Pulmonary vascular remodeling and fibrosis are the critical pathological characteristics of hypoxic pulmonary hypertension. Our previous study demonstrated that hypoxia is involved in the functional alteration of lung fibroblasts, but the underlying mechanism has yet to be fully elucidated. The aim of the present study was to investigate the effect of mast cells on the proliferation, function and phenotype of fibroblasts under hypoxic conditions. Hypoxia facilitated proliferation and the secretion of proinflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6, in human mast cells (HMC-1). RNA sequencing identified 2,077 upregulated and 2,418 downregulated mRNAs in human fetal lung fibroblasts (HFL-1) cultured in hypoxic conditioned medium from HMC-1 cells compared with normoxic controls, which are involved in various pathways, including extracellular matrix organization, cell proliferation and migration. Conditioned medium from hypoxic HMC-1 cells increased the proliferation and migration capacity of HFL-1 and triggered phenotypic transition from fibroblasts to myofibroblasts. A greater accumulation of collagen type I and III was also observed in an HFL-1 cell culture in hypoxic conditioned medium from HMC-1 cells, compared with HFL-1 cells cultured in normoxic control medium. The expression of matrix metalloproteinase (MMP)-9 and MMP-13 was upregulated in HFL-1 cells grown in hypoxic conditioned medium from HMC-1 cells. Similar pathological phenomena, including accumulation of mast cells, activated collagen metabolism and vascular remodeling, were observed in a hypoxic rat model. The results of the present study provide direct evidence that the multiple effects of the hypoxic microenvironment and mast cells on fibroblasts contribute to pulmonary vascular remodeling, and this process appears to be among the most important mechanisms underlying hypoxic pulmonary hypertension.
Pulmonary hypertension generally results from hypoxic lung diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis and bronchiectasis (
The pulmonary vascular wall is a heterogeneous three-layered structure composed of adventitia, media and intima. Pulmonary artery remodeling is a complicated pathological process involving disorders of all three layers of the vascular wall, including adventitial thickening, medial hypertrophy, neointima formation and plexiform lesions (
The aim of the present study was to investigate the effect of mast cells on the viability, function and phenotype of fibroblasts under hypoxic conditions. It was hypothesized that hypoxia-activated mast cells affect the characteristics of lung fibroblasts and are involved in pulmonary vascular remodeling. To test this hypothesis, the viability and secretion of cytokines by human mast cells (HMC-1) under hypoxia were first examined. Subsequently, the effects of hypoxic conditioned medium from HMC-1 cells on human fetal lung fibroblasts (HFL-1) were examined using RNA sequencing (RNA-seq) analysis and molecular biology experiments. Finally, using a rat model of hypoxic pulmonary hypertension, the distribution of mast cells, extracellular matrix remodeling and myofibroblast transition in the lung were explored.
The human mast cell line HMC-1 (Cellbio) was cultured in Iscove's Modified Dubecco's Medium (IMDM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin and 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA). HMC-1 cells were incubated at 37°C in a 5% CO2 atmosphere at 95% relative humidity. For hypoxic exposure, HMC-1 cells were placed in a modulator incubator in an atmosphere of 94% N2, 5% CO2 and 1% O2, and normoxic conditions were defined as 21% O2. Subsequently, cells and supernatants were collected for subsequent experiments. For the preparation of conditioned medium, the supernatants were centrifuged for 10 min at 500 × g at room temperature to remove detached cells and used directly. HFL-1 cells (Type Culture Collection Center, Chinese Academy of Sciences) were cultured in Ham's F-12K (Kaighn's) medium with 10% FBS, 100 mg/ml streptomycin and 100 U/ml penicillin until confluent.
