FormalPara Key Summary Points

Sodium-glucose cotransporter-2 (SGLT2) inhibitors function through a novel mechanism of reducing renal tubular glucose reabsorption by inhibiting target SGLT2 receptors present in the renal tubule.

All studies which have reported experimental effects of SGLT-2 inhibitors suggest that this class of drug may exert ‘off-target’ cardiovascular benefits by modulating vascular endothelial cell activation and improving endothelial cell dysfunction, a critical early step in atherogenesis.

Chronic and acute treatment with dapagliflozin led to a significant endothelial-dependent vasorelaxation in the aorta of diabetic mice, which some studies suggest may be due to a direct effect on vascular cells.

The ex vivo and in vitro studies reviewed here support a possible class effect of SGLT-2 inhibitors on the regulation of endothelial function.

Anti-inflammatory effects of SGLT-2 inhibitors have been observed in diabetic nephropathy models, via a suppression of the advanced glycation endproducts (AGEs)-receptor pathway, as well as in in vitro studies, thereby implicating anti-inflammatory effects that are independent of glucose-lowering.

Systemic administration of SGLT-2 inhibitors markedly reduced expression of pro-inflammatory adhesion markers and cytokines in diabetic rodent models.

Arguably, the evidence from the experimental studies reported in this review points towards SGLT-2 inhibitors exerting additional benefits beyond their primary receptor targets in the renal tubule as well as acting independently of glucose control.

Introduction

Diabetes is a major public health problem [1] with an increasing economic burden worldwide and a strong link to cardiovascular disease [2]. The insulin resistance associated with type 2 diabetes (T2D) contributes to hyperglycemia, dyslipidemia, and hypertension, all of which significantly increase cardiovascular risk [3]. Such cardiovascular risk factors are associated with impaired endothelial function [4], a significant contributing factor to the pathogenesis of atherosclerotic vascular disease in patients with T2D [5,6,7]. Possible mechanisms of hyperglycemia-induced endothelial damage include insulin resistance and inflammation [8]. Nitric oxide (NO) [produced by endothelial NO synthase (eNOS)] exerts cardio-protective effects by inducing the relaxation of smooth muscle cells, thereby preventing several cascades of events, including migration of leukocytes into arteries, platelet adhesion, and smooth muscle cell proliferation [4, 9]. In diabetes, however, reduced eNOS activity and/or elevated reactive oxygen species (ROS) production reduces NO bioavailability and increases the production of harmful endothelial-derived vasoactive mediators such as endothelin-1 and ROS, leading to the progression of atherosclerosis and hypertension [4].

In recent years landmark clinical studies have reported the beneficial effects of the antidiabetic agents glucagon-like peptide-1 (GLP-1) receptor agonists and sodium-glucose co-transporter-2 (SGLT-2) inhibitors in reducing the risk of cardiovascular mortality in patients with T2D [10,11,12]. The findings of a recent network meta-analysis revealed that although both classes of antidiabetic drugs demonstrated cardiovascular benefits, SGLT-2 inhibitors were superior in reducing cardiovascular events compared to GLP-1 receptor agonists which was more beneficial in reducing the risk of nonfatal stroke [13].

SGLT-2 expression occurs in both the small intestine and kidney. These transporters act by modulating glucose reabsorption in the proximal renal tubules [14, 15]. Inhibition of SGLT-2 in kidneys facilitates excess urinary glucose excretion and lowers circulating blood glucose levels in patients with diabetes. Although SGLT-2 expression has not been reported in cardiac and vascular tissues, recent clinical evidence has shown considerable reductions in blood pressure and improvement in endothelial function associated with their use. However, it is unclear if these effects are just due to the indirect glucose-lowering effect of these antidiabetic drugs [16, 17]. There is accumulating evidence on the effect of SGLT-2 inhibitors and their underlying mechanisms in animal and in vitro models of endothelial dysfunction [10]. Collectively, these indicate that SGLT-2 inhibitors are likely to have a direct influence on the cardiovascular system.

