Between January 2018 and December 2023, four patients (three male, one female; age range, 44-54 years; median age, 47.5 years) diagnosed as heterozygous HTRA1mcs and four healthy controls (HCs; three male, one female; age range, 43-53 years; median age, 48 years) were recruited from the Department of Neurology, First Hospital of Shanxi Medical University (Taiyuan, China). The inclusion criteria were as follows: i) confirmation of a heterozygous HTRA1 mutation by genetic testing; ii) presence of typical CSVD-related neuroimaging features on brain MRI; iii) age between 18 and 60 years; and iv) provision of written informed consent. The exclusion criteria were as follows: i) other hereditary or acquired causes of CSVD; ii) presence of severe systemic diseases; iii) history of stroke not attributable to CSVD; and iv) any contraindication to MRI. Inclusion criteria for HCs were: i) absence of HTRA1 mutations confirmed by genetic screening; ii) no clinical symptoms or neuroimaging features suggestive of CSVD; iii) age and sex matched to the patient group; and iv) provision of written informed consent. Exclusion criteria for HCs were: i) any history of neurological disorders; ii) significant systemic illnesses; or iii) any contraindications to MRI; iv) any abnormal findings on brain MRI suggestive of CSVD or other intracranial pathologies.

All patients exhibited typical CSVD imaging features, with brain magnetic resonance imaging (MRI) lesions corresponding to the clinical symptoms. The brain MRI scans were performed using a 3.0-Tesla Siemens MAGNETOM Prisma scanner. T2-weighted fluid-attenuated inversion recovery (FLAIR) sequences were acquired with the following parameters: Repetition time, 9,000 msec, echo time, 94 msec, and inversion time, 2,500 msec. Clinical and imaging data were assessed from the probands and their family members. Cognitive function was evaluated using the Montreal Cognitive Assessment (MoCA) and the Mini-Mental State Examination (MMSE). The study was approved by the ethics committee of the First Hospital of Shanxi Medical University (approval no. KYLL-2024-161), and all participants provided written informed consent. Peripheral blood samples were collected from patients for genetic analysis and HCs.

Genomic DNA was analyzed via Sanger sequencing of DNA extracted from peripheral blood collected from the probands, family members and HCs in anticoagulant EDTA-coated tubes. Genomic DNA was extracted using the Ezup Column Blood Genomic DNA Extraction kit (Sangon Biotech, cat. no. B518253) according to the manufacturer's instructions. PCR was performed using Taq Plus DNA Polymerase (Sangon Biotech, cat. no B600090). Thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min; followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, and elongation at 72°C for 30 sec; with a final extension at 72°C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis. Following purification by gel extraction, the products were sequenced on a 3730XL sequencer (Applied Biosystems). Sanger sequencing and primer synthesis (Table I) were performed by Sangon Biotech Co., Ltd. The pathogenicity of the HTRA1 heterozygous mutations c.854C>T (p.P285L) and c.905G>A (p.R302Q) was assessed using bioinformatics tools, including Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), Sorting Intolerant From Tolerant (/sift.bii.a-star.edu.sg), Mutation Taster (https://www.mutationtaster.org), Combined Annotation Dependent Depletion (https://cadd.gs.washington.edu/score), non-synonymous single nucleotide polymorphism (nsSNP) Analyzer (https://snpanalyzer.uthsc.edu) and Mutation Assessor (http://mutationassessor.org/r3). According to the Ensembl database (https://www.ensembl.org), both mutations were identified as single nucleotide polymorphisms, specifically c.854C>T (rs587776446) and c.905G>A (rs2133449474). The aforementioned tools predicted that both mutations were deleterious and likely pathogenic, indicating a notable impact on HTRA1 function and potential involvement in associated phenotypic mechanisms.

Total RNA was extracted using TRNzol Universal Reagent (cat. no. DP424; TIANGEN Biotech Co., Ltd.) according to the manufacturer's instructions. RNA integrity was assessed using the Agilent 5400 Fragment Analyzer System (Agilent Technologies), and the RNA integrity number (RIN) was determined for each sample. Sequencing libraries were prepared and subjected to paired-end (PE) sequencing with a read length of 150 bp (2×150 bp) on the Illumina NovaSeq X Plus platform (Illumina, Inc.) using the NovaSeq X Series 25B Reagent Kit (300 cycles; cat. no. 20104706; Illumina, Inc.). The final library loading concentration was measured by quantitative PCR (qPCR) and was 4.03 nM for patient samples and 4.24 nM for normal controls. RNA-seq was performed by Novogene Co., Ltd. Raw reads were filtered to remove adapters and low-quality sequences, aligned to the human genome (GRCh38: obtained from the NCBI, assembly accession: GCF_000001405.26) using HISAT2 (daehwankimlab.github.io/hisat2/), and quantified using feature counts. Differential expression analysis [DESeq2 (https://pubmed.ncbi.nlm.nih.gov/25516281), |log₂ FC|>1, FDR-adjusted P<0.05] was used to identify significant genes, which were visualized using volcano or heatmaps. Principal Component Analysis (PCA) was performed on the normalized expression data of all detected genes using the prcomp function in R software (version 4.3.1). Gene Ontology (GO, http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; kegg.jp/) pathway enrichment analyses were conducted using cluster Profiler (FDR <0.05) (version 4.19.6; https://doi.org/10.1089/omi.2011.0118).

