Atherosclerosis, as the underlying mechanism of cardiovascular and cerebrovascular diseases, is caused by many complicated risk factors, such as lipid accumulation and abnormal biomechanics (1, 2). These disordered microenvironments in the vasculature result in vascular inflammation, excessive proliferation and migration, fibrosis, and extensive necrosis, which contribute to atherogenesis and the formation of vulnerable plaques (3). Biomechanics, as an emerging field of cell and developmental biology, is considered as a regulator of atherosclerosis through mechanosensitive channels. A recent study used the computed tomography angiography of human circumflex coronary artery to construct computational simulation model and revealed a causal link between low-density lipoprotein (LDL) transportation and wall shear stress in the coronary artery, in which flow patterns altered the influx and efflux of cholesterol (4). Moreover, endocytosis regulates the transportation and degradation of lipids and apoptotic cell debris in atherosclerotic plaques, which controls the interaction between cells and their microenvironment (5). Due to the important role of the mechanical environment and membrane trafficking in vascular dysfunction and atherosclerosis, it is important to investigate the changes in endocytosis induced by abnormal biomechanics.

Endocytosis-mediated intracellular transport and the positive regulation of signalling cascades are the key regulators of cholesterol homeostasis, which play a casual role in atherogenesis (6). Endocytosis and the subsequent intracellular itinerary are based on the encapsulation of the fluid plasma membrane, selective receptors and vesicle-associated proteins. This selective route allows endothelial cells (ECs) to act as a vascular barrier to regulate the transport of hydrophilic and hydrophobic substances in blood and prevent harmful substances in tissues (7, 8). Meanwhile, the influx and efflux of cholesterol are specifically controlled by endocytosis with lipoprotein receptors including low-density lipoprotein receptor family and scavenger receptor family (Table 1) (6, 9). Recent studies have revealed that the scavenger receptors-mediated transcytosis, which rely on caveolae-based intracellular vesicles, play an important role in the delivery and accumulation of LDL in artery (10, 11). The accumulation of LDL in the sub-endothelial area will further stimulate excessive uptake and exhausted metabolism of lipid in vascular smooth muscle cells (VSMCs) and macrophages, which will result in unexpected inflammation, polarization/phenotype transformation, autophagy and the formation of foam cells (12, 13).

During the development of atherosclerosis, biomechanics, including shear stress, tensile force, and stiffness, play an important role in vascular inflammation, oxidative stress and lipid transportation (23, 24). Low shear stress (LSS) and oscillatory shear stress (OSS) generated by disturbed flow have been identified as hazard factors of atherosclerosis in hypercholesterolemic mini-pigs that correlate plaque growth with vulnerable features (25). These LSS and OSS will result in unexpected uptake of LDL and promote the development of atherosclerosis (26). Meanwhile, studies have also revealed that tensile force activation of stretch-related ion channels clinically contributes to arterial remodeling and relevant vascular dysfunction (27, 28). Moreover, recent research has also suggested that arterial stiffness is associated with artery atherosclerosis stroke in human with GWAS analysis that comprises 127,121 individuals of European ancestry (29). Thus, it is important to discuss the relationship between biomechanics and endocytosis in atherosclerosis.

In this review, we summarized the major types of endocytosis in atherosclerosis, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis and micropinocytosis. We then focused on the role of endocytosis in responding to biomechanical forces and describe the signal transduction involved in lipid transportation in the biomechanical microenvironment. The integration of biomechanics and endocytosis might improve our understanding of the transportation of high-risk lipoprotein and contribute to a better understanding of the formation of atherosclerotic plaques.

