# Shear-Induced ITGB4 Promotes Endothelial Cell Inflammation and Atherosclerosis

Authors: Xiangquan Kong; Siyu Chen; Shuai Luo; Aiqun Chen; Liguo Wang; Haoyue Tang; Feng Wang; Zhimei Wang; Xiaofei Gao; Guangfeng Zuo; Wenying Zhou; Yue Gu; Zhen Ge; Junjie Zhang
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

The local heterogeneity in the distribution of atherosclerotic lesions is caused by local flow patterns. The integrin family plays crucial regulatory roles in diverse biological processes, but knowledge of integrinβ4 (ITGB4) in shear stress-induced atherosclerosis is limited. This study clarified that low shear stress (LSS) regulates the generation of ITGB4 in endothelial cells with atheroprone phenotype to identify ITGB4’s role in atherosclerosis. We found that LSS led to an increase in ITGB4 protein expression both in vitro and in vivo. ITGB4 knockdown attenuated inflammation and ROS generation in human umbilical vein endothelial cells (HUVECs) and reduced atherosclerotic lesion areas in ApoE-/- mice fed with HFD, largely independent of effects on the lipid profile. Mechanistically, ITGB4 knockdown altered the phosphorylation levels of SRC, FAK, and NFκB in HUVECs under LSS conditions. In addition, the knockdown of NFκB inhibited the production of ITGB4 and SRC phosphorylation, and the knockdown of SRC downregulated ITGB4 protein expression and NFκB activation. These data demonstrate a critical role of ITGB4 in atherosclerosis via modulation of endothelial cell inflammation, and ITGB4/SRC/NFκB might form a positive feedback loop in the regulation of endothelial cell inflammation.


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## Body

## 1. Introduction

Atherosclerosis is currently one of the most frequent causes of mortality worldwide and is characterized by a chronic inflammatory disease of the vascular wall involving most arteries of the whole body, such as the aorta, carotid arteries, and coronary arteries [1]. The development of atherosclerosis is accompanied by endothelial cell dysfunction, smooth muscle cell proliferation and mononuclear macrophage infiltration, internalizing modified lipoproteins to become foam cells, and lipid deposition in parts of the artery, eventually developing into an atherosclerotic plaque [2]. Among these pathological processes, endothelial cell dysfunction is an initial factor [3]. The regulatory mechanism of endothelial cell dysfunction remains incompletely understood.The shear stress is defined as a hydrodynamic force generated by blood flow, which is determined by the diameter of the vessel, flow plasticity, blood viscosity, and flow velocity [4]. Areas of arteries with low shear stress (<4 dyne/cm2), such as inner curvatures of the aortic arch, are prone to develop atherosclerotic lesions, whereas areas with normal shear stress (10-24 dyne/cm2), such as straight vessels, largely prevent the progression of atherosclerosis [5]. Integrins are heterodimeric transmembrane receptors, and at least 18 α and 8 β subunits are known in humans, generating a large family of approximately 24 heterodimers that regulate diverse and important cellular processes, including cell anchorage, migration, survival, lineage commitment, and the expression of differentiated phenotypes [6]. Many previous studies of shear stress-induced atherosclerosis have focused on the effect of members of the integrin adhesion receptor family [7]. Previous studies have shown that atheroprotective unidirectional shear stress inhibits the activation of endothelial YAP/TAZ by modulating the integrin–Gα13–RhoA pathway [8]. Recent studies have pointed out that atheroprone flow-induced integrin α5 translocation and activation of integrin in ECs is mediated by the Piezo1-Ca2+-PTP1B-ANXA2 pathway [9]. However, few studies have examined the effects of ITGB4 on shear stress-associated atherosclerosis.ITGB4 (also known as CD104) was initially identified as a member of the integrin superfamily, which is a superfamily of cell adhesion receptors. Previous studies have reported that the pathological and physiological functions of integrinα6β4 can be mediated entirely by the ITGB4 cytoplasmic domain [10]. Multiple studies have shown that ITGB4 attenuates human lung endothelial inflammatory responses by regulating various signaling pathways, such as CCL17, SHP-2, and MAPK [11, 12]. ITGB4 knockdown depresses the expression of caveolin-1 and Ras and attenuates HUVEC senescent features [13]. However, the relationship between ITGB4 and shear stress-associated vascular inflammation and the underlying regulatory mechanism remain unclear.In view of the key role of ITGB4 in endothelial dysfunction, in this study, we first detected the change in ITGB4 protein levels under LSS bothin vitro and in vivo. Next, we investigated the effect of ITGB4 knockdown on endothelial cell inflammation, ROS generation, and atherosclerosis progression. Finally, we identified the molecular mechanism by which ITGB4 regulates endothelial inflammation in HUVECs stimulated by LSS. Together, these findings highlight a novel regulatory role for ITGB4 in endothelial cell inflammatory responses and atherosclerosis and identify novel targets for the development of innovative therapies to treat atherosclerosis.


## 2. Material and Methods

### 2.1. Regents

Antibodies against ITGB4 (sc-514426) and p-FAK (sc-81493) were purchased from Santa Cruz Biotechnology (USA). Antibodies against FAK (3285S), SRC (2109S), p-SRC (59548S), and HRP-linked antibody (7076, 7074) were purchased from Cell Signaling Technology (USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GB11002) were purchased from Servicebio (Wuhan, China). Endothelial cell growth medium and 1% penicillin/streptomycin were purchased from Gibco (USA), and fetal bovine serum (FBS) was from Thermo Fisher Scientific (USA).


### 2.2. Cell Culture

HUVECs were purchased from the American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in endothelial cell medium (Gibco) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco) at 37°C and 5% CO2.


### 2.3. Flow Apparatus

Before simulation with shear stress, HUVECs were placed on plates with fibronectin (10μg/mL) and grown in ECM with 5% CO2. Then, HUVECs were subjected to shear stress in a parallel-plate flow chamber designed by Naturethink (Shanghai, China). The chamber was connected with a peristaltic pump and placed in a constant temperature enclosure with 5% CO2. The shear stress was estimated as τ=4μV/D, where V is the flow velocity, μ is the viscosity of the perfusate, and D is the lumen diameter of the rubber tube. In parallel, cells in the low shear stress condition were exposed to media flow at 2 dyne/cm2, and cells in the static condition were grown in plates.


