Hcy accelerates the biological dysfunction of ANA-1 macrophages and vascular injure via Rap1A/MAPK signal

doi:10.21203/rs.3.rs-4880995/v1

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Hcy accelerates the biological dysfunction of ANA-1 macrophages and vascular injure via Rap1A/MAPK signal

https://doi.org/10.21203/rs.3.rs-4880995/v1

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Hyperhomocysteinemia (HHcy) is a kind of risk factor for various vascular diseases. The dysfunction of macrophages plays a crucial role in the development of HHcy-aggravated vascular injury. However, macrophages injure is still unclear. In this study, we propose the hypothesis that the Rap1A/ERK1/2 signal pathway is responsible for mediating the dysfunction of macrophages in HHcy-induced vascular diseases. We found that Cell proliferation, migration and invasion ability were increased after Hcy treatment. In addition, the mRNA levels of CD80 and CD86, and protein expression of Rap1A, iNOS were also increased in Hcy group. Interference of Rap1A reversed Hcy-induced cell proliferation, migration and invasion, and also reduced macrophage inflammatory response and M1 polarization. Phosphorylation of ERK1/2 was inhibited after Rap1A knockdown. Suppression of ERK1/2 alleviated Hcy-induced macrophage dysfunction. Together, Rap1A mediates Hcy-induced macrophage dysfunction via ERK1/2 signaling, which highlights a new mechanism in the pathogenesis and development of HHcy-aggravated vascular diseases.

Biological sciences/Cell biology

Biological sciences/Developmental biology

Biological sciences/Molecular biology

Biological sciences/Physiology

Homocysteine

Macrophages

Rap1A

ERK1/2

Vascular injury

Homocysteine (Hcy) is a metabolite of the body's methionine cycle and is typically maintained at low levels. HHcy has been identified as an independent risk factor for various diseases, including arteriosclerosis, cerebrovascular disease (Grande, Hooshmand et al., 2023), and kidney injury (Zhang, Li et al., 2022). Particularly coronary heart disease (Kaplan, Tatarkova et al., 2020; Zhu, Li et al., 2022; Grande, Hooshmand et al., 2023) and stroke (Chen, Zou et al., 2022). The pathogenesis of these conditions is linked to the promotion of vascular smooth muscle cell proliferation (Balint, Jepchumba et al., 2020), increased platelet aggregation (Carrizzo, Iside et al., 2022), vascular endothelium injury (Balint, Jepchumba et al., 2020), and activation of the immune response (Liu, Yan et al., 2021; Xiong, Ma et al., 2021).It has been reported that dysfunction of macrophages promotes monocyte-macrophage inflammation (Xiong, Ma et al., 2021), M1 polarization (Gao, Wang et al., 2014), cell proliferation (Liu, Yan et al., 2021), migration (Zhu, Li et al., 2022), and activation of associated signaling pathways (Yang, Ren et al., 2022). However, the mechanism by which macrophages damage blood vessels remains unclear.

The Ras-associated protein 1A (Rap1A), as a member of the small G-protein oncogene Ras superfamily, is involved in cell-matrix adhesive interactions, junction formation, polarity, and proliferation (Shah, Brock et al., 2018; Cao, 2019). Additionally, Rap1 regulates macrophage function. We also observed that in Rap1 knock-down mice, epithelial proliferation was significantly decreased compared to WT controls (Cao 2019). In PCa cells, knock-down of Rap1A inhibits cell viability. Targeting Rap1A with siRNA significantly reduces cervical cancer cell proliferation, invasion, and migration (Cao 2019). Therefore, Rap1A is involved in disease development by affecting cellular dysfunction. In pancreatic cancer, an increase in Rap1A expression was observed along with an increase in phosphorylation of kinase p38 mitogen-activated protein kinase (MAPK) (Shah, Brock et al. 2018). Rap1A has the ability to activate extracellular signal-regulated p38, which in turn promotes the proliferation, migration, and invasion of ovarian cancer cells (Lu, Wang et al. 2016). Additionally, Cathepsin C has the ability to promote M1 polarization of macrophages through the p38-MAPK pathway (Lu, Wang et al. 2016). However, it is currently unknown whether the Rap1A/p38 signal pathway regulates the proliferation, migration, and M1 polarization of macrophages in Hcy.

In short, the effect of Rap1A/p38 signal pathway can regulation Hcy-induced macrophage dysfunction and provide a further strategy of treatment on HHcy related diseases.