Endocytosis, as an important part of the cellular trafficking system, is a conserved channel for internalizing molecules and macromolecules through membrane deformation (30). The typical process of endocytosis contains four fundamental steps: (1) molecules specifically bind to receptors or the cell membrane; (2) coating proteins, cytoskeleton and membrane fusion proteins are rearranged to drive cell membrane-encapsulating molecules; (3) molecules are encapsulated in trafficking vesicles, such as endosomes and phagosomes; and (4) vesicles are transported to subcellular organelles (Figure 1). Due to the entry mechanism, endocytosis can be classified into pinocytosis and phagocytosis. The pinocytosis can further be classified into clathrin-mediated endocytosis and clathrin-independent endocytosis, such as caveolae-mediated endocytosis, caveolae- and clathrin- independent endocytosis and macropinocytosis, which can form differently sized vesicles (31) (Figure 1). Meanwhile, some specific inhibitors are also widely used to detect the influence of endocytosis to cell functions (Table 2). These engulfment processes drive lipid transportation and apoptotic cell clearance in the vasculature, which are inexorable events in atherosclerosis (37, 38). Here, we briefly discussed the role of different endocytosis mechanisms in atherosclerosis.

Shear stress is a blood flow-generating frictional force that is closely associated with vascular dysfunction and atherosclerosis. Several studies have demonstrated that atherosclerotic plaque usually emerge in near branches and bends of arteries that are exposed to disturbed flow, generating LSS or OSS (71, 72). These blood flow-induced vascular dysfunctions are linked to the changes of several signalling pathways, such as Klf2/4, Hippo–Yap–Taz, and Wnt/β-catenin, which participate in maintaining vascular integrity and tissue homeostasis (72–74). A recent study has revealed that shear stress not only mediates signal transduction but also increases LDL coverage on the endothelial glycocalyx, which controls the transportation of LDL across the vascular wall (75). Thus, it is essential to discuss the influence of shear stress on cell endocytosis during atherosclerosis (Figure 2).

ECs, as the barrier of vessel and blood flow, are equipped with various mechanosensitive channels that transfer biomechanical signals to modulate cellular function and behaviour, such as inflammation, endothelial-to-mesenchymal transition, and endocytosis (2). These shear stress-sensitive signals are mostly membrane receptors, cell‒cell junctional proteins and cell-matrix adhesion proteins (76). Moreover, Exposure of ECs on 10 dynes/cm² shear stress will result an increased endocytosis and can be reduced by inhibiting reactive oxygen species (ROS) (77). Meanwhile, the accumulation of NO and ROS in LSS or OSS areas will result in the increased stabilization and expression of Cav1, which may enhance Cav1-mediated transcytosis (78). The surface density of caveolae in the cell membrane, which has been implicated in haemodynamic forces, is necessary for mechanotransduction and arterial remodelling (79). Ramírez et al. found that Cav1 is highly expressed in the “athero-prone” area and controls the transcytosis of LDL in atherosclerosis. Moreover, mice lacking Cav1 expression show less accumulation of lipids in athero-prone areas and atherosclerotic plaques (56). Our laboratory's recent study has revealed that LSS- and OSS-induced ROS can promote the internalization of extracellular vesicles in vascular ECs. We also observed that extracellular vesicles accumulated in the artery arch that underwent LSS and OSS (80). Abnormal shear stress can also activate multiple Ca²⁺ channels, including Piezo1, P2ry2, connexin and transient receptor potential (TRP) channels, which are considered mechanosensors and are significantly associated with inflammation in ECs during atherosclerosis (72, 81, 82). Meanwhile, Ca²⁺ influx can initiate activity-dependent bulk endocytosis and participate in the engulfment of LDL (83, 84).

Ongoing studies of OSS and LSS in ECs also report the role of mechano-transduction in endocytic receptors. Due to numerous researchers have performed transcriptome data under different shear stress, these analyses reveal that shear stress can mediate multiple transcriptional processes of endocytic receptors, such as Cd36 and Scarb1 (85). The overexpression of these lipoprotein receptors in arteries promotes the uptake of macromolecules into cells, which participates in the formation of atherosclerosis (6). Meanwhile, shear stress also plays an important role in the reorganization of Cdc42-dependent actin polymerization, which is important for LDL endocytosis (86, 87). Macrophage cortical F-actin depolymerization is required for actin polymerization to form a hydrolytic compartment-the lysosomal synapse, which digests aggregated LDL via Cdc42 Rho GTPase and GEF pathway. In summary, shear stress regulates multiple processes of endocytosis that control the accumulation of LDL and other macromolecules in the vasculature, thereby critically contributing to the development of atherosclerosis near branches and bends of arteries.

