The novel anthraquinone compound Kanglexin prevents endothelial-to-mesenchymal transition in atherosclerosis by activating FGFR1 and suppressing integrin β1/TGFβ signaling

Abstract

Endothelial-mesenchymal transition (EndMT) disrupts vascular endothelial integrity and induces atherosclerosis. Active integrin β1 plays a pivotal role in promoting EndMT by facilitating TGFβ/Smad signaling in endothelial cells. Here, we report a novel anthraquinone compound, Kanglexin (KLX), which prevented EndMT and atherosclerosis by activating MAP4K4 and suppressing integrin β1/TGFβ signaling. First, KLX effectively counteracted the EndMT phenotype and mitigated the dysregulation of endothelial and mesenchymal markers induced by TGFβ1. Second, KLX suppressed TGFβ/Smad signaling by inactivating integrin β1 and inhibiting the polymerization of TGFβR1/2. The underlying mechanism involved the activation of FGFR1 by KLX, resulting in the phosphorylation of MAP4K4 and Moesin, which led to integrin β1 inactivation by displacing Talin from its β-tail. Oral administration of KLX effectively stimulated endothelial FGFR1 and inhibited integrin β1, thereby preventing vascular EndMT and attenuating plaque formation and progression in the aorta of atherosclerotic Apoe^−/− mice. Notably, KLX (20 mg/kg) exhibited superior efficacy compared with atorvastatin, a clinically approved lipid-regulating drug. In conclusion, KLX exhibited potential in ameliorating EndMT and retarding the formation and progression of atherosclerosis through direct activation of FGFR1. Therefore, KLX is a promising candidate for the treatment of atherosclerosis to mitigate vascular endothelial injury.

Keywords

atherosclerosis / EndMT / integrin β1 / FGFR1 / MAP4K4 / Kanglexin

1 Introduction

Atherosclerosis serves as the underlying pathology for ischemic stroke and coronary artery disease, and it is the primary instigator of acute myocardial infarction. This condition arises from an excessive accumulation of lipids and inflammatory cells beneath the arterial endothelium, giving rise to the development of atherosclerotic plaques. These plaques subsequently induce vasoconstriction and ischemic injury to vital organs. The presence of inflammatory cells promotes foam cell formation, which aggregates and ultimately culminates in necrotic core formation, rendering the plaques unstable. The rupture of these vulnerable plaques can precipitate thrombosis and acute cardiovascular events [1–3]. The arterial endothelium is composed of vascular endothelial cells intricately interconnected to establish a protective barrier that separates the bloodstream from the vascular wall [4,5]. Under the influence of factors such as hypoxia, hypertension, and exposure to smoke, endothelial cells undergo a phenotypic transformation known as endothelial-to-mesenchymal transition (EndMT). This process is characterized by the development of stress fibers within endothelial cells and the secretion of the extracellular matrix (ECM), which enhances endothelial permeability and triggers lipid deposition, inflammatory cell adhesion, and atherosclerotic plaque formation. EndMT leads to the upregulation of mesenchymal cell markers, including vimentin, α-SMA, and N-cadherin, accompanied with a downregulation of endothelial cell markers such as VE-cadherin and CD31 [6–9]. In addition to its role in initiating atherosclerosis, EndMT is crucial in the pathogenesis of various conditions including renal fibrosis, cardiac fibrosis, pulmonary fibrosis, and cancer [10–13].

Transforming growth factor-beta (TGFβ) is the most potent inducer of EndMT because of its major isomers, including TGFβ1, TGFβ2, and TGFβ3; it effectively triggers EndMT [14]. TGFβ initiates EndMT by directly binding to TGFβ receptor 1 and 2 (TGFβR1 and TGFβR2) on the endothelial cell membrane, subsequently promoting the phosphorylation of Smad2/3. Once activated, Smad2/3 forms a heteropolymer complex with Smad4, and this complex moves to the nucleus, ultimately resulting in the overexpression of key regulators such as Twist, Snail, and Slug. These factors then inhibit endothelial gene expression but stimulate mesenchymal gene expression to foster EndMT [15,16]. Integrins play a pivotal role in integrating and coordinating various cellular components, including the ECM, cytoskeleton, and intracellular signaling pathways. Moreover, they possess diverse functions in cell differentiation, migration, and adhesion. Notably, active integrin β1 serves as a crucial promoter of TGFβ/Smad signaling and EndMT [17,18]. MAP4K4 assumes the responsibility of inactivating integrin β1 by inducing Moesin phosphorylation within endothelial cells, thereby playing a pivotal role in the defense mechanism against EndMT in endothelial cells [19,20]. The activity of MAP4K4 in endothelial cells has been demonstrated to be regulated by fibroblast growth factors (FGFs) and fibroblast growth factor receptor 1 (FGFR1) signaling. Upon activation, FGFR1 interacts with and activates MAP4K4, leading to the inhibition of integrin β1 and TGFβ signaling [20]. However, the expression of FGFR1 diminishes in endothelial cells exposed to pro-EndMT stimuli. Moreover, endothelial deletion of FGFR1 leads to an increase in total plaque burden in Apoe^−/− mice fed a high-fat diet (HFD). Clinical studies have also demonstrated a strong correlation between the severity of coronary atherosclerosis and the loss of endothelial FGFR1 expression. In summary, FGFR1 is a key inhibitor of EndMT and represents a crucial therapeutic target in the context of atherosclerosis [21–23].

FGFs are endogenous peptide ligands for FGFR1 [24]. In clinical practice, peptide administration primarily involves local delivery or injections, so it is often plagued by suboptimal patient compliance. Atherosclerosis is a chronic progressive disease with long-term implications. Therefore, there is an urgent need to develop oral medication targeting FGFR1 to address the challenges associated with using peptides in atherosclerosis treatment and improve patient compliance. Previous studies have demonstrated the potential of Kanglexin (KLX), an anthraquinone compound (Fig.1), in activating the AMPK/eNOS signaling pathway for endothelial protection [25]. Furthermore, KLX has shown promise in promoting angiogenesis and facilitating diabetic wound healing by activating FGFR1 and ERK1/2 signaling [26]. Building upon these previous investigations, this study aimed to investigate whether KLX can protect against TGFβ1-induced EndMT and ameliorate atherosclerosis in Apoe^−/− mice subjected to HFD. Additionally, we explored the mechanisms underlying KLX’s suppression of integrin β1, TGFβ/Smad signaling, and EndMT by activating FGFR1 and MAP4K4. These findings introduce KLX as a promising candidate drug for preventing and treating vascular EndMT and atherosclerosis.

