Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

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First Author: Zhu Huaxin, Hao Zheng

Corresponding Author: Li Meihua

Affiliation: Department of Neurosurgery, First Affiliated Hospital of Nanchang University

[REF: Zhu H, Hao Z, Xing Z, Tan J, Zhao Y, Li M. Impinging Flow Induces Expression of Monocyte Chemoattractant Protein-1 in Endothelial Cells Through Activation of the c-Jun N-terminal Kinase/c-Jun/p38/c-Fos Pathway [published online ahead of print, 2022 May 14]. World Neurosurg. 2022;S1878-8750(22)00663-5. doi:10.1016/j.wneu.2022.05.032] PMID: 35580782

Abstract

Background: Monocyte Chemoattractant Protein-1 (MCP-1) is an important regulatory factor in the formation and development of intracranial aneurysms. This study explores the molecular mechanisms related to the induction of MCP-1 and inflammatory factor expression in Human Umbilical Vein Endothelial Cells (HUVECs) under hemodynamic conditions.
Methods: A modified T-shaped flow chamber was used to simulate fluid flow and wall shear stress at arterial bifurcations. HUVECs under static conditions without impinging flow served as the control group. Western blot was used to detect the protein expression levels of ERK, JNK, p38, AP-1, and MCP-1, while qRT-PCR was used to measure the mRNA expression levels of MCP-1, IL-1β, and IL-6.
Results: Under impinging flow, the phosphorylation levels of ERK, JNK, and p38, as well as the protein levels of MCP-1, c-Jun, and c-Fos increased. The mRNA expression of MCP-1, IL-1β, and IL-6 was elevated. Pre-treatment of HUVECs with specific inhibitors of JNK and p38 significantly attenuated the increased expression of MCP-1, IL-1β, and IL-6, while the ERK-specific inhibitor did not show a significant effect.
Conclusion: Impinging flow regulates MCP-1 and inflammatory factors through the JNK/c-Jun/p38/c-Fos pathway, participating in the inflammatory response of endothelial cells.

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Illustration showing how impinging flow regulates endothelial dysfunction in HUVECs. Under impinging flow, MCP-1 and inflammatory factors are regulated via the JNK/c-Jun/p38/c-Fos pathway, contributing to endothelial dysfunction.
Keywords: HUVECs, MCP-1, JNK, p38, Hemodynamics

Introduction

Intracranial aneurysm (IA) is one of the most common cerebrovascular diseases, with an incidence of approximately 1-3% in the general population[50]. IA typically refers to the abnormal bulging of the arterial wall. After rupture, it can cause intracranial hemorrhage and hematoma due to mass effect, leading to complications such as intracranial vasospasm, brain tissue damage, and cerebral edema, which severely threaten the life and health of patients[17,56]. IA is currently believed to be related to several factors, including hemodynamic changes, age, genetics, hypertension, and environment[31]. The mechanisms underlying IA formation require further in-depth research.

Through continuous in-depth studies, the importance of hemodynamics in the occurrence, development, and rupture of IA has been confirmed from multiple aspects[36,37,64]. Numerous clinical and basic studies have shown that IA often occurs at bifurcations or curved sections of intracranial vessels. When blood flows through these vessels, impinging flow and complex hemodynamics occur, leading to endothelial injury, as well as wall inflammation and remodeling[21,34,36,47]. These changes are the main pathophysiological processes in aneurysm formation and development. Endothelial dysfunction is considered an important factor in IA formation[22,43]. When blood flows through vessels, its viscosity generates friction, termed wall shear stress (WSS), which plays a crucial role in maintaining normal cell morphology[16]. High WSS leads to vessel wall dilation, ultimately disrupting endothelial function, thereby promoting IA development and rupture[15]. Studies have confirmed that patients with ruptured IA have higher WSS[9-11]. The WSS generated by impinging flow at vascular bifurcations is complex and variable.
IA is accompanied by infiltration of various inflammatory cells, with monocyte macrophages as the main type of inflammatory cells; inflammatory infiltration is associated with high expression levels of various inflammatory factors, including interleukins (IL)-6 and IL-8, which can weaken or destroy arterial wall tissue[1,42]. In human IA specimens and rodent IA models, significant macrophage infiltration has been observed in the aneurysm wall, and inhibiting macrophages can significantly reduce the incidence of animal IA, confirming the importance of macrophages in IA formation[23,25,44,63]. Monocyte Chemoattractant Protein-1 (MCP-1) is a member of the C-C chemokine family. It is located on chromosome 17 q11.2-q21, containing three exons and two introns, and the mature MCP-1 protein consists of 76 amino acids, strongly inducing chemotaxis of monocyte macrophages[26]. MCP-1 has been confirmed to be highly expressed in both ruptured and unruptured IA walls, and downregulating MCP-1 expression can significantly reduce the incidence of animal IA[27,59]. These findings collectively indicate that MCP-1 plays a crucial role in the occurrence and development of IA.
Mitogen-activated protein kinases (MAPKs) are expressed in various cells, including endothelial cells (ECs). MAPKs play an essential role in intracellular signal transduction and can be activated by various stimuli, including ischemia, shear stress, and vasoactive substances. Moreover, MAPKs regulate a series of critical cellular physiological functions, including inflammation, differentiation, proliferation, and apoptosis, and also play a vital role in regulating cell survival[28]. Important members of the MAPK family include extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), which are involved in cellular processes induced by cytokines, bacteria, physical and chemical stressors, and other stimuli[5,18]. Activator protein-1 (AP-1) is a dimeric complex mainly assembled and synthesized by Jun and Fos family proteins[35]. As a transcription factor, AP-1 participates in various biological processes, including cell proliferation, differentiation, inflammation, and apoptosis, and is influenced by activation of the MAPK pathway[3,39,57].
The mechanisms by which impinging flow induces endothelial injury remain to be elucidated. This study investigates the role of the MAPK pathway in the inflammatory response of ECs under impinging flow and explores the regulatory patterns of MCP-1 and inflammatory factors.