Cell culture

ANA-1 murine macrophages were purchased from the Cell Center of Chinese Academy of Medical Sciences (Beijing, China) and maintained in RIMP1640 (Gibco, Australia) with 7% FBS and 1% penicillin and streptomycin in 37°C humidified air containing 5% CO₂ (Heraeus, German). When the macrophages were in the logarithmic growth phase, cells were seeded into two groups: control group, Hcy group (cells were treated with 100uM Hcy). After 24h, cells were collected and used for subsequent experiments.

Cell viability assay

ANA-1 cells were seeded with 3000 cells per well, 10 µL of CCK8 (P0010, Beyotime, China). The OD value was measured by microplate reader (mµlISKANMK3, Thermo, USA).

Enzyme-linked immunosorbent assay (ELISA)

The IL-6 and TNF-α cytokines were measured using ELISAs (Biotech, Beijing, China) following the manufacturer’s protocol. ANA-1 cells and its supernatants were obtained from cell-conditioned medium and cell lysate.

Cell proliferation assay

In this study, the EdU assay was performed to evaluate the cell proliferation. In brief, cells in each group were incubated on a 20 µmol/L EdU (5-Ethynyl-2′-Deoxyuridine) (Ruibo, Guanghzou, China) medium for 2 h and fixed on 4% paraformaldehyde for 30 min. After 5% Triton X-100 was incubated at room temperature for 30 min, 200 µL Apollo staining solution was added and incubated at room temperature in dark for 30 min, Hoechst-33342 reaction solution was added after PBS washing. Inverted Fluorescence microscope (IX51, OLYMPUS, Japan) detects and calculates the percentage of EdU positive cells.

Statistical analysis

Data analysis was performed using GraphPad 6.0 Software (GraphPad, San Diego, CA, USA). The statistical comparisons (P-values) were calculated using Student's t-test and one-way and/or two-way analysis of variance (ANOVA).

Hcy promoted the biological dysfunction of ANA-1 macrophages

Inflammatory monocytes and tissue-resident macrophages are major regulators of tissue repair, regeneration, and proliferation. To determine the effect of Hcy on proliferation and migration of ANA-1 macrophages, we first compared the cell viability to different concentration of Hcy. The cell viability was increased in different concentration of Hcy, including 0, 25, 50, 100, 200, and 500µM, as compared with negative control (Fig. 1A). After macrophages stimulated with 100 Hcy, the protein expression of PCNA increased while P27 decreased both in transcriptional and translational level (Fig. 1B). Meanwhile, the EdU assay also showed DNA replication activity was increased (Fig. 1C). Cell scratch test and transwell test are important methods to analyze cell migration. When ANA-1 cells treated with Hcy, cell migration was obviously enhanced. (Fig. 1D, E). Macrophages are heterogeneous, and their phenotype and function are regulated by many factors. M1 macrophages have an inflammatory phenotype while M2 macrophages have anti-inflammatory phenotype. When ANA-1 cells treated with Hcy for 24h, CD80 and CD86 mRNA expression was increased significantly (Fig. 1F), and contents of TNF-α and IL-6 were also increase (Fig. 1G, H). Moreover, the protein expression of iNOS was autumented in Hcy group (Fig. 1I). Therefore, Hcy accelerated the biological dysfunction of ANA-1 macrophages.

Hcy increased upregulation of Rap1A

Multiple genes abnormal expression is involved in pathogenesis of Hcy, we analyzed the gene expression profiling of Hcy in ANA-1 cells by RNA sequencing. Histogram of KEGG showed that cellular processes and immune diseases pathway enrichment (Fig. 2A). Heatmap and clustering analysis was found that 16 genes were down-regulated and 1 gene ENSMUSG00000068798 which encoded Rap1A was up-regulated in the Hcy group compared with control group (Fig. 2B). In case that Rap1A has taken part in multiple pathophysiological processes in mammals, we assayed Rap1A expression after Hcy treatment of ANA cells. The expression of Rap1A were increased in Hcy group (Fig. 2C).

