Metformin in cardiovascular diabetology: a focused review of its impact on endothelial function

As a first-line treatment for diabetes, the insulin-sensitizing biguanide, metformin, regulates glucose levels and positively affects cardiovascular function in patients with diabetes and cardiovascular complications. Endothelial dysfunction (ED) represents the primary pathological change of multiple vascular diseases, because it causes decreased arterial plasticity, increased vascular resistance, reduced tissue perfusion and atherosclerosis. Caused by “biochemical injury”, ED is also an independent predictor of cardiovascular events. Accumulating evidence shows that metformin improves ED through liver kinase B1 (LKB1)/5'-adenosine monophosphat-activated protein kinase (AMPK) and AMPK-independent targets, including nuclear factor-kappa B (NF-κB), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), forkhead box O1 (FOXO1), krüppel-like factor 4 (KLF4) and krüppel-like factor 2 (KLF2). Evaluating the effects of metformin on endothelial cell functions would facilitate our understanding of the therapeutic potential of metformin in cardiovascular diabetology (including diabetes and its cardiovascular complications). This article reviews the physiological and pathological functions of endothelial cells and the intact endothelium, reviews the latest research of metformin in the treatment of diabetes and related cardiovascular complications, and focuses on the mechanism of action of metformin in regulating endothelial cell functions.

Patients with diabetes suffer from a higher incidence and rate of mortality of cardiovascular disease (CVD) compared with nondiabetic subjects [1]. Diabetes per se is also a high-risk factor for CVD [2]. According to the disease statistic report from the International Diabetes Federation, the global prevalence of diabetes was 9.3% in 2019 estimated and is projected to reach 10.2% by 2030 [3]. Numerous studies have shown that there is a positive correlation between glucose levels and the incidence of CVD. It is reported that the incidence of heart failure increases by 15% when glycosylated hemoglobin increases by 1%, and the incidence of myocardial infarction decreases by 16% if anti-hyperglycemic drugs are used intensively [4,5]. With multiple pathophysiological components, including hyperglycemia, insulin resistance (IR), hyperinsulinemia and hyperlipidemia, diabetes changes the vascular responsiveness to Ivyspring International Publisher various vasoconstrictors and vasodilators and is a vital factor in the occurrence and development of the vascular complications of diabetes [5]. Hyperglycemia is the primary determinant factor of thrombosis in the coronary vessels through the modification of microbiota thrombus colonization in hyperglycemic patients with ST-segment elevation myocardial infarction (STEMI) [6], and via the direct modification of atherosclerotic plaque instability and rupture with consequent acute thrombosis (in STEMI) and worse prognosis [7,8]. The overexpression of inflammatory and pro-thrombotic markers is linked to the hyperglycemic levels in the serum, the coronary thrombus [9] and the peri-coronary fat [10]. This is evidence of excessive inflammation and prothrombotic effects of hyperglycemia in patients with atherosclerotic plaque instability, rupture, acute thrombosis, and STEMI.
Various diabetes-related pathological changes vary with the size and location of blood vessels, leading to decreased arterial plasticity, increased vascular resistance, reduced tissue perfusion, and atherosclerosis [11,12]. In the process of atherosclerotic plaque formation, circulating monocytes first adhere to the lesion area of the vascular wall, then infiltrate into the wall, and differentiate into macrophages. These macrophages take up modified lipids through phagocytosis and scavenger receptor mediated processes, and leading to the formation of foam cells, which represent the hallmark of arterial plaques [13][14][15][16]. Based on these pathological changes, the development and progression of atherosclerosis plays a critical role in the underlying pathological basis of most patients with stable coronary disease [17].
Ross and colleagues proposed the "endothelial cell injury" model of atherosclerosis ("response to injury") sort of assuming that atherosclerosis results from physical damage to the endothelium [18]. It was subsequently found that biochemical molecules [19,20], including turbulent blood flow and inflammatory cytokines/chemokines, actually serve as physiochemical injury. We propose the hypothesis that "biochemical injury" is the cause of endothelial dysfunction (ED). ED is related to a series of pathological events in diabetes and its complications [21]. In patients with stable coronary disease, with the increase of IR and carbohydrate intolerance, the functional coronary circulation abnormalities of endothelium-dependent vascular movement will gradually worsen [22]. Impaired NO-dependent vasodilatation is a predictor of future cardiac events and the development of coronary ED [23]. Moreover, coronary ED is as a cause of ischemic heart disease (IHD) linked worse prognosis in diabetic patients with stable coronary disease [24,25]. This is evidenced in stable IHD patients during angiography of coronary vessels and invasive measurements of ED. It is noteworthy that the occurrence of ED and the development of atherosclerosis is very rapid in patients with diabetes in the absence of an effective pharmacological intervention [26]. ED is also linked to insulin resistant states including obesity, diabetes, and the metabolic dysfunction [27]. Hyperglycemia is the major causal factor in the development of ED in patients with diabetes. Hyperglycemia is also a main risk factor for CVD, particularly IHD alone and in combination with diabetes. Indeed, hyperglycemia is a risk factor for CVD/IHD independently from diabetes.
Diabetes can be classified into several categories, including type 1 diabetes (T1D), type 2 diabetes (T2D), gestational diabetes mellitus, and specific types of diabetes due to other causes. Accounting for 90 -95% of all diabetes, T2D is generally due to a progressive loss of β-cell insulin secretion frequently on the background of IR [28]. The established relationship between T2D and CVD provides a therapeutic framework that anti-diabetic drugs may have beneficial effects on cardiovascular risk factors and help reduce the atherosclerotic process in diabetic patients. In addition to hypoglycemic benefits, whether anti-diabetic drugs can prevent or treat ED and improve atherosclerosis and CVD is an important research direction [29,30]. When choosing pharmacological interventions for patients with the comorbidities of diabetes and CVD, the hypoglycemic effects and potential cardiovascular benefits should be considered simultaneously. These considerations are critical to our understanding of the potential role of antidiabetic medications in reducing the development and progression of atherosclerosis and CVD in patients both with and without diabetes.
Because the treatment of hyperglycemia changes many factors other than circulating glucose levels, clinical and experimental data support the hypothesis that factors other than hyperglycemia are the culprit [31]. As the first-line drug for most patients with T2D, metformin is indicated because of its benefits on glycemia management but also affects several other pathways/mechanisms including oxidative stress, inflammation, lipoprotein metabolism and thrombosis [32,33]. For example, metformin provides a protective effect on cardiovascular function by reducing weight gain and serum-free insulin level, according to the report from UK Prospective Diabetes Study (UKPDS) [34]. Moreover, various studies have emphasized on the anti-inflammatory and antioxidant role of antidiabetic medications, with multiple mechanisms, which activation of 5'-adenosine monophosphat-activated protein kinase (AMPK) by metformin has had a key role in many of them [35][36][37]. Notably, the pleiotropic effects of antidiabetic medications have been seen for metformin and new classes of antidiabetic drugs and incretins. To date, metformin shows pleiotropic effects to reduce the peri-coronary inflammatory stress [10], including anti-thrombotic and anti-atherosclerotic properties, which are also played by incretins in STEMI [38] and non-STEMI [39] patients. Therefore, this evidences the protective effects played by incretins on the atherosclerotic plaque for patients with acute coronary syndrome, in the condition of acute coronary thrombosis [38] and multivessel disease with and ED [39]. For a detailed review of cardiovascular actions and molecular targets of glucagon-like peptide-1 receptor agonists (GLP-1RAs) [36] and sodium glucose cotransporter 2 inhibitors (SGLT2i) [37], please refer to our recent reviews. As mentioned above, ED can be an independent predictor of cardiovascular events and the first step toward coronary arteriosclerosis [40]. Therefore, evaluating the effects of metformin on improving ED would deepen our knowledge of the therapeutic potential of metformin. In this review, we aim to provide a comprehensive account of the physiological and pathological functions of endothelial cells and the intact endothelium, and summarize the latest research on metformin in diabetes and associated cardiovascular complications, with particular emphasis on the role of metformin on regulating endothelial cell functions. The healthy endothelium serves diverse biological functions in physiological state, including 1) serving as a physiological barrier and site of innate immunity; 2) regulation of vascular tone; 3) mediating mechanotransduction through mechanical sensors/ mechanosensitive complexes; 4) keeping endothelial integrity by an effect on both endothelial injury and the capacity for endothelial repair; 5) keeping the balance of anticoagulant and procoagulant function; 6) regulating angiogenesis; 7) as an essential cell type for the metabolic and synthetic function.

