Introduction
Treatment of type 2 diabetes should aim at improving vascular structure and function in the micro- and macrocirculation besides metabolic control [1]. Arterial stiffness, a key parameter of vascular changes, is characterized by an increased pulse wave velocity along the arterial tree of both the forward and backward (reflected) pulse wave leading to increased central systolic blood pressure and elevated central pulse pressure [2, 3]. Central systolic blood pressure is the integral of various components of arterial stiffness, an important surrogate parameter of afterload, and strongly linked to future cardiovascular outcome [4, 5]. Likewise, central pulse pressure has been shown to be superior in the prediction of cardiovascular events compared to measurements of pulse pressure at the brachial level, and there is evidence for an association between both forward and backward wave amplitudes and increased risk for incident cardiovascular disease and all-cause mortality [5,6,7].
In the EMPA-REG OUTCOME study (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients) treatment with the selective sodium-glucose cotransporter 2 inhibitor (SGLT2-inhibitor) empagliflozin reduced the primary combined cardiovascular end point as well as secondary end points of hospitalization due to heart failure, cardiovascular morbidity, total mortality and renal end points [8]. The underlying pathophysiologic mechanisms are currently under intensive discussion, but the crucial question about the pivotal mechanism causing the reduced cardiovascular death rate and total mortality still remains to be elucidated. Interestingly, the benefits observed in the EMPA-REG OUTCOME study were documented in a population in whom cardiovascular risk factors, including blood pressure and dyslipidemia were well treated with the use of renin–angiotensin–aldosterone system inhibitors, statins and acetylsalicylic acid. The authors of the EMPA-REG OUTCOME study mention changes in arterial stiffness among others as possible mechanisms [8]. Most recently we have shown that empagliflozin improves arterial stiffness in a double blind, placebo controlled, crossover clinical trial including 71 patients with type 2 diabetes mellitus [9]. The aim of the current analysis is to identify potential determinants for the improvement of arterial stiffness observed during empagliflozin therapy.
Methods
Study design
This is a prespecified analysis of patients, who participated in an investigator initiated prospective, double blind, randomized, placebo-controlled, cross-over, interventional single center study conducted at the Clinical Research Center of the Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Germany (http://www.crc-erlangen.de) (http://www.ClinicalTrials.gov: NCT02471963). The principal findings of the clinical trial have been previously published [9]. Participants were recruited by advertising in local newspapers in the area of Erlangen-Nuremberg, Germany, and eligible participants were enrolled consecutively. Written informed consent was obtained before study inclusion. The study protocol was approved by the local ethics committee (University of Erlangen-Nuremberg), and the study was conducted in accordance with the Declaration of Helsinki and the principles of good clinical practice guidelines.
Analysis of changes in variables
On the basis of evidence from previous studies [10] the following mediators involving several mechanistic categories have been chosen for analysis: Glucose control (HbA1c, fasting plasma glucose), volume status (copeptin, hematocrit), sympathetic activation (heart rate), lipids (LDL-cholesterol), vascular tone (systolic 24-h ambulatory blood pressure), inflammation [high sensitive CRP (hsCRP)] and other (uric acid).
Study population
Characteristics of the study population have been previously published [9]. In brief, female and male patients aged between 18 and 75 years with diagnosed type 2 diabetes mellitus, defined by fasting glucose ≥ 126 mg/dl or HbA1c ≥ 6.5% (48 mmol/mol) or on blood glucose lowering medication, were included in the study. Estimated glomerular filtration rate (eGFR) had to be ≥ 60 ml/min/1.73 m2. Patients who used insulin, glitazone, gliptine or SGLT-2 inhibitor therapy within the past 3 months and patients with more than one oral blood glucose lowering medication were excluded. Patients on any antidiabetic agent had at least a 4 weeks wash-out phase prior to the baseline examination. Other key exclusion criteria were HbA1c ≥ 10% (86 mmol/mol), fasting plasma glucose > 240 mg/dl, any history of stroke, transient ischemic attack, instable angina pectoris or myocardial infarction within the last 6 months prior to study inclusion, uncontrolled hypertension (office blood pressure ≥ 180/110 mmHg), congestive heart failure (CHF) NYHA stage III and IV, use of loop diuretics and pregnancy. Eight patients from the original study cohort (71 patients) were excluded because they presented with hsCRP values above 5 mg/l. Another five patients from the original study cohort showed a clinical infect correlate such as cystitis, vaginal infection, cold or gout. Even though these patients did not present with hsCRP above 5 mg/dl, they were excluded from our analysis based on the clinical investigation. Conventional blood pressure and heart rate measurements in the office and during 24-h were carried in standard fashion by validated devices.