Fig.1 Kanglexin (KLX) suppresses EndMT in HUVECs induced by TGFβ1. (A) Chemical structure of KLX. (B) KLX mitigated the morphological changes and abnormal intercellular connectivity in HUVECs induced by TGFβ1 (10 ng/mL, 24 h); n = 4. (C) KLX reduced collagen synthesis and secretion in TGFβ1-induced HUVECs; n = 6. (D) KLX (10 μM) attenuated TGFβ1-induced abnormal morphology and distribution of F-actin in HUVECs. Scale bar represents 20 μm; n = 9. (E) Protein abundance of endothelial cell markers VE-cadherin and CD31 and mesenchymal cell markers N-cadherin and vimentin in HUVECs; n = 4–5. (F–I) Statistical graphs of protein abundance of VE-cadherin, CD31, N-cadherin, and vimentin in HUVECs; n = 4–5. (J) Fluorescence intensity of endothelial cell markers VE-cadherin and mesenchymal cell marker vimentin in HUVECs. HUVECs were treated with TGFβ1 (10 ng/mL) for 24 h to induce EndMT. Scale bar represents 20 μm; n = 3. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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2 Methods

2.1 Chemicals and reagents

KLX, with a purity exceeding 99%, was synthesized by the Department of Medicinal Chemistry and Natural Medicine Chemistry at Harbin Medical University, located in Harbin, Heilongjiang, China. The chemical synthesis of KLX has been thoroughly documented in a previous study [27].

Primary antibodies were sourced from various reputable providers. CD31 (#ab222783), vimentin (#ab20346), phospho-integrin β1 (Thr788/799; #ab5189), integrin β1 (#ab179471), GAPDH (#ab8245), and PD173074 (#ab141117) were procured from Abcam, Cambridge, England. Primary antibodies including VE-cadherin (#D87F2), N-cadherin (#13116), phospho-FGR1 (Tyr653/654; #52928), FGFR1 (#9740), phospho-Smad 2/3 (Ser465/467; #D27F4), Smad2/3 (#D7G7), and α-tubulin (#11H10) were acquired from Cell Signaling Technology in Danvers, USA. Primary antibodies TGFβR1 (#bs-0638R), TGFβR2 (#bs-0117R), Snail (#bs-1382R), Twist (#bs-2441R), and Slug (#bs-1382R) were purchased from Bioss Antibodies in Beijing, China. Additionally, the primary antibody for Samd4 was sourced from Santa Cruz Biotechnology in Texas, USA. Anti-HRP antibodies (#926–32211, #926–32212) were obtained from LI-COR Biosciences, Lincoln, USA, and the 9EG7 antibody was procured from R&D Systems, Minnesota, USA.

2.2 Culture and treatment of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVECs) obtained from Otwo Biotech, Shenzhen, China (Catalog No. HTX1922), were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under controlled conditions at 37 °C with 5% CO₂ and 95% air. Cells were passaged when their confluence exceeded 90%, and HUVECs from passages 3 to 8 were selected for subsequent experiments. HUVECs were uniformly seeded in six-well plates and cultured until they reached a confluence of 90%. Prior to the experiment, HUVECs were pretreated with 10 μM KLX for 1 h. Subsequently, they were exposed to 10 ng/mL TGFβ1 for 24 h to induce EndMT, followed by further assessments.

2.3 Establishment of atherosclerosis animal model and KLX treatment

Apoe^−/− mice (weighing 18–20 g) were obtained from Vital River Laboratory Animal Technology in Beijing, China. All animal protocols were approved by the Ethics Committee of Harbin Medical University (specifically, the Institutional Review Board of the College of Pharmacy at Harbin Medical University) and adhered to the ARRIVE guidelines and recommendations for ethical care and use of experimental animals. Following a one-week period of adaptive feeding, the Apoe^−/− mice were randomly allocated into several groups, including an atherosclerosis model group, KLX treatment groups (at doses of 5, 10, and 20 mg/kg/day), and atorvastatin (ATV) treatment group (at a dose of 5 mg/kg/day). Group assignments were based on the original bodyweights of the mice. The Apoe^−/− mice were then subjected to HFD for a duration of 16 weeks to induce atherosclerosis, whereas wild-type mice receiving a normal diet (ND) served as the normal control group. The atherosclerosis model group was administered with KLX solvent via intragastric administration once daily. The mice in the KLX and ATV treatment groups received corresponding doses of KLX and ATV through intragastric administration once daily for the entire 16-week period. These dosages were determined based on preliminary experiments. At the end of the study, assessments and analyses were conducted in accordance with the established research plan.

2.4 Serum biochemical analysis

Upon completing the experiment, the mice underwent a 12-h fasting period to prepare for serum biochemical analysis. Blood samples were collected from the inner canthus of the mice and processed to isolate serum. The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum of each mouse were determined by using an automatic biochemical analyzer (Hitachi, Japan) with appropriate kits (Medical System, Ningbo, China).

2.5 Oil red O staining of aorta and aortic root

At the end of the experiment, the mice were humanely euthanized, and the entire aorta was meticulously isolated. The aortas were submerged in PBS, and meticulous removal of excess adipose and connective tissue surrounding them was conducted under a microscope. Subsequently, the aortas were fixed in 4% paraformaldehyde overnight, followed by immersion in a 30% sucrose solution overnight. The sunken thoracic aorta was incubated and immersed in an oil red O solution (0.5 g of oil red O powder dissolved in 100 mL of isopropyl alcohol) on a shaker for 30 min. Any non-specific staining was effectively removed through rinsing with 60% isopropyl alcohol. The staining results were observed and documented by using a microscope (Leica, Germany).

For frozen sections of the aortic root, they were briefly immersed in a 60% isopropyl alcohol solution for 20 s. Subsequently, the sections were placed in an ice-water mixture for 10 s and submerged in an oil red O solution for 15 min. To remove any excess dye, we dipped the sections in 60% isopropyl alcohol for 3 s and soaked them in ice water for 10 s. The sections were stained with Meyer’s hematoxylin solution for 3 min and submerged in water for 10 min to regain a blue hue. Excess water was blotted away using filter paper, and the sections were sealed with glycerol jelly mounting medium. The staining results were observed and recorded by using a microscope (Leica, Germany).

2.6 Hematoxylin–eosin staining of aortic root

The hematoxylin–eosin staining procedure was conducted in accordance with the guidelines provided in the hematoxylin–eosin stain kit (Solarbio Life Science, Beijing, China). Frozen sections of the aortic root were initially fixed with Davidson’s fixative for 1 min at room temperature. Excess Davidson’s fixative was subsequently rinsed away under running water. The sections were then immersed in hematoxylin solution for 6 min, followed by a 10 min rinse in running water to restore the tissue to a blue hue. Tissue differentiation was achieved via 2 min of treatment with acid alcohol, and subsequent staining with eosin solution was performed for 2 min. Any surplus staining solution was thoroughly removed with deionized water. Subsequently, the tissue was dehydrated with a series of ethanol solutions with concentrations of 75%, 85%, 95%, and 100% and rendered transparent using xylene. The sections were finally sealed with glycerol jelly mounting medium, and the staining results were observed and recorded by using a microscope (Leica, Germany).

