Silk fibroin promotes H3K9me3 expression and chromatin reorganization to regulate endothelial cell proliferation

B. SF treatment increases the level of H3K9me3 in ECs

To determine the differences in gene expression between SF-treated HUVECs and controls, we conducted a comparative RNA sequencing (RNA-seq) analysis of HUVECs exposed to 0.4% SF for 2 days. Differential expression analysis revealed 593 downregulated and 669 upregulated genes (cutoff: adjusted P value < 0.05, fold change ≥ 1.2) [Fig. 2(a)]. Downregulated genes are represented in blue, while upregulated genes are represented in red. To explore the differences in gene expression between the two culture conditions, we conducted the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, which revealed a preference for the upregulation of multiple cell cycle pathways in SF-cultured cells (Fig. S4). Additionally, gene ontology (GO) term analysis demonstrated that, compared with the control treatment, SF treatment led to the upregulation of genes associated with cell cycle progression [Fig. 2(b)].

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FIG. 2.

SF treatment increases the level of H3K9me3 in ECs. (a) Heatmap showing the changes in gene expression in the SF group (n = 3) compared with the control group (n = 3). The expression of significantly upregulated (n = 669, red) or downregulated (n = 593, blue) genes (adjusted P value < 0.05, FC ≥ 1.2) is indicated. The legend for the color scale is shown. (b) Bar plot of GO biological process terms for the significantly upregulated genes. (c) Heatmap showing that 26 genes related to histone modification were upregulated under SF treatment conditions. (d) Western blot analysis was performed to detect H3K9me3 levels in HUVECs treated with 0.2% or 0.4% SF solution for 2 days. (e) Immunofluorescence staining was used to detect H3K9me3 levels in control and SF-treated HUVECs after 2 days (scale bar = 50 μm). Hoechst was used as a DNA counterstain. (f) The mean fluorescence intensity of H3K9me3 was compared (n > 150). (g) The observed reorganization of H3K9me3-marked chromatin in the SF groups (scale bar = 2 μm). (h) HUVECs were treated with 0.2% or 0.4% SF solution for 2 days and stained for H3K9me2 (scale bar = 10 μm). Hoechst was used as a DNA counterstain. (i) The mean fluorescence intensity of H3K9me2 was compared (n > 150). The mean ± SEM is presented based on more than three separate experiments. Statistical significance is indicated by ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001, while 'ns' indicates no statistical significance.

Subsequently, we leveraged our RNA-seq data to identify gene expression changes related to chromosome organization and histone modification. Notably, SF treatment primarily increased the expression of H3K9me3-related genes, including HDAC1, EGR1, UHRF1, RCOR2, and SUV39H1 [Figs. 2(c) and S5]. As a result, we hypothesized that SF treatment would elevate H3K9me3 levels and contribute to cell proliferation. To validate this hypothesis, HUVECs were treated with DPBS, 0.2% SF, or 0.4% SF for 2 days. Western blot analysis revealed that SF treatment upregulated H3K9me3 levels [Fig. 2(d)]. In parallel, HUVECs treated with SF exhibited a significantly greater H3K9me3 level [Figs. 2(e) and 2(f)]. SF solution treatment also increased the H3K9me3 level in mouse brain endothelial bEnd.3 cells (Fig. S6).

Moreover, cells treated with SF displayed an accumulation of H3K9me3-modified chromatin within the nuclear inner cavities on day 2 compared to that in the control group [Fig. 2(g)]. Furthermore, immunofluorescence staining analysis of HUVECs following 2 days of SF treatment revealed no significant difference in the level of H3K9me2 compared to those in the control groups [Figs. 2(h) and 2(1)]. In summary, these findings suggest that the upregulation of H3K9me3 in EC following SF treatment may be linked to cell proliferation.

C. H3K9me3 is responsible for SF-induced EC proliferation

To investigate whether the increase in H3K9me3 influenced the proliferation of HUVECs in the SF-treated group, we assessed the impact of reducing H3K9me3 levels using TSA, a small molecule inhibitor of histone deacetylase (HDAC).¹⁹ HUVECs were exposed to 0.4% SF, 100 nM TSA, or a combination of 100 nM TSA with 0.4% SF for two days. The negative control sample was treated with DMSO. Western blot analysis was also conducted under identical conditions, revealing that TSA reduced the level of H3K9me3 in a manner consistent with the pattern observed in SF-induced H3K9me3 [Fig. 3(a)]. Subsequently, we performed immunofluorescence staining and quantified the mean fluorescence intensity of H3K9me3. The levels of H3K9me3 were greater in the TSA-treated group treated with SF than in the TSA-treated group [Figs. 3(b) and 3(c)]. Treatment with both TSA and SF resulted in increased cell proliferation compared to treatment with TSA alone [Fig. 3(d)]. These results strongly suggest that SF treatment leads to an increase in the level of H3K9me3, subsequently promoting cell proliferation.

