Endothelial cells (ECs) form the inner lining of blood vessels that play an important role in maintaining vascular integrity and homeostasis. They provide a physical barrier between the blood and vessel walls or tissues, which is essential in regulating homeostasis and coordinating responses to blood vessel injury and inflammation. As ECs are constantly exposed to mechanical, chemical, and biological stresses, their plasma membrane is frequently disrupted and repaired. Failure to repair the disrupted membrane may cause endothelial cell death, which impairs the barrier function of blood–tissues or vessel walls, leading to vasculopathy, acute organ injury, and the initiation of atherosclerosis ^[1]. The capacity and mechanisms of cells to repair wounded plasma membranes have been studied in a number of cell types, especially skeletal muscle and cardiomyocytes [2,3,4,5]^[2][3][4][5]. Although the exact mechanisms underlying the membrane repair or resealing in these cells are still not fully understood, shreds of evidence available so far indicate that plasma membrane resealing is an autonomous and intrinsic process. In addition, although frequent disruption and resealing are commonly observed in mechanically exposed tissues, including skeletal muscle and the endothelium, the study of endothelial cell membrane repair is limited. 

2. Endothelial Cell Membrane Damage

The ECs in vivo are constantly exposed to mechanical, chemical, and biological stresses generated by hemodynamic forces, toxic environmental substances, and metabolites and pathogen-derived pore-forming toxins under physiological and pathological conditions. Those extracellular and intercellular factors can constantly damage endothelial cell membrane. Earlier studies found that on average, 6.5% of the endothelial cells of a rat aorta show transient and resealable plasma membrane disruption ^[1]. Interestingly, these wounded endothelial cells were particularly abundant around vascular bifurcation characterized by turbulent and disturbed flow, which is the area prone to atherosclerosis. These results suggest that the disruption and resealing of the endothelial plasma membrane may frequently happen, and failure to repair the wounded cells may be an important mechanism leading to the formation of early atherosclerotic lesions ^[1].

2.1. Mechanical Stresses

ECs are constantly subjected to a great amount of dynamic changed mechanical forces, including shear stress and mechanical stretch, under physiological or pathological conditions. Those mechanical stresses can directly damage endothelial cell membranes and induce cell death. For instance, under hypertension, pressure-induced stretching of the vascular wall may cause EC membrane damage, similar to exercise-induced skeletal muscle membrane damage. Other mechanical forces may also cause EC membrane damage. For example, the EC plasma membrane may be damaged by interaction with flashed blood cells or inner substances (protein aggregates or inhaled nanoparticles) [28]^[6].

2.2. Chemical Stresses

Oxidation of low-density lipoprotein (oxLDL) is believed to play a key role in the pathogenesis of atherosclerosis. It was demonstrated that a high dose of oxLDL can directly damage the cell membrane and cause cell death [29]^[7]. Oxidative stress and intracellular iron promote lipid peroxidation of poly-unsaturated fatty acids, leading to the removal of damaged fragments and destabilization of the plasma membrane [30]^[8]. In addition, lipid peroxidation can be induced by reactive oxygen species (ROS). During hypoxia or ischemia/reperfusion (I/R) injury, an inadequate blood supply leads to mitochondrial dysfunction and the generation of ROS, which is further exacerbated upon re-oxygenation [28]^[6]. In addition, membrane lipids are subject to enzymatic damage by host or foreign phospholipases. Alterations in membrane fluidity through interactions with amphipathic molecules or cholesterol extraction can make membranes easy to injure [28]^[6].

2.3. Biological Stresses

Pathogen infection can induce plasma membrane damage by several mechanisms. For example, some bacteria can breach the plasma membrane and enter the cells. To breach the plasma membrane, the bacteria can secrete virulence factors, such as secreted sphingomyelinases and phospholipases [31]^[9]. In addition, the membrane damage can also be achieved by pore-forming toxins, the largest class of bacterial toxins [32]^[10]. Although distinct from bacterial pore-forming toxins, viruses similarly compromise host membrane integrity through transmembrane channels termed viroporins [33]^[11]. There is also a large group of endogenous pore-forming proteins that can cause cell membrane damage. Perforin and complement factors are a major weapon of immune killing by pore-forming activities [34]^[12]. Other pore-forming proteins, such as gasdermins and MLKL (mixed-lineage kinase domain-like pseudokinase), which are end products of pyroptosis and necroptosis pathways, respectively, cause cell lysis by forming pores at the cell membrane [35,36]^[13][14].