Methods

Modified T-shaped Flow Chamber

In this study, a modified T-shaped flow chamber was used to simulate fluid flow at arterial bifurcations, as well as WSS and wall shear stress gradient (WSSG)[24,60-62]. Figure 1A is a schematic diagram of the improved T-shaped flow chamber. Fluent software (Ansys, Canonsburg, PA, USA) was used to simulate the flow chamber, flow rate, WSS, WSSG, and turbulence intensity. The flow rate used in our experiments was 500 ml/min, with WSS ranging from 18-36 dynes/cm² and WSSG from 0-140 dynes/cm³ (Figure 1D, E). The experimental device operated at 37°C, with impinging flow acting for 0, 3, or 6 hours. Based on the distribution of WSS, human umbilical vein endothelial cells (HUVECs) on the glass slides were divided into three different regions. Region 1 was subjected to low to moderate WSS and abrupt WSSG, Region 2 to high WSS and abrupt WSSG, and Region 3 to moderate WSS and low, stable WSSG (Figure 1B, C). HUVECs not subjected to impinging flow served as the static control group.

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 1. Schematic diagram of the modified T-shaped flow chamber system: 1. Pulsation damper, 2. Rubber tube, 3. Threaded interface, 4. T-shaped flow chamber, 5. Pressure gauge, 6. Flow regulator; 7. Reservoir (A[24]). Based on the distribution of WSS, HUVECs on the same glass slide were divided into three regions (B, C). Region 1 was influenced by low to normal WSS and high WSSG, Region 2 by high WSS and WSSG, and Region 3 by normal WSS and low WSSG (C, D, E).

Cell Culture

HUVECs were purchased from Shanghai Fuheng Biotechnology Co., Ltd. Cell culture was prepared and maintained according to standard operating procedures. HUVECs were cultured in endothelial cell medium (ScienCell Research Laboratories, Carlsbad, CA, USA), supplemented with 1% endothelial cell growth supplement, 5% fetal bovine serum, and 1% penicillin/streptomycin, and incubated in a 37°C, 5% CO2 incubator. HUVECs were cultured in endothelial cell medium containing the respective inhibitors (JNK inhibitor-SP600125 (10μM); p38 inhibitor-SB203580 (10μM); ERK inhibitor-PD98059 (10μM)) for 6 hours before exposure to impinging flow. All inhibitors were purchased from Abcam. For quantification of cell density, cells in each region were counted under an optical microscope.

Western Blot Analysis (WB)

