Effects of Notch signalling pathway on the relationship between vascular endothelial dysfunction and endothelial stromal transformation in atherosclerosis

Abstract

At present, with the improvement of living standards and population aging, the incidence of cardiovascular and cerebrovascular disease is on the rise and has been a serious threat to human health. Statistics show that the current death caused by cardiovascular and cerebrovascular disease has become the first cause of death has been increasing year by year. Therefore, studies on coronary heart disease and atherosclerosis (AS) have become a hot topic in clinical and basic research. In this study, the question of the effect of Notch signalling pathway on the relationship between endothelial dysfunction and endothelial stromal transformation in AS was studied in depth. Based on our results, we drew conclusions as follows. First, the Notch signalling pathway was activated in the atherosclerotic model; secondly, the Notch signalling pathway was demonstrated to enhance AS by promoting vascular endothelial dysfunction; thirdly, it was demonstrated that the Notch signalling pathway was mediated by promoting endothelial and to enhance AS; finally, we confirmed the endothelial function through the Notch signalling pathway to affect the transformation of endothelial stroma to achieve synergistic AS effect. The results of this study have a good guiding significance for the important role of Notch signalling in AS and indicate the ability to influence endothelial function and endothelial stromal transformation by intervening Notch signalling pathway and can affect the relationship between them, and thus eventually achieve the treatment of AS.

Keywords:

• Notch signalling pathway
• AS
• vascular endothelial dysfunction
• endothelial stromal transformation

Introduction

At present, with the improvement of living standards and population aging, the incidence of cardiovascular and cerebrovascular disease is on the rise and has been a serious threat to human health [1]. Statistics show that the current death caused by cardiovascular and cerebrovascular disease has become the first cause of death and increasing trend year by year. Therefore, studies on coronary heart disease and AS have become a hot topic in clinical and basic research [2,3].

Normal vascular endothelium is a combination of perception and effect of the organ with a variety of physiological effects, and in the blood pressure changes in the cycle of various chemical media and hormone levels at the same time. This change can make the appropriate response mainly through the secretion of endothelial cells, physiological state regulation of vasomotor status, anticoagulation, antiplatelet aggregation and thrombosis [4]. Pathological conditions can promote inflammation, smooth muscle cell proliferation and thrombosis [5]. Its dysfunction in the development of AS plays an important role in the development process, which has been confirmed by a large number of clinical and epidemiological data [6,7]. The detection of vascular endothelial function is important for the early diagnosis and evaluation of cardiovascular disease. The balance between vasoconstrictor factors and vasoconstrictor factors produced by endothelial cells is a sign of normal function of endothelial cells [8]. Once this balance lost, cell will be under dysfunction, the most important of which is endothelium-dependent vasodilatation dysfunction. The evaluation methods of this dysfunction include: no viability determination, detection of vasoconstrictor factor, peripheral vascular (brittle artery) endothelial function ultrasound, coronary artery endothelial function test, detection of endothelial cell expression of anticoagulant/procoagulant factors, cytokines, adhesion molecules and other changes [9]. In recent years, a large number of studies have shown that aspirin, statins, ACEI could simultaneously improve endothelial function and reduce the incidence of cardiovascular disease and mortality. However, antioxidant and hormone replacement therapy could improve vascular endothelial function, but not necessarily able to reduce the heart morbidity and mortality of vascular disease [10,11]. Therefore, it is of great theoretical and practical significance to further explore the protective measures of vascular endothelial function and its prognostic value.

Notch signalling pathway was first discovered by Mohr in studying the gene function of Drosophila, which is a highly conserved intercellular communication pathway in biological evolution and plays an important role in many organ development processes [12]. The study found that there are four Notch receptors in mammals, Notch1, Notch2, Notch3 and Notch4, respectively. Notch ligands are Jagged1, Jagged2 and Delta1, Delta3, Delta4. All receptors and ligands are transmembrane proteins, and adjacent cells can pass through the binding of the receptor to the ligand. Notch1, Notch3, Notch4, Delta-like4, Jagged1 and Jagged2 were expressed on the arterial endothelium [13]. The Notch signalling pathway is a conserved and important signal transduction pathway that affects cell fate, which involves almost all cell proliferation and differentiation activities, and plays an important role in regulating cell differentiation, proliferation and apoptosis, and a series of physiological pathologies [14]. Notch signalling pathway family members include ligands, receptors, nuclear transcription factors, regulatory molecules and downstream target molecules and other components, which play a classic and conservative role in adult individuals and embryonic development process [15]. The results of this research mentioned above is related to embryonic development, tumorigenesis, degeneration of the immune system, immune system function regulation, bone marrow hematopoietic stem cell expansion, and other physiological and pathological processes.

