Notch4 mediates vascular remodeling via ERK/JNK/P38 MAPK signaling pathways in hypoxic pulmonary hypertension

A group of surgical lung tissues were collected from 4 PH patients including 2 patients with PH secondary to chronic obstructive pulmonary disease and 2 patients with PH related to interstitial lung disease, and 4 patients without PH in the control group. According to the previous method in the study on PH [30], lung tissues from the control patients were commonly collected at a site remote from tumor foci.

All patients were enrolled from Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China). This study was approved by the Ethics Committee of Tongji Hospital. Written informed consent was obtained from each patient.

HPASMCs were obtained from Procell Life Science & Technology Corporation. All cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and maintained in a humidified incubator with 5% CO₂ at 37 °C. The hypoxic HPASMCs were cultured in a 5% O₂ incubator (Galaxy R; RS Bitotech, Alloa, UK) continually gassed with 5% CO₂ and 90% N₂.

The antibodies against proliferating cell nuclear antigen (PCNA), Bcl-2 associated X (Bax), survivin, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), β-Actin and normal rabbit or mouse immunoglobulin G (IgG) were obtained from Proteintech (Wuhan, Hubei, China). B cell leukemia/lymphoma 2 (Bcl-2) antibody was obtained from Boster (Wuhan, Hubei, China). P38, p-P38, JNK, p-JNK, ERK, p-ERK antibodies, and Protein G magnetic beads were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Notch4 was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). HRP-conjugated anti-Rabbit IgG and HRP-conjugated anti-Mouse IgG were obtained from Servicebio (Wuhan, Hubei, China). HRP-conjugated anti-Rabbit IgG light chain and HRP-conjugated anti-mouse IgG light chain were obtained from Abbkine Scientific (Redlands, CA, USA). U0126, SP600125 and SB203580 were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Small interfering RNAs (siRNAs) against Notch4 and negative control siRNA were synthesized by RiboBio (Guangzhou, China). HPASMCs were transfected with siRNAs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacture’s instruction. The siRNA against Notch4 sequence was as follows: CAACGGGCACUGUGAGAAA. When reaching 40–60% of confluence, the cells were transfected with 50 nmol siRNA using Lipofectamine 3000. Notch4 overexpression plasmid and negative control plasmid were synthesized by GeneChem (Shanghai, China). The mammalian expression plasmid for Flag-tagged Notch4 was constructed by molecular cloning. The construct was confirmed by DNA sequencing. When reaching 80–90% of confluence, the cells were transfected with 1 μg purified plasmid. Finally, the mRNA and protein levels of cells were analyzed by quantitative real-time PCR and western blotting at 48 h and 72 h after transfection, respectively.

Cell viability was measured using the Cell Counting Kit-8 assay. HPASMCs were seeded into 96-well plates at the cell density of 3000 cells per well for 24 h. Then the medium was replaced with different transfection mixture. After 6 h incubation, the transfection mixture was replaced with medium supplemented with 10% fetal bovine serum, and the cells were further incubated for 24 h. After that, cells were incubated under normoxic or hypoxic conditions for 24 h. Finally, 10 μL Cell Counting Kit-8 (Dojindo, Japan) reagent was added into per well and optical density in each well was determined by an ELx800 Universal Microplate Reader (BioTek Instruments, Winooski, VT, USA).

HPASMCs were seeded into 96-well plates (3000 cells per well) and cultured in normoxic or hypoxic conditions as mentioned above. Cell proliferation was assessed using Edu Cell Proliferation Assay Kit (RiboBio, Cell‐Light™ Edu Apollo^®643 In Vitro imaging kit), according to the manufacture’s instruction. The cells were added with 100 μL Edu (diluent reagent A with a complete medium by 1:1000) and incubated for 2 h at 37 °C. Then cells were fixed in 4% paraformaldehyde for 15–30 min and incubated with 1 × Apollo^® solution for another 30 min at room temperature. Cell nuclei were stained with 100 μL 1 × Hoechst33342 for 30 min. Finally, the cells were examined under a fluorescent microscope (Olympus, Japan). Data were shown as fold-change increase in the percentage of Edu-incorporating cells in treated cells, compared with negative controls.

