Genetic endothelial systems biology of sickle stroke risk

Abstract

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n ‫؍‬ 11) or not-at-risk (n ‫؍‬ 9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1␤/tumor necrosis factor␣. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation. IntroductionMany human diseases present in a clinically variable manner, yet the basis for the biologic phenomenon of phenotypic heterogeneity, the variation in presentation of any given disease, is generally unknown. We have used a specific example of this phenomenon to address our overarching hypothesis that genetic, inherited differences in endothelial biology can underlie the phenotypic heterogeneity of human vascular disease.Sickle cell anemia, caused by inherited homozygosity for the mutant sickle hemoglobin, is a disease characterized by anemia, vascular occlusions, and chronic organ damage. It has an exceedingly complex pathophysiology and incredibly diverse clinical complications. 1 Among these, there are 3 stroke syndromes: clinically silent strokes occurring in children resulting from multifocal small vessel disease; hemorrhagic strokes occurring in adults; and clinical ischemic stroke, the classical stroke syndrome of sickle cell anemia.Notably, approximately 10% of children with sickle cell anemia develop classic ischemic stroke, with peak age being approximately 5 years. 2,3 Risk factors include elevated white count, low blood hemoglobin, hypertension, and a prior neurologic event. [2][3][4][5] However, the primary risk factor is occlusive disease at the circle of Willis (CoW), 6,7 the encircling structure of medium to large vessels at the base of the brain. CoW disease is thought to be causal, as the strokes tend to be due to thrombosis occurring over the area of vessel wall abnormality, and the extent of stroke correlates with degree of CoW stenosis. 2,8 Stroke pathogenesis does not simply involve sickling in the vasa vasorum because vessels in the CoW do not have vasa vasorum. 9 Our hypothesis ...

Proinflammatory phenotype with imbalance of KLF2 and RelA: Risk of childhood stroke with sickle cell anemia

“…The etiology of stroke in children with SCA is incompletely understood, but is known to be due to obstruction of the large intracranial vessels [2]. Systems analysis of causative factors of childhood stroke using RNA microarray data from blood outgrowth endothelial cells (BOECs) indicated the importance of the inflammation signaling pathway as a risk factor [3]. The transcription factor NFjB appears vital to this signaling, because NFjB interacts with many downstream and upstream genes that contributed to the statistical significance of the inflammatory pathway.…”

Cell adhesion molecule 1: a novel risk factor for venous thrombosis

“…From each culture, RNA was isolated and prepared exactly as previously described, 29 and more than 15 g of fragmented RNA was available. Samples were applied to the Affymetrix U133A chip, and hybridization and raw expression analysis were performed by the University of Minnesota Biomedical Imaging and Processing Facility.…”

“…As previously described, 29 raw microarray data were analyzed and tested for significance of single-gene differences by the use of both the Welch t test and statistical analysis of microarray (SAM), a test that uses false discovery rate. The Welch test assumes a normal distribution of data; the SAM test was specifically developed for microarray data and small sample numbers, where normality is not known and multiple comparisons are taken into account.…”

“…The latter data have already been publicly posted. 29 Original microarray data for this study have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus and are accessible through GEO Series accession numbers GSE17078 and GSE9877 (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?accϭGSE17078 and http://www.ncbi.…”

Individualized identification of disturbed pathways in sickle cell disease

“…Genome-wide analyses have discovered several genes influencing the likelihood of developing SCD [4,6,7]. Since most biological processes arise from integrated activities among many genes, interpreting the consequences on a pathway level contributes to understanding how gene perturbations account for disease [8].…”