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Research progress of endothelial-mesenchymal transition and vulnerable plaque
Research progress of endothelial-mesenchymal transition and vulnerable plaque. Atherosclerosis (AS) vulnerable plaque rupture is the common pathological basis of adverse cardiovascular and cerebrovascular events such as acute myocardial infarction, stroke, and sudden cardiac death in patients with cardiovascular and cerebrovascular diseases.
Vulnerable plaque refers to a plaque that has a tendency to rupture, is prone to thrombosis, and may progress rapidly. Various stimulating factors in the bloodstream can induce endothelial cells to undergo endothelial-mesenchymal transition (EndMT), thereby accelerating the process of AS plaque.
EndMT participates in the occurrence and outcome of AS, and is closely related to plaque vulnerability. This article focuses on the concept of EndMT and its mechanism of action in vulnerable plaques, in order to provide new ideas and theoretical basis for the prevention and treatment of vulnerable plaques.
Atherosclerosis (AS) is a pathological process driven by the accumulation of intimal lipids and chronic inflammation, and eventually a series of ischemic changes in the organs are caused by the thickening and stiffening of the vessel wall and the narrowing of the lumen. In addition to systemic risk factors such as metabolic abnormalities and circadian rhythm disorders, local biochemical and biomechanical changes also accelerate the process of AS.
In coronary artery AS lesions, the vulnerable plaque ruptures secondary to platelet activation and thrombosis, and eventually blocks the lumen. Acute coronary syndrome (ACS) caused by this is the number one killer of cardiovascular disease patients’ health . Plaque rupture and plaque erosion are the two most common types of vulnerable plaques.
Numerous studies have shown that the endothelial-mesenchymal transition (EndMT) plays an important role in the development and outcome of AS [1, 2, 3]. Therefore, exploring the potential regulatory mechanism of EndMT on vulnerable plaques will be helpful For early intervention and prevention of the occurrence of ACS.
1. The concept of EndMT
EndMT refers to the process in which endothelial cells (ECs) lose their original characteristics and transform into mesenchymal cells under the action of a variety of stimulating factors. The result is that ECs undergo major changes in polarity, morphology, and function. Variety.
In order to obtain the mesenchymal phenotype, ECs inhibited endothelial-specific markers such as vascular endothelial-cadherin (VE-cadherin) and platelet endothelial cell adhesion molecule-1 (platelet endothelial cell adhesion molecule-1) to varying degrees. , PECAM-1 (ie CD31), the expression of angiopoietin receptor 1/2, and activate mesenchymal markers such as α-smooth muscle actin (α-smooth muscle actin, α-SMA), vimentin and adult Expression of Fibroblast Specific Protein 1, etc.
In this process, the cells change from a compact cobblestone-like shape to a spindle shape, their anti-platelet production ability is impaired, and the characteristics of mesenchymal cells such as invasion and migration are enhanced . In addition to producing a permanent mesenchymal phenotype, ECs can also remain in the intermediate stage of transdifferentiation for a long time.
This process is called partial EndMT. At this time, the cells acquire a mesenchymal phenotype without losing endothelial-specific markers and present progenitor-like characteristics . This intermediate form of phenotypic transformation is unstable and can be reversed under certain conditions, thus becoming a potential target for treatment .
A variety of signaling pathways are involved in the regulation of EndMT, including the classic transforming growth factor-β (transforming growth factor-β, TGF-β) signaling pathway and the subsequent discovery of Notch, Wnt/β-catenin, Hippo pathways, etc. [7, 8 , 9, 10].
These signals ultimately regulate the expression of endothelial and mesenchymal genes by affecting the activity of transcription factors Snail, Slug, Twist and Zeb . EndMT is related to the development of embryonic heart valves and atrioventricular septum, and is involved in the development of a variety of cardiovascular diseases, including pulmonary hypertension, valvular disease, myocardial fibrosis and AS [11, 12, 13].
2. The basic characteristics of vulnerable plaques
Vulnerable plaque is an important pathological basis for inducing ACS. At present, all plaques with a tendency to rupture, thrombosis, and rapid progress are collectively referred to as vulnerable plaques, which can be divided into three types: easily ruptured plaques, erodible plaques, and calcified nodules . Among them, EndMT is mainly involved in the pathogenesis of ruptured plaques and erodible plaques.
