GLP-1 receptor agonists (GLP-1RAs): cardiovascular actions and therapeutic potential

Type 2 diabetes mellitus (T2DM) is closely associated with cardiovascular diseases (CVD), including atherosclerosis, hypertension and heart failure. Some anti-diabetic medications are linked with an increased risk of weight gain or hypoglycemia which may reduce the efficacy of the intended anti-hyperglycemic effects of these therapies. The recently developed receptor agonists for glucagon-like peptide-1 (GLP-1RAs), stimulate insulin secretion and reduce glycated hemoglobin levels without having side effects such as weight gain and hypoglycemia. In addition, GLP1-RAs demonstrate numerous cardiovascular protective effects in subjects with or without diabetes. There have been several cardiovascular outcomes trials (CVOTs) involving GLP-1RAs, which have supported the overall cardiovascular benefits of these drugs. GLP1-RAs lower plasma lipid levels and lower blood pressure (BP), both of which contribute to a reduction of atherosclerosis and reduced CVD. GLP-1R is expressed in multiple cardiovascular cell types such as monocyte/macrophages, smooth muscle cells, endothelial cells, and cardiomyocytes. Recent studies have indicated that the protective properties against endothelial dysfunction, anti-inflammatory effects on macrophages and the anti-proliferative action on smooth muscle cells may contribute to atheroprotection through GLP-1R signaling. In the present review, we describe the cardiovascular effects and underlying molecular mechanisms of action of GLP-1RAs in CVOTs, animal models and cultured cells, and address how these findings have transformed our understanding of the pharmacotherapy of T2DM and the prevention of CVD.


Introduction
Over 90% of diabetes is classified into two main types being Type 1 diabetes mellitus (T1DM) and T2DM. T1DM is defined as an autoimmune disease, resulting in insulin deficiency. T2DM is a metabolic disease caused by acquired endocrine disorders. T2DM is the underlying cause of many complications, among which, cardiovascular diseases (CVDs) are mainly responsible for the high rates of morbidity and mortality. Atherosclerotic CVD manifest as heart attacks and strokes, is responsible for up to 85 per cent of cardiovascular deaths [1]. Atherosclerosis emerges as the "invisible killer" lurking in the cardiovascular system. With the gradual progression of atherosclerotic plaques, atherosclerosis will induce angina pectoris, or cerebral ischemia, leading to severe clinical manifestations such as heart failure, myocardial infarction (MI), cerebral infarction and lower limb ischemia and amputation [2].
Multi-country, multi-center long-term cardiovascular outcome trials (CVOTs) confirmed that GLP-1RAs decrease cardiovascular mortality and provide cardiovascular benefits to reduce the Ivyspring International Publisher incidence of MI or non-fatal stroke [3]. For example, in the LEADER trial, when compared with placebo, liraglutide decreased the incidence of major cardiovascular events (MACE) by 13%, cardiovascular death by 22% and all-cause death by 15% among T2DM patients with CVD, on a standard treatment basis [4]. In addition, liraglutide provided multiple additional benefits by reducing the incidence of hypoglycemia, lowering HbA1c and functioning through multiple mechanisms to better control multiple causes of MACE, such as blood lipids, BP as well as body weight (BW).
GLP-1 is a gut-derived insulin agonist with the ability to suppress glucagon release and stimulate insulin secretion through targeting α-cells and β-cells, respectively [5]. Likewise, GLP-1RAs can lower postprandial glycemia predominantly by slowing gastric emptying and promoting weight loss [6,7]. GLP-1RAs may improve endothelial cell function via anti-inflammatory and vasodilatory properties. Also, anti-proliferative effects on smooth muscle cells and anti-inflammatory effects on macrophages may contribute to the protection against the development and progression of atherosclerosis [8][9][10]. In consideration of the dual roles of GLP-1RAs in hypoglycemic effects and preventing CVD among T2DM patients, we reviewed the cardiovascular effects and molecular mechanisms of GLP-1RAs in CVOTs, animal models and cultured cells, and discuss how these findings transform pharmacotherapies for T2DM, and even expand the indication of this important category of anti-diabetic drugs to CVD patients without diabetes.

