siLOXL2 Inhibits Endothelial Inflammatory Response and EndMT Induced by ox-LDL

Atherosclerosis (AS) is a prominent cause of mortality and morbidity. It is characterized by an inflammatory pathophysiology that leads to arterial wall sclerosis, thickening, and plaque formation [1]. The primary lipoprotein implicated in cholesterol-induced AS is low-density lipoprotein (LDL). Oxidized LDL (ox-LDL) possesses significant pro-oxidative stress and proinflammatory effects [2]. ox-LDL plays a substantial role in the onset and progression of AS by leading to endothelial cell (EC) dysfunction, increasing leukocyte adhesiveness, and triggering the expression of leukocyte and monocyte adhesion substances on the endothelial surface, namely, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectins [3‒5]. The transition from nitric oxide signaling to reactive oxygen species (ROS) signaling is a key determinant in the activation of ECs. Nitric oxide plays a crucial role in promoting homeostasis and preserving the quiescent state of the vascular wall through various mechanisms, such as inhibiting proinflammatory cytokine secretion, immune cell extravasation, smooth muscle cell proliferation, thrombosis, and vascular leakage [6]. On the other hand, ROS triggers NFκB signaling, which serves as the primary regulator of inflammation. This transition toward oxidative stress represents a fundamental process underlying EC dysfunction in response to various pathophysiological triggers [7].

Macrophages, which are derived from monocyte cells [1], can phagocytize ox-LDL to produce lipid-rich foam cells, trigger the production of ROS, secrete proinflammatory molecules, and lead to inflammation of vascular walls and their subsequent repair, which results in the formation and growth of plaques [8, 9]. The proteolytic enzyme matrix metalloproteinase 9 (MMP-9) can weaken plaque fibrous caps by degrading the extracellular matrix (ECM), which leads to plaque instability [10, 11]. MMP-9 is highly expressed in unstable plaques [12]. However, the macrophage secretion of MMP-9 can be induced by ox-LDL [13].

A member of the lysyl oxidase family, which also includes the enzymes lysyl oxidase (LOX) and lysyl oxidase-like 1–4 (LOXL1–LOXL4), is lysyl oxidase-like 2 (LOXL2). Like the other LOX family members, LOXL2 has a conserved catalytic domain that catalyzes the oxidative deamination of lysine residues to cross-link collagen and elastin [14]. In addition to its function in the remodeling of the ECM through the cross-linking of collagen and elastin, LOXL2 controls several intracellular signaling pathways involved in cell division, proliferation, tumor metastasis and the epithelial-to-mesenchymal transition (EMT) [15‒18].

EC has the capability to develop myofibroblast-like characteristics via a specific type of EMT referred to as endothelial-to-mesenchymal transition (EndMT). Throughout the process of EndMT, the expression levels of endothelial markers such as VE-cadherin and CD31 are diminished, while there is an acquisition of mesenchymal markers like α-smooth muscle actin (α-SMA), N-cadherin, and calponin [19, 20]. The occurrence of EndMT involves the disruption of cell-cell interactions and cell polarity, leading to a morphological transformation into a spindle-shaped structure and the development of a migratory and invasive phenotype accompanied by increased ECM synthesis [21‒23]. However, a previous study reported that LOXL2 has been demonstrated to control EMT through several methods, including the regulation of transcription factors that control both EMT and EndMT [24]. Therefore, we thus hypothesize that LOXL2 could regulates ox-LDL-induced migration, inflammation, oxidative stress, and monocyte adhesion by inhibiting the EndMT pathway.

Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO₂. Cells were passaged every 3–4 days after reaching 60–70% confluence. The cells were treated with ox-LDL at different concentrations (0, 10, 25, 50, 100 μg/mL) and incubated for 24 h.

The sequences for siRNA targeting human LOXL2 were as follows: LOXL2-siRNA1: forward, 5′-GGA UUC CUG GGU UCA AAU UTT-3′, reverse, 5′-AAU UUG AAC CCA GGA AUC CTT-3′; LOXL2-siRNA2: forward, 5′-GGC CCU UGG AAG UAC AAA UUU-3′, reverse, 5′-AUU UGU ACU UCC AAG GGC CUU-3′); LOXL2-siRNA3: forward, 5′-GCA AUG AGA AGU CCA UUA UTT-3′; reverse, 5′-AUA AUG GAC UUC UCA UUG CTT-3′; negative control siRNA for LOXL2 (siNC sequence: forward, 5′-AAA CAU GCC AAG CGC AAA CAU-3′; reverse, 5′-AUG UUU GCG CUU GGC AUG UUU-3′) were commercially ordered from GenePharma Company (China). siRNAs (siLOXL2 and siNC) were transfected into HUVECs (5 × 10⁵ cells) with Lipofectamine 2000 (Invitrogen, USA). After 48 h, real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to examine the interference efficiency.

