mRNA sequencing reveals the distinct gene expression and biological functions in cardiac fibroblasts regulated by recombinant fibroblast growth factor 2

Materials

Recombinant histidine-tagged LMW-FGF2 protein (18 kDa) was used as FGF2 protein drug in this study. Recombinant FGF2 was expressed by the prokaryotic expression system (E. coli strain: BL21; Transgen Biotech, Beijing, China) and purified by heparin affinity gel (Bio-Rad, Hercules, California, USA) and nickel affinity gel (GE Healthcare Life Sciences, Chicago, Illinois, USA), as described previously (Sun et al., 2015, 2019). For primary rat cardiac fibroblast isolation and culture, the following materials were used: collagenase type II (Gibco, Thermo Fisher Scientific, Shanghai, China), Dulbecco’s modified Eagle’s medium (DMEM) containing 1,000 mg/L glucose and 4 mM L-Glutamine (HyClone Laboratories, Logan, UT, USA), fetal bovine serum (FBS, Gibco, Billings, Montana, USA), phosphate buffered saline (PBS, Solarbio, Beijing, China), trypsin (Solarbio, Beijing, China), and penicillin-streptomycin (Solarbio, Beijing, China), cell culture dishes and plates (Corning, Oneonta, NY, USA). For RNA isolation and quantification, RNAiso Plus and TB Green Premix Ex Taq II were purchased from Takara Biomedical Technology (Beijing, China), and RevertAid First Strand cDNA Synthesis kit was ordered from Thermo Fisher Scientific. Antibodies, including Anti-DDR2 antibody (ab221812), Anti-alpha smooth muscle Actin (ab7817), anti-collagen type III antibody (ab7778), and Alexa Fluor® 488 conjugated antibodies (ab150113 for anti-mouse and ab150077 for anti-rabbit) were purchased from Abcam (Shanghai, China). Propidium iodide (PI, Beyotime, Shanghai, China) was used to stain nuclei and 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma, Steinheim, Germany) was applied to stain the cells for fluorescence imaging.

CFs isolation and culture

Neonatal Sprague-Dawley rats (10–15 rats, 3 days old) were obtained from Experimental Animal Centre of Xinxiang Medical University and the animal studies were approved by the Xinxiang Medical University Animal Supervision Committee (approval no. XYLL-2020078). The neonatal rats were decapitated using sterile scissors, and hearts were extracted from the body and immediately washed with DMEM medium. The isolated hearts were cut into small pieces (approximately 1 mm³). The small tissues were digested by collagenase type II (0.05% w/v) and trypsin (0.06% w/v) in DMEM for 10 min at 37 °C, and the supernatant was transferred into DMEM with 10% (v/v) FBS. The digestion was repeated for three times. The cells and undigested tissues were separated by filtration and the isolated primary cells were pelleted by centrifugation at 1,000 × g for 5 min. The primary cells were seeded into a tissue culture plate with DMEM containing 10% FBS and 1% (v/v) penicillin/streptomycin for fibroblast adhesion, and after 45 min the unattached cells were removed to purify CFs. CFs were subcultured for three passages (P0-P3) to detected their differentiation in vitro. The primary CFs were split into six well plates for RNA isolation and 48 well plates for immunofluorescence. To determine the effect of FBS on CFs activation, the cells were cultured with DMEM containing 0%, 2.5%, 5% and 10% (v/v) FBS for 24 h. The bright field images were captured by an inverted microscope (T-DH; Nikon, Tokyo, Japan).

Treatment CFs with FGF2

Different concentrations of FGF2 (1, 3, 10, 30, 100 ng/mL) were added into DMEM with 2.5% FBS to determine their regulatory functions on CFs growth and activation. Subsequently, 30 ng/mL FGF2 was used to treat CFs for determination of its functions. As CFs attached to the plate, CFs in control group (CFs-CON) were treated with DMEM containing 2.5% FBS and CFs in FGF2-treated group (CFs-FGF2) were treated with DMEM containing 2.5% FBS and 30 ng/mL FGF2. The treated cells were collected for mRNA quantification and immunofluorescence imaging. For detection of the expression of collagen type III, CFs-CON and CFs-FGF2 were incubated for 5 days and the culture medium was refreshed every 2 days.