3. Endothelial Cell Membrane Repair

Despite the fundamental importance of membrane repair for cell survival and tissue integrity, studies on endothelial cell membrane repair are limited. Current studies suggest that endothelial cell membrane repair may share some general mechanisms with other cell types, such as skeletal muscle, but also have specific components to fulfill the specific situations that endothelial cells encounter.

3.1. Dysferlin and Myoferlin

All known mammalian ferlin gene products (dysferlin, myoferlin, and otoferlin) are essential for the trafficking of intracellular vesicles and may be involved in cell membrane repair. Both dysferlin and myoferlin are abundantly expressed in skeletal muscles and are important components of repairing machinery for muscles [37]^[15]. Loss of dysferlin or myoferlin activity causes limb–girdle muscular dystrophy through impaired skeletal muscle repair after physical injury [38,39]^[16][17]. Otoferlin is a specific member strongly expressed in the auditory inner hair cells that are responsible for transmitting auditory information to the central nervous system. Mutations of otoferlin cause autosomal recessive deafness in humans, and genetic inactivation of the otoferlin gene impairs Ca²⁺-dependent exocytosis in mouse auditory inner hair cells and causes profound deafness [40]^[18]. Proteomic profiling showed that both dysferlin and myoferlin are localized at the endothelial membrane and lipid rafts [41]^[19]. Western blot confirmed their expression in human primary endothelial cells. Moreover, recent studies show that dysferlin may participate in endothelial cell membrane repair by mediating lysosome fusion with the endothelial cell membrane [42]^[20], and myoferlin is required for efficient endothelial membrane repair after mechanical injury [43]^[21]. Nevertheless, the importance and mechanisms of dysferlin and myoferlin in endothelial cell membrane repair in vitro and in vivo need to be further determined.

3.2. Annexins and S100A10/11

Annexins constitute a multigene family of Ca²⁺-dependent phospholipid-binding proteins, with members of the family being expressed in animals, plants, fungi, and protists. This family can be divided into five classes (A–E) based on their biological origins. In vertebrates, class A is composed of 12 members, AnxA1–AnxA13 (Anx12 is currently unassigned). Annexins have two structural domains: a conserved C-terminal domain with 4 repeats of 70–80 amino acids (8 in the case of AnxA6) and a variable N-terminal domain. Each repeat contains a Ca²⁺ binding motif, allowing the annexins to rapidly translocate to the plasma membrane or intracellular membranes by binding to negatively charged phospholipids [44]^[22]. The functions of annexins include Ca²⁺-regulated endocytic and exocytic events, the maintenance and regulation of membrane–cytoskeleton contacts, and membrane domain organization [45]^[23]. AnxA2 is the most abundant member in endothelial cells [46]^[24]. A1 and A5 are also expressed in endothelial cells. Recent studies show that AnxA1, A2, A5, and A6 all rapidly move to the wounded sites after injury in endothelial cells. They can form a protein complex with S100A11, which is an EF hand-type Ca²⁺-binding protein. The knocking down of AnxA2 or S100A11 proved that both of them are required for efficient repair of endothelial cell membrane wounds. Further studies show that the S100A11 C-terminal extension (residues 93–105) can interact with AnxA1 and AnxA2 in a Ca²⁺-dependent manner. In addition, synaptotagmin-1 also binds to S100A11 in the presence of Ca²⁺, and depletion of synaptotagmin-1 interferes with wound site recruitment of S100A11 and proper membrane resealing in endothelial cells [47]^[25].

