A. Four groups of patients with type 2 diabetes mellitus with high or very high cardiovascular risk or heart failure with preserved ejection fraction (HFpEF) will be studied before and at 6 and 12 months of treatment:

• 60 patients treated with a combination of GLP1 analogue and SGLT2 inhibitor ± metformin
• 60 patients treated with GLP-1 agonist as a second step after metformin
• 60 patients treated with SGLT2 inhibitor as a second step after metformin
• 60 patients treated with a combination of insulin and other antidiabetic agents (metformin - DPP4 inhibitors) Individuals will be equal distributed as far as age, gender and body mass index concerned. In addition, patients suffered from kidney disease and retinopathy are excluded.

Disordered glucose tolerance in patients with type 2 diabetes mellitus is multifactorial and includes dysfunction not only of β-cells of the pancreas, liver and muscles, but also of the adipose tissue, gastrointestinal tract, kidney, α-cells of the pancreas and the brain. Therefore, it is likely that the use of drugs that act on multiple aspects of the pathophysiology of diabetes will have beneficial effects on glycemic control and outcome of patients.In addition to the many possible causes of glucose deregulation, people with type 2 diabetes have comorbidities such as hypertension, hyperlipidemia and obesity resulting in increased cardiovascular risk. Optimal treatment of hyperglycaemia and cardiovascular risk factors is necessary to reduce adverse events. Although the initial approach is the modification of dietary factors (physical activity, diet, smoking cessation), the progressive nature of type 2 diabetes ultimately leads to the use of multiple therapies. Guidelines for the treatment of patients with type 2 diabetes with high HbA1c are initial double-combined therapy or triple combination if glycemic control is not achieved after 3 months. Although insulin is usually included in such combinations, additional treatment options are required for patients with high HbA1c who also need to lose weight and avoid hypoglycaemia. It is likely that the outcome of patients could be improved by using drugs targeting different aspects of the disease (glycemia, body weight, blood pressure, lipids).

Over the last decade, two classes of antidiabetic drugs have been added to weight loss, improved cardiovascular risk factors and a low risk of hypoglycemia: glucagon-like peptide-1 agonists (GLP-1, glucagon like peptide-1) and inhibitors of the sodium glucose subtype 2 (SGLT2, sodium-glucose co-transporters). Recent studies have shown that these two drugs have cardioprotective effects possibly through different mechanisms of action, while their complementary mechanical, pharmacological and clinical effects make their combination (in addition to metformin) potentially beneficial in type 2 diabetes patients. Specifically, SGLT2 inhibitors manifest their cardioprotective activity by improving hemodynamic parameters, while GLP-1 agonists through antiatherogen / anti-inflammatory mechanisms.

The increased arterial stiffness with the associated increase in the range of reflected pressure waves, determines not only the augmentation of systolic pressure but also the rate of increase which depends on age. Increased levels of pulse pressure (indirect atherosclerotic index) and pulse wave velocity (PWV), a reliable measure of arterial stiffness, predict future cardiovascular events. Endothelial dysfunction is associated with the presence of atherosclerosis and is also considered as an indicator of the early changes preceding it. The role of impaired endothelial function of large arteries seems to be significant in the pathogenesis of cardiovascular disease. Endothelial glycocalyx is a glycoprotein layer that covers the surface of endothelial cells and regulates arterial wall permeability and the interaction of endothelial cells with circulating blood cells. Inflammatory or atherogenic stimuli, such as hyperglycemia, lead to glucocalyx disorder and increased vascular sensitivity to further atherogenic stimuli. The two-dimensional speckle tracking ultrasound allows accurate estimation of left ventricular distortion, which is disordered in diabetics in relation to healthy subjects.

Hypothesis of the proposed study:

Endothelial function, arterial stiffness and left ventricular deformation are improved in patients with type 2 diabetes with high or very high cardiovascular risk or heart failure with preserved ejection fraction (HFpEF) treated with GLP-1 agonist and SGLT2 inhibitor compared to either one or both combination of insulin and other antidiabetic tablets.

Aim of the study:

The aim of this study is to investigate the effect of GLP-1 agonist, SGLT2 inhibitor and their combination on endothelial function, arterial stiffness, central hemodynamic characteristics and left ventricular deformation in patients with type 2 diabetes with high or very high cardiovascular risk or HFpEF and compared with the combination of insulin and other antidiabetic tablets.