### 2.4. Animals

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH), Bethesda, MD, USA) and the local ethics review board. Model animal research center of Nanjing University (Nanjing, China) provided us with male ApoE-/- mice at 8 weeks of age. All mice were euthanized with a lethal dose of sodium pentobarbital. Aortic arch inner curvatures and descending aortas were separated and fixed in 4% paraformaldehyde.


### 2.5. Adeno-Associated Virus (AAV) Injection

Endothelial ITGB4 knockdown AAVs were constructed by inserting ITGB4 shRNA into rAAV-tie2-mCherry-5′miR-30a-shRNA (ITGB4)-3′miR-30a-WPREs. Next, 100 μL volumes of rAAV-tie2-ITGB4 vector or rAAV-tie2-control vector were injected into the tail vein of 8-week-old ApoE-/- mice. The virus titer was 2×1012 genomes per milliliter. In order to construct coronary atherosclerosis models, two groups (n=10) of mice were fed with a high-fat and high-cholesterol diet after two weeks. After 8 weeks of feeding, mice were euthanized for subsequent studies.


### 2.6. Proteomics

The samples of cells were collected by centrifugation at25,000×g for 15 min at 4°C. Then, polypeptide preparation and iTRAQ quantification were performed by Luming Biotechnology Company.


### 2.7. Western Blotting

Total protein from HUVECs was lysed by a buffer containing RIPA and a protease inhibitor mixture (PMSF, aprotinin). Concentrations of protein were determined by a BCA Protein Assay Kit (23250, Thermo Fisher). Equal amounts of protein lysate (20μg) were separated by 5% SDS–PAGE (75 V, 0.5 hours; 110 V, 1 hour) and electrotransferred (110 V, 1 hour) at 4°C onto polyvinylidene difluoride membranes. Then, the cells were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 hours at room temperature (RT) and incubated with primary antibody overnight at 4°C. After washing three times with TBST, the blots were incubated with secondary antibody for 2 hours at RT. The membranes were washed 3 times again and assessed by chemiluminescence (PK10003, Proteintech) using a sys imaging system.


### 2.8. Small Interfering RNA (siRNA) and Cell Transfection

Cells maintained at 60% to 70% confluency were transfected with 100 nM ITGB4, SRC, and NFκB siRNA or scrambled siRNA (Gene Pharma, Shanghai, China) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001). For cells used for western blotting or ROS staining, cells were collected 48 h after transfection. Cells 48 h posttransfection were treated with LSS for 2 h for further analysis. The targeting sequences of siRNAs are shown in Supplement Table 1.


### 2.9. Luciferase Reporter Gene Assays

The interaction between ITGB4 and NFκB was detected by dual-luciferase reporter assay. The ITGB4 promoter was amplified and cloned into the pGL3 basic vector (Promega, USA). ITGB4 promoter-constructed plasmid and the NFκB overexpression plasmid were cotransfected into HEK 293 T cells (5×104 cells per well in 24-well plates). After 48 h of transfection, luciferase activity was determined by dual-luciferase assay kit (E1910, Promega, US) according to the manufacturer’s instructions.


### 2.10. Immunofluorescence Staining

Tissue sections and HUVECs on slides were washed three times with PBS and incubated in 4% paraformaldehyde for 15 minutes. The samples were infiltrated with 0.1% Triton X-100/PBS for 10 minutes, and the nonspecific immune response was blocked with 3% BSA at room temperature for 1 hour, washing the samples with PBS three times between each step. Next, the samples were incubated with the primary antibody at 4°C overnight. Then, after washing with PBS, the samples were incubated with the fluorescent conjugated secondary antibody in the dark for 2 hours. The nuclei were stained with DAPI (Beyotime, China). The images were collected by a laser scanning confocal microscope (LSM 710; Carl Zeiss, Germany), and the fluorescence intensity of the images was quantified using Image Pro Plus 6.0 software.


### 2.11. Quantitative Real-Time PCR

Total RNA was extracted using TRIzol reagent. We used the HiScript III first strand cDNA synthesis kit (Cat # R312-01/02, Vazyme, China) to reverse transcribe the 1μg RNA extracted from endothelial cells into complementary DNA strands. ChamQ Universal SYBR qPCR Master Mix (Cat 35; Q711-02, Vazyme, China) was used for RT qPCR. GAPDH was used as an internal control for quantification. The software is 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2-∆∆ Ct method was used to calculate the relative mRNA expression. The primers of genes are shown in Supplementary Table 2.


### 2.12. H&E Staining

The plaque area was examined with hematoxylin eosin (HE) staining. Firstly, dehydrate the sections with ethanol and xylene. After being briefly washed with distilled water, the nucleus was stained with 5% hematoxylin solution for 10 minutes. After being washed with distilled water for 5 minutes, incubate the sections with 0.1% hydrochloric acid ethanol for 30 seconds. Finally, the sections were stained with eosin solution for 2 minutes. After washing, the HE-stained sections were imaged with microscopy (BX43, Olympus).


### 2.13. Oil Red O Staining

Oil Red O staining of the aorta was performed as described in a previous study. Briefly, after staining with a freshly prepared Oil Red O working solution, aortas were differentiated by 60% isopropanol, washed with ddH2O, and then observed by a bright-field microscope (Olympus).


### 2.14. ROS Detection by DHE

A collection of HUVECs was cleaned three times with PBS, fixed with paraformaldehyde for 15 minutes, incubated in 30μM DHE (Invitrogen) in PBS for 30 min at room temperature in the dark, washed with 1× PBS twice, and immediately imaged with confocal microscopy.


### 2.15. Statistical Analysis

Data were generated from at least three independent experiments. Quantitative results are expressed asmean±SEM. The statistical analysis was performed using the GraphPad Prism software (Version 8.0). Statistical differences were determined by Student’s t-test for comparison between two groups and one-way ANOVA followed by Bonferroni’s test for comparison among multiple groups. P<0.05 was considered statistically significant.