Thin-cap fibrous atherosclerotic plaque is a precursor of plaque rupture, usually with a large number of inflammatory cell infiltration and rich in lipid necrotic core . Degradation of the fiber cap and enlargement of the lipid core are the main causes of plaque rupture. Local hypoxia and inflammatory stimulation accelerate angiogenesis in the plaque and cause bleeding in the plaque.
In addition, the point-like calcification near the fiber cap increases the local stress, which can easily lead to plaque rupture under the action of abnormal fluid shear stress. Vasodilatory remodeling is also common in thin cap fibrous atherosclerotic plaques, which can compensate for the encroachment of the lumen due to plaque formation or intimal hyperplasia, maintain the lumen patency, but also hide larger necrotic cores and more Many inflammatory cells and microcalcification foci.
Once the fibrous cap ruptures, the lipids and tissue factors in the necrotic core are exposed to the bloodstream, which can activate the coagulation cascade, leading to thrombosis.
Compared with thin-cap fibrous atherosclerotic plaques, erodible plaques have complete fibrous caps, smaller necrotic cores, and rare infiltration and calcification of inflammatory cells, but ECs are severely stripped . Disturbance of blood flow mediates apoptosis and exfoliation of ECs by activating Toll-like receptor 2 is the main factor leading to plaque erosion.
At this stage, the widespread application of intensive lipid-lowering drugs still cannot reduce the high incidence of ACS, suggesting that it is necessary to explore the formation mechanism of vulnerable plaques and find new therapeutic targets.
3. EndMT participates in the formation and rupture of vulnerable plaques
Normal ECs can maintain vascular barrier, coordinate leukocyte transport and prevent thrombosis. Affected by EndMT, the tight adhesion connection between ECs is gradually lost, and the expression of leukocyte adhesion molecules increases, which will destroy the selective permeability of the blood vessel wall and drive the infiltration of inflammatory cells, leading to endothelial dysfunction. Chen et al.  confirmed that EndMT can promote neointimal hyperplasia, and can induce ECs to change to pro-inflammatory, procoagulant and other undesirable phenotypes.
Another study found that compared with fibrous calcified plaques, the number of ECs with EndMT in complex plaque lesions increased significantly, and it was inversely proportional to the thickness of the fibrous cap . It can be seen that EndMT can not only initiate AS, but is also associated with plaque instability.
The formation of vulnerable plaques is related to many factors, including local biochemical changes such as oxidative stress, active inflammation, and hypoxia [17, 18], and systemic risk factors such as metabolic abnormalities and circadian rhythm disorders [19, 20]. In addition, abnormal hemodynamics is also an important cause of accelerated plaque rupture . These factors can affect the formation and rupture of vulnerable plaques by regulating EndMT.
1) Oxidative stress and EndMT:
Oxidative stress is pathological damage induced by the increase of reactive oxygen species (ROS) in the body. As we all know, elevated blood lipids will lead to the deposition of lipids, mainly low-density lipoproteins, under the inner membrane. Low-density lipoprotein is oxidized and modified by ROS to form oxidized low-density lipoprotein (ox-LDL), which is an important factor leading to the occurrence and progression of AS.
Studies have found that the combination of ox-LDL and hemagglutinin-like oxidized low-density lipoprotein receptor can not only increase the expression of EndMT key transcription factor Snail in ECs and inhibit its ubiquitination degradation , but also activate phosphoinositol ( PKC) signal, which initiates EndMT by increasing the expression of matrix metalloproteinases (MMPs) and endogenous TGF-β in ECs .
In addition, Zhu et al.  found that the vulnerability of human carotid artery plaque was positively correlated with the activation of EndMT. Further studies have shown that the circadian clock protein brain and muscle arnt-like 1 (BMAL1) expression is absent in vulnerable plaques, and subsequently activated bone morphogenetic protein (BMP) The signaling pathway triggers EndMT by promoting the accumulation of ROS. Thus, oxidative stress can mediate vulnerable plaque formation through EndMT.
2) Inflammation and EndMT:
AS is a chronic inflammatory reaction of the blood vessel wall, from lipid streaks to the formation, rupture and thrombosis of vulnerable plaques, it is always accompanied by the participation of a variety of inflammatory factors and inflammatory cells. Chen et al.  found that the expression of interferon-γ, tumor necrosis factor-α and interleukin-1β in AS plaques increased. Subsequently, they used these three cytokines to synergistically stimulate human umbilical vein endothelial cells (HUVECs) to simulate the inflammatory microenvironment within the plaque.