All three regions of HUVECs were collected and prepared for Western blot (WB) analysis. Cells were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and protein phosphatase inhibitors (Beijing Solarbio Technology Co., Ltd.) at 4°C for 30 minutes. The cell lysates were centrifuged at 14,000×g at 4°C for 15 minutes, and the supernatant was collected. The protein concentration of each sample was determined using a BCA protein assay kit (Beijing Solarbio Technology Co., Ltd.). Cell lysates were mixed with SDS-PAGE loading buffer (Beijing Solarbio Science & Technology) in equal proportions and heated in a water bath at 100°C for 5-10 minutes to denature the proteins. After cooling to room temperature, the mixture was centrifuged at 12,000×g for 2-5 minutes. Denatured proteins were analyzed by SDS-PAGE and transferred to a polyvinylidene fluoride membrane for immunoreaction. The membrane was incubated for 1 hour in blocking solution (5% milk and 0.1% Tween20 in Tris-buffered saline), followed by overnight incubation at 4°C with primary antibodies (ERK [ab184699] (1:1000), p-ERK [ab201015] (1:1000), JNK [ab179461] (1:1000), p-JNK [ab124956] (1:1000), p38 [ab170099] (1:2000), p-p38 [ab195049] (1:1000), c-Fos [ab184666] (1:1000), c-Jun [ab32137] (1:1000), MCP-1 [ab214819] (1:1000), and β-actin [ab8226] (1:1000); all purchased from Abcam). The membrane was washed three times (5 minutes each) with TBS containing 0.1% Tween20 and incubated at room temperature for 2 hours with secondary antibodies (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein blots were detected using the Pierce ECL Plus Western Blotting Kit (ThermoFisher Scientific, Waltham, MA, USA).

Gene Expression Analysis

Following the manufacturer’s instructions, total RNA was extracted from HUVECs using TRIzol reagent (Invitrogen) and stored at -80°C. RNA was reverse transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (Invitrogen). The expression of target genes was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the iQ5 instrument (Bio-Rad) and ChamQ Universal SYBR qPCR Master Mix Kit (Invitrogen).

Statistical Analysis

Results are expressed as mean ± standard error. Data were analyzed using SPSS software (version 19.0; IBM). The statistical significance of differences between groups was assessed using one-way ANOVA, while the chi-square test was used for categorical variables. A P-value < 0.05 was considered statistically significant. Each experiment was performed in triplicate to ensure the overall accuracy of the data.

Results

Changes in HUVECs Morphology

Under impinging flow, there were no significant changes in cell density in Regions 1 and 3, while there was a marked decrease in endothelial cell density in Region 2, with increased gaps between endothelial cells and diverse cell morphologies. Furthermore, as the duration of impinging flow increased, the cell density in Region 2 progressively decreased (Figure 2).

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 2. Human Umbilical Vein Endothelial Cells (HUVECs) under impinging flow. As the duration of impinging flow increased, cell density in Region 2 decreased (A-B) (scale bar, 50μm). Data are presented as mean ± standard error. Each experiment was performed at least three times. *P<0.05, ***P<0.001.

MAPK Activation and Expression of MCP-1 and Inflammatory Factors

HUVECs were exposed to impinging flow for 3 or 6 hours, and total protein was extracted from all three regions for WB analysis. Compared to the static control group, phosphorylation of ERK, JNK, and p38 increased over time. The expression levels of c-Fos, c-Jun, MCP-1, and inflammatory factors were significantly increased compared to the static control group (P < 0.05) (Figure 3).

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 3. Activation of Mitogen-Activated Protein Kinases (MAPK) and expression of MCP-1 and inflammatory factors. Under impinging flow, phosphorylation of ERK, JNK, and p38 increased over time (A-C), and expression levels of MCP-1, c-Jun, and c-Fos increased (D, E). mRNA expression of MCP-1, IL-1b, and IL-6 increased (F-H). Data are presented as mean ± standard error. *P<0.05, each experiment performed at least three times. *P<0.05, **P<0.01. ns, not significant.

JNK/c-Jun Pathway Regulates Expression of MCP-1 and Inflammatory Factors

Prior to exposure to impinging flow for 6 hours, HUVECs were treated with the JNK inhibitor SP600125 (10μM). Total protein was extracted for WB analysis. Compared to the untreated group, phosphorylation of JNK was inhibited by SP600125, and expression levels of c-Jun, MCP-1, and inflammatory factors significantly decreased (P<0.05) (Figure 4). There were no significant changes in ERK and p38 phosphorylation or c-Fos expression.

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 4. JNK/c-Jun pathway regulates expression of MCP-1 and inflammatory factors. HUVECs were pre-treated with the JNK inhibitor SP600125 (10μM) before exposure to impinging flow for 6 hours; phosphorylation of JNK decreased (B), while phosphorylation of ERK (A) and p38 (C) did not change. Expression levels of MCP-1 (D) and c-Jun (E) decreased, while expression of c-Fos (E) did not change. mRNA levels of MCP-1 (F), IL-1b (G), and IL-6 (H) decreased. Data are presented as mean ± standard error. Each experiment was performed at least three times. *P<0.05, **P<0.01. ns, not significant.

p38/c-Fos Pathway Regulates Expression of MCP-1 and Inflammatory Factors

Before exposure to impinging flow for 6 hours, HUVECs were treated with the p38 inhibitor SB203580 (10μM). This led to inhibition of p38 phosphorylation activation, and expression levels of c-Fos, MCP-1, and inflammatory factors significantly decreased (P < 0.05) (Figure 5). There were no significant changes in ERK and JNK phosphorylation levels or c-Jun expression levels.

