Role of C3a Receptors and C5a Receptors on Mesenchymal Stem Cells

描述（由申请人提供）：心肌梗死和随后的充血性心力衰竭是最具挑战性和最普遍的健康问题之一。干细胞移植有可能使心脏修复，间充质干细胞（MSC）是心脏再生的有前途的细胞类型之一，尽管在干细胞导致治愈性治疗之前仍存在许多挑战，目前更现实的目标是干细胞介导的改善血运重建而不是心肌细胞替代。一种可能的方法是在体外指导MSC以增加其归巢和植入患病心脏的倾向。我们假设过敏毒素C3 a和C5 a就是这样一种手段。我们的初步研究令人惊讶地表明，C3 a受体（C3 aR）在MSC中易位到细胞核，而不是在其他细胞类型中。有趣的是，其他G蛋白偶联受体的核转位与信号通路和转录激活的长期激活相关，这可能导致心脏保护，并且实际上用C3 a刺激的MSC上调几种营养因子和血管生成因子。目标1：为了确定C3 a和C5 a对MSC功能的影响，这与它们的心脏修复潜力有关：假设：C3 a和C5 a对MSC具有影响，这将提高它们作为心脏修复的可移植细胞的潜力。这是基于C3 a和C5 a都引起与MSC扩增、心脏保护生长因子的产生和向心肌细胞分化相关的信号传导途径（ERK和Akt）的持久激活的观察。MSC功能的重要方面（细胞迁移、营养因子的产生、增殖和分化）将在用C3 a或C5 a刺激的MSC中确定。这些功能将在体外和体内测定。目标二：研究C3 a刺激的MSC中C3 aR和相关信号分子的核转位，并确定核转位和延长的ERK 1/2和Akt激活对MSC功能的影响。目标2.1：假设：MSC中的C3 aR易位到细胞核，这与信号通路的延长激活和转录激活有关。将在C3 a刺激的人MSC的核提取物中测定C3 aR、磷酸化ERK 1/2、磷酸化Akt和β-抑制蛋白的核易位。将使用C5 a进行比较。将使用抑制C3 aR核转位的方法来确定核转位对细胞信号传导（Aim 2.1）和细胞功能（Aim 2.2）的作用。目的2.2：研究核转位和C3 a和C5 a介导的ERK 1/2和Akt激活对MSC功能的影响。假设：ERK 1/2和/或Akt通路的长期激活-以及在C3 aR的情况下，该受体的核转位-负责C3 a和C5 a的功能效应。将评估ERK 1/2和Akt的抑制以及C3 aR的核转位对特定C3 a或C5 a介导的功能的影响，以确定哪种信号传导途径介导哪种影响。 公共卫生相关性：心肌梗死和随后的充血性心力衰竭仍然是最具挑战性和普遍的健康问题。干细胞移植有可能使心脏修复，间充质干细胞（MSC）是心脏再生的有前途的细胞类型之一。一种可能的方法是在体外以增加其植入和归巢到患病心脏的潜力的方式指导MSC。我们假设趋化因子C3 a和C5 a就是这样一种手段。在这个提议中，C3 a和C5 a对MSC的功能作用将被调查。

DESCRIPTION (provided by applicant): Myocardial infarction and subsequent congestive heart failure are among the most challenging and prevalent health problems. Stem cell transplantation has the potential to allow cardiac repair, and mesenchymal stem cells (MSC) are among the promising cell types for cardiac regeneration, although many challenges remain, before stem cells will lead to curative therapy, and it is currently more realistic to aim for stem cell-mediated improved revascularization than for cardiomyocyte replacement. One possible approach is to instruct MSC in vitro in ways which will increase their propensity for homing and engraftment to the diseased heart. We hypothesize that the anaphylatoxins C3a and C5a are one such means. Our preliminary studies surprisingly showed that the C3a receptor (C3aR) is translocated to the nucleus in MSC, but not in other cell types. Interestingly, nuclear translocation of other G-protein-coupled receptors has been associated with prolonged activation of signaling pathways and transcriptional activation, which may result in cardioprotection, and indeed MSCs stimulated with C3a up-regulate several trophic and angiogenic factors. Aim 1: To determine the effect of C3a and C5a on MSC functions which are relevant to their cardiac repair potential: Hypothesis: C3a and C5a have effects on MSCs that will improve their potential as transplantable cells for cardiac repair. This is based on the observation that C3a and C5a both cause long-lasting activation of signaling pathways (ERK and Akt) that are associated with MSC expansion, production of cardioprotective growth factors and differentiation toward cardiomyocytes. Important facets of MSC function (cell migration, production of trophic factors, proliferation and differentiation) will be determined in MSCs stimulated with C3a or C5a. These functions will be determined both in vitro and in vivo. Aim 2: To characterize the nuclear translocation of the C3aR and associated signaling molecules in C3a-stimulated MSCs and to determine the effect of the nuclear translocation and the prolonged ERK1/2 and Akt activation on MSC function. Aim 2.1: Hypothesis: The C3aR in MSCs translocates to the nucleus, which is associated with prolonged activation of signaling pathways and transcriptional activation. Nuclear translocation of the C3aR, phospho-ERK1/2, phospho-Akt and beta-arrestin will be determined in nuclear extracts of C3a-stimulated human MSCs. C5a will be used for comparison. Means of inhibiting nuclear translocation of the C3aR will be used to determine the role of the nuclear translocation on cell signaling (Aim 2.1) and cell function (Aim 2.2). Aim 2.2: To determine the effect of the nuclear translocation and the prolonged C3a and C5a-mediated ERK1/2 and Akt activation on MSC function. Hypothesis: The prolonged activation of ERK1/2 and/or Akt pathways - and in the case of the C3aR, the nuclear translocation of this receptor - are responsible for the functional effects of C3a and C5a. The effect of inhibition of ERK1/2 and Akt and of nuclear translocation of the C3aR on specific C3a or C5a mediated functions will be assessed in order to determine which signaling pathway mediates which effect. PUBLIC HEALTH RELEVANCE: Myocardial infarction and subsequent congestive heart failure remain among the most challenging and prevalent health problems. Stem cell transplantation has the potential to allow cardiac repair, and mesenchymal stem cells (MSCs) are among the promising cell types for cardiac regeneration. One possible approach is to instruct MSCs in vitro in ways which will increase their potential for engraftment and homing to the diseased heart. We hypothesize that the chemotactic factors C3a and C5a are one such means. In this proposal the functional role of C3a and C5a on MSC will be investigated.