Endothelial cell functions
There are three layers of blood vessels, and the innermost layer is the intima composed of a monolayer of endothelial cells and the underlying internal elastic lamella [41]. In the physiological state, the healthy endothelium serves diverse biological functions ( Figure 1) and creates an adaptive life support system that extends to almost every tissue and organ [42].
Among all the functions of the intact endothelium [43], the first is its role as a physiological semi-permeable barrier and an emerging component of innate immunity. As the barrier and site of innate immunity between blood and tissues, the endothelium actively regulates the exchange and transport of small and large molecules and defends against invading pathogens and infections [44,45]. In addition, as a carbohydrate-rich layer lining the vascular endothelium [46], the glycocalyx acts as a barrier between blood, the endothelium and underlying tissues and plays an essential role in maintaining the barrier integrity and consequently homeostasis and quiescence of the vasculature [47].
A pivotal function of the endothelium is to regulate vascular tone through secreting substances that relax and constrict blood vessels. Nitric oxide (NO), a classical mediator of vasodilation, is produced by endothelial cells and glycocalyx. NO regulates the relaxation of vascular smooth muscle cells (VSMCs) mainly by stimulating soluble guanylyl cyclase to produce cyclic guanosine monophosphate [46,48]. By reacting with the Cys thiol group and reversibly regulating S-nitrosylation, NO can play a role in relaxing blood vessels [49]. Endothelial nitric oxide synthase (eNOS) is the leading source of most vascular NO, and is also a critical enzyme to protect endothelium against vascular damage [50,51]. Besides NO, vasodilators include other dissolved gasses such as hydrogen sulfide (H2S) and carbon monoxide (CO), which are also secreted by endothelial cells [52][53][54]. The endothelium also produces vasoconstrictor molecules, such as angiotensin II (AngII), endothelin 1 (ET-1), thromboxane A2 (TXA 2 ), thrombin, and others [55]. The dynamic balance between vasoconstrictor and vasodilator mediators is an important foundation for endothelial cells to maintain homeostasis. On the other hand, the autonomic nervous system and various neurotransmitters play an essential role in the regulation of vascular tone [56]. The endothelial cells express a variety of receptors that specifically interact with neurotransmitters from the nerve terminal. Among various neurotransmitters, norepinephrine (NE), adenosine triphosphate (ATP) and neuropeptide Y (NPY) relax blood vessels, while acetylcholine (Ach) and calcitonin gene-related peptide (CGRP) act as vasodilators. The endothelial cells are triggered to produce NO and other vasoactive gases when the adrenergic endothelial receptors are activated [57].
The endothelium senses mechanical stress and mediates mechano-transduction through mechanosensors or mechanosensitive complexes [58]. Endothelial cells convert mechanical stress into intracellular biochemical signals, such as increased NO, H2S, and calcium concentration [59][60][61]. Thus, these responses of vascular mechano-transduction serve to prevent vascular cells from damage induced by mechanical injury [58]. Endothelial integrity, another primary function of the endothelium, is affected by the degree of injury and depends on the integrity of the endogenous repair ability of the vessels [62]. When an adjacent endothelial cell is damaged, the local mature endothelial cell can replicate to replace the lost and damaged cell. However, if endothelial cells only rely on local replication function, it is far from enough to resist external damage [63]. A recently study shows that the protection is driven by a transient activation of extracellular-signal-regulated kinase (ERK) in endothelial cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of endothelial cells in clusters [64]. Endothelial progenitor cells (EPCs) in the circulation participate in another repairing mechanism for maintaining the integrity of the endothelium, which has been clearly explained [65]. Therefore, mechano-transduction and circulating EPCs may play an essential role in the pathogenesis of vascular disease by affecting endothelial injury and repair.
Maintaining the balance between anticoagulant and procoagulant status of the vessel wall and blood is a critical function of the endothelium. Although endothelial cells have close contact with platelets, there is no harmful reaction in the blood vessels of a healthy organism [66]. Plasminogen activator (PA) and plasminogen activator inhibitor (PAI) are synthesized and secreted by endothelial cells, providing anticoagulant and procoagulant regulatory mechanisms, respectively [67]. There are several mechanisms involved in the regulation process, such as enhanced plasminogen activation by tissue-PA (t-PA) or urokinase-PA (u-PA), reduced inhibition of plasmin by α2-antiplasmin or of PAs by PAIs, and enhanced conversion of single-chain u-PA to two chain u-PA [68].
Angiogenesis, depending on endothelial cell migration, is another basis function of the endothelium [69]. Endothelial cells regulate angiogenesis through producing several factors, including vascular endothelial growth factor (VEGF), NO, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and triggers [44,70]. On this basis, endothelial cells have the ability to adjust their number and arrangement to meet local needs.
As a sizeable endocrine organ, endothelial cells have a metabolic (large source of energy form glycolysis) and synthetic function. In addition to vasoactive substances, endothelial cells can also secrete various mediators, thus affecting the whole-body cell function [44]. Endothelial cells are also processing factories of important metabolites, such as glucose, fatty acids, amino acids, lipids, ATP, acetyl coenzyme A, and lactate, and endothelial cells regulate the supply of nutrition and oxygen to all tissues of the body [44,71]. The angiocrine and angiogenic function of endothelial cells underlie its importance in regulating tumor metastasis and neovascularization in states of myocardial infarction.
In summary, these physiological functions of the intact endothelium complement each other, and each role is crucial to maintaining the homeostasis of the vasculature.

