Pericyte Microvesicles as Plasma Biomarkers Reflecting Brain Microvascular Signaling in Patients With Acute Ischemic Stroke

Abstract

Background: Blood-based biomarkers have the potential to reflect cerebrovascular signaling after microvascular injury; yet, the detection of cell-specific signaling has proven challenging. Microvesicles retain parental cell surface antigens allowing detection of cell-specific signaling encoded in their cargo. In ischemic stroke, the progression of pathology involves changes in microvascular signaling whereby brain pericytes, perivascular cells wrapping the microcapillaries, are one of the early responders to the ischemic insult. Intercepting the pericyte signaling response peripherally by isolating pericyte-derived microvesicles may provide not only diagnostic information on microvascular injury but also enable monitoring of important pathophysiological mechanisms.

Methods: Plasma samples were collected from patients with acute ischemic stroke (n=39) at 3 time points after stroke onset: 0 to 6 hours, 12 to 24 hours, and 2 to 6 days, and compared with controls (n=39). Pericyte-derived microvesicles were isolated based on cluster of differentiation 140b expression and quantified by flow cytometry. The protein content was evaluated using a proximity extension assay, and vascular signaling pathways were examined using molecular signature hallmarks and gene ontology.

Results: In this case-control study, patients with acute ischemic stroke showed significantly increased numbers of pericyte-derived microvesicles (median, stroke versus controls) at 12 to 24 hours (1554 versus 660 microvesicles/μL; P=0.0041) and 2 to 6 days after stroke (1346 versus 660 microvesicles/μL; P=0.0237). Their proteome revealed anti-inflammatory properties mediated via downregulation of Kirsten rat sarcoma virus and IL (interleukin)-6/JAK/STAT3 signaling at 0 to 6 hours, but proangiogenic as well as proinflammatory signals at 12 to 24 hours. Between 2 and 6 days, proteins were mainly associated with vascular remodeling as indicated by activation of Hedgehog signaling in addition to proangiogenic signals.

Conclusions: We demonstrate that the plasma of patients with acute ischemic stroke reflects (1) an early and time-dependent increase of pericyte-derived microvesicles and (2) changes in the protein cargo of microvesicles over time indicating cell signaling specifically related to inflammation and vascular remodeling.

Keywords: microvesicles; pericytes; secretome; signaling; stroke.

Figure 1.

Patients with stroke included in the study. CONSORT diagram showing the screening of eligible patients included in the study.

Figure 2.

Early increase of pericyte-derived microvesicles (MVs) after ischemic stroke. A, Scatter plots showing the quantification of pericyte-derived MVs^CD140b+ from the plasma of control subjects (ctrl group) and patients with stroke (acute phase between 0 and 6 h, 12 and 24, and 2 to 6 d after stroke onset). Statistical analysis was performed using Kruskal-Wallis nonparametric test with Dunn test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

Patients MV^CD140b+ protein composition and corresponding hallmark pathways. A, Volcano plot showing the differentially expressed proteins between MVs^CD140b+ collected from patients with stroke compared with control subjects between 0 and 6, 12 to 24 h, and 2 to 6 d after ischemic stroke onset. Statistical testing was performed using 2-way ANOVA with Tukey´s multiple comparison. B, The corresponding top 10 enriched functional pathways obtained from the molecular signature database (MSigDG). The data from the enrichment analysis are ranked based on the most significant adjusted P value. Blue bars represent significance, while gray bars represent nonsignificant P values. P<0.05 was considered significant and log₂-fold change cutoff was set to (−1:1). IL-6 indicates interleukin-6; KRAS, kirsten rat sarcoma virus; NF-kB, nuclear factor kappa B; STAT3, signal transducers and activators of transcription 3; and TNF-α, tumor necrosis factor alpha.

Figure 4.

Timeline of the impact of pericyte-derived microvesicles (MVs) protein composition and function. A, The pericyte-derived MVs protein composition showing reduced inflammation at the hyperacute phase between 0 and 6 h. This time point after stroke is usually accompanied by pericyte detachment and migration into the brain parenchyma. B, Between 12 and 24 h after stroke, the MV-content was predominantly angiogenic and moderately proinflammatory were this time point is usually associated with basement membrane degradation and vascular leakage. C, Between 2 and 6 d, the MV protein content correlated to angiogenesis, vascular maturation, and mild proinflammation. Downregulated proteins are marked blue, while upregulated proteins are marked in red. Created with www.BioRender.com. CD40 indicates cluster of differentiation 40; CXCL5, C-X-C motif chemokine5; FGF-21, fibroblast growth factor-21; GDNF, glial cell line–derived neurotrophic factor; IL-17C, interleukin-17C; MCP-4, monocyte chemoattractant protein-4; MMP-1, matrix metalloprotease-1; PD-L1, programmed death-ligand 1; SIRT2, sirtuin 2; UPA, urokinase-type plasminogen activator; and VEGFA, vascular endothelial growth factor A.