Gene expression analysis of the microvascular compartment in multiple sclerosis using laser microdissected blood vessels

Abstract

The blood brain barrier (BBB) is formed by capillary endothelial cells with inter-endothelial cell tight junctions and other cells such as pericytes and astrocytes present. Previous studies have shown a role for tight junction abnormalities in BBB leakage in multiple sclerosis (MS) brain. This marks a key stage in the development of inflammatory demyelination in MS. The aim of this study was to identify aberrantly expressed genes involved in BBB changes in MS lesions. A focused endothelial cell biology microarray, capable of detecting changes in expression of 113 endothelial cell-specific genes, was employed to analyse endothelial cell mRNA extracted from post-mortem control white matter, MS normal appearing white matter (NAWM), chronic active or inactive lesions by laser capture microdissection. Microarray analysis found 52 genes out of 113 analysed, predominantly in the activation functional group, to be differentially expressed in lesions compared to control or NAWM (p < 0.01). The majority of the differentially expressed genes were validated by quantitative real time PCR. In addition, the protein expression profiles of ICAM2, MMP2, and VEGFR1 were examined by immunofluorescent staining of selected tissue blocks. ICAM-2 was expressed at a higher level in chronic inactive lesions than control or NAWM, corresponding with the increased mRNA measured by microarray and real time PCR. The data shown, presenting a number of differentially expressed genes in the microvascular compartment of MS lesions, may shed light on the molecular mechanisms that are involved in the breakdown of the BBB. This moves us a step closer to the identification of potential therapeutic targets for repair of the compromised BBB.

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role

“…An increasing number of studies in a range of neurological diseases have used this approach to identify disease-relevant, cell-specific gene expression changes (Boone et al, 2013, Chu et al, 2009, Kumar et al, 2013, Pietersen et al, 2009, Simpson et al, 2011, Baker et al, 2015. To date, only two studies have employed LCM in the investigation of human MS cases: one identifying gene expression changes in total extracts from NAWM, peri-lesional and lesional regions compared to control WM (Mycko et al, 2012) and the other identifying transcriptomic changes in the vascular compartment in MS (Cunnea et al, 2010); with neither study specifically assessing changes in the astrocyte transcriptome.…”

“…To date, only two studies have combined LCM with microarray analysis in MS research, including analysis of the gene expression profile of blood vessels (Cunnea et al, 2010) and LCM-ed areas of MS lesions and NAWM (Mycko et al, 2012).…”

Roles of the Cyclooxygenase 2 Matrix Metalloproteinase 1 Pathway in Brain Metastasis of Breast Cancer

“…Despite promising hope in metalloproteinases as therapeutic targets, use of general inhibitors of metalloproteinases in clinical trials did not yield effective outcome (16), indicating that more detail and systematic analysis * This work was supported, in whole or in part, by National Institutes of Health of the role of metalloproteinases is necessary. Interestingly, brain ischemia is reported to stimulate the permeability of the BBB by promoting disruption of tight junction complex via activation of MMP2 and -9 (17)(18)(19)(20). In other studies, MMP3, -8, and -9 were shown to be able to cleave Occludin, a key protein controlling the tight junction (21)(22)(23).…”

Angiogenesis in multiple sclerosis and experimental autoimmune encephalomyelitis

“…Endothelial α5β1 integrin, involved in endothelial proliferation in hypoxic conditions [87], is transiently upregulated in EAE [40]. Gene expression analysis of the lasercaptured microvascular compartment of active lesions from MS autopsy samples has shown an increased expression of matrix metalloprotease-14 (MMP-14), MMP-2, ADAM17, VEGF-A, and VEGF-R1 [88]. Other inflammatory mediators such as TNF-α, IL-8, transforming growth factor-β (TGF-β), and MMP-9 released by immune cells induce angiogenesis [51] and, in turn, new vessel walls are easily permeable to immune cell transmigration and foster adhesion and cytokine molecules expression [89].…”