Immunofluorescence imaging

The cultured CFs in 48 well plates were fixed with 4% (w/v) paraformaldehyde (PFA) for 15 min and permeabilized with 0.3% (v/v) Triton X-100 for 15 min at room temperature. The cells were blocked with goat serum for 1 h. The purity of isolated cells was quantified by detecting the expression of discoidin domain receptor 2 (DDR2, antibody concentration: 2 μg/mL). To detect CFs activation, expression of α-SMA (antibody concentration: 1 μg/mL) was determined with immunofluorescence staining. Expression of collagen type III (antibody concentration: 2 μg/mL) was detected to validate the mRNA sequencing result. The fluorescent secondary antibodies (1:500 dilution) were applied to display fluorescence for imaging. Cell nuclei were stained with PI. Fluorescent images were captured by a laser scanning confocal microscope (FV1000; Olympus, Tokyo, Japan). Each imaging experiment was performed with three biological replicates and two technical replicates.

Real-time polymerase chain reaction

CFs-CON and CFs-FGF2 were collected with RNAiso Plus, and total RNA was extracted and quantified by Nanodrop ND-100 (Thermo Fisher Scientific, Shanghai, China). Reverse transcription was performed according to RevertAid First Strand cDNA Synthesis kit protocol. The primers used in this study were designed with Oligo 6 (Molecular Biology Insights, Cascade, CO, USA), and the primer synthesis was performed by GENEWIZ (Suzhou, China). The sequences of primers are listed in Table S1. RT-PCR was carried out with TB Green Premix Ex Taq II on a QuantStudio^TM 7 Flex Real-Time PCR system (Applied Biosystems, Life Technologies, Waltham, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as the endogenous standard for subsequent analysis. Three biological replicates were performed for RT-PCR experiments.

RNA sequencing and data analysis

CFs-CON and CFs-FGF2 were collected with Trizol and mRNA was extracted by RNeasy Mini Kit (Qiagen, Shanghai, China). The total RNA was quantified and qualified by Nanodrop and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Library preparation and RNA sequencing with Illumina NovaSeq 6000 were processed by GENEWIZ. HISAT2 (v2.0.1) was used to align the collected gene sequences to Rattus genome (Rattus_norvegicus.Rnor_6.0.dna.toplevel) and HTSeq (v0.6.1) estimated gene expression levels from the pair-end clean data. The mapped reads were normalized to fragments per kilobase of exon model per million mapped fragments (FPKM) for comparison of gene expression levels. Three biological replicates were used in each group and the Pearson correlation coefficient is over than 0.92 (Fig. S1). The statistical power of this experimental design, calculated in RNASeqPower is 0.929. DESeq2 Bioconductor package was performed to analyze the difference of gene expression. Volcano plot and GeneExpression-FoldChange plot (scatter plot of gene expression level against fold change) were displayed in Matlab (2019a) to visualize the significantly regulated genes (log2(foldchange) ≥ 0.75 or log2(foldchange) ≤ −0.75) with limited expression levels (log10(baseMean) ≥ 2) and q values (q < 0.05, p values adjusted by false discovery rate (FDR) method). The MATLAB code for volcano plot and GeneExpression-FoldChange plot can be downloaded from GitHub at https://github.com/hscsun/ScatterFoldChanges.git. Gene ontology (GO) enrichment was analyzed and displayed with clusterProfiler package in R (3.6) and Metascape (Wu et al., 2021b; Zhou et al., 2019).

Cell adhesion assay

CFs were incubated in a 6-well plate at a density of 5 × 10⁵ cells/well with and without FGF2 for 24 h. Both CFs-CON and CFs-FGF2 were trypsinized for 30 s and immediately fixed with 4% PFA. After washing with PBS, the cells were stained with CFSE for 1 h at room temperature. Nuclei were stained with PI. Images were taken by a laser scanning confocal microscope.