## 2.1. Regents

Antibodies against ITGB4 (sc-514426) and p-FAK (sc-81493) were purchased from Santa Cruz Biotechnology (USA). Antibodies against FAK (3285S), SRC (2109S), p-SRC (59548S), and HRP-linked antibody (7076, 7074) were purchased from Cell Signaling Technology (USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GB11002) were purchased from Servicebio (Wuhan, China). Endothelial cell growth medium and 1% penicillin/streptomycin were purchased from Gibco (USA), and fetal bovine serum (FBS) was from Thermo Fisher Scientific (USA).


## 2.2. Cell Culture

HUVECs were purchased from the American Type Culture Collection (Manassas, VA, USA). HUVECs were cultured in endothelial cell medium (Gibco) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco) at 37°C and 5% CO2.


## 2.3. Flow Apparatus

Before simulation with shear stress, HUVECs were placed on plates with fibronectin (10μg/mL) and grown in ECM with 5% CO2. Then, HUVECs were subjected to shear stress in a parallel-plate flow chamber designed by Naturethink (Shanghai, China). The chamber was connected with a peristaltic pump and placed in a constant temperature enclosure with 5% CO2. The shear stress was estimated as τ=4μV/D, where V is the flow velocity, μ is the viscosity of the perfusate, and D is the lumen diameter of the rubber tube. In parallel, cells in the low shear stress condition were exposed to media flow at 2 dyne/cm2, and cells in the static condition were grown in plates.


## 2.4. Animals

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH), Bethesda, MD, USA) and the local ethics review board. Model animal research center of Nanjing University (Nanjing, China) provided us with male ApoE-/- mice at 8 weeks of age. All mice were euthanized with a lethal dose of sodium pentobarbital. Aortic arch inner curvatures and descending aortas were separated and fixed in 4% paraformaldehyde.


## 2.5. Adeno-Associated Virus (AAV) Injection

Endothelial ITGB4 knockdown AAVs were constructed by inserting ITGB4 shRNA into rAAV-tie2-mCherry-5′miR-30a-shRNA (ITGB4)-3′miR-30a-WPREs. Next, 100 μL volumes of rAAV-tie2-ITGB4 vector or rAAV-tie2-control vector were injected into the tail vein of 8-week-old ApoE-/- mice. The virus titer was 2×1012 genomes per milliliter. In order to construct coronary atherosclerosis models, two groups (n=10) of mice were fed with a high-fat and high-cholesterol diet after two weeks. After 8 weeks of feeding, mice were euthanized for subsequent studies.


## 2.6. Proteomics

The samples of cells were collected by centrifugation at25,000×g for 15 min at 4°C. Then, polypeptide preparation and iTRAQ quantification were performed by Luming Biotechnology Company.


## 2.7. Western Blotting

Total protein from HUVECs was lysed by a buffer containing RIPA and a protease inhibitor mixture (PMSF, aprotinin). Concentrations of protein were determined by a BCA Protein Assay Kit (23250, Thermo Fisher). Equal amounts of protein lysate (20μg) were separated by 5% SDS–PAGE (75 V, 0.5 hours; 110 V, 1 hour) and electrotransferred (110 V, 1 hour) at 4°C onto polyvinylidene difluoride membranes. Then, the cells were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 hours at room temperature (RT) and incubated with primary antibody overnight at 4°C. After washing three times with TBST, the blots were incubated with secondary antibody for 2 hours at RT. The membranes were washed 3 times again and assessed by chemiluminescence (PK10003, Proteintech) using a sys imaging system.


## 2.8. Small Interfering RNA (siRNA) and Cell Transfection

Cells maintained at 60% to 70% confluency were transfected with 100 nM ITGB4, SRC, and NFκB siRNA or scrambled siRNA (Gene Pharma, Shanghai, China) using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001). For cells used for western blotting or ROS staining, cells were collected 48 h after transfection. Cells 48 h posttransfection were treated with LSS for 2 h for further analysis. The targeting sequences of siRNAs are shown in Supplement Table 1.


## 2.9. Luciferase Reporter Gene Assays

The interaction between ITGB4 and NFκB was detected by dual-luciferase reporter assay. The ITGB4 promoter was amplified and cloned into the pGL3 basic vector (Promega, USA). ITGB4 promoter-constructed plasmid and the NFκB overexpression plasmid were cotransfected into HEK 293 T cells (5×104 cells per well in 24-well plates). After 48 h of transfection, luciferase activity was determined by dual-luciferase assay kit (E1910, Promega, US) according to the manufacturer’s instructions.


## 2.10. Immunofluorescence Staining

Tissue sections and HUVECs on slides were washed three times with PBS and incubated in 4% paraformaldehyde for 15 minutes. The samples were infiltrated with 0.1% Triton X-100/PBS for 10 minutes, and the nonspecific immune response was blocked with 3% BSA at room temperature for 1 hour, washing the samples with PBS three times between each step. Next, the samples were incubated with the primary antibody at 4°C overnight. Then, after washing with PBS, the samples were incubated with the fluorescent conjugated secondary antibody in the dark for 2 hours. The nuclei were stained with DAPI (Beyotime, China). The images were collected by a laser scanning confocal microscope (LSM 710; Carl Zeiss, Germany), and the fluorescence intensity of the images was quantified using Image Pro Plus 6.0 software.


## 2.11. Quantitative Real-Time PCR

Total RNA was extracted using TRIzol reagent. We used the HiScript III first strand cDNA synthesis kit (Cat # R312-01/02, Vazyme, China) to reverse transcribe the 1μg RNA extracted from endothelial cells into complementary DNA strands. ChamQ Universal SYBR qPCR Master Mix (Cat 35; Q711-02, Vazyme, China) was used for RT qPCR. GAPDH was used as an internal control for quantification. The software is 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2-∆∆ Ct method was used to calculate the relative mRNA expression. The primers of genes are shown in Supplementary Table 2.