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FIG. 3.

H3K9me3 is responsible for SF-induced EC proliferation. (a) Western blotting was used to detect the expression levels of H3K9me3 in the control, SF, TSA, and TSA treated with SF groups. (b) Immunofluorescence staining was used to detect the level of H3K9me3 in the control, SF, TSA, and TSA-treated SF groups (scale bar = 50 μm). (c) The fluorescence intensity of H3K9me3 in each group was analyzed and compared (n ≥100). (d) Proliferation of HUVECs cultured with DMSO, SF, TSA, or TSA and treated with SF for 2 days. The means ± SEM are presented based on more than three separate experiments. Compared with control, statistical significance is indicated by ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001. Compared with the TSA-treated group, statistical significance is indicated by ##p ≤ 0.01 and ###p ≤ 0.001. The “ns” indicates no statistical significance. The statistical significance was estimated using a two-tailed t-test. The statistical significance was estimated using a two-tailed t-test.

D. SF regulates the reorganization of nuclear chromatin

Notably, our analysis revealed that, according to the GO cellular component term, genes related to nuclear components were predominantly upregulated, including the nucleus, nucleoplasm, chromosome, and condensed chromosome [Fig. 4(a)]. Furthermore, the GO molecular functional term analysis demonstrated that SF treatment induced the upregulation of genes associated with nuclear transcriptional regulation, such as genes related to nucleoside-triphosphatase activity, chromatin binding, and histone binding (Fig. S7). To assess chromatin distribution, we established an H2B-eGFP HUVEC line and treated the cells with DPBS, 0.2%, or 0.4% SF for 2 days. As anticipated, cells treated with SF exhibited an accumulation of H2B-marked overall chromatin at the nuclear inner cavities on day 2, in contrast to the control group [Figs. 4(b) and 4(c)]. Additionally, compared with those of the control, the morphology of HUVECs treated with SF exhibited distinct changes, as indicated by a larger nuclear area (Fig. S8). Furthermore, compared with cells in the control group, cells treated with SF demonstrated an accumulation of H2B-marked overall chromatin within the nuclear inner cavities from 0 to 48 h [Figs. 4(d) and 4(e)]. In summary, these results illustrate that SF treatment can influence the organization of chromatin.

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FIG. 4.

SF regulates the reorganization of nuclear chromatin. (a) GO cellular component term analysis was conducted for the upregulated genes. (b) H2B-eGFP transgenic HUVECs were treated with 0.2% or 0.4% SF solution for 48 h (scale bar = 10 μm). The H2B concentration along the white line was analyzed via ImageJ software. H2B-marked overall chromatin was highly localized at the nuclear center and formed large chromatin domains in the SF groups. (c) Normalized fluorescence intensity of H2B-eGFP in each shell in control (n = 31) and SF-treated cells (n = 32). (d) SF regulates the reorganization of nuclear chromatin. H2B-eGFP transgenic HUVECs were cultured with 0.2% or 0.4% SF solution for 0, 12, 24, or 48 h (scale bar = 2 μm). (E) Normalized fluorescence intensity of H2B-eGFP in each shell in control (n = 36) and SF-treated cells (n = 34).