Role of Rap1A in Hcy-induced biological dysfunction of ANA-1 macrophages

Previous study has showed that Rap1A enhanced cell proliferation and migration. Knocking down of Rap1A were to reduced this phenomenon. To further analysis the role of Rap1A on the biological dysfunction of ANA-1 cells(Figure S1). We observe that the cell viability and proliferation was reduced by siRap1A (Fig. 3A,B and C). Moreover, cell migratory and invasion abilities were suppressed in macrophages transfection with Lv-Rap1A (Fig. 3D, E). These results indicated that down-regulation of Rap1A decreased the proliferation, invasion and migration of macrophages. For further investigate the role of Rap1A in the M1 phenotype transformation of macrophages induced by Hcy, M1 phenotype related indicators were measured after Rap1A silencing. As shown in Fig. 3F, CD86 and CD86 mRNA level were obvious decreased after Rap1A knockdown as compared to Hcy group. IL-6 (Fig. 3G) and TNF-α (Fig. 3H) content in supernatant were decreased companied with reduced iNOS expression (Fig. 3I) after Rap1A knockdown. These data indicated that Rap1A was involved in Hcy-induced M1 phenotype transformation of macrophages.

Rap1A suppressed mitogen-activated protein kinase phosphorylation

Rap1 binds to GTP or GDP and acts as a "molecular switch" in cellular biological functions. Phosphorylation regulation is an important downstream signaling pathway for Rap1A, so we examined Rap1A downstream pathway with phosphorylated proteomics. The volcano plot analysis of phosphorylated protein between Lv-Rap1A and Hcy groups were displayed in Fig. 4A. GO and KEGG show that phosphorylated protein were enriched in cellular process and catalytic activity (Fig. 4B-C). in addition we also found that down-regulation of Rap1A could obviously decrease the phosphorylation level of ERK1/2. These data indicated that Rap1A suppressed MAPK phosphorylation (Fig. 4D-F).

Rap1A/ERK1/2 signaling was involved in biological dysfunction of ANA-1 macrophages

On the basis of previous studies showing that ERK signaling pathways could regulate macrophages proliferation, invasion and migration. Then we treated macrophages with ERK1/2 specific inhibitor, SCH772984 (Figure S2). In order to clarify the role of Rap1A/ERK1/2 signal in regulating biological function of macrophages induced by Hcy, ANA-1 cells were intervened with Lv-Rap1A and SCH772984. Co-treatment with Lv-Rap1A and SCH772984 significant alleviated cell proliferation (Fig. 5A-C), invasion (Fig. 5D) and migration (Fig. 5E) when compared with Lv-Rap1A group. In case that ERK1/2 pathway has participated in macrophages polarization, we further addressed the effect of Rap1A/ERK1/2 signal in M1 phenotype transformation. As shown in Fig. 5F, mRNA level of CD86 and CD80 were significant decreased when Rap1A and ERK1/2 both inhibition. Moreover, TNF-α and IL-6 contents in supernatant were also decreased (Fig. 5G-H), and protein expression of iNOS was decreased (Fig. 5I) after Lv-Rap1A and SCH772984 co-treatment. These data indicated that Rap1A/ERK1/2 signaling pathways potentially facilitate the Hcy-induced macrophages biological dysfunction.

Vascular diseases are often accompanied by inflammation, such as arteriosclerosis(Grinan, Escola-Gil et al. 2022), stroke and arthritis. Serum Hcy levels in arteriosclerosis and rheumatoid arthritis are usually elevated, and Hcy exacerbates inflammation by increasing macrophages proliferation, migration and invasion. In this study, our results provided new evidence that Rap1A augumented ANA-1 macrophages activation, proliferation, adhesion, invasion, and M1 polarization via p38 signal in the presence of Hcy.

Macrophages, derived from monocytes and belonging to immune cells, participate in both nonspecific and specific defenses in the body, with deformational movement and phagocytosis(Gonzalez, Lu et al. 2023). Macrophages usually exist in the tissues of the body as usual macrophages, which become activated macrophages after being stimulated, and then secrete a large number of soluble pro-inflammatory factors, accelerating the pathological process of the disease. Results of this study showed after Hcy intervention, ANA-1 macrophages cells viability and proliferation ability was significant increased, as well as a significant increase in cell migration and invasion ability, consistent with literature reports. When macrophages undergo M1 polarization, the expression of inducible nitric oxide synthase (iNOS) is significantly increased. In addition, CD80 and CD86 are classical indicators of M1 polarization. In this study, the content of TNF-α and IL-6 in the supernatant of macrophages treated with Hcy was significantly increased, accompanied with increased mRNA expression of CD80 and CD86 and protein expression of iNOS. These results indicated that Hcy might cause macrophages dysfunction characteristic with augmented cell viability, proliferation, migration, invasion and M1 polarization.

