**1. Introduction**

The prevalence of type 2 diabetes (T2D) is increasing worldwide, and it is expected to affect over 500 million adults worldwide by 2030 [1]. T2D is an important contributor to adverse cardiovascular complications, which are the leading causes of morbidity and mortality in Western countries [2].

Prevention of complications in T2D is closely linked to long-term control of hyperglycemia [3] since metabolic consequences extending beyond impaired glucose metabolism can affect almost every tissue and organ system of the body [4]. Despite the tendency in patients with good metabolic control to have a significantly reduced risk of developing complications, vascular disease can continue to develop and progress even under intensive treatment regimens due to the phenomenon known as "glycemic memory" [5]. Increased glucose levels can lead to metabolic derangements associated with vision loss, peripheral neuropathy, myocardial infarction, strokes, foot ulcers, and end-stage renal disease, which may cause permanent disability [6].

Despite the advancement of technologies to monitor blood glucose, for the vast majority of patients with diabetes, glycated hemoglobin (HbA1c) provides an excellent measure of glycemic control [7]. Nontraditional serum markers for short-term glucose control may enhance the ability to monitor hyperglycemia in people with diabetes. Fructosamine, glycated albumin, and 1,5-anhydroglucitol are of recent interest, especially in populations where the interpretation of HbA1c may be problematic such as in the setting of anemia, hemolysis, renal disease or pregnancy [8]. Most studies confirm a close linear relationship between HbA1c and mean blood glucose [9]. This suggests that HbA1c may be used not only as a diagnostic marker for the presence and severity of hyperglycemia during the preceding 4–12 weeks before the test but also over time as a "biomarker for a risk factor", i.e., hyperglycemia as a risk factor for diabetic retinopathy (DR), diabetic nephropathy (DN), and other vascular complications [4]. A 1% increase in absolute concentrations of HbA1c is associated with about 10–20% increase in cardiovascular disease risk [10]. The American Diabetes Association (ADA) now recommends the use of HbA1c to diagnose T2D with a cut-off value of ≥6.5%. Individuals with HbA1c levels of 5.7–6.4% are considered to be prediabetic. The ADA also recommends in patients with T2D, values of HbA1c less than 7% to prevent long-term complications associated with the disease [11,12].

As in the general population, in patients with diabetes, the treatment and prevention of cardiovascular disease require the use of specific biomarkers to predict risk. Most of these biomarkers are focused on already known pathophysiological pathways and mechanisms affecting the cardiovascular system. In the diabetic population the advanced glycation end products (AGEs), endothelin-1 (ET-1), matrix metalloproteinases (MMPs), high-sensitivity C-reactive protein (hsCRP), N-terminal fragment of brain natriuretic peptide (NT-proBNP), high-sensitivity troponin T (hsTnT), lipids, and albuminuria can be useful in predicting of cardiovascular disease [13,14]. In this regard, the markers for glucose-induced vascular damage, such as AGEs and urinary microalbumin levels, may be particularly useful in predicting the risk in individuals with diabetes [13].

Important factors in the development of vascular complications in T2D are the increased glycation, degradation, and/or accumulation of elastin and collagen in the vascular wall [15]. MMPs, which hydrolyze the protein components of the vascular extracellular matrix, are actively involved in this process. The subgroup of MMPs known as gelatinases, in particular gelatinase A (MMP-2) and gelatinase B (MMP-9), can degrade collagen (COL), denatured COL (gelatin), elastin (EL), laminin, fibronectin, and other substrates [16]. Dysregulation of gelatinase activity is associated with vascular inflammation, remodeling, and fibrosis and may contribute to the pathophysiology of diabetic complications [17]. In a previous study of patients with hypertension and T2D, we showed that elevated serum levels of MMP-2 and MMP-9 may reflect early structural changes in the vascular extracellular matrix [14]. Unlike the other MMPs, MMP-2 and MMP-9 differ in that they contain three type II fibronectin repeats that have a high binding affinity for collagen. These repeats direct the catalytic pocket of the gelatinases close to the collagen, thereby enhancing the rate of their hydrolysis [18]. The enhanced proteolytic activity of MMP-2 and MMP-9 is accompanied by the release of COL, EL and their derivatives (e.g., EL-derived peptides (EDPs), COL type IV (CIV)-derived peptides (CIV-DP)) in blood circulation, which is followed by the production of specific anti-elastin (AE) and anti-collagen (AC) antibodies (Abs) from IgM, IgG, and IgA classes (AEAbs IgM, AEAbs IgG, AEAbs IgA, ACAbs IgM, and ACAbs IgG) against their epitopes. These autoantibodies can serve as valuable control biomarkers for the turnover of protein components in the vascular extracellular matrix (ECM). Elevated levels of anti-CIV (ACIV) Abs IgG (ACIVAbs IgG) in hypertensive patients with T2D may indicate increased degradation of CIV [19], which is the most abundant structural component in the basement membrane (BM) of the small vessels [20]. Similarly, elevated levels of AEAbs IgA may indicate increased degradation of the elastic fibers in the vessel wall as a sign of microvascular [21] and/or macrovascular [22] disease in T2D.