E. SF promotes EC proliferation through downregulation of the MAPK signaling pathway

Next, we found that SF solution treatment downregulated the MAPK signaling pathway in EC through KEGG pathway enrichment analysis [Fig. 5(a)]. To further confirm the involvement of MAPK signaling in SF-induced EC proliferation effect, chromium picolinate, an activator of MAPK was used to activate the MAPK signaling pathway. Immunofluorescence staining analysis showed that the amount of fluorescence of H3K9me3 in SF-treated EC was significantly reduced by chromium picolinate treatment (50 μM) [Figs. 5(b) and 5(c)]. Next, we observed that chromium picolinate treatment inhibited EC proliferation and also reduced the SF solution-promoted EC proliferation [Fig. 5(d)]. These results confirmed that the activation of the MAPK signaling pathway decreased proliferative activity and H3K9me3 levels in SF-treated EC. We further investigated whether the downregulation of MAPK in SF-treated EC was related to chromatin reorganization. We observed that EC treated with both SF and chromium picolinate exhibited an accumulation of chromatin at the nuclear inner cavities when compared to treatment with chromium picolinate alone [Figs. 5(e) and 5(f)]. We also observed that the chromatin organization of EC treated with chromium picolinate and SF displayed an accumulation of H3K9me3-marked chromatin at the nuclear inner cavities when compared to the chromium picolinate treatment alone (Fig. S9). These observations revealed that SF treatment increased proliferative activity, H3K9me3 level, and chromatin reorganization through downregulation of the MAPK signaling pathway in EC.

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FIG. 5.

SF promotes EC proliferation through downregulation of the MAPK signaling pathway. (a) KEGG pathway analysis was conducted for the downregulated genes. (b) Immunofluorescence staining was used to detect the levels of H3K9me3 in the chromium picolinate (50 μM) and chromium picolinate-treated SF groups (scale bar = 50 μm). (c) The fluorescence intensity of H3K9me3 in each group was analyzed and compared (n ≥100). (d) Proliferation of HUVECs cultured with chromium picolinate (CP) and chromium picolinate treated with SF for 2 days. (e) H2B-eGFP transgenic HUVECs were treated with chromium picolinate (50 μM) or chromium picolinate treated with 0.4% SF solution for 48 h (scale bar = 10 μm). (f) Normalized fluorescence intensity of H2B-eGFP in each shell in chromium picolinate-treated cells (n = 30) and both chromium picolinate- and SF-treated cells (n = 32). The mean ± SEM is presented based on more than three separate experiments. Compared with control, statistical significance is indicated by ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, and ^****p ≤ 0.0001. Compared with the SF-treated group, statistical significance is indicated by #p ≤ 0.05. The “ns” indicates no statistical significance. The statistical significance was estimated using a two-tailed t-test.

B. Cell culture

The human umbilical vein endothelial cell (HUVEC) line was obtained from ScienCell Research Laboratories. Mouse brain endothelial bEnd.3 cells (bEnd.3) were obtained from the Baidi Biotechnology Co., Ltd (Zhejiang, China). Cells were cultivated in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin antibiotic solution (P/S). HUVECs were incubated in 6 cm culture dishes at 37 °C in an atmosphere containing 5% carbon dioxide (CO₂) with 95% humidity. The culture medium was replaced every 2 days. Once the confluence reached 80%–90%, HUVECs were expanded via trypsinization using 0.25% trypsin containing 0.02% EDTA and subsequently resuspended in a fresh growth medium.

C. Cell proliferation assay

To assess the effect of SF treatment on EC proliferation and viability, we employed the cell counting kit 8 (CCK-8; abbkine; Wuhan, Hubei, China) assay in accordance with the manufacturer's guidelines.² HUVECs were initially seeded at a density of 5 × 10³ cells per well in 96-well plates (Jet Biofil, Guangzhou, Guangdong, China) and were allowed to incubate overnight at 37 °C in a 5% CO₂ incubator. These cells were cultured in DMEM supplemented with 10% FBS and 1% P/S. After the removal of the medium, 100 μL of fresh DMEM containing varying concentrations of SF (0, 0.2%, 0.4%, 0.8%, 1.6%, 3.2%, and 6.4%) was added to the HUVECs, after which the cells were cultivated for periods of 1, 2, and 3 days. Following the incubation period, the SF treatment medium was aspirated, and the cells were cultured with 100 μl of fresh FBS-free medium containing CCK-8 reagent (at a ratio of 10:1) for 30 min at 37 °C. Ultimately, the optical density was determined at 450 nm using a microplate reader (Bio-Tek, Winooski, VT, USA).

D. Construction of the H2B-eGFP stable cell line and imaging

The H2B-eGFP HUVEC line was established through the transduction of HUVECs with retroviral constructs encoding H2B-eGFP variants. Subsequently, these cells were sorted based on eGFP expression using flow cytometry (Sony Biotechnology, Tokyo, Japan). Following sorting, the cells were cultured in 24-well plates within a CO₂ chamber maintained at 37 °C. These cells were subjected to treatment with 0.2% or 0.4% SF for 0, 12, 24, or 48 h. The resultant samples were then subjected to imaging through laser scanning confocal microscopes (Nikon AX, Japan) (Zeiss LSM980, Germany).