Clinical GLP-1RAs and their pharmacological basis of action
GLP-1RAs, consist of short-acting and long-acting agents. Short-acting drugs include exenatide twice daily, lixisenatide once daily and oral semaglutide once daily, while long-acting drugs include liraglutide, semaglutide, exenatide, albiglutide and dulaglutide all being administered once weekly. The primary pharmacodynamic distinctions between long-acting and short-acting GLP-1RAs are that long-acting agonists augment insulin production and suppress glucagon generation to lower postprandial glucose and fasting plasma glucose, while short-acting agonists chiefly lower postprandial glucose by delaying gastric emptying. Long-acting GLP-1RAs also have other benefits including improved gastrointestinal tolerability, moderate plasma drug concentration fluctuations, and more convenient dosage regimens which may improve medication adherence and treatment outcomes [11] (Table 1). Exendin-4 was originally found in the saliva of the venomous lizards whose synthetic version is exenatide. Exendin-4 inhibits the remodeling in the remote myocardium of rats following acute MI by attenuating β-catenin activation and activating glycogen synthase kinase-3, β-arrestin-2 and protein phosphatase 2A [12]. In addition, exendin-4 protects against cardiac ischemia/reperfusion injury in rats by enhancing antioxidant levels and inhibiting c-Jun NH2-terminal kinase (JNK)/p 66 Shc/NADPH oxidase axis [13]. GLP-1RAs, such as lixisenatide, not only rely on β-cell function and glucagon suppression, but also assist to lower glucose by other (insulin-independent) mechanisms such as delayed gastric emptying, underlying the clinical utility of GLP-1RAs as adjuvant therapy to basal insulin in longstanding T2DM [14]. Further, liraglutide protects against vascular oxidative stress via attenuating eNOS (endothelial NO synthase) s-glutathionylation (a hallmark of eNOS uncoupling) and increasing NO bioavailability [15].
The GLP-1 analogs, semaglutide and liraglutide, delay the progression of atherosclerosis by regulating inflammatory pathways in low-density lipoprotein receptor-deficient (LDLr -/-) and apolipoprotein E-deficient (ApoE -/-) mice [16]. Oral semaglutide reduces systolic blood pressure (SBP), BW and blood glucose levels. Nevertheless, it is associated with increased incidence of gastrointestinal adverse reactions [17]. Exenatide ameliorates intramyocellular lipid deposition through improving insulin sensitivity and activating the AMP-activated protein kinase (AMPK) signaling pathway without BW reduction [18]. Compared with twice-daily exenatide, dulaglutide treatment reduces glycated hemoglobin and fasting plasma glucose (FPG) to a greater extent [19]. Taspoglutide prevents apoptosis in vitro, promotes β-cell proliferation, and exerts multiple protective effects of β-cell in Zucker Diabetic Fatty rats [20]. Compared with both placebo and liraglutide at 26 weeks, orally bioavailable semaglutide is superior in reducing BW and non-inferior to liraglutide concerning for decreasing HbA1c [21].

Cardiovascular outcome trials of GLP-1RAs
In the past decade, CVOTs with GLP-1RAs have been conducted with at least seven agents including liraglutide, lixisenatide, exenatide, semaglutide, dulaglutide, albiglutide and oral semaglutide ( Table  2). In CVOTs, liraglutide, semaglutide and dulaglutide demonstrated appreciable cardiovascular and renal protective effects, among which liraglutide had an appreciable effect on mitigating the risk of cardiovascular death [4], while semaglutide and dulaglutide conferred a risk reduction on non-fatal stroke [22,23]. Moreover, liraglutide also had effects on reducing the incidence of MI in high-risk patients for T2DM, and may improve the clinical outcomes of MI [24].
To date, the REWIND study has demonstrated for the first time that primary cardiovascular prevention strategies can significantly reduce cardiovascular adverse events in patients with T2DM, regardless of whether or not these patients are co-diagnosed with CVD [23]. In this regard, dulaglutide is an ideal hypoglycemic drug for patients with T2DM due to its reported utility in primary prevention and secondary prevention. By February 21, 2020, dodecaplatin ® (dulaglutide) is the first and only FDA-approved anti-diabetic drug for primary and secondary prevention to alleviate the risk of MACE [25].

Hyperglycemia
All five long-acting GLP-1RAs can reduce glucose levels and BW [4,22,23,26,27]. In SUSTAIN 3, SUSTAIN 7 and SUSTAIN 10, the GLP-1RAs semaglutide reduced HbA1c by up to 1.8%, achieved the glycemic control target of HbA1c in 80% of the intervention group, and reduced BW by up to 6.5 kg, outcomes which were superior to liraglutide, exenatide and dulaglutide [28][29][30]. In the LEAD-5 trial, liraglutide (1.8 mg once daily) reduced HbA1c more than insulin glargine during the treatment period (26 weeks) [31]. The results of AWARD-CHN2 showed that the reduction of FPG by dulaglutide was similar to insulin glargine. In addition, post-prandial blood glucose reduction by dulaglutide was superior to insulin glargine. In addition to this, dulaglutide can greatly stabilize and decrease blood glucose levels [32].

Atherosclerosis and coronary artery disease
The levels of HbA1c, dyslipidemia, BW and BP are several of the risk factors for the progression of atherosclerosis and CVD in patients with T2DM. Several clinical trials have demonstrated that most of GLP-1RAs improve these parameters and thus attenuate the development and progression of atherosclerosis, especially concerning low-density lipoprotein cholesterol (LDL-C) levels [33]. In 2015, a randomized controlled trial showed that taspoglutide can reduce total cholesterol, LDL-C and triglycerides [34]. Among new-onset patients with diabetes receiving standard statin therapy, liraglutide together with metformin improved lipid distribution of LDL and C-reactive protein (CRP) in atherosclerosis [35]. Studies have also reported that under the conditions of comparable glycemic control, liraglutide (1.2 mg/d) alone was more effective than liraglutide with metformin or metformin alone on lipid metabolism and cardiovascular protection [36].

Hypertension
In the LEAD series of studies, clinical data demonstrated that treatment with liraglutide for 26 weeks can reduce SBP [4]. Exenatide improved the BP and blood lipid level of T2DM patients for 7 consecutive years in a DURATION open-extension study [37]. In the REWIND study and AWARD5 study, dulaglutide consistently improved BP and lipid levels in patients [23,38]. Mechanistically, GLP-1 can lower BP through a diuretic mechanism, and other effects on the kidneys [39,40]. At another level, GLP-1RAs also improve systemic insulin sensitivity to lower arterial hypertension in T2DM by decreasing the concentration of angiotensin II (Ang II) [41,42].