2.7 Masson staining of aortic root

The Masson staining assay was performed according to the operating instructions of the Modified Masson’s Trichrome Stain Kit (Solarbio Life Science, Beijing, China). Frozen sections of aortic root were fixed with Davidson’s fixative for 1 min at room temperature. Excess Davison’s fixative was washed with running water, and the sections were stained with hematoxylin solution for 10 min. Excessive staining solution was removed by deionizing water, and the sections were differentiated with acidic ethanol for 5–10 s. After the differentiation solution was washed away with deionized water, the sections were stained with Masson blue solution for 1 min. The slices were stained with Ponceau S solution for 2 min to discolor (bright positive red is preferred). After the sections were differentiated with phosphomolybdic acid solution for 1 min, the liquid was washed away with a weak acid solution. The sections were dyed with aniline blue for 1 min, and the aniline blue dye was washed away with a weak acid working solution. The sections were sealed with glycerol jelly mounting medium, and the staining results were observed and recorded by using a microscope (Leica, Germany).

2.8 Determination of collagen content

The collagen content was determined according to the instructions provided by the Sircol Soluble Collagen Assay Kit (Biocolor, the UK; #S1000). Fresh cell culture supernatant (600 μL) was mixed thoroughly with 120 μL of pre-cooled isolation concentration reagent. The mixture was incubated at 4 °C for 48 h and centrifuged at 12 000 r/min for 10 min. Subsequently, the supernatant was discarded and replaced with 1 mL of Sircol staining reagent. The tube was gently inverted every 5 min and slowly mixed for a total of 30 min. Afterward, it underwent another round of centrifugation at 12 000 r/min for an additional 10 min. Following the removal of the supernatant, pre-cooled Acid-Salt Wash Reagent (750 μL) was added to the precipitate-containing tube, which underwent centrifugation at 12 000 r/min for another 10 min before discarding the supernatant once again. Upon the addition of alkali reagent (250 μL), the precipitation was dissolved by vortex. About 10 μL of sample solution was diluted with 180 μL of alkali reagent and added to the 96-well plate. The absorbance of each sample was detected at 555 nm.

2.9 Immunofluorescence

HUVECs were inoculated in glass bottom dishes with a confluence of 70%. After treatment with different factors, the cells were moistened with PBS for 3 min and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were permeated using 1 mL of 0.2% Triton X-100 for 10 min. The cells were rinsed with PBS, blocked with 2% BSA solution for 50 min, and incubated with a diluted primary antibody overnight at 4 °C in darkness. On the second day, after PBS washing, the cells were incubated with the second antibody at room temperature for 50 min. The nuclei were then labeled with DAPI. The images were obtained by laser confocal microscopy (FV1000, Olympus, Japan).

Frozen sections of the aortic root (10 μm) were infiltrated with pre-cooled acetone for 10 min. After being washed with PBS, the sections were permeated with 0.1% Trixon-100 for 20 min. The sections were rinsed with PBS again and sealed with 1% BSA–PBS solution at room temperature for 1 h. After rinsing with PBS, each section was treated with 100 μL of diluted primary antibody and incubated at 4 °C overnight. On the second day, after being washed with PBS, the sections were incubated with the second antibody at room temperature for 1 h. The nuclei were then labeled with DAPI. The images were captured by laser confocal microscopy (FV1000, Olympus, Japan).

2.10 Duolink proximity ligation assay

The Duolink proximity ligation assay was performed according to the instructions provided in the Duolink® proximity ligation assay kit (Sigma–Aldrich, Darmstadt, Germany, #DUO921012). Frozen sections of the aortic root were fixed with 4% paraformaldehyde solution for 10 min, washed with PBS, and permeated with 0.2% Triton X-100 for 10 min. After discarding the permeabilization solution, the sections were washed again with PBS and incubated at 37 °C for 30 min in a blocking solution. Subsequently, the sections were incubated with a primary antibody solution prepared with Duolink antibody diluent overnight at 4 °C. Following this step, the primary antibody solution was discarded, and the sections were washed twice at room temperature for 5 min each time using washing buffer A. PLA probe solution was added to the sections, and they were incubated at 37 °C for 100 min. The sections were cleaned with wash buffer A and incubated with the connecting solution for 30 min at 37 °C. After another round of washing using wash buffer A, amplified solution was applied to the sections and they were incubated once more at 37 °C for 100 min. After washing with wash buffer B, the sections were sealed with Duolink® in situ sealer containing DAPI. The results were recorded by confocal microscopy, and statistical analysis was conducted by Image J software.

2.11 Western blot analysis

Western blot analysis was performed in accordance with the previous method. Protein should be extracted under ice-cold conditions. The cells or tissues were lysed using ice-cold protein lysates (RIPA:phosphatase inhibitor:protease inhibitor = 100:10:1), and the lysed mixture was centrifuged at 16 000 g and 4 °C for 15 min. The supernatant was collected, and the protein concentration was measured (Beyotime, Shanghai, China; # P0012). The protein samples were heated in a metal bath at 95 °C for 10 min. The samples were cooled and subjected to gel electrophoresis. Protein samples were added to the gel wells for separation. After complete separation, protein was transferred to an NC membrane and blocked with 5% skim milk for 2 h to block nonspecific bands. The NC membrane was washed with PBST on a shaker for 30 min and incubated with diluted primary antibody with CD31, VE-cadherin, vimentin, N-cadherin, P-FGFR1, FGFR1, integrin β1, P-integrin β1, TGFβR1, TGFβR2, P-Smad2/3, Smad2/3, Smad4, Snail, Twist, Slug, α-tubulin, β-actin, GAPDH (1:1000) at 4 °C overnight. The NC membrane was washed with PBST on a shaker for 30 min and incubated with the anti-HRP antibody (1:10 000) for 50 min in the dark at room temperature. The NC membrane was washed with TBST on a shaker in darkness for 30 min. The expression of the target protein was detected by an infrared fluorescence scanning system, and the optical density integral value of the protein bands was analyzed by Image J software.

2.12 TRITC phalloidin staining

HUVECs were inoculated in glass bottom dishes with a confluence of 50%. After treatment with different factors, the cells were moistened with PBS for 3 min and fixed with 4% paraformaldehyde for 10 min. After washing with PBS, the cells were permeated with 0.5% Triton X-100 for 5 min, followed by another rinse with PBS. The cells were incubated with TRITC Phalloidin solution for 30 min under dark conditions and rinsed again with PBS. The nuclei were stained with DAPI for 30 s, and the cells were rinsed with PBS. The results were recorded by confocal microscopy (FV1000, Olympus, Japan), and statistical analysis was performed by Image J software according to the following equation: F-actin (+) area % = Area of F-Actin(+)/Total image area × 100%.

2.13 Immunoprecipitation

Protein extraction should be performed under cold conditions. HUVECs were lysed using ice-cold protein lysates (RIPA:phosphatase inhibitor:protease inhibitor = 100:10:1), and the resulting lysate was centrifuged at 14 000× g and 4 °C for 5 min. About 100 μL of supernatant was collected and incubated with anti-FGFR1 antibody or immunoglobulin G working solution on a rotating mixer at 4 °C overnight to form an antigen–antibody complex. The remaining supernatant served as the input control. Subsequently, 100 µL of the antigen–antibody complex was incubated with 100 µL of magnetic beads suspended with TBS for 1 h at room temperature. The sample tube was then placed on a magnetic rack to collect the bead–antigen–antibody complex. After washing the bead–antigen–antibody complex with lysate, the complex was diluted with SDS-PAGE loading buffer and heated at 95 °C for 5 min. Finally, the sample was placed on a magnetic rack to separate the magnetic beads, and the supernatant was used for Western blot analysis.