Cells at 70% confluence were serum-starved in 0.1% FBS for 24 h for synchronization, plated at 5×103 cells per well in 96-well plates and cultured for 6 h at 37℃ in 5% CO2 in order to allow the cells to attach. Subsequently, HMC-1 cells were incubated with fresh IMDM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS under hypoxic conditions, and HFL-1 cells were cultured in conditioned medium from hypoxic or normoxic HMC-1 cells for different times. The Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.) was used to determine cell proliferation at various time points. For the CCK-8 assay, cells were incubated with 10
Secreted cytokines from HMC-1 cells, including tumor necrosis factor (TNF)-α (cat. no. KE00068, ProteinTech Group, Inc.), interleukin (IL)-1β (cat. no. KE00021, ProteinTech Group, Inc.), IL-6 (cat. no. KE00007, ProteinTech Group, Inc.) and IL-15 (cat. no. KE00102, ProteinTech Group, Inc.) were measured with the use of ELISA kits. ELISA was performed according to the manufacturer's protocol (ProteinTech Group, Inc.). Briefly, the supernatants or standards were added to 96-well microplates and incubated for 60 min at room temperature. After washing 3 times with wash buffer, the corresponding detection antibodies were added for 60 min at 37°C. The plate was washed with wash buffer, 100
Total RNA was extracted from HFL-1 and HMC-1 cells by use of TRIzol reagent (Tiangen), and cDNA was acquired by use of reverse transcriptase (Thermo Fisher Scientific, Inc.). qPCR with the use of Maxima SYBR-Green I qPCR Master Mix (Thermo Fisher Scientific, Inc.) was performed as follows: 95°C for 5 min, followed by 35 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 1 min. cDNA was amplified with the primer sequences for TNF-α (forward 5′-ccttcctctctccagatgtttc-3′, reverse 5′-cccggtctcccaaataaataca-3′), IL-1β (forward 5′-cctg-gactttcctgttgtctac-3′, reverse 5′-aagtgagtaggagaggtgagag-3′), IL-6 (forward 5′-cagctatgaactccttctccac-3′, reverse 5′-cgtcgag-gatgtaccgaattt-3′), IL-15 (forward 5′-ccactgtgtccggaattgat-3′, reverse 5′-gaacccaccagaaggaagaaa-3′), matrix metallopro-teinase (MMP)-9 (forward 5′-ggtaaggagtactcgacctgta-3′, reverse 5′-cggcactgaggaatgatctaag-3′), MMP-13 (forward 5′-ggcgacttc-tacccatttga-3′, reverse 5′-cttggagtggtcaagacctaag-3′), tissue inhibitor of metalloproteinase 1 (TIMP-1; forward 5′-gatg-gtgggtggatgagtaatg-3′, reverse 5′-gggttctctggtgtctctct-3′), collagen type I (forward 5′-cccagccaagaactggtatag-3′, reverse 5′-ggtgatgttctgagaggcatag-3′) and III (forward 5′-ttggaagtcctg-gtccaaag-3′, reverse 5′-caccaccttcacccttatctc-3′), α-smooth muscle actin (α-SMA; forward 5′-ccgaatgcagaaggagatca-3′, reverse 5′-gtggacagagaggccaggat-3′) and β-tubulin (forward 5′-gaggctgagagcaacatgaa-3′, reverse 5′-cagttgagtaagacg-gctaagg-3′). For quantification, target gene expression was normalized to that of β-actin in each sample. Data analysis involved the -ΔΔCq method (
Total proteins were extracted from treated cells using lysis buffer containing protease inhibitors (Beyotime Institute of Biotechnology). The protein concentration of the lysates was quantified with a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Total protein in lysates (50
RNA integrity was examined by 1% formaldehyde denaturing gel electrophoresis. RNA-seq was performed by Illumina HiSeq sequencer (Illumina, Inc.) at CapitalBio Corporation. The raw data of RNA-seq were aligned to the human genome version hg19. Differentially expressed genes with absolute fold change >2.0 and P<0.05 were considered statistically significant in the RNA-seq analysis. To better understand the roles of the differentially expressed mRNAs, biological process analysis was performed using the Gene Ontology (GO;
HFL-1 cells were seeded at 5×106 cells per well in 6-well plates and then incubated at 37℃ in 5% CO2 until confluence. Following preincubation with F12K medium with 0.1% FBS for 24 h for synchronization, a scratch was created in each well by using a 200-
The slides of HFL-1 cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with PBS three times, they were permeabilized with 0.1% Triton X-100 in PBS for 5 min on ice and then blocked with 3% BSA in PBS for 30 min at room temperature. Subsequently, HFL-1 cells were incubated with rabbit anti-α-SMA antibody (dilution, 1:1,000; cat. no. 55135-1-AP, ProteinTech Group, Inc.) or corresponding serum as a negative control at 4℃ overnight. Unbound antibodies were removed by washing with PBS three times, and the slides were incubated with goat anti-rabbit IgG-FITC antibody (1:500 dilution; cat. no. ab6717, Abcam) in the dark for 60 min. After washing with PBS three times, HFL-1 cells were stained with 4',6-diamidino-2-phenylindole (DAPI; 10
All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and approved by the Peking University First Hospital Ethical Review Committee (approval no. J201533). Male Sprague-Dawley rats (n=12, age, 8-10 weeks, weight, 220±10 g) were obtained from Vital River Laboratory Animal Technology Co. Ltd. and randomly divided into normoxic and hypoxic groups (n=6/group). A hypoxic rat model was established as described in our previous study (
The lung specimens were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5-
Statistical analysis involved the use of SPSS 16.0 (SPSS, Inc.). Quantitative data are presented as the mean ± standard deviation of at least 3 independent experiments. Student's t-test or one-way ANOVA followed by Scheffe's post hoc test was used to compare multiple groups. P<0.05 was considered to indicate statistically significant differences.
