p66Shc gene expression in peripheral blood mononuclear cells and progression of diabetic complications

Patients were recruited at the Diabetes Outpatient Clinic of the University Hospital of Padova between 2011 and 2012. Inclusion criteria were: a diabetes diagnosis according to ADA criteria, age 18–80, both genders. Exclusion criteria were: acute disease or infection; recent (within 3 months) surgery, trauma or cardiovascular event at baseline; immune disorders or organ transplantation; cancer; advanced liver (cirrhosis) or kidney (uremia) disease; pregnancy or lactation; inability to provide informed consent.

We collected the following baseline data for each patient: age, sex, BMI, diabetes duration, HbA1c, systolic and diastolic blood pressure, urinary albumin creatinine ratio (UACR), serum creatinine, lipid profile (total, LDL, and HDL cholesterol and triglycerides), and medications. The estimated glomerular filtration rate (eGFR) was calculated according to the CKD-EPI formula [18]. Hypertension was defined as a systolic blood pressure ≥ 140 mmHg or a diastolic blood pressure ≥ 90 mmHg, or the use of anti-hypertensive medications.

We recorded detailed information on diabetic complications, as follows. Retinopathy was defined on the basis of a digital funduscopic examination and scored remotely by an experienced ophthalmologist, according to the ETDR study [19]. Somatic neuropathy was defined in the presence of typical sensory or motor symptoms, confirmed by clinical examination (vibratory perception threshold and monofilament sensitivity) and eventual determination of neural conduction velocity. Autonomic neuropathy was screened annually using four routine cardiovascular autonomic function tests: deep breathing, lying-to-standing, Valsalva manoeuvre (Neurotester system) and orthostatic hypotension. Nephropathy was defined as either an albumin excretion rate of 30 mg/g creatinine or higher, or as an eGFR of 60 mL/min/1.73 mq or lower. Microangiopathy was defined as the presence of either retinopathy, neuropathy, or nephropathy or a combination thereof.

Coronary artery disease (CAD) was defined as a history of myocardial infarction or angina, or evidence of significant coronary artery disease at coronary angiography. Peripheral arterial disease (PAD) was defined as a history of claudication or rest pain, or significant stenosis in leg arteries. Cerebrovascular disease (CerVD) was defined as symptomatic or asymptomatic stenosis carotid arteries (at least 30% lumen narrowing) or the presence of a past history of stroke/transient ischemic attack. Macroangiopathy was defined as the presence of CAD, PAD, or CerVD, or a combination thereof. At baseline, all patients also underwent additional cardiovascular characterization by a carotid ultrasound with recording of maximal carotid intima-media thickness (IMT) and degree of stenosis, a determination of the ankle-brachial index (ABI), and an echocardiography with recording of systolic dysfunction (an ejection fraction of 50% or lower) and diastolic dysfunction (defined as E:A reversal).

PBMCs were collected from 20 mL of blood over Histopaque-1077 (Sigma-Aldrich, Milano, Italy) as follows. Blood (20 mL) was drawn into tubes containing EDTA, transferred to a 40-mL plastic centrifuge tube, diluted with 20 mL of phosphate buffer saline (PBS) and gently mixed. The diluted blood was gently layered on 5 mL of Ficoll-Paque in a centrifuge tube and centrifuged at 360 g for 50 min at room temperature; the supernatant was carefully aspirated and discarded. The mononuclear cells at the interface with the plasma were pipetted into a plastic centrifuge tube, washed with PBS, and centrifuged twice at 170 g for 10 min. The mononuclear fraction contained 90–93% lymphocytes and 3–5% PBMCs when evaluated by Wright staining.

The primary outcome was a first expanded MACE (major adverse cardiovascular event), defined as cardiovascular death, non-fatal acute myocardial infarction (AMI), non-fatal stroke, unstable angina, unplanned revascularization, or hospitalization for heart failure.

Event definition and adjudication was performed as in our previous pseudo-prospective studies [20]. The cause of death was determined by the principal condition and was considered to be cardiovascular in case of: sudden death; death occurring up to 14 days after an acute myocardial infarction; death occurring in the context of clinically worsening symptoms and/or signs of heart failure; death occurring up to 30 days after a stroke; death due to another documented cardiovascular cause (e.g. dysrhythmia, pulmonary embolism, or intervention). Any death not attributed to a non-cardiovascular cause were presumed to be cardiovascular. Nonfatal myocardial infarction was defined in the presence of at least 2 of the following 3 criteria: cardiac biomarker elevation; electrocardiographic changes consistent with new ischemia; imaging evidence of new non-viable myocardium or new wall motion abnormalities. Nonfatal stroke was defined as the rapid onset of a focal/global neurological deficit (change in level of consciousness, hemiplegia, hemiparesis, numbness or sensory loss affecting one side of the body; dysphasia/aphasia; hemianopia, other new neurological sign/symptom), with a duration of ≥ 24 h (< 24 h if the event was associated with pharmacologic treatment, or in the presence of available brain imaging showing new haemorrhage or infarct, or resulting in death), and confirmed by a neurology specialist or by brain imaging. Unstable angina was defined as resting, new onset, or worsening angina, in the absence of elevation in cardiac biomarkers, and in the presence of new or worsening ST-T changes on ECG, or evidence of ischemia by cardiac imaging, or angiographic evidence of ≥ 70% stenosis in an epicardial coronary artery. Heart failure was defined in the presence of typical clinical manifestations or their worsening (dyspnoea, orthopnoea, paroxysmal nocturnal dyspnoea, oedema, pulmonary basilar crackles, jugular venous distension, third heart sound or gallop rhythm, radiologic evidence of worsening heart failure), needing new therapy or up-titration of doses (diuretics, inotropes, vasodilators), eventually supported by changes in biomarkers (e.g. brain natriuretic peptides). Unplanned coronary, peripheral, or carotid revascularization was considered if occurred > 6 months after baseline.