## 2.12. H&E Staining

The plaque area was examined with hematoxylin eosin (HE) staining. Firstly, dehydrate the sections with ethanol and xylene. After being briefly washed with distilled water, the nucleus was stained with 5% hematoxylin solution for 10 minutes. After being washed with distilled water for 5 minutes, incubate the sections with 0.1% hydrochloric acid ethanol for 30 seconds. Finally, the sections were stained with eosin solution for 2 minutes. After washing, the HE-stained sections were imaged with microscopy (BX43, Olympus).


## 2.13. Oil Red O Staining

Oil Red O staining of the aorta was performed as described in a previous study. Briefly, after staining with a freshly prepared Oil Red O working solution, aortas were differentiated by 60% isopropanol, washed with ddH2O, and then observed by a bright-field microscope (Olympus).


## 2.14. ROS Detection by DHE

A collection of HUVECs was cleaned three times with PBS, fixed with paraformaldehyde for 15 minutes, incubated in 30μM DHE (Invitrogen) in PBS for 30 min at room temperature in the dark, washed with 1× PBS twice, and immediately imaged with confocal microscopy.


## 2.15. Statistical Analysis

Data were generated from at least three independent experiments. Quantitative results are expressed asmean±SEM. The statistical analysis was performed using the GraphPad Prism software (Version 8.0). Statistical differences were determined by Student’s t-test for comparison between two groups and one-way ANOVA followed by Bonferroni’s test for comparison among multiple groups. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. LSS Upregulates ITGB4 ExpressionIn Vitro and In Vivo

To identify potential proteins that may inform the mechanism underlying the progression of shear stress-induced atherosclerosis, we stimulated HUVECs with low shear stress for 24 h and subjected them to isobaric tags for relative and absolute quantitation- (iTRAQ-) based quantitative proteomic analysis. 234 differentially expressed genes were screened, including 151 upregulated genes and 83 downregulated genes (FigureS1A). Among the integrin family, the most significantly changed was ITGB4 (Figure 1(a)). To validate the proteomic results, we analyzed the ITGB4 protein content of HUVECs stimulated by LSS for 24 h via western blotting. ITGB4 protein expression initially increased with increasing stimulation time and peaked at approximately 12 h. Qualification of western blotting showed that the change in ITGB4 was significant (Figure 1(b)). Immunofluorescence staining further confirmed that LSS treatment markedly increased the expression of ITGB4 compared with static cells (Figure 1(c)). To further assess whether ITGB4 expression is associated with LSS in vivo, we detected ITGB4 protein expression in the lesser curvature of the aortic arch, low shear stress regions, greater curvature of the aortic arch, and normal shear stress regions by enface staining. Consistent with these in vitro results, the expression level of ITGB4 was augmented at proatherogenic sites compared with antiatherosclerotic sites (Figure 1(d)). These data showed that LSS significantly increased the expression of ITGB4 protein in vitro and in vivo.Figure 1
LSS upregulates ITGB4 expressionin vitro and in vivo. (a) Heat map of the expression of integrin family members. (b, c) HUVECs were exposed to static conditions or LSS for 0-24 h. The content of ITGB4 was detected by (b) western blot and (c) immunofluorescence. (d) Enface staining of the ITGB4 content of the aortic arch (AA) and thoracic aorta (TA) in C57Bl/6 mice. Scale bar: 20 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


### 3.2. LSS Promotes Atheroprone Gene Expression and ROS Generation via ITGB4In Vitro

To determine whether ITGB4 affects the development of atherosclerosis, we treated HUVEC with scrambled siRNA or ITGB4 siRNA under LSS. For knockdown experiments, three specific siRNAs targeting the ITGB4 gene were designed. We validated the knockdown efficiency for all ITGB4 siRNAs by western blotting. As shown in results, the abundance of ITGB4 was significantly downregulated by ITGB4 siRNAs (Figure2(a)). It has been reported that the expression of proinflammatory genes ICAM-1 and VCAM-1 and the production of reactive oxygen species (ROS) are upregulated by LSS. In this study, LSS increased the protein expression level of proinflammatory factors ICAM-1 and VCAM-1, while ITGB4 siRNA treatment weakened this effect (Figures 2(b) and 2(c)). Consistently, qPCR revealed that the mRNA expression of ICAM-1 and VCAM-1 was eliminated by ITGB4 siRNA (Figure 2(d)). At the same time, in order to further demonstrate the role of ITGB4 on the expression of proinflammatory genes, we used the constructed plasmid based on pcDNA3.1 to overexpress ITGB4. The effectiveness of ITGB4 overexpression was verified by western blotting (Figure S2A). The results showed that overexpression of ITGB4 further exacerbated the increased expression of proinflammatory factors ICAM-1 and VCAM-1 caused by LSS (Figure S2B). Next, we measured the generation of ROS in HUVECs exposed to LSS with or without ITGB4. LSS-induced oxidative stress promotes endothelial dysfunction, which is one of the early predictors of atherosclerosis and heart disease. The results of DHE staining show that LSS significantly upregulated the expression of ROS, which was significantly reversed by the deletion of ITGB4 (Figure 2(e)). Taken together, these results indicated that ITGB4 plays an essential role in the proatherogenic effect of LSS in vitro.Figure 2
LSS promotes atheroprone gene expression and ROS generation via ITGB4in vitro. (a) HUVECs were transfected with scr siRNA and ITGB4 siRNA for 48 h. The expression of ITGB4 was detected by western blot. (b, c) Western blot detection of the expression of VCAM-1 and ICAM-1 in HUVECs from the scr siRNA group and ITGB4 siRNA group. (d) The RNA expression of VCAM-1 and ICAM-1 was measured by RT–qPCR and normalized against GAPDH mRNA. (e) ROS generation of HUVECs transfected with scr siRNA and ITGB4 siRNA was detected by DHE staining. Photographs were taken and counted using a confocal microscope. Scale bar: 50 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)


