Stroke, the 5th leading cause of death in the United States, is responsible for approximately 129,000 deaths per year (AHA Statistics). It is well recognized that cerebrovascular homeostasis is vital for the brain's functional and structural integrity, and alteration of endothelial cell functions has been inferred in the pathogenesis of stroke, even when the cause was unknown. Recently we discovered that sphingolipid de novo biosynthesis plays a very important role in preserving endothelial cell functions, to impact hypertension, inflammation and heart failure. However, direct evidence of a causative role of endothelial dysfunction in the pathogenesis cerebrovascular diseases, especially ischemic stroke, is lacking. In this regard, we generated a mouse that lacks SPT specifically in the endothelium (ECKO-Sptlc2), and report that this mouse develops spontaneous ischemic stroke. Based on our findings, we hypothesize that blocking the de novo sphingolipid biosynthesis by genetically excision of the SPT enzyme specifically in the endothelium, will lead to endothelial dysfunction with the onset of a pro-inflammatory and pro-thrombotic state, sufficient to trigger the ischemic stroke. To test this hypothesis we propose two aims: 1) characterize ECKO-Sptlc2 mice as a novel model of ischemic stroke triggered by the altered sphingolipid pathway in the vascular endothelium. In this aim we will characterize the extent and distribution of the ischemic lesions; measure the cerebral blood flow (CBF); assess the endothelial-dependent vasodilation by measuring local changes in CBF via a laser-Doppler probe (Perimed); and map the location of thrombi in the ischemic brains of ECKO-Sptlc2 mice.
Aim 2) to define the dysfunctional state of EC lacking Sptlc2 and the changes in sphingolipid levels contributing to this condition. We will measure sphingolipid levels in ECKO-Sptlc2 and Sptlc2f/f cerebral tissue and primary brain EC in culture; assess in vivo blood brain barrier (BBB) permeability in ECKO-Sptlc2 and control mice; measure murine brain endothelial barrier function in basal and following pro-permeability stimuli. Circulating inflammatory and thrombotic markers, will be measured in ECKO-Sptlc2 and control mice by using Luminex. Histological analysis of coronal brain and heart sections for endothelial inflammatory (e.g., ICAM-1, VCAM) and pro-thrombotic markers (e.g., VWF, PAI-1) will be performed; FACS analysis for inflammatory and pro- thrombotic activation markers in ECKO-Sptlc2 and control brain and heart endothelial cell. The major goal of the study is to systematically characterize this novel animal model of endothelial dysfunction- triggered ischemic stroke, in order to build future preclinical and translational studies on a solid foundation.
The overall goal of this proposal is to characterize a novel mouse model of ischemic stroke triggered by endothelial dysfunction following the decrease of sphingolipid levels, specifically in the endothelial cells. This proposal explores a novel pathway in regulating vascular functions to impact cerebrovascular diseases, including ischemic stroke, a growing public health issue for which the current pharmacological therapies are inadequate to control the problem. These proposed studies will contribute to understand the role of locally produced sphingolipids on endothelial cell functions and its impact on stroke, and may lead to new pharmacological approaches for the treatment of cerebrovascular diseases. !