Hyperglycemia and ED
ED occurs when the production and secretion of vasodilators and vasoconstrictors are out of balance, leading to impaired endothelium-dependent vasodilation. Hyperglycemia, as the defining clinical feature of diabetes mellitus, is known to cause ED through several mechanisms [72], including those involving advanced glycation end products (AGEs), hexosamine, glycocalyx reduction, protein kinase C (PKC), oxidative stress, proliferative dysfunction, ascorbic acid and aldose reductase [73][74][75][76][77][78]. Hyperglycemia induces changes in a variety of cellular signaling and metabolic pathways including altered hemodynamics due to decreased eNOS activity and increased ET-1 synthesis, increased oxidative stress and inflammation, basement membrane thickening caused by transforming growth factor-β (TGF-β) mediated increased synthesis of collagen IV and fibronectin, extracellular matrix changes, VEGF-induced damage to VSMC vascular permeability, cellular growth, inhibition of protein kinase B (PKB, also known as Akt) activation, angiogenesis, and decrease of Na + -K + -ATPase activity [79][80][81]. These changes can further lead to endothelial function injury. In addition, fluctuating plasma glucose levels in people with diabetes can also cause endothelial cell damage [82]. It is speculated that endoglin is involved in the regulation of ED, and oxidative stress may play a role in this regulation [83]. Through suppression of phosphatidylinositol 3 kinase (PI3K)-Akt signalling and activation of FOXO1 in cultured primary endothelial cells, hyperglycemia stimulated placenta growth factor (PlGF) secretion which is a pro-inflammatory angiogenic mediator [84]. Hyperglycemia drives ED via different mechanisms, including excessive oxidative stress, over activated inflammation and reduced NO bioavailability (see Figure 2). Mechanistically, metformin improves ED through liver kinase B1 (LKB1)/AMPK and AMPK-independent targets [85]. The mechnisms involved can interact with each other and amplify the influence, which may bring a vicious circle.
Previous studies have shown that hyperglycemia-induced oxidative stress could cause deoxyribonucleic acid (DNA) damage and induce endothelial cell senescence, injury, apoptosis, and endothelial-to-mesenchymal transition (EndoMT) [86][87][88]. Upon DNA damage, the nuclear enzyme poly ADP-ribose polymerase (PARP) is activated, promoting the production of adenosine diphosphate (ADP)-ribose polymers. Then, hyperglycemiainduced overproduction of superoxide also inhibits glyceraldehyde phosphate dehydrogenase (GAPDH) activity [89]. Furthermore, glycolytic intermediates produce to diacylglycerol, which can activate both classical and novel PKC pathways [81] involved in vascular hyperpermeability, cell growth, neovascularization and other vascular abnormalities of diabetes. There are at least 11 PKC isoforms, and the β-and δ-isoforms appear to be activated preferentially by the diabetic milieu [81]. High glucose-induced nuclear factor-kappaB (NF-κB) DNA binding activity may also induce a low-grade pro-inflammatory state and mediate this inhibition of cellular migration by regulating the NO levels in endothelial cells [90,91]. In addition, the release of endothelial cell-derived vasoconstrictor factors and the disruption of the endothelial glycocalyx both increase in a high-glucose environment [92,93]. The main precursor of AGEs, methylglyoxal (MGO), a reactive dicarbonyl generated is believed to play a critical role in ED. As presented in a previous study, after treatment with MGO for three months, it was observed that the formation of AGEs and the expression of monocyte chemoattractant protein 1 (MCP1) and the receptor for AGEs (RAGE) were increased compared to the matched control group [94], while the bioavailability of NO was decreased significantly concurrent with increased level of superoxide anion. Therefore, MGO scavengers have the potential to attenuate hyperglycemia-induced ED and MGO through ATP-sensitive potassium (KATP)/ mitogen-activated protein kinase (MAPK) pathway activation [95].