Chronic kidney disease (CKD)
CKD is clinically characterized by a decline in glomerular filtration rate (GFR) and proteinuria for diabetic nephropathy, which is determined by the pathological changes in the structure and function in the kidney of patients with T2DM. Several recent CVOTs have shown that GLP-1RAs can reduce the decline of estimated GFR (eGFR) and delay the onset of the occurrence of proteinuria in patients with T2DM [4,22,26,46,47].
In the ELIXA trial, lixisenatide ameliorated macroalbuminuria assessed as a decline in the urinary albumin-to-creatinine ratio. An analysis of the ELIXA trial data indicated that short-term GLP-1RAs may also have renal protective effects, analogous to long-acting GLP-1RAs [47]. Analysis of the LEADER trial data demonstrated that liraglutide exerted a 22% decline in the morbidity of newly diagnosed or aggravated nephropathy [4]. While in SUSTAIN-6, a sharper decline (46%) in macroalbuminuria seems to be entirely due to the semaglutide treatment than a 26% fall in macroalbuminuria by liraglutide [22]. The AWARD-7 trial, which was the first clinical trial among moderate-to-severe CKD and T2DM displayed convincing protective effects on eGFR after treatment with GLP-1RAs [46]. In addition, the analysis of the EXSCEL trial revealed that a composite end-point of new macroalbuminuria, 40% eGFR decline or renal death was reduced by the addition of exenatide in T2DM [26].
To summarize several meta-analyses of CVOTs conducted with GLP-1RAs, it isconfirmed that based on 43 [48]. Further, GLP-1RAs significantly reduce the risk of MI and stroke, but have a neutral effect on hospitalization for heart failure upon 5 available CVOTs [49]. Meta-analysis of 7 CVOT pooled demonstrated that GLP-1RAs were associated with a similar decrease in MACE regardless of gender [50]. From one hundred and thirteen available trials, the safety of GLP-1RAs was confirmed in pancreatitis. Conversely, treatment with GLP-1RAs can increase cholelithiasis mobility [51].

Effects of GLP-1RAs on atherosclerosis and atherosclerosis in the setting of diabetes
Atherosclerosis with multiple and diverse drivers is a dominant complicating process in T2DM [53]. Exposure to western diets pathologically alters the expression of many genes leading to atherosclerosis [16]. According to a recent study, GLP-1R is mainly expressed in the macrophage enrichment region of atherosclerotic plaques, which provide us a new perspective on pharmacological modification of GLP-1R upon signal in atherosclerosis [54].
Preclinical studies have shown that large doses of GLP-1RAs can reduce lipid deposition and plaque volume on the aortic surface of hyperglycemic mice via an AMPK-independent action in streptozotocin (STZ)-induced hyperglycemic and hyperlipidemic mice or type 1 diabetic rats [55]. GLP-1RAs have direct anti-atherosclerotic effects in euglycemic mice, ApoE -/mice or Watanabe heritable hyperlipidemic (WHHL) rabbits in a GLP-1R-dependent manner [56][57][58]. Therefore, these studies demonstrated that GLP-1RAs have an impact on preventing and stabilizing atherosclerotic vascular disease, which may translate into its protective effects against MACE in human subjects [59].
GLP-1RAs attenuate the activation and recruitment of macrophages into the endothelium [63]. Treatment with GLP-1RAs can reduce monocyte/macrophage accumulation in the arterial wall by inhibiting the adhesion of monocytes to activated endothelium from the arteries of C57BL/6 mice as well as in ApoE -/mice [64]. GLP-1RAs can also exert anti-inflammatory effects on macrophages as well as regulating macrophage phenotype [65]. For example, treatment of murine macrophages with GLP-1RAs in vitro inhibits monocyte chemoattractant protein-1 gene expression and p65 as well as tumor necrosis factor (TNF)-α expression. Moreover, GLP-1RAs can suppress atherosclerosis in T2DM (STZ treated) rats by inhibiting macrophage infiltration and apoptosis [66]. The above studies suggest that GLP-1RAs may be a valuable strategy for the treatment of atherosclerosis ( Figure 1) ( Table 3).

Effects of GLP-1RAs on hypertension
Hypertension and accompanying cardiovascular remodeling are amongst the leading drivers of atherosclerosis and causes of CVD. Endogenous GLP-1R signaling exerts a physiologically relevant effect on BP control, which may be attributable, in part, to its tonic actions on proximal tubule Na/H mediated sodium reabsorption, and the intrarenal renin-angiotensin II system [67]. In spontaneously hypertensive rats, GLP-1 treatment mediates renal vasodilation and attenuates renal autoregulatory responses [68]. In addition, acute renal infusion with GLP-1 can augment renal blood flow and promote natriuresis and dieresis [69]. In summary, GLP-1 or GLP-1RA treatment can make a positive impact on reducing the impact of hypertension in affected patients ( Figure 1).