### 3.3. LSS Promotes Atherosclerosis via ITGB4In Vivo

In order to further investigate the effect of ITGB4 on the progression of atherosclerosisin vivo, we constructed an adeno-associated virus- (AAV-) based vector to support targeted knockdown of the ITGB4 gene in vascular endothelial cells. Mice in the ApoE-/- backgrounds were divided into three groups: the first group (n=8) was a negative control group fed a chow diet for 8 weeks; the second group (n=8) was injected with vector virus (ITGB4vectorApoE–/–) and fed a high-fat and high-cholesterol diet (HCD) for 8 weeks; and the third group (n=8) was injected with ITGB4 AAV (ITGB4KDApoE–/–) and fed a HFD for 8 weeks. Then, the models were further evaluated. Using a blood lipid detection kit, we found that the levels of total cholesterol (TC), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) showed no difference with or without ITGB4 knockdown (Figure S3A). At the same time, we stained the atherosclerotic plaque of the mouse aorta and found that compared with ITGB4vectorApoE–/– mice, the expression of ICAM-1 and VCAM-1 related to the inflammatory response at the plaque areas of ITGB4KDApoE–/– mice was reduced (Figure S3B). Oil Red O staining revealed that, compared with ITGB4vector ApoE–/– mice, the total atherosclerotic areas were significantly decreased in the aorta of ITGB4KDApoE–/– mice (Figure 3(a)). H&E staining of aortic roots showed that HFD strikingly enhanced the size of atherosclerotic plaque areas, while ITGB4 knockdown attenuated the size of atherosclerotic plaque areas (Figure 3(b)). Additionally, Oil Red O staining revealed significantly mitigated lipid deposition in plaques of the aortic root in ITGB4KDApoE–/– mice compared to ITGB4vector ApoE–/– mice (Figure 3(c)). These outcomes indicate that ITGB4 influences plaque development and lipid deposition in the progression of atherosclerosis.Figure 3
LSS promotes atherosclerosis via ITGB4in vivo. ApoE-/- mice were randomly divided into 3 groups: NCD+ITGB4vector, HCD+ITGB4vector, and HCD+ITGB4KD. (a) The representative images and quantification of lesion areas were assayed by Oil Red O staining. Scale bar: 1 mm. (b) The representative images and quantification of lesion areas were assayed by H&E staining. Scale bars: 50 μm. (c) The representative images and quantification of lesion areas were assayed by Oil Red O staining. Scale bars: 50 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)


### 3.4. ITGB4 Mediates LSS-Induced FAK, SRC, and NFκB Activation in HUVECs

To investigate the mechanism by which ITGB4 may induce endothelial cell inflammation and ROS generation, we first examined the expression of several genes known to be highly implicated in the integrin signaling pathway. The results showed that the phosphorylation levels of FAK and SRC increased in a time-dependent manner (Figure4(a)). Next, we examined whether blocking ITGB4 could abolish FAK and SRC phosphorylation in HUVECs. We found phosphorylation of FAK and SRC was significantly elevated after LSS stimulation in control cells, whereas no significant increase in the phosphorylation of FAK and SRC was detected in cells transduced with ITGB4 siRNA after stimulation (Figure 4(b)). Previous studies revealed that LSS increases the abundance of NFκB and promotes cytoplasm-to-nucleus translocation of NFκB. As expected, LSS upregulated the phosphorylation of NFκB and promoted NFκB shuttling from the cytosol to the nucleus (Figures 4(b) and 4(c)). Both the increase in the phosphorylation level of NFκB and the enhanced nuclear translocation of NFκB by LSS were blunted by ITGB4 siRNA (Figure 4(c)). In summary, these data show that LSS induces activation of the FAK/SRC/NFκB signaling pathway via ITGB4 in vitro.Figure 4
ITGB4 mediates LSS-induced FAK, SRC, and NFκB activation in HUVECs. (a) The protein levels of p-FAK, FAK, p-NFκB, NFκB, p-SRC, and SRC were measured by western blot in HUVECs treated with LSS for 0-24 h. (b) The protein levels of p-FAK, FAK, p-NFκB, NFκB, p-SRC, and SRC were measured by western blot isolated from static or low shear stress conditions transfected with scr siRNA or ITGB4 siRNA. (c) The representative images and location of NFκB in HUVECs were assayed by immunofluorescence staining under static or low shear stress conditions transfected with scr siRNA or ITGB4 siRNA. Scale bars: 20 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)


### 3.5. LSS Instigates Positive Feedback Regulation between ITGB4/SRC and NFκB

To further clarify the relationship between the increase in ITGB4 protein expression and activation of NFκB, JASPAR CORE database (http://jaspar.genereg.net/) was used to analyze transcription factor binding sites (TFBS) in the ITGB4 promoter. There were three predicted binding sites: -148~-138 (E1), -238~-228 (E2), and -1891~-1881 (E3). Then, a dual-luciferase reporter assay was conducted by transfection with a dual-luciferase reporter containing the promoter sequence of ITGB4 to validate the binding activity between NFκB and the ITGB4 promoter (Figure 5(a)). The result reveals that NFκB can regulate the transcriptional activity of ITGB4 via the binding of NFκB to the ITGB4 promoter (Figure 5(b)). To analyze the possibility that NFκB affects the protein content of ITGB4 and SRC under LSS stimulation, we detected the ITGB4 protein level and phosphorylation level of SRC when NFκB was silenced. The transfection efficiency of NFκB siRNAs is verified (Figure 5(c)). Western blotting showed that transfection with NFκB siRNA reversed the increase of ITGB4 and p-SRC caused by LSS (Figure 5(d)) but had no effect on p-FAK expression (Figure S4A). In addition, the protein content of ITGB4 and NFκB activity in HUVECs transfected with scrambled siRNA or SRC siRNA were also investigated. The knockdown efficiency of SRC is verified (Figure 5(e)). LSS upregulated ITGB4 expression and NFκB phosphorylation levels, whereas SRC knockdown attenuated this alteration (Figure 5(f)) but SRC knockdown had no influence on the expression of p-FAK (Figure S4B). Taken together, these data suggest that an ITGB4-positive feedback loop promotes NFκB activation via SRC in response to LSS stimulation (Figure 6).Figure 5
LSS instigates positive feedback regulation between ITGB4/SRC and NFκB. (a) The JASPAR database predicts transcription factors that may bind to the ITGB4 promoter. (b) Dual-luciferase assay kit was used to detect the luciferase activity of HEK293T cells that were cotransfected with ITGB4 promoter plasmids and vector or NFκB plasmids. (c) Detection of three NFκB siRNAs. (d) The protein expression of ITGB4, p-SRC, and SRC in HUVECs under static or low shear stress conditions transfected with scr siRNA or NFκB siRNA. (e) Detection of three SRC siRNAs. (f) The protein expression of ITGB4, p-NFκB, and NFκB in HUVECs under static or low shear stress conditions transfected with scr siRNA or SRC siRNA. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)Figure 6
Shear-induced ITGB4 promotes endothelial cell inflammation and atherosclerosis. Low shear stress increased the expression of ITGB4, and ITGB4 activated FAK and SRC. SRC promotes the nuclear translocation of NFκB and increases the expression of ICAM-1 and VCAM-1 and the generation of ROS. In turn, the activation of NFκB promotes ITGB4 transcriptional activity.