To examine the effects of hypoxia on the viability of human mast cells, HMC-1 cells were cultured under hypoxic conditions and their proliferation was detected. As shown in
To explore the changes in cytokines in human mast cells under hypoxia, we next cultured HMC-1 cells under hypoxia for the indicated time periods. The expression of IL-1β and IL-6 increased substantially after 12 h and peaked after 24 h, by almost three- and six-fold, respectively, in the medium of HMC-1 cells exposed to hypoxia compared with the normoxic controls. The expression of TNF-α mRNA was increased in a time-dependent manner, while the expression of IL-15 mRNA did not exhibit obvious changes (
To evaluate the effects of mast cells on lung fibroblasts under hypoxia, the HFL-1 cells were cultured in hypoxic and normoxic conditioned medium from HMC-1 cells and RNA-seq analysis was performed to compare the global mRNA expression profile. A box plot was drawn to visualize the distribution of the intensities of all the samples after normalization and the distribution of log2 ratios was found to be similar in all the samples (
The proliferation and migration of lung fibroblasts plays an important role in hypoxic pulmonary hypertension. To assess the effect of the secretion of paracrine factors from mast cells on human lung fibroblasts, HFL-1 cells were cultured in hypoxic and normoxic conditioned medium from HMC-1 cells and their proliferation and the migration were examined. Compared with normoxic conditioned medium from HMC-1 cells, hypoxic conditioned medium significantly increased the proliferation of HFL-1 cells (
To further characterize the role of mast cells in fibrogenesis, the expression of collagen I and III was first detected in HFL-1 cells under different culture conditions. The mRNA expression levels of collagen type I and III were both higher in the hypoxic conditioned medium compared with those in the control medium, and the protein expression displayed the same trend (
We investigated whether human mast cells affect the constitutive characteristics of lung fibroblasts under hypoxia. The expression of α-SMA mRNA and protein was upregulated in HFL-1 cells cultured in hypoxic conditioned medium from HMC-1 cells compared with HFL-1 cells cultured in normoxic conditioned medium (
Histological analysis revealed that the thickness of pulmonary vessels in rats in the hypoxic group was increased compared with the normoxic controls, while their vascular lumens were narrowed (
The normal pulmonary vascular system is well-organized and exhibits high compliance, but its physical structure is destroyed by pulmonary vascular remodeling in patients with pulmonary hypertension, which is characterized by increased vascular stiffness and reduced pulmonary arterial compliance (
Mast cells are derived from CD34-expressing hematopoietic stem cells and have traditionally been recognized as sentinel cells in allergic and non-allergic immune responses under physiological conditions (
Under physiological conditions, the balance between proliferation and apoptosis of vascular cells contributes to the maintenance of the thickness of the vascular wall. If the equilibrium is disturbed, the overproliferating cells may obstruct the vessel lumen, resulting in vascular remodeling and increased pulmonary pressure. The adventitial fibroblast is a pivotal regulator of vascular wall function and performs biological functions, including proliferation, differentiation, synthesis and secretion of extracellular matrix and other mediators (
The imbalance of extracellular matrix synthesis and degradation also contributes to the structural remodeling of peripheral pulmonary arteries and aggravates the pathological process (
The phenotypic alteration of lung fibroblasts activated by hypoxic conditioned medium from mast cells was consistent with our hypothesis that the multiple effects of the hypoxic microenvironment and mast cells on fibroblasts contribute to pulmonary vascular remodeling, and this process is likely one of the important mechanisms underlying the development of hypoxic pulmonary hypertension.
tumor necrosis factor-α
interleukin
matrix metalloproteinase
chronic obstructive pulmonary disease
Iscove's modified Dulbecco's medium
fetal bovine serum
Cell Counting Kit-8
enzyme-linked immunosorbent assay
tissue inhibitor of metalloproteinase
collagen type I
collagen type III
hypoxia-inducible factor
pulmonary arterial hypertension
α-smooth muscle actin
Gene Ontology
Kyoto Encyclopedia of Genes and Genomes
hematoxylin and eosin
phosphate-buffered saline
4′,6-diamidino-2-phenylindole
Not applicable.