Anticoagulant-coated blood collection tubes were used to collect 3-5 ml venous blood from the HTRA1mc and HC groups. Following standing at 25°C for 30 min, the blood was centrifuged at 1,200 × g for 10 min at 4°C to obtain plasma, divided into microcentrifuge tubes, and centrifuged at 3,200 × g for another 10 min at 4°C to remove platelets. The expression levels of HTRA1 (cat. no. HM11176) and NADPH oxidase 4 (NOX4; both Bioswamp Life Science Lab; Wuhan Bienle Biotechnology Co., Ltd.; cat. no. HM11179) were measured using ELISA kits, in accordance with the manufacturer's instructions.

The lentiviral vectors for OE included mCherry red fluorescent LV8N (EF-1a/mCherry&Puro) and non-fluorescent LV6 (EF-1a/Puro) shuttle plasmid vectors. For knockdown, the short hairpin (sh)HtrA1-RNA lentiviral vectors included GFP green fluorescent LV3 (H1/GFP&Puro) and non-fluorescent LV2 (U6/Puro) shuttle plasmid vectors (GenePharma). The coding and protein sequences of mouse HtrA1 were obtained from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). The HtrA1 gene fragments were cloned into the NotI/NsiI sites of the LV8N and LV6 shuttle plasmid vectors. Following verification via Sanger sequencing, the HtrA1 gene OE plasmid was constructed. The following shRNA sequences were incorporated into the LV3 and LV2 shuttle plasmids for the mouse HtrA1 gene: shRNA-HtrA1-mus-749, 5'-GGGTCAAGGTTGAGCTGAAGA-3'; shRNA-HtrA1-mus-885, 5'-GCTGAGACCTGGAGAATTTGT-3'; shRNA-HtrA1-mus-1079, 5'-GCGAGGTGATTGGGATTAACA-3'; and negative control (NC), 5'-TTCTCCGAACGTGTCACGT-3'.

Recombinant viral plasmids for HtrA1 OE and knockdown, along with three auxiliary packaging plasmids (PG-P1-VSVG, PG-P2-REV and PG-P3-RRE) (GenePharma), were subjected to high-purity non-toxic endotoxin extraction. Plasmid DNA was extracted using a high-purity midi-prep kit (Shanghai GenePharma Co.) and dissolved in sterile double-distilled water. The concentration and purity of plasmid DNA were determined by UV spectrophotometry, and only samples with an A260/A280 ratio between 1.8 and 2.0 were used for subsequent experiments. For lentivirus production, the third-generation lentiviral packaging system was used. 293T cells (GenePharma) were co-transfected with RNAi-Mate reagent (GenePharma, cat. no. G04001) at 37°C. In total, 20 μg plasmid DNA was used, composed of the recombinant HtrA1-OE or HtrA1-shRNA plasmid and three helper plasmids (PG-P1-VSVG, PG-P2-REV, PG-P3-RRE) at a 1 : 1 : 1 : 1 mass ratio (2.5 μg each). After 48 h, the lentiviral supernatant was collected, filtered (0.45 μm), and concentrated. The final lentivirus titer was determined to be ≥1×10⁸ TU/ml (GenePharma). bEnd.3 cells were infected at an MOI of 125 (Table II). The virus-containing medium was removed after 24 h and replaced with fresh medium. Following a 48-h recovery, transduced cells were selected and maintained in medium containing 5 μg/ml puromycin.

