Effects of incretin treatment on cardiovascular outcomes in diabetic STEMI-patients with culprit obstructive and multivessel non obstructive-coronary-stenosis

Background No proper data on prognosis and management of type-2 diabetic ST elevation myocardial infarction (STEMI) patients with culprit obstructive lesion and multivessel non obstructive coronary stenosis (Mv-NOCS) exist. We evaluated the 12-months prognosis of Mv-NOCS-diabetics with first STEMI vs.to non-diabetics, and then Mv-NOCS-diabetics previously treated with incretin based therapy vs. a matched cohort of STEMI-Mv-NOCS never treated with such therapy. Methods 1088 Patients with first STEMI and Mv-NOCS were scheduled for the study. Patients included in the study were categorized in type 2 diabetics (n 292) and non-diabetics (n 796). Finally, we categorized diabetics in current-incretin-users (n 76), and never-incretin-users (n 180). The primary end point was all cause deaths, cardiac deaths, and major adverse cardiac events (MACE) at 12 months of follow up. Results The study results evidenced higher percentage of all cause deaths (2.2% vs. 1.1%, p value 0.05), cardiac deaths (1.6% vs. 0.5%, p value 0.045), and MACE (12.9% vs. n 5.9%), p value 0.001) in diabetic vs. no diabetic patients at 12 months follow up. Among diabetic patients, the current vs never-incretin-users, did not present a significant difference about all cause of deaths, and cardiac deaths through 12-months. The MACE rate at 1 year was 7.4% in diabetic incretin-users STEMI Mv-NOCS patients vs. 12.9% in diabetic never-incretin-users STEMI-Mv-NOCS patients (p value 0.04). In a risk-adjusted hazard analysis, MACE through 12 months were lower in diabetic STEMI-Mv NOCS incretin-users vs never-incretin-users patients (HR 0.513, CI [0.292–0.899], p 0.021). Consequently, lower levels of glucagon-like peptide 1(GLP-1) were predictive of MACE at follow up (HR 1.528, CI [1.059–2.204], p 0.024). Conclusion In type 2 diabetic patients with STEMI-Mv-NOCS, we observed higher incidence of 1-year mortality and adverse cardiovascular outcomes, as compared to non-diabetic STEMI-Mv-NOCS patients. In diabetic patients, never-incretin-users have worse prognosis as compared to current-incretin-users. Trail registration Clinical trial number: NCT03312179, name of registry: clinicaltrialgov, URL: clinicalltrialgov.com, date of registration: September 2017, date of enrollment first participant: September 2009


Background
In general population, non-obstructive (< 50% stenosis diameter and flow fractional reserve > 0.80) noninfarcted related coronary diseases was common among patients presenting with ST-segment elevation myocardial infarction (STEMI), and were no associated with a significant increase in mortality [1]. In diabetic patients, there is a higher prevalence of multivessel disease, and of non obstructive coronary artery lesions [2,3]. To date, STEMI diabetic patients with culprit obstructive lesion and multivessel non obstructive coronary stenosis (Mv-NOCS) represent a conundrum because no proper data regarding their prognosis and management exist. So far, incretin-based therapies have shown a broad range of unique cardiovascular actions translating into cardiovascular protection [4]. Therefore, given the paucity of data in this setting, we evaluated the 12-months prognosis of Mv-NOCS-diabetics with STEMI as compared with a matched cohort of non-diabetic patients. In this research we studied clinical outcomes after first STEMI event in STEMI-Mv-NOCS diabetics vs. non-diabetics, and then divided in diabetic incretin-users vs. diabetic never-incretin-users. As first, we compared number all cause of deaths, cardiac deaths, and of major adverse cardiac events (MACE) through 12 months in diabetic STEMI-Mv-NOCS patients vs. non-diabetic STEMI-Mv-NOCS patients. Secondary, we divided diabetic STEMI-Mv-NOCS incretin users vs. never-incretin-users, and we assessed all cause deaths, cardiac deaths, and MACE through 12-months of follow up. Our study hypothesis was that, diabetics STEMI-Mv-NOCS may have worse prognosis after first STEMI event as compared to non diabetics. Secondary, STEMI-Mv-NOCS diabetics current-incretin-users may present a significantly lower rate of MACE through 12 months as compared to a matched cohort of STEMI-Mv-NOCS-diabetics never treated with such therapy. Therefore, incretin therapy may represent a validate and innovative treatment to reduce worse prognosis in a population of STEMI-Mv-NOCS diabetics. Indeed, incretin therapy may improve clinical outcomes, ameliorating the prognosis of STEMI-Mv-NOCS diabetic patients.