Figure 2. Role of endothelial dysfunction in diabetes and its cardiovascular complications.
Hyperglycemia is the leading and common characteristic of diabetes and its cardiovascular complications, including insulin resistance and glucose fluctuation. Hyperglycemia drives endothelial dysfunction via different mechanisms, such as 1) Decreased bioavailability of NO and increased secretion of vasoconstrictors, e.g., ET-1 and Ang II; 2) Increased level of MGO/AGE/RAGE; 3) Enhanced inflammatory response, which is also affected by increased glucose fluctuation; 4) Enhanced oxidative stress; 5) Disruption of endothelial glycocalyx; 6) The expression of epigenetic, such as the level some miRNA, lncRNA. The role of endothelial dysfunction is reflected in the following aspects: 1) The vasodilation decreases and the vasoconstriction increases, and then the vascular homeostasis is broken; 2) Leukocytes adhesion and migration, and platelet aggregation increase; 3) The injury/apoptotic process of endothelial cells is increased; 4) The process of EndoMT is promoted; 5) The endothelial senescence is increased; 6) The permeability of endothelial cells increases. Abbreviations: AMPK, 5'-adenosine monophosphateactivated protein kinase; AGEs, advanced glycation end products; Ang II, angiotensin II; EndoMT, endothelial-to-mesenchymal transition; ET-1, endothelin 1; ICAM1, intercellular adhesion molecule 1; LncRNAs, long noncoding RNAs; MGO, methylglyoxal; MiRNAs, microRNAs; NF-κB, nuclear factor-KappaB; PARP, poly ADP-ribose polymerase; Akt, also known as PKB, protein kinase B; RAGE, receptor for AGEs; SIRT1, sirtuin 1; VCAM1, vascular cell adhesion molecule 1.

Insulin resistance (IR) and ED
In diabetes mellitus, hyperinsulinemia and hyperglycemia are both reported to selectively impair receptor-dependent, endothelium-dependent relaxation [96]. The metabolic disorders of IR, hyperglycemia and excessive circulating free fatty acids cause vascular tissue oxidative stress, mild inflammation, platelet hyperactivity, and ED. In 1997, Pinkney et al. proposed that the IR syndrome was accompanied by many aspects of atherogenesis, which could be viewed as the different consequences of the effect of ED on different vascular beds [97]. In a rat model of IR, the endothelium-dependent relaxation is impaired due to the defect in NO/ prostanoid-independent relaxation [98]. Associated with lipotoxicity, glucotoxicity, and inflammation, IR may damage the endothelial cell and thus trigger and accelerate atherosclerosis and CVD [30, [99][100][101][102].
Insulin activates the PI3K-Akt pathway, phosphorylates eNOS at Serine 1177 site, thereby promoting the synthesis of NO in endothelial cells under normal physiological conditions [103]. However, when the body is in a state of IR, the PI3K-Akt pathway is impaired, as well as the process of NO production. Alternatively, the MAPK pathway causes the synthesis of inflammatory markers, including PAI-1, intercellular adhesion molecule 1 (ICAM1), E-selectin, and vascular cell adhesion molecule 1 (VCAM1), thereby leading to ED [104]. Therefore, the pathogenic factors of ED induced by obesity, one of the main causes of IR, in the early stages will further impair the insulin signaling pathways of endothelial cells, resulting in reduced vasodilatation, abnormal capillary recruitment, and the transfer of insulin substrate to the target tissues [105]. Even after blood glucose levels have returned to normal, the known phenomenon of 'metabolic memory' persists and the harmful effects of hyperglycemia are perpetuated. In addition, molecular and vascular abnormalities involving NO persisted for several months even though the biochemical parameters of IR and ED were normalized, confirming the long-term effects of metabolic memory [106]. Increasing evidence shows that chronic inflammation of metabolic tissues plays a causal role in obesity-induced IR. However, it is still unclear how specific endothelial factors affect metabolic tissues [107].

Advanced glycation end products (AGEs) and ED
The reactive oxygen intermediates (ROI), the reactive chemical entities accelerating the formation of AGEs, activate multiple mechanisms that are potentially involved in the development of vascular complications, such as the activation of NF-κB, PKC and upregulation of the hexosamine pathway [108]. The binding of AGEs to RAGE increases intracellular enzymatic superoxide production [109] and induces the expression of heparanase in human umbilical vein endothelial cells (HUVECs), via activation of the forkhead box O4 (FOXO4) [110]. In a cohort study, independent of other cardiovascular risk factors, the elevated serum AGEs levels in patients with T2D was shown to be associated with ED [111]. AGEs impair endothelial-dependent vascular relaxation and the mechanism is primarily due to reduced bioavailability and activity of NO [112] and activation of p38 and ERK1/2 by reducing eNOS expression and increasing the level of reactive oxygen species (ROS) production [113]. Of note, serum AGEs do not follow the same time course even though glycated hemoglobin A1c and markers of endothelial function improve under insulin therapy [114], suggesting that there may be other factors affecting AGEs, in addition to hyperglycemia, such as oxidative stress.

