Chronic cerebral hypoperfusion alters the CypA-EMMPRIN-gelatinase pathway: Implications for vascular dementia

Abstract

Chronic cerebral hypoperfusion (CCH) is postulated to underlie multiple pathophysiological processes in vascular dementia (VaD), including extracellular matrix dysfunction. While several extracellular matrix proteins, namely cyclophilin A (CypA), extracellular matrix metalloproteinase inducer (EMMPRIN) and gelatinases (matrix metalloproteinases, MMP-2 and -9) have been investigated in acute stroke, their involvement in CCH and VaD remains unclear. In this study, CypA-EMMPRIN-gelatinase proteins were analysed in a clinical cohort of 36 aged, cognitively unimpaired subjects and 48 VaD patients, as well as in a bilateral carotid artery stenosis mouse model of CCH. Lower CypA and higher EMMPRIN levels were found in both VaD serum and CCH mouse brain. Furthermore, gelatinases were differentially altered in CCH mice and VaD patients, with significant MMP-2 increase in CCH brain and serum, whilst serum MMP-9 was elevated in VaD but reduced in CCH, suggesting complex CypA-EMMPRIN-gelatinase regulatory mechanisms. Interestingly, subjects with cortical infarcts had higher serum MMP-2, while white matter hyperintensities, cortical infarcts and lacunes were associated with higher serum MMP-9. Taken together, our data indicate that perturbations of CypA-EMMPRIN signalling may be associated with gelatinase-mediated vascular sequelae, highlighting the potential utility of the CypA-EMMPRIN-gelatinase pathway as clinical biomarkers and therapeutic targets in VaD.

Keywords: Cerebrovascular disease; chronic cerebral hypoperfusion; cyclophilin A; gelatinase; vascular dementia.

Figure 1.

Serum concentrations of CypA and ECM proteins, and their respective ROC curves in detecting VaD in a clinical study cohort. Bar graphs of (a) CypA, (b) EMMPRIN, (c) MMP-2, (d) MMP-9 serum concentrations (mean ± SEM in ng/mL, with white dots indicating individual measurements) in a cohort of NCI (n = 36) and VaD (n = 48) subjects and (e) ROC analyses indicating the diagnostic performance of each serum biomarker as well as that of a composite panel of all four serum biomarkers in detecting VaD. AUC: area under curve; NCI: no cognitive impairment; ROC: receiver operating characteristic; VaD: vascular dementia. *p < 0.05 and ***p < 0.001 indicate significant differences between NCI and VaD groups (Mann-Whitney U tests). ^p < 0.05 indicates significantly better goodness-of-fit compared to individual markers (Likelihood ratio tests).

Figure 2.

Associations of serum gelatinases with cerebrovascular diseases in a clinical study cohort. Bar graphs of (a–c) MMP-2 and (d–f) MMP-9 serum concentrations (mean ± SEM in ng/mL, with white dots indicating individual measurements) with absence or presence of (a,d) confluent white matter hyperintensities (as measured by Fazekas score ≥2), (b,e) lacunes or (c,f) cortical infarcts in a combined cohort (n = 84). *p < 0.05 and **p < 0.01 indicate significant differences between groups (Mann-Whitney U tests).

Figure 3.

Effects of BCAS-induced chronic hypoperfusion on CBF and white matter integrity. (a) Representative CBF images with ROIs between bregma and lambda across the area of both hemispheres of the brain, (b) bar graphs showing mean CBF (±standard error of the mean, SEM) measured before (“Basal”) and immediately after the procedure (“Post-Surgery”) in both the sham-operated and BCAS C57BL/6J mice and (c) Representative images of LFB staining in sham-operated and BCAS mice 30 days after procedures demonstrated white matter rarefaction and formation of vacuoles (indicated by arrows) in the corpus callosum (paramedian and medial, regions “#1” and “#2”), caudoputamen (region “#3”), internal capsule (region “#4”) and optic tract (region “#5”) following BCAS. Coronal sections were imaged at 4x magnification, scale bar 500μm; zoomed in images were imaged at 60x magnification, scale bar 20μm. Images were taken under identical exposures and conditions. BCAS: bilateral carotid artery stenosis; CBF: cerebral blood flow; LFB: Luxol Fast Blue; ROI: region of interest. *p < 0.05 indicates significant difference compared to the basal value (Student’s T tests, n = 3 in each group).

Figure 4.

Effects of BCAS-induced chronic hypoperfusion on immunoreactivities of CypA and ECM proteins in mouse cortex and hippocampus. Representative immunoblots and bar graphs of immunoreactivities (in mean ± SEM arbitrary units normalized to β-actin, with white dots indicating individual measurements) of (a,b) CypA, (c,d) EMMPRIN, (e,f) MMP-2 and (g,h) MMP-9 in cortical and hippocampal tissues of sham-operated or BCAS mice 30 days after the procedures. Molecular weights (in kDa) are indicated on the right of each blot (all unedited blots can be found in Supplementary Figure S2). CypA: cyclophilin A; ECM: extracellular matrix; EMMPRIN: extracellular matrix metalloproteinase inducer; MMP: matrix metalloproteinase. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences between Sham-operated and BCAS groups (Student’s T tests, n = 4–6 in each group).