Treatment
Patients underwent a run-in/wash-out phase of 4 weeks if pretreated with any antidiabetic agent, or 2 weeks if not pretreated with any antidiabetic agent and afterwards were randomized to either empagliflozin 25 mg orally once daily or placebo. Following 6 weeks of treatment with either of these drugs, the patient underwent a wash-out phase of 1 week. Then the patient received the other substance for another 6 weeks of intervention (cross-over).
Assessment of vascular function and central hemodynamics
To derive the central (aortic) arterial waveform, a validated system (SphygmoCorTM System; AtCor Medical, Sydney, Australia) was applied [5, 7, 20] by recording radial artery waveforms from the radial artery at the wrist, using high-fidelity applanation tonometer (Millar Instruments, Houston, Tex.) [5, 6, 20]. Corresponding central (aortic) waveforms were then automatically generated from the radial artery waveform by a validated transfer function [5, 7]. This allows obtainment of the following parameters: central systolic pressure, central pulse pressure, central augmentation pressure, central augmentation index (cAIx), cAIx normalized to a heart rate of 75 beats per minute (cAIx@75), pulse pressure amplification, as well as forward and backward reflected wave amplitude.
Assessment of blood pressure and potential determinants of vascular function
Office blood pressure measurement was performed in a standardized fashion according to guideline recommendations [4]. During 24-h ambulatory daily-life conditions, brachial systolic and diastolic blood pressure, pulse pressure and heart rate were measured by the Mobilograph (IEM, Aachen, Germany). The technology has been validated previously [5, 11, 12].
All blood samples were measured centrally at the biochemistry laboratory of the University of Erlangen-Nuremberg according to established methods. In particular, hsCRP was measured via particle-reinforced nephelometry. Copeptin was analysed by lab MVZ Dr. Limbach GbR using Time Resolved Amplified Cryptate Emission method. Coefficient of variation of measurements was below 10%.
Statistical methods
Normal distribution of data was confirmed by histogram and Kolmogorov–Smirnov test prior to further analysis. Data were compared by paired and unpaired t-tests and expressed as mean ± standard deviation (SD) in text and tables. A two-sided p-value < 0.05 was considered statistically significant. Bivariate correlation analyses were performed using Pearson’s test. Multivariate regression analysis was performed including the parameters sex, age and change of the following parameters under treatment with empagliflozin: HbA1c (model 1), copeptin concentration, hematocrit, 24-h ambulatory heart rate, LDL-cholesterol, uric acid, 24-h ambulatory blood pressure and hsCRP. A second multivariate regression analysis model included besides the other previously mentioned parameters fasting plasma glucose instead of HbA1c (model 2). Vascular stiffness parameters entered our model as an independent variable, namely as first change in central systolic blood pressure, second change in pulse pressure, third change in forward wave amplitude and fourth change in reflected wave amplitude. A separate multiple regression analysis was performed for each of the four independent variables mentioned. Potential collinearity between the dependent variables in our model has been excluded by calculating correlation coefficients between the dependent variables. There is no correlation between change in systolic 24-h ambulatory blood pressure and sex (r = − 0.006, p = 0.967), age (r = − 0.008, p = 0.955) and change of the following parameters: uric acid (r = 0.104, p = 0.443), hsCRP (r = − 0.027, p = 0.844), LDL-cholesterol (r = − 0.204, p = 0.129), fasting plasma glucose (r = 0.165, p = 0.220), hematocrit (r = 0.93, p = 0.490) and copeptin (r = − 0.074, p = 0.591). All analyses were performed using IBM SPSS Statistics 22 (SPSS Inc, Chicago, IL/USA).
Results
Study population
Characteristics of the study cohort have been previously published [9]. In brief, the study cohort comprised 58 patients with type 2 diabetes mellitus (all Caucasians, 59% male) with mean age of 62 ± 7 years, HbA1c level of 6.69 ± 0.8% (50 ± 8.7 mmol/mol), office blood pressure 128 ± 13/78 ± 7.2 mmHg, 24-h ambulatory blood pressure 129 ± 10/79 ± 6.3 mmHg, body weight 87.7 kg and body mass index of 29.5 ± 3.9 kg/m2. None of the patients were on any antidiabetic medication (85% were on metformin prior to study inclusion), whereas 50 patients received antihypertensive medications at baseline (84% received an angiotensin receptor blocker or an ACE-inhibitor), without any changes in medication throughout the study period.