We performed quantitative analyses of chromatin changes following the established protocol detailed in previous studies.³⁵ We measured H2B-eGFP plot profiles across the nucleus diameter of the equatorial focal plane with ImageJ software. The individual plot profiles were normalized on the average intensity and divided into five shells with equal distance from the nuclear periphery to the interior.

E. Flow cytometry analysis

For the proliferation assay, HUVECs were labeled with CellTrace Violet (CTV; Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.¹⁷ Subsequently, the labeled cells were seeded in 6-well culture plates (Corning, NY, USA) and allowed to incubate overnight at 37 °C in a 5% CO₂ incubator. Following this incubation period, the cells were exposed to either 0.2 or 0.4 (wt. %/vol. %) SF or Dulbecco's phosphate-buffered saline (DPBS) as a control. After two days, the labeled cells were trypsinized and collected in a fresh growth medium. Flow cytometry analysis was performed using Beckman Coulter equipment (Brea, CA, USA), and FlowJo software was used to analyze the cell proliferation data.

F. Scratch assays of cell migration

ECs were initially plated in 6-well culture plates and maintained at 37 °C in a 5% CO₂ incubator. Once the cells reached confluence, a scratch was generated using a 20 μl micropipette tip, followed by two washes with DPBS solution. Subsequently, each well was replenished with fresh DMEM supplemented with either 0.2% or 0.4% SF, while DPBS served as the control. The cells were then incubated for a period of up to 12 h or 24 h at 37 °C in a 5% CO₂ incubator. Microscopy images were captured at both the 0-h and 12- or 24-h time points using a phase-contrast microscope. To assess cell migration, cell-free areas in the microphotographs were quantified using ImageJ software for comparative analysis.

G. Immunofluorescence staining and quantification

For immunofluorescence experiments, HUVECs were cultured on a 35 mm confocal dish (Biosharp, Hefei, China). The cells were fixed with 4% paraformaldehyde (PFA; Biosharp, Hefei, China) for 15 min at room temperature, followed by three washes with phosphate-buffered saline (PBS; BBI, Shanghai, China) for 5 min each. Subsequently, permeabilization was carried out using 0.25% Triton X-100 (Biosharp, Hefei, China) in PBS for 10 min at room temperature, followed by another three washes with PBS. The cells were then blocked with 5% goat serum (Invitrogen, Grand Island, NY) in PBS at room temperature for 2 h. Following blocking, primary antibodies were applied to the cells in PBS (with 1% goat serum), which were subsequently incubated overnight at 4 °C. The primary antibodies utilized in this study included anti-H3K9me3 antibodies (CST, 13969, diluted 1:1000), anti-H3K9me2 antibodies (CST, 4658, diluted 1:1000), anti-PCNA antibodies (Santa Cruz, sc-56, diluted 1:500), and anti-ICAM-1 antibodies (Proteintech, 16174-1-AP, diluted 1:800). After washing three times for 5 min each with PBST (0.1% Triton X-100 in PBS), the cells were incubated with secondary antibodies diluted in blocking buffer for two hours at room temperature. The secondary antibodies used were goat anti-mouse IgG Alexa 488 (CST, 4408S, diluted 1:1000), goat anti-rabbit IgG 594 (CST, 8889S, diluted 1:1000) and goat anti-rabbit IgG 488 (Proteintech, SA00013-2, diluted 1:1000). After another three washes with PBS, Hoechst 33342 (Thermo Fisher Scientific, diluted 1:1000), or DAPI (Thermo Fisher Scientific, diluted 1:1000) was used to stain the cell nuclei for 15 min. Subsequently, the cells were subjected to three additional 5-min washes with PBS before being mounted with the mounting medium from Thermo Fisher Scientific. The samples were imaged utilizing a laser scanning confocal microscope (Olympus FV3000, Japan) (Nikon AX, Japan). Relative fluorescence units were quantified using ImageJ software.