HUVECs (2 × 10³ cells/well) were seeded into 96-well plates and then treated with different concentrations of ox-LDL (0, 10, 25, 50, 100 μg/mL) for 24 h. CCK-8 (10 μL, C0037, Beyotime) solution was added to each well of the 96-well plates. After 3 h of incubation, a microplate reader examined cell absorbance at 450 nm.

The migration capability of HUVECs was assessed by the transwell assay. The cell seeding density in transwell chambers was 1 × 10⁵ cells/mL. The stained migrated cells were counted under a light microscope (magnification, ×100) from 5 fields to calculate the average number of migrated cells.

HUVECs were stained with DCFH-DA (1:1,000) at 37°C for 30 min in the dark. Then, the samples were washed three times with PBS. The fluorescence was measured at 485 nm (excitation) and 535 nm (emission) using a microplate reader (IX71, Olympus, Japan).

The HUVECs were homogenized to obtain cell lysate and kept in the refrigerator at −80°C. The activities of MDA (S0131S, Beyotime, Shanghai, China) content, superoxide dismutase (SOD) (S0109, Beyotime) activity, CAT (S0051, Beyotime) activity were determined using the commercial kit and previously reported protocol was followed for the oxidative stress level [25].

Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines in the culture medium of HUVECs cells. After various treatments, supernatants were collected, and levels of interleukin-1β (IL-1β, DLB50, R&D Systems), interleukin-6 (IL-6, D6050, R&D Systems), and tumor necrosis factor-alpha (TNF-α, DTA00D, R&D Systems) were evaluated by ELISA kits (R&D Systems Inc., Minneapolis, MN, USA). The concentrations were expressed as pg/mL.

To assess the adhesion of THP1 to HUVECs, a monocyte-endothelium adhesion assay was performed using a plate from Baiao Leibo Technology Co., Ltd, Beijing, China. After applying various treatments, HUVECs were mixed with FITC-stained THP1 monocytes and incubated for 1 h. Non-adherent cells were then removed, and we observed the adhesion of the green fluorescence-labeled THP1 cells to the HUVECs using a microscope (IX51, Olympus, Japan). The number of adhered THP1 cells was counted from at least five random fields to obtain the average value.

Total RNA was isolated from HUVECs using TRizol (Invitrogen, USA). Total RNA was reverse-transcribed into complementary DNA (cDNA). SYBR Green reagent (TaKaRa, Japan) was used in an ABI Prism 7700 Real-Time PCR equipment (Applied Biosystems, USA) to amplify the mRNA by RT-qPCR. The relative gene expression was normalized to the internal control, GAPDH, using the 2^-ΔΔCt formula [26]. The primers for LOXL2, VEGFA, MMP-9, IL-1β, IL-6, TNF-α, ICAM-1, VCAM-1, α-SMA, vimentin, CD31, von Willebrand factor (vWF), and GAPDH were designed by the NCBI Primer-Blast Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), which is listed in Table 1. The Biological replicates were three, and the technical replicates were six.

Total protein was extracted using RIPA lysis buffer, and nuclear protein was extracted using Extraction Reagents (Pierce Biotechnology, Inc., Rockford, IL. USA). Protein concentrations were measured using a BCA protein assay kit (Beyotime Biotechnology, China). Protein samples (50 μg) were separated on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% low-fat milk and incubated with primary antibodies LOXL2 (1:500, sc-293427; mouse monoclonal, Santa Cruz), α-SMA (1:500, sc-53142, mouse monoclonal, Santa Cruz), vimentin (1:500, sc-32322, mouse monoclonal, Abcam), CD31 (1:400, ab9498, mouse monoclonal, Abcam), vWF (1:500, ab154193, rabbit monoclonal, Abcam), and GAPDH (1:1,000, ab9485, rabbit polyclonal, Abcam). The membranes were incubated with HRP-linked secondary antibodies. The β-actin (1:1,000, Cat# ab8227, Abcam) was used as an internal control. The bands were visualized by ECL (Thermo, Waltham, MA, USA) and were analyzed using the ImageJ software.