Methods
Consecutive 796 non diabetic and 292 diabetic patients with first STEMI and no-altered fractional flow reserve (FFR > 0.80) of Mv-NOCS (20-49% luminal stenosis), referred for coronary angiography within 12 h of clinical presentation of the clinical event, were entered in a database prospectively. STEMI was diagnosed according to international guidelines by evidence of myocardial injury (defined as an elevation of cardiac troponin values with at least one value above the 99th percentile upper reference limit), associated to symptoms consistent with myocardial ischemia, as persistent chest discomfort or other symptoms suggestive of ischemia (shortness of breath, nausea/vomiting, fatigue, palpitations, or syncope), and ST-segment elevation in at least two contiguous leads ≥ 2.5 mm in men < 40 years, ≥ 2 mm in men ≥ 40 years, or ≥ 1.5 mm in women in leads V2-V3 and/or ≥ 1 mm in the other leads [5]. In these patients, we performed an early, and immediate coronary angiography followed by percutaneous coronary intervention to have a rapid restoration of epicardial blood flow in the infarct related artery [5].Therefore, patients with no coronary disease detected by coronary angiography, presence of obstructive and Mv-obstructive stenosis, left ventricular ejection fraction less than 25%, previous myocardial infarction, previous PCI or/and coronary by-pass grafting, Tako-tsubo cardiomyopathy, myocarditis, acute or chronic infection or inflammatory diseases, hematologic disorder, malignancies, end-stage liver or renal disease, and use of steroid therapy or chemotherapy were excluded. Subjects were categorized in non-diabetic and diabetic patients [6]. Furthermore, the diabetic patients answered a specific questionnaire about medicines used for diabetes treatment before the beginning of the study, the dates of the beginning and the end of treatment, the route of administration, and the duration of use. Information from the medicine inventory during the study and this specific questionnaire was used to classify the subjects. The patients with diabetes who never used incretin, such as glucagon-like peptide 1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, were classified as "never-incretin-users. " The patients with Conclusion: In type 2 diabetic patients with STEMI-Mv-NOCS, we observed higher incidence of 1-year mortality and adverse cardiovascular outcomes, as compared to non-diabetic STEMI-Mv-NOCS patients. In diabetic patients, neverincretin-users have worse prognosis as compared to current-incretin-users. Trail registration Clinical trial number: NCT03312179, name of registry: clinicaltrialgov, URL: clinicalltrialgov.com, date of registration: September 2017, date of enrollment first participant: September 2009 Keywords: Type 2 diabetes, STEMI, Non-obstructive coronary stenosis diabetes who had already used, for at least 6 months, GLP-1 agonists or DPP-4 inhibitors were classified as "current incretin-users". Therefore, upon emergency wards admission, all patients were assigned to undergo prompt coronary angiography. This was a multi center prospective "real world" study conducted at University of Campania "Luigi Vanvitelli", Cardarelli hospital, and Monaldi hospital (Naples, Italy), between July 2009 and July 2016. The study was conducted in accordance with the Declaration of Helsinki. The Ethics Committees of all participating institutions approved the protocol (Ethic Committee University of Campania "Luigi Vanvitelli" number: 1177). All patients were informed about the study nature, and gave their written informed, and signed consent to participate in the study. The study was retrospectively registered.

Study protocol Laboratory analysis
After an overnight fast, plasma glucose and HbA1c levels were measured by enzymatic assays in the hospital chemistry laboratory. GLP-1 levels (Active GLP-1  Specific ELISA Kit; Epitope Diagnostics) were measured after an overnight fast (at 8:00 A.M.) and after breakfast in diabetic patients. A standardized hospital breakfast for ACS patients contained 419 kcal (57% carbohydrate, 17% protein, and 26% fat). After breakfast, blood samples for the measurement of GLP-1 were obtained every 30 min over a 2-h period. The mean of the four GLP-1 evaluations was defined as the postprandial GLP-1 value. The standardized meal tolerance test and baseline evaluations were performed 5 days after STEMI.