2.14 Statistical analysis

All values are presented as the mean ± standard error. The sample sizes are provided in the figure legends, and each data point represents an independent test of a biological sample. Statistical analyses were conducted utilizing GraphPad Prism software and involved t-tests, paired t-tests, one-way ANOVA, and two-way ANOVA analyses as appropriate. A P value less than 0.05 was considered statistically significant.

3 Results

3.1 KLX suppresses EndMT in TGFβ1-induced HUVECs

To investigate the effect of KLX on EndMT, we treated HUVECs with TGFβ1 (10 ng/mL) for 24 h to induce EndMT. EndMT induces morphological changes in endothelial cells and reduces intercellular connections [8]. Under normal conditions, HUVECs exhibit a pebble-like shape with tight intercellular connections. Following TGFβ1 treatment, HUVECs transformed into a spindle shape and displayed significantly reduced intercellular connections. However, treatment with KLX (10 μM) alleviated the changes in endothelial cell morphology and restored intercellular connectivity disrupted by TGFβ1 (Fig.1). EndMT stimulates the collagen synthesis signaling pathway, thereby enhancing the synthesis and secretion of ECM [28]. TGFβ1 increased collagen secretion in HUVECs, but KLX (10 μM) attenuated this effect (Fig.1). EndMT leads to remodeling of F-actin skeleton protein in endothelial cells, causing abnormal morphology and distribution of F-actin, as well as the formation of stress fibers, pseudopodia, and lamellipodia [29]. TRITC phalloidin staining was used to examine the morphology and distribution of F-actin in HUVECs. As depicted in Fig.1, F-actin was predominantly concentrated around the cell membrane with a typical pebble-like shape in normal HUVECs. No stress fibers were observed within the cytoplasm or obvious pseudopods around the cell membrane. Following treatment with TGFβ1, F-actin disappeared from around the cell membrane, but numerous stress fibers formed within the cytoplasm along with pseudopods and lamellipods near the cell membrane. Pretreatment of HUVECs with KLX (10 μM) mitigated these aberrant changes induced by TGFβ1. KLX alone helped maintain the intrinsic morphology and distribution of F-actin under normal culture conditions for HUVECs (Fig.1). EndMT induces the upregulation of mesenchymal cell markers vimentin and N-cadherin but suppresses the expression of endothelial cell markers VE-cadherin and CD31 [8]. As shown in Fig.1‒1I, TGFβ1 treatment downregulated VE-cadherin and CD31 expression but promoted vimentin and N-cadherin expression in HUVECs. These effects were mitigated by KLX. Moreover, KLX administration increased the levels of endothelial cell markers and decreased mesenchymal cell markers in HUVECs without TGFβ1 treatment, indicating its potential to protect cultured endothelial cells from differentiation. Immunofluorescence analysis corroborated these findings. As shown in Fig.1 and 1K, KLX alleviated the downregulation of VE-cadherin and upregulation of vimentin triggered by TGFβ1. Notably, KLX enhanced VE-cadherin and decreased vimentin expression in HUVECs without TGFβ1 treatment, thereby preserving their phenotypic stability as endothelial cells. Taken together, these results indicated that KLX effectively suppressed EndMT in TGFβ1-induced HUVECs.

3.2 KLX alleviates aortic EndMT and atherosclerosis

Aberrations in vascular endothelial cell structure or function help promote atherogenesis. EndMT disrupts the tight connection between endothelial cells and increases the permeability of vascular endothelium, resulting in lipid deposition, inflammatory cell adhesion, and atherosclerotic plaque formation [30,31]. Our findings demonstrated that KLX alleviated EndMT in HUVECs. We further investigated whether KLX plays a beneficial role in the formation and progression of atherosclerotic plaques. Apoe^−/− mice were fed HFD for 16 weeks to induce atherosclerosis, and different doses of KLX were administered intragastrically to evaluate its anti-atherosclerosis effect. Mice treated with ATV, which is a widely used lipid-regulating agent in clinical practice for preventing atherosclerosis, served as positive controls. First, the serum lipid profile of mice in each group was determined. We found that KLX did not exhibit any beneficial effects on dyslipidemia in Apoe^−/− mice fed a long-term HFD; however, ATV effectively improved this dyslipidemia. Moreover, KLX significantly attenuated the elevation of serum transaminase levels caused by long-term HFD, whereas ATV exacerbated the increase of the transaminase content, indicating its adverse effect on liver function (Tab.1). To evaluate the effect of KLX on lipid deposition in the aortic wall, we separated and stained the whole aorta and aortic root tissues of mice with oil red O. En face oil red O staining revealed the formation of atherosclerotic plaques in the aortic arch and thoracic aorta of HFD mice. Oral administration of KLX (10 and 20 mg/kg) or ATV (5 mg/kg) effectively reduced plaque formation in both regions (Fig.2 and 2B). Pronounced results were observed through oil red O staining of aortic roots. As shown in Fig.2 and 2F, HFD mice developed a large area of lipid-deposited plaques in the aortic roots. KLX significantly reduced plaque formation and lipid deposition. In addition, H&E staining was used to evaluate the area of total atherosclerotic plaque and necrotic core in the aortic roots of Apoe^−/− mice. As illustrated in Fig.2 and 2E, HFD mice displayed an increased number of atherosclerotic plaques and a substantial presence of necrotic cores within plaque regions. KLX (10 and 20 mg/kg) treatment substantially reduced total plaque area and the area and number/size of necrotic cores, suggesting that KLX stabilized existing atherosclerotic plaques and delayed disease progression (Fig.2 and 2H). The secretion of collagen in the atherosclerotic plaque within the aortic roots of Apoe^−/− mice was evaluated by Masson staining. As shown in Fig.3 and 3C, a significant increase in collagen secretion was observed in the aortic plaques of HFD mice, whereas oral administration of KLX (20 mg/kg) resulted in reduced collagen secretion. Immunofluorescence analysis was performed to assess the protein expression levels of EndMT markers within the aortic plaques. Co-staining for CD31 (red) and vimentin (green) demonstrated decreased CD31 levels and increased vimentin levels within the aortic intima of HFD mice. Treatment with KLX attenuated HFD-induced alterations in CD31 and vimentin expression within the aortic endothelium, suggesting that KLX effectively impeded the progression of aortic EndMT (Fig.3, 3D, and 3E). Collectively, these findings indicated that KLX alleviated aortic EndMT and atherosclerosis.