The bEnd.3 cells (Procell Life Science) were cultured in high-glucose DMEM (HyClone; Cytiva; cat. no. SH30022.01) supplemented with 10% FBS (Wuhan Boster Biological Technology, Ltd.; cat. no. PYG0001) at 37°C in a 5% CO₂ atmosphere. Upon reaching ~50% confluence, the cells were exposed to serum-free high-glucose DMEM along with lentiviral supernatant overexpressing HtrA1 [multiplicity of infection=125]. After 24 h incubation, the medium was replaced with complete medium containing 10% FBS. After 72 h, 5 μg/ml puromycin was used for screening of HtrA1 OE and knockdown cells for 96 h. The groups were as follows: Untransfected (UT)-OE; NC-OE (bEnd.3 cells infected with LV8N-NC or LV6-NC lentiviral supernatant); HtrA1-OE (bEnd.3 cells infected with LV8N-HtrA1 or LV6-HtrA1 lentiviral supernatant); sh-UT; sh-NC (bEnd.3 cells infected with LV3-NC or LV2-NC lentiviral supernatant); and sh-HtrA1 (bEnd.3 cells infected with LV3 or LV2 lentiviral supernatant). Cells in the sh-HtrA1 group were treated with 5 μM GLX351322 (MedChemExpress; cat. no. HY-100111) for 24 h at 37°C to downregulate the expression of NOX4.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, cat. no. 15596-026), followed by chloroform and isopropanol precipitation. RNA concentration and purity were assessed using a Take3 micro-detection plate (BioTek, RNA samples were reverse transcribed into cDNA using the PrimeScript RT reagent kit with gDNA Eraser according to the manufacturer's protocol (Takara Bio, Inc.; cat. no. RR047A). The obtained cDNA was amplified using a Roche Diagnostics LightCycler480II. The 20 μl reaction mixture comprised 10.0 μl TB Green Premix Ex TaqII (Takara Bio, Inc.; cat. no. RR820A), 2.0 μl cDNA, 6.4 μl diethylpyrocarbonate water and 0.8 μl (10 μM) primer. Thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, and annealing/extension at 60°C for 30 sec. GAPDH was used as an endogenous reference for the normalization of mRNA expression. The HtrA1 mRNA levels were determined using the 2^−ΔΔCq method (24). The primer sequences (Sangon Biotech Co., Ltd.) were as follows: HtrA1 forward, 5'-tgacggcgggcatctccttc-3' and reverse, 5'-tcttggtgacagctttccctttgg-3' and GAPDH forward, 5'-ccctggccaaggtcatccat-3' and reverse, 5'-tcacgccacagctttccaga-3'.

bEnd.3 cells and mouse brain tissue samples were lysed in RIPA lysis buffer (cat. no. AR0102-100) containing broad-spectrum protease inhibitors (both Wuhan Boster Biological Technology Co.; cat. no. AR1182). Protein concentration was determined using a BCA protein assay kit (Wuhan Boster Biological Technology Co.; cat. no. AR0146) following the manufacturer's instructions. Equal amounts (30 μg) of protein were separated by 10% SDS-PAGE, followed by transfer onto a 0.22 μM polyvinylidene fluoride membrane. The membranes were blocked at room temperature for 2 h with 5% (w/v) non-fat dry milk in 0.1% TBST. After blocking for 2 h, the membranes were incubated overnight with primary antibodies at 4°C as follows: Rabbit anti-HtrA1 (1:250, Wuhan Boster Biological Technology Co.; cat. no. A01801-1), anti-occludin (cat. no. A2601), anti-zona occludens (ZO)1 (both 1:1,000, both ABclonal Biotech Co., Ltd.; A0659), anti-claudin (CLDN)5 (1:250, BIOSS; cat. no. bs-10296R), anti-NOX4 (Wuhan Boster Biological Technology Co.; cat. no. BM4135), anti-caspase3 (both 1:1,000, ABclonal Biotech Co., Ltd.; cat. no. A2156), anti-GAPDH (1:5,000, Shanghai Abways Biotechnology Co., Ltd.; cat. no. AB0037) and anti-β-actin (1:5,000, ABclonal Biotech Co., Ltd.; cat. no. AC038). After washing the membrane with TBST, it was incubated with goat anti-rabbit IgG-HRP secondary antibody (1:5,000; Wuhan Boster Biological Technology Co., Ltd.; cat. no. BA1054) for 90 min at 25°C. The luminescent solution was prepared using the ECL Luminescent Substrate kit (Wuhan Boster Biological Technology Co., Ltd.; cat. no. AR1171) and the bands were visualized using an imaging analysis system (Bio-Rad Laboratories, Inc.; Chemidoc MP). The gray value of the target proteins was analyzed using the ImageJ software (V1.8.0; National Institutes of Health).

A total of 5×10³ cells was seeded in a 96-well plate. After 12, 24, 48 and 72 h, cells were treated with working solution containing 10 μl CCK-8 solution (MedChemExpress; cat. no. HY-K0301) and 90 μl DMEM with 10% FBS. After 2 h incubation in the dark, the absorbance was measured at 450 nm using a multifunctional microplate reader (BioTek; Agilent Technologies, Inc.; Synergy H1).

RTCA was performed using the RTCA S16 system (ACEA Biosciences, Inc.). A total of 50 μl DMEM with 10% FBS in E-plate16 wells was used for determining the baseline. bEnd.3 cells were seeded at a density of 5×10³ cells/well in the wells of E-plate16. Following 30 min incubation at 37°C, RTCA was performed for 72 h, with impedance measured at 15-min intervals.

