In recent years, several studies have confirmed that EndMT in endothelial cells can promote the progression of AS plaques(Qiu et al., 2022),(Wang et al., 2021). The role of shear stress in EndMT has received close attention, but the precise mechanism by which OSS activates EndMT and how it affects the progression of AS plaques need to be further elucidated. In this study, we confirmed that OSS promotes the formation of early and lately AS plaques in ApoE-/- mice by oil red staining analysis and immunohistochemical and immunofluorescence staining found that OSS activates the EndMT process and regulates ID1 protein expression. Multiple studies have shown that TGF-β signalling is enriched in the OSS region(Deng et al., 2021),(Suwittayarak et al., 2022), which is consistent with our immunohistochemical results. We used TGF-β1 to act on HAECs to simulate a low oscillating shear stress environment in vitro. Our results showed that TGF-β1 also activated the EndMT process and up-regulated the expression of ID1 protein.Furthermore, in vivo, the ID1 protein inhibitor AGX51 inhibited OSS-mediated EndMT and subsequent AS progression. The intervention of ID1 expression by molecular and pharmacological methods in vitro can deter EndMT and restore endothelial cell migration ability. On the contrary, ID1 overexpression promotes EndMT in ECs.These studies suggest that ID1 is involved in OSS-mediated EndMT and AS plaque formation. We also found that inhibition of ID1 abrogated low oscillating shear stress-induced EndMT signalling, including activation of specific transcription factors Snail and Wnt/β-catenin signalling.
EndMT plays an essential role in various cardiovascular diseases, and its research on the occurrence and development of AS plaques is also deepening(Wang et al., 2021). Studies have found that there is a class of cells in the subendothelial layer of human AS plaques that have both endothelial and mesenchymal cell markers, so it is speculated that EndMT is involved in the progression of atherosclerosis(Moonen et al., 2015). Several studies have observed endothelial cells with EndMT within AS plaques(Evrard et al., 2016), and the loss of endothelial cells on the plaque surface may be related to their migration into the plaque(Chen et al., 2021). Vascular endothelial dysfunction is an essential feature of atherosclerosis, and biomechanical factors also play an irreplaceable role in the process of AS(Mazzi et al., 2021).In adults, AS lesions are preferentially located at arterial branches and bends, which produce low oscillating shear stress (OSS) on the vessel in the range of 0.5 ± 4 Dyn/cm2.OSS is one of the leading causes of atherosclerotic plaque formation and affects the development of vulnerable plaques, putting plaques at a higher risk of rupture, leading to unstable angina, myocardial infarction, and stroke(Timmins et al., 2017). OSS in atherosclerotic areas reduces protective endothelial FGFR1 signalling while activating TGF-β, suggesting that low oscillating shear stress is an activation signal for EndMT(Chen et al., 2015).In addition, OSS can also lead to ROS generation and inflammatory signalling during atherosclerosis, both of which promote the development of EndMT(Krenning et al., 2016). In this study, we confirmed that OSS could promote the occurrence and development of EndMT and AS plaques. In our present study, we found that OSS induces EndMT in ApoE-/- mice endothelial cells and promotes AS plaque progression but only induces upregulation of the mesenchymal gene α-SMA and preserve the expression of the endothelial gene CD31; that is, ECs are undergoing partial EndMT.This subset of ECs may be more easily reversible under certain circumstances, such as cardiac fibroblasts that can derive endothelial cells after acute ischemic heart injury(Ubil et al., 2014). The occurrence and development of AS plaques are more common in arterial bifurcations, and blood flows in disordered areas. We speculate that EndMT is involved, which causes a part of the endothelial layer to be destroyed. The blood continuously contacts the intimal surface lacking endothelial cells, which eventually leads to plaque formation and rupture. Consistent with this, Evrard et al.(Evrard et al., 2016) used an endothelial lineage tracing system and found that EndMT caused endothelial cells on the plaque surface to migrate into the plaque interior, further damaging the endothelial cell layer. The above findings suggest that the relationship between OSS, EndMT, and AS can promote EndMT, thereby affecting the occurrence and development of AS.