## 3.1. LSS Upregulates ITGB4 ExpressionIn Vitro and In Vivo

To identify potential proteins that may inform the mechanism underlying the progression of shear stress-induced atherosclerosis, we stimulated HUVECs with low shear stress for 24 h and subjected them to isobaric tags for relative and absolute quantitation- (iTRAQ-) based quantitative proteomic analysis. 234 differentially expressed genes were screened, including 151 upregulated genes and 83 downregulated genes (FigureS1A). Among the integrin family, the most significantly changed was ITGB4 (Figure 1(a)). To validate the proteomic results, we analyzed the ITGB4 protein content of HUVECs stimulated by LSS for 24 h via western blotting. ITGB4 protein expression initially increased with increasing stimulation time and peaked at approximately 12 h. Qualification of western blotting showed that the change in ITGB4 was significant (Figure 1(b)). Immunofluorescence staining further confirmed that LSS treatment markedly increased the expression of ITGB4 compared with static cells (Figure 1(c)). To further assess whether ITGB4 expression is associated with LSS in vivo, we detected ITGB4 protein expression in the lesser curvature of the aortic arch, low shear stress regions, greater curvature of the aortic arch, and normal shear stress regions by enface staining. Consistent with these in vitro results, the expression level of ITGB4 was augmented at proatherogenic sites compared with antiatherosclerotic sites (Figure 1(d)). These data showed that LSS significantly increased the expression of ITGB4 protein in vitro and in vivo.Figure 1
LSS upregulates ITGB4 expressionin vitro and in vivo. (a) Heat map of the expression of integrin family members. (b, c) HUVECs were exposed to static conditions or LSS for 0-24 h. The content of ITGB4 was detected by (b) western blot and (c) immunofluorescence. (d) Enface staining of the ITGB4 content of the aortic arch (AA) and thoracic aorta (TA) in C57Bl/6 mice. Scale bar: 20 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


## 3.2. LSS Promotes Atheroprone Gene Expression and ROS Generation via ITGB4In Vitro

To determine whether ITGB4 affects the development of atherosclerosis, we treated HUVEC with scrambled siRNA or ITGB4 siRNA under LSS. For knockdown experiments, three specific siRNAs targeting the ITGB4 gene were designed. We validated the knockdown efficiency for all ITGB4 siRNAs by western blotting. As shown in results, the abundance of ITGB4 was significantly downregulated by ITGB4 siRNAs (Figure2(a)). It has been reported that the expression of proinflammatory genes ICAM-1 and VCAM-1 and the production of reactive oxygen species (ROS) are upregulated by LSS. In this study, LSS increased the protein expression level of proinflammatory factors ICAM-1 and VCAM-1, while ITGB4 siRNA treatment weakened this effect (Figures 2(b) and 2(c)). Consistently, qPCR revealed that the mRNA expression of ICAM-1 and VCAM-1 was eliminated by ITGB4 siRNA (Figure 2(d)). At the same time, in order to further demonstrate the role of ITGB4 on the expression of proinflammatory genes, we used the constructed plasmid based on pcDNA3.1 to overexpress ITGB4. The effectiveness of ITGB4 overexpression was verified by western blotting (Figure S2A). The results showed that overexpression of ITGB4 further exacerbated the increased expression of proinflammatory factors ICAM-1 and VCAM-1 caused by LSS (Figure S2B). Next, we measured the generation of ROS in HUVECs exposed to LSS with or without ITGB4. LSS-induced oxidative stress promotes endothelial dysfunction, which is one of the early predictors of atherosclerosis and heart disease. The results of DHE staining show that LSS significantly upregulated the expression of ROS, which was significantly reversed by the deletion of ITGB4 (Figure 2(e)). Taken together, these results indicated that ITGB4 plays an essential role in the proatherogenic effect of LSS in vitro.Figure 2
LSS promotes atheroprone gene expression and ROS generation via ITGB4in vitro. (a) HUVECs were transfected with scr siRNA and ITGB4 siRNA for 48 h. The expression of ITGB4 was detected by western blot. (b, c) Western blot detection of the expression of VCAM-1 and ICAM-1 in HUVECs from the scr siRNA group and ITGB4 siRNA group. (d) The RNA expression of VCAM-1 and ICAM-1 was measured by RT–qPCR and normalized against GAPDH mRNA. (e) ROS generation of HUVECs transfected with scr siRNA and ITGB4 siRNA was detected by DHE staining. Photographs were taken and counted using a confocal microscope. Scale bar: 50 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)


