Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection and were cultured in endothelial cell medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. HUVECs were treated with 5 ng/ml Ang-II (Sigma-Aldrich; Merck KGaA). A total of 24 h later, cells were harvested and used for further experiments.

Cells were treated with 20 µM DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl]- S-phenylglycine t-butyl ester) (Sigma-Aldrich; Merck KGaA), a Notch pathway inhibitor, for 24 h at 37°C.

Cells (5×10³) were seeded in 96-well plates. After treatment with Ang-II (5 ng/ml) for 24 h, cell viability was determined with a CCK-8 assay (Beyotime Institute of Biotechnology), according to the manufacturer's instructions.

Apoptosis analysis was performed using a FITC-Annexin V apoptosis detection kit (BD Biosciences). Cells (1×10⁶) were harvested and washed once with PBS. Then, cells were incubated with propidium iodide and FITC-Annexin V for 15 min at 4°C. After washing with PBS three times, cells were suspended in binding buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc.) and FlowJo software (version 10; FlowJo LLC). Early + late apoptosis was assessed.

A total of 12 female Sprague-Dawley (SD) rats (age, 6 weeks; weight, 153±12.4 g) were randomly divided into two groups: Hypertension model group (n=6) and sham operation group (sham group, n=6). One rat in the hypertension model group died, the cause of death was cerebral infarction, it was found with symptoms of hemiparalysis and the dissection was performed after death. The duration of the animal experiment was 1 month. Animal health and behavior were monitored once a day post-surgery. All the animals were kept in the specific-pathogen-free animal laboratory at 24±1°C with 50±10% humidity, 12-h light/dark cycles, and free access to food and water. The weight of the SD rats was measured before surgery. Rats were anesthetized with pentobarbital sodium (30 mg/kg). Under sterile conditions, an incision was made in the mid scapular region and an osmotic pump containing angiotensin II (infusion rate, 0.7 mg/kg per day) was implanted. Blood pressure was measured once before surgery, and 1 week and 3 weeks after surgery. Sham-operated rats underwent the same surgical procedure after anesthesia, except that no osmotic pump was implanted. Rats were euthanized by pentobarbital sodium (150 mg/kg), and the heart rates were detected to verify death. All experimental protocols were approved by the Committee of Animal Care and Use at Sun Yat-sen University (approval no. SYSU-IACUC-2019PGY260K; Guangdong, China).

Protein from cells or tissues were isolated and lysed in RIPA lysis buffer (Sigma-Aldrich; Merck KGaA), containing protease inhibitors (Sigma-Aldrich; Merck KGaA). Protein concentration was determined by the Braford method (BioRad Laboratories, Inc.). Proteins (50 µg) were separated via SDS-PAGE on a 10% gel, and subsequently transferred onto PVDF membranes. The membrane was blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4°C with anti-collagen (COL)-I (cat. no. ab260043), anti-matrix metalloprotease (MMP)2 (cat. no. ab92536), anti-MMP9 (cat. no. ab38898), anti-cav-1 (cat. no. ab18199), anti-Notch1 (cat. no. ab167441) and GAPDH (cat. no. ab9485) antibodies (all 1:1,000; all purchased from Abcam). Subsequently, the membrane was incubated with a HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (1:5,000; cat. no. ab6721; Abcam) for 2 h at room temperature. The proteins were detected by ECL chemiluminescence (EMD Millipore) and analyzed using ImagePro Plus software (version 6.0; Media Cybernetics, Inc.).

siRNAs (si-1, si-2, and si-3) targeting cav-1 and control siRNA were synthesized by Shanghai GenePharma Co., Ltd. siRNA transfection (50 nmol) was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 48 h post-transfection, cells were collected and used for further experiments.

Total RNA from cells and tissues was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The following temperature protocol was used for reverse transcription: 37°C for 2 min, 23°C for 10 min, 55°C for 10 min and 85°C for 10 min. RT-qPCR was conducted on a PRISM 7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR-Green (Tiangen Biotech Co., Ltd.). The following thermocycling conditions were used for qPCR: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The expression was calculated using the 2^−ΔΔCq method (20). GAPDH was used as a control. Primers used are shown in Table I.

Tissues were fixed with 4% paraformaldehyde overnight at room temperature. Paraffin-embedded tissue slices (thickness, 4 µm) were dewaxed and rehydrated. Slices were blocked with 10% goat serum (Thermo Fisher Scientific, Inc.) for 30 min at room temperature, and then incubated with anti-cav-1 (1:1,000; cat. no. ab17052; Abcam) at 4°C overnight. After washing with PBS, slices were incubated with Alexa 555-conjugated secondary antibody (1:5,000, cat. no. A28180; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. Diamidino-phenyl-indole (DAPI) was utilized to stain the nuclei. Images were obtained using a fluorescence microscope (Nikon Corporation) and analysed using ImagePro Plus software (version 6; Media Cybernetics, Inc.).

