Biochemical Mechanisms of Vasospasms

描述（改编自申请人摘要）：长期目标 本申请旨在发现引起脑血管痉挛的分子 蛛网膜下腔出血（SAH）。血管痉挛是一种常见的原因， SAH患者中的迟发性缺血性卒中有人提出，氧化的 SAH后胆红素产生抑制蛋白磷酸酶的化合物， 这种抑制作用会导致血管收缩和血管增生 在血管痉挛患者中可见。我们已经确定了候选分子， 是胆红素的过氧化片段， 对颈动脉血管平滑肌细胞增殖的影响。 此外，这些分子在体外对血管产生代谢作用， 与血管痉挛患者脑脊液产生的相同。最后， 过氧化胆红素分子存在于患者的CSF中， 血管痉挛本申请中的前两个目标将决定 在血管痉挛患者的CSF中发现过氧化形式的胆红素; 哪些片段与临床血管痉挛的存在相关;以及 胆红素的过氧化形式引起血管收缩和血管收缩。 体外增殖。这些研究将采用生物化学净化 分离胆红素氧化形式的程序和鉴别方法 他们的结构。对化合物的鉴定可能使人们有可能 做一个血管痉挛测试使用体外颈动脉环测定法来 评估耗氧量、等长收缩力、高能磷酸盐， 有和无血管痉挛患者CSF磷酸酶活性。文化 将使用平滑肌细胞的量来确定来自患有 血管痉挛和过氧化胆红素化合物刺激增殖， 与对照溶液相比，体外细胞中的这种增加是否 细胞增殖与蛋白磷酸酶的抑制有关。第三 Aim将测试血管收缩是否可能是由于抑制 过氧化胆红素片段对平滑肌磷酸酶的影响，如果是这样， 这发生在哪个亚细胞区室，以及哪些磷酸酶是 压抑最后一项目标将确定是否有来自血管痉挛患者的CSF 和纯化的氧化胆红素分子（当注入蛛网膜下腔时 啮齿动物的空间）在该体内模型中引起血管痉挛和脑损伤。 该模型将用于在未来的研究中筛选可能的治疗方法。的 该项目的最终长期目标是确定 血管痉挛，以制定有效的诊断，治疗和 这种脑血管疾病的预防方法。

DESCRIPTION (Adapted from applicant's abstract): The long-term objective of this application is to discover the molecule(s) that cause cerebral vasospasm following subarachnoid hemorrhage (SAH). Vasospasm is a frequent cause of delayed ischemic stroke in SAH patients. It is proposed that oxidation of bilirubin following SAH produces compounds that inhibit protein phosphatases, and that this inhibition causes the vasoconstriction and vascular proliferation seen in patients with vasospasm. We have identified candidate molecules that are peroxidized fragments of bilirubin that appear to produce vasoconstriction of carotid vessels and proliferation of vascular smooth muscle cells in vitro. In addition, these molecules produce metabolic effects on vessels in vitro that are identical to those produced by CSF from patients with vasospasm. Lastly, the peroxidized bilirubin molecules are present in the CSF of patients with vasospasm. The first two aims in this application will determine which peroxidized forms of bilirubin are found in the CSF of patients with vasospasm; which fragments correlate with the presence of clinical vasospasm; and which peroxidized forms of bilirubin cause vascular constriction and vascular proliferation in vitro. These studies will employ biochemical purification procedures to isolate the oxidized forms of bilirubin and methods to identify their structures. Identification of the compounds may make it possible to develop a test for vasospasm. An in vitro carotid artery ring assay is used to assess the oxygen consumption, isometric forces, high-energy phosphates, and phosphatase activity of CSF from patients with and without vasospasm. Cultures of smooth muscle cells will be used to determine whether CSF from patients with vasospasm and the peroxidized bilirubin compounds stimulate proliferation of the cells in vitro compared to control solutions, and whether this increase in cell proliferation is related to inhibition of protein phosphatases. The third Aim will test whether the vascular constriction might be due to inhibition of smooth muscle phosphatases by the peroxidized bilirubin fragments, and if so which subcellular compartment this occurs in, and which phosphatases are inhibited. The last Aim will determine whether CSF from patients with vasospasm and purified oxidized bilirubin molecules (when injected into the subarachnoid space of rodents) causes vasospasm and cerebral injury in this in vivo model. This model will be used to screen for possible therapies in future studies. The ultimate long-term goal for this project is to define the molecular causes of vasospasm in order to develop effective diagnostic, therapeutic and preventative approaches for this cerebral vascular disease.