Identification of Clinically Relevant Brain Endothelial Cell Biomarkers in Plasma

Abstract

Background: Proteins expressed by brain endothelial cells (BECs), the primary cell type of the blood-brain barrier, may serve as sensitive plasma biomarkers for neurological and neurovascular conditions, including cerebral small vessel disease.

Methods: Using data from the BLSA (Baltimore Longitudinal Study of Aging; n=886; 2009-2020), BEC-enriched proteins were identified among 7268 plasma proteins (measured with SomaScanv4.1) using an automated annotation algorithm that filtered endothelial cell transcripts followed by cross-referencing with BEC-specific transcripts reported in single-cell RNA-sequencing studies. To identify BEC-enriched proteins in plasma most relevant to the maintenance of neurological and neurovascular health, we selected proteins significantly associated with 3T magnetic resonance imaging-defined white matter lesion volumes. We then examined how these candidate BEC biomarkers related to white matter lesion volumes, cerebral microhemorrhages, and lacunar infarcts in the ARIC study (Atherosclerosis Risk in Communities; US multisite; 1990-2017). Finally, we determined whether these candidate BEC biomarkers, when measured during midlife, were related to dementia risk over a 25-year follow-up period.

Results: Of the 28 proteins identified as BEC-enriched, 4 were significantly associated with white matter lesion volumes (CDH5 [cadherin 5], CD93 [cluster of differentiation 93], ICAM2 [intracellular adhesion molecule 2], GP1BB [glycoprotein 1b platelet subunit beta]), while another approached significance (RSPO3 [R-Spondin 3]). A composite score based on 3 of these BEC proteins accounted for 11% of variation in white matter lesion volumes in BLSA participants. We replicated the associations between the BEC composite score, CDH5, and RSPO3 with white matter lesion volumes in ARIC, and further demonstrated that the BEC composite score and RSPO3 were associated with the presence of ≥1 cerebral microhemorrhages. We also showed that the BEC composite score, CDH5, and RSPO3 were associated with 25-year dementia risk.

Conclusions: In addition to identifying BEC proteins in plasma that relate to cerebral small vessel disease and dementia risk, we developed a composite score of plasma BEC proteins that may be used to estimate blood-brain barrier integrity and risk for adverse neurovascular outcomes.

Keywords: blood-brain barrier; cerebral small vessel disease; dementia; endothelial cells; neuroimaging; proteomics; single-cell gene expression analysis.

Figure 1.

Overview of study design. Key: BEC; brain endothelial cell; MRI; magnetic resonance imaging.

Figure 2.

Discovery of brain endothelial cell (BEC) proteins in plasma associated with cerebral small vessel disease. A) Heatmap showing the identification of endothelial cell enriched transcripts using an automated annotation algorithm of Human Protein Atlas expression data across 76 cell types. B) Venn diagram displaying a subset of transcripts empirically defined as BEC-specific in one or more single cell and/or single nuclei RNA-seq studies. C) Volcano plot showing the associations of BEC proteins as well as a composite score of BEC proteins with white matter lesion volumes and ADRD biomarkers. Results derived from linear regression models adjusted for age, sex, race, education, APOEε4, eGFR-creatinine and an index of eight comorbid conditions (obesity, hypertension, diabetes, cancer, ischemic heart disease, chronic heart failure, chronic kidney disease, chronic obstructive pulmonary disease); white matter lesion analyses additionally adjusted for total white matter volumes. βs reflect adjusted differences in white matter lesion volumes per each log2 increase in protein level. Red indicates statistical significance (unadjusted p<.05); gray indicates marginal significance (unadjusted p=.051). Significant positive and negative associations with ADRD biomarkers are indicated by up and down arrows, respectively. Key: Aβ ratio; Amyloid Beta_42/40; BEC; brain endothelial cell; GFAP, glial fibrillary acidic protein; NfL, neurofilament light; pTau-181, phosphorylated Tau 181.

Figure 3.

Associations of candidate BEC biomarkers and a BEC protein composite score with white matter lesion volumes in BLSA participants. Scatterplots and lines of best fit showing predicted changes in white matter lesion volumes associated with A) CDH5 B) CD93 C) ICAM2 D) GP1BB and E) RSPO3. Results (β’s, p-values) display predicted white matter lesion volumes per each log2 increase in protein level derived from linear regression models adjusted for age, sex, race, education, APOEε4, eGFR-creatinine, total white matter volumes and an index of eight comorbid conditions. F) Stratified boxplot illustrating changes in white matter lesion volumes according to BEC protein composite score quartiles. Results (β’s, p-values) were derived from unadjusted linear regression models. Key: BEC; brain endothelial cell; RFU, relative florescence units.

Figure 4.

External validation of candidate BEC biomarkers and a BEC composite score using clinically- and pathologically-relevant outcome measures. A) Volcano plot showing the associations of BEC proteins and BEC composite score with matter lesion volumes, cerebral microhemorrhages and lacunar infarcts. Results derived from linear or logistic regression models adjusted for age, sex, race-center, education, APOEε4, eGFR-creatinine, total intracranial volume, and cardiovascular risk factors (BMI, diabetes, hypertension, and current smoking status). βs reflect adjusted differences in white matter lesion volumes or cerebral microhemorrhage/lacunar infarct odds per each log2 increase in protein level. Red indicates statistical significance (unadjusted p<.05); gray indicates marginal significance (unadjusted p=.052). B) Stratified boxplot illustrating changes in white matter lesion volumes according to BEC composite score quartiles. Results (β’s, p-values) were derived from unadjusted linear regression models. C) Forest plot showing 25-year dementia risk associated with candidate BEC biomarkers and BEC composite score. Hazard ratios reflect adjusted differences in dementia risk per each log2 increase in BEC protein and BEC composite score levels. Red indicates statistical significance (unadjusted p<.05). D) Kaplan-Meier curves (and a corresponding at-risk table) displaying cumulative risk of incident dementia over 25 years according to bottom and upper quartiles that reflect log2 increases in BEC composite score. Dementia risk results (HRs, β’s, p-values) were derived from Cox proportional hazards regression models adjusted for age, sex, race-center, education, APOEε4, eGFR-creatinine, and cardiovascular risk factors. Key: BEC; brain endothelial cell; HR, hazard ratios; RFU, relative florescence units; CSVD, cerebral small vessel disease.

Figure 5.

Summary of findings from the current study.