"Liquid Biopsy" of White Matter Hyperintensity in Functionally Normal Elders

Abstract

Background and Objective: In the aging brain, increased blood-brain barrier (BBB) leakage and white matter hyperintensity (WMH) on MRI are frequently presumed secondary to cerebral small vessel disease (cSVD) or endotheliopathy. We investigate this association in vivo by quantifying protein cargo from endothelial-derived exosomes (EDE), and comparing levels between two groups of functionally normal elders with and without WMH. In addition, we study associations of EDE proteins with upstream and downstream factors, such as inflammation and neurodegenerative changes, respectively. Methods: Twenty six neurologically normal older adults completed general health questionnaires, neuropsychological and physical examinations, and brain MRI. WMH was visually graded with modified Fazekas score of 2 or greater used to classify 11 subjects as cases, and 15 without WMH as controls. Plasma total exosomes were precipitated and EDEs enriched by sequential immuno-precipitations. In addition, we quantified three inflammatory cytokines from plasma and imaging variables on MRI. Group means were compared, the discriminant functions of biomarkers calculated, and the association of EDE biomarkers with plasma inflammatory markers, cognition, and imaging outcomes assessed via regression modeling. Results: Plasma levels of EDE cargo proteins GLUT1, LAT1, P-GP, and NOSTRIN were significantly higher in subjects with WMH in comparison to those without. In contrast, EDE levels of the marker with low expression in brain (VCAM1) were equal between groups. The effect sizes for each of the brain-expressed cargo proteins (GLUT1, LAT1, and P-GP) were such that age-adjusted logistic regressions revealed areas under the curve (AUC) with range of 0.82-0.89, differentiating subjects with WMH from those without. VCAM1 poorly discriminated between groups (AUC:0.55). Higher levels of all brain-expressed EDE proteins were also associated with lower cognitive function, unrelated to burden of WMH. Levels of LAT1 and P-GP were significantly inversely associated with global gray matter volumes, and EDE GLUT1, LAT-1, and P-GP concentrations were significantly associated with systemic IL-6 levels. Conclusion: In a case control study of clinically normal adults with and without WMH, concentrations of EDE proteins were significantly higher in subjects with WMH in comparison to controls. This work is a first step toward in vivo dissection of molecular changes in endothelia of functionally normal subjects with radiographic evidence of age-associated white matter disease.

Keywords: biomarkers; cerebral small vessel disease; exosomes; extracellular vesicles; inflammation; white matter.

FIGURE 1

Accuracy of EDE cargo proteins in preclinical cSVD.

FIGURE 2

Regression models with global gray matter volume. Associations of EDE cargo proteins with total gray matter volumes. This figure shows leverage residuals plots of global gray matter volumes regressed on normalized EDE cargo biomarkers. Units for gray matter volumes entered into the model are mm³. Age in years and total intracranial volumes in mm³ were controlled for in all regressions. R² values are adjusted for number of predictors in regression model. All values are rounded to two significant digits. GM, gray matter; blue stars, controls; purple dashes, cases.

FIGURE 3

Regression models with processing speed. Leverage residuals plots of normalized levels of EDE cargo biomarkers associated with cognition (processing speed) where units are in seconds. Distributions were normalized via log transformation. All regressions were adjusted for age. R² values are adjusted for number of predictors in regression model. All values are rounded to two significant digits. Blue stars: controls; purple dashes: cases.

FIGURE 4

Levels of CD81, the exosomal marker used to normalize EDE cargo are not significantly different between groups (p = 0.69).