Atherosclerosis refers to the accumulation of cholesterol, and fatty, fibrous, and inflammatory substances in the arteries intima. The term atherosclerosis comes from the Greek word for “gruel” or “porridge,” reflecting the appearance and shape of the lipid material core of the typical atherosclerotic lesion (1). Approximately one-third of global deaths are attributed to atherosclerotic cardiovascular diseases (CVDs) (2). Atherosclerotic arteries lead to acute coronary syndromes, ischemic strokes, aneurysms, intermittent claudication, ulceration, and gangrene (1). Atherosclerosis is characterized by the development of lesions in the wall of the artery, and a disorder in any of the arteries is likely to be a starting factor of atherogenesis. As a main barrier of the artery, the endothelium plays a vital role in the development of atherosclerosis (3, 4). Small GTPases activated by Rho guanine nucleotide exchange factors (GEFs) partly or completely regulate endothelial cell migration and proliferation and adherens junctions of impermeable surfaces. In addition to the endothelium, inflammatory, or anti-inflammatory macrophages controlled by Rho GEFs, the small GTPase RhoA and its downstream effector, and Rho-associated coiled-coil containing kinases (ROCK) are also critical components for the development, regression, or stabilization of the atherosclerotic plaque (5). The mechanism of atherosclerosis is not fully elucidated. It is well-known that the Rho GTPase family, especially Rho A, has a vital function in the development and progression of arteriosclerosis. Rho GEFs, the upstream activators of Rho GTPase, are also involved in the atheromatous pathological process. In this mini review, we will briefly discuss the role of Rho GEFs and their potential target in atherosclerosis.

During 2009 to 2019, CVD was the number one cause of death (222.58 deaths per 1,00,000 in 2009; 239.9 death per 1,00,000 in 2019) (6). Atherosclerosis is the leading cause of CVD worldwide (1). Globally, more than 75% of CVD deaths occur in low- and middle-income countries (7). CVDs cause 10% of disability-adjusted life years lost in low- and middle-income countries and 18% of disability-adjusted life years lost in high-income countries (8). These statistics indicate that atherosclerosis places a heavy economic burden on developing countries. Although the specific mechanism of atherosclerosis remains unknown, a consensus is that multiple factors bring about atherosclerosis (9). These factors can be divided into genetic and acquired, and they work together to drive the development and progression of atherosclerosis. From another perspective and according to the progress in research, atherosclerosis is also considered a chronic disease (10). Inflammation and lipoprotein metabolism remain the focus of atherosclerosis research (11).

Here, we provide a brief introduction to the etiology of atherosclerosis. The artery wall provides the lining and multiple factors that conspire to produce atherosclerosis (Figure 1). In addition to traditional risk factors, novel risk factors (e.g., exposure to air pollution and noise, sleep deprivation, psychosocial stress, and intestinal microbiome) could be acquired to promote and affect atherosclerosis at different stages (12). Convincing evidence from genetic, epidemiologic, and clinical studies suggests that low-density lipoprotein cholesterol (LDL-C) causes atherosclerosis (13). LDL-C is a crucial factor in the initial stage of atherosclerosis. In this stage, LDL-C fluxes into the arterial wall and is kept within the intimal layer to provoke atherosclerosis. The transendothelial movement of LDL impairs the arterial wall and barrier function of the endothelium (14). LDL particles gradually gather in the subendothelial space and attach to intimal proteoglycans. The smooth muscle cells and macrophages become engulfed with lipid and contribute to foam cell formation and lesion development (15). In the progression stage, smooth muscle cells that migrate from the media into the intima as well as monocyte-derived macrophages accumulate under the artery lining, and foam cell numbers increase as the atherosclerotic plaque grows (16, 17). Macrophages and smooth muscle cells activate programmed cell death, which accumulates in the lipid-enriched nidus or necrotic core (18). During the development of atherosclerosis, calcification lesions form and accumulate in many arterial regions (19). The mechanism of Rho GEF involvement in atherosclerosis will be described in section The Emerging Role and Interaction Mechanisms of Rho GEF in Atherosclerosis.

The current research shows that the human genome of the Rho GEF family encodes 20 GTPase and 82 RhoGEFs (20). There are two groups of Rho GEFs, dedicator of cytokinesis (DOCK)-related proteins and diffuse B-cell lymphoma (Dbl)-like families (21). In humans, Dbl-like families are the most prominent Rho family GEFs, which comprise 71 members and can be structured in 20 subfamilies (22). Sharing a 170–190 amino acid Dbl homology (DH) domain is the common characteristic of Dbl-like Rho GEFs. Most Dbl-like Rho GEFs have a conserved core that is composed of DOCK-homology region (DHR)-1/C2 and DOCK homology region 2 (DHR2) domains, either single or connected with single SH3 or pleckstrin homology (PH) domains (21). Other than the DH domain, Dbl-like Rho GEFs contain domains that mediate interaction with membranes, proteins or phosphorylated amino acids, PH domains, or diverse enzymatic activities such as kinases, phosphatases, GEFs, or GAP. All known DH domains of Rho GEFs have related C-terminal PH domains involved in targeting and regulatory functions. The active site of DH domains is located near the junction between the DH domain and the PH domain (23). The DH and PH domains work in tandem, which allows the catalysis of Rho proteins (24). DOCK proteins are unrelated to the Dbl family in structure and mechanical properties, affecting Cdc42 and Rac, but not Rho A. The characteristics of DOCK proteins are a conserved catalytic domain (DHR2) and a phospholipid-binding domain (DHR1), which allow the GEFs to target the membrane (25).