To observe the alteration of the cerebral vein following the induction of hypertension, rat brain tissues were sliced into ~1 mm³ and the white-matter region with small veins were incubated in 2.5% glutaraldehyde for 12 h at 4°C. After washing with PBS, tissues were fixed with 1% osmic acid for 3 h at 4°C. Tissues were dehydrated and embedded in epoxy resin for 4 h at room temperature. Then, the tissue was cut into 70-nm sections. After staining with uranyl acetate at room temperature for 2 h and lead citrate at room temperature for 15 min, tissues were observed using a transmission electron microscope (Leica Microsystems, Inc.).

Statistical analysis was carried out using SPSS 19.0 software (IBM Corp.). Comparisons were analyzed by one-way ANOVA and followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

In order to understand the molecular mechanism of cav-1 in the pathogenesis of hypertension, an Ang-II-induced hypertension model was established in rats. As shown in Fig. 1A, rats infused with Ang-II (the hypertension group) showed a significantly lower venous flow velocity compared with the sham-operated rats. In contrast, the blood pressure in the hypertension group was significantly higher compared with the sham-operated group (Fig. 1B). Moreover, the thickness of the vessel walls was significantly higher compared with the sham-operated animals (Fig. 1C). Together, these results suggested that the rat hypertension model induced by Ang-II was successfully established.

The expression levels of cav-1 and Notch1 were examined in rat brain tissues. The protein levels of cav-1 and Notch1 were significantly increased in the brain tissue of the hypertension group compared with the sham-operated group, as characterized by immunofluorescence staining and western blotting, respectively (Fig. 2A and B). Consistent with these results, RT-qPCR analysis showed that the mRNA levels of cav-1 and Notch1 in the brain tissues of hypertensive rats were significantly higher compared with the sham-operated animals (Fig. 2C). Also, the mRNA expression of Notch3 was examined in the two groups, but there was no significant difference between them (Fig. S1).

To provide further support for the in vivo results, cultured HUVECs were utilized. A CCK-8 assay was carried out to determine the effect of Ang-II on HUVEC viability. The results showed that the viability of Ang-II-treated HUVECs was significantly decreased compared with the control cells (Fig. 3A). A flow cytometry assay revealed that Ang-II exposure significantly increased the apoptotic rate of HUVECs (Fig. 3B). In addition, RT-qPCR and western blotting demonstrated that Ang-II treatment significantly increased the mRNA and protein levels of collagen-1, MMP2 and MMP9, which are important regulators of the extracellular matrix (Fig. 3C and D). Therefore, these results indicated that Ang-II regulated HUVEC viability and altered extracellular matrix-associated protein expression.

Increased expression of cav-1 was also observed in HUVECs, following the administration of Ang-II (Fig. 4A). Thus, it was investigated whether cav-1 might contribute to Ang-II-mediated HUVEC cell viability and extracellular matrix alteration. To assess this, cav-1 expression was knocked down by siRNAs. As shown in Fig. 4B, the expression levels of cav-1 were significantly downregulated after transfection with cav-1 siRNAs (si-1, si-2, and si-3), as si-2 was shown to be the most effective it was selected for further experiments. It was found that the silencing of cav-1 restored the Ang-II-inhibited viability of HUVECs (Fig. 4C) Additionally, the enhancement of apoptosis in HUVECs induced by Ang-II was also reversed by cav-1 knockdown (Fig. 4D). Moreover, the depletion of cav-1 downregulated the mRNA and protein levels of collagen-1, MMP2 and MMP9, which were increased by Ang-II (Fig. 4E and F). Together, these results suggested that cav-1 reversed Ang-II-induced viability and extracellular matrix alteration in HUVECs.

The upregulation of Notch1 in Ang-II-infused mice allowed for the investigation of whether cav-1 may regulate vascular remodeling via Notch signaling. Indeed, consistent with the in vivo results, treatment with Ang-II increased the expression of Notch1, whereas knockdown of cav-1 decreased the protein expression of Notch1 (Fig. 5A). To confirm the involvement of Notch signaling activation in vascular remodeling, the Notch pathway inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester), was applied, which was demonstrated to decrease the expression of Notch1 (Fig. 5B). It was observed that while Ang-II treatment decreased the viability of HUVECs, DAPT treatment reversed this effect (Fig. 5C). Consistently, the increased apoptosis observed in Ang-II-treated HUVECs was decreased following DAPT administration (Fig. 5D). Moreover, treatment with DAPT reversed the Ang-II-mediated upregulation of Notch1, collagen-1, MMP2 and MMP9 (Fig. 5E and F). Overall, these results implied that cav-1/Notch functions in vascular remodeling.