A total of 5×10³ bEnd.3 cells was seeded in 24-well plates. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Following washing with PBS, 0.5% Triton X-100 (Beyotime Biotechnology; cat. no. P0096-500 ml) was added at room temperature for permeabilization of cell membrane. Blocking was performed using 5% BSA (Bossis; cat. no. AR1006) for 2 h at 25°C. The cells were then incubated overnight at 4°C with the primary antibodies as follows: Rabbit anti-CLDN5 (1:100, ABclonal Biotech Co., Ltd.; A10207), anti-occludin (1:150, Bossis; cat. no. bs-10011R), anti-ZO1 (1:150, ABclonal Biotech Co., Ltd.; cat. no. A0659) and anti-NOX4 (1:100, Wuhan Boster Biological Technology Co. Ltd.; BM4135). Cells were washed three times with PBS with 0.1% Tween-20 (PBST) and incubated with Alexa Fluor 555-labeled donkey (1:500, cat. no. A0453) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L; 1:200, both Beyotime Biotechnology; cat. no. A0423) at 25°C for in the dark for 2 h. The cells were washed three times with PBST for 15 min and imaged under a confocal microscope (Leica GmbH). Fluorescence intensity was analyzed using ImageJ software (V1.8.0; National Institutes of Health).

A total of 2×10⁵ cells were seeded in the upper chamber of a 24-Transwell system (8 μm, Falcon; Corning Life Sciences; cat. no. 353097) and incubated at 37°C in a humidified 5% CO₂ incubator for 24 h. The upper chamber was filled with 200 μl DMEM (HyClone; Cytiva) with 10% FBS (Boster, cat. no. PYG0001), whereas the lower chamber contained 600 μl PBS. Following 12 h incubation at 37°C, the medium from the upper chamber was removed and the cells were washed three times with PBS. A total of 100 μl 1 μg/ml FITC-dextran (40,000 MW, Sigma-Aldrich; Merck KGaA; cat. no. SLCB8958) was added to the upper chamber. The medium in the lower chamber was discarded and replaced with 600 μl PBS. After the 1 h incubation at 37°C, the fluorescence intensity was measured using the SpectraMax i3x detection system at excitation/emission (Ex/Em) wavelengths of 485/528 nm (Molecular Devices, LLC).

bEnd.3 cells (1.5×10⁵) were seeded in a 6-well plate and cultured for 24 h at 37°C. DCFH-DA (MedChemExpress; cat. no. HY-D0940) staining was performed when the cell density reached 80%. The culture medium was discarded and the cells were washed three times with PBS. Subsequently, the cells were incubated with 1 ml DCFH-DA working solution at a concentration of 10 μM for 20 min at 37°C in the dark. Images were captured under a fluorescence microscope (Ex/Em=488/525 nm) and the fluorescence intensity was analyzed using ImageJ software (V1.8.0, National Institutes of Health).

The cells were detached using 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. T1300). The cells were lysed using RIPA lysis buffer (Beijing Solarbio, cat. no. R0010) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The protein concentration was measured using the BCA method. The activity of SOD and MDA were determined using the SOD (cat. no. A001-3) and MDA Detection kits (both Nanjing Jiancheng Bioengineering Institute; cat. no. A003-1-2), according to the manufacturer's instructions.

Apoptosis (early + late apoptotic cells) was detected via flow cytometry using the Annexin V-FITC/propidium iodide (PI) staining kit (Dalian Meilun Biology Technology Co., Ltd.; cat. no. MA0220) following the manufacturer's instructions. Briefly, 2×10⁵ bEnd.3 cells were seeded in 6-well plates and cultured for 24 h at 37°C. Cells were detached using 0.25% trypsin (Wuhan Boster Biological Technology Ltd.; cat. no. PYG0065) and incubated with 5 μl Annexin V-FITC/PI staining solution for 15 min at 25°C in the dark. Flow cytometry was performed using a NovoCyte 3130 instrument (ACEA Biosciences, Inc.). Red and green fluorescence signals were recorded and analyzed using the NovoExpress software (Version 1.5.0, ACEA Biosciences, Inc.).

