Benefits of Eliminating Cell Death Pathways in Health and Disease

说明(申请人提供)：消除细胞死亡途径在健康和疾病中的益处影响血管健康的潜在过程在很大程度上仍未确定，使得目前用于治疗症状的治疗干预措施缺乏对潜在的、与生理相关的疾病和再生途径的准确了解。我们建议通过消除非生命细胞死亡和相关的炎症过程来限制炎症，并协调造血和心血管组织的修复和再生。我们的发现是，小鼠caspase 8(Caspase8)缺乏的胚胎致死是由释放的RIP3坏死造成的，这为利用消除这些途径的好处开辟了道路。Casp8-/-RIP3-/-(DKO)小鼠发育为对炎性侮辱不敏感的可存活和有生育能力的成体 但保留了足够的免疫功能来控制病毒感染，促使试点努力利用这种环境来改善组织修复、同种异体植入和组织再生。Casp8和RIP3通路进化到对抗病毒编码的细胞死亡抑制因子，但被释放出来导致炎症性疾病，破坏组织修复、动态平衡和再生。这项建议使用了遗传和小分子方法来消除细胞死亡途径，所有这些都是由DKO小鼠建模的，以及我们创造的另一个小鼠品系。我们已经证明，即使总体伤口修复率看起来正常，dko小鼠仍能抵抗急性炎症、炎症性癌症和缺血再灌注损伤，这引发了一个非常重要的问题：“在缺乏外源性细胞死亡途径的情况下，如何在临床上利用S的生命”？DKO细胞的植入和分化效率高于WT细胞。值得注意的是，Casp8-/-RIP3-/-小鼠在异基因(MHC不匹配)骨髓移植中存活下来，这会导致WT C57BL/6小鼠发生急性移植物抗宿主病(GvHD)。令人惊讶的是，DKO成纤维细胞支持核重新编程，比WT细胞效率更高。我们建议通过基因操作消除细胞死亡的有害后果，使这一领域更接近于改善造血和心血管组织修复和再生的治疗干预策略。我们建议调查小鼠和小鼠细胞的遗传缺陷，并开发恒河猴和人成纤维细胞的基因敲除策略，以结合新描述的RIP3激酶小分子抑制剂和商业上可获得的caspase抑制剂，以改善组织修复、同种异体移植和核重新编程。我们的目标是：(1)优化同种异体植入，(2)通过消除外源性凋亡而增强核重新编程，而不会引发坏死。通过这些研究获得的知识将对当前的组织修复和移植方法以及未来的再生医学策略产生广泛的影响。

DESCRIPTION (provided by applicant): Benefits of Eliminating Cell Death Pathways in Health and Disease the underlying processes influencing vascular health remain largely undefined, leaving current therapeutic interventions to treat symptoms without a precise understanding the underlying, physiologically relevant disease and regenerative pathways. We propose to restrict inflammation and orchestrate repair and regeneration of hematopoietic and cardiovascular tissues by eliminating nonvital cell death and associated inflammatory processes. Our discovery that embryonic lethality of caspase 8 (Casp8)-deficiency in mice results from unleashed RIP3 necrosis opens the path to harnessing benefits of eliminating these pathways. Casp8-/- Rip3-/- (DKO) mice develop into viable and fertile adults that lack a susceptibility to inflammatory insult but retain sufficient immune function to control viral infection, prompting pilot efforts to harnes this setting to improve tissue repair, allogeneic engraftment and tissue regeneration. The Casp8 and RIP3 pathways evolved to counteract virus-encoded cell death suppressors, but become unleashed to cause inflammatory disease and undermine tissue repair, homeostasis and regeneration. This proposal employs genetic as well as small molecule approaches to eliminate cell death pathways, all modeled by DKO mice, as well as a additional mouse strains we have created. We have already shown that DKO mice resist acute inflammation, inflammatory cancer and ischemia reperfusion injury even though overall wound repair rates appear normal, prompting the very important question, "How can benefit(s) from life in the absence of extrinsic cell death pathways be harnessed in the clinic"? DKO cells engraft and differentiate with higher efficiencies than WT cells. Remarkably, Casp8-/-Rip3-/- mice survive allogeneic (MHC mismatched) bone marrow engraftment that causes acute graft versus host disease (GvHD) in WT C57BL/6 mice. Surprisingly, DKO fibroblasts support nuclear reprogramming with improved efficiency over WT cells. We propose to eliminate deleterious consequences of cell death through genetic manipulation, moving this area closer to a therapeutic intervention strategy to improve hematopoietic and cardiovascular tissue repair and regeneration. We propose to investigate genetic deficiency in mice and mouse cells, and develop knock-out strategies on rhesus macaque and human fibroblasts to combine with newly described small molecule inhibitors of RIP3 kinase and commercially available caspase inhibitors, to improve tissue repair, allogeneic transplantation and nuclear reprogramming. Our Aims are to: (1) optimize allogeneic engraftment, and (2) enhance nuclear reprogramming by eliminating extrinsic apoptosis without the disadvantage of triggering necrosis. The knowledge gained through these studies will have broad impact in current tissue repair and transplantation approaches, as well as future regenerative medicine strategies.