Ubiquitin C-terminal hydrolase 1: A novel functional marker for liver myofibroblasts and a therapeutic target in chronic liver disease.

BACKGROUND & AIMS: Ubiquitination is a reversible protein modification involved in the major cellular processes that define cell phenotype and behaviour. Ubiquitin modifications are removed by a large family of proteases named deubiquitinases. The role of deubiquitinases in hepatic stellate cell (HSC) activation and their contribution to fibrogenesis are poorly defined. We have identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, determined its function in activated HSC and its potential as a therapeutic target for fibrosis. METHODS: Deubiquitinase expression was determined in day 0 and day 10 HSC. Increased UCHL1 expression was confirmed in human HSC and in an alcoholic liver disease (ALD) patient liver. The importance of UCHL1 in hepatic fibrosis was investigated in CCl4 and bile duct ligation injured mice using a pharmacological inhibitor (LDN 57444). The effects of UCHL1 inhibition on HSC proliferation were confirmed by Western blot and 3H thymidine incorporation. RESULTS: Here we report that pharmacological inhibition of UCHL1 blocks progression of established fibrosis in CCl4 injured mice. UCHL1 siRNA knockdown, LDN 57444 treatment, or HSC isolated from UCHL1(-/-) mice show attenuated proliferation in response to the mitogen, platelet-derived growth factor. Additionally, we observed changes in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb) in the absence of UCHL1 highlighting a potential mechanism for the reduced proliferative response. CONCLUSIONS: UCHL1 expression is highly upregulated upon HSC activation and is involved in the regulation of HSC proliferation. This study highlights therapeutic opportunities for pharmacological targeting of UCHL1 in chronic liver disease.

Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis.

Subspecialty training in the ambulatory clinic: a preliminary investigation of an endocrinology curriculum.

PURPOSE: There is scant literature on education in the ambulatory subspecialty clinic. This preliminary study was conducted to investigate subspecialty education, using endocrinology as a model. METHOD: In 2002, a questionnaire was mailed to 90 former University of Virginia internal medicine (IM) residents who completed training between 1998 and 2001. The questionnaire asked how well residency prepared them to take care of patients with various endocrine disorders, and to state the best and worst parts of their endocrine training. In 2001-02, 11 fourth-year medical students and 26 senior IM residents rotating on the endocrinology service were asked to record their patient encounters in logbooks. This information was compared to the perceived strengths and weaknesses of endocrine training. RESULTS: Sixty-one residents (68%) completed questionnaires. Significant differences were found in perceived preparedness to care for patients with various endocrine disorders, with diabetes, hypothyroidism, and osteoporosis as strengths and reproductive disorders representing a significant weakness. Fifteen residents (58%) and ten students (91%) completed logs. The logbooks revealed that current learners were well exposed to patients with diabetes and thyroid disorders during the block rotation, but saw relatively few patients with other endocrine disorders. CONCLUSION: Former IM residents felt well prepared by residency to manage certain common endocrine problems, but felt significantly less prepared for other problems, most notably reproductive disorders. University of Virginia's curriculum was modified using information from this study to improve the educational experience in endocrinology. More studies are needed to define optimal strategies for teaching and learning in the subspecialty clinic.

Multigenerational epigenetic adaptation of the hepatic wound-healing response.

We investigated whether ancestral liver damage leads to heritable reprogramming of hepatic wound healing in male rats. We found that a history of liver damage corresponds with transmission of an epigenetic suppressive adaptation of the fibrogenic component of wound healing to the male F1 and F2 generations. Underlying this adaptation was less generation of liver myofibroblasts, higher hepatic expression of the antifibrogenic factor peroxisome proliferator-activated receptor γ (PPAR-γ) and lower expression of the profibrogenic factor transforming growth factor ß1 (TGF-ß1) compared to rats without this adaptation. Remodeling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for the histone variant H2A.Z and trimethylation of histone H3 at Lys27 (H3K27me3) at PPAR-γ chromatin. These modifications to the sperm chromatin were transmittable by adaptive serum transfer from fibrotic rats to naive rats and similar modifications were induced in mesenchymal stem cells exposed to conditioned media from cultured rat or human myofibroblasts. Thus, it is probable that a myofibroblast-secreted soluble factor stimulates heritable epigenetic signatures in sperm so that the resulting offspring better adapt to future fibrogenic hepatic insults. Adding possible relevance to humans, we found that people with mild liver fibrosis have hypomethylation of the PPARG promoter compared to others with severe fibrosis.