The data were presented as mean ± standard deviation, and the analysis was conducted using SPSS 20.0 statistical software (IBM Analytics, New York, NY, USA). Differences between three or more groups were statistically analyzed using one-way ANOVA. A p value of less than 0.05 was considered statistically significant.

To investigate the expression levels of LOXL2 in ox-LDL-induced ECs, HUVECs were treated with ox-LDL at several concentrations (0, 10, 25, 50, 100 μg/mL) for 24 h. CCK-8 assessed the cell activity of HUVECs. With increasing the dose, the cell viability was potentially reduced after 25 μg/mL of dose (Fig. 1a). The mRNA expression of LOXL2 in ox-LDL-induced HUVECs was enhanced by increasing the dose (Fig. 1b). Further, the protein expression of LOXL2 was investigated by Western blot analysis. The LOXL2 expression was improved with the increasing dose (Fig. 1c), and the same expression trends were observed after quantification analysis (Fig. 1d). The results demonstrated that LOXL2 protein expression was increased in ox-LDL-induced ECs, suggesting a potential role for LOXL2 in the cellular response to ox-LDL.

To assess the potential effect of LOXL2 on the ox-LDL-induced migration and ECM gene expression in HUVECs, LOXL2 siRNA (siLOXL2), and control siRNA (siNC) were transfected to HUVECs for 48 h. Western blot analysis was performed to investigate protein levels of LOXL2, and siLOXL2-2 showed a significant inhibitory effect of LOXL2 expression (Fig. 2a). Quantification analysis of the grayscale value of protein bands in Western blot revealed the same pattern of LOXL2 expression (Fig. 2b). In addition, HUVECs were transfected with LOXL2 siRNA or control siRNA, followed by incubation with 100 μg/mL ox-LDL for a further 24 h. HUVECs were stained with 0.1% crystal violet. ox-LDL + siLOXL2 significantly reduced the migration ability of HUVECs (Fig. 2c). Quantification analysis showed that ox-LDL + siLOXL2 consistently inhibits the migration ability of HUVECs (Fig. 2d). Furthermore, the RT-qPCR was carried out to determine the mRNA expression of VEGFA and MMP-9 in HUVECs, and ox-LDL + siLOXL2 potentially inhibited the expression of VEGFA and MMP-9 (Fig. 2e, f). Therefore, the results showed that LOXL2 silencing suppresses the migration ability of HUVECs with ox-LDL and ECM gene expression in HUVECs.

To study the potential impact of LOXL2 on the intracellular ROS generation and oxidative stress in HUVECs with ox-LDL, HUVECs were stained with DCFH-DA. The ox-LDL + siLOXL2 significantly reduced intracellular ROS production (Fig. 3a). Quantification analysis of the DCFH-DA fluorescence intensity in HUVECs showed a similar pattern of ROS production in HUVECs (Fig. 3b). In addition, ox-LDL + siLOXL2 significantly inhibited the expression of oxidative stress marker gene Malondialdehyde (MDA) (Fig. 3c), whereas it potentially enhanced the marker genes SOD (Fig. 3d) and catalase (CAT) (Fig. 3e). Therefore, the results investigated that LOXL2 inhibition potentially reduced the intracellular ROS generation and oxidative stress in HUVECs with ox-LDL.

We carried out an ELISA assay and RT-qPCR to investigate the potential effect of LOXL2 on the ox-LDL-induced inflammation in HUVECs. Supernatant levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and TNF-α were markedly increased after ox-LDL treatment, and ox-LDL + siLOXL2 co-treatment significantly inhibited the levels of IL-1β, IL-6, and TNF-α (Fig. 4a–c). In addition, mRNA levels of IL-1β, IL-6, and TNF-α potentially elevated after ox-LDL treatment, and IL-1β, IL-6, and TNF-α levels were significantly decreased after ox-LDL + siLOXL2 co-treatment (Fig. 4d–f). Therefore, the results suggested that LOXL2 inhibition significantly attenuated the production of proinflammatory cytokines.