All animal experiments were approved by the Animal Welfare and Ethics Committee of the First Hospital of Shanxi Medical University (approval no. DWYJ-2025-304, Taiyuan, China). Male C57BL/6J mice (Spef Biotechnology Co., Ltd.; age, 6-8 weeks) were housed in a specific pathogen-free animal facility at 25±2°C, 50-60% relative humidity and a 12/12-h light/dark cycle with free access to food and water (n=24, weight, 20-25 g). Humane endpoints were as follows: i) loss of more than 20 % of initial body weight within 24 h, ii) inability to reach food or water, iii) persistent hunched posture with ruffled fur, iv) moribund state and v) respiratory distress. Animals reaching any of these endpoints were immediately euthanized by cervical dislocation. None of the animals reached the predefined humane endpoints during the study. The endothelial cell-specific HtrA1-shRNA mouse model was created by injecting AAV HBAAV2/BI30-m-HtrA1-shRNA-EGFP (Hanbio Biotechnology Co., Ltd., cat. no. 80062053) into the tail vein of C57BL/6J mice. Mice were divided into three groups (n=8): Vehicle (mice injected with saline), AAV/B130-NC (mice injected with a virus control) and AAV/B130-shHtrA1 group (mice injected with HtrA1-interfering AAV). Anesthesia was induced with 5% isoflurane using a compact small animal anesthesia machine (RWD Life Science Co., Ltd.), followed by maintenance with 1.5-2.5% isoflurane. After 4 weeks, mice were sacrificed by cervical dislocation and brain tissue was isolated. To investigate the effects of oxidative stress on AAV/B130-shHtrA1 C57BL/6J mice were intraperitoneally administered GLX351322 (5 mg/kg/day) for 4 weeks.

Brain tissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C. Tissue was sectioned into 15 μm slices on a cryostat (Leica, RM2235). Antigen retrieval was performed by heating the sections in EDTA antigen retrieval solution (Zhongshan Jinqiao Biotechnology Co., Ltd.; cat. no. ZLI-9069) at 95°C for 2 min in a water bath. After cooling to room temperature, the sections were blocked with 5% BSA (Solarbio, cat. no. SW3015) for 30 min. The sections were incubated with primary antibodies overnight at 4°C and with the secondary antibodies for 1 h at room temperature. The primary antibodies were as follows: Mouse-anti-α-smooth muscle actin (SMA; Zenbio, Inc.; cat. no. L29JLZC) and rabbit-anti-HtrA1 (both 1:50, Proteintech Group, Inc.; cat. no. 00129131). The secondary antibodies were Alexa Fluor 488-labeled goat anti-mouse (cat. no. A0428) and Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L; both 1:500, both Beyotime; cat. no. A0453). The nuclei were counterstained with DAPI (Beijing Solarbio Science & Technology Co., Ltd.; C0065) for 10 min at 25°C. Fluorescent images were captured using a confocal microscope (Leica GmbH).

Mice were intravenously injected with 0.5% Evans blue (Absin, cat. no. Abs47002009) in saline at 3 ml/kg body weight and euthanized 2 h after dye circulation. The weight of each cerebral hemisphere was recorded and Evans blue extraction was performed by incubating the brain tissue in acetone for 24 h at 25°C, followed by centrifugation at 12,000 × g for 30 min at 4°C. Evans blue staining was quantified by measuring the absorbance at 620 nm using a spectrophotometer (Thermo Fisher Scientific, Inc.).

Each mouse underwent the OFT once to avoid habituation or stress-associated behavioral changes. A plain 50×50×40 cm open-field arena was used to assess the locomotor activity and anxiety-like behavior. Following 30 sec habituation, the total distance traveled and time spent in the center arena (16.7×16.7 cm) was recorded in a 5 min session. The frequencies of rearing and defecation in the apparatus were recorded. At the end of each experimental trial, the apparatus was thoroughly cleaned and disinfected by spraying 75% ethyl alcohol to eliminate residual olfactory cues. Mouse behavior was analyzed using VisuTrack software (XR-VT version, Shanghai Xinruan Information Technology Co., Ltd.).

Mice were placed in an open field with two identical objects to familiarize them with their environment. Following 6 h, the mice were returned to the same open field for testing. One of the familiar objects was replaced with a novel object that had a unique color and shape. In both phases of the experiment, each mouse faced the wall of the apparatus and was allowed to explore the objects freely for 5 min. Before each trial, the apparatus was cleaned using 75% ethanol solution. Mouse behavior was recorded using a video tracking system and analyzed using the VisuTrack software. NOR memory was evaluated using the NOR index (NOI) as follows: NOI=(time spent exploring the novel object)/(total exploration time for both objects) ×100%.

The MWM test was conducted in a white circular tank, measuring 120 cm in diameter and 50 cm in height, filled with tap water maintained at 22±3°C, and divided into four equal virtual quadrants. Non-toxic white paint was added to ensure the water was opaque. During the place navigation-training phase, which consisted of four trials of 120 sec/day over 4 consecutive days, a circular escape platform (diameter, 10 cm) was submerged 1 cm below the water surface in a designated quadrant. Each mouse was released from different starting positions, facing the tank wall and allowed to locate the escape platform. Upon reaching the platform, the mouse was allowed to stay on it for 3 sec. Latency to escape onto the platform was recorded. If a mouse failed to locate the platform within 120 sec, it was guided to the platform and allowed to remain there for 30 sec. On day 5, a probe test was conducted, wherein the mice swam freely without the platform for 120 sec. The proportion of time spent in the target quadrant and the number of platform crossings were recorded. Swimming paths were monitored and analyzed using VisuTrack software.

