Matrix Metalloproteinases in Hepatic Ischemia and Reperfusion Injury

描述（由申请人提供）：肝缺血再灌注损伤（IRI）发生在所有移植肝脏、创伤、休克和肝脏血液供应暂时中断的择期手术中。IRI导致高达10%的早期移植失败，并可导致急性和慢性排斥反应的发生率显著升高。每年都有成千上万的病人在等待捐赠器官的过程中死亡。这种令人沮丧的情况，加上人群中肥胖的显著流行，导致在移植中使用亚理想脂肪变性肝脏的需求增加，因为它们对IRI的高度易感性，导致功能障碍风险增加。在之前的资助期内，我们的研究结果表明，特异性基质金属蛋白酶-9 （MMP-9）抑制可显著改善正常肝脏的IRI，并揭示了MMP-2和TIMP-1在肝损伤中的潜在关键作用。本提案(i)探讨了MMP-9抑制在边缘性脂肪变性肝IRI中的肝保护特性，（ii）分析了MMP-2和TIMP-1在正常肝和脂肪变性肝ir诱导损伤中的功能。我们期望所提出的工作将为关键MMPs/TIMPs的个体功能提供基本见解，从而导致新的治疗方法的发展，从而最大限度地减少其有害影响，同时最大化其在正常和脂肪变性肝IRI中的有益功能。建立正常和脂肪变性小鼠部分肝脏IRI模型，以及离体冷脂肪变性肝缺血后的大鼠异体移植模型，将用于解决以下目标：(1)解剖基质金属蛋白酶-9特异性抑制影响边缘脂肪变性肝脏IRI结果的机制。使用MMP-9-/-缺陷小鼠和针对MMP-9的特异性治疗操作，我们将分析特异性MMP-9抑制是否(i)保护边缘脂肪变性肝脏免受i /R损伤，（ii）破坏白细胞交通和趋化因子释放/激活，我们将（iii）确定导致脂肪变性肝脏IRI中MMP-9表达的细胞内信号机制；(2)分析基质金属蛋白酶-2活性影响正常肝和脂肪肝IRI结果的机制。我们将确定选择性MMP-2抑制是否(i)影响肝功能/保存，（ii）导致MMP-9表达改变，（iii）干扰白细胞迁移和细胞因子/趋化因子激活模式，此外，我们将（iv）确定MMP-2的来源及其在正常和脂肪变性肝IRI中的相对贡献；(3)剖析金属蛋白酶组织抑制剂-1影响正常肝脏和脂肪变性肝脏IRI的机制。我们将通过确定TIMP-1缺乏对(i)肝功能/保存、（ii）肝脏再生和凋亡、（iii） MMP激活以及（iii）白细胞募集和促炎网络的影响来评估TIMP-1在正常和脂肪变性肝IRI中的功能。此外，我们将评估Timp-1过表达作为部分肝脏IRI和脂肪变性OLT的治疗方法的功能。

DESCRIPTION (provided by applicant): Hepatic ischemia reperfusion injury (IRI) occurs in all transplanted livers, in trauma, shock, and in elective surgery where blood supply to the liver is temporary interrupted. IRI causes up to 10% of early transplant failures and can lead to significantly higher incidence of acute and chronic rejections. Thousands of patients die every year while waiting for a donor organ. This gloomy scenario, together with the significant prevalence of obesity in the population, has led to an increased need of using suboptimal steatotic livers in transplantation at elevated risks of dysfunction based on their high susceptibility to IRI. In the previous funding period, our results demonstrated that specific matrix metalloproteinase-9 (MMP-9) inhibition profoundly ameliorates IRI in normal livers, and unveiled potential key roles for MMP-2 and TIMP-1 in liver injury. This proposal (i) explores the hepatoprotective properties of MMP-9 inhibition in marginal steatotic liver IRI and (ii) dissects the functions of MMP-2 and TIMP-1 in IR-induced damage in both normal and steatotic livers. We expect that the proposed work will provide fundamental insights on the individual functions of key MMPs/TIMPs, leading to the development of novel therapeutic manipulations that can minimize their detrimental effects while maximizing their beneficial functions in normal and in steatotic liver IRI. Well-established models of partial liver IRI in normal and in steatotic mice, and of ex vivo cold steatotic liver ischemia followed by iso-transplantation in rats will be used to address the following aims: (1) To dissect mechanisms by which Matrix Metalloproteinase-9 specific inhibition affects the IRI outcome in marginal steatotic livers. Using MMP-9-/- deficient mice and specific therapeutic manipulations against MMP-9, we will dissect whether specific MMP-9 inhibition (i) protects marginal steatotic livers from the I/R insult, (ii) disrupts leukocyte traffic and chemokine release/activation, and we will (iii) identify intracellular signaling mechanisms leading to MMP-9 expression in steatotic hepatic IRI; (2) To analyze mechanisms by which Matrix Metalloproteinase-2 activity affects the IRI outcome in normal and in steatotic livers. We will determine whether selective MMP-2 inhibition (i) affects liver function/preservation, (ii) results in altered expression of MMP-9, (iii) interferes with patterns of leukocyte migration and of cytokine/chemokine activation and, furthermore, we will (iv) identify the sources of MMP- 2 and their relative contribution in normal and steatotic liver IRI; and (3) To dissect the mechanisms by which tissue inhibitor of metalloproteinases-1 affects IRI in normal and in steatotic livers. We will assess the function of TIMP-1 in normal and in steatotic liver IRI by determining the impact of TIMP-1 deficiency on (i) liver function/preservation, (ii) liver regeneration and apoptosis, (iii) MMP activation, and on (iii) leukocyte recruitment and pro-inflammatory networks. Moreover, we will assess the function of Timp-1 overexpression as a therapeutic approach in partial liver IRI and in steatotic OLT.