CFs isolation and culture in vitro

Cardiac tissues were dispersed to isolate cardiac cells and the isolated cells were seeded into a plate. After 45 min, the unattached cells were removed by medium replacement to purify CFs and the purified CFs were subcultured to detect their activation in vitro (Fig. 1A). The immunofluorescence result shows the isolated cells express DDR2 indicating the isolated cells are mainly cardiac fibroblasts (Fig. 1B). Bright field imaging shows the morphology of primary CFs is more solid and after one passaging the morphology of CFs is relatively flat (Fig. 1C), which is consistent with the previous findings (Santiago et al., 2010). The immunofluorescence results also show the expression of α-SMA in CFs was increased as cultured in vitro (Fig. 1D). Since the isolated CFs were rapidly activated during incubation in vitro, the first passage of CFs was applied for drug treatment and determination of their biological activities.

CFs were treated with DMEM containing 0%, 2.5%, 5% and 10% of FBS for 24 h and fixed with 4% PFA. Expression of α-SMA of fixed CFs was determined to reveal the regulatory effect of FBS on CFs activation. The results show that CFs treated with 2.5% FBS express more α-SMA than CFs treated with 5% and 10% FBS (Fig. 2), suggesting CFs activation was more obvious in low serum medium. The nucleic acid staining of CFs treated without FBS was weaker than that of other groups, indicating the essential role of FBS in DNA synthesis (Fig. 2). To reduce the effect of FBS on CFs gene expression, DMEM containing 2.5% FBS was used for treatment of CFs with FGF2.

Regulatory effect of FGF2 on CFs activation

CFs were treated with induction medium containing various concentrations of FGF2 (0, 1, 3, 10, 30, 100 ng/mL) for 24 h to determine the effects of FGF2 on CFs activation. Expression of α-SMA protein was detected to analyse the level of CFs activation. It shows that incubation with FGF2 (3–30 ng/mL) could significantly reduce the expression of α-SMA in CFs and 30 ng/mL FGF2 displayed a better suppressive effect than 3–10 ng/mL FGF2 (Fig. 3A). RT-PCR results show that FGF2 could significantly supress the expression of myofibroblast marker genes (Acta2 and Tagln) and ECM genes (Col3 and Ccdc80) and higher concentration was more efficient (Fig. 3B). These indicate that FGF2 is a potential protein drug to inhibit CFs activation and the inhibitory effect is sensitive and concentration-dependent. Thus, 30 ng/mL FGF2 was applied to treat CFs to detect its effect on CFs, and the bright field imaging results show that the cellular morphologies of CFs-FGF2 are slenderer than CFs-CON, indicating FGF2 has a significant regulatory effect on CFs (Fig. 3C).

Sequencing analysis of CFs gene expression

Highly expressed genes in CFs

To explore the regulation of FGF2 on gene expression, CFs-FGF2 and CFs-CON respectively incubated with and without FGF2 were collected for mRNA sequencing. The mapped genes in CFs-CON were sorted by gene expression level (FPKM), and it shows that the top 60 highly expressed genes contain many genes of ECM proteins, e.g., Col1a1, Col3a1, Sparc, Fn1, Actb, Acta2, Tagln, Tpm4 and Myl6 (Fig. 4A). Some of these are up-regulated or down-regulated by FGF2, e.g., Actb, Acta2, Col3a1, Sparc and Ctgf (Fig. 4A). Enriched functions of the top 500 highly expressed genes were analysed by Metascape. The results demonstrate that the high expressed genes are enriched in ribosome, metabolic process, homeostasis, ECM organization, wounding, supramolecular fiber organization, cell motility, cell-substrate adhesion, smooth muscle contraction, etc., which are consistent with the biological activities of fibroblasts (Fig. 4B). In these enriched functions, ECM organization, collagen metabolic process, wounding, supramolecular fiber organization, cell-substrate adhesion and focal adhesion are strongly related functions, which are involved in ECM organization and fibrosis (Fig. 4B).