Tab.1 Serum lipid contents in Apoe–/– mice

Serum lipid(mmol/L)	ND	HFD	HFD
			ATV 5 mg/kg	KLX 5 mg/kg	KLX 10 mg/kg	KLX 20 mg/kg
Cholesterol	3.40 ± 0.13	29.32 ± 5.75***	22.30 ± 3.40#	27.89 ± 4.58	29.83 ± 5.28	27.08 ± 4.20
Triglyceride	0.74 ± 0.06	0.99 ± 0.14	1.03 ± 0.37	1.06 ± 0.16	1.11 ± 0.27	1.10 ± 0.27
LDLC	0.68 ± 0.24	13.28 ± 4.18***	8.23 ± 2.53#	12.23 ± 3.40	14.00 ± 5.57	12.03 ± 3.23
HDLC	2.02 ± 0.11	0.41 ± 0.18***	0.80 ± 0.33#	0.50 ± 0.06	0.39 ± 0.15	0.39 ± 0.11
ALT	66.20 ± 7.66	100.00 ± 20.25 **	170.00±145.82	109.00 ± 40.73	65.40 ± 20.60#	63.60 ± 15.26#
AST	23.20 ± 4.09	66.00 ± 26.77 **	70.00 ± 20.72	65.20 ± 8.40	31.00 ± 2.65#	25.60 ± 12.70#

LDLC, low-density lipoprotein cholesterol; HDLC, high-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase. ** P < 0.01, *** P < 0.001 vs. ND, # P < 0.05 vs. HFD, n = 5.

Fig.2 KLX delays the formation and progression of atherosclerosis. (A) Oil red O staining of the whole aortas. Scale bar: 5 mm; n = 4. (B) Percentage of plaque area/total area in the aorta, aortic arch, and thoracic aorta. AA, aortic arch; TA, thoracic aorta. (C) Oil red O staining of the aortic roots of Apoe−/− mice. Scale bar: 200 μm; n = 4. (D) H&E staining of the aortic roots of Apoe−/− mice; scale bar: 200 μm; n = 4. (E) H&E staining of the aortic roots of Apoe−/− mice. Scale bar: 100 μm; n = 4. (F) Quantification of Oil red O staining of aortic roots; n = 4. (G) Quantification of plaque area of aortic roots; n = 4. (H) Quantification of necrotic core area of aortic roots; n = 4. ATV, atorvastatin. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig.3 KLX alleviates EndMT in the aortic intima of Apoe−/− mice. (A) Masson staining of aortic roots. Scale bar: 200 μm; n = 4. (B) Immunofluorescence staining of CD31 (red) and vimentin (green) in the aortic roots of Apoe−/− mice. DAPI (blue) represents the nucleus. Scale bar: 40 μm; n = 3. (C) Quantification of collagen content in aortic roots; n = 4. (D) Quantification of immunofluorescence intensity of CD31; n = 3. (E) Quantification of immunofluorescence intensity of vimentin; n = 3. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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3.3 KLX inhibits the activation of TGFβR/Smad signaling in HUVECs

The data presented above demonstrated that KLX effectively inhibited EndMT in TGFβ1-induced HUVECs and in HFD-induced Apoe^−/− mouse aortas. To further elucidate the underlying mechanisms behind KLX’s inhibitory effects on EndMT, we investigated its impact on the TGFβR/Smad signaling pathway. Activation of TGFβR/Smad signaling is a pivotal component of TGFβ1-induced EndMT [15,18,20]. TGFβR1 and TGFβR2 are essential receptors that enable TGFβ1 to phosphorylate downstream Smad2 at Ser465/467 and Smad3 at Ser423/425 residues. Phosphorylated Smad2 (Ser465/467) and Smad3 (Ser423/425) form a heterocomplex with Smad4 and translocate to the nucleus, where they regulate the expression of EndMT-related genes. To assess the impact of KLX on the activity of TGFβR/Smad signaling, we analyzed the protein levels of TGFβR1 and TGFβR2 and their interactions in TGFβ1-induced HUVECs. Duolink in situ proximity ligation assay demonstrated close proximity between TGFβR1 and TGFβR2 in HUVECs treated by TGFβ1 (Fig.4). Moreover, TGFβR1 and TGFβR2 proteins were upregulated in response to TGFβ1 stimulation; however, this upregulation was effectively attenuated by KLX pretreatment (Fig.4). Furthermore, TGFβ1 induced the phosphorylation of Smad2/3 and the nuclear translocation of Smad4 in HUVECs; KLX preconditioning mitigated these effects (Fig.4 and 4D). The nuclear translocation of the Smad2/3/4 complex contributes to the expression of transcriptional factors such as Twist, Snail, and Slug, which subsequently promote EndMT [32]. Our findings indicated that Slug, Twist, and Snail expression levels were elevated in TGFβ1-stimulated HUVECs; however, KLX pretreatment led to a dose-dependent reduction in these elevations (Fig.4). In conclusion, KLX effectively inhibited the activation of TGFβR/Smad signaling in TGFβ1-stimulated endothelial cells.

Fig.4 KLX inhibits the activation of TGFβR/Smad signaling in HUVECs. (A) Duolink in situ analysis for TGFβR1 and TGFβR2 in HUVECs; n = 5. (B) Western blot analysis of TGFβR1 and TGFβR2 in HUVECs; n = 4. (C) Western blot analysis of phospho-Smad2/3 (p-Smad2/3) and total-Smad2/3 (T-Smad2/3) in HUVECs; n = 4. (D)Western blot analysis of cytoplasmic and nuclear Smad4 in HUVECs; n = 4. (E) Western blot analysis of transcriptional factors Snail, Slug, and Twist in HUVECs; n = 4–5. HUVECs were treated with TGFβ1 (10 ng/mL) for 24 h to induce EndMT. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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3.4 KLX inhibits TGFβR/Smad signaling by suppressing integrin β1

Integrins play a pivotal role in the pathogenesis of EndMT. Integrin β1 activation induces EndMT and organ fibrosis by initiating the TGFβ/Smad signaling pathway [33]. Inhibition of integrin β1 effectively blocks TGFβR/Smad signaling and halts fibrosis progression, whereas activation of integrin β1 has been associated with induction of EndMT [15,18,20]. To explore the relationship between KLX-mediated inactivation of TGFβ signaling and integrin β1, we investigated the impact of KLX on integrin β1 levels. Western blot analysis revealed that integrin β1 and phosphorylated integrin β1 (T788 + T789) levels increased in HUVECs upon TGFβ1 stimulation; however, this increase was reversed following KLX pretreatment (Fig.5).

