Etiology of microvascular changes in Gram-positive sepsis: mechanisms and therape

描述(申请人提供)：耐甲氧西林金黄色葡萄球菌(MRSA)感染已成为社区和医院的严重健康问题。在美国，每年约有2万人死于耐甲氧西林金黄色葡萄球菌，这一比例高于死于艾滋病的人数。本提案的目的是描述MRSA肺炎/脓毒症期间微血管高通透性的新的机制方面，并提供将降低发病率和死亡率的新的有效治疗方案。我们的一般假设是，刺激精氨酸加压素V1a受体(V1aR)将缓解MRSA脓毒症引起的血管高通透性并逆转低血压，而刺激精氨酸加压素V2受体(V2R)将增加血管高通透性，不影响低血压。我们建议阐明下游机制，重点是V1aR、V2R、活性氮(RNS)与强大的血管生成生长因子(如血管生成素-2(Ang-2)和血管内皮生长因子(VEGF)之间的相互作用。目的：在绵羊MRSA脓毒症模型中，建立严重血管高通透性的机制，重点研究V1aR和V2R对潜在通透性因子Ang-2释放和微血管高通透性的相反作用。假设：1)V1aR激活通过抑制Ang-2释放降低血管高通透性；2)V2R刺激通过促进Ang-2释放增加血管高通透性；3)选择性V1aR激动剂在预防心血管衰竭方面优于V1aR/V2R混合激动剂精氨酸加压素(AVP)。目的：在绵羊MRSA脓毒症模型上，确定新型选择性V1aR激动剂通过抑制过量一氧化氮的产生而阻止RNS的形成，从而减轻严重的血管渗漏。RNS如何导致血管渗漏的下游机制也将被确定。假说：1)RNS引起的血管高通透性是由Ang-2释放和VEGF表达介导的；3)RNS引起V1aR的低反应性：3)V1aR激动剂通过抑制过量的一氧化氮产生而减少RNS的形成。目的：在人类细胞培养研究中，探讨MRSA诱导的严重血管高通透性的分子机制，特别是V2R激活引起血管高通透性的下游机制，以及V1aR激动剂如何抑制MRSA败血症的血管高通透性。假设：1)V2R刺激通过MAPK依赖途径促进Ang-2的释放；2)V1aR激动剂抑制V2R，从而阻止Ang-2的释放；3)V1aR激动剂调节Ang-2与Tie-2受体的相互作用，很可能是通过抑制Tie-2的激活；4)VEGF通过其直接作用或通过V2R刺激促进Ang-2的释放；5)在MRSA脓毒症中，V2R的激活是由RNS介导的；6)V1aR被RNS氧化/硝化。为了实现这些目标，这项研究将利用一种新的绵羊MRSA败血症模型和先进的细胞培养技术。我们尤其乐观的是，拟议的研究将导致选择性V1aR和过氧亚硝酸盐调节剂激动剂的翻译临床研究。

DESCRIPTION (provided by applicant): Methicillin-resistant Staphylococcus aureus (MRSA) infection has become a serious health problem both in the community and in hospitals. ~20,000 people die annually in the U.S. because of MRSA, the rate is greater than those die from AIDS. The goal of the present proposal is to characterize novel mechanistic aspects of microvascular hyper-permeability during MRSA pneumonia/sepsis and offer novel and efficient therapeutic options that will reduce morbidity and mortality. Our general hypothesis is that stimulation of arginine vasopressin V1a receptors (V1aR) will mitigate MRSA sepsis-induced vascular hyper-permeability and reverse the hypotension, whereas the stimulation of the arginine vasopressin V2 receptors (V2R) will augment the vascular hyper- permeability and will not affect the hypotension. We propose to elucidate the downstream mechanisms, focusing on the interaction between V1aR, V2R, reactive nitrogen species (RNS), and potent angiogenic growth factors such as angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF). Aim 1: In an ovine MRSA sepsis model, establish mechanisms of severe vascular hyper-permeability, focusing on opposing effects of V1aR and V2R on release of potent permeability factor Ang-2 and microvascular hyper- permeability. Hypotheses: 1) V1aR activation reduces vascular hyper-permeability by inhibiting Ang-2 release; 2) V2R stimulation increases vascular hyper-permeability by promoting Ang-2 release; 3) a selective V1aR agonist is superior to a mixed V1aR/V2R agonist arginine vasopressin (AVP) for preventing cardiovascular collapse. Aim 2: In an ovine MRSA sepsis model, determine that novel and selective V1aR agonist will mitigate severe vascular leakage by preventing RNS formation through inhibiting excessive nitric oxide production. The down- stream mechanisms of how RNS cause vascular leakage will be also determined. Hypotheses: 1) The vascular hyper-permeability caused by RNS is mediated by Ang-2 release and VEGF expression; 3) RNS cause hypo- reactivity of V1aR: 3) V1aR agonist reduces RNS formation by inhibiting excessive nitric oxide production. Aim 3: In human cell culture studies, determine the molecular mechanisms of MRSA-induced severe vascular hyper-permeability with especial reference to downstream mechanisms of how V2R activation causes, and of how V1aR agonist inhibits, vascular hyper-permeability in MRSA sepsis. Hypotheses: 1) V2R stimulation pro- motes Ang-2 release in a MAPK-dependent pathway; 2) The V1aR agonist suppresses V2R and thus prevents Ang-2 release; 3) V1aR agonist modulates interaction of Ang-2 with Tie-2 receptors, most probably by sup- pressing the Tie-2 activation; 4) VEGF promotes Ang-2 release by its direct action or via V2R stimulation; 5) V2R activation in MRSA sepsis is mediated by RNS; 6) V1aR is oxidized/nitrated by RNS. To achieve the aims, the study will utilize a novel ovine model of MRSA sepsis and sophisticated cell culture techniques. We are particularly optimistic about the possibility that the proposed research will lead to translational clinical studies for agonists of the selective V1aR and peroxynitrite modulators.