Endothelial NOX4‑driven oxidative stress inhibition reverses HtrA1 deficiency‑induced blood‑brain barrier disruption and cognitive impairment

Heterozygous high temperature requirement serine peptidase A1 (HTRA1) mutations are associated with autosomal dominant cerebral small vessel disease (CSVD), but their pathogenic mechanisms remain elusive. In the present study, clinical data were collected from two families carrying heterozygous HTRA1 mutations, with pathogenic mutations verified through Sanger Sequencing. Between January 2018 and December 2023, four patients with CSVD were recruited from the Department of Neurology, First Hospital of Shanxi Medical University (Taiyuan, China). Whole blood RNA Sequencing (RNA‑seq) was performed to identify differentially expressed genes. Lentiviral vectors were constructed for HtrA1 overexpression and knockdown in mouse brain microvascular endothelial bEnd.3 cells to assess cell viability, oxidative stress, tight junction integrity and Apoptosis. Adeno‑associated virus (AAV) technology was used to assess how HtrA1 gene interference affects the function of cerebral vascular endothelial cells in mice. Finally, the NOX4 Inhibitor GLX351322 was administered to investigate its regulatory effects on cell permeability and Apoptosis. Behavioral changes were assessed through open field and novel object recognition test and Morris water maze experiments to evaluate its impact on cognitive behavior in mice. The present study analyzed clinical data from two enrolled families with heterozygous HTRA1 mutations [c.854C>T (p.P285L) and c.905G>A (p.R302Q)] and observed stroke, cognitive decline and gait disturbances. RNA‑seq of patient blood revealed downregulated HTRA1, occludin‑like protein 1 and Claudin 5, alongside upregulated NOX4, with apoptotic pathways prominently enriched. HtrA1 overexpression in bEnd.3 cells enhanced viability, decreased oxidative stress and Apoptosis and elevated tight junction protein expression, whereas HtrA1 knockdown exacerbated these effects. In mice, AAV‑mediated HtrA1 suppression in cerebrovascular endothelial cells increased NOX4 and caspase3 levels, disrupted blood‑brain barrier (BBB) integrity and induced anxiety‑ and depressive‑like behaviors, measured by the open field test, along with cognitive and memory impairment evaluated using the novel object recognition and Morris water maze tests. The NOX4 Inhibitor GLX351322 partially restored endothelial function, mitigated BBB damage and alleviated behavioral impairment. The present findings demonstrated that heterozygous HTRA1 mutations promoted CSVD via NOX4‑mediated oxidative stress, endothelial dysfunction and BBB breakdown. Targeting the HTRA1‑NOX4 interaction using GLX351322 rescued cerebrovascular and cognitive pathology, offering preclinical validation for therapeutic intervention.