In this study, we established the model of AS in mice, we firstly studied the changes in the expression of Notch signalling pathway in the diseased part, then explored the existence of vascular endothelial dysfunction and Notch signalling pathway in the process of disease. After that, we explored the correlation between endothelial stromal transformation and Notch signalling pathway. Finally, the correlation analysis was conducted to explore the effect of Notch signalling pathway on the changes of vascular endothelial dysfunction and endothelium in the development of AS.

Material and methods

Mice

C57/B6 mice purchased from the Nanjing University Institute of animal models. MxCre mice and RBP-JFlox mice were kept and preserved from this laboratory. All animal experiments were carried out in accordance with the regulations on the Administration of Laboratory Animals promulgated by the National Science and Technology Commission and the regulations on the Administration of Laboratory Animals in the Animal Center of the University.

As model establishment

To establish a model of local AS in mice, and provide a model for the use of various genetically modified mice for deep mechanism studies, the carotid arteries were chemically damaged by saturated ferric chloride. After 8 weeks, the carotid arteries of the hypercholesterolemia mice with apolipoprotein E gene knockout had vascular intimal hyperplasia, atherosclerotic plaque formation, success rate reaching one hundred percent. In the same way, apolipoprotein E and lipoprotein lipase gene double knockout mice carotid artery caused more severe endometrial hyperplasia, indicating that the local lack of lipoprotein lipase gene would promote the proliferation of arterial intima. Localized ferric chloride stimulation could induce intimal hyperplasia of the carotid artery in mice. The formation of atherosclerotic plaque for the use of genetically modified mice for AS research provided a simple and reliable model.

Analysis of vascular endothelial function

In order to objectively and quantitatively evaluate the degree of vascular disease, we developed a vascular image measurement system using modern computer graphics and computer image processing analysis techniques. Quantitative measurement of vascular intima thickness, medium thickness and endometrial atherosclerotic plaque percentage of the entire area of blood vessels was also conducted.

Analysis of endothelial stromal transformation

Morphological characteristics of cell growth after primary culture and passage were observed under inverted phase contrast microscope. The expression of CD31 and ⧠factors was detected by immunofluorescence assay. CMVECs were identified. The process was as follows: (i) when the cell layer was covered with the bottom of the culture dish, it was digested and passed down to the 24-well culture plate containing the tablet and incubated overnight at 37° C in a 5% CO₂ incubator; (ii) the cells were placed in 24-well cell culture plate, washed with PBS 3 times, each time 1 min; (iii) the cell climbing tablets were placed on the ice plate, with 4% paraformaldehyde for 30 min, rinse with PBS 3 times, each 5 min; (iv) with 0.5% Triton placed at 37 °C drilling 10 min, the cells were washed with PBS 3 times for 5 min; (v) 1% BSA at 37 °C for 30 min, the cells were washed with PBS 3 times for 1 min; (vi) 1: 200 at 37° C for 1 h, the PBS was rinsed three times, each time the sample was washed with PBS twice a day for 5 min, and the control group was replaced with PBS for 5 min; (vii) DAPI was stained at 37 °C for 15 min, and the cells were washed with PBS 3 times for 5 min, wherein the CD31 primary antibody was a mouse source, the secondary antibody was FITC-labelled goat anti-mouse IgG, the VIII factor primary antibody was a rabbit source, and the secondary antibody was a TRITC-labelled goat anti-rabbit IgG; (viii) dropping glycerine seal, the cells were immediately placed under a fluorescence microscope to observe, take pictures; and (ix) the cytoplasm was red, and green fluorescence was taken as positive cells.

Western blotting

RIPA lysate was dissolved to obtain appropriate lysate and PMSF was added (final concentration 1 mM) before use. The cell culture solution was removed and washed with PBS. The lysate was added to each well to obtain 200 μL of lysate per well, and mixed several times with a pipette to allow the solution to be in contact with the lysate cells. After full cleavage and 12,000g centrifugation for 5 min, the supernatant was taken for protein quantification. Loading buffer (5×) was added to the quantified protein sample and cooked for 10 min in boiling water. The sample could be detected by Western blotting.

