The Role of Fibrocytes in Liver Fibrosis

描述(申请人提供)：慢性肝损伤导致肝纤维化，其特征是广泛的细胞外基质蛋白(ECM)沉积，转化生长因子-β1和循环内毒素(脂多糖)水平增加，以及产生胶原的肌成纤维细胞的激活。肝纤维化的发展伴随着骨髓来源的纤维细胞的募集，这些细胞是CD45和胶原A1(I)表达的细胞，以前参与了肺、皮肤和肾脏的纤维化的发病机制。我们已经证明，纤维细胞通过在纤维化的肝脏中组成4-6%的胶原产生细胞并分化为致纤维化的肌成纤维细胞来促进肝纤维化。尽管数量很少，但我们的初步数据表明，天然抑制剂血清淀粉样蛋白P(SAP)抑制纤维细胞功能(S)，导致纤维细胞流向纤维化肝脏的阻断，并显著减轻肝纤维化。目前的研究方案利用四氯化碳(CCl4)和胆管结扎(BDL)两种肝损伤模型，研究SAP在肝纤维化过程中对肝纤维化、纤维细胞募集和分化为肌成纤维细胞的影响，以及炎症信号的调节。我们预测纤维细胞在肝纤维化的发病机制中起重要作用。因此，纤维细胞可能是一个有吸引力的抗纤维化靶点。 心理治疗。为了支持我们的假设，我们开发了一种补充方法来研究纤维细胞在肝纤维化中的作用。利用Cre-loxP重组技术，我们获得了无纤维细胞的转基因小鼠(AIM-2)。体内纤维细胞的消融是通过在胶原A1(I)+CD45+纤维细胞中过表达白喉毒素-a(DTA)或受体(DTR)来实现的。纤维细胞的缺失还没有报道，这将为了解纤维细胞在肝纤维化发病机制中的作用提供宝贵的见解。我们认为，纤维细胞可能成为抗纤维化治疗的新靶点。此外，用于肺和皮肤纤维化的临床治疗的纤维细胞抑制剂SAP可能为肝纤维化患者提供一种潜在的新疗法。在这里，我们确定了骨髓来源的纤维细胞(造血细胞或间充质细胞)的来源，并确定了它们与普通髓系祖细胞(CMP)和单核细胞(AIM 3)的关系。我们预测，纤维细胞起源于c-kit+SCA-1+造血干细胞，但在造血过程中发育先于CMP。与之一致的是，纤维细胞具有高度的可塑性，在对肝损伤的反应中，不仅迁移到纤维化的肝脏，还迁移到脾，在那里它们介导了抗菌反应。目前尚不清楚胶原蛋白的表达如何影响纤维细胞的功能。我们通过产生胶原突变的纤维细胞来解决这个问题，这些细胞下调胶原A1(I)的产生(5‘SL)，或者表达更稳定的不可切割的Col1a1rr蛋白(AIM4)。对突变纤维细胞的体内功能进行了研究，并与野生型纤维细胞进行了比较。我们预计突变的胶原缺失纤维细胞在迁移、分化为肌成纤维细胞方面存在缺陷，而Col1a1rr突变的纤维细胞有望具有高度纤维化的表型。

DESCRIPTION (provided by applicant): Chronic liver injury results in hepatic fibrosis, which is characterized by extensive deposition of extracellular matrix proteins (ECM), increased levels of TGF-¿1 and circulating endotoxin (lipopolysaccharide, LPS), and activation of collagen producing myofibroblasts. Development of liver fibrosis is accompanied by recruitment of bone marrow (BM)-derived fibrocytes, that are CD45 and collagen-a1(I) expressing cells, previously implicated in the pathogenesis of fibrosis in lungs, skin, and kidneys. We have demonstrated that fibrocytes contribute to liver fibrosis by comprising 4-6% of collagen producing cells in the fibrotic liver and differentiating into fibrogenic myofibroblasts. Despite of low numbers, our preliminary data suggest that inhibition of fibrocyte function(s) by the natural inhibitor Serum Amyloid P (SAP), results in the blockage of fibrocyte flux into fibrotic liver, and significantly attenuates liver fibrosis. Current proposal investigates the effect of SAP on liver fibrosis, fibrocyte recruitment and differentiation into myofibroblasts, and mediation of inflammatory signals in the course of liver fibrosis using two models of liver injury, carbontetrachloride (CCl4 and bile duct ligation (BDL). We anticipate that fibrocytes play an important role in pathogenesis of liver fibrosis (AIM 1). Therefore, fibrocytes may present an attractive target for anti-fibrotic therapy. To support our hypothesis, we developed a complimentary approach to study the role of fibrocytes in liver fibrosis. Using Cre-loxP recombination technique, we generate transgenic mice devoid of fibrocytes (AIM 2). Ablation of fibrocytes in vivo is achieved by overexpression of Diphtheria toxin-a (DTA) or receptor (DTR) in Collagen-a1(I)+CD45+ fibrocytes. Deletion of fibrocytes has not been reported, and will provide the invaluable insight into the role of fibrocytes in pathogenesis of liver fibrosis. We believe that fibrocytes may become a novel target for anti-fibrotic therapy. Futhermore, fibrocyte inhibitor SAP, which is used in clinical trals for lung and skin fibrosis, may present a potentially new treatment for patients with liver fibrosi. Here we determine the origin of BM-derived fibrocytes (hematopoietic or mesenchymal), and define their relationship to common myeloid progenitors (CMP) and monocytes (AIM 3). We predict that fibrocytes originate from c-kit+Sca-1+ hematopoietic stem cells, but developmentally precede CMP during hematopoiesis. In concordance, fibrocytes possess high plasticity, and in response to liver injury migrate to fibrotic liver, but also to spleen where they mediate antimicrobial response. It remains unclear how expression of collagen affects functions of fibrocytes. We address this question by generation of collagen-mutant fibrocytes, which downregulate collagen-a1(I) production (5'SL), or express a more stable uncleavable Col1a1rr protein (AIM 4). The functions of mutant fibrocytes are studied in vivo and compared to the wild type fibrocytes. We expect that mutant collagen-deficient fibrocytes exhibit a defect in transmigration, differentiation into myofibroblasts, while Col1a1rr-mutant fibrocytes are expected to possess a highly fibrogenic phenotype.