Differentially expressed genes (DEGs) regulated by FGF2

The mapped genes were scattered into five groups, low expression genes (log10(baseMean) < 2), not significant genes (q ≥ 0.05), low fold change genes (−0.75 < log2(fold change) < 0.75), up-regulated genes and down-regulated genes. Of these, 430 up-regulated genes and 391 down-regulated genes are considered to be the differently expressed genes regulated by FGF2 (Figs. 5A and 5B). The heatmap of differently expressed genes indicates that gene expressions of the repeated samples are consistent, while the expression levels of CFs-CON and CFs-FGF2 are obviously different (Fig. 5C). To analyse the effect of FGF2 on CFs biological functions, the differently expressed genes were applied to GO enrichment analysis. The enriched functions of biological process (BP), molecular function (MF) and cellular component (CC) were clustered and the major functions with high gene counts and low similarity were displayed to recognise the significantly regulated functions (Fig. 5D). Angiogenesis, protein kinase activity, cell-substrate adhesion, actin filament organization and extracellular matrix organization are involved in BP group (Fig. 5D). The MF group contains functions including receptor regulator activity, enzyme activator activity, actin binding, collagen binding, etc. (Fig. 5D). Differently expressed genes related to cell components are enriched in ECM, adherens junction, receptor complex, membrane microdomain, actin cytoskeleton, site of polarized growth and apical part of cell (Fig. 5D). Actin filament organization is enriched in all these three groups, and both ECM organization and receptor complex are also strongly enriched functions. These indicate FGF2 significantly regulates transcription of receptor-ligand genes, ECM component genes and actin cytoskeleton genes. To determine the accuracy of the mRNA sequencing, we quantified some significantly regulated genes, including Acta2, Col3a1, Ccdc80, Eln, Gas6, Itgb1, Tagln and Thbs2, and the results show that the RT-PCR results are consistent with mRNA sequencing results (Fig. 5E), suggesting the mRNA sequencing result is reliable and could be used to analyse the effects of FGF2 on CFs gene expression and biological function.

The principle enriched functions regulated by FGF2 in cellular component cluster

Cellular component enrichment presents the enriched genes and functions in cellular component organization. The GO enrichment network of cellular component shows that the highly enriched functions are involved in membrane organization (pink labelled functions), pericellular and extracellular matrix organization (blue labelled functions) and cytoskeleton organization (brown labelled functions) (Fig. 6A). Of these, ECM, adherens junction, actin cytoskeleton, which are fundamental for CFs activation and tissue fibrosis. In actin cytoskeleton group, many genes were strongly decreased, e.g., Myh7a, Myh9a, Myh10, Myh11, Cald1, Lmod1, and Daam1 (Fig. 6B). A large number of genes related to ECM, including genes of ECM protein (Col3a1, Col11a1, Cthrc1, Postn, Eln, Fmod, Ccdc80, Gpc4, Gpc6, etc.) and genes of signalling proteins (Wnt4, Tgfb2, Tgfb3, etc.), were decreased, although some genes were increased, e.g., Col7a1, Mmp16 and Col8a1 (Fig. 6B). In adherens junction cluster, a bunch of genes, e.g., Plekha7, Jag1, Itgbl1 and a bunch of genes, e.g., Plekha7, Jag1 and Itgb1, were decreased (Fig. 6B).

The differently expressed genes were also enriched in clusters of membrane microdomain, basolateral plasma membrane and transport vesicle, and these functions were up-regulated by the relevant genes (Fig. S2). Gene set enrichment analysis (GSEA) results indicate actin cytoskeleton was suppressed by FGF2, while axon guidance was promoted, and many genes in cell leading edge function were increased as listed suggesting FGF2 might promote the polarized growth of CFs (Fig. S3).

GSEA was also applied to quantify effects of FGF2 on ECM organization and cell adhesion of CFs. The analysis results indicate both functions were down-regulated by FGF2 (p < 0.05), suggesting FGF2 could suppress these cell functions (Fig. 7A). Expression of collagen type III in CFs-CON and CFs-FGF2 were stained, and it shows collagen type III could be expressed and accumulated by CFs-CON, while it was not strongly accumulated in CFs-FGF2 (Fig. 7B). These demonstrated that activated CFs expressed large amounts of collagen and induced collagen accumulation, but FGF2 could to some extent suppress this expression and accumulation. To determine the adhesion of CFs to substrate, the cells were stained with CFSE to visualize the adhesion area of the cells. The results show that the adhesion areas of CFs-FGF2 are much smaller than those of CFs-CON, indicating FGF2 could decrease the adhesion of CFs to substrate (Fig. 7C).