RT-PCR

Total RNA from BMDM was extracted with RNAiso Plus (Takara, Dalian, China), and cDNA synthesized with the PrimeScript RT reagent Kit (Takara, Dalian, China). Quantitative PCR was performed using a SYBR Premix Ex TaqTM II (Takara, Dalian, China). Data were collected on an ABI StepOne real-time PCR system (Applied Biosystems, Carlsbad, CA). The PCR primers used in this study are listed in Table S1. The expression levels of target genes were normalized to the housekeeping gene Gapdh (ΔCt), and the results were calculated with 2^−ΔΔCtt method.

Statistical analysis

Data were expressed as mean (± SE) and analyzed by a SPSS software package (SPSS Standard version 13.0, SPSS Inc., Chicago, IL). Differences between variables were assessed by the chi-square test. Survival analysis of patients with colorectal cancer was calculated by Kaplan–Meier analysis. A log-rank test was used to compare different survival curves. A Cox proportional hazards model was used to calculate univariate and multivariate hazard ratios for the variables. Unpaired Student’s t-test and one-way ANOVA were used as appropriate to assess the statistical significant of difference. p-Values under .05 were considered statistically significant.

Results

Notch signalling pathway is active in the AS model

AS is the chronic inflammation and immune response of the arterial wall to damage and irritation. In the early formation of AS, monocytes in the injured site are under aggregation, adhesion, migration and through the vascular endothelium, and then activated and differentiated into macrophages. Macrophages in AS play a central role in the whole process of development. After the formation of lipid cells, the formation of AS is the starting factor [16]. Macrophage phagocytes uptake the accumulated normal and modified lipoproteins and then differentiate into foam cells that persist in the plaques, which contributes to the formation of thrombotic necrotic core rendering a propensity to intravascular blood clot that underlies myocardial infarction and stroke [17]. Vascular smooth muscle cells (VSMC) moved into the intima, phagocytic lipid-forming myogenic foam cells, hyperplasia migration of oxidized low-density lipoprotein (ox-LDL) to disintegrate the above two kinds of foam cells, the formation of sporadic necrosis and atherosclerotic plaque formation. In this process, Notch signal pathway is from many aspects of the role [18].

Notch signalling pathway is extensively involved in the differentiation of inflammatory cells, and the components of this pathway are upregulated in chronic inflammatory lesions. It is reported that Notch signalling pathway is widely present throughout the immune system cell development options, particularly in the development and differentiation of lymphocytes and monocytes, and regulate the function of mature immune cells. The relationship between Notch signalling and inflammation is complex and well-established. It is reported that the production of cytokines such as tumour necrosis factor (TNF) and interferon-γ (IFN-γ) in the process of inflammation can regulate the expression and activation of Notch receptors and ligands in cells. But when the cytokines are different, the specific regulation of the Notch signal is also different. In turn, the Notch signal regulates the inflammatory response in vivo (seen in Figure 1).

Figure 1. Expression of Notch receptor mRNA in AS mice model induced by ox-LDL. *p < .05.

In addition, the Notch signalling pathway can also regulate the production of cytokines produced by immune cells, increase the secretion of proinflammatory cytokines and increase the secretion of anti-inflammatory factors and different receptor ligands in different immune cells [19]. Notch signalling pathway is through the regulation of these immune cells and cytokines to influence AS. In this lesion, different immune cells have different effects. It has been reported that monocytes, macrophages, dendritic cells, T cell subsets, NK cells, neutrophils, platelets, etc. may play a role in causing AS; regulatory T cells and B cells may play AS effect. IL-1, IL-1, IL-2, IL-3, IL-6, CXCL8, IL-10, IL-12, IL-15, IL-18, IFN-γ, TGF-β1, TGF-β2 and TGF-β3 may be prone to AS blood vessels (seen in Figure 2). A large number of experiments have shown that the activation of Notch signalling pathway in immune cells can aggravate AS lesions, especially the Notch signalling pathway in macrophages, and play an important role in the development of inflammation and AS. This is because that macrophages for the occurrence and development of AS have a decisive role. Studies have found that macrophages in the gradual accumulation of lesions for vascular remodelling and plaque formation make a significant contribution, while macrophages can also exacerbate the inflammation of the AS plaque and plaque structural instability.