ECs and VSMCs are sensitive to changes in physiological mechanical stretch and tension force that contain circumferential and axial stresses of approximately 100 kPa (1 × 10⁶ dyne/cm²) from periodical distention and relaxation in arteries (88). Arteries under hypertension also inherit axial stress, which is associated with blood pressure and periodic distention and relaxation of arteries (89) (Figure 3). Meanwhile, VSMCs can be the engine of artery showing stronger contractile ability to generate and sustain stress on artery that is important in maintaining vessel tone and blood pressure (102). Elastic lamellae comprising elastin and collagens, which attaches and sandwiches VSMCs in the middle through focal adhesion complex, affects VSMCs contraction and relaxation (103, 104). Furthermore, ECs and VSMCs subjected to cyclic stretch display an elongated spindle morphology and show reorganization of the cytoskeleton. Studies have defined that 5%–10% strain is the physiological stretch, while excessive strain (15%–20%) is the pathological stretch in artery (105, 106). The unexpected stretch and tension force on vascular cells including ECs and VSMCs will activate multiple signalling and result excessive proliferation, migration and apoptosis (Figure 3) (107, 108). The VSMCs response to cycle stretch can express integrin αVβ3, which can inhibit ox-LDL-induced apoptosis although PINCH-1 in the progression of atherosclerosis (109, 110). Moreover, the cardiovascular cells response to cyclic stretch increase the accumulation and update of extracellular matrix (111). These studies suggest that normal physiological stretch and tension are important signals for cardiovascular function and contribute to the homeostasis and development of vessels.

Pressure-derived stretch and tension force, which derived from the vascular deformation caused by blood pressure on the vessel wall, can influence the transportation of albumin and LDL. Studies have used 4 mm, 5 mm and 6 mm sleeves to restrict vessel from in New Zealand White rabbit and pressurized at 70, 120, or 160 mm Hg blood pressure, which control the deformation of vessel. The results showed that the accumulation of LDL in vessel was increased between 120 and 160 mm Hg with 5 mm sleeves, which explains the relationship between pressure-derived stretch and atherosclerosis (112). Stretch force also promotes the oxidation of LDL and accelerates the accumulation of ox-LDL in VSMCs (113). Meanwhile, cells stimulated with cyclical tensile stretch highly express the ox-LDL receptor Lox1, which is essential for internalizing ox-LDL (114). Moreover, membrane tension is also an important regulator of clathrin-mediated endocytosis through controlling the formation of clathrin-coated pits. Joseph et al. found that high tension interrupts the process of flat membrane-to-clathrin-coated structure transition by inhibiting the recruitment of epsin to the plasma membrane (44). Moreover, a recent study found that Torc2, a rapamycin-mediated protein kinase, can regulate plasma membrane tension to affect the reorganization of the actin cytoskeleton and vesicle fission to endocytosis sites (115). These results suggest that stretch and tension forces are essential for endocytic processes that regulate the transportation of multiple lipids and proteins in the vasculature (Figure 4).