Oxidative stress, inflammation and ED
Oxidative stress is a state of imbalance between antioxidant systems and exposure to toxic ROS and related chemical species. It has been established that a low concentration of intracellular ROS is essential in maintaining antioxidant/redox homeostasis, vascular integrity and function. However, under pathological conditions such as occuring during the development of atherosclerosis, injury after ischemia-reperfusion and hypoxia, the ROS level is elevated in response to adverse stimuli [120]. ROS are produced by a variety of oxidase enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), lipoxygenase, xanthine oxidase, cyclooxygenase, glucose oxidase, eNOS uncoupling, and mitochondrial electron transport (cytochromeP450 monooxygenase) [121][122][123][124][125][126][127][128]. It follows that proteins and lipids are oxidized by excessive ROS (especially free radicals), which leads to the overexpression of redox genes, intracellular calcium overload and DNA fragmentation, and consequently damage to VSMCs, endothelial cells and myocardial cells [129]. In the early stage of atherosclerosis, the number of inflammatory cells (monocytes/macrophages/ T-lymphocytes) and the process of arteriosclerosis are all associated with enhanced serum levels of inflammatory parameters [130]. As seen in chronic inflammation, the arteriosclerotic artery produces different adhesion molecules, cytokines, and growth factors [20,131]. When endothelial cells are activated by inflammation, the increased expression of VCAM1, selectins and ICAM1 will promote monocyte adhesion to the vessel wall [132]. Many cytokines, including circulating inflammatory cytokines, tumor necrosis factor-α (TNF-α), ROS, oxidized low-density lipoprotein (oxLDL), and traditional risk factors can directly or indirectly induce abnormal functioning of endothelial cells, resulting in impaired vasodilation, increased endothelial permeability, increased leukocyte adhesion and thrombosis [133][134][135].
Over-inflammatory/oxidative stress causes ED in patients with and without diabetes [136,137]. Indeed, in young women without IHD and low risk of CVD ("low risk subjects"), the over-inflammatory/ oxidative stress may be the main determinant factors of ED and major adverse cardiovascular events (MACE) [137]. It is relevant to note that ED could be linked to other non-classical CVDs' risk factors as adipose tissue deposition and excess. This is observed in women [137] with different classes of breast fat density (lowest breast density) and increased risk of pre-diabetes and diabetes. On the other hand, it has been remarked in overweight patients with altered glycemia and IR, as those with pre-DM and without first clinical manifestation of CVD. Excess of abdominal adipose tissue in these overweight pre-diabetics patients with IR is a chronic source of inflammatory molecules that cause ED and reduction of cardiac performance and worse prognosis [138,139].
In 2002, a study from Ceriello et al. [74] showed that oxidative stress was a common mediator of postprandial hyperglycemia and hypertriglyceridemia on endothelial function. Hyperglycemia increases intracellular ROS through the interrogation mechanism between cytosolic and mitochondrial ROS production [140], which once again shows that the decrease in NO production and the imbalance of increased ROS production may be related to the impaired endothelium-dependent vasodilation in patients with CVD. Conditions such as hyperlipidemia can also induce increased levels of AGEs, leading to increased ROS and oxLDL accumulation in diabetic animals [141]. Cytokines and oxLDL stimulate endothelial cell permeability, and NF-κBdependent inflammatory gene expression and ROS appear to play a central role in mediating both responses [72]. Therefore, the vicious cycle among ED, oxidative stress and inflammation will lead to atherosclerosis in diabetes and associated conditions [128].

Endothelial-to-mesenchymal transition (EndoMT) and ED
EndoMT is an extremely complex pathophysiological process during which endothelial cells lose their specific markers and acquire the phenotypes of mesenchymal cells or myofibroblasts [142]. In hyperglycemia, it is deduced that high glucose levels induce EndoMT through ERK, Smad2/3, nucleotidebinding oligomerization domain-containing protein 2 (NOD2), ROCK1, and serum response factor in glomerular endothelial cells -this sequence results in increased expression of mesenchymal markers in different types of endothelial cells [143,144]. Recently, evidence has suggested that EndoMT is a critical link in the interactions between ED and inflammation. Through this process, varieties of inflammatory mediators activate endothelial cells and transform them into mesenchymal-like cells. The factors implicated in this response are interleukin-1beta (IL-1β), TNF-α, NF-κB transcription factor, and endotoxins [145][146][147]. In addition to inflammation and hyperglycemia, hypoxia [148], dyslipidemia [149], miRNAs and lncRNAs [150] and other abnormalities are related to the induction of EndoMT. Notwithstanding these findings, the totalities of the mechanisms involved in EndoMT are not fully understood.