HPASMCs were seeded into a 6-well plate (100,000 cells per well). Firstly, cells were starved in serum-free for 24 h. After transfection for 48 h, cells were exposed to normoxic or hypoxic conditions for another 24 h. Then cells were collected and resuspended in 200 μL binding buffer per well. The cells were labeled with 5 μL Annexin-V and propidium iodide (PI) to assess cell apoptosis using an Annexin-V/PI detection kit (Keygen Biotech, Nanjing, China). Finally, apoptotic cells were analyzed using flow cytometry (BD Biosciences, SanJose, CA). Apoptotic cells including early apoptotic cells (Annexin-V positive and PI-negative) and late apoptotic cells (Annexin-V positive and PI-positive) were shown.

Cell migration assay was performed in a 24-well plate (Corning, MA, USA) according to the manufacture’s instruction. Firstly, 200 μL cell suspensions (10,000 cells totally) containing 1% fetal bovine serum were seeded into the upper chamber, while 600 μL medium containing 15% fetal bovine serum were added into lower chambers. The cells were cultured in normoxic or hypoxic condition for 24 h. Then cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Non-migrated cells were removed by scraping the membranes with a cotton swab from the upper surface. Finally, cells were counted under an optical microscope (Zeiss, Oberkochen, Germany).

Total RNA was isolated using Trizol (Takara, Dalian, China) according to the manufacture’s instruction. RNA concentration was determined by Nanodrop analysis. cDNA was synthesized from total RNA using PrimeScript RT reagent kit (Takara, Dalian, China). Quantitative RT-PCR was performed using SYBR Green Mix (Takara, Dalian, China). The primer sequences were as follows: Notch4 forward, 5′-CGTACCCCACTTCACACTGC-3′, reverse, 5′-AGGTGTAGTCCCGTCGTCTG-3′.

The cycling conditions were as follows: initial denaturation for 10 min at 95 °C followed by 40 cycles of denaturation (15 s at 95 °C), annealing and elongation (30 s at 60 °C). The relative expression of gene was calculated using 2^−△△Ct method using β-Actin as the reference gene.

Total cellular protein was extracted by RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, PH7.4) supplemented with phenylmethylsulfonyl fluoride, cocktail, and phosphorylation protease inhibitor. Cell lysis was centrifuged at 12,000 rpm for 15 min and the supernatants were collected for determination of the protein concentration by BCA assay. All steps were performed at 4 °C. Then 50 μg of protein were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by western blotting. The signals were detected using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). Relative expressions of target proteins were quantified by Image J software.

Co-immunoprecipitation experiments were performed according to a standard protocol. Briefly, cells were harvested and lysed in IP-lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40) and protease inhibitor. Supernatants were collected by microcentrifuge (14,000 rpm, 10 min, 4 °C), and pre-cleared with 20 μL Protein G Magnetic beads (Cell Signaling Technology) for 2 h. Then the pre-cleared supernatants were incubated with primary antibodies (2 μg/mL) rotated overnight at 4 °C to form an immunocomplex. Then transferred immunocomplex solution to the tube containing pre-washed magnetic beads and incubated rotated for 3–4 h at 4 °C. Finally, the immunocomplex was washed five times with 500 μL of 1 × cell lysis buffer and analyzed by western blotting analysis.

Formalin-fixed, paraffin-embedded tissue sections were de-waxed and antigen retrieval with citrate buffer (PH = 6.0). Then sections were incubated in 3% hydrogen peroxide for 20 min at room temperature to block endogenous peroxidase, followed by blocked in 5% bovine serum for 40 min. Primary antibodies were added to sections and incubated overnight at 4 °C in a humidified chamber, then incubated with secondary antibody for 40 min at room temperature. For immunohistochemistry analysis, the sections were further incubated with the avidin–biotin-peroxidase complex, which was detected with diaminobenzidine. Finally, sections were counterstained by hematoxylin, dehydrated in graded ethanol, and mounted with Permount.