Stiffness, also known as elasticity, is the mechanical force to resist deformation and is important for cell proliferation, migration and signal transduction. Arterial stiffness is closely associated with degenerative and remodelling changes in the extracellular matrix, resulting in vascular calcification, hypertension, and atherosclerosis (116). A recent Multi-Ethnic study with 6,814 men and women aged 45–84 years found that high load-dependent carotid artery stiffness is associated with a higher incidence of subclinical atherosclerosis and thus contributes to multiple cardiovascular events, such as stroke and hypertension (117). The stiffness of the artery is determined by their physiological conditions during atherosclerosis, such as the lipid core/necrotic core (1 kPa), fibrous plaque (35.5–54 kPa) and calcification (80–300 kPa), compared with normal artery (10–50 kPa) (118, 119). Meanwhile, the changing matrix stiffness also mediates the mechanical signal transduction and phenotypic transformation of vascular cells, which is closely related to cell fate and vascular function (118, 120). Recent studies have revealed that changing matrix stiffness in the vasculature promotes the adhesion of monocytes on ECs by enhancing the expression of miR-126 (targeting VCAM-1) and miR-222 (targeting ICAM-1) (121). Meanwhile, 1 kPa (soft) and 100 kPa (hard) substrate stiffnesses also increase cholesterol efflux in VSMCs and increase the expression of the macrophage marker CD68, suggesting that substrate stiffness can regulate the phenotypic switching of VSMCs (Figure 3) (122).

Endocytosis, as an important route of lipid metabolism, can also be regulated by cell and matrix stiffness. The surface topography of substrates regulates cell stiffness by activating mechanical signalling pathways. Li et al. found that a nanostructure stiff substrate can alter cell stiffness and behaviour to enhance clathrin-dependent endocytosis through its nano-topographical effect on integrin receptors. It has also been revealed that cells on a glass-based nanostructure stiff substrate respond similarly to 1 kPa soft hydrogels, which can reduce cell stiffness and membrane tension force (123). Furthermore, the cytoplasmic stiffness that influences deformability and membrane invagination can modulate the endocytic ability of cells. The high cortex stiffness of the subcellular structure will affect cell deformability, resulting in less phagocytic ability on macrophages (124). Because the stiffness of the necrotic core and lipid core in atherosclerotic plaques are nearly 1 kPa, we suspect that cells in atherosclerotic plaques will engulf more lipoprotein and apoptotic cells, resulting in the formation of foam cells.

A recent study revealed that macrophages prefer to take up native LDL and ox-LDL on 1 kPa soft substrates but with no difference in proliferative activity on soft and stiff substrates in an inflammatory microenvironment (125). However, Li et al. found that macrophages on soft 4 kPa PA hydrogels promote cell apoptosis and have less ox-LDL phagocytosis than those on 30 kPa substrates (126). Moreover, TRPV4 calcium-permeable channels that respond to matrix stiffness promote the transmembrane transport of Ca²⁺ and regulate the endocytosis of ox-LDL although CD36 (127). They also found that TRPV4, known as a mechano-sensor, plays an important role in regulating macrophage foam cell formation (127). Furthermore, Cav1 as the important part of caveolae will stimulate by soft substrates to modulate YAP activity through controlling actin polymerization and mediate caveolae internalization (128). These mechanotransducting property of Cav1 is also associated with cell stiffness and caveolae-based endocytosis. Le et al. discovered that disturber flow will increase the elastic modulus of ECs by increasing the expression of Cav1, which is positive correlation with the uptake of oxLDL (129). Recent studies have also revealed that the membrane tension force is important for Cav1-based vesicular trafficking, which is regulated by the stiffness of the extracellular matrix and depends on the mechano-transduction of the integrin/RhoA axis to stimulate a Cav1-dependent manner (130). These studies suggest that matrix and cell stiffness may regulate the transportation of lipids and proteins, which may contribute to the progression of atherosclerosis (Table 3).

Nanomedicine have widely applied in detecting and treating atherosclerosis with designing multiple responded or targeting molecule. Whereas, there are few researches focusing on the transporting route of nanoparticles into cells during treatment. Researchers have considered that nanoparticles firstly will closely contact with targeted cells and induce cell membrane to generate forces. Then, cell membrane will further encapsule nanoparticles through endocytic route and internalize nanoparticles (131). Thus, according to the endocytic route, we can design engineered nanoparticles to target diseased cells or even loading nano-drugs into living cells to assist drug delivery (132). Hu et al. design a tetrapod needle-like PdH nanozyme that can be internalized and storage into macrophages. By using the inflammatory response property, macrophages as the vesicles can deliver the nanozyme to atherosclerotic area and inhibit ROS (133). Furthermore, the biomechanics also can influence the endocytic process. Qin et al. have found that disturbed flow in artery will accelerate the internalized process of nano-sized extracellular vesicles through activating ROS in ECs (80). Due to the important role in avoiding the phagocytosis from immunity, researchers have used macrophages-membrane to encapsule nanoparticles and can efficiently and safely inhibit the progression of atherosclerosis (134). Thus it is important to develop nanomedicine based on the mechanism of intracellular endocytosis.