Current status of metformin in the amelioration of ED
Metformin is a biguanide derivative that exerts an anti-hyperglycemic effect with minimal risk of hypoglycemia. Metformin is currently the first choice for the majority of patients with newly diagnosed T2D requiring medical therapy. The primary status of metformin has arisen from the results of clinical evidence from the UKPDS [34]. The reduction of glucose production in liver cells and increased uptake in skeletal muscle are considered the primary processes mediating the hypoglycemic effect of metformin although multiple other actions have been suggested [151].
As for the efficacy of SGLT2i [37] and GLP-1RAs [36], numerous clinical studies have shown that metformin can improve vascular endothelial function and reduce CVD in patients with diabetes (see Table 1 [152-167]). A study reported in 2001, utilizing a moderate dose of metformin (500 mg twice daily) administered for 12 weeks and compared to placebo, showed improvement in Ach-stimulated flow (and IR) was observed among diet-treated T2D patients. In this study IR was the sole predictor of endothelium-dependent blood flow after metformin treatment [152]. Similar benefits were observed with longer-term and higher doses of metformin. Fifteen T2D patients took 1700 mg per day for three months, and t-PA, VCAM1 and ICAM1 levels were significantly reduced [155]. For obese patients who have previously received medical nutrition guidance and regular exercise, after taking metformin for 12 weeks (average dosage 1381 ± 85 mg/d), a decrease in PAI-1 and VEGF was observed, effects which were independent of favourable effects of metformin on body mass index (BMI) and glycemic control [156]. Among T2D patients with stable coronary heart disease, treatment with metformin affects VCAM1 and asymmetric dimethylarginine (ADMA) levels [166]. Still, it does not impair the endothelial healing of drug-eluting stents in these patients with or without insulin use [168]. In the long-term treatment (4.3 years) with metformin plus insulin, most of the inflammatory biomarkers, such as the von Willebrand factor (vWF), soluble VCAM1, t-PA, and ICAM1, were significantly reduced even after adjusting for baseline differences in age, sex, smoking and severity of previous CVD [161]. When comparing the effect with the other antihyperglycemic agents, at equivalent levels of glycemic control, metformin was more effective in reducing biomarkers reflecting inflammation and improving endothelial functions than repaglinide (an insulin secretagogue) [157], and only rosiglitazone (the insulin-sensitizing agent) improved endothelium-dependent vasodilatation and insulin sensitivity more than metformin [169]. In addition, with lipid-lowering agents, such as atorvastatin or fenofibrate, used in combination among T2D patients, metformin is demonstrated not only to prevent the glucose-induced impairment of endothelial functions partly but also potentiates lymphocyte-suppressing, endothelial protective and systemic anti-inflammatory effects of fenofibrate, superior to monotherapy [158,170]. A recent randomized controlled trial showed that through distinct or complementary mechanisms of action on the vascular wall, metformin was able to improve functional capillary density during post-occlusive reactive hyperemia in obese newly diagnosed drug-naive women with T2DM [165].
The traditional paradigms of T2D occurring only in adults and T1D only in children are no longer accurate, as both diseases occur in both age-groups. There is less data available about metformin affects on the endothelium among patients with T1D in whom increasing evidence suggests the deleterious effect of glycemic variability and fluctuations on CVD. The Reversing with Metformin Vascular Adverse Lesions (REMOVAL) trial, the largest and longest trial of metformin in T1D to date, has demonstrated that metformin does not reduce either the carotid intima-media thickness (IMT) or the reactive hyperemia index (RHI) after treatment with metformin for approximately three years [167]. However, among the uncomplicated subjects with T1D, metformin is shown to improve the flow mediated dilation (FMD) and the biomarker of oxidative stress (urinary 8-iso-prostaglandin F2α) after 6-month treatment, irrespective of its effects on glycemic control and BMI [160]. There was a cardioprotective effect of metformin for those without overt CVD through improving circulating EPCs, pro-angiogenic cells, and circulating endothelial cells count and function independently [171]. In addition, with the combination of metformin and the SGLT2i, empagliflozin, used in patients with T1D, arterial stiffness and endothelial function assessed by FMD and RHI were significantly improved in the combination therapy group compared to those on monotherapy [164].
Furthermore, metformin reduces the inflammatory biomarkers, such as vWF, sVCAM1, and sICAM1, among volunteers with impaired glucose tolerance [172]. Moreover, metformin may improve vascular function and decrease myocardial ischemia in nondiabetic women with chest pain and angiographically normal coronary arteries [173]. Among women with polycystic ovary syndrome (PCOS), those resulting from the critical pathophysiologic component contributing to hyperandrogenisms and the reproductive metabolic features with IR, conflicting results have been reported regarding the effects of metformin on endothelial function [174]. Metformin (850-1700 mg daily) therapy could reduce the baseline diameter of the brachial artery, FMD, IMT, and serum VEGFB after reactive hyperemia among women with PCOS, and even improve the FMD values, plasma ET-1 after 6-month treatment [175][176][177][178][179]. In one study which compared the characteristics among nonobese adolescents with PCOS and healthy age-matched volunteers, only the surrogate markers of cardiovascular risk such as blood pressure, levels of C-reactive protein (CRP), and PAI-1, whereas the deterioration of vascular structure and function has also been undetected even after 6-month treatment with metformin [180]. Metformin therapy reduces the high risk of cardiovascular events in pre-DM patients by reducing coronary ED with two-year therapy [181].  Nutritive microvascular reactivity↑; Functional capillary density during post-occlusive reactive hyperemia↑ Metformin was able to improve vascular function in obese newly diagnosed drug-naive T2DM women, through distinct or complementary mechanisms of action on the vascular wall.