The results showed that ECs up-regulated the expression of mesenchymal marker proteins and transcription factors Zeb2, Slug, and Snail, and showed stronger chemotaxis of inflammatory cells. Persistent inflammation can lead to the loss of the protective FGF signal in ECs, and the subsequently activated TGF-β signal induces EndMT, which leads to aggravation of local inflammation and expansion of the neointima, and ultimately promotes plaque progression . Macrophages are one of the most important inflammatory cells in plaques.
They can not only secrete proteolytic enzymes to degrade the extracellular matrix and weaken the thickness of the fibrous cap, but also promote the disintegration of the outer elastic membrane, participate in the positive remodeling of blood vessels, and affect the plaques. Block stability. Macrophages accumulate in AS plaques and act as a new type of EndMT inducer . A recent study found that foam cells derived from M1 macrophages can induce EndMT in a paracrine manner, mainly by up-regulating the expression of chemokine 4 to increase the permeability and adhesion of ECs .
3) Hypoxia and EndMT:
Hypoxia exists in AS plaques, especially in the necrotic core of the plaques. In addition to the thickening of the intima and reducing the ability to diffuse oxygen, the high metabolic activity of local inflammatory cells also aggravates the degree of hypoxia of the plaque. Evrard et al.  found that as AS progresses, the percentage of hypoxic cells in the plaque intima increases significantly.
At the same time, the study showed that although hypoxia up-regulated the expression of ECs mesenchymal marker genes and transcription factors Snail and Slug, the regulation of endothelial markers was uncertain. Hypoxia down-regulated the effects of vasodilation and endothelial protection. The expression of endothelial nitric oxide synthase has no obvious effect on the adhesion molecule CD31 between endothelial cells, suggesting that hypoxia can induce part of EndMT.
It is worth noting that part of EndMT has been proved to be related to angiogenesis , and hypoxia can also promote angiogenesis by up-regulating hypoxia-inducible factor-1. Whether the two have an interaction in regulating plaque angiogenesis is worth further the study.
4) Abnormal hemodynamics and EndMT:
Blood flow disorder is the key factor that causes plaque rupture, and even the fuse of plaque rupture. Fluid shear stress refers to the frictional force produced by blood flow on the blood vessel wall, and is closely related to the phenotype and functional regulation of ECs.
Studies have found that blood flow disturbances such as arterial branches, bends, and stenosis are affected by low and/or oscillating shear stress and are prone to form plaques, while uniform high-level flow shear stress has the function of anti-AS. Low shear stress can enhance the expression and activity of MMP-2, 9, 12 and cathepsin K and S in plaques, promote the degradation of extracellular matrix and rupture of intravascular elastic membrane, accelerate the infiltration of inflammatory cells, and promote the vulnerability of plaques Process . Chen et al.  found that under oscillating shear stress, the content of Twist1 in HUVECs increased, and the expression of fibroblast growth factor receptor 1 (fibroblast growth factor receptor 1, FGFR1) decreased.
Twist is the key transcription factor of EndMT, and FGFR1 has been proven to protect ECs and inhibit EndMT . Another study found that low shear stress can up-regulate the expression of Twist1 and GATA4 in ECs, and activating the GATA4-Twist1 signal axis can induce EndMT to promote flow-mediated endothelial dysfunction and increased vascular permeability [30, 31]. Therefore, abnormal hemodynamics can promote EndMT and induce plaque rupture.
5) Systemic risk factors and EndMT:
In addition to common AS risk factors such as abnormal glucose and lipid metabolism and hypertension, smoking and circadian rhythm disorders can also promote the development of stable plaques to vulnerable plaques by inducing EndMT. Smoking is an independent risk factor for AS, and the active ingredient nicotine in its smoke can cause ApoE-/- mice to produce a larger area of AS. Under nicotine stimulation, ECs up-regulate the expression of Snail by activating the ERK1/2 signal and induce EndMT .
Circadian rhythm disorders can lead to a variety of chronic inflammation and metabolic-related diseases, and are closely related to the occurrence of ACS. The lack of the clock protein BMAL-1 in the vascular endothelium can induce endothelial dysfunction of microvessels and large blood vessels . Recent studies have shown that the vulnerability of human carotid plaques is related to the loss of BMAL-1 expression in the plaques. BMAL-1 can inhibit the accumulation of ROS and EndMT induced by ox-LDL, while the lack of BMAL-1 can aggravate oxidative stress damage by activating BMP signals and promote the formation of vulnerable plaques .
