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.
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