H. Western blot analysis

The cultured cells were resuspended in 100 μl of RIPA lysis and extraction buffer (Pierce, Rockford, IL, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Subsequently, the cell lysates were sonicated to fragment genomic DNA and subjected to centrifugation at 15 000 × g for 15 min at 4 °C. The protein concentration in the resulting supernatants was determined using a bicinchoninic acid (BCA) protein assay. The denatured proteins were then separated on an SDS–polyacrylamide gel and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Following the transfer, the membranes were blocked with 5% nonfat milk for 2 h at room temperature. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies diluted 1:1000 in 5% nonfat milk in PBS. The primary antibodies used were anti-H3K9me3 (CST, 13969; diluted 1:1000) and anti-PCNA (Santa Cruz, sc-56; diluted 1:500). The membranes were subjected to three washes with 0.1% Tween-20 in Tris-buffer saline and subsequently incubated with horseradish peroxidase-conjugated IgG secondary antibodies (CST, 7074 or 7076, diluted 1:1000) at room temperature for 2 h. Finally, the immunoreactive bands were visualized utilizing an Omni-ECL™ Femto Light Chemiluminescence Kit (EpiZyme, Shanghai, China) with a ChemiDoc MP Imaging System (Bio-Rad, Singapore). The gray values of the blots were quantified using ImageJ software.

I. RNA sequencing

RNA was extracted from HUVECs treated with 0.4% SF on day 2 utilizing Total RNA Kit I reagent (Omega Bio-Tek, GA, USA) following the manufacturer's guidelines. The integrity of the isolated RNA was assessed using an Agilent 2100 Bioanalyzer. Subsequently, library preparation and sequencing were performed on the DNESEQ-T7 platform, generating 150 bp paired-end reads. To process the RNA reads Trim Galore v0.6.10 was used to eliminate adapters and filter out low-quality reads. The processed reads were subsequently aligned to the GRCh38/hg38 reference genome utilizing STAR v2.7.3a software.³⁶ Expression quantification was carried out using the feature counts (subread v2.0.0) tool³⁷ with gene annotation obtained from Homo sapiens Ensembl v110. Differential expression analysis was conducted employing the DESeq2 v1.32.0 tool.³⁸ Genes that exhibited a fold change greater than 1.2 and an adjusted p-value less than 0.05 were considered differentially expressed. Furthermore, functional annotation enrichment analysis was performed using the bioinformatics resources provided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID).³⁹

J. TSA treatment and antioxidative activity assay

HUVECs were subjected to TSA treatment following established protocols detailed in prior studies.^9,16,19 The cells were initially seeded at a density of 5 × 10³ cells per well in 96-well plates and allowed to incubate overnight. Subsequently, the cells were exposed to different conditions, including 0.4% SF, 100 nM TSA, or a combination of 100 nM TSA with 0.4% SF, for 2 days. A negative control sample was treated with dimethyl sulfoxide (DMSO). Cell survival was assessed using the CCK-8 assay.

The objective of the present study was to evaluate the influence of SF on endothelial cell death induced by oxidative damage caused by varying concentrations of hydrogen peroxide (H₂O₂). HUVECs were seeded at a density of 5 × 10³ cells per well in 96-well plates. These cells were pretreated with H₂O₂ at concentrations of 0, 250, 500, and 1000 μM for 1 h. Subsequently, the cells were cultured in fresh medium containing 0.4% (wt/vol) SF for 24 h at 37 °C in a 5% CO₂ incubator. Following the treatment, the SF-containing medium was removed, and the cells were cultured in 100 μl of fresh medium devoid of FBS. Cell survival was determined utilizing the CCK-8 assay.

K. Chromium picolinate treatment assay

HUVECs were subjected to chromium picolinate treatment following established protocols detailed in prior studies.⁴⁰ The cells were initially seeded at a density of 5 × 10³ cells per well in 96-well plates and allowed to incubate overnight. Subsequently, the cells were exposed to different conditions, including 0.4% SF, chromium picolinate (50,100 μM), or a combination of chromium picolinate with 0.4% SF for 2 days. A negative control sample was treated with dimethyl sulfoxide (DMSO). Cell survival was assessed using the CCK-8 assay.

This work was supported by grants from the Fundamental Research Funds for the National Key R&D Project (No. 2022YFF0710700), the Natural Science Foundation of Chongqing (No. CSTB2023NSCQ-MSX0918), the Fundamental Research Funds for Central Universities (No. 2023CDJKYJH082), and the Shenzhen Science and Technology Program (JCYJ20220530144415035).