Statistical analysis was performed using the GraphPad Prism 9 (GraphPad Software Inc.; Dotmatics) and R (version 4.3.1; R Foundation for Statistical Computing) software. Data are presented as the mean ± standard deviation of three independent experiments. Comparisons were made using one-way ANOVA followed by Tukey's, LSD t or Dunnett's post hoc test. Differentially expressed genes were analyzed using the DESeq2 package. The Wald test was used to analyze the differences between two groups, with the filtering conditions set at |log₂ FC|>1 and an adjusted P-value <0.05. The significance of enrichment analysis was assessed using a hypergeometric distribution test. P<0.05 was considered to indicate a statistically significant difference.

Sanger sequencing was performed to analyze the carrier status of the pathogenic variants in two families (Fig. 1A and B). In family 1 (F1), the proband (F1 III-5), his elder brother (F1 III-1) and younger brother (F1 III-7) were found to carry a heterozygous HTRA1 mutation (c.854C>T, p.P285L). In F2, the proband (F2 III-1), two sisters (F2 III-2 and -4) and a brother (F2 III-6) carried a heterozygous HTRA1 mutation (c.905G>A, p.R302Q). The proband (F2 III-1) and their sister (F2 III-2) and brother (F2 III-6) all died of cerebrovascular diseases.

The predicted pathogenicity of the HTRA1 c.854 C>T and c.905 G>A mutations is shown in Table III. The phenotypic characteristics exhibited a notable degree of homogeneity, with the clinical presentation, including subcortical ischemic events and progressive cognitive decline, being characteristic of CSVD (Table IV). The pedigrees of families with heterozygous HTRA1 mutations are shown in Fig. 1A.

F1III-1 was a 49-year-old male patient who presented with recurrent strokes and cognitive decline for 20 years. The patient experienced a first stroke, manifesting as left-sided weakness and dysphagia. The patient is bedridden because of limb weakness, gait disturbance and pseudobulbar palsy. The patient had a medical history of alopecia and spondylosis deformans. Brain MRI, with fluid-attenuated inversion-recovery (FLAIR) sequences, revealed multiple lacunar infarcts and confluent white matter hyperintensity (WMH) in the periventricular regions (Fig. 1C). Cognitive evaluation at the time of diagnosis demonstrated substantial impairment. The patient achieved a Montreal Cognitive Assessment (MoCA) score of 13/30 (severe impairment; normal range ≥26) and a Mini-Mental State Examination (MMSE) score of 16/30 (moderate impairment; normal range ≥24).

F1III-5 was a 46-year-old male patient who presented with right-sided weakness, dysarthria, history of cerebral infarction, and cognitive decline. The patient had alopecia and spondylosis deformans with lumbar disc surgery. Brain MRI with FLAIR sequences revealed multiple lacunar infarcts and WMH in the periventricular regions (Fig. 1C). Cognitive assessment revealed a notable decline (MoCA score, 15; MMSE score, 18).

F1III-7 was a 44-year-old male patient who presented with recurrent dizziness and gait unsteadiness. The patient exhibited hypertension and diabetes. Brain MRI indicated multiple lacunar infarcts and WMH (Fig. 1C). Cognitive assessment showed a mild decline (MoCA score, 25; MMSE score, 24).

F2III-4 was a 54-year-old female patient who presented with recurrent dizziness, diplopia and impaired movement of the right limbs, with a history spanning 8 years. Brain MRI showed multiple subcortical lacunar infarcts and WMH (Fig. 1C), with cognitive assessment indicating a mild decline (MoCA score, 23; MMSE score, 21).

PCA revealed significant differences between the HTRA1mc and HC groups (Fig. 1D). In total, 489 differentially expressed genes were identified, of which 238 were up- and 260 were downregulated (Figs. 1E and 2A). In the HTRA1mc group, mRNA expression of HTRA1, occludin-like protein 1 (OCEL1) and CLDN5 was downregulated, whereas that of NOX4 and Bcl-2 associated X (BAX) was upregulated (Fig. 1E). The KEGG enrichment analysis showed significant enrichment in 'ribosome', 'P53 signaling pathway', 'apoptosis', 'thyroid cancer', 'endometrial cancer', 'breast cancer', and gastric cancer (Fig. 1F). GO enrichment analysis revealed significant enrichment in 'defense response to virus', 'leukocyte-mediated immunity', 'lymphocyte mediated immunity', and 'cell killing' (Fig. 2B). The HTRA1mc group showed decreased plasma HTRA1 (Fig. 2C) and elevated NOX4 protein (Fig. 2D) levels compared with the HC group. These findings indicated that HTRA1 mutations may increase oxidative stress and apoptosis, thereby contributing to CSVD. Additionally, the downregulation of tight junction-associated genes, such as OCEL1 and CLDN5, may impair the blood-brain barrier (BBB) function.