The mechano-environments of atherosclerotic plaques are complex and involve multiple factors for vascular homeostasis that control cell transcription and biological function. Endocytosis is a fundamental process in which vascular cells internalize nutrients, lipids and other molecules. However, the unexpected endocytosis of lipids and inflammatory molecules accelerates atherosclerosis via lipid accumulation and the formation of foam cells (56, 135). It is urgent to fully elucidate the interactions between biomechanical force and vascular endocytosis. Recent studies of the single-cell transcriptome and functional screening of membrane receptors have already revealed the role of biomechanics in vascular cell endocytosis in atherosclerosis. They presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and gene expression signatures. Long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Cav1-positive early endosomes, highlighting Cav-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress (85, 136). However, a challenge remains regarding how the signal transduction of biomechanical force to endocytosis in atherosclerosis is regulated via different pathways, responsive molecules and endocytosis-related proteins. Nevertheless, some researchers have explored whether biomechanical force regulates multiple pathways that are associated with mechanosensitive transcription factors that typically participate in the transcription of lipoprotein-transported proteins (137, 138). Moreover, cells are prefer to engulfing stiff nanoparticles which can easily achieve full wrapping (131). The size and stiffness of LDL also will decrease in acidic condition or oxidation which suggests cells may hard to engulf LDL particles (139, 140). Therefore, it is important to identify novel mechanosensitive pathways and dynamic processes of endocytosis in arteries.

The material transported by endocytosis is also essential for nanomedicine, which provides a targeted route for nanoparticles to enter and deliver cargo in cells. Due to the intracellular delivery of nanoparticles relying on vascular physiology, microenvironment and cell phenotype, it is important to select and design suitable nanomaterials to deliver drugs. Several studies of nanomedicine have found that the accumulation of nanoparticles in cells is influenced by the biomechanical environment in the vasculature. The engulfment of nanoparticles is associated with the shear stress of the vessels, and physiological changes in ECs under disturbed flow increase the accumulation of nanoparticles (141). Meanwhile, disturbed flow inducing oxidative stress can also promote the uptake of nanoscale materials (80). This flow-dependent accumulation is important for drug delivery during atherosclerosis, which is usually present at the vascular branch and downstream of curved regions. Researchers have also found that physiological cyclic stretch promotes the internalization of silica nanoparticles, which is related to cell stress and exocytotic events (142). Moreover, the changes in plasma membrane morphology by cyclic stretch will affect the distribution of actin and reduce the internalization of nanoparticles in VSMCs (143). Thus, understanding the role of nanoscale materials in endocytosis is important for investigating intracellular delivery and providing more targeted points for nanomedicine.

Overall, biomechanical force is an essential feature to regulate endocytosis that accommodates molecule and macromolecule trafficking and lipid metabolism in atherosclerosis. Endocytosis is not only important for nutrient uptake but also a primary route by which lipid particles enter cells. Here, we summarized the main endocytic process in atherosclerosis and described the interaction role of biomechanics and endocytosis in atherosclerosis. A consistent conclusion from these studies is that the changing biomechanics of the vasculature will result in disordered endocytosis, which is typically associated with lipoprotein transportation. Therefore, further analysis of pathophysiological endocytosis under biomechanical force will improve our understanding of the development of atherosclerosis and lead to the discovery of new therapeutic drugs and targets in atherosclerosis.