Consequently, a number of large clinical studies have been conducted to investigate endothelial function in patients treated with SGLT-2 inhibitors. The DEFENCE study found a significant improvement in endothelial function in patients with T2D receiving dapagliflozin 5 mg/day as an add-on therapy to metformin 750 mg/day [18]. Similarly, T2D patients with ischemic heart disease showed a significant reduction in surrogate markers for endothelial function following 12 weeks of dapagliflozin monotherapy [19]. An investigation by Solini et al. suggested that dapagliflozin might have a protective cardioprotective effect in preserving vasodilating capacity [20]. Similarly, Lunder et al. showed that empagliflozin 25 mg/day as add-on therapy to metformin therapy 2000 mg/day significantly reduced arterial stiffness in patients with type 1 diabetes [21]. Furthermore, the findings from a recent systematic review found that only SGLT-2 inhibitors significantly improved endothelial function, as assessed by flow-mediated dilation, in comparison to other classes of antidiabetic agents [22].

This systematic review was therefore undertaken to evaluate the effect of SGLT-2 inhibitors on endothelial function in preclinical models. The evidence from animal and in vitro studies will enhance our understanding of the underlying molecular mechanisms of SGLT-2 inhibitors on endothelial dysfunction and in turn lead to the development of novel approaches to improved management of T2D patients, especially those with high risk of cardiovascular events.

Methodology

Database Search

This systematic review was performed following the Preferred Reporting Items for Systematic Review (PRISMA) guidelines [23]. Both the PubMed and EMBASE (including MEDLINE) electronic databases were searched from inception to May 2020 for publications reporting in vivo, ex vivo, or in vitro evaluation of the effect of SGLT-2 inhibitors on endothelial function using the following keywords: “SGLT-2”, “sodium-glucose co-transporter inhibitor,” “dapagliflozin,” “canagliflozin,” “empagliflozin,” “ibragliflozin,” “luseogliflozin,” “endothelium,” “vascular,” “endothelial,” “endothelia,” “experimental study,” “in vitro,” “in vivo,” “animal,” “mice,” “cell,” and “ex-vivo.” The searches were carried out by two independent researchers. In addition, the references of relevant articles were manually searched for any additional relevant articles.

This article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors.

Study Selection and Data Extraction

Eligible studies were included if they were published in English, involved cell culture or animal models, assessed endothelial function evaluated by any method, and used SLGT-2 inhibitors as the intervention at any dose or duration. Studies were excluded if they were not in English, involved human clinical trials, did not use SGLT-2 inhibitors, used other antidiabetic medications or combined therapies, or if the study design was non-experimental. Two independent reviewers performed the extraction of relevant data.

Quality and Risk of Bias Assessment

Each eligible study was assessed for quality and risk of bias by two independent reviewers. For methodological quality assessment, data on randomization method, study duration, random sequence generation, allocation concealment, and blinding as well as other risks of bias information relating to the reporting and attrition bias from each study were extracted. Assessment of the risk of bias was completed using the SYRCLE tool [24] for the in vivo experiments. The internal validity of each study was assessed by performance bias, detection bias, and selection bias. The external validity of the animal population, drug interventions, and outcomes were assessed. Since the included studies did not report pre-defined primary and secondary outcomes, the reporting bias domain (selective outcome reporting) was not used in the assessment of the risk of bias. The scoring was as follows: low risk of bias (reported) was denoted as ‘L’; high risk of bias (not reported), as ‘H’; unclear or unknown risk of bias, as ‘?’. Since the experimental reporting on the methodology for animal models was generally considered to be relatively poor [25], other bias domains of reporting of randomization and blinding were included. Calculation of sample size and temperature control reporting were also included as other risks of bias domains. These items were scored as ‘yes’ if reported, and ‘no’ if not reported. Due to the substantial heterogeneity of the studies in terms of design and interventions, as well as limitations in the methodology used, a meta-analysis was not possible, and we present here only a qualitative systematic review.

Results

Search Findings

A flow chart of the identification of eligible studies is shown in Fig. 1. A total of 144 articles were identified through searches of the PubMed, EMBASE, and MEDLINE electronic databases. After removal of duplications, 82 articles remained. A further 43 articles were excluded because they did not meet the inclusion criteria. Therefore, we evaluated the full text of 39 potentially relevant studies, of which 24 eligible experimental studies were selected for the final analysis, all of which met the inclusion criteria.