The present study was supported by the National Natural Science Foundation of China (grant no. 81670043).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
XW, LL and XL conceived and designed the experiment. XW, XC, YW and YL performed the experiments. XW and XC performed data analysis. XW, LL and XL prepared and wrote the manuscript. All authors have read and approved the final manuscript.
All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the Peking University First Hospital Ethical Review Committee (approval no. J201533). All efforts were made to minimize animal suffering and the number of animals used.
Not applicable.
The authors declare that they have no competing interests.
Effects of hypoxia on the proliferation of HMC-1 cells. (A) Following culture under hypoxic conditions, the expression of HIF-1α in HMC-1 cells was determined by western blotting and used to confirm that cells exist within a hypoxic environment. (B) The proliferation of HMC-1 cells under hypoxic and normoxic conditions was examined by the Cell Counting Kit-8 assay for the indicated time periods. There were 4 biological replicates/condition. Error bars represent the standard deviation. *P<0.05, **P<0.01. HMC-1, human mast cells; HIF-1α, hypoxia inducible factor-1α; OD, optical density.
Effects of hypoxia on the secretion of cytokines by HMC-1 cells. (A) HMC-1 cells were cultured under hypoxic conditions for the indicated time periods and then subjected to reverse transcription-quantitative polymerase chain reaction to detect the expression of cytokines IL-1β, IL-6, IL-15 and TNF-α. β-tubulin was used as an internal control. (B-E) Following culture under hypoxic conditions, the levels of the secreted cytokines IL-1β, IL-6, IL-15 and TNF-α in the conditioned medium from HMC-1 cells were determined by ELISA. There were 4-6 biological replicates/condition. Error bars depict the standard deviation. *P<0.05, **P<0.01, NS, not significant. IL, interleukin; TNF, tumor necrosis factor; HMC-1, human mast cells.
Differential expression of mRNA in HFL-1 cells cultured in hypoxic and normoxic conditioned medium from HMC-1 cells. (A) Box plot of RNA sequencing dataset after normalization. (B) Hierarchical clustering of differentially expressed mRNAs in HFL-1 hypoxic group and normoxic controls. (C) Scatter plots demonstrated differential mRNA expression between hypoxic and normoxic samples. The red circles indicate upregulated genes, the green circles represent downregulated genes, and black circles indicate unchanged genes. (D) Volcano plots were generated to visualize the mRNA expression profiles between hypoxic and normoxic samples. (E and F) GO enrichment and KEGG analysis of differentially expressed mRNAs in HFL-1 cells cultured in the hypoxic and normoxic conditioned medium from HMC-1 cells. HFL-1, human lung fibroblasts; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; H, hypoxia; N, normoxia; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.
Effects of conditioned medium from HMC-1 cells on the proliferation and migration of HFL-1 cells. (A) After treatment with normoxic or hypoxic conditioned medium from HMC-1 cells, the proliferation of HFL-1 cells was assessed by the Cell Counting Kit-8 assay. (B) Migration of HFL-1 cells after culture in normoxic or hypoxic conditioned medium from HMC-1 cells was determined by scratch assay. Representative images before and after inflicting the wound are shown and quantified. There were 6 biological replicates/condition. Error bars depict the standard deviation. *P<0.05, **P<0.01, NS, non-significant. Bar, 1 mm. N-CM, normoxic conditioned medium; H-CM, hypoxic conditioned medium; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.