Materials and methods :

A. Four groups of patients with type 2 diabetes mellitus with high or very high cardiovascular risk or HFpEF will be studied before and at 6 and 12 months of treatment:

• 60 patients treated with a combination of GLP1 analogue and SGLT2 inhibitor ± metformin
• 60 patients treated with GLP-1 agonist as a second step after metformin
• 60 patients treated with SGLT2 inhibitor as a second step after metformin
• 60 patients treated with a combination of insulin and other antidiabetic agents (metformin - DPP4 inhibitors) Individuals will be equal distributed as far as age, gender and body mass index concerned. In addition, patients suffered from kidney disease and retinopathy are excluded.

B. All patients will be submitted to an echocardiographic study in order to estimate the total left ventricular and atrial myocardial global longitudinal strain (GLS) as well as the twisting-untwisting of the left ventricle using speckle tracking imaging. In addition, pulse wave velocity (PWV, measured in m/s), pulse wave augmentation index [AIx %, which is calculated by the formula (P2-P1)/PP x 100, where P1 stands for peak systolic pressure, P2 stands for second peak systolic pressure due to wave reflection and PP stands for Pulse Pressure], central systolic blood pressure (SBPao, measured in mm Hg) and central pulse pressure (PPao, measured in mm Hg) with Arteriograph, Mobilograph and Complior, and perfused boundary region (PBR) of sublingual vessels (5-25 μm in size) using a high-resolution camera with Sideview Darkfield Imaging technique (Microscan, Glucockeck). PBR consists the cell-free space which is formed from the separation of red blood cells from plasma at the surface of the endothelial glycocalyx. Increased PBR is considered to be an accurate indicator of the reduction of endothelial glycocalyx thickness due to plasma penetration into the glycocalyx.

Μalondialdehyde (MDA), protein carbonyls (PC) as oxidative stress markers, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, thrombomodulin, nitrites and nitrates, N-terminal pro B-type natriuretic peptide (NT-proBNP), growth differentiation factor (GDF)-15, mitochondrial-derived peptide-c (MOTS-c), blood glucose, glycosylated hemoglobin HbA1c and a full lipidemic profile will be measured before and at 6 months and at 12 months of treatment.

Also, the incidence of non-fatal major cardiovascular events (myocardial infarction, heart failure hospitalization, ischemic stroke) will be nored via performing telephonic follow-up during a long-term follow-up of six years. Accordingly, a cost analysis will be conducted to evaluate and compare the economic outcomes of the four treatment arms.

The examination will be carried out at the Laboratory of Preventive Cardiology of the 2nd Department of Cardiology of the University of Athens at "Attikon" Hospital. Τhe investigators will also compare the participants with history of coronary artery disease with those without hisotry of coronary artery disease.

C. For the statistical analysis of the results, the significance of each intervention will be measured by t-test and the 4 groups will be compared by ANOVA (post-hoc comparisons with Bonferroni correction). Non-parametric tests will be used for non-Gaussian distributions. Forty individuals should be included in each group in order the results being statistically significant. In a previous study, myocardial global longitudinal strain of the left ventricle showed normal distribution with standard deviation 10. A true difference in mean left ventricular myocardial distortion between the intervention group (GLP-1 agonist compined with SGLT2 inhibitor) and a control one (combination of insulin and other antidiabetic tablets) which is calculated of -6.344% or 6.344% will be calculated with probability 0.8. The probability of a type I error if the null hypothesis is that the mean difference between the two groups is equal, is equivalent to 0.05.

Significance/future prospects:

This study is expected to open the horizons of better understanding the cardiovascular benefits of GLP-1 agonist, SGLT2 inhibitor and their combination in patients with type 2 diabetes mellitus. Moreover, the contribution of the investigator's results, in a clinical-level could be the use of new drug therapies that reduce both microvascular complications by reducing glycemia and macrovascular complications (myocardial infarction, stroke, cardiovascular mortality) as the first treatment line in patients with type 2 diabetes with high or very high cardiovascular risk.

Inclusion Criteria:

• Subject has type 2 diabetes mellitus
• Subject has high or very high cardiovascular risk
• Subject has heart failure with preserved ejection fraction

Exclusion Criteria:

• Subject has type 1 diabetes mellitus
• Subject has kidney disease
• Subject has retinopathy

240 individuals (60 subjects in each group)