Fig. 1
figure 1

Flow chart of the identification of eligible studies

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Study Characteristics

The characteristics of the included studies are summarized in Tables 1 and 2. Findings on the SGLT-2 inhibitors canagliflozin, ipragliflozin, empagliflozin, dapagliflozin, tofogliflozin, and luseogliflozin were reported.

Table 1 The effect of sodium-glucose co-transporter-2 inhibitors on endothelial function in animal models
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Table 2 The direct effect of sodium-glucose co-transporter-2 inhibitors on endothelial function in cell culture
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Animal Models

A total of 18 studies reported in vivo data (13 in mice and four in rats, and one in rabbit; Table 1). Twelve studies used diabetic models, and four studies were performed in atherogenic models or an obese model. The SGLT-2 inhibitors used were empagliflozin (1–30 mg/kg/day), dapagliflozin (0.1–7 mg/kg/day), ipragliflozin (0.1–3 mg/kg/day), canagliflozin (2 studies, 1–30 mg/kg/day), tofogliflozin, and luseogliflozin (1–10 mg/kg/day). The duration of treatments ranged from 4 to 12 weeks. The rodents were aged between 5 weeks and 11 months at the beginning of the studies. All intervention administrations were oral gavage except one which was via intragastric route.

Three of the ex vivo studies included isolated pulmonary arteries from the thoracic aorta, one used the abdominal aorta, and one study used both the pulmonary and coronary arteries. Three of the ex vivo studies used dapagliflozin (1.0 nM–10 μM), four used empagliflozin (dose not reported), and two studies used canagliflozin (100 pmol/l–100 μmol/l).

In Vitro Studies

The in vitro studies (n = 9; Table 2) were performed in human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), murine endothelial cells, or porcine coronary artery endothelial cells. The drug interventions included empagliflozin (6 studies; 0.1–10 µM, 24 h, 3 days or 30 min), dapagliflozin (3 studies; 1–100 nM, 3 days or 24 h) and canagliflozin (2 studies; 10 µM for 15 or 30 min). Before SGLT-2 inhibitors were added to the cell culture, the cells were stimulated with agents, such as tumor necrosis factor-α (TNFα), palmitate, high glucose, interleukin-1β (IL-1β), or acetylcholine, for various durations ranging from 1 to 24 h to induce damage to the cells/tissue so that any protective effects of the SGLT-2 inhibitors could be assessed.

Experimental Outcomes of SGLT-2 Inhibitors

The Effect of SGLT-2 Inhibitors on Endothelial Function

It has been shown in both diabetic and nondiabetic models that SGLT-2 inhibitors improve endothelial function in euglycemic and hyperglycemic conditions, as summarized in Table 1. The vascular response was examined in different studies to investigate the effects of SGLT-2 inhibitors on endothelial dysfunction and the proposed mechanism by which they reduce oxidative stress, namely, the formation of advanced glycation endproducts (AGEs) and their receptors (RAGEs), which is involved in AGE/RAGE signalling and inflammation.

The Direct Effect of SGLT-2 Inhibitors on Endothelial Cells

The direct involvement of SGLT-2 inhibitors in different cell lines was assessed, and the results are summarized in Table 2.

Qualitative and Risk of Bias Assessment

The overall and individual scores for the qualitative and risk of bias assessment of in vivo studies are summarized in the ESM (ESM Table S1; ESM Figure S1). Most of the included studies did not report sufficient information for the assessment of the risk of bias.

Although randomization was reported in six studies (50%), no detail was reported. Consequently, random allocation, housing, and assessment of outcome risk of bias were mostly unclear. Four studies (33%) reported blinding during the experimentation, three of which reported blinding of outcome assessor and one reported blinding of the examiners to the allocation of animals. Therefore, risk of bias related to blinding of the examiner and assessor as well as allocation concealment was assessed as unclear. Measures to reduce random housing (performance bias) were reported in two studies (16%), with a further two studies not reporting random housing (high risk of bias) and the remaining eight studies unclear. Only in five of the studies (42%) were the baseline characteristics between the control and intervention group reported. Since the reporting was quite poor for animal experiments, most of the possible bias sources were scored as unclear risk (ESM Table S1). Four studies (33%) reported details of dropouts to score risk of attrition bias as low level. None of the studies reported sample size calculation to explain and justify the number of animals used per group.