Rho GEFs activate Rho GTPases by catalyzing the exchange of GDP for GTP (26). RhoGEFs, RhoGAPs, and RhoGDIs work together to regulate Rho GTPases. During this course, GTP-bound active conformation and GDP-bound inactive conformation of Rho GTP form a cycle that coordinates the activity of Rho GTP to activate effector proteins and elicit a cellular response (27). In response to extracellular stimuli, many Rho GEFs interact with specific proteins that Rho GTPases target and coordinate the Rho GTPase signaling network at specific sites in cells (28). The Rho GTPase regulation and Rho GTPase function in different vascular cells are illustrated in Figure 2 (29–33). A noted example is a complex comprising the Rho GEF β-PIX (RAC/CDC42-specific) with the RAC/CDC42 acting on PAK, which conducts turnover of integrin-containing focal adhesions (28).

Another example is the P-REX1, which brings RAC1 together with its effector FLI1 to promote cell migration (34). Information regarding this aspect, however, is scarce. Rho GEF proteins make up a complex interactive network that accurately activates Rho GTPases in translational modifications (35). In the status of the active conformation of Rho GTPases, the Rho GTPases interact with a variety of effector proteins, including kinases, adaptor proteins, and actin regulators, thereby activating cellular responses and leading to changes at the cellular level, which depend on the stimulus effectiveness and cell type (35).

A growing body of evidence suggests that the over-activation of Rho proteins is the shared pathogenesis of several cardiovascular disorders such as atherosclerosis, hypertension, and diabetes (36). Rho GEFs can activate Rho proteins by catalyzing the exchange of GDP for GTP and regulating Rho protein activity (36). In addition, some Rho GEFs have been identified as susceptibility genes for CVDs. Using next generation sequencing (NGS), bioinformatics, and ARHGEF17-deficient zebrafish, our research group found that ARHGEF17 is a candidate gene for intracranial aneurysm (IA) (37). In the aforementioned study, rs2298808 of ARHGEF17 was shown to be related to IA in the Chinese cohort, but arhgef17 knockdown of zebrafish caused bleeding and endothelial lesion in the brain region. Some Rho GEFs are more expressed in rat aorta and mesenteric arteries than in other arteries, such as PDZ-Rho GEF at mRNA and protein levels (38, 39). Vascular smooth muscle cells (VSMCs) and vascular endothelial cells are vital components of the artery, and regulation of the functions of these two types of cells by GEFs may be a participant in artery function. Angiotensin II (Ang II) regulates VSMC function by inducing tyrosine phosphorylation of Vav. PSD95-discs large-ZO1 (PDZ)-Rho GEF has been shown to be tyrosine-phosphorylated by temporary stimulation with Ang II (40). However, whether Rho GEFs are involved in the Ang II-regulated regulation of VSMCs remains unknown. Tumor endothelial marker-4 (TEM4) can also be called ARHGEF17 or p164-RhoGEF, and it regulates the integrity of the intercellular junctions and endothelial cell function (41). Rho GEF TEM4, which supports the persistence of cell migration by adjusting actin fibers and cell adhesions in protruding membranes, regulates the migration of endothelial cells.

Without a doubt, Rho proteins have a significant effect on the cardiovascular system (83, 84). Rho GEFs, as activators and the most direct upstream molecules of Rho proteins, are expressed in cardiovascular cells and are suitable candidate targets for the drug therapy of atherosclerotic disorders (Figure 3).

Some drugs targeting Rho proteins require complex approaches or processes to treat atherosclerosis, such as statins, which require multiple steps to lower LDL (82). In this process, the more steps and multiple effects that are involved can cause more side effects. Currently, although statins have a very positive effect, various statin-associated symptoms, including statin-associated muscle symptoms, diabetes mellitus, and central nervous system complaints, have been reported (85). Statins competitively and reversibly inhibit HMG-CoA reductase by their lactone ring and side chains that help them bind to the enzyme's active site (86), which contributes to the inhibition of cholesterol synthesis and brings about decreasing cholesterol production and upregulating LDL receptor (87). Statins can reduce the ROCK activity of white cells independent of LDL reduction (88). In human aortic endothelial cells, statins delay tissue factor induction by thrombin in a Rho/ROCK-dependent manner (89). Regulating the Rho/ROCK pathway with Rho GEFs could achieve the same therapeutic effect as statins while avoiding its many side effects. Hence, the authors suggest the regulated target of Rho proteins by Rho GEFs as a potential therapeutic target in atherosclerosis disease.

Compared with the current drugs targeting Rho proteins, the use of Rho GEFs to directly regulate the Rho proteins will be more effective and have fewer side effects. This progress in cardiovascular medicine is a sterling example of how the clinical application of scientific discoveries benefits patients. The findings of current research open a door of understanding into the development of atherosclerosis. The discovery of prosaposin as a potential therapeutic target may lead to the development of new therapeutics, which is precisely what precision medicine advocates (90). Of course, more research on Rho GEF regulation of atherosclerosis is needed.

DL, XY, and ZW designed and conceptualized the article. MLi and WX collected material and prepared the manuscript, figures, and tables. All authors significantly contributed to the writing of the manuscript and provided important intellectual content.

This study was supported by (1) the Youth Project of National Natural Science Foundation of China (No. 82000477); (2) Tianjin Key Research and Development Plan, Key Project of Science and Technology Support (No. 20YFZCSY00010); (3) Scientific Research Program Project of Tianjin Education Commission (No. 2018ZD03); and (4) the Natural Science Foundation of Tianjin, China (Grant No. 20JCZDJC00300).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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