Fig.5 KLX inhibits TGFβR/Smad signaling by inhibiting integrin β1. (A) Western blot analysis of integrin β1 and phospho-integrin β1 in HUVECs; n = 5. (B) Western blot analysis of TGFβR1 and TGFβR2 in HUVECs; n = 4. (C) Western blot analysis of P-Smad2/3 in HUVECs; n = 5. (D) Western blot analysis of Snail and Twist in HUVECs; n = 4. (E) TRITC phalloidin staining of F-actin in HUVECs. Scale bar: 20 μm; n = 3. (F) Western blot analysis of VE-cadherin and vimentin in HUVECs; n = 4‒5. (G) Immunofluorescence staining analysis of phospho-integrin β1 in atherosclerotic plaque area; n = 3. HUVECs were treated with TGFβ1 (10 ng/mL) for 24 h to induce EndMT; 9EG7 (1 mg/mL, 6 h) was used to activate integrin β1 in HUVECs. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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To establish the pivotal role of integrin β1 in KLX’s inhibition of TGFβ signaling and EndMT, HUVECs were exposed to an integrin β1-activating antibody (9EG7) designed to activate integrin β1 [18,34]. Western blot analysis demonstrated that KLX almost completely suppressed the upregulation of TGFβR1 and TGFβR2 triggered by TGFβ1, and this inhibitory effect was fully counteracted by 9EG7. Conversely, treatment with control immunoglobulin G (IgG) did not interfere with KLX’s inhibitory effect on TGFβR1/2 protein levels (Fig.5). Similarly, the inhibitory impact of KLX on Smad2/3 phosphorylation was entirely reversed by 9EG7 (Fig.5). Additionally, 9EG7 nullified KLX’s suppression of Snail and Twist expression (Fig.5). Finally, we investigated the influence of integrin β1 activation on KLX’s inhibition of the EndMT phenotype. We observed that the protective effect of KLX on F-actin morphology and distribution indicated by TRITC phalloidin staining was entirely reversed by 9EG7 (Fig.5). Western blot analysis further suggested that KLX’s inhibitory effect on changes in VE-cadherin and vimentin expression induced by TGFβ1 was fully abrogated by 9EG7 (Fig.5). Immunofluorescence staining of atherosclerotic aortic roots yielded consistent results, with phosphorylated integrin β1 (T788 + T789) levels elevated in atherosclerotic aortic roots and subsequently suppressed by KLX treatment (Fig.5). These findings demonstrated that KLX inhibited TGFβR/Smad signaling by suppressing integrin β1.

3.5 KLX activates the FGFR1/MAP4K4 signaling pathway in HUVECs

MAP4K4 serves as a critical regulator of integrin β1 activity within endothelial cells. Upon activation, MAP4K4 phosphorylates Moesin, leading to the displacement of Talin from the intracellular domain of integrin β1 and consequent inactivation of integrin β1 [19,35]. Notably, knockdown of MAP4K4 results in the activation of integrin β1 and Smad2/3, ultimately promoting EndMT [20]. To investigate the relationship between KLX’s inhibition of integrin β1 and MAP4K4, we performed Western blot analysis, which demonstrated that exposure of HUVECs to KLX for only 0.5 h led to the activation of MAP4K4 (Fig.6). KLX induced the phosphorylation of Moesin during the same time (Fig.6). P-Moesin effectively displaced Talin by binding to the intracellular region of integrin β1 and deactivated it [34,35]. This result was confirmed by examining the interaction between Talin and integrin β1. As indicated in Fig.6, TGFβ1 induced an interaction between talin and phospho-integrin β1, which consequently activated integrin β1 in endothelial cells. However, KLX significantly mitigated these effects. Furthermore, in vivo research revealed a significant reduction in the protein abundance of phospho-MAP4K4 (Ser629) within atherosclerotic plaque areas compared with normal vascular endothelium; however, this reduction was restored following KLX treatment (Fig.6 and 6E). Collectively, these findings suggested that KLX activated MAP4K4 in endothelial cells, thereby inactivating integrin β1.

Fig.6 KLX activates FGFR1/MAP4K4 signaling in endothelial cells. (A) Western blot analysis of MAP4K4 and phospho-MAP4K4 in HUVECs; n = 3. (B) Western blot analysis of Moesin and phospho-Moesin in HUVECs; n = 3. (C) Immunofluorescence staining analysis of phospho-integrin β1 and Talin in HUVECs; n = 3. (D) Immunofluorescence staining analysis of phospho-MAP4K4 in atherosclerotic plaque area; n = 5. (E) Quantification of phospho-MAP4K4 in atherosclerotic plaque area; n = 5. (F) Western blot analysis of MAP4K4 and phospho-MAP4K4 in HUVECs pretreated by PD173074 (10 μM, 1 h); n = 4. (G) Western blot analysis of FGFR1 and phospho-FGFR1 in HUVECs; n = 4. (H) Immunofluorescence staining of phospho-FGFR1 in atherosclerotic plaque area; n = 3. PD173074 (10 μM, 1 h) was used to inhibit the activation of FGFR1. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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FGFR1 plays a crucial role in inhibiting TGFβ/Smad signaling and serves as a key target for EndMT. Previous studies have identified FGFR1 as an indispensable factor for the activation of MAP4K4 in endothelial cells. Inhibition of FGFR1 significantly decreases the levels of phospho-MAP4K4 (Ser629), whereas its activation has been associated with the induction of phospho-MAP4K4 (Ser629) [22,23,27]. Consequently, we sought to explore whether KLX’s inhibition of integrin β1 and activation of MAP4K4 are mediated by FGFR1. Western blot analysis indicated that the upregulation of phospho-MAP4K4 (Ser629) induced by KLX was effectively blocked when HUVECs were treated with the FGFR1 inhibitor PD173074 (Fig.6). Further investigation demonstrated that exposure to KLX for 30 min induced phosphorylation at Tyr653/654 residues on FGFR1 in HUVECs (Fig.6). KLX restored the expression of phospho-FGFR1 (Tyr653/654) in the vascular endothelium within the atherosclerotic plaque area (Fig.6 and 6I). These findings highlight the role of KLX in activating the FGFR1/MAP4K4 signaling pathway in endothelial cells.

3.6 FGFR1 activity is essential for KLX to inhibit integrin β1/TGFβR/Smad signaling and EndMT

To investigate whether FGFR1 is a crucial factor in KLX-mediated inhibition of integrin β1/TGFβR/Smad signaling and EndMT, we conducted a series of experiments. Western blot analysis revealed that pretreatment with PD173074 effectively reversed the inhibitory effects of KLX on integrin β1 phosphorylation, TGFβR induction, and Smad2/3 activation (Fig.7). Additionally, PD173074 counteracted KLX’s inhibition of Snail and Twist expression in HUVECs (Fig.7). Western blot analysis and immunofluorescence staining demonstrated that PD173074 nullified KLX’s capacity to restore the abnormal protein levels of VE-cadherin and vimentin in TGFβ1-induced HUVECs (Fig.7 and 7C). In terms of endothelial cell morphology and intercellular connectivity, PD173074 revoked the protective effects exerted by KLX (Fig.7). TRITC phalloidin staining also showed that PD173074 completely reversed KLX’s beneficial effects on F-actin morphology and distribution in HUVECs (Fig.7). These results collectively support that KLX’s inhibition on integrin β1/TGFβR/Smad signaling and EndMT was dependent on FGFR1 activity.

