Pericyte loss and endothelial activation define distinct vascular states in cerebral small vessel disease

Cerebral small vessel disease (cSVD) is a leading cause of stroke and vascular cognitive impairment, yet the cellular mechanisms underlying microvascular dysfunction in human disease remain incompletely understood. In particular, the relationship between pericyte alterations and endothelial activation, two key features of blood–brain barrier dysfunction, remains unresolved. Here, we performed a quantitative single-vessel analysis of the human cortical microvasculature across increasing cSVD severity and ageing. Using multiplex immunohistochemistry combined with spectral unmixing and automated image analysis, we analysed 11,409 microvascular fragments from post-mortem brain tissue derived from 20 cases. Endothelial cells, pericytes, and endothelial activation were assessed using PECAM-1, PDGFRβ, and VCAM-1, respectively. Microvascular density and diameter differed between cortical grey matter and the underlying white matter, with white matter vessels being less dense and wider in controls. While vessel diameter remained stable across cSVD severity groups, microvascular density increased with cSVD severity and age in the white matter. At the molecular level, PDGFRβ signal decreased markedly with increasing cSVD severity, consistent with reduced pericyte coverage. This reduction was observed in both grey and white matter and correlated with disease severity and age. Notably, intermediate severity groups displayed marked heterogeneity, with vessels exhibiting either preserved or markedly reduced PDGFRβ signal. In parallel, endothelial markers PECAM-1 and VCAM-1 increased significantly with disease severity, reflecting endothelial activation. Unsupervised Gaussian mixture clustering of marker expression identified three vascular states characterised by (i) preserved PDGFRβ signal with low endothelial activation, (ii) reduced PDGFRβ signal without endothelial activation, and (iii) reduced PDGFRβ signal combined with endothelial activation. These clusters broadly aligned with neuropathological severity but revealed heterogeneity not captured by post-mortem diagnosis alone. Together, these findings suggest that pericyte alterations and endothelial activation are partially dissociated processes in human cSVD and identify distinct microvascular phenotypes across disease severity. These cross-sectional observations provide a framework for future longitudinal and experimental studies to determine their temporal and mechanistic relationships.