Gene chip results showed that Hcy intervention in macrophages, representative cellular processes and immune response-related pathways were enriched, accompanied by up-regulation of Rap1A expression, which was also confirmed in in vitro experiments. Deletion of the Rap1A gene reduced macrophage migration capacity and inhibited angiogenesis, impairing endothelial cell migration and proliferation. Our research found that the proliferation, migration and invasion of macrophages induced by Hcy could be partially reversed by transfection of lentivirus carrying the interfering fragment of Rap1A. Studies have shown that over-production of Rap1A is the main cause of vascular remodeling and inflammatory reaction, and leads to the development of vascular-related diseases. Rap1A activation is involved in the repair of damaged endometrium through epithelial cell polarity remodeling. In the present study, inhibition of Rap1A expression could significantly reduce Hcy-induced increase of TNF-α and IL-6 contents and M1polarization. Taken together, these data support the idea that Rap1A is involved in Hcy-induced macrophage dysfunction.

Rap1A can phosphorylate and activate downstream proteins. Rap1A activates JAK/STAT inflammatory signaling pathway mediated by fatty acid binding protein 4(Xu, Zhang et al. 2022). Rap1A interact with the lncRNA TGFB3-AS1 to promote inflammatory responses in macrophages through the Wnt/β-catenin signaling pathway(Zhang, Hao et al. 2021). These evidences prove that Rap1A can affect downstream phosphorylation pathway. Then we analyzed the phosphorylation signaling pathways downstream of Rap1A with the phosphorylated proteome and found that Rap1A can regulate multiple phosphorylation pathways, including the mitogen activated protein kinases (MAPK) pathway.Rap1A has been shown to activate ERK/MAPK pathway, which promotes cell migration and inhibits cell differentiation(Shah, Brock et al. 2018, Jin, Di et al. 2019).Therefore, regulation of MAPK pathway is an important mechanism of Rap1A. Activation of upstream proteins of JNK and p38 creates conditions for RAS/RAF activity. Therefore we also speculate Rap1A may be involved in the regulation of RAS/RAF/MEK/ERK signaling pathway.

Knockdown Rap1A reduces ERK1/2 phosphorylation levels, while JNK and P38 have no significant change, which is consistent with our conjecture literature. These findings indicated that Rap1A suppressed ERK1/2 phosphorylation. To further explore the effect of ERK1/2 on ANA-1macrophage dysfunction, we intervened the cells with ERK1/2-specific blockers. In the presence of Hcy, co-inhibition of Rap1A and ERK1/2 significantly reduced the proliferation, migration and invasion of macrophages, and relieved M1 polarization and inflammation to some extent. These data suggest that Rap1A-ERK1/2 signaling pathway is involved in Hcy-induced macrophage proliferation, migration, invasion, M1 polarization and inflammation.

Collectively, the reported reveal that Hcy accelerates macrophage proliferation, migration, invasion, M1 polarization and inflammation through regulating Rap1A-ERK1/2 signal pathway. The discovery of this novel mechanism provides forward-looking and in-depth insight on Hcy-induced macrophage dysfunction and brings hope that inhibition of Rap1A-ERK1/2 signal may be used in the field of vascular injury disease.

Author contributions: All the authors contributed to the writing and and data analysis.

Funding：This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81960063 and 81760095). Ningxia Medical University National Major Science and Technology Projects and scientific and technological achievements "Project" (XJKF230105)

Declarations:The authors declare that we have no conflict of interests.

Ethical approval: Not applicable.

Informed consent :All the authors consent for the publication of the article.

Conflict of Interest：The authors declare that they have no conflict of interest.

Data availability：All data are provided by the corresponding author.

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You are reading this latest preprint version

Hcy accelerates the biological dysfunction of ANA-1 macrophages and vascular injure via Rap1A/MAPK signal

Status:

Version 1

Abstract

Figures

Introduction

Material and method

Cell culture

Cell viability assay

Enzyme-linked immunosorbent assay (ELISA)

Cell proliferation assay

Statistical analysis

Results

Hcy promoted the biological dysfunction of ANA-1 macrophages

Hcy increased upregulation of Rap1A

Role of Rap1A in Hcy-induced biological dysfunction of ANA-1 macrophages

Rap1A suppressed mitogen-activated protein kinase phosphorylation

Rap1A/ERK1/2 signaling was involved in biological dysfunction of ANA-1 macrophages

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1