In the present study, we investigate the clinical significance of HbA1c as a predictive biomarker for hyperglycemia-induced vascular damages in T2D, based on the statistical relationships between HbA1c levels and corresponding levels of MMP-2, MMP-9, AEAbs (IgM, IgG, and IgA), ACIVAbs IgM, and the levels of CIV-DP, reflecting CIV and EL turnover in the vascular wall.

#### **2. Materials and Methods**

#### *2.1. Study Population and Design*

The study was approved by the University Research Ethics Committee and conducted in accordance with the Declaration of Helsinki (IRB approval no. 314-REC/Prot. 29). The study population consisted of 79 persons: 59 patients with T2D treated at the University Hospital Georgi Stranski, Pleven, and 20 healthy control subjects. Two groups were formed: Group I (*n* = 20): control group (Control); Group II (*n* = 59): patients with T2D. The clinical characteristics of the groups are shown in Table 1.


**Table 1.** Clinical characteristics of the groups in the study population.

\* *p* < 0.05, \*\*\* *p* < 0.001; <sup>1</sup> Mean ± SEM; <sup>2</sup> N/A, not available; BMI: body mass index; TC: total cholesterol; LDL–C: low-density lipoprotein cholesterol; HDL–C: high-density lipoprotein cholesterol; TG: triglyceride; CRP: C-reactive protein; SBP: systolic blood pressure; DBP: diastolic blood pressure.

Selected control individuals were without diabetes mellitus, hypertension, or other vascular diseases, with a mean age of 61.5 ± 2.9 years. The mean age of patients with T2D was 60.7 ± 1.9 years. The patients were screened for microangiopathy using ophthalmoscopy and assessment of 24-h urine albumin excretion. Macroangiopathy was evaluated on the basis of clinical evidence for coronary artery disease, cerebrovascular disease, peripheral arterial disease, and/or history for acute arterial vascular events. Controls were screened for microangiopathy using ophthalmoscopy, and for macroangiopathy by physical examination, blood pressure measurement, electrocardiogram testing, measuring cholesterol levels, data on obesity and smoking, family history. The incidence of microangiopathy in the T2D group (*n* = 50) was 58%, and the incidence of macroangiopathy (*n* = 18) was 31%. Nine patients had both micro- and macrovascular diseases (Table 1).

According to the study design, our first aim was to compare the levels of MMP-2, MMP-9, AEAbs (IgM, IgG, and IgA), ACIVAbs IgM, and CIV-DP between patients and healthy controls. Our second aim was to compare within the patient group the levels of tested markers distributed below and above the different cut off values of HbA1c in the range between 6.0% and 8.0% (6.0%–6.5%–7.0%–7.5%–8.0%). All patients were divided into two subgroups according to these five cut-off values of HbA1c and we compared the levels of the markers between these subgroups (≤6.0% vs. >6.0%; ≤6.5% vs. >6.5%; ≤7.0% vs. >7.0%; ≤7.5% vs. >7.5%; ≤8.0% vs. >8.0%; see Table 2).


**Table 2.** Statistical significance between the levels of test markers in T2D subgroups at cut-off HbA1c values of 6.0%, 6.5%, 7.0%, 7.5%, and 8.0%.

\* *p* < 0.05, \*\* *p* < 0.01, NS—not significant; S—significant; MMP-2: matrix metalloproteinase-2; MMP-9: matrix metalloproteinase-9; AEAbs: anti-elastin antibodies; ACIVAbs: anti-collagen IV antibodies; CIV-DP: CIV-derived peptides.