Effects of conditioned medium from HMC-1 cells on the production of collagens and MMPs by HFL-1 cells. (A) Following culture in normoxic or hypoxic conditioned medium from HMC-1 cells, the mRNA expression of collagen type I and III, MMP-9, MMP-13 and TIMP-1 in HFL-1 cells were assessed by reverse transcription-quantitative polymerase chain reaction analysis, normalized to the expression of β-tubulin, and expressed as fold change relative to the expression of the control group. (B) Western blot analysis of collagen type I and III, MMP-9, MMP-13 and TIMP-1 protein expression in HFL-1 cells cultured in conditioned medium from HMC-1 cells. There were 4 biological replicates/condition. Error bars depict the standard deviation. *P<0.05, **P<0.01, NS, not significant. COL I, collagen type I; COL III, collagen type III; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of metalloproteinase-1; N-CM, normoxic conditioned medium; H-CM, hypoxic conditioned medium; HMC-1, human mast cells; HFL-1, human fetal lung fibroblasts.
Effects of conditioned medium from HMC-1 cells on the transition of HFL-1 cells from fibroblasts to myofibroblasts. (A) After treatment with nor-moxic or hypoxic conditioned medium from HMC-1 cells, the expression of α-SMA mRNA was determined by reverse transcription-quantitative polymerase chain reaction analysis. β-tubulin was used as an internal control. (B) Western blotting was performed to assess the level of α-SMA protein in HFL-1 cells cultured in normoxic or hypoxic conditioned medium from HMC-1 cells. (C) After treatment with conditioned medium from HMC-1 cells, HFL-1 cells were stained for immunofluorescence for α-SMA. Nuclei were counterstained with DAPI. There were 4-6 biological replicates/condition. Error bars represent the standard deviation. *P<0.05, **P<0.01. Bar, 50
Effects of hypoxia on mast cells and ECM remodeling in rat lungs. (A) The pathological changes of pulmonary vessels in the rat lungs after exposure to hypoxia were examined by hematoxylin and eosin staining (magnification, ×200 and 400). (B) Toluidine blue staining demonstrated increased accumulation of mast cells in the lungs of hypoxic rats (magnification, ×200 and 400). (C and D) The expression of collagen I, collagen III, MMP-9, MMP-13 and TIMP-1 in the lungs of rats after exposure to hypoxia was determined by immunohistochemistry staining (magnification, ×200). ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; N, normoxic; H, hypoxic.
Top 20 upregulated and downregulated mRNAs in human fetal lung fibroblasts (HFL-1) cultured in hypoxic conditioned medium from human mast cells (HMC-1) and normoxic controls.
A, Upregulated mRNAs
| ||
---|---|---|
mRNAs | P-value | log2 fold change |
TNFSF8 | 0.000 | 9.4533 |
IL36RN | 0.020 | 9.1130 |
CYP19A1 | 0.000 | 8.5295 |
BEST3 | 0.001 | 7.0775 |
IL24 | 0.000 | 7.0084 |
WTAPP1 | 0.000 | 6.7566 |
STC1 | 0.000 | 6.6111 |
ESM1 | 0.002 | 6.6003 |
CCL3L3 | 0.002 | 6.5711 |
MMP10 | 0.002 | 6.5556 |
IL36B | 0.002 | 6.5535 |
THBD | 0.000 | 6.4407 |
CSF2 | 0.026 | 6.3719 |
DCSTAMP | 0.000 | 6.3002 |
IL1B | 0.000 | 6.2982 |
SHISA2 | 0.005 | 6.2159 |
LCE1C | 0.000 | 6.1842 |
EREG | 0.000 | 6.1640 |
PTGS2 | 0.000 | 6.1336 |
SERPINB2/10 | 0.000 | 6.0740 |
| ||
B, Downregulated mRNAs | ||
| ||
mRNAs | P-value | log2 fold change |
| ||
MUC19 | 0.038 − | 8.1010 |
USH2A | 0.000 − | 7.3792 |
TACR3 | 0.000 − | 7.3221 |
ADH1B | 0.000 | −7.0604 |
RSPO2 | 0.000 | −6.8458 |
CDSN | 0.000 | −6.6503 |
ITIH5 | 0.000 | −6.6445 |
SMTNL2 | 0.000 | −6.6222 |
RNU2-52P | 0.000 | −6.5828 |
PTGDR2 | 0.000 | −6.5349 |
ASTN1 | 0.000 | −6.5022 |
SPTB | 0.000 | −6.4692 |
OGN | 0.000 | −6.4411 |
LAD | 0.000 | −6.3950 |
NFE2 | 0.000 | −6.3828 |
GPR20 | 0.000 | −6.2826 |
MUC22 | 0.000 | −6.2617 |
TMEM37 | 0.000 | −6.2537 |
ABCA9 | 0.000 | −6.2412 |
DNAH2 | 0.000 | −6.2343 |