Frozen lung sections were fixed with 4% paraformaldehyde for 15 min. Primary antibodies were added to sections and incubated overnight at 4 °C in a humidified chamber after blocked with 5% donkey serum albumin for 1 h. Alexa 594-labeled anti-mouse and Alexa 488-conjugated anti-rabbit antibody (Invitrogen, CA, USA) were used as secondary antibodies. Nuclei were counterstained with DAPI for 5 min.

All animal experiments were performed in compliance with the guidelines for animal testing and research, with ethical approval from Tongji Hospital. Adult male Sprague–Dawley rats weighing 200–250 g were fed standard chow and water ad libitum. The rats were exposed to either normoxia or chronic hypoxia (10% O₂) 8 h daily for 5 weeks with a 12 h light–dark cycle. Hypoxic rats were randomly divided into four groups: hypoxic group (n = 5); AAV1-si-NC group (100 μL, n = 5); AAV1-si-Notch4 prophylactic group (100 μL, n = 7); AAV1-si-Notch4 therapeutic group (100 μL, n = 6). Hypoxic therapeutic group were intratracheal delivery of AAV1-si-Notch4 after exposed to a hypoxic atmosphere for two weeks. In the prophylactic group, AAV1-si-Notch4 was delivered at an equivalent dose to rats before exposed to the hypoxic chamber. At 5 weeks, rats were anesthetized with 1% pentobarbital injection (120 mg/kg ip). RVSP was measured via the right jugular vein using a 1.2-F pressure catheter. Then hearts and pulmonary vasculature were perfused with cold saline injection into the right ventricle. Hearts were excised and measured by Fulton’s Index (ratio of right ventricle weight to left ventricle plus septum weight, RV/[LV + S]). Right lungs were snap-frozen in liquid nitrogen and stored at – 80 °C for biochemical measurements. Left lungs were perfused with 4% paraformaldehyde, followed by paraffin embedding. Sections were stained with hematoxylin and eosin and immunohistochemistry for α-smooth muscle actin. The medial wall thickness index was calculated as follows: Wall thickness (%) = 100 × (external diameter − internal diameter)/external diameter.

Statistical analyses were performed using GraphPad Prism version 8.0. Results were plotted as means ± SD. The Student’s t-test was used for two-sample analyses, and normal distributions were assumed. One-way analysis of variance with Tukey’s post hoc test was used for more than two-sample analyses. All experiments were performed at least in triplicate. Significance was considered at the level of P < 0.05.

To investigate the expression of Notch4 in normal and pulmonary hypertensive lungs, we examined lung tissues from 4 individuals with HPH undergoing lung transplantation compared with lung tissues from 4 non-PH individuals (Tables 1, 2). The protein levels of Notch4 had a tendency to increase in HPH patients compared with non-PH patients (Fig. 1A). Immunofluorescent staining of human lung tissues showed that Notch4 was localized in the media and intimal layer of small pulmonary arterials. Higher level of Notch4 staining was seen in lung tissues with HPH compared to matched control lung tissues (Fig. 1B). Vascular smooth muscle cells (vSMCs) are the predominant constituents of media. The excessive vSMCs proliferation, migration and apoptosis resistance plays a key role in vascular remodeling in HPH. Therefore, we focus on the function and the underlying mechanisms of Notch4 in HPASMCs isolated from human small pulmonary arteries. Analysis of cultured HPASMCs exposed to normoxia or hypoxia demonstrated that Notch4 mRNA and protein levels were increased under hypoxic conditions and peaked at 3 h and 24 h, respectively (Additional file 1: Fig. S1A and S1B). Meanwhile, increased mRNA level of Delta-like 4 was also observed in HPASMCs exposed to hypoxia (Additional file 1: Fig. S1D), which may hint that hypoxia induces Notch4 signaling by increasing the levels of Delta-like 4 in HPASMCs.