Inflammatory markers
Routine analyses and inflammatory status, as ratio between macrophages 1 (CD68) and macrophages 2 (soluble-CD163) (M1/M2 ratio), and high sensitivity C-reactive protein (hs-CRP), were obtained on admission before coronary angiography and before full medical therapy was started.

Quantitative coronary angiography
Upon emergency wards admission, all patients were assigned to undergo prompt coronary angiography. The analyses of all angiographic data before were performed by three interventional cardiologists (M.F., M.C. and C.P.), and followed by percutaneous coronary intervention (PCI) with angioplasty and direct stenting of culprit vessel lesion [6]. Coronary stenting of culprit coronary vessel lesion was the technique of choice for all admitted patients [6]. Therefore, admitted STEMI diabetic and non diabetic patients received preferably primary PCI (92%, n 1001). On other hand, a low percentage of STEMI patients (8%, n 87) were diagnosed in non-PCI-capable hospitals, and they did not receive primary PCI. In these patients, physicians performed a thrombolytic reperfusion therapy. Moreover, 69 patients (79%) received rescue PCI, and 56 patients (65%) were treated by stent implantation. After that, these cardiologists blinded to patient categorization, reviewed selecting cases with Mv-NOCS, as coronary vessels with no-altered fractional flow reserve (FFR > 0.80), and associated to 20-49% luminal stenosis [5,7].

Coronary care unit/intensive cardiac care unit
All treated patients were then monitored and managed in Intensive Care Unit following reperfusion, by continuous monitoring, and specialized care [6] for STEMI and related acute complications (arrhythmias, heart failure, etc.) treatment.

Echocardiographic assessment
At admission patients underwent two-dimensional echocardiography as previously described [8]. This exam was used to asses heart chambers morphology, volumetry, wall contraction, cardiac valves morphology and function, and ejection fraction [8]. To asses heart chambers wall contractility we used scheme as previously described [8]. This exam was used at admission to confirm STEMI diagnosis, and during follow up to stage STEMI disease progression (6 and 12 months after STEMI).

Follow-up
After discharge from the hospital, all patients were managed and followed quarterly for 12 months after event, as outpatients, to perform clinical evaluation, routine analyses and cardiovascular evaluation (ECG, exercise ECG, echocardiography, exercise myocardial scintigraphy), as well as with the goal to maintain HbA1c level at < 7%, fasting blood glucose level of 90-140 mg/dl, and postprandial blood glucose level of < 180 mg/dl. The mean follow-up was 16 ± 3 months. Follow-up visits were performed in our outpatients clinic.

Cardiovascular endpoints
The study end point was all cause deaths, cardiac deaths, and major adverse cardiac events (MACE) at 12 months follow up.

Statistical analysis
SPSS version 23.0 (IBM statistics) was used for all statistical analyses. Categorical variables were presented as frequencies (percentages) and continuous variables as mean ± SD. For the general population of diabetics and non diabetics we calculated a sample size using a power of 80% and confidence of 95%. For comparison among diabetic never-incretin-users and diabetic current-incretin-users, a propensity score matching (PSM) was developed from the predicted probabilities of mortality and MACE by a multivariable logistic regression model. Diabetic never-incretin-users were matched to diabetic current-incretin-users on the basis of PSM. In all matched patients, the balancing property was satisfied. Overall survival and event-free survival were presented using Kaplan-Meier survival curves and compared using the log-rank test. Univariable Cox models were then used to compare event risks. Within all the diabetic and nondiabetic groups, all cause of deaths, cardiac deaths, and MACE were assessed by using multivariable Cox models with adjustment for statistically different variables at baseline and follow-up: hypertension, dyslipidemia, current smoking, ace-inhibitors, calcium inhibitors, thiazide diuretics, aspirin, statin, BMI, heart rate, HDLcholesterol, LDL-cholesterol, triglycerides levels, hs-CRP, M1/M2, and GLP-1 levels. The resulting hazard ratios (HRs) and 95% confidence intervals (CIs) were reported. To investigate the effects of GLP1 levels on cardiovascular endpoints, we evaluated STEMI outcomes at 1-year follow-up stratified by GLP-1 quartiles. A 2-tailed p value < 0.05 was considered statistically significant.