Effects of GLP-1RAs on myocardial infarction (MI)
MI and its consequent tissue damage leading to heart failure are mainly responsible for the fatality in patients with T2DM. Evidence indicates that GLP-1RAs can reduce the occurrence of MI [70]. Among patients with acute MI undergoing percutaneous interventions, GLP-1 treatment can reduce infarct size, together with improving left ventricular ejection fraction (LVEF) [71]. GLP-1RA (such as exendin-4) treatment can reduce the size of MI impacted cardiac volume in rats, inhibit ventricular dilation, myocardial fibrosis and hypertrophy in vivo [72]. Mechanically, GLP-1RAs regulate insulin-like growth factor-1/2 and upregulate α-estrogen receptors to exert cardioprotective actions in isoprenaline-induced MI [73]. In addition, another report demonstrated the reparative role of GLP-1RAs in MI via modulating sirtulin (SIRT)1/Parkin/mitophagy [74]. Interestingly, even in obese swine, GLP-1R activation can improve cardiac function and augment cardiac output after MI [75]. Accordingly, it is suggested that GLP-1RAs have properties of alleviating MI and repairing damaged cardiac tissue to improve cardiac function ( Figure 1).

Effects of GLP-1RAs on heart failure
Myocardial remodeling has a critical role in heart failure following MI. Disturbances in cardiac calcium metabolism are a hallmark of heart failure. GLP-1RAs prevent post-MI remodeling by having an impact on the extracellular matrix changes rather than dependency on its glycemic control [76]. There is another mechanism showing that GLP-1RA treatment regulates Ca 2+ levels due to reductions in the ryanodine receptor (RyR)2 phosphorylation and suppression of the activation of calmodulindependent protein kinase (CaMK)-II [77]. Moreover, a favorable impact of GLP-1RAs on cardiac remodeling can occur through activation of the eNOS/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) nexus [72].
Cardiac hypertrophy leads to heart failure at the later decompensated stage of the disease process, which is a primary cause of morbidity and mortality. Evidence has recently revealed that GLP-1RAs inhibit cardiac hypertrophy by upregulating GLP-1R expression and activating the AMPK/mechanistic target of the rapamycin (mTOR) signaling pathway [78].
Mitochondrial dysfunction, apoptosis and oxidative stress are in part due to methylglyoxal accumulation. Therefore, the attenuation of methylglyoxal-induced mitochondrial abnormalities may suppress the progression of heart failure. In cultured H9c2 cells, GLP-1RAs attenuate the production of mitochondrial and intracellular reactive oxygen species (ROS) induced by methylglyoxal. As a result, GLP-1RAs display suppressive effects on mitochondrial functions and oxidative stress on heart failure [79].
Although GLP-1RAs can play a favorable role in improving heart failure based on pre-clinical studies, whereas some clinical evidence relevantly have reported that GLP-1RAs treatment could be associated with adverse heart failure events, eg. increasing heart rate, reducing LVEF [43][44][45]. Therefore, overall, the benefit of GLP-1RAs for heart failure has been difficult to confirm (Figure 1).

Effects of GLP-1RAs on cardiomyopathy
Diabetic cardiomyopathy is an important cardiac complication which is accompanied by cardiac hypertrophy [78]. Chronic inflammation results in the loss of cardiomyocytes in diabetic hearts. GLP-1RAs have the potential to inhibit cardiac inflammation via retarding the production of pyroptotic cytokines, caspase-1 and the AMPK-thioredoxin interacting protein (TXNIP) pathway in high-fat diet-fed rats [80]. Specifically, GLP-1RAs exert protective effects on exercise-induced cardiomyopathy via increasing autophagy, reducing inflammation-related proteins and ROS generation via restoring the expression of catalase and manganese superoxide dismutase (MnSOD) [81].
GLP-1RAs attenuate apoptosis in cardiomyocytes induced by hyperglycemia via inhibiting receptor of advanced glycation end products (RAGE) receptor [82]. In H9c2 cardiomyoblasts, GLP-1RAs protect against cell apoptosis and inhibit the expression of tumor protein P53 (p53). GLP-1RAs not only mediate the inhibition of myocardial apoptosis, but also improve cardiac energy metabolism. These studies illustrate that GLP-1RAs may confer protective effects on the progression of cardiomyopathy [83].
GLP-1RA treatment also mitigates experimental diabetic cardiomyopathy by the indirect mechanisms of augmenting glucose oxidation in cardiomyocytes [84]. In terms of mechanisms, GLP-1RAs can block C/EBP homologous protein (CHOP)-mediated endoplasmic reticulum (ER) stress via attenuating the irreversible electroporation (IRE)-α unfolded protein response (UPR) [85]. There is a further action that the reduction of HOX transcript antisense RNA (HOTAIR) expression gives rise to cardiomyopathy. As a result, GLP-1RAs demonstrate a new therapeutic action in cirrhotic rats with cardiomyopathy through the protective role of HOTAIR via SIRT1 activation [86].
Abnormal autophagy and mitochondrial injury are involved in diabetic cardiomyopathy. The cardioprotective effects of mesenchymal stem cells, GLP-1RAs or pioglitazone may improve mitochondrial function via regulating autophagy and inflammatory signaling [87]. Several studies revealed that GLP-1RAs relieve myocardial damage and glucose toxicity by promoting autophagy, which is associated with decreasing mTOR phosphorylation and increasing AMPK phosphorylation [88]. Furthermore, a new orally bioavailable GLP-1RA, oral hypoglycemic peptide 2 (OHP2), can confer protective effects on diabetic cardiomyopathy through rescuing cardiac lipotoxicity by inhibiting Rho (the small GTPase) kinase (ROCK)/PPARα pathway [89,90] ( Figure 1).