4. EndMT regulates the morphological structure of vulnerable plaques
In addition to the influence of biochemical and biomechanical factors, the structure and composition of the plaque itself also determine its stability. As the microenvironment changes, the local and overall structure of the plaque changes accordingly, showing unique morphological features, including large lipid cores, thin fiber caps, positive vascular remodeling, waterfall-like inflammatory activities, and necrotic core edges. Neovascularization and plaque microcalcifications on the shoulder, etc. Studies have shown that EndMT accelerates the development of vulnerable plaques by affecting the morphological structure of plaques.
1) EndMT participates in the degradation of extracellular matrix, leading to the thinning of the fibrous cap and the positive remodeling of blood vessels:
The dynamic balance between the production and degradation of extracellular matrix is an important factor in maintaining the stability of the fibrous cap and vascular wall structure. Studies have shown that EndMT can mediate the formation of vulnerable plaques by affecting the stability of the extracellular matrix. Evrard et al.  used ECs lineage tracing experiments to prove that in complex plaques with unstable characteristics, the proportion of fibroblasts transformed from EndMT is higher, and the number is inversely proportional to the thickness of the fiber cap. Further research found that compared with the high expression of collagen in normal fibroblasts, the expression of MMPs in these cells increased while the expression of collagen decreased. This breaks the original balance between collagen and MMPs in the extracellular matrix, and makes plaques develop towards vulnerable plaques lacking collagen and thinning of the fibrous cap.
Positive vascular remodeling is another important feature of vulnerable plaque. It is vividly compared to a wolf in sheep’s clothing. It seems to be an important compensatory mechanism for maintaining coronary blood flow, but it is not beneficial to the clinical prognosis. Intravascular ultrasound shows that plaques with positive remodeling can hide larger eccentric lipid nuclei, more inflammatory cells and microcalcifications . Low shear stress is a common cause of positive vascular remodeling and plaque rupture , and the induced EndMT may be the common pathological basis of the two.
Low shear stress not only reduces the expression of FGFR1 in ECs, but also activates the TGF-β signal to induce EndMT . During this process, the expression of leukocyte adhesion molecules on the surface of ECs increases, which will promote circulating monocytes to enter the subendothelial layer and differentiate into macrophages. On the one hand, macrophages engulf ox-LDL and transform into foam cells to maintain the expansion of the lipid core of the plaque. On the other hand, over-infiltrated macrophages secrete a large amount of MMPs to degrade basement membrane and extracellular matrix, leading to thinning of the fibrous cap and enlargement of the lumen.
In short, low shear stress induces EndMT to show uncontrolled inflammation and matrix degradation of the plaque, and the enlarging lumen will further aggravate the low shear stress and form a vicious circle.
2. EndMT mediates intraplaque angiogenesis:
intraplaque angiogenesis and the resulting intraplaque hemorrhage is an important mechanism to accelerate the rupture of vulnerable plaques. New blood vessels in plaques mostly originate from adventitia to nourish blood vessels, which are important channels for transporting inflammatory mediators and regulating plaque metabolism.
Due to the lack of pericytes and basement membrane coverage, the new blood vessel has a simple structure, high permeability, and high expression of leukocyte adhesion molecules, which is easy to drive inflammatory cell infiltration. The rupture of fragile capillaries can release a large amount of free cholesterol and promote the rapid expansion of the necrotic core .
Studies have pointed out that the apical cells that grow at the tip of new blood vessels in a budding manner exhibit some of the characteristics of EndMT. The cells break away from the original monolayer of endothelium, extend filopodia and migrate through the extracellular matrix, but still retain contact with neighboring cells. Connect .
The ECs and apical cells that have part of EndMT lack top-bottom polarity, and the expression of Snail and Slug in the cells is up-regulated [37, 38]. In addition to promoting angiogenesis, these transcription factors are also involved in the regulation of EndMT-related genes.
In fact, many studies have confirmed the important role of some EndMT in pathological angiogenesis, including cerebrovascular malformations, pulmonary hypertension and intratumoral angiogenesis [39, 40, 41].