Discussion

The aim of this systematic review was to assess existing evidence on the protective effects of SGLT-2 inhibitors on endothelial function in preclinical models. All of the studies published to date which have reported on the experimental effects of SGLT-2 inhibitors suggest that this class of drug may exert cardiovascular benefits by modulating vascular endothelial cell activation and improving endothelial cell dysfunction, a critical early step in atherogenesis.

These beneficial effects are likely due to (1) glucose-lowering effects, thereby preventing downstream glucotoxicity, such as AGE formation, AGE/RAGE signalling, reduction of oxidative stress, and inflammation and impairment of vascular function, and (2) direct effects on vascular endothelial cells (as summarized in Fig. 2).

Fig. 2
figure 2

Summary of potential mechanisms involved in the protective effects of sodium-glucose co-transporter-2 (SGLT-2) inhibitors on endothelial function. Akt Protein kinase B, EDHF endothelium-derived hyperpolarizing factor, NLRP3 NLR family pyrin domain containing 3, SNP sodium nitroprusside; see footnotes to Tables 1 and 2 for other definitions

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Chronic and acute treatment with dapagliflozin led to a significant endothelial-dependent vasorelaxation in the aorta of diabetic mice [26, 27]. These data are consistent with results from studies that used ipragliflozin [28], empagliflozin [29,30,31], and canagliflozin [32, 33] in diabetic mice. Likewise, the findings from ex vivo studies indicated that various SGLT-2 inhibitors could induce direct vasorelaxation [26, 34,35,36]. This beneficial effect on endothelial cell function points to several potential mechanisms of vasodilation, including modulation of adhesion molecules, attenuation of inflammation, activation of eNOS phosphorylation, potassium/calcium channel mediation independent of the endothelium;,decreased cardiac macrophage infiltration, and reduced oxidative stress. This beneficial effect could be due to the sustained reduction in plasma glucose concentration, which was observed even during shorter treatment periods, suggesting the reliability and effectiveness of glucose-lowering therapy with SGLT-2 inhibitors. However, given that the ApoE−/− mice used in the study by Gaspari et al. [26] were nondiabetic and did not exhibit a change in glucose metabolism induced by dapagliflozin, it remains uncertain whether this class of drug exerts a direct effect on blood vessels [26]. A recent study found a significant increase in sodium-glucose transporter-1 (SGLT1) and SGLT-2 expression in high glucose-treated porcine endothelial cells [37]. Thus, it is possible that the direct effect of SGLT-2 inhibitors, via inhibition of SGLT-2 in the vascular wall, contributes to improvement in endothelial function.

The ex vivo and in vitro studies reviewed support a possible class effect of the SGLT-2 inhibitors on the regulation of endothelial function that includes the reduction of inflammatory cytokine secretion and hyperglycemia-mediated protein expression, as well as improved viability of hyperglycemic endothelial cells. Overall, the beneficial effects of SGLT-2 inhibitors on endothelial dysfunction appear to be consistent across all of the outcomes measured, such as vascular function and oxidative stress. None of the studies investigated reported any adverse effects related to treatment with SGLT-2 inhibitors. Oxidative stress is known to lead to cell and tissue damage via the production of ROS, such as active oxygen and free radicals, with oxidative stress markers, including thiobarbituric acid reactive substances (TBARS), reported to be significantly elevated in people with T2D with non-alcoholic fatty liver disease [38]. In vitro experiments have demonstrated attenuation of protein kinase B (Akt) and eNOS phosphorylation in HUVECs treated with methylglyoxal, the precursor of AGEs, indicating that this effect seems to be at least partially attributable to the improvement in eNOS function in a hyperglycemic state [28]. It has been argued that this anti-oxidant effect is most likely due to the inhibition of NADPH oxidase activity and a decreased serum level of the AGE precursor methylglyoxal [31, 39]. However, the glucose-lowering effect of SGLT-2 inhibitors in diabetic mice significantly decreased oxidative stress, as determined by urinary excretion of 8-oxo-2′-deoxyguanosine and in cardiac and vascular tissue, whole blood, aorta, and the heart [28].