Molecular mechanisms of GLP-1RAmediated cardiovascular actions
Lowering blood glucose levels by enhancing insulin secretion T2DM is characterized by the presence of chronic fluctuating hyperglycemia, obesity-associated insulin resistance as well as the impaired secretion of insulin and dysfunction of the incretin system. Although endogenous GLP-1 lowers blood glucose levels, it is degraded quickly, which results in loss of its hypoglycemic effects. It follows that long-acting GLP-1RAs have a longer half-life and show prolongation of the glucose-lowering effects [3].
GLP-1RAs can improve β cell function in patients with early T2DM via coordinated regulation of the fate of α and β cells. There is evidence showing that GLP-1RAs can reduce the expression of β cell apoptotic markers Bcl-2-associated x (Bax) and caspase-3 while upregulating Bcl-2 [99,100], as well as protecting against lipotoxic stress via phosphoinositide 3-kinase (PI3K)/Akt/forkhead box proteins O1 (FoxO1) pathway [101]. GLP-1RAs increase the sensitivity of β cells to glucose and positively reduce insulin resistance to further promote insulin secretion [102]. GLP-1RAs stimulate insulin secretion and suppress glucagon secretion, which may modify cardiac energy metabolism in T2DM patients [103]. Based on the above mechanisms of β cells, GLP-1RA treatment exerts favorable effects on hyperglycemia ( Figure 2).

Reversing endothelial dysfunction by GLP-1RAs
The cardiovascular phenomenon of endothelial dysfunction involves a series of structural and functional abnormalities, including abnormal secretory capacity and properties of the endothelium and impaired vascular tone, as well as an impaired endothelial barrier function. GLP-1RAs stimulate NO production and eNOS activation, eliciting vasorelaxation in arterial endothelium in vitro [104]. GLP-1RAs make a direct protective impact on the endothelium by the GLP-1R-dependent AMPK/Akt/ eNOS pathway [105]. GLP-1RAs also promote endothelial barrier integrity via activating protein kinase A (PKA)-and Ras-related C3 botulinum toxin substrate 1 (Rac1) [106]. Moreover, GLP-1 exerts stabilizing effects on the endothelial contraction and barrier for AGE-treated endothelium via activating GLP-1R/cyclic adenosine monophosphate (cAMP)/ PKA and inhibiting mitogen-activated protein kinase (MAPK) signaling pathways together with RAGE/ Rho/ROCK [97,98]. Through the above mechanisms, GLP-1RAs can mediate the function of regulating endothelial-mediated contraction and vasodilation, as well as maintaining the stability of the endothelial barrier.
GLP-1RAs can improve the efficacy of protection against the adhesion of inflammatory cells to the endothelium. Specifically, GLP-1RAs downregulate activation of VCAM-1, the intracellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor type 1 (PAI-1) in endothelial cells [57]. Further, GLP-1RAs block the adhesion of monocytes to endothelium induced by oxidized LDL via extracellular signal-regulated kinase (ERK) 5-Kruppel-like factor (KLF)2 signaling pathway to prevent endothelial permeability [107,108]. Likewise, GLP-RAs inhibit platelet aggregation and prevent thrombosis [109]. These findings indicate that GLP-1RAs have anti-adhesive and anti-thrombotic properties in the endothelium that may delay the development of atherosclerosis [110].
GLP-1RAs play a key role in regulating the response to inflammation of the endothelium. Specifically, GLP-1RAs block NLRP3 (Nod-like receptor protein 3h) inflammasome via SIRT1 activation [111]. GLP-1RAs also suppress the nuclear factor-κB (NF-κB) pathway and free fatty acid-induced cellular ROS in endothelium [112]. GLP-1RAs also downregulate the expression of MMPs and tissue inhibitors of MPs (TIMPs) for anti-inflammation [110]. Furthermore, noted that GLP-1RAs can increase cholesterol excretion and reduce cholesterol accumulation by promoting the expression of ATP-binding cassette transporter A1 (ABCA1) in the endothelium. Therefore, treatment with GLP-1RAs shows favorable effects on anti-inflammation through the suppression of these proinflammatory cytokine and inflammatory signal pathways, as well as augmenting cholesterol efflux in the endothelium.
Under high glucose conditions, endothelial cells acquire the characteristics of fibroblasts, via endothelial-mesenchymal transition (EndMT). EndMT promotes diabetic cardiac fibrosis [117]. EndMT relies on activating the Smad2/3-Slug pathway at an early stage [118]. GLP-1 treatment protects against EndMT through inhibiting the activation of poly (ADP-ribose) polymerase 1 (PARP-1) which can elicit Smad3 and transforming growth factor (TGF)-β1 responses. Moreover, GLP-1RAs can reverse EndMT induced by high glucose and IL-1β via activating the AMPK pathway, which was evidenced by augmenting an endothelium marker expression (CD31), as well as alleviating mesenchymal markers expression (SM22α (sensitive 22 kDa actin-binding protein of the calponin), vimentin and Snail) [119]. These results indicate that GLP-1RAs have therapeutic effects against EndMT, an important process in atherosclerosis and fibrotic diseases.
To summarize these mechanisms and their corresponding efficacy with GLP-1RAs or GLP-1, GLP-1RAs show the endothelial protective properties and thus owe to modulating vascular tone and maintaining endothelial barrier integrity, as well as the anti-adhesive and anti-thrombotic actions. In addition, the inhibition of EndMT, the attenuation of inflammation and apoptosis are also responsible for reversing endothelial dysfunction with GLP-1RA treatment ( Figure 2).

