Dupuytren's disease, a benign fibroproliferative disorder of the palmar fascia, represents an ideal model to study tissue fibrosis. Transforming growth factor-β
1
(TGF-β
1
) and its downstream Smad signalling system is well established as a key player during fibrogenesis. Thus, targeting this basic pathomechanism seems suitable to establish new treatment strategies. One such promising treatment involves the substance N-acetyl-L-cysteine (NAC), shown to have antifibrotic properties in hepatic stellate cells and rat fibroblasts. In order to investigate antifibrotic effects of N-acetyl-L-cysteine (NAC), fibroblasts were isolated from surgically resected fibrotic palmar tissues (Dupuytren fibroblasts, DF) and exposed to different concentrations of NAC and recombinant TGF-β
1
. Fibroblasts isolated from tendon pulleys served as controls (control fibroblasts, CF). Smad signalling was investigated by a Smad binding element driven reporter gene analysis. Both cell types express TGF-β
1
, indicating autocrine signalling in DF and CF. This was confirmed by comparing reporter gene activity from LacZ and Smad7 adenovirus infected cells. NAC treatment resulted in abrogation of Smad mediated signalling comparable to ectopically overexpressed Smad7, even when the cells were stimulated with recombinant TGF-β
1
or ectopically expressed a constitutively active TGF-β receptor type I. Additionally, NAC dose-dependently decreased expression of three major indicators of impaired fibrotic matrix turnover, namely alpha-smooth muscle actin (α-SMA), α 1 type I procollagen (CollA1), and plasminogen activator inhibitor-type I (PAI-1). Our results suggest that TGF-β signalling and subsequent expression of fibrogenesis related proteins in Dupuytren's disease is abrogated by NAC thus providing a basis for a therapeutic strategy in Dupuytren's disease and other fibroproliferative disorders.
Background/Aims:
Profibrogenic TGF-β signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-β effects provide promising antifibrotic results, however,
in vivo
data regarding Smad activation during fibrogenesis are scarce.
Methods:
Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting.
Results:
Cholestasis induces TGF-β signaling
via
Smad3
in vivo
, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. Conclusions: Hepatocytes of damaged liver tissue display increased TGF-β signaling
via
Smad3. Further, negative feedback regulation of TGF-β signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.
Epidermal regeneration is a complex process, strongly influenced by growth factors, including keratinocyte growth factor (KGF). The objective of this study was to establish immortalized HaCaT keratinocytes and KMST-6-fibroblasts stably expressing KGF. Transfection efficiency, genomic integration, and functionality of the transgene were determined by ELISA and PCR, and KGF-expressing clones were selected using an air-liquid interface test system. HaCaT cells displayed stronger transgene expression compared to transfected fibroblasts, and the most effective HaCaT clone was incubated on a membrane carrier to form a "membrane cell graft." Twenty-one superficial second-degree burn wounds were created in each of three pigs, and wound healing capacity of the generated "polypeptide cell delivery system" after grafting was examined. Untransfected HaCaT keratinocytes and membrane-covered and untreated burn wounds served as controls. Histological and macroscopical follow-up revealed that grafting of transfected HaCaT cells resulted in complete reepithelialization within 5 days, while wounds covered with untransfected cells needed 2 days longer. At untreated sites, a thin epithelium was detectable after 10 days. The results indicate that wound healing processes can be stimulated distinctly by growth factors secreted from HaCaT cells, with a prominent role for transgenic KGF.
The authors suggest that distinct proliferative properties of Dupuytren-derived fibroblasts are dependent on the cells' origin. Conclusions about the cause or pathogenesis cannot yet be drawn. Further investigation concerning their apparently different fibroproliferative properties is necessary.
There is an increased risk for burn injuries associated with home oxygen therapy of patients with chronic obstructive pulmonary disease since 10 to 50 % of these patients continue to smoke. Enzymatic eschar removal of facial burns is gaining popularity but intubation of this specific patient group often leads to prolonged weaning and can require tracheostomy. This study dealt with the question if enzymatic debridement in these patients can also be performed in analgosedation. A selective review of the literature regarding burn trauma associated with home oxygen use in patients with COPD was performed, as well as a retrospective analysis of all patients with burn injuries associated with home oxygen use and chronic obstructive pulmonary disease that were admitted to the study clinic. In the literature 1746 patients with burns associated with home oxygen use are described, but none of them received enzymatic debridement. In this study seventeen patients were included. All three patients in this study with facial full-thickness burn injuries received enzymatic debridement. The mortality rate in this cohort was 17.6 % (3/17). Up to date, there is limited experience performing regional anesthesia debridement in patients with COPD. This is the first manuscript describing the use of enzymatic debridement in patients with COPD and home oxygen therapy. We could confirm other studies that intubation of these patients leads to prolonged ventilation hours and increases the probability for poor prognosis. Therefore, we described the treatment of enzymatic debridement in analgosedation without intubation.
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