Christopher J. Parsons

Molecular mechanisms of hepatic fibrogenesis

Liver fibrosis, a wound‐healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM) proteins, of which type I collagen predominates. This alters the structure of the liver leading to organ dysfunction. The activated hepatic stellate cell (HSC) is primarily responsible for excess collagen deposition during liver fibrosis. Two important aspects are involved in mediating the fibrogenic response: first the HSC becomes directly fibrogenic by synthesizing ECM proteins; second, the activated HSC proliferates, effectively amplifying the fibrogenic response. Although the precise mechanisms responsible for HSC activation remain elusive, substantial insight is being gained into the molecular mechanisms responsible for ECM production and cell proliferation in the HSC. The activated HSC becomes responsive to both proliferative (platelet‐derived growth factor) and fibrogenic (transforming growth factor‐β[TGF‐β]) cytokines. It is becoming clear that these cytokines activate both mitogen‐activated protein kinase (MAPK) signaling, involving p38, and focal adhesion kinase–phosphatidylinositol 3‐kinase–Akt–p70 S6 kinase (FAK‐PI3K‐Akt‐p70S6K) signaling cascades. Together, these regulate the proliferative response, activating cell cycle progression as well as collagen gene expression. In addition, signaling by both TGF‐β, mediated by Smad proteins, and p38 MAPK influence collagen gene expression. Smad and p38 MAPK signaling have been found to independently and additively regulate α1(I) collagen gene expression by transcriptional activation while p38 MAPK, but not Smad signaling, increases α1(I) collagen mRNA stability, leading to increased synthesis and deposition of type I collagen. It is anticipated that by understanding the molecular mechanisms responsible for HSC proliferation and excess ECM production new therapeutic targets will be identified for the treatment of liver fibrosis.

Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats.

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase‐1 (TIMP‐1), which blocks matrix metalloproteinase (MMP) activity. TIMP‐1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP‐1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl4), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti–TIMP‐1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl4‐treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle α‐actin (α‐SMA). Compared to controls, rats administered anti–TIMP‐1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti–TIMP‐1 resulted in a marked decrease in α‐SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP‐2. In conclusion, administration of a TIMP‐1 antibody attenuated CCl4‐induced liver fibrosis and decreased HSC activation and MMP‐2 activity. (HEPATOLOGY 2004.)

Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct–ligated rats

Recent evidence indicates that the renin–angiotensin system (RAS) plays a major role in liver fibrosis. Here, we investigate whether the circulatory RAS, which is frequently activated in patients with chronic liver disease, contributes to fibrosis progression. To test this hypothesis, we increased circulatory angiotensin II (Ang II) levels in rats undergoing biliary fibrosis. Saline or Ang II (25 ng/kg/h) were infused into bile duct–ligated rats for 2 weeks through a subcutaneous pump. Ang II infusion increased serum levels of Ang II and augmented bile duct ligation–induced liver injury, as assessed by elevated liver serum enzymes. Moreover, it increased the hepatic concentration of inflammatory proteins (tumor necrosis factor α and interleukin 1β) and the infiltration of CD43‐positive inflammatory cells. Ang II infusion also favored the development of vascular thrombosis and increased the procoagulant activity of tissue factor in the liver. Livers from bile duct–ligated rats infused with Ang II showed increased transforming growth factor β1 content, collagen deposition, accumulation of smooth muscle α‐actin–positive cells, and lipid peroxidation products. Moreover, Ang II infusion stimulated phosphorylation of c‐Jun and p42/44 mitogen‐activated protein kinase and increased proliferation of bile duct cells. In cultured rat hepatic stellate cells (HSCs), Ang II (10−8 mol/L) increased intracellular calcium and stimulated reactive oxygen species formation, cellular proliferation and secretion of proinflammatory cytokines. Moreover, Ang II stimulated the procoagulant activity of HSCs, a newly described biological function for these cells. In conclusion, increased systemic Ang II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. (HEPATOLOGY 2005;41:1046–1055.)

Inhibition of transforming growth factor‐β/Smad signaling improves regeneration of small‐for‐size rat liver grafts

Transforming growth factor‐β (TGF‐β) is a potent inhibitor of cell proliferation. This study investigated whether overexpression of Smad7, which blocks TGF‐β–induced activation of Smad2/3, could prevent the suppression of regeneration of small‐for‐size liver grafts. Rats were intravenously given adenoviruses (2 × 1010 pfu/rat) carrying the LacZ gene or the Smad7 gene (Ad‐Smad7) 3 days prior to liver harvesting. Half‐size livers were implanted into recipients of the same weight or twice the donor weight, and this resulted in half‐size or quarter‐size liver grafts. Cell proliferation, detected by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation, increased to 23% in half‐size grafts at 38 hours after implantation but was only 4% in quarter‐size grafts. Graft weight did not increase after 38 hours in full‐size and quarter‐size grafts but increased 28% in half‐size grafts. Ad‐Smad7 restored BrdU labeling to 32%, and the graft weight increased to 43% in quarter‐size grafts. Serum total bilirubin increased approximately 30‐fold after the implantation of quarter‐size grafts. Ad‐Smad7 blunted hyperbilirubinemia by 80%. The basal hepatic TGF‐β1 level was 7 ng/g of liver wet weight, and this increased to 30 ng/g at 1.5 hours after the transplantation of full‐size grafts but decreased rapidly afterwards. After the transplantation of quarter‐size grafts, however, TGF‐β1 progressively increased to 159 ng/g in 38 hours. Nuclear phosphorylated Smad2/3 was barely detectable, and p21Cip1 expression was negligible in full‐size grafts but increased markedly in quarter‐size grafts. Ad‐Smad7 blocked Smad2/3 activation and expression of p21Cip1. Together, these data show that TGF‐β is responsible, at least in part, for the defective liver regeneration in small‐for‐size grafts by activating the Smad signaling pathway. Liver Transpl 16:181–190, 2010.

Mutation of the 5′-Untranslated Region Stem-Loop Structure Inhibits α1(I) Collagen Expression in Vivo

Type I collagen is a heterotrimeric extracellular matrix protein consisting of two α1(I) chains and one α2(I) chain. During liver fibrosis, activated hepatic stellate cells (HSCs) are the major source of the type I collagen that accumulates in the damaged tissue. Expression of α1(I) and α2(I) collagen mRNA is increased 60-fold compared with quiescent stellate cells and is due predominantly to post-transcriptional message regulation. Specifically, a stem-loop structure in the 5′-untranslated region of α1(I) collagen mRNA may regulate mRNA expression in activated HSCs through its interaction with stem-loop binding proteins. The stem-loop may also be necessary for efficient production and folding of the type I collagen heterotrimer. To assess the role of the stem-loop in type I collagen expression in vivo, we generated a knock-in mouse harboring a mutation that abolished the stem-loop structure. Heterozygous and homozygous knock-in mice exhibited a normal phenotype. However, steady-state levels of α1(I) collagen mRNA decreased significantly in homozygous mutant MEFs as well as HSCs; intracellular and secreted type I collagen protein levels also decreased. Homozygous mutant mice developed less liver fibrosis. These results confirm an important role of the 5′ stem-loop in regulating type I collagen mRNA and protein expression and provide a mouse model for further study of collagen-associated diseases.

HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase

The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.