Regulation of vascular tone
Impaired endothelium-dependent vasodilation is a common characteristic in arteries in both T1D and T2D. NO is the major vasodilator synthesized from its precursor L-arginine via eNOS [182]. Metformin (60 mg/kg/d) treatment improves endothelial functions in the T2D rat model (Goto-Kakizaki rats) and significantly improves NO bioavailability in rats [183]. Metformin (300 mg/kg/d) therapy does not effectively correct hyperglycemia; however, the reduction of vasodilation and total eNOS activity in a T1D rat model (streptozotocin, STZ-induced rats) can be corrected by metformin treatment [184]. In a 2006 study, AMPK was found to be necessary for metformin to enhance eNOS activation in vivo [35]. There is a positive correlation between the concentrations of metformin (50-500 mmol/L) and the levels of eNOS with serine-1179 (Ser1179) phosphorylation and heat shock protein 90 (HSP90), resulting in increased eNOS activation and NO bioactivity [35]. Henceforth, AMPK activation became one main recognised mechanisms of the vascular protective effects of metformin. Recently, the eNOS activating effect of metformin (250 mg/kg/d) on EPCs of STZ-induced diabetic mice has also been demonstrated [185].
Metformin affects the expression of several vasoconstrictive molecules such as ET-1, AngII, and tissue factors to regulate vascular tone. In both clinical and basic research, metformin treatment reduces ET-1 levels in patients with PCOS (Diamanti-Kandarakis et al. 2001) and insulin-resistant human endothelial cells [193].
Metformin inhibits PKC membrane translocation and activity induced by AngII [194]. AngII stimulates vascular NOX and is activated through the AngII receptor AT1R-PKC pathway [195], and is closely related to vascular oxidative stress in ED under diabetic conditions [196]. In addition, by inhibiting vasoconstrictor prostanoids, metformin also improves the endothelial function of mesenteric arteries in a T2D rat model [197].

Inhibition of inflammation and leukocyte adhesion to endothelial cells
Metformin inhibits the pro-inflammatory changes induced by cytokines, which may be one mechanism for its vascular actions beyond the hypoglycemic effect. Like the AMPK activator AICAR, which can inhibit the increase in NF-κB reporter gene expression incubated with TNF-α in HUVECs [198], metformin (2-10 mM) is been found to inhibit inflammation through the AMPK-dependent IKK/IKBα/ NF-κB inhibitory pathway (Figure 3) [199]. Except for inhibition of NF-κB through blockade of the PI3K-Akt pathway in human saphenous vein endothelial cells [200], metformin also inhibits TNF-α induced production of Interleukin-6 (IL-6) [201] and several other inflammatory molecules responsible for monocyte adhesion to activated endothelial cells including VCAM1, MCP1, E-selectin, and ICAM1, by up-regulating B-cell lymphoma 6 (BCL6) and AMPK-induced phosphorylation of PARP1 at ser-177 [202], and the phosphorylation of histone deacetylase 5 (HDAC5) at serine 498 [203].

Inhibition of endothelial cell senescence
Endothelial cell senescence leads to ED and contributes to the progression of age-associated vascular disorders [218]. Endothelial cell senescence is characterized by cell cycle arrest and pro-inflammatory gene expression [219][220][221] and is driven by many factors, including high glucose, ROS, inflammatory cytokines, ionizing radiation, and telomere dysfunction [222,223]. High glucose induces endothelial cell senescence by inhibiting sirtuin 1 (SIRT1) expression, which can be attenuated with metformin (50-250 μM) treatment by modulating the SIRT1 downstream targets forkhead box O1 (FOXO1) and p53/p21 [224,225]. In ApoE -/mice, metformin (50 mg/kg/d) therapy significantly reduces vascular aging and inhibits atherosclerotic plaque formation through AMPK activation leading to SIRT1/Dot1-like protein (DOT1L)/ histone H3 lysine 79 trimethylation (H3K79me3)-induced upregulation of sirtuin 3 (SIRT3) levels [226]. Metformin increases fenestrations in liver sinusoidal endothelial cells isolated form old and young mice [227]. Calcification is a vascular disease marker and a prognostic factor of CVD, which is related to the aging of the vascular system [228]. It is speculated that metformin, acting via AMPK dependent mechanisms, may be a potential target for the treatment of vascular calcification [229]. Indeed, clinical trials have been designed to assess the potential benefits of metformin as an anti-aging drug that enhances healthspan and extends lifespan [230]. The beneficial effects of metformin on aging and healthspan are primarily indirect via its effects on cellular metabolism and result from its anti-hyperglycemic action, enhancing insulin sensitivity, reduction of oxidative stress and protective effects on the endothelium and vascular function [230].

Inhibition of EndoMT
EndoMT is a driver event in the pathogenesis of atherosclerosis, diabetes associated myocardial remodeling, pulmonary hypertension and others. A study in 2018 explored the effects of different flow patterns on the EndoMT in endothelial cells, indicating that pharmacological activation of AMPK or SIRT1 could attenuate oscillatory shear stress (OS)induced EndoMT [237]. The beneficial therapeutic effect of metformin on atherosclerosis may be mediated at least in part by inhibiting the EndoMT in the vasculature. The expression of Krüppel-like factor 4 (KLF4), a major transcription factor showing endothelial protection and maintenance of vascular homeostasis, and cholesterol-25-hydroxylase (Ch25h) decreases in high glucose level [238], while metformin (200 mg/kg/d) can increase the expression of KLF4 and Ch25h to suppress EndoMT [239].

Inhibition of the permeability of the endothelium
The increased permeability of the endothelium is accompanied by inflammation-mediated reversible cell rounding and inter-endothelial gap formation [240] and high glucose-activated PKC pathway [241]. Metformin (0.33 mg/ml, two weeks) treatment is associated with improved glycocalyx barrier properties and indicates reduced vascular permeability in mice [242]. Metformin (400 mg/kg/d, 100 mg/kg/d, respectively) administration is associated with reduced lung endothelial hyperpermeability and systemic inflammatory response in STZ-induced diabetic mice [243] and alleviates microvascular ED by suppressing hypoxia inducible factor-1 alpha (HIF-1α)/Profilin-1 (PFN1) signaling, which mediates endothelial cell permeability during diabetic retinopathy [244]. Moderate doses of metformin (0.1-1.0 mM) are shown to produce an increase in the trans-endothelial resistance of endothelial monolayers and enhance the vascular barrier integrity [245]. By activation of AMPK signaling pathway and regulation of oxidant/anti-oxidant balance, metformin abrogates the disruptive effect of polyphosphate which induce hyper-permeability and inflammatory responses through mTOR pathway in endothelial cells [246].