## 3.3. LSS Promotes Atherosclerosis via ITGB4In Vivo

In order to further investigate the effect of ITGB4 on the progression of atherosclerosisin vivo, we constructed an adeno-associated virus- (AAV-) based vector to support targeted knockdown of the ITGB4 gene in vascular endothelial cells. Mice in the ApoE-/- backgrounds were divided into three groups: the first group (n=8) was a negative control group fed a chow diet for 8 weeks; the second group (n=8) was injected with vector virus (ITGB4vectorApoE–/–) and fed a high-fat and high-cholesterol diet (HCD) for 8 weeks; and the third group (n=8) was injected with ITGB4 AAV (ITGB4KDApoE–/–) and fed a HFD for 8 weeks. Then, the models were further evaluated. Using a blood lipid detection kit, we found that the levels of total cholesterol (TC), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) showed no difference with or without ITGB4 knockdown (Figure S3A). At the same time, we stained the atherosclerotic plaque of the mouse aorta and found that compared with ITGB4vectorApoE–/– mice, the expression of ICAM-1 and VCAM-1 related to the inflammatory response at the plaque areas of ITGB4KDApoE–/– mice was reduced (Figure S3B). Oil Red O staining revealed that, compared with ITGB4vector ApoE–/– mice, the total atherosclerotic areas were significantly decreased in the aorta of ITGB4KDApoE–/– mice (Figure 3(a)). H&E staining of aortic roots showed that HFD strikingly enhanced the size of atherosclerotic plaque areas, while ITGB4 knockdown attenuated the size of atherosclerotic plaque areas (Figure 3(b)). Additionally, Oil Red O staining revealed significantly mitigated lipid deposition in plaques of the aortic root in ITGB4KDApoE–/– mice compared to ITGB4vector ApoE–/– mice (Figure 3(c)). These outcomes indicate that ITGB4 influences plaque development and lipid deposition in the progression of atherosclerosis.Figure 3
LSS promotes atherosclerosis via ITGB4in vivo. ApoE-/- mice were randomly divided into 3 groups: NCD+ITGB4vector, HCD+ITGB4vector, and HCD+ITGB4KD. (a) The representative images and quantification of lesion areas were assayed by Oil Red O staining. Scale bar: 1 mm. (b) The representative images and quantification of lesion areas were assayed by H&E staining. Scale bars: 50 μm. (c) The representative images and quantification of lesion areas were assayed by Oil Red O staining. Scale bars: 50 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)


## 3.4. ITGB4 Mediates LSS-Induced FAK, SRC, and NFκB Activation in HUVECs

To investigate the mechanism by which ITGB4 may induce endothelial cell inflammation and ROS generation, we first examined the expression of several genes known to be highly implicated in the integrin signaling pathway. The results showed that the phosphorylation levels of FAK and SRC increased in a time-dependent manner (Figure4(a)). Next, we examined whether blocking ITGB4 could abolish FAK and SRC phosphorylation in HUVECs. We found phosphorylation of FAK and SRC was significantly elevated after LSS stimulation in control cells, whereas no significant increase in the phosphorylation of FAK and SRC was detected in cells transduced with ITGB4 siRNA after stimulation (Figure 4(b)). Previous studies revealed that LSS increases the abundance of NFκB and promotes cytoplasm-to-nucleus translocation of NFκB. As expected, LSS upregulated the phosphorylation of NFκB and promoted NFκB shuttling from the cytosol to the nucleus (Figures 4(b) and 4(c)). Both the increase in the phosphorylation level of NFκB and the enhanced nuclear translocation of NFκB by LSS were blunted by ITGB4 siRNA (Figure 4(c)). In summary, these data show that LSS induces activation of the FAK/SRC/NFκB signaling pathway via ITGB4 in vitro.Figure 4
ITGB4 mediates LSS-induced FAK, SRC, and NFκB activation in HUVECs. (a) The protein levels of p-FAK, FAK, p-NFκB, NFκB, p-SRC, and SRC were measured by western blot in HUVECs treated with LSS for 0-24 h. (b) The protein levels of p-FAK, FAK, p-NFκB, NFκB, p-SRC, and SRC were measured by western blot isolated from static or low shear stress conditions transfected with scr siRNA or ITGB4 siRNA. (c) The representative images and location of NFκB in HUVECs were assayed by immunofluorescence staining under static or low shear stress conditions transfected with scr siRNA or ITGB4 siRNA. Scale bars: 20 μm. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)


## 3.5. LSS Instigates Positive Feedback Regulation between ITGB4/SRC and NFκB

To further clarify the relationship between the increase in ITGB4 protein expression and activation of NFκB, JASPAR CORE database (http://jaspar.genereg.net/) was used to analyze transcription factor binding sites (TFBS) in the ITGB4 promoter. There were three predicted binding sites: -148~-138 (E1), -238~-228 (E2), and -1891~-1881 (E3). Then, a dual-luciferase reporter assay was conducted by transfection with a dual-luciferase reporter containing the promoter sequence of ITGB4 to validate the binding activity between NFκB and the ITGB4 promoter (Figure 5(a)). The result reveals that NFκB can regulate the transcriptional activity of ITGB4 via the binding of NFκB to the ITGB4 promoter (Figure 5(b)). To analyze the possibility that NFκB affects the protein content of ITGB4 and SRC under LSS stimulation, we detected the ITGB4 protein level and phosphorylation level of SRC when NFκB was silenced. The transfection efficiency of NFκB siRNAs is verified (Figure 5(c)). Western blotting showed that transfection with NFκB siRNA reversed the increase of ITGB4 and p-SRC caused by LSS (Figure 5(d)) but had no effect on p-FAK expression (Figure S4A). In addition, the protein content of ITGB4 and NFκB activity in HUVECs transfected with scrambled siRNA or SRC siRNA were also investigated. The knockdown efficiency of SRC is verified (Figure 5(e)). LSS upregulated ITGB4 expression and NFκB phosphorylation levels, whereas SRC knockdown attenuated this alteration (Figure 5(f)) but SRC knockdown had no influence on the expression of p-FAK (Figure S4B). Taken together, these data suggest that an ITGB4-positive feedback loop promotes NFκB activation via SRC in response to LSS stimulation (Figure 6).Figure 5
LSS instigates positive feedback regulation between ITGB4/SRC and NFκB. (a) The JASPAR database predicts transcription factors that may bind to the ITGB4 promoter. (b) Dual-luciferase assay kit was used to detect the luciferase activity of HEK293T cells that were cotransfected with ITGB4 promoter plasmids and vector or NFκB plasmids. (c) Detection of three NFκB siRNAs. (d) The protein expression of ITGB4, p-SRC, and SRC in HUVECs under static or low shear stress conditions transfected with scr siRNA or NFκB siRNA. (e) Detection of three SRC siRNAs. (f) The protein expression of ITGB4, p-NFκB, and NFκB in HUVECs under static or low shear stress conditions transfected with scr siRNA or SRC siRNA. Data are presented as the mean±SEM. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)Figure 6
Shear-induced ITGB4 promotes endothelial cell inflammation and atherosclerosis. Low shear stress increased the expression of ITGB4, and ITGB4 activated FAK and SRC. SRC promotes the nuclear translocation of NFκB and increases the expression of ICAM-1 and VCAM-1 and the generation of ROS. In turn, the activation of NFκB promotes ITGB4 transcriptional activity.