Sánchez-Duffhues et al.  confirmed that in advanced AS plaques, the adventitious vascular ECs, which are the main source of neovascularization in the plaque, can show CD31/α-SMA double positive, suggesting that some EndMT has occurred. Blood flow disturbances, inflammation and hypoxia can all promote angiogenesis in plaques by inducing EndMT. Studies have confirmed that low shear stress can induce partial EndMT in HUVECs . This may be the underlying mechanism of plaque shoulder neovascularization induced by low shear stress.
In addition, the inflammation and hypoxic microenvironment in the plaques increased the expression of the key EndMT transcription factors Slug and Snail in ECs. Slug up-regulates membrane matrix metalloproteinase 1, and mediates vascular sprouting in a way that does not depend on the destruction of cell adhesion . The lack of Snail inhibits the migration, proliferation and germination of ECs . When exposed to hypoxia, ECs intermediate mesenchymal markers increased significantly, but the expression of endothelial markers related to cell connections such as VE-cadherin and CD31 did not decrease .
This suggests that in addition to increasing the expression of VEGF, hypoxia can also promote angiogenesis in the plaque by mediating part of EndMT.
3) EndMT accelerates the formation of calcification in the plaque:
microcalcification and punctate calcification are the active stages of vascular calcification, which are closely related to plaque rupture. Point-like calcifications located in the fiber cap or plaque shoulder significantly increase local stress and stretching, making this area more susceptible to rupture under the impetus of abnormal fluid shear forces.
Plaque calcification belongs to endometrial calcification, which is similar to endochondral osteogenesis and is a programmed mineralization process initiated by inflammatory factors . At present, a variety of mineralization markers have been found in AS plaques, including BMP, osteopontin, and bone sclerostin . Vascular smooth muscle cells have long been considered an important source of osteoblasts in vascular calcification, but recent studies have shown that ECs can also induce AS calcification through EndMT . Many studies have confirmed the association between EndMT and vascular calcification.
During AS calcification, the expression of BMP signal increases. Matrix gla protein (MGP) is an extracellular peptide that inhibits vascular calcification by blocking BMP signaling. Yao et al.  found that in MGP-/- mice, ECs had EndMT before transdifferentiation into osteoblasts, which was manifested by the co-localization of endothelial markers and pluripotent stem cell markers.
Further studies have shown that the absence of MGP leads to an increase in elastase and kininase activity, and EndMT is mediated by Sox2 . Once ECs acquire an osteogenic phenotype through EndMT, mineralization can proceed in the extracellular matrix. In AS plaques, inflammation and calcification are complementary. Common inflammatory factors in AS such as tumor necrosis factor-α and interleukin-1β can also drive EndMT, making ECs more sensitive to osteogenic differentiation induced by BMP9.
The mechanism depends on the down-regulation of bone morphogenetic protein receptor 2 in ECs and the subsequent decrease in JNK signal activity . The study also found that there is an EndMT phenomenon in the adventitia nourishing blood vessels, which means that the osteogenic signals initiated by the adventitia can be transmitted to the inner membrane through the nourishing blood vessels, and these ECs have the potential for osteogenic differentiation.
4) EndMT and plaque erosion:
Plaque erosion is another important mechanism that causes ACS after plaque rupture. It is characterized by intact fibrous cap, rich in vascular smooth muscle cells and proteoglycan matrix, but ECs has serious apoptosis  . Studies have found that there are a large number of infiltrating neutrophils in erosive plaques.
These cells adhere to the surface of the inner membrane and form a unique extracellular network structure by releasing intracellular granular proteins and chromatin, called neutrophils. External trapping nets (neutrophil extracellular traps, NETs). NETs mediate oxidative stress and ECs apoptosis through Toll-like receptor 2, causing plaque erosion and thrombus formation .
A recent study showed that EndMT is induced when the content of NETs exceeds the phagocytic capacity of ECs. Neutrophil elastase produced by NETs disrupts cell-to-cell connections by hydrolyzing VE-cadherin and activates β-catenin signal. β-catenin participates in the transcription of EndMT-related genes, including vimentin, MMP9 and Snail .
EndMT is a complex and flexible phenotypic transformation form of ECs, which participates in the development of AS and mediates the formation of vulnerable plaques. It is worth noting that there are still some unavoidable problems in EndMT research.
First, there is currently a lack of highly specific endothelial and mesenchymal markers.
Secondly, in different experimental models, there is no uniform standard for which markers must be detected and to what extent each marker can be considered effective.
With the development of genetic pedigree tracing and single-cell gene sequencing technology, future research on EndMT will become more and more standardized.
(source:internet, reference only)