Figure 2. Expression of Notch receptor genes in monocyte-derived macrophages in AS Model. **p < .01; ***p < .001.

Notch signalling pathway enhances AS by promoting vascular endothelial dysfunction

The expression level of Dll4 is lower in the vascular network of adult individuals, but the expression of D114 is significantly upregulated in atherosclerotic vascular tissue [20]. For example, in the vascular tissue of renal cell carcinoma, the expression level of Dll4 is more than 9 times that of renal tissue, and its expression is synergistic with VEGF and β-FGF. In clinical breast cancer specimens, AS within the endothelial cells almost obtains 100% expression of Dll4. In addition, the high expression of Dll4 is closely related to the prognosis of AS, and the expression of Dll4 in breast cells is progressing more rapidly [21]. However, in colon cancer, which is in contrast to hypoxia, VEGF can upregulate the expression of Dll4 in colon cancer, and Dll4 can downregulate the expression of VEGFR2 by promoting methylation of VEGFR2. Dll4 blockade can significantly promote angiogenesis in AS, increasing vascular sprouting and vascular branch formation [22]. But the new blood vessels are ineffective, due to AS neovascularization and functional abnormalities leading to AS tissue haemoperfusion decreased, and hypoxia increased more than 7 times, can inhibit the growth of most AS and can inhibit the growth of AS that is not sensitive to VEGF antibodies [23]. Jagged1-induced activation of Notch signalling pathway can promote the proliferation of neuroblastoma, and Notch signalling pathway in different AS also has different effects [24]. Our previous study by Dr. Hu Xingbin showed that the exclusion of Notch signalling core RBP-J could slow the growth of some AS but also promote the growth of some AS [25]. Atherosclerotic cells overexpress the soluble Notch receptor and can inhibit VEGF-induced angiogenesis, affecting the vitality of AS. The role of Notch in the angiogenesis of AS may be partly due to the induction of AS in the internal hypoxic environment. Studies have shown that HIF-1α can interact with the intracellular segment of Notch1 to increase cellular response to hypoxia when exposed to atherosclerotic cells in hypoxic conditions [26,27]. In addition, the upregulation of endothelial cell surface Dll4 is likely to be directly regulated by HIF-1α and hypoxia responses to the Dll4 promoter [28]. These results suggest that Notch signalling pathway plays a key role in the adaptation of AS to hypoxic conditions. Notch1 also plays an important role in the inhibition of hemangioma production, and some studies have shown that Notch1 knockout mice in the liver leads to the formation of hemangioma, and the survival rate of mice was significantly reduced [29]. Mitogen activator kinase (MAPK) can induce Jagged1 expression to activate Notch signalling of adjacent endothelial cells and promote angiogenesis in AS (seen in Figure 3).

Figure 3. Left column: Notch signalling pathway regulates the expression of endothelial cell junction molecules; right column: Notch signalling regulates the expression of endothelial cell linker molecule VE-Cadherin.

Notch signalling pathway enhances AS by promoting endothelial stromal transformation

Notch, like TGF-β, is able to induce interstitial cell transformation in endothelial cells culture in vitro. It mediates End MT by modulating the expression of transcription factors, Snail, Slug and ZEB1 [30]. Notch increases the expression of Snail and Slug proteins in endothelial cells. Snail and Slug are transcription factors known to inhibit the expression of VE-cadherin in endothelial cells [31]. VE-cadherin plays a key role in maintaining the stability of endothelial cells. If its expression is inhibited, the tight junction of the endothelial cells will be destroyed, and the cells will be more prone to phenotypic changes. Early studies have shown that Notch signalling is activated in the atrioventricular ducts, inducing the guanosine cyclase isoform subunit Gucy1a3 and Gucy1b3 transcription while promoting endothelial cell secretion of integrin A, activation of cells within the eNOS, PI3K/AKT so that eNOS phosphorylation of NO, NO and the receptor Gucy1a3 and Gucy1b3 interaction, regulation of endothelial cell transformation, to promote the development of ventricular calculi [32]. In this process, the Notch signalling pathway and the TGF-β signalling pathway are synergistic. After Notch signalling pathway activation, the soluble NICD is released, the intracellular Smad3 mRNA is upregulated, and the Smad3 protein is stabilized, both of which regulate the End MT.