Fig.7 FGFR1 activity is crucial for KLX-mediated inhibition of integrin β1/TGFβR/Smad signaling and EndMT. (A) Western blot analysis of P-integrin β1, TGFβR1/2, P-Smad2/3, Snail, and Twist in HUVECs; n = 4–5. (B) Western blot analysis of VE-cadherin and vimentin in HUVECs; n = 3–5. (C) Morphological observation and immunofluorescence staining of VE-cadherin and vimentin in HUVECs; n = 3. (D) TRITC phalloidin staining of F-actin in HUVECs; Scale bar: 20 μm; n = 3. HUVECs were treated with TGFβ1 (10 ng/mL, 24 h) to induce EndMT; PD173074 (10 μM, 1 h) was used to inhibit the activation of FGFR1. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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3.7 KLX activates FGFR1 and promotes the interaction of p-FGFR1 and p-MAP4K4

The preceding results highlight that KLX suppresses integrin β1/TGFβs/Smad signaling and EndMT by activating FGFR1. Therefore, we sought to elucidate the mechanism underlying KLX-mediated activation of FGFR1. To confirm the activation of KLX to FGFR1 on endothelial cell membranes, we synthesized a fluorescently labeled derivative of KLX by attaching Cy5 to its side chain, enabling the visualization of KLX localization in endothelial cells (Fig.8). Immunofluorescence staining revealed the co-localization of Cy5-KLX with FGFR1 on the cytomembrane of HUVECs after incubation with Cy5-KLX (Fig.8). Small molecular compounds exhibit competitive occupation when they interact with their targets. As shown in Fig.8, pre-treatment of cells with KLX (Cy5-free, 10 μM) resulted in a reduction in fluorescence intensity of Cy5-KLX and its interaction with FGFR1, indicating a competitive relationship between KLX and Cy5-KLX. Previous studies have shown that direct interaction between FGFR1 and MAP4K4 is crucial for inducing MAP4K4 phosphorylation by FGFR1 [20]. Thus, we investigated the impact of KLX on this interaction between FGFR1 and MAP4K4. Co-immunoprecipitation analysis demonstrated that treatment with KLX triggered an increased interaction between phosphorylated forms of both proteins (P-FGFR1 and P-MAPKK), whereas PD173074 attenuated this interaction in HUVECs (Fig.8). Duolink in situ proximity ligation analysis further confirmed a reduction in proximity between P-FGFR1 and P-MAPK4K4 within atherosclerotic plaque areas in mice; however, oral administration of KLX restored their close proximity (Fig.8). In summary, these results indicated that KLX acted as a potential activator of FGFR1 and activated MAP4K4 by facilitating the direct interaction between P-FGFR1 and P-MAP4K4.

Fig.8 KLX activates FGFR1 by promoting the interaction between p-FGFR1 and p-MAP4K4. (A) Chemical structures of Cy5-KLX. (B) Immunofluorescence staining analysis of FGFR1 and Cy5 in HUVECs; n = 3. Cells were treated with Cy5-KLX at 37 °C for 1 h. The red channel represents Cy5, the green channel represents FGFR1, and the nucleus is labeled with DAPI. (C) Co-immunoprecipitation analysis of phospho-FGFR1 and phospho-MAP4K4 in HUVECs; n = 3. (D) Duolink in situ proximity ligation analysis of phospho-FGFR1 and phospho-MAP4K4 in aortic root of mice; Scale bar represents 150 μm, n = 3. PD173074 (10 μM, 1 h) was used to inhibit the activation of FGFR1. Statistically significant differences between the specified groups are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

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4 Discussion

Vascular endothelial cells undergo a phenotypic transformation into mesenchymal cells when exposed to hypoxia, nicotine, modified lipoproteins, or TGFs [36]. EndMT induces the redistribution of F-actin and the formation of stress fibers in endothelial cells, leading to an increase in intercellular space and lipid deposition. Consequently, EndMT marks the initial step toward atherosclerotic plaque formation [9,37]. Among the signaling factors involved, TGFβ plays a pivotal role as a potent inducer of EndMT. In mammals, the TGFβ superfamily comprises three subtypes that share 70% homology and exhibit similar biological activities in vitro for inducing EndMT [38]. Notably, TGFβ1 is widely expressed in various tissues and strongly associated with vascular diseases [39]. In this study, we observed that TGFβ1 treatment induced a range of mesenchymal phenotypic characteristics in HUVECs. Morphologically, HUVECs transformed from a pebble-like shape to a spindle-like shape with F-actin redistribution forming stress fibers and resulting in increased intercellular space and permeability. Functionally, TGFβ1 stimulation enhanced the production of ECM by endothelial cells. In terms of EndMT marker gene expression, TGFβ1 downregulated endothelial cell marker genes and upregulated mesenchymal cell marker genes in HUVECs. KLX effectively mitigated the morphological, functional, and gene expression abnormalities induced by TGFβ1 in endothelial cells. Notably, HUVECs possess significant differentiation potential even without TGFβ1 stimulation and display differentiation tendencies over time. KLX was found to upregulate the expression of endothelial marker genes, thereby preserving the normal structure and morphology of endothelial cells and exerting protective effects on them independent of TGFβ1 stimulation. In summary, KLX exhibited a robust anti-EndMT effect in endothelial cells, regardless of whether they are stimulated by TGFβ1 or not.

Our findings suggested that KLX inhibited EndMT by blocking the activation of the TGFβ signaling pathway. The activation of Smad2/3 represents a critical step in TGFβ1-induced EndMT. Upon stimulation by TGFβ1, heterodimerization between TGFβ1 and TGFβR2 occurs, recruiting and activating Smad2/3. These activated Smad proteins form heteropolymers with Smad4 before translocating into the nucleus where they induce an excessive expression of key transcription factors including Twist, Snail, and Slug [40,41]. KLX impedes the formation of TGFβR1/2 heteropolymers and reduces the protein abundance of TGFβR1/2 in TGFβ1-treated HUVECs. Consequently, these changes suppress the phosphorylation of Smad2/3 and inhibit the nuclear translocation of the Smad2/3/4 complex, resulting in the reduced expression of nuclear transcription factors that promote EndMT. Thus, these mechanisms elucidate KLX’s ability to prevent the upregulation of vimentin and N-cadherin, as well as the downregulation of CD31 and VE-cadherin induced by TGFβ1. In conclusion, KLX exerted its anti-EndMT effect by inhibiting the TGFβ signaling pathway.