IDs are closely related to tumours and cardiovascular diseases(Ling et al., 2014).In 1990, Benezra et al.(Benezra et al., 1990) first discovered that ID1 is widely present in mammalian cells and is known for its involvement in inhibiting nuclear transcription factors binding to DNA, including the inhibition of atomic transcription of proto-oncogenes(Jen et al., 1992). So there is a lot of research on ID1 protein and cancer. Atherosclerosis is similar to cancer in many ways, including angiogenesis, inflammation, and Epithelial-Mesenchymal Transition(EMT)/EndMT.In addition, several studies have shown that the ID1 protein is an essential force-sensitive factor(Ni et al., 2010). Through immunohistochemistry and immunofluorescence staining, we confirmed that the expression of ID1 protein in the common carotid artery of ApoE-/- mice were down-regulated under the action of OSS ensuring that ID1 protein is regulated by shear stress. In cell experiments, HAECs were treated with TGF-β1, and the corresponding changes in the expression of endothelial marker CD31 and mesenchymal marker α-SMA were detected by WB to confirm that the EndMT cell model was successfully established. However, the expression of ID1 protein is up-regulated under the stimulation of TGF-β1.In vitro and in vivo experiments seem to be contradictory. Still, in the complex pathological process of AS, inflammatory factors, oxidized LDL, or stress are all involved in the regulation of ID1, which may counteract part of the induction of ID1 expression by TGF-β. We have reason to believe that ID1 is a force-sensitive factor, and OSS plays a leading role in the regulation of ID1(Gadomski et al., 2020),(Edhayan et al., 2016),(Qiu et al., 2011). This is also an exciting aspect. Later, our experimental group will use a flat flow chamber device to truly explore the effect of OSS on ID1 at the cellular level. According to recent studies, IDs are closely related to the progression of atherosclerosis, and ID inhibitors successfully prevent the passage of atherosclerosis(Avecilla et al., 2017).Wojnarowicz et al.(Wojnarowicz et al., 2019) found that an ID1 protein inhibitor, AGX51, inhibits retinal pathological neovascularization by inhibiting the Id1-E47 interaction, resulting in ubiquitin-mediated ID1 degradation.Our study found that AGX51 can reduce the occurrence and development of OSS-mediated AS plaques. EndMT is considered a new therapeutic target for AS. Many studies have explored the relationship between ID1 and tumour EMT(Zhao and Liu, 2020). However, the regulatory role of ID1 on EndMT in AS has not been reported so far. In the present study, we confirmed that the ID1 protein inhibitor AGX51 inhibited partial OSS-mediated EndMT in the left common carotid artery of ApoE-/- mice using double immunofluorescence staining and immunohistochemistry of CD31 and α-SMA.In vitro, the WB assay found that ID1 silencing by AGX51 or sh-RNA inhibited part of the EndMT process in TGF-β1-treated HAECs.Statins reduce the concentration of CCR-5, the receptor for the proinflammatory cytokine CCL-4, decrease EndMT, and inhibit subsequent AS lesion formation(Yang et al., 2017). Increased cell migration ability is a feature of EndMT cells(Phan et al., 2021), we evaluated the migration of HAECs using scratch assay and Transwell, and the results showed that inhibition of ID1 protein could attenuate the TGF-β1-induced increase of HAECs migration ability. Paeoniflorin inhibits platelet-derived growth factor-bb (PDGF-BB)-stimulates increased proliferation and migration of human pulmonary artery smooth muscle cells, and inhibits EndMT(Yu et al., 2022).In addition, forced overexpression of ID1 can down-regulate the expression of endothelial markers (CD31, VE-cadherin) and up-regulate the expression of mesenchymal markers (α-SMA, SM22α). Further promotion of EndMT.Taken together, ID1 regulates OSS-mediated EndMT and atherosclerosis.
The canonical Wnt/β-Catenin signalling pathway acts on the proliferation, differentiation, and migration of cardiomyocytes, cardiac endothelial cells, and primordial valve cells in different stages of cardiac development. It regulates the endothelial-mesenchymal process during the formation of the endocardial cushion—plasma cell transformation (EndMT) process(Tyson et al., 2020).ID1 protein is one of the downstream targets of Wnt/β-Catenin signalling regulation, and the positive feedback between ID1 protein and Wnt signalling plays a vital role in cardiac differentiation(Chien et al., 2004).Wnt/β-Catenin signalling drives EndMT by increasing Snail expression. Snail, a key transcription factor of EndMT, is regulated by shear stress and is preferentially expressed in the OSS region. Promotes AS plaque progression by promoting endothelial cell proliferation and migration and enhancing the permeability of the endothelial monolayer to macromolecules(Mahmoud et al., 2017). We observed upregulation of Snail and β-catenin expression in ApoE-/- mouse common carotid artery endothelial cells and TGF-β1-induced HAECs exposed to OSS, and inhibition of ID1 reduced OSS-mediated Snail and Wnt/β- Activation of Catenin and overexpression of ID1 increased the expression of these EndMT essential signalling pathway proteins. These results suggest that ID1 regulates the OSS-mediated EndMT process by Ctrlling the EndMT key transcription factor Snail and the canonical Wnt/β-Catenin signalling pathway.
Taken together, ID1 is involved in OSS-mediated EndMT and the progression of atherosclerosis. Biomechanical factors-induced EndMT, which promotes the development of atherosclerotic plaques, is currently a hotspot.ID1 protein inhibitors reduce the effect of atherosclerotic plaques by inhibiting OSS-mediated Snail and Wnt/β-Catenin signalling pathways and inhibiting EndMT.Research on endogenous EndMT antagonists is still in its infancy, and our study provides valuable information for ID1 inhibitors in treating EndMT and atherosclerosis.ID1 is expected to be a potential therapeutic target for treating AS patients.