Discussion
The main results were as follows: first, in a contemporary sample of type 2 diabetic patients with STEMI-Mv-NOCS, we observed higher cumulative incidence of 1-year mortality and adverse cardiovascular outcomes as compared to non-diabetic STEMI-Mv-NOCS patients; second, in PSM diabetic patients, diabetic never-incretin-users have higher number of MACE as compared to diabetic current-incretin-users. The prognosis of patients with NOCS has been evaluated, by a recent study [9], which evidenced that among individuals without known CAD and obstructive CAD, non obstructive plaque presence enhances risk prediction of incident mortality. Moreover, [9,10] among patients with type 2 diabetes, non obstructive and obstructive stable CAD were associated with higher rates of all-cause mortality and major adverse cardiovascular events at 5 years, and this risk was significantly higher than in non-diabetic subjects. However, these studies did not provide any evidence about the influence of STEMI-Mv-NOCS management on outcomes following the cardiac event in diabetic patients. In our study after STEMI, we observed an increased

Table 2 Study endpoints in diabetics vs. overall study population, and in incretin-users vs. never-incretin-users
MACE is for major adverse cardiac events; the symbol "/" is indicating not statistical significant (p value > 0.05) incidence of cardiovascular disease in STEMI-Mv-NOCS patients, both after adjustment for baseline, and follow up cardiovascular risk factors. In this context, the poor outcomes of diabetic STEMI-Mv-NOCS as compared to non-diabetic Mv-NOCS-STEMI, observed in our study, might be explained by an abruptly increment of atherosclerosis in diabetics as compared to a more slow progression of coronary atherosclerosis extension in non-diabetics [11]. In this scenario, the diabetic status may affects several pathogenetic mechanisms, that favor the plaque instability and subsequently plaque rupture in the absence of obstructive coronary stenosis, including inflammation, endothelial dysfunction with the inability to augment coronary flow in response to stress, and coronary vasospasm. Accordingly, our data evidenced more inflammatory cells and CPR levels in diabetic than in Fig. 1 a Kaplan-Meier curve for all cause deaths. In left part all cause deaths cumulative survival curve at 360 days follow up comparing diabetic (green color) vs. non diabetic patients (blue color). In right part all cause deaths cumulative survival curve at 360 days follow up comparing diabetic incretin users (green color) vs. diabetic never-incretin-users patients (blue color). There is a statistical significant higher number of events comparing diabetic vs.non diabetic patients (p value < 0.05). b Kaplan-Meier curve for cardiac deaths. Kaplan-Meier curve for cardiac deaths. In left part all cause deaths cumulative survival curve at 360 days follow up comparing diabetic (green color) vs. non diabetic patients (blue color). In right part all cause deaths cumulative survival curve at 360 days follow up comparing diabetic incretin users (green color) vs. diabetic never-incretin-users patients (blue color). There is a statistical significant higher number of events comparing diabetic vs.non diabetic patients (p value < 0.05) no-diabetic patients ( Table 1). The present findings also show a protective effect of incretin therapies on cardiovascular outcomes in Mv-NOCS diabetic patients after STEMI. Without conditioning cardiac mortality, and all cause of deaths, incretin therapy may affect MACE at 12 months follow up. Indeed, diabetic patients treated with incretin therapies had the lowest incidence of cardiovascular events at the same level of blood glucose levels vs. never-incretin-users. In human randomized, double-blind clinical studies, DPP-4 inhibitors did not appear to reduce the risk of major adverse cardiovascular events among patients with type 2 diabetes without and with established cardiovascular disease [12][13][14]. However, definitive proof of an effect of DPP-4 inhibitors in patients with acute coronary syndrome, as well as in patients with DPP-4 inhibitors therapy before the cardiovascular event is currently lacking. In our study after STEMI, the 1-year follow-up results show a higher reduction in the MACE endpoint in patients previous treated with incretin as compared to patients without incretintherapy despite a similar severity of atherosclerotic disease (coronary stenosis < 50%; FFR > 0.80) at baseline. Moreover, both at baseline and at follow-up the currentincretin-users presented lower levels of inflammatory cells, as reported by a M1/M2 ratio and inflammatory markers as CRP, and higher GLP-1 values (Table 1). Accordingly, human studies showed that sitagliptin, vildagliptin and exenatide [15][16][17], even at a single dose, exert a potent anti inflammatory effect, and that many of these effects were persistent over a period of 12 weeks, thus suggesting that the anti-inflammatory effects of GLP-1-based therapies could help to reduce atherosclerosis progression. This concept has been recently investigated by authors [18], reporting that, in acute coronary syndromes, the cardiovascular outcomes were strictly correlated to postprandial GLP-1 levels independently from endogenous (DPP-4 inhibitors) vs exogenous (GLP-1 agonist) treatments. Therefore, patients assigned to incretin therapy may have a lesser plaque progression to an unstable phenotype, than patients assigned to other anti-diabetic therapies [18]. In our study we evidenced that patients with higher GLP-1 levels had lower number of events. Moreover, we may report a protective cardiovascular effect of GLP-1 agonist therapy on atherosclerotic plaques of patients with diabetes, as previously described [19]. However, these results may be due to the small sample size of study population, and the short time of follow-up duration, and future clinical trial have to assess this research topic.