Improving vascular smooth muscle cell (VSMC) dysfunction by GLP-1RAs
VSMC dysfunction can be defined as the occurrence of excessive proliferation, autophagy, fibrosis, senescence, and phenotypic transformation. Accordingly, VSMC dysfunction ultimately aggravates vascular injury and accelerates the development of atherosclerosis. GLP-1RAs suppress hyperglycemia-induced proliferation, migration and apoptosis of VSMCs via suppressing the ERK1/2 and PI3K/Akt pathways [10]. In terms of proliferation, liraglutide attenuates VSMC proliferation induced by Ang II through eliciting cell cycle arrest and activating AMPK [120]. Furthermore, the number of neuron-derived orphan receptor 1 (NOR1) which can modulate VSMC proliferation, is decreased by GLP-1RAs in a dose-dependent manner in mice [121].
VSMC senescence induced by Ang II is another important aspect of VSMC dysfunction. In this regard, GLP-1RAs can provide the protection against VSMC senescence via increasing the nuclear factor erythroid 2-related factor 2 (Nrf2) activity [122]. In addition, GLP-1RAs inhibit superoxide anion formation and ensuing VSMC senescence through suppressing Rac1 activation via cAMP/PKA pathways [123]. VSMC phenotypic switching is characterized by transformation from a quiescent contractile to an activated synthetic phenotype, promoting proliferation and leading to the formation of the neointima, the pathological vascular compartment critical for the development of atherosclerosis. In rat coronary artery smooth muscle cells, GLP-1RAs can inhibit phenotypic modulation induced by AGEs through blocking the NF-κB signaling pathway [124]. Further, GLP-1RAs promote VSMC redifferentiation via AMPK/SIRT1/forkhead box O (FOXO3a) pathways, where the expression of contractile VSMCs markers (SM22α and Calponin) is increased [125]. Likewise, GLP-1 inhibits VSMC dedifferentiation by regulating mitochondrial dynamics via enhancing adiponectin (APN) expression, which in turn promotes the phosphorylation of AMPK and SIRT1 in VSMC [126,127].
Osteoblastic differentiation of VSMCs plays an important cytopathological role in arterial calcification. By inhibiting osteoblast differentiation of VSMCs, GLP-1RAs can ameliorate phosphateinduced vascular calcification, which is mediated by the AMPK/mTOR pathway or AMPK/receptor activator of nuclear factor kappa B ligand (RANKL), as occurs in the osteoblast differentiation of VSMCs [128,129].
Arterial restenosis and vascular remodeling are a response to vascular injury, usually manifested as neointimal hyperplasia. GLP-1RAs can inhibit neointimal hyperplasia by suppressing VSMC proliferation and migration as mentioned above. GLP-1 treatment can exert the direct action on reversing vascular remodeling by downregulating MMP1 expression via alleviating the ERK1/2/NF-κB pathway in Ang II-induced rat aortic VSMCs [130]. Furthermore, GLP-1R activation can prevent arterial restenosis through inhibiting VSMC proliferation and migration via stress-associated endoplasmic reticulum protein 1 (SERP1) [131].
As a result, GL-1RAs play a role in reversing or improving VSMC dysfunction by suppressing VSMCs proliferation, migration, apoptosis, as well as inhibiting VSMC phenotypic switching, especially inhibiting osteoblastic differentiation. GLP-1RAs have an impact in reversing vascular remodeling and attenuating the progression of VSMC senescence (Figure 2).