mCherry red fluorescence carried by lentivirus was observed in both the NC-OE and HtrA1-OE groups (Fig. 3A). RT-qPCR and western blot analyses showed that the HtrA1 mRNA (Fig. 3B) and protein (Fig. 3C) levels were significantly higher in the HtrA1-OE group than in the NC-OE group. No differences were observed between the UT-OE and NC-OE groups (Fig. 3B and C). In the CCK-8 assay, cells in the HtrA1-OE group exhibited enhanced viability at both 48 and 72 h compared with those in the NC-OE group (Fig. 3D). These findings were supported by the RTCA results (Fig. 3E), which suggested that HtrA1 OE improved the viability of bEnd.3 cells. DCFH-DA, SOD and MDA assays were performed to assess oxidative stress. The HtrA1-OE group showed decreased NOX4 expression (Fig. 3C and F) and reactive oxygen species (ROS) levels (Fig. 3G) compared with the NC-OE group, whereas the SOD activity was higher and MDA levels were lower in the HtrA1-OE group (Fig. 3H). These results indicated that HtrA1 OE decreased oxidative stress and exerted a protective effect on bEnd.3 cells.

HtrA1 OE in bEnd.3 cells significantly increased the expression of tight junction proteins ZO1, occludin and CLDN5 in the HtrA1-OE group compared with that in the NC-OE group (Fig. 4A). These results were corroborated by immunofluorescence, with higher expression levels of these proteins observed in the HtrA1-OE group (Fig. 4B and C). The FITC-dextran experiment revealed decreased cell permeability in the HtrA1-OE group compared with that in the NC-OE group (Fig. 4D), indicating strengthened tight junctions. Western blotting revealed a significant decrease in caspase3 expression in the HtrA1-OE group compared with that in the NC-OE group (Fig. 4E). Flow cytometry also indicated decreased apoptosis in the HtrA1-OE group compared with the NC-OE group (Fig. 4F). These findings indicated that HtrA1 OE strengthened the integrity of tight junctions and decreased apoptosis in bEnd.3 cells.

Both the sh-NC and sh-HtrA1 groups showed green fluorescence, confirming successful establishment of a stable HtrA1 knockdown cell line (Fig. 5A). RT-qPCR and western blot analyses revealed significantly decreased HtrA1 mRNA and protein levels in the shRNA-HtrA1-mus-1079 group compared with those in the sh-NC group (Fig. 5B and C). As the shRNA-HtrA1-mus-1079 group showed the highest interference efficiency, it was selected as the sh-HtrA1 group for subsequent experiments. The CCK-8 assay showed decreased viability of cells in the sh-HtrA1 group at 72 h compared with that in the sh-NC group (Fig. 5D). Consistently, RTCA (Fig. 5E) demonstrated decreased cell viability following HtrA1 knockdown. The expression of NOX4 was increased in the sh-HtrA1 group compared with that in the sh-NC group, as evidenced by western blot analysis (Fig. 5F) and immunofluorescence (Fig. 5G). ROS levels were higher in the sh-HtrA1 group compared with sh-NC group (Fig. 5H), whereas the SOD activity and MDA levels were lower and higher, respectively (Fig. 5I). These findings indicated that HtrA1 knockdown increased oxidative stress in bEnd.3 cells.

Following HtrA1 knockdown in bEnd.3 cells, western blot analysis showed decreased expression of the tight junction proteins ZO1, occludin and CLDN5 in the sh-HtrA1 group compared with that in the sh-NC group (Fig. 6A). Confocal microscopy confirmed this reduction (Fig. 6B and C). The FITC-dextran assay revealed increased permeability in the sh-HtrA1 group (Fig. 6D), indicating compromised tight junction integrity and enhanced cell permeability following HtrA1 knockdown. Western blot analysis revealed elevated levels of caspase3 in the sh-HtrA1 group compared with the sh-NC group (Fig. 6E). Flow cytometry confirmed increased apoptosis in the sh-HtrA1 group (Fig. 6F). These findings indicated that HtrA1 knockdown increased apoptosis in bEnd.3 cells, emphasizing the role of HtrA1 in regulating tight junctions and cell barrier properties.