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 5. p38/c-Fos pathway regulates expression of MCP-1 and inflammatory factors. HUVECs were pre-treated with the p38 inhibitor SB203580 (10μM) before exposure to impinging flow for 6 hours; phosphorylation of p38 (C) decreased, while phosphorylation of ERK (A) and JNK (B) did not change. Expression levels of MCP-1 (D) and c-Fos (E) decreased, while expression of c-Jun (E) did not change. mRNA levels of MCP-1 (F), IL-1b (G), and IL-6 (H) decreased. Data are presented as mean ± standard error. Each experiment was performed at least three times. *P<0.05, **P<0.01. ns, not significant.

Inhibition of ERK Phosphorylation Does Not Significantly Affect Expression of MCP-1 and Inflammatory Factors

Before exposure to impinging flow for 6 hours, HUVECs were treated with the ERK1/2 inhibitor PD98059 (10μM). Compared to the untreated group, phosphorylation of ERK was inhibited by PD98059, while phosphorylation of p38 and JNK, as well as expression levels of c-Jun, c-Fos, MCP-1, and inflammatory factors did not significantly change (Figure 6).

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Figure 6. Inhibition of ERK phosphorylation does not significantly affect expression levels of MCP-1 and inflammatory factors. HUVECs were pre-treated with the ERK inhibitor PD98059 (10μM) before exposure to impinging flow for 6 hours; phosphorylation of ERK (A) decreased, while phosphorylation of JNK (B) and p38 (C) did not change. Expression levels of MCP-1 (D), c-Jun, and c-Fos (E) also did not change, similar to the mRNA levels of MCP-1 (F), IL-1b (G), and IL-6 (H). Data are presented as mean ± standard error. Each experiment was performed at least three times. *P<0.05, **P<0.01, ***P<0.001. ns, not significant.

Discussion

IA is a saccular arterial protrusion primarily caused by local thinning and dilation of the vascular wall. Rupture of IA is the leading cause of subarachnoid hemorrhage (SAH), resulting in high mortality and morbidity[48]. The mechanisms of IA formation and rupture have not been fully elucidated, but many studies link them to endothelial dysfunction, extracellular matrix remodeling, and vascular smooth muscle cell (VSMC) phenotypic changes that cause pathological alterations in the vascular system[4,19,20,29,30]. The lifecycle of IA has three distinct stages: formation, growth, and rupture. In each stage, blood flow plays a crucial role, converting mechanical stimuli into biological signals[45,49]. The fact that most lesions are located at arterial bifurcations or curves supports the importance of hemodynamics in the biology of IA[6,12]. The complex flow patterns determine the localization of EC dysfunction.[40,51].

ECs, forming a monolayer, are an important part of regulating vascular health; the endothelial layer is regularly arranged throughout the vascular network, forming a molecular barrier that is crucial for various physiological functions[49]. Under normal physiological conditions, vascular ECs become spindle-shaped, aligning with the flow of blood to regulate vascular tension, cell adhesion, and proliferation of smooth muscle cells, thereby maintaining vascular homeostasis[7]. Abnormal hemodynamic conditions trigger EC dysfunction and injury; subsequently, ECs secrete matrix metalloproteinases (such as MMP2 and MMP), damaging the extracellular matrix[41,47,54]. Flow-mediated endothelial dysfunction, followed by VSMC phenotypic transition from contractile to synthetic (thereby enhancing the inflammatory response), is a key step in the pathophysiological development of IA[49]. The synthetic phenotype promotes inflammation in many vascular diseases by creating an environment conducive to inflammation and remodeling, which in turn maintains and expands endothelial dysfunction and recruitment of immune cells[38,46]. Thus, endothelial dysfunction is a core pathogenesis of IA and may indeed be the initial step in IA formation. We simulated hemodynamics at arterial bifurcations using a modified T-shaped flow chamber to explore how impinging flow affects the behavior of ECs[60]. Our results indicate that impinging flow disrupts cell-cell junctions in HUVECs, activates intracellular inflammatory responses, and subsequently triggers endothelial dysfunction.