The secondary outcome was development of new microangiopathy or macroangiopathy. New-onset macroangiopathy was defined as the new occurrence of CAD, PAD, or CerVD in patients who were free from any macroangiopathy at baseline. New-onset microangiopathy was defined as the new occurrence of retinopathy, neuropathy, or nephropathy in patients who were free from any microangiopathy at baseline.

Based on the baseline clinical characteristics of patients, we assumed an annual rate of cardiovascular events and death (MACE) of at least 4%. Thus, with n = 100 patients and a follow-up of 5 years, we estimated that at least 20 MACE had to be recorded: the study had 80% power to detect a significant higher incidence of MACE in the group with above median p66Shc expression (n = 50) versus those with below median p66Shc expression (n = 50) if the true relative risk was > 3.0.

Normality of continuous data was checked using the Kolmogorov–Smirnov test. Normal continuous variables are expressed as mean ± standard deviation and non-normal variables were log-transformed before analysis. Categorical variables are expressed as percentage. Comparisons in continuous variables between two groups were analysed using unpaired 2-tail Student’s t test, whereas comparisons in categorical variables were analysed with the Chi square test. The Bonferroni correction was used to adjust alpha for multiple testing. Correlations were checked using the Pearson’s r coefficient. To detect independent determinants of the outcome, we performed a multivariable logistic regression analysis, where a dichotomous endpoint was the dependent variable and confounders were entered as independent (explanatory) variables. SPSS version 23 or higher was used and statistical significance was accepted at p < 0.05.

p66Shc gene expression was determined in peripheral blood mononuclear cells of 100 patients with diabetes. Clinical characteristics of the study population are reported in Table 1. We included 16 patients with type 1 diabetes and 84 with type 2 diabetes. Patients were about 60 years old, with an average diabetes duration of 10 years, and an overall poor glycaemic control (HbA1c 7.8%).

We first divided patients according to low or high p66Shc gene expression in PBMCs (Table 1), based on the median value (1.17 relative expression [ΔΔ^Ct]). We detected no differences in all clinical and biochemical variables between the two groups. Marginal differences in the use of some medications did not survive after Bonferroni correction.

We then evaluated p66Shc expression according to the presence or absence of complications. We detected no significant differences in the average p66Shc expression between patients with and without coronary artery disease, peripheral arterial disease, cerebrovascular disease, any macroangiopathy, cardiac dysfunction, retinopathy, neuropathy, chronic kidney disease, micro/macroalbuminuria, and any microangiopathy (Fig. 1).

After the baseline determination of p66Shc gene expression, patients were followed-up for a median of 5.6 (IQR 5.0–6.4) years. Information on 11 patients could not be recalled; baseline clinical characteristics of patients lost to follow-up did not differ from those of patients with complete follow-up data (not shown). During this period, 6/89 patients died (3 for cancer, 2 for cardiovascular causes, and 1 for respiratory failure), 20/83 alive patients experienced at least one major adverse cardiovascular event (MACE), and 13 patients experienced more than one MACE. Among the 20 patients with incident MACE, the first event was acute myocardial infarction in 1, stroke or TIA in 4, unstable angina in 3, hospitalization for heart failure in 9, and unplanned revascularization in 4 (Table 2).

Gene expression of p66Shc did not differ significantly in patients with an adverse cardiovascular outcome (cardiovascular death and MACE, n = 22, annual rate of 4.4%) compared to those without (n = 77; Fig. 2a). The composite outcome of cardiovascular death or MACE occurred in 13 of 46 patients with low p66Shc expression and in 9 of 43 patients with high p66Shc expression (28.3% vs. 20.9%, p = 0.469; Fig. 2b, c).

During follow-up, 22/89 patients (24.7%) developed new complications: 15 macroangiopathy, 11 microangiopathy, and 4 both. In patients who developed micro- or macroangiopathy, p66Shc expression was about 30% higher than in those who did not develop or already had baseline complications (p = 0.032; Fig. 2d). The association was significant for new macroangiopathy (p = 0.011; Fig. 2e), but not for new microangiopathy (p = 0.155; Fig. 2f). Also among patients who were free from macroangiopathy at baseline (n = 34/89), those who developed macroangiopathy had higher p66Shc expression than those who did not (1.24 ± 0.11 versus 1.87 ± 0.26; p = 0.023). New macroangiopathy occurred in 4/46 patients with low p66Shc expression and in 13/43 patients with high p66Shc expression (8.7% vs 30.2%; p = 0.047; Fig. 2g).

Among patients who were free from macroangiopathy at baseline, those who developed new macroangiopathy differed at p < 0.1 from those who did not only for higher baseline p66Shc expression (p = 0.023) and lower ABI (p = 0.088). Upon a multivariable analysis, the association between p66Shc expression and new development of macroangiopathy was independent from ABI (p = 0.048).