Mario Zierden

Effects and Regulation of Autoreactive CD8+ T Cells in a Transgenic Mouse Model of Autoimmune Hepatitis

BACKGROUND & AIMS Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease of unknown etiology. Autoreactive T cells are thought to mediate liver injury, but the pathogenesis of AIH is poorly understood because of the lack of suitable animal models. We established a mouse model to investigate liver-specific T-cell responses and assess the effects and regulation of autoreactive CD8(+) T cells in the pathogenesis of AIH. METHODS We generated transgenic mice expressing the influenza virus hemagglutinin (HA) autoantigen under control of mouse albumin regulatory elements and alpha-fetoprotein enhancers (Alb) specifically in the liver (Alb-HA mice); they were crossed with mice that express a specific T-cell receptor (TCR) (CL4-TCR). CL4-TCR transgenic CD8(+) T cells were also adoptively transferred into Alb-HA mice. We investigated immunologic mechanisms of CD8(+) T cell-induced liver damage and of counteracting peripheral tolerance. RESULTS The double-transgenic mice (Alb-HA/CL4-TCR) spontaneously developed chronic, autoimmune-mediated hepatitis, characterized by necroinflammatory lesions, hepatic fibrosis, and increased levels of aminotransferase; these features resembled those of AIH. Interestingly, most liver-infiltrating, HA-specific CD8(+) T cells had an anergic phenotype; regulatory CD4(+)CD25(+)Foxp3(+) T cells accumulated in the inflamed liver. CONCLUSIONS The continuous development of a few, HA-specific CD8(+) effector T cells is sufficient to induce chronic hepatitis. Peripheral tolerance mechanisms such as induction of T-cell anergy and accumulation of regulatory CD4(+)CD25(+)Foxp3(+) T cells protect the liver from severe damage. Our mouse model that spontaneously develops chronic autoimmune-mediated hepatitis provides a new tool to investigate autoantigen-specific T-cell responses and regulatory mechanisms involved in the prevention and pathogenesis of AIH.

Genetic Ablation of PDGF-Dependent Signaling Pathways Abolishes Vascular Remodeling and Experimental Pulmonary Hypertension

Objective— Despite modern therapies, pulmonary arterial hypertension (PAH) harbors a high mortality. Vascular remodeling is a hallmark of the disease. Recent clinical studies revealed that antiremodeling approaches with tyrosine–kinase inhibitors such as imatinib are effective, but its applicability is limited by significant side effects. Although imatinib has multiple targets, expression analyses support a role for platelet-derived growth factor (PDGF) in the pathobiology of the disease. However, its precise role and downstream signaling events have not been established. Approach and Results— Patients with PAH exhibit enhanced expression and phosphorylation of &bgr; PDGF receptor (&bgr;PDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLC&ggr; at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of &bgr;PDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated &bgr;PDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLC&ggr; (&bgr;PDGFRF3/F3). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in &bgr;PDGFRF3/F3 pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in &bgr;PDGFRF3/F3 mice compared with wild-type littermates. These alterations led to protection from hypoxia-induced PAH and right ventricular hypertrophy. Conclusions— By means of a genetic approach, our data provide definite evidence that the activated &bgr;PDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLC&ggr; activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.

Class IA Phosphatidylinositol 3-Kinase Isoform p110α Mediates Vascular Remodeling

Objective—Neointima formation after vascular injury remains a significant problem in clinical cardiology, and current preventive strategies are suboptimal. Phosphatidylinositol 3′-kinase is a central downstream mediator of growth factor signaling, but the role of phosphatidylinositol 3′-kinase isoforms in vascular remodeling remains elusive. We sought to systematically characterize the precise role of catalytic class IA phosphatidylinositol 3′-kinase isoforms (p110&agr;, p110&bgr;, p110&dgr;), which signal downstream of receptor tyrosine kinases, for vascular remodeling in vivo. Approach and Results—Western blot analyses revealed that all 3 isoforms are abundantly expressed in smooth muscle cells. To analyze their significance for receptor tyrosine kinases–dependent cellular responses, we used targeted gene knockdown and isoform-specific small molecule inhibitors of p110&agr; (PIK-75), p110&bgr; (TGX-221), and p110&dgr; (IC-87114), respectively. We identified p110&agr; to be crucial for receptor tyrosine kinases signaling, thus affecting proliferation, migration, and survival of rat, murine, and human smooth muscle cells, whereas p110&bgr; and p110&dgr; activities were dispensable. Surprisingly, p110&dgr; exerted noncatalytic functions in smooth muscle cell proliferation, but had no effect on migration. Based on these results, we generated a mouse model of smooth muscle cell–specific p110&agr; deficiency (sm-p110&agr;−/−). Targeted deletion of p110&agr; in sm-p110&agr;−/− mice blunted growth factor–induced cellular responses and abolished neointima formation after balloon injury of the carotid artery in mice. In contrast, p110&dgr; deficiency did not affect vascular remodeling in vivo. Conclusions—Receptor tyrosine kinases–induced phosphatidylinositol 3′-kinase signaling via the p110&agr; isoform plays a central role for vascular remodeling in vivo. Thus, p110&agr; represents a selective target for the prevention of neointima formation after vascular injury, whereas p110&bgr; and p110&dgr; expression and activity do not play a significant role.