Our study demonstrated that KLX blocked the activation of TGFβ signaling by inactivating integrin β1, a transmembrane protein composed of integrin α and β1 subunits. Integrin β1 plays a crucial role in fibrosis as it activates TGFβ and induces EndMT, making it a promising target for treating multi-organ fibrosis. Previous studies have shown that inhibiting integrin β1 diminishes the TGFβ-induced formation of the TGFβR1/2 complex, upregulation of TGFβR1/2, and EndMT [15,18,20]. In our study, we confirmed the crucial role of integrin β1 for KLX to inhibit TGFβ activation. We observed that KLX downregulated the expression and phosphorylation of integrin β1. Furthermore, we found that the 9EG7 antibody (an integrin β1-activating antibody) reversed KLX’s inhibition on TGFβ signal transduction and EndMT induction. These findings strongly suggested that KLX suppressed TGFβ1-mediated EndMT by inhibiting the activation process of integrin β1, which involves conformational changes in its extracellular domains leading to separation between intracellular α and β1 domains. Talin serves as a key regulator that activates integrin β1 by binding to its intracellular β-tail, whereas MAP4K4 acts as a direct suppressor of integrin β1. In endothelial cells, active MAP4K4 induces Moesin phosphorylation, which subsequently binds to the intracellular β tail of integrin β1, displacing Talin and leading to the inactivation of integrin β1 [19,42,43]. KLX inhibits the binding of Talin to integrin β1 and deactivates integrin β1 by activating MAP4K4 and phosphorylating Moesin, functioning as a small-molecule inhibitor of integrin β1. Compared with the RGD peptide, small-molecule inhibitors targeting integrin β1 exhibit less impact on other RGD receptor integrins, thereby holding greater promise for the clinical treatment of fibrosis-related diseases [44,45].

Despite KLX’s clear inhibitory effect on integrin β1, our study demonstrated that FGFR1 was the critical target through which KLX suppressed integrin β1 and TGFβ signaling. FGFR1 is a crucial inhibitor of TGFβ/Smad signaling and EndMT in endothelial cells [20,46]. The activation of MAP4K4 in endothelial cells critically depends on FGFR1. Inhibition of FGFR1 significantly reduces phospho-MAP4K4 levels, whereas the activation of FGFR1 is associated with the upregulation of phospho-MAP4K4 [20]. Previous investigations conducted by our research team have already shown that KLX activates FGFR1 in endothelial cells to promote angiogenesis [26]. Based on this information, we hypothesized that KLX can function as a small-molecule activator for FGFR1, thereby activating MAP4K4 and inhibiting integrin β1. Our findings substantiated this hypothesis by demonstrating the rapid activation of FGFR1 in endothelial cells by KLX. Furthermore, the inactivation of FGFR1 completely overturned the KLX-induced activation of MAP4K4 and its inhibition of integrin β1 and TGFβ signal transduction, thereby impeding the KLX-mediated suppression of EndMT in endothelial cells. These results suggested that FGFR1 was essential for KLX to suppress integrin β1 and prevent EndMT. Concerning the mechanism of MAP4K4 activation, our study revealed that KLX promoted the interaction between P-FGFR1 and MAP4K4, thereby inducing phosphorylation of MAP4K4. In summary, KLX inhibited EndMT by activating FGFR1 and MAP4K4, consequently inhibiting integrin β1 and its downstream TGFβ/Smad signal transduction (Fig.9).

Fig.9 Molecular mechanism of KLX in suppressing EndMT. In this illustration, we elucidate the molecular mechanism through which KLX exerts its inhibitory effect on EndMT. KLX initiates a cascade of events by activating FGFR1, leading to the induction of a critical interaction between phosphorylated FGFR1 and phosphorylated MAP4K4. This interaction results in the inactivation of integrin β1 and the TGFβR/Smad signaling pathway. VE-cad, VE-cadherin.

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EndMT plays a pivotal role in vascular development and the pathogenesis of various vascular diseases, including endothelial dysfunction and the formation of atherosclerotic plaques [6,7]. Considering the efficacy of KLX in mitigating EndMT, our study aimed to investigate the potential benefits of KLX in preventing atherosclerosis. Our results confirmed that oral administration of KLX effectively restrained vascular EndMT and hindered atherosclerotic progression. In our study, oral administration of KLX at a dose of 10 mg/kg/day led to a significant reduction in lipid deposition and plaque formation within the aortic arch, thoracic aorta, and aortic root. This effect was comparable with that of ATV, a recognized treatment for atherosclerosis. Additionally, KLX led to a decrease in the number and size of necrotic cores within the plaques, indicating improved plaque stability and reduced risk of rupture. KLX exhibited the ability to reduce abnormal collagen secretion and the expression of EndMT marker genes within atherosclerotic plaque areas. These findings were consistent with our in vitro experiments, which revealed that KLX deactivated integrin β1 through FGFR1 and MAP4K4 phosphorylation, thereby enhancing their proximity within atherosclerotic plaques.

In the present clinical landscape, lipid-regulating drugs are commonly employed to delay plaque formation or progression. However, there is currently a lack of approved anti-atherosclerotic drugs specifically targeting vascular protection in clinical practice. Although our previous studies demonstrated the effectiveness of KLX in improving dyslipidemia in HFD-fed hamsters and rats, they did not exhibit a significant beneficial effect on hyperlipidemia in Apoe^−/− mice subjected to an extended duration of HFD (Tab.1) [27,47]. Therefore, the primary anti-atherosclerotic effect of KLX stems from its protective impact on vascular endothelial cells. Furthermore, unlike ATV, which is associated with liver-related adverse effects, KLX was observed to reduce elevated serum transaminase levels caused by long-term consumption of HFD, indicating its hepatoprotective effect. In summary, KLX stands out in comparison with ATV by exerting an anti-atherosclerotic role through the preservation of vascular endothelial integrity. KLX exhibits promising pharmacological effects in the prevention and treatment of atherosclerosis while presenting few hepatotoxic side effects.

5 Conclusions

In conclusion, this study provides robust evidence supporting the preventive efficacy of KLX against vascular EndMT and atherosclerosis. KLX mitigated EndMT by inhibiting integrin β1 and intercepting the TGFβR/Smad signaling pathway within endothelial cells, which was attributed to KLX’s activation of FGFR1 and MAP4K4. Oral administration of KLX significantly reduced lipid deposition, suppressed collagen secretion, and prevented plaque formation in the aortas of Apoe^−/− mice. The compelling combination of effects, which encompassed the prevention of vascular EndMT and atherosclerosis, as well as the stimulation of FGFR1, positions KLX as a promising novel anti-atherosclerotic agent.

Yixiu Zhao et al

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Acknowledgements

This work was financially supported by the Basic Research Support Program for Outstanding Young Teachers of Heilongjiang Province (No. YQJH2023038), the National Natural Science foundation of China (Nos. 82170431, 82070312, 82373868, 82330011, and U21A20339) and the Scientific Research Project of Provincial Scientific Research Institute of Heilongjiang Province (No. CZKYF2022-1-B007).

Compliance with ethics guidelines

Conflicts of interest Yixiu Zhao, Zhiqi Wang, Jing Ren, Huan Chen, Jia Zhu, Yue Zhang, Jiangfei Zheng, Shifeng Cao, Yanxi Li, Xue Liu, Na An, Tao Ban, Baofeng Yang, and Yan Zhang declare that they have no conflict of interest. Baofeng Yang is a member of the Editorial Board of Frontiers of Medicine, who was excluded from the peer-review process and all editorial decisions related to the acceptance and publication of this article. Peer-review was handled independently by the other editors to minimise bias.

All institutional and national guidelines for the care and use of laboratory animals were followed.

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