Inflammation has been implicated in vascular dysfunction in diabetes [40]. As a result, several studies have examined the ideal approach to prevent and manage this inflammatory response [41, 42]. Anti-inflammatory effects of SGLT-2 inhibitors were previously observed in diabetic nephropathy models, and subsequently proposed to occur via a suppression of the RAGE pathways [43]. This, in turn, has led to many further potential pathways being proposed, ranging from direct effects on the expression/release of pro-inflammatory mediators or indirect effects via oxidative balance, hyperglycemia-induced cytokine production, immune system function, and/or obesity‐related inflammation [44]. Systemic administration of the SGLT-2 inhibitors empagliflozin, ipragliflozin, or luseogliflozin led to markedly reduced expression of pro-inflammatory adhesion markers and cytokines, such as IL-6, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) in diabetic rodent models [28, 45,46,47]. Canagliflozin administration inhibited inflammation in metabolic tissue of mice fed a high-fat diet. In keeping with these previous observations, an in vitro study showed that dapagliflozin treatment inhibited TNFα induction of ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) protein expression in human endothelial cells [45]. Similarly, dapagliflozin inhibited IL-1β-stimulated MCP-1 and IL-6 secretion [26]. Taken together these in vivo and in vitro results strongly suggest direct underlying mechanisms of action of SGLT-2 inhibitors on endothelial function that are independent of beneficial glucose-lowering effects [26].

The doses of SLGT-2 inhibitors used in these studies were determined from the therapeutic dose required to reach a peak concentration, or in the case of in vitro studies, the concentration that reflected the maximum plasma concentration. Therefore, for example, canagliflozin, which reaches a peak concentration of about 7 μmol/l in human plasma, was employed at a therapeutically relevant concentration of 10 μmol/l, while dapagliflozin and empagliflozin, which produce peak concentrations of 1–2 μmol/l, were employed at doses of 1 μmol/l [45].

These observations from animal and cell model experiments are supported by clinical studies, as a reduction in biomarkers of cardiovascular inflammation (namely, highly sensitive C-reactive protein) was observed in diabetic patients treated with dapagliflozin [48]. The cardiovascular benefits observed in multiple clinical trials were seen to occur independently of glycemic or lipid control [49]. The underlying pathophysiological mechanisms of the improved cardiovascular outcomes observed was hypothesized to be due to the diuretic (and, therefore, anti-hypertensive) effects of SGLT-2 inhibitors [50]. In addition, the ‘thrifty substrate’ hypothesis proposes a shift in metabolism from glucose and lipids to ketone bodies as a direct effect of SGLT-2 inhibition [51]. Mudaliar et al. [49] argue that although diuretic and vascular endothelium effects could have an impact on cardiovascular improvements, such considerable benefits are highly unlikely in the short period of 3 months. Notably, the vascular dysfunction associated with T2D was initially thought to occur due to hyperglycemia, as a result of interactions between various pathways [52]. Arguably, the evidence from the experimental studies reported in this review point towards SGLT-2 inhibitors exerting additional benefits beyond glucose control.

Limitations

Due to insufficient or poor-quality methodological reporting of the included studies, as well as considerable heterogeneity, the observations reported should be interpreted with caution. Furthermore, although well-established tools exist for the assessment of methodology quality of human clinical trials, there are currently no validated or standardized grading scales available for the assessment of methodological quality in animal studies. Another limitation of our review may be the inclusion of only published studies, which may be a potential source of publication bias [53]; unpublished results or conference abstracts may have reported negative findings.

Conclusions

In this systematic review, we have demonstrated that experimental evidence suggests that SGLT-2 inhibitors are effective at attenuating vascular dysfunction in preclinical models, with complex underlying mechanisms that may act independently to hyperglycemia-related benefits. The evidence from these preclinical studies supports the demonstrated improved vascular functional outcomes of human clinical trials. Further studies are needed to clarify how these compounds are acting in the vasculature and to discover their target sites of action.