The chemokine MCP-1 has a powerful effect on both monocytes and macrophages. Significant macrophage infiltration has been observed in both ruptured and unruptured IAs, and inhibiting macrophages can significantly reduce IA formation[23]. Hemodynamics associated with high WSS activate pro-inflammatory signals in vascular ECs, leading to the accumulation of macrophages at sites exposed to high WSS and triggering inflammatory responses[14,21]. Previous studies have found high expression of MCP-1 in IA walls, and knockout of MCP-1 significantly reduced macrophage infiltration, confirming its important regulatory role in IA formation[2]. In this study, we simulated fluid flow in vitro and found that MCP-1 expression in HUVECs significantly increased under impinging flow; this provides evidence for MCP-1’s involvement in the early stages of endothelial dysfunction.

The MAPK signaling pathway plays a crucial role in signal transduction from the cell surface to the interior; it is also involved in cell proliferation, differentiation, migration, and apoptosis, as well as stress and inflammation. The MAPK pathway transmits extracellular signals to cells through receptors (including G protein-coupled receptors), protein kinases, and transcription factors[18]. We found that phosphorylation of ERK, JNK, and p38 was activated by impinging flow. We confirmed using specific inhibitors that JNK and p38 regulate the expression of MCP-1 and inflammatory factors via c-Jun and c-Fos, respectively. Although ERK plays a limited role in the production of MCP-1 and inflammatory factors, phosphorylation/activation of ERK triggers downstream signaling pathways involved in regulating cell growth, proliferation, differentiation, and migration, leading to widespread cellular changes[8]. After comparing differentially expressed genes in IA and normal superficial temporal artery tissues, it was found that the ERK pathway may play an important role in the formation and development of IA. Therefore, further research is needed to determine how ERK may be involved in regulating endothelial dysfunction (through other biological pathways)[58].

Impinging flow regulates the inflammatory response of HUVECs by activating JNK and p38, which may participate in the formation and development of IA by modulating endothelial dysfunction. During IA formation, JNK and p38 regulate the phenotypic transition of VSMCs[55]. Compared to unruptured aneurysms, phosphorylation of JNK and p38 is enhanced in ruptured IA tissues and positively correlates with IA size[32,33]. Increased levels of ANXA3 and enhanced JNK signaling activity can be observed in IA; silencing ANXA3 can inhibit the phenotypic transition of VSMCs in IA by suppressing phosphorylation of the JNK signaling pathway[52]. MiR-21 promotes the production of inflammatory-related factors through the JNK signaling pathway, participating in the formation and rupture of IA[13]. In a rabbit IA model, upregulation of KLF2 increased phosphorylation of p38 and regulated IA progression[53]. Although many studies have found that JNK and p38 are involved in the occurrence and development of IA, the cellular processes regulating their activity are very complex. It remains unclear whether impinging flow participates in IA by modulating MCP-1 and inflammation (mediated by JNK and p38).

Our work has certain limitations. First, the cells from the three regions affected by impinging flow were collected together, and we did not analyze the cells from different regions separately. Considering the limited size of the slides and the limited number of cultured cells, the number of cells may be too small for subsequent analyses due to the reduction caused by impinging flow. If the cells were collected by region, their numbers might be too few for further analysis. However, given the advancements in sequencing technology, particularly single-cell sequencing, we may analyze cells from different regions in the future to strengthen our findings on the effects of impinging flow on HUVECs and related signaling pathways in vitro. Furthermore, this study lacks validation from animal IA models. Third, vascular endothelial cells constitute only part of the arterial wall, and other components of the artery still need further investigation to determine their impact on IA.

Conclusion

Discussion

Under impinging flow, MCP-1 and inflammatory factors are regulated via the JNK/c-Jun/p38/c-Fos pathway, participating in endothelial dysfunction. However, further in vivo experimental validation is necessary.

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Corresponding Author Profile

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

Professor Li Meihua

First Affiliated Hospital of Nanchang University

  • Director of Neurosurgery, Chief Physician, Associate Professor, Doctoral Supervisor

  • Outstanding Young and Middle-aged Expert in Jiangxi Province Health System, Jiangxi Province Talent Program, Jiangxi Province Backbone Teacher and Young and Middle-aged Discipline Leader

  • In 2016, awarded the second prize of Science and Technology Progress Award for “Clinical Application of Lateral Cranial Base Approach for Complex Cranial Base Tumors” (first contributor) and in 2020, awarded the first prize of Jiangxi Province Science and Technology Progress Award for “Construction and Application of New System for Diagnosis and Treatment of Craniopharyngioma” (second contributor)

Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

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Impinging Flow Induces MCP-1 Expression in Endothelial Cells via JNK/c-Jun/p38/c-Fos Pathway

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