Metformin on endothelial cell metabolism
Evidence has demonstrated the importance of endothelial cells in the maintenance of homeostasis across the entire vascular system. The initiation and progression of atherosclerosis may originate from the disorder of endothelium intracellular metabolism that can be detected at the earliest stages of developing the syndrome [269]. Endothelial cell metabolism is important for maintaining metabolic health, such as glucose, lipid and amino acid, as they have been most extensively studied [270]. The study identifies a metabolic program that promotes the acquisition of a quiescent endothelial state and emphasizes the role of metabolites as signaling molecules in endothelial cells [271]. The activity and migration of endothelial cells decrease when the enzymes related to glucose metabolisms, such as G6P dehydrogenase and transketolase, are inhibited [272]. Previous studies showed that metformin affects FABP4 mediated endothelial fatty acid metabolism [273,274]. Endothelial cells internalize chylomicrons, which trigger lipid accumulation in aortic of lipoprotein lipase-deficient mice [275]. Recently, metformin (100 μM) is shown to reduce saturated fatty acid-induced lipid accumulation and inflammatory response by restoring autophagic flux in endothelial cells [205]. In addition, metformin (1-5 mM) takes part in regulation of hexose transport [276], reversing glucose starvation-induced ER stress [277] in endothelial cells. Metformin, GLP-1RAs, and SGLT2i are the only drugs screened in seven classes of anti-hyperglycemic drugs which reduce ER stress caused by pharmacological or hyperglycemic conditions in human coronary artery endothelial cells (HCAECs) [214]. In endothelial cells arginine, glutamine, leucine and valine play a role in the metabolism of endothelial cells [278][279][280][281].

Metformin on mitochondrial dynamics
Mitochondria are critical integrators that participate in signal transduction, energy production, ROS generation, and cell apoptosis. Recent studies have highlighted the importance of mitochondrial dynamics, especially mitochondrial fusion and fission in mitochondrial homeostasis [282]. In endothelial cells, more fragmented structures caused by fission are associated with mitochondrial dysfunction, contributing to ED with an unclear mechanism [283]. Mitochondrial division is triggered when Dynaminrelated protein 1 (DRP1), a cytosolic guanosine-5'triphosphatase, binds with fission 1 (FIS1) or mitochondrial fission factor (MFF) on mitochondria [282]. Metformin (300 mg/kg/d) is observed to suppress the progression of atherosclerosis through the inhibition of DRP1-mediated mitochondrial fission in STZ-induced diabetic ApoE -/mice [284]. In addition to inhibiting mitochondrial division, promoting mitochondrial fusion may also benefit endothelial cell function and represent a potential therapeutic approach for diabetes patients with CVD, which is an area that needs further study. In terms of the efficacy of metformin, studies have also shown that metformin (0.01-2 mmol/L) can promote mitochondrial biogenesis by activating AMPK and inducing PGC1α in HUVECs [213].

Therapeutic potential of metformin in COVID-19 patients with diabetes
Coronavirus disease 19 , caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), is associated with multiple factors, such as cytokine storm, hyperactive inflammatory and immune responses, and coagulation disorders [285]. Among COVID-19 patients with diabetes, the intensive care hospitalization rate and mortality rate are two to three times higher than that of patients without diabetes [286]. Diabetes has been identified as an independent risk factor for poor prognosis of COVID-19 [287,288]. The underlying pathogenic links between COVID-19 and diabetes include effects on inflammation, glucose homeostasis, immune status changes and renin angiotensin aldosterone system (RAAS) activation [289]. Metformin treatment is associated with reduced mortality in COVID-19 patients with diabetes [290][291][292]. Patients who used metformin before hospitalization also showed lower severity parameters at admission [293]. During SARS-CoV-2 infection, in addition to controlling the glucose level, metformin may attenuate ED, inhibit viral entry and infection, resist oxidative stress, and change inflammatory and immune responses that would explain its ability to achieve cardiovascular protection in COVID-19 [294,295]. Therefore, metformin may represent a drug candidate for treating COVID-19 and gain the advantage of fighting SARS-CoV-2 induced immune storm in COVID-19 patients with diabetes [296].

Metformin on other transcriptional factors
Recent data has shown that islet ED in db/db mice was evident compared with non-diabetic mice [297]. Metformin/empagliflozin treatment (6 weeks) could reduce the expression of dysfunction marker genes and increase insulin release in vivo [297]. It is speculated that metformin may improve the blood glucose level of T2D patients by enhancing the function of islet endothelial cells. Metformin (100 mg/kg/d) can also promote the development of pulmonary vessels and up regulate the expression of glioma-associated oncogene homolog 1 (Gli1) in pulmonary vascular endothelial cells of hyperoxia newborn mice [298].

Conclusion
Similar as SGLT2i and GLP-1RAs, numerous clinical studies have shown that metformin can improve vascular endothelial functions and reduce CVD in patients with diabetes. Mechanistically, metformin improves ED through LKB1/AMPK and AMPK-independent targets. In addition to several common mechanisms, including increasing NO production, inhibiting inflammation and oxidative stress, metformin can protect endothelial cell functions and the intact endothelium by reducing EndoMT and increasing the differentiation ability of EPCs. Based on the protective effects of metformin on blood vessels summarized in these basic and clinical studies, we suggest that metformin is also worthy of attention for potential treatments for CVD and other panvascular diseases in which ED plays a fundamental role.