3.3. Caveolins

Caveolins are major components associated with caveolar lipid rafts in the plasma membrane and are implicated in many aspects of cellular functions. There are three members of caveolins: caveolin-1, caveolin-2, and caveolin-3. Caveolin-1 is the primary member of caveolins and is ubiquitously expressed in all tissues, including endothelial cells. Caveolin-3 (Cav3) is strictly expressed in skeletal muscles. Mutation of Caveolin-3 is linked to muscular dystrophy in patients [28]^[6]. Cav3 is essential for the repair of muscle membrane damage. Cav3 can form a protein complex with MG53 and dysferlin. Cav3 mutants that cause retention of Cav3 in the Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair [48]^[26]. These results suggest that Cav3 at the membrane is required for correct localization and retention of MG53 and dysferlin on muscle membranes. While Cav3 is specifically expressed in muscles, Cav1 is expressed in many tissues, including endothelial cells. Cav1 forms a protein complex with myoferlin in endothelial cells. Both Cav1 and myoferlin are critical for efficient endothelial membrane repair after injury [43]^[21].

3.4. Weibel–Palade Bodies

Weibel–Palade bodies (WPB) are unique secretory organelles in endothelial cells that undergo evoked exocytosis following intracellular Ca²⁺ or cAMP elevation, thereby supplying the vasculature with factors controlling hemostasis. The major components stored within WPB are the multimeric glycoprotein von Willebrand factor (VWF) and the adhesion receptor P-selectin. WPBs move to the endothelial cell membrane when there is a blood vessel injury and fuse with the plasma membrane. VWF is released into the lumen of the blood vessel and provides platelet adhesion sites. Low levels of VWF can lead to a bleeding tendency, such as in von Willebrand disease, and elevated levels of VWF are associated with an increased risk for thrombosis and cardiovascular disease [49]^[27]. A recent study observed that complement-mediated endothelial membrane injury can be mitigated by the mobilization of WPBs along with the secretion of VWF. Endothelial cells lacking WPBs were not resistant to complement-mediated damage but became resistant when transfected to express VWF. The study suggests that WPB exocytosis in response to endothelial plasma membrane damage may be an essential mechanism for endothelial cell membrane repair [50]^[28]. Interestingly, several other studies show that annexin A2 and S100A10 complex is required for WPB exocytosis [51,52]^[29][30]. In a recent study, the Annexin A2/S100A10 was confirmed to be required for efficient endothelial cell membrane repair after mechanical injury [46]^[24]. How WPB repairs the endothelial cell membrane still needs further study.

3.5. Other Proteins

Theoretically, any proteins that regulate processes such as cytoskeleton, exocytosis, and endocytosis may affect cell membrane repair. For example, Rab3a, Rab10, myosin 11A, 11B, SNARE, and ESCRT III (endosomal sorting complexes required for transport III) are reported to be involved in the regulation of cell membrane repair [53,54,55,56]^{[31][32][33][34]}. However, whether they are also involved in endothelial cell membrane repair needs to be determined. Anoctamin 5 (ANO5, also known as TMEM16E) is a member of the transmembrane 16 (TMEM16) family of Ca²⁺-activated ion channels and phospholipid scramblases. Human mutations of ANO5 cause adult-onset limb–girdle muscular dystrophy 2L (LGMD2L) and Miyoshi muscular dystrophy (MMD3). In mice, ANO5 is implicated in muscle differentiation and membrane repair [57]^[35]. ANO5 is primarily expressed in muscle and bone. It is also expressed in endothelial cells; however, its role in endothelial cells remains unclear. Endophylin-A2, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ~40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin 2, a ~100 kDa GTPase that is essential for endocytic membrane scission. Both endophilin-A2 and dynamin 2 are involved in the regulation of cell membrane repair [58]^[36]; however, whether those proteins are also involved in endothelial cell membrane repair remains to be studied. MG53 (also known as TRIM72) is a protein member of the TRIM family that acts as a key component of cell membrane repair machinery in muscles [14]^[37]; however, MG53 is not expressed in human endothelial cells. The role of MG53 may be fulfilled by another TRIM protein or non-TRIM protein in endothelial cells, but this needs to be determined.