulated angiogenesis in the chick embryo CAMs. The CAMs vessels entered the xenografts and anastomosed with the newly formed xenografts vessels (CD34+ and CD105+) those containing nucleated chick erythrocytes. Myofibroblasts (α-SMA+) and macrophages (CD68+) were readily recognized in the xenograft thickness. We concluded that the xenografts of Dupuytren's fibromatosis transplanted onto chick embryo CAMs continued to develop and preserved the histological and immunohistological features.

Autologous transplantation of culture-born myofibroblasts into intact and injured rabbit ligaments.

PURPOSE: The myofibroblast, a contractile fibroblastic cell expressing α-smooth muscle actin (α-SMA), has been reported to play a role in ligament healing. The aim of this study was to evaluate the feasibility of transplanting culture-derived myofibroblasts in injured rabbit medial collateral ligaments (MCL) and in intact anterior cruciate ligaments (ACL). METHODS: Fibroblasts isolated from the iliotibial band were cultured in the presence of transforming growth factor beta-1 (TGF-ß1) for five days and analysed for α-SMA expression. In a concentration of TGF-ß1 ≥ 10 ng/ml, the differentiation rate into myofibroblast was 90%. After labelling with PKH26, α-SMA -positive cells were transplanted in intact ACL and in injured MCL of ten rabbits. RESULTS: Survival of PKH-26+ cells was seen in all intact and damaged ligaments one day after injection. The density of PKH-26+ cells had decreased at seven days postinjection in both ligaments. Double-positive PKH-26+/α-SMA+ cells were only observed in injured MCL at seven days postinjection. Moreover, we found that genetically modified fibroblasts differentiate into myofibroblasts and can be transplanted into ligaments. CONCLUSIONS: Our data demonstrate that culture-born myofibroblasts survive and maintain α-SMA expression up to one week after transplantation. This study provides the first insight into the feasibility of transplanted mechanically active cells for ligament reconstruction.

Ureteral reconstruction using autologous tubular grafts for the management of ureteral strictures and defects: an experimental study.

OBJECTIVE: To investigate whether the peritoneal cavity could function as a bioreactor to produce autologous tubular grafts for ureteral reconstruction in beagles. MATERIALS AND METHODS: 8-Fr Silastic tubes were implanted into the peritoneal cavities of 6 female beagles. At 3 weeks, the tubes were harvested and the tubular tissue covering the tubes was gently everted. A segment 3 cm in length of the right mid-ureter, involving two thirds of its diameter, was removed parallel to the ureteral axis, leaving a third of the ureteral wall. A 5-Fr double-J stent was inserted into the ureter through the created defect, and two thirds of the graft were anastomosed to both edges of the ureteral defect. One third of the graft was overlapped with the retained normal ureter and anastomosed to the external surface of the lumens. Thus, the graft was partly encapsulated by the remainder of ureteral wall. The stent was maintained for 6 weeks and removed. Excretory urography was performed at 8 (n = 3) and 12 weeks (n = 3), postoperatively. Meanwhile, the neoureter was harvested and analyzed. The left ureter served as the control and a simple intubated ureterotomy was performed. RESULTS: Histological analysis of the tubular tissue demonstrated transversely arranged myofibroblasts and an outer layer of mesothelium. The tissue was easily everted and transplanted as a ureteral graft. Eight weeks postoperatively, the neoureter demonstrated normal ureteral architecture, composed of multilayers of urothelium surrounded by smooth muscle bundles, which became increasingly organized with time. Excretory urography indicated no stenosis or hydronephrosis. CONCLUSIONS: These results show that autologous tubular tissue grown within the recipients' peritoneal cavity can be used for ureteral reconstruction in the beagle model.

Bone marrow cells in murine colitis: multi-signal analysis confirms pericryptal myofibroblast engraftment without epithelial involvement.

BACKGROUND: The contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions. METHODS: In attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract. PRINCIPAL FINDINGS: In rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels. CONCLUSIONS: We conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.

The influence of early-phase remodeling events on the biomechanical properties of engineered vascular tissues.

OBJECTIVES: During the last decade, the use of ex vivo-derived materials designed as implant scaffolds has increased significantly. This is particularly so in the area of regenerative medicine, or tissue engineering, where the natural chemical and biomechanical properties have been shown to be advantageous. By focusing on detailed events that occur during early-phase remodeling processes, our objective was to detail progressive changes in graft biomechanics to further our understanding of these processes. METHODS: A perfusion bioreactor system and acellular human umbilical veins were used as a model three-dimensional vascular scaffold on which human myofibroblasts were seeded and cultured under static or defined pulsatile conditions. Cell function in relation to graft mechanical properties was assessed. RESULTS: Cells doubled in density from approximately 1 × 10(6) to 2 ± 0.4 × 10(6) cells/cm ringlet, whereas static cultures remained unchanged. The material's compressive stiffness and ultimate tensile strength remained unchanged in both static and dynamic systems. However the Young's modulus values increased significantly in the physiologic range, whereas in the failure range, a significant reduction (66%) was shown under dynamic conditions. CONCLUSIONS: As pulse and flow conditions are modulated, complex mechanical changes are occurring that modify the elastic modulus differentially in both physiologic and failure ranges. Mechanical properties play an important role in graft patency, and a dynamic relationship between structure and function occurs during graft remodeling. These investigations have shown that as cells migrate into this ex vivo scaffold model, significant variation in material elasticity occurs that may have important implications in our understanding of early-stage vascular remodeling events.