Reducing macrophage inflammation, foam cell formation, and polarization by GLP-1RAs
Under pathological stress, macrophages contribute to inflammatory responses, which drive insulin resistance and T2DM. In this regard, GLP-1RAs (exendin-4, for example) not only alleviate macrophage infiltration, but also suppress IL-1β, IL-6 and TNF-α expression.
The transformation of monocyte/macrophages into foam cells, is the hallmark of atherogenesis. Vascular located monocytes/macrophages engulf oxidized-LDL (ox-LDL), generating foam cells which release a plethora of pro-inflammatory mediators. The process of foam cell formation occurs via the imbalanced influx and efflux of modified lipoproteins. After lipid uptake, acyl-CoA: cholesterol acyltransferase 1 (ACAT1) catalyzes cholesterol esterification. GLP-1RAs can suppress foam cell formation by activating GLP-1R signaling induced autophagy [134], reducing ACAT1 expression/ activity [93], as well as inhibiting the PKA/CD36 pathway to inhibit ox-LDL uptake [135]. Although in foam cells, GLP-1RAs also can protect against oxidative stress and steatosis via modulation of AMPK/sterol regulatory element binding transcription factor 1 (SREBP1) [136]. Therefore, the reduction of foam cells with GLP-1RAs treatment is speculated to inhibit vascular senescence and delay the progression of atherosclerosis.
The activation of macrophages by GLP-1 also contributes to anti-inflammatory factor secretion and M2 polarization. In this regard, GLP-1 treatment leads to decreased expression of macrophage-specific markers for M1 (inducible NOS (iNOS), IL-6 and TNF-α) and enhances expression level of M2-marker genes, including arginine-1, macrophage galectin-1 (MGL-1), IL-10, as well as mannose receptor-1 (MRC-1). Mechanistic evidence has indicated that GLP-1RAs promote bone marrow-derived macrophage polarization into the M2 phenotype via the cAMP-PKA-signal transducers and activators of the transcription 3 (STAT3) pathway [137,138]. It is acknowledged that activation of STAT3 which is crucial to M2 phenotype transformation, while STAT1 activity which is a key determinant for the M1 subtype. Hereby, GLP-1RAs can diminish STAT1 activity [91]. By modulating macrophage polarization towards an M2 subtype, GLP-1RAs can inhibit inflammation occurring in the vessel lumen, an action which would alleviate the progression of atherosclerosis (Figure 2).
In addition, the inhibition of NLRP3 activation by GLP-1RAs requires regulation of the mitochondrial pathway. GLP-1RAs augment the mitophagy pathway to suppress NLRP3 inflammasome and ameliorate oxidative stress-induced injury in the liver [140]. Hereby, the suppression of inflammatory injury with GLP-1RAs treatment can be eliminated by 3-methyladenine/the mitochondrial kinase (PINK1) siRNA [141]. Moreover, what is clinically noteworthy, is that GLP-1RA treatment can reduce intimal hyperplasia after stent implantation by modulating the above pathway [142]. Combined with abovementioned data, GLP-1RAs show a positive impact on the suppression of NLRP3 inflammasome ( Figure 2).

Modulating immune cell function and inflammation by GLP-1RAs
The development of diabetes and obesity is underpinned by chronic inflammation [143]. In this context, GLP-1RAs can inhibit the secretion of IL-2 and interferon (IFN)-γ, thereby regulating the immune microenvironment and preventing diabetes [144]. GLP-1RA can inhibit the progression of atherosclerosis in the early phase by regulation of immune cell phenotypes. Particularly, liraglutide attenuates pre-established atherosclerosis throughout the aorta and aortic root in ApoE -/mice via increasing macrophage polarization into anti-inflammatory macrophages and by reducing the number of pro-inflammatory macrophages [92]. By doing so, in atherosclerotic plaques from murine aortas, GLP-1RAs regulate proinflammatory mediators and the phenotype of immune cells.
The immuno-modulatory effects of GLP-1RAs also suggest their potential in T1DM. In non-obese diabetic (NOD) mice which are a murine model of T1DM, GLP-1RAs can improve immune functions and inflammatory responses as well as mitigating against islet cell damage, an action which may be related to the decrease in the expression of miR-19b [145]. Similarly, GLP-1RAs exert the potential protection against inflammatory responses in T1DM through decreasing IFN-γ secretion and downregulating PD-1 expression in T cells by attenuating the Janus kinase (JAK)-STAT pathway [146,147]. Thus, GLP-1RAs play an underlying role in immune-regulation in T1DM (Figure 2).

Inhibitory effects on vascular aging by GLP-1RAs
Aging is a crucial component of the pathogenesis of CVD, as reflected by increased oxidative stress and impaired antioxidant defense, responses which may be induced by psychosocial stress [122,148]. ROS and inflammation are main mediators of vascular senescence. GLP-1RAs alleviate vascular aging under chronic stress via increasing adiponectin (APN) levels and inhibiting aortic MMP-9 and MMP-2 expression in ApoE -/mice [96,149]. Excessive autophagy is another cause of vascular aging. GLP-1 treatment can attenuate abnormal autophagy induced by ROS and inflammation through upregulating HDAC6 via the GLP-1R-ERK1/2 pathway [150]. ER stress and DNA damage are also responsible for vascular senescence. Regarding this, GLP-1RAs inhibit ER stress and promote protein folding via endoplasmic reticulum oxidoreductase (ERO1α) in an AMPK-dependent manner, as well as mediating the process of mitochondrial fusion [115,151]. For senescence of VSMCs, GLP-1RAs activate Nrf2 and inhibit Rac1 through cAMP/PKA pathways to prevent Ag II-induced senescence [122,123]. In brief, all of the mechanisms and actions that have protective efficiency on endothelium and VSMCs, can play a role in preventing vascular senescence ( Figure 2).