The monocyte-endothelium adhesion assay was carried out to explore the effect of LOXL2 on the adhesion of THP1 monocytes to HUVECs. THP1 monocytes were stained with FITC and cocultured with HUVECs. The adhesion of THP1 to HUVECs was observed under an inverted fluorescence microscope (IX71, Olympus, Japan). The results showed that ox-LDL treatment increased the adhesion cell number of THP1 monocytes to HUVECs, whereas ox-LDL + siLOXL2 treatment significantly reduced the adhesion cell number (Fig. 5a). The same decreased adhesion cell number trends were observed after quantification analysis (Fig. 5b). Moreover, the mRNA expression of adhesion genes ICAM-1 and VCAM-1 were determined by the RT-qPCR analysis in HUVECs. The obtained results showed that ICAM-1 and VCAM-1 gene expression was enhanced after the ox-LDL treatment, and ox-LDL + siLOXL2 significantly inhibited ICAM-1 and VCAM-1 gene expression (Fig. 5c, d). Therefore, the results demonstrated that LOXL2 inhibition significantly attenuated the adhesion of THP1 monocytes to HUVECs.

We examined the expression of EndMT marker genes, including mesenchymal cell-specific genes (α-SMA and vimentin) and EC-specific genes (CD31 and vWF) in HUVECs using RT-qPCR and Western blot analysis. The results showed that α-SMA and vimentin gene expressions were elevated, and CD31 and vWF gene expressions were reduced with ox-LDL treatment, whereas the ox-LDL + siLOXL2 treatment reversed the gene expression patterns (Fig. 6a, b). In addition, we investigated the gene expression of EndMT markers using Western blot analysis. The results demonstrated that ox-LDL treatment increased the α-SMA and vimentin and decreased the CD31 and vWF gene expressions, whereas the reversed expression pattern was observed after ox-LDL + siLOXL2 treatment (Fig. 6c). After quantification analysis, we observed similar trends of expression patterns for the EndMT markers gene (Fig. 6d, e). The results demonstrated that LOXL2 inhibition potentially reduced the ox-LDL-induced EndMT in HUVECs.

The present study investigated the potential effect of LOXL2 on ox-LDL-induced migration, inflammation, oxidative stress, and monocyte adhesion by suppressing the EndMT pathway. Our obtained results demonstrated that LOXL2 expression is increased in ox-LDL-induced ECs, LOXL2 attenuation decreases the ox-LDL-induced migration and ECM gene (VEGFA and MMP-9) expression, suppresses the ox-LDL-induced intracellular ROS production and oxidative stress in HUVECs, inhibits ox-LDL-induced inflammation of HUVECs, and decreases the adhesion of THP1 monocytes to HUVECs. Furthermore, α-SMA and vimentin gene expressions were increased, while CD31 and vWF gene expressions were reduced with the ox-LDL treatment. However, the gene expression patterns were reversed with the ox-LDL + siLOXL2 therapy. Therefore, our results speculate that LOXL2 could be a potential target for treating AS significantly.

To study LOXL2 expression levels in ox-LDL-induced ECs, HUVECs were treated for 24 h with various doses of ox-LDL. After 25 μg/mL of dosage, we observed a potential reduction in cell viability with increasing dose (Fig. 1a). Interestingly, increasing the dosage led to higher LOXL2 mRNA expression in ox-LDL-induced HUVECs (Fig. 1b). We then conducted Western blot analysis was carried out to verify increased LOXL2 protein expression in ox-LDL-induced HUVECs. As shown in Figure 1c, d, the results demonstrated a clear trend of enhanced LOXL2 expression with increased doses. These findings indicate that ECs stimulated by ox-LDL express more LOXL2 protein.

A previous study reported that through the let-7b-5p/HOXA1 axis, which controls the expression of genes involved in cell proliferation, apoptosis, and ECM remodeling, lncRNA ROR enhanced the biological properties of ox-LDL-induced HUVECs [27]. Another study showed that the expression of genes involved in cell cycle, migration, and ECM production was altered by miR-200c-3p’s stimulation of ox-LDL-induced EndMT in HUVECs through the SMAD7/YAP pathway [28]. However, our study uncovered a new aspect. We found that ox-LDL + siLOXL2 potentially attenuated the migration ability of HUVECs (Fig. 2c). The ECM-related gene, including VEGFA and MMP-9 expression, is significantly regulated by the treatment of ox-LDL + siLOXL2 (Fig. 2e, f). Therefore, the results of the study demonstrated that LOXL2 silencing inhibits HUVECs’ migrating capacity with ox-LDL and ECM gene expression, introducing a fresh perspective to the field.