An endothelial cell-specific HtrA1 knockdown mouse model was constructed by injecting AAV2/B130-TIE carrying a specific endothelial promoter into the tail vein of C57BL/6J mice. After 4 weeks, immunofluorescence staining of HtrA1 in mouse brain slices showed notable HtrA1 expression in cerebrovascular endothelial cells, colocalizing with the vascular marker α-SMA (Fig. 7A). The green fluorescent protein marker in the viral vector confirmed the successful transfection of endothelial cells (Fig. 7B). RT-qPCR and western blotting revealed significantly reduced HtrA1 mRNA (Fig. 7C) and protein levels (Fig. 7D) in the AAV/B130-shHtrA1 group, indicating effective gene knockdown. Expression of tight junction proteins ZO1, occludin and CLDN5 decreased, whereas that of NOX4 and caspase3 was increased in the AAV/B130-shHtrA1 compared with that in the AAV/B130-NC group (Fig. 7D).

Functional rescue experiments using GLX351322 were performed following HtrA1 knockdown in bEnd.3 cells. The sh-HtrA1 + GLX351322 group exhibited a significant decrease in NOX4 expression compared with the sh-NC group (Fig. 8A). The present study evaluated expression levels of tight junction proteins, ZO1, occludin and CLDN5. Western blot analysis did not indicate any significant differences between the sh-HtrA1 + GLX351322 and sh-NC groups (Fig. 8A). In the FITC-dextran experiment, no significant increase in permeability was observed for the sh-HtrA1 + GLX351322 compared with the sh-NC group (Fig. 8B). There was no significant difference in levels of caspase3 in the sh-HtrA1 + GLX351322 compared with the sh-NC group (Fig. 8A). Moreover, flow cytometry showed that the apoptosis rate in the sh-HtrA1 + GLX351322 group was similar to that in the sh-NC group (Fig. 8C). These findings indicated that inhibiting NOX4 expression enhanced the expression of tight junction proteins, decreased cell permeability and mitigated apoptosis. Western blot analysis (Fig. 8D) showed elevated protein expression of NOX4 in the AAV/B130-shHtrA1 compared with the AAV/B130-NC mice. GLX351322 treatment significantly decreased the NOX4 levels, indicating effective inhibition. Additionally, GLX351322 increased the expression of tight junction proteins ZO1, occludin and CLDN5 and decreased the expression of caspase3 compared with that in the AAV/B130-shHtrA1 group. The Evans Blue assay revealed decreased BBB permeability following GLX351322 injection (Fig. 8E).

In the OFT, the AAV/B130-shHtrA1 mice exhibited significantly decreased total distance traveled and decreased time spent in the central region compared with the AAV/B130-NC mice (Fig. 9A-C). However, GLX351322 significantly increased the total distance travelled and increased time spent in the central region in the AAV/B130-shHtrA1 mice (Fig. 9A-C). AAV/B130-shHtrA1 mice showed decreased rearing and increased defecation frequency compared with the AAV/B130-NC mice (Fig. 9D and E). By contrast, the AAV/B130-shHtrA1 + GLX351322 mice exhibited increased rearing and decreased defecation frequency (Fig. 9D and E). These results indicated that GLX351322 effectively reversed depressive immobility and anxiety in the AAV/B130-shHtrA1 mice.

In the NORT (Fig. 10A), no significant difference in the exploration time for two identical objects during the familiarization period was observed between groups (Fig. 10B). During the test phase, the NOI in AAV/B130-shHtrA1 mice was significantly lower than that in AAV/B130-NC mice, whereas intraperitoneal injection of GLX351322 reversed this decline (Fig. 10C). These results indicated that the intraperitoneal injection of GLX351322 rescued memory deficit in the AAV/B130-shHtrA1 mice. MWM test was used to evaluate spatial learning and memory in mice. No difference in mean swimming speed was observed between the groups during the entire MWM test (Fig. 10D). The escape latency of AAV/B130-shHtrA1 mice was markedly longer on training days 3 and 4 than that of the AAV/B130-NC group, whereas the intraperitoneal injection of GLX351322 significantly shortened the escape latency of AAV/B130-shHtrA1 mice on day 4 during the place navigation-training phase (Fig. 10E), indicating GLX351322 relieved the spatial learning deficit in AAV/B130-shHtrA1 mice. In the probe test, the AAV/B130-shHtrA1 mice demonstrated significantly decreased percentage of swimming time in the target quadrant (Fig. 10F) and number of platform crossings (Fig. 10G) compared with the AAV/B130-NC mice, whereas these decreases were reversed in AAV/B130-shHtrA1 + GLX351322 mice, indicating GLX351322 proficiently improved the spatial reference memory in AAV/B130-shHtrA1 mice. Representative trajectories on training day 4 (Fig. 10H) and during the probe test (Fig. 10I) are shown.