Conclusion
The novelty of this research is to show "real world data" about clinical outcomes in diabetic STEMI-patients with culprit obstructive lesion and Mv-NOCS treated by incretins vs. standard hypoglycemic drugs. Moreover, diabetics current-incretin-users vs. never-incretin-users presented a significantly lower rate of MACE through 12 months, as represented by the evident significant abrupt decreasing of Kaplan-Meier survival curves free from MACE (Fig. 1). This study result supports incretin therapy as the best treatment of diabetics STEMI-Mv-NOCS patients. Therefore, incretin effect on the control of hyperglycemia homeostasis may be associated to other pleiotropic effects, than playing a decisive rule in the control of atherosclerotic plaque progression, and functionality in diabetic STEMI-Mv-NOCS patients. In conclusion, diabetic STEMI-Mv-NOCS patients show unacceptable rates of adverse cardiovascular events, that may be controlled, and/or reduced by incretin therapy. Indeed, tailored strategies, including incretin-based therapies, should be considered in the treatment of these patients.
(See figure on previous page.) Fig. 2   We have considered as statistical significant a p value < 0.005, with hazard ratio (HR) at 95% of confidence of interval (CI). At multivariable analysis the parameter associated with a statistical significant value (p value < 0.005) has been marked with the symbol*. Bas.Lesion length is indicating basal lesion length; HsCRP is for high sensitivity C reactive protein; Low GLP-1 is indicating lower terzile of GLP-1 (glucagon-like peptide 1) values, as < 20 pg/ml; LVEF is for left ventricle ejection fraction; M1/M2 ratio is the ration between macrophage 1 and macrophage 2 cells. VD-3 is indicating a multivessel coronary disease with 3 coronary vessels (B) Univariate and multivariate analysis of factors to predict cardiac deaths at follow up. We have considered as statistical significant a p value < 0.005, with hazard ratio (HR) at 95% of confidence of interval (CI). At multivariable analysis is the parameter associated with a statistical significant p value (p value < 0.005) has been marked with the symbol*. Bas.Lesion length is indicating basal lesion length; HsCRP is for high sensitivity C reactive protein; Low GLP-1 (glucagon-like peptide 1) is indicating lower terzile of GLP-1values, as < 20 pg/ml; LVEF is for left ventricle ejection fraction; M1/M2 ratio is the ratio between macrophage 1 and macrophage 2 cells. VD-3 is indicating a multi vessel coronary disease with 3 coronary vessels (C) Univariate and multivariate analysis of factors to major adverse cardiac events (MACE) at follow up. We have considered as statistical significant a p value < 0.005, with hazard ratio (HR) at 95% of confidence of interval (CI). At multivariable analysis is the parameter associated with a statistical significant value (p value < 0.005) has been marked with the symbol*. Bas.Lesion length is indicating basal lesion length; HsCRP is for high sensitivity C reactive protein; Low GLP-1 (glucagon-like peptide 1) is indicating lower terzile of GLP-1 values, as < 20 pg/ml; LVEF is for left ventricle ejection fraction; M1/M2 ratio is the ratio between macrophage 1 and macrophage 2 cells. VD-3 is indicating a multivessel coronary disease with 3 coronary vessels