ROS refers to chemical species that contain reactive oxygen, such as peroxides, superoxide, hydroxyl radicals, and singlet oxygen. ROS plays an important role in cell signaling and homeostasis in a biological setting. However, oxidative stress significantly boosts the generation of ROS, which can severely damage cell structures [29]. Previous studies demonstrated that the generation of ROS and ox-LDL increases as AS progresses [30]. Extracellular/intracellular oxidative stress and endothelial damage have both been linked to excessive ROS generation [31, 32]. In the present study, our focus was on LOXL2, a protein involved in regulating ECM and cell adhesion. We reported that ox-LDL + siLOXL2 significantly decreased intracellular ROS production (Fig. 3a). In addition, ox-LDL + siLOXL2 markedly reduced the expression of the oxidative stress marker gene Malondialdehyde (MDA) (Fig. 3c). At the same time, it is increased the expression of SOD (Fig. 3d) and CAT (Fig. 3e). Therefore, the results demonstrated that LOXL2 silencing significantly reduced ox-LDL-induced intracellular ROS production and oxidative stress in HUVECs.

ox-LDL can facilitate the adhesion of monocytes, neutrophils, and lymphocytes to ECs by stimulating the production of ICAM-1, VCAM-1, E-selectin, and P-selectin [33]. Under pathological circumstances, ECs generate cytokines, including IL-1β, IL-6, and TNF-α, leading to inflammatory reactions. These inflammatory cytokines can also increase the expression of adhesion molecules on the surface of ECs, stimulating EC adhesion and consequent injury [34]. However, our study showed that supernatant levels of IL-1β, IL-6, and TNF-α were significantly increased after ox-LDL treatment, and ox-LDL + siLOXL2 treatment potentially reduced the levels of IL-1β, IL-6, and TNF-α (Fig. 4a–c). Moreover, the mRNA expression of adhesion genes ICAM-1 and VCAM-1 gene expression was improved after the ox-LDL treatment, and ox-LDL + siLOXL2 significantly reduced ICAM-1 and VCAM-1 gene expression (Fig. 5c, d), shedding light on the molecular mechanisms involved. Thus, the results demonstrated that LOXL2 suppression significantly inhibited the proinflammatory cytokines secretion and adhesion of THP1 monocytes to HUVECs.

The transformation of ECs into mesenchymal-like cells (EndMT) under pathological settings provided evidence that ECs are heterogeneous and highly adaptable [35]. A key factor in this transformation is ox-LDL, which our study focuses on. Recent studies have revealed that interferon regulatory factor 2-binding protein 2 inhibits ox-LDL-induced inflammation and EndMT by upregulating krüppel-like factor 2, attenuating macrophage-mediated inflammation and susceptibility to AS [36]. In addition, it has been demonstrated that the drug pyrogallol-phloroglucinol-6, 6-bieckol inhibits ox-LDL-induced EndMT by upregulating vWF and PECAM-1 expression while downregulating α-SMA and vimentin expression [37]. However, our study showed that the mesenchymal cell-specific (α-SMA and vimentin) gene expressions were elevated, and the EC-specific (CD31 and vWF) gene expressions were reduced with ox-LDL treatment. In contrast, the ox-LDL + siLOXL2 treatment altered the gene expression patterns (Fig. 6a, b).

This study showed that the EndMT pathway was inhibited by LOXL2 downregulation, which is protected against ox-LDL-induced EC dysfunction. This suggests that LOXL2 could be a potential therapeutic target for treating AS. However, further research is needed to fully understand and validate these findings in vivo settings.

Ethical approval is not required for this study in accordance with local or national guidelines.

The authors declared no conflict of interest with other people or organizations.

This study was supported by the Science and Technology Development Fund of Shanghai Pudong New Area (Grant No. PKJ2022-Y49).

Zhongsheng Zhu designed the project, supervised the project, and revised the manuscript. Jing Ma performed experiments and wrote the first draft of the manuscript. Jia Ling and Rui Tong helped perform the experiments and collect data. Jiefen Guo analyzed the data and performed statistical analysis.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author, Dr. Zhongsheng Zhu (email: [email protected]).