After binding to the receptor between the adjacent Notch signalling pathways, the α-converting enzyme can be activated, and the extracellular domain of the receptor is digested, followed by the combination of γ-secretase protease and ADAM family enzyme, and the release of the active Notch receptor intracellular domain active form of NICD. NICD enters the nucleus and binds to the recombinant signal binding protein J (RBP-1), and the transcription of the downstream target gene is activated by the action of the transcriptional activator. Notch signal transduction process without the second messenger and protein kinase involved in the cell differentiation process play a fine regulatory role (seen in Figure 4). A number of studies have shown that Notch channels are involved in the process of myocardial fibrosis, whereas Notch channels, Notch3, Notch4, Delta-like4, Jagged1 and Jagged2 are all expressed on the arterial endothelium, and studies have shown that Notch signalling pathway involved in the regulation of EMT process Jagged-1 activation of Notch signalling in epithelial cells can induce mesenchymal cell-specific protein upregulation, accompanied by epithelial cell morphology, function to interstitial cell differentiation [33–35]. Sahlgren et al. found that Notch signalling was involved in the regulation of hypoxia-induced EMT [36].

Figure 4. Simulated hypoxia conditions induced by End MT-induced CMVE.

Vascular endothelial dysfunction affects the transformation of endothelial stroma to achieve synergistic AS effect through the notch signalling pathway

Normal endothelial cells can regulate blood vessel tension, maintain vascular structure, secrete anticoagulant, antiplatelet substances and fibrinolytic protein, and have anti-inflammatory effect, including preventing neutrophils, monocytes and other inflammatory cells to the blood vessel wall adhesion aggregation [37]. Vascular endothelial dysfunction refers to endothelial cells in the pathological factors (such as hyperlipidaemia, oxygen-free radicals, smoking, high blood flow shear stress) to stimulate the occurrence of endothelial dysfunction. Vascular tension regulation disorder and adhesion molecule expression abnormalities are two important manifestations of endothelial dysfunction. Vascular endothelium-dependent relaxation response weakened or even disappeared, and the reason is the release of endothelial cells released endothelium-derived relaxation factor (EDRF) has changed. EDRF is the main component of NO secretion and decreased activity, resulting in vasospasm, abnormal contraction, thrombosis and angiogenesis, not only the formation of AS early, but the development of AS also plays a very important role. Abnormal expression of adhesion molecules can lead to abnormal adhesion of endothelial cells, adhesion of endothelial cells to the arterial subcutaneous space, differentiation, absorption of lipid into foam cells, and promote the occurrence and development of AS. NO is based on L-arginine as a matrix, through the NO synthase (NOS) role in the formation. Inducible nitric oxide synthase (iNOS) is mainly distributed in macrophages, mast cells, neutrophils, etc., but in the physiological conditions, they do not express. NOS is an enzyme related to the production of diastolic factors. NO produced by endothelium, mainly in endothelial synthesis, and its catalyzed synthesis of NO through various pathways plays anti-AS effect: including strong vasorelaxation, inhibition of platelet adhesion and aggregation in the vascular endothelium, cytoprotective effect, inhibition of smooth muscle cell (SMC) proliferation and inhibiting endothelial monocyte adhesion. In the AS state or condition of inflammatory cytokines such as tumour necrosis factor (TNF-α) and low-density lipoprotein (LDL) stimulation, the expression of NOS was significantly reduced to reduce NO synthesis. The relevant animal experiments found that given NOS inhibitor will obviously accelerate the AS process. The reverse regulation of NO is endothelin ET-1, which is mainly secreted by vascular endothelial cells (VECs). Plasma ET-1 levels are positively correlated with the severity of AS. After VEC injury, ET release increased, and vasoconstriction increased, while promoting SMC hyperplasia, causing atherosclerotic plaque formation and expansion. NO biological activity decrement leads to a relatively increase in ET, causing vasoconstriction, vascular remodelling and dysfunction. Studies have confirmed that plasma ET concentrations of patients with AS were significantly higher than normal, which indicated increased degree and the degree of disease. And a large number of ET caused by coronary artery strong and persistent contraction, increased myocardial ischemia, induced angina or myocardial infarction.