Improving cardiomyocyte/cardiac fibroblast dysfunction by GLP-1RAs
Cardiomyocyte/cardiac fibroblast dysfunction is related to abnormal proliferation, apoptosis, hypertrophy, as well as inflammation and fibrosis, which may be caused by mitochondrial dysfunction and metabolic disorders. Persistent hyperglycemia can mediate hypoxic or ischemic injury, as well as elicit cardiomyocyte contractile dysfunction and the NF-κB signaling pathway activation, thus inducing ER stress [152]. GLP-1RA can help maintain a balance of energy metabolism in cardiomyocytes through activating PI3K/Akt as well as p38 MAPK pathways as well as activating Nrf-2/heme oxygenase-1 (HO-1), contributing to augmented adenosine triphosphate formation and glucose ingestion by cardiomyocytes [153][154][155]. Based on these pathways, GLP-1RAs improve metabolic disorders to alleviate cardiomyocyte dysfunction.
Cardiac inflammation, fibrosis and hypertrophy are the main manifestations of myocardial dysfunction.
Hyperglycemia-mediated cardiomyocyte damage is associated with inflammation. GLP-1RAs can have effects on inflammation and defending ER stress via suppressing NF-κB in diabetic cardiomyocyte models [152]. GLP-1R can also suppress cardiac hypertrophy induced by Ang II through attenuating the Nox4-histone deacetylase 4 (HDAC4) axis [162]. Apart from these actions, GLP-1RAs ameliorate cardiac fibrosis induced by abdominal aortic constriction via interfering with Ang II type 1 receptor signaling [163]. Therefore, GLP-1RAs can provide protective effects on the above undesirable physiological phenomena in cardiomyocytes.
GLP-1RAs can modulate intracellular calcium homeostasis to prevent reperfusion injury directly in cardiomyocytes [164]. GLP-1RAs also exert anti-arrhythmic effects via reducing calcium leak from the sarcoplasmic reticulum in ventricular arrhythmia, which has the potential to decrease phosphorylation of the type 2 ryanodine receptor (RyR2) and alleviate the activity of CaMK-II [77]. Moreover, GLP-1R activation counteracts the effects of β-adrenoceptor stimulation on cardiac ventricular excitability and reduces ventricular arrhythmic potential [165]. Apart from maintaining calcium channel homeostasis, GLP-1RAs also activate ATPsensitive potassium channels to attenuate cardiac apoptosis and hypertrophy induced by pressure overload [166].
To sum up, GLP-1RAs improve cardiomyocyte/ cardiac fibroblast dysfunction by making balancing energy metabolism, and multiple pathways related to AMPK to inhibit cardiac apoptosis. Moreover, the suppression of cardiac inflammation, fibrosis and hypertrophy with GLP-1RA treatment provide a clinically favorable impact on cardiac dysfunction.

Heterogeneous effects of GLP-1RAs in CVOTs
In the GLP-1RA family of agents, the results of CVOT have been inconsistent. The baseline characteristics of the study populations, study drugs and study designs may be considered as possible reasons for the variability [170]. Furthermore, a real-world retrospective study comparing the compliance of patients receiving different GLP-1RAs for the treatment of hyperglycemic found that even if GLP-1RA weekly preparations were used, there were still differences in compliance [171]. Such differences in compliance between GLP-1RA preparations are mainly due to the differences between injection devices [172]. Ultimately, the duration of GLP-1RA treatment seems to be the factor that explains the heterogeneity of the clinical studies involving GLP-1RA.
Although some promising observations have been reported in various animal models, the impact of GLP-1RAs on human myocardial function is more heterogeneous. In a randomized controlled study, exenatide administered once a week for 18 months did not change carotid plaque volume or composition [173]. Further, while GLP-1RAs have a positive impact on the LVEF, it seems to be inconsistent in most patients with heart failure and effects are quite limited [174]. However, it is currently unclear and to what extent the differences observed in CVOT reflect the heterogeneous pharmacological properties of each drug in the GLP-1RA category. In order to enhance the clinical interpretation of these CVOTs, researchers have quantified the absolute treatment effect over time based on the number of treatments required to avoid a MACE, thus revealed the difference between GLP-1RA [175]. Also, due to the important role of endothelial dysfunction in CVD, the effects of GLP-1RA on flow-mediated vasodilation (FMD) in patients remain to be investigated for their contribution to the CVD actions of GLP-1RAs.

New pharmacological effects and molecular targets of GLP-1RAs
Our understanding of the disease mechanisms and molecular targets of drugs has been facilitated by new biotechnology advances, such as RNA sequencing. RNA-seq can also be used as a useful tool to dissect the molecular targets of drugs in CVD [176]. Therefore, the use of new technological advances such as single cell RNA-seq can shed new light on the GLP-1RA-dependent and independent effects and targets in animal models and humans [177].

The theranostic potential of new modifiers of GLP-1RAs
Due to the high expression profile of GLP-1R in pancreatic β-cells, GLP-1 analogs (such as exendin-4) have been modified by different methods (to prolong half-life, maintain receptor binding affinity and reduce renal drug accumulation) for theranostic purposes and serve as excellent templates for future cardiovascular drug development [178][179][180][181][182][183][184]. More importantly, these modified GLP-1 analogues have shown hypoglycemic [185,186] and cardiovascular protective effects [180,183] in different animal models. Further exploration of these modified agents in animal models of cardiometabolic diseases will deepen our understanding of the potential cardioprotective mechanisms of GLP-1 and GLP-1RAs and potentially provide future directions for precise therapeutic intervention [180].
GLP-1RAs are a class of agents with pleiotropic activities additional to the lowering of blood glucose. These pleiotropic effects include the reduction of BW and BP, the improvement of lipid metabolism and a spectrum of cardioprotective effects. The current comprehensive management strategy of diabetes has gradually shifted from "hypoglycemic as the focus" to "emphasizing both the control of blood glucose and the improvement of MACE and cardiovascular outcomes" [187,188]. In this regard, GLP-1RAs have shown promising efficacy as to the improvement of hard cardiovascular outcomes with cardiovascular safety.