In our study, hypoxia promoted HPASMCs proliferation and migration, and inhibited cell apoptosis. To assess the role of Notch4 involved in the process in HPASMCs, siRNA against Notch4 were transfected into HPASMCs. The mRNA and protein expression of Notch4 were dramatically decreased following transfection with siRNA against Notch4 in HPAMSCs (Additional file 1: Fig. S2). The knockdown of Notch4 partially abrogated the excessive cell proliferation and migration, and suppressed cell apoptosis resistance under hypoxia (Fig. 2A–E). The levels of proteins associated with cell proliferation (survivin and PCNA), apoptosis (Bax and Bcl-2), and migration (MMP9) were measured in HPASMCs transfected with si-Notch4 or si-NC. Consistently, up-regulation of survivin, PCNA, MMP9, and decreased ratio of Bax/Bcl-2 were observed in hypoxic HPASMCs and partially abrogated by transfection with siRNA against Notch4 (Fig. 2F). Taken together, these findings indicate that hypoxia-induced HPASMCs proliferation, migration and apoptosis resistance involves Notch4 signaling.

The experiments described above implicate that Notch4 signaling mediate the effect of hypoxia on HPASMCs proliferation, apoptosis, and migration. We next explored the molecular mechanisms in the process. The level of Notch4 overexpression was determined by qRT-PCR and Western blotting (Additional file 1: Fig. S3). Notch4 overexpression promoted HPASMCs proliferation and migration, and inhibited cell apoptosis (Fig. 3B–H). The interaction between Notch4 and MAPK pathway in hypoxia HPASMCs was investigated. Hypoxia upregulated the phosphorylation of ERK, JNK, and P38 in HPASMCs, which were partially abrogated by transfection of siRNA against Notch4 (Fig. 3A). Meanwhile, the specific inhibitors of ERK, JNK, and P38 pathways (U0126, SP600125, or SB203580) partially abrogated the excessive proliferation and migration, and apoptosis resistance of HPASMCs induced by Notch4 overexpression (Fig. 3B–H). Furthermore, we assessed the expression of proteins associated with cell proliferation, apoptosis, and migration. Consistently, Notch4 overexpression increased protein expressions of survivin, PCNA, MMP2, and decreased the ratio of Bax/Bcl-2, which were partly restored by co-treatment with U0126, SP600125, or SB203580 (Fig. 4A–C). Taken together, our results demonstrate that MAPK pathway is responsible for mediating the effect of Notch4 on HPASMCs proliferation, apoptosis, and migration under hypoxia. The interaction between Notch4 and ERK/JNK/P38 was confirmed using co-immunoprecipitation. The above results provide some clues for a novel crosstalk mechanism between non-canonical Notch signaling and MAPK pathway via interaction between Notch4 and ERK/JNK/P38 (Fig. 5A and B).

Our previous study has reported a rat model of pulmonary vascular remodeling exposed to hypoxia for 2 weeks [31]. An adeno-associated virus (AAV) 1- encoded si-Notch4 was intratracheally delivered into the rats on day 0 and week 2 after hypoxic exposure in the prophylactic group and therapeutic group, respectively. We showed that AAV1-si-Notch4 can cross biological barriers and effectively achieve targeted pulmonary vascular media (Additional file 1: Fig. S4) and vSMCs-specifically silence Notch4 (Fig. 7B). The results showed that silencing Notch4 significantly reduced the RVSP of HPH rats measured by right cardiac catheterization (Fig. 6A and B). There was no significant difference in systematic arterial blood pressure among all the groups (Fig. 6C).

The prevention and attenuation of hypoxia-induced vascular remodeling by airway luminal delivery of AAV1-si-Notch4 was assessed by hematoxylin and eosin staining, immunohistochemistry staining for α-smooth muscle actin (Fig. 7A), RV hypertrophy assessed by RV/(LV + S) weight and RV/BW weight (Fig. 6D and E). Consistently, the protein levels of p-ERK, p-JNK, and p-P38 were decreased in the pulmonary artery in HPH rats treated with AAV1-si-Notch4 compared with negative controls (Fig. 7C).

All animal experiments were performed in compliance with the guidelines for animal testing and research, with ethical approval from Tongji Hospital. All patients were enrolled from Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China). This study was approved by the Ethics Committee of Tongji Hospital and. Written informed consent was obtained from each patient.

Not applicable.

All authors declare that they have no competing interests.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.