In order to explore the molecular mechanism by which Notch signalling regulates the formation of vascular structures, we decided to screen the target genes of Notch signalling by using gene expression profiles. We also used neonatal mice retinal angiogenesis model, which was conducted by giving P3 newborn mice the DMSO or GSI injection, stripping the mouse retina in P7, separation of retinal vascular tissue, extract RNA samples and being sent to company to do gene expression using gene chip. We used Me V-TM4 (http://mev.tm4.org/) software to analyze the data and carried out cluster analysis to identify 231 significant differences in the gene. In addition, because our sample only detected three pairs, the sample size was small, and we used GSEA software to find 113 changes in the more obvious differential genes. We divided this total of 344 differential genes by DAVID online software analysis (https://david.ncifcrf.gov/); according to functional classification, these genes are divided into 43 categories, of which we were interested in cell adhesion and extracellular matrix-related genes, respectively (seen in Figure 5). We designed HUVECs in vitro and synthesized real-time quantitative primers to detect these genes. The results showed that there were significant changes in four genes: LAMB1, HSPG2, ANGPTL4 and PCDH18 (seen in Figure 6).

Figure 5. Twenty-six different genes associated with cell adhesion and extracellular matrix.

Figure 6. Real-time quantitative PCR was used to detect differentially expressed genes. *p < .05; **p < .01.

Discussions

AS is the pathological basis of coronary heart disease. According to inflammation theory, AS induced by coronary heart disease is caused by the local vascular and systemic inflammatory response [38]. Endothelial cell damage, monocyte and granulocyte activation, the production of related cytokines and boobies, leukocyte attachment and infiltration, mononuclear cell infiltration to macrophage differentiation and activation, the formation of foam cells, smooth muscle migration, etc., eventually lead to lipid deposition and reduction of atherosclerotic plaque formation. We used Western blotting to detect the expression of tight junction protein ZO-1 and adhesion protein EE-cadherin, β-catenin and N-cadherin. The results showed that the change of Notch signalling pathway could significantly affect the expression of VE-cadherin and β-catenin. And the expression of ZO-1 and N-cadherin was not statistically significant [39]. Notch signalling can downregulate the expression of N-cadherin by regulating the expression of N-cadherin, a core of the Notch signalling core transcription factor. At the animal level, we used Mx-Cre; RBP-Jflox mice to observe the changes in blood retinal barrier and blood–brain barrier after removal of the Notch signalling core RBP-J by vascular endothelial cells. Innovatively, in the world, there are still no such reports. In addition, we found that hD1R could promote neonatal rat retinal vascular cell coverage and, in other words, promote the mouse retinal vascular maturity. In order to study the molecular mechanism of Notch signalling pathway in regulating vascular structure and function, we did a gene chip. We used Me V-TM4 software to perform cluster analysis for screening out 231 differential genes. But, due to less sample size, only three pairs were available which affected the accuracy of the data, so we used GSEA software (http://software.broadinstitute.org/gsea/index.jsp) to find the rest 113 genes with significant changes, but no statistically significant differences were observed. We used the DAVID online database to identify 17 differentially expressed genes associated with cell adhesion and the 9 differential genes associated with the extracellular matrix from 344 differential genes.

Conclusions

In this study, the question of the effect of Notch signalling pathway on the relationship between endothelial dysfunction and endothelial stromal transformation in AS was studied sufficiently through a variety of experimental means to draw a strong conclusion. Firstly, the Notch signalling pathway was active in the atherosclerotic model. Secondly, the Notch signalling pathway was demonstrated to enhance AS by promoting vascular endothelial dysfunction. Thirdly, it was demonstrated that the Notch signalling pathway was mediated by promoting endothelial and could enhance AS. Finally, we confirmed the endothelial function through the Notch signalling pathway affecting the transformation of endothelial stroma to achieve synergistic AS effect. The results of this study have a good guiding significance for the important role of Notch signalling pathway in AS, indicate the ability to influence endothelial function and endothelial stromal transformation by intervening Notch signalling pathway, can also affect the relationship between the two courses and thus eventually realize the purpose of treatment of AS.

Disclosure statement

No potential conflict of interest was reported by the authors.