## 4. Discussion

Integrins are glycoproteins on the surface of cells that are composed ofα and β subunits with noncovalent bonds. Previous research has revealed that there are 18 kinds of alpha and 8 kinds of beta subunits forming 24 kinds of heterologous dimers. ITGB4 differs from other β subunits in that it binds only to the α6β4 subunit to form the α6β4 dimer and has a long intracellular domain containing 1088 amino acids [14]. Tyrosine in the intracellular domain tail often phosphorylates and enforces the adaptor protein Shc, which further activates the downstream signaling component [15]. Some studies have shown that it is involved in the progression of diseases such as intestinal tumors and epidermolysis bullosa letalis and is expressed in tumor-related blood vessels [16, 17]. Li et al. used four different NCBI GEO datasets and TCGA datasets to analyze the relationship between ITGB4 level expression and overall survival in tumors. The high expression of ITGB4 in intestinal tumor cells was significantly associated with adverse overall survival, suggesting that ITGB4 is a prognostic factor for colon cancer [18]. Overexpression of ITGB4 promotes epithelial-mesenchymal transformation and enhances the ability to invade and metastasize [19]. It has not been reported whether ITGB4 plays a role in atherosclerosis. In the present study, we showed that ITGB4 was activated under LSS both in vitro and in vivo. In animal studies, ITGB4 knockdown also showed that ITGB4 plays an important role in the development of atherosclerosis. In addition, our findings establish a new association between ITGB4, SRC, and NFκB in HUVECs and emphasize that this pathway may be a potential target to prevent and treat atherosclerosis.Coronary atherosclerotic heart disease mainly involves the subepicardial coronary artery, and its pathological basis is chronic inflammatory and fibrous hyperplasia, characterized by lumen stenosis, resulting in myocardial ischemia and hypoxia [20]. Although the entire coronary tree is exposed to atherosclerotic systemic conditions, atherosclerotic stenosis occurs at specific sites, such as the medial side of the curved segment, near the bifurcation, and lateral side of the bifurcation segment [20]. Endothelial shear stress is the tangential stress generated by the friction between blood and vascular intima. The shear stress is generally greater than 10 dyne/cm2 under stable laminar flow, which is characterized by uniform flow in a single direction and has an antiatherosclerotic effect [21]. LSS (less than 4 dyne/cm2) resulted in extremely reduced endothelial shear force, resulting in vascular remodeling, smooth muscle cell proliferation, and lumen stenosis. Therefore, LSS is a driving factor of atherosclerosis.As a nonreceptor protein tyrosine kinase, SRC participates in regulating various physiological and pathological processes, including bone formation and remodeling, and bone metastases in breast, prostate, and lung cancers [21, 22]. Notably, via interacting with integrin, E-cadherin, and focal adhesion kinase, SRC family kinases play an important role in regulating cell migration and motility [23]. In addition to this, pathways for cell survival and proliferation as well as gene expression are also regulated by SRC [24]. It is worth mentioning that two major integrins α6 and β4 have been found to be involved in the progression, invasion, and metastasis of breast cancer [25]. The binding of ligands to integrins activates several intracellular signaling molecules, such as FAK and SRC [26, 27]. In response to mutual activation of FAK/SRC, multiple downstream signaling pathways are initiated via phosphorylation by various adapter proteins, resulting in important cellular responses, including motility, proliferation, survival, migration, and invasion [27, 28]. In this study, we found that LSS upregulates the protein level of ITGB4 and further promotes the phosphorylation of SRC and FAK.NFκB has always been considered as a prototypical proinflammatory signaling pathway, for the reason that the activation of NFκB is mediated by proinflammatory cytokines, chemokines, and adhesion molecules, such as interleukin 1 (IL-1) and tumor necrosis factor a (TNFα) [29]. Recently, the regulation of SRC and NFκB has been widely reported. LPS promotes inflammation and apoptosis by activating the SRC-mediated NFκB p65 and MAPK signaling pathways, resulting in AKI in mice [30, 31]. Activated NFκB p65 translocates into the nucleus and regulates the transcription of a variety of inflammatory genes. Meanwhile, we found increased phosphorylation of NFκB p65 in HUVECs with LSS treatment, which was reversed by SRC knockdown. These findings corroborated an earlier study in which laminar shear stress attenuated NFκB p65 nuclear translocation and DNA binding activity and attenuated TNFα-induced inflammation [32].


## 5. Conclusion

In summary, these results demonstrated that the screened ITGB4 is associated with atherosclerosis. LSS upregulated ITGB4 both invitro and in vivo. ITGB4 gene knockout attenuated atherosclerotic lesions in ApoE-/- mice, which is largely independent of the effect on lipid profile, but through decreasing endothelial cell inflammation and ROS generation. In addition, we also found a positive feedback loop in ITGB4/SRC/NFκB, further revealing the critical role of ITGB4 in atherosclerosis. Therefore, ITGB4 is expected to become an effective therapeutic target for coronary atherosclerosis in the future.



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*Source: 5842677-2022-10-25.xml*