*3.1. Comparison of the Tested Markers between the T2D and Control Groups*

Patients with T2D showed statistically significantly higher serum levels of MMP-2 (30.68 ± 1.87 vs. 36.22 ± 1.50; *p* = 0.026), MMP-9 (25.84 ± 2.83 vs. 38.48 ± 2.69; *p* = 0.002), and AEAbs IgA (0.29 ± 0.03 vs. 0.55 ± 0.05; *p* < 0.001) than healthy controls (Figures 1A,B and 2A).

**Figure 1.** (**A**) Serum levels of MMP-2 in T2D group vs. control group. (**B**) Serum levels of MMP-9 in T2D group vs. control group. (**C**) Serum levels of MMP-2 in patients with HbA1c values ≤7.5% vs. patients with HbA1c values >7.5%. (**D**) Serum levels of MMP-9 in patients with HbA1c values ≤7.5% vs. patients with HbA1c values > 7.5%. Data are represented as mean ± SD. \* *p* ≤ 0.05, \*\* *p* < 0.01.

**Figure 2.** (**A**) Serum levels of AEAbs IgA in T2D group vs. control group (**B**) Serum levels of AEAbs IgA in patients with HbA1c values ≤7.5% vs. patients with HbA1c values >7.5%. (**C**) Serum levels of AEAbs IgM in the T2D group vs. control group. (**D**) Serum levels of AEAbs IgG in the T2D group vs. control group. Data are represented as mean ± SD. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001, n.s.—not significant.

Serum levels of AEAbs IgM were significantly lower in T2D group than in controls (0.34 ± 0.03 vs. 0.18 ± 0.01; *p* = 0.001; Figure 2C). The levels of AEAbs IgG were also lower in the T2D group than in controls, but the difference was not statistically significant (0.33 ± 0.02 vs. 0.31 ± 0.04; *p* = 0.697; Figure 2D). The levels of ACIVAbs IgM (0.18 ± 0.02 vs. 0.12 ± 0.01; *p* = 0.016) and CIV-DP (1.16 ± 0.05 vs. 0.74 ± 0.03; *p* < 0.001) in patients with T2D were significantly lower than in controls (Figure 3A,B).

**Figure 3.** (**A**) Serum levels of ACIVAbs IgM in T2D group vs. control group (**B**) Serum levels of CIV-DP in T2D group vs. control group. Data are represented as mean ± SD. \* *p* < 0.05, \*\*\* *p* < 0.001.

#### *3.2. Comparison of the Tested Markers between T2D Subgroups at Cut-O*ff *Values of HbA1c from 6.0 to 8.0%*

Comparison of the tested markers levels at different HbAc cut-off values showed the most significant indication for vascular change at a cut-off HbA1c value of 7.5%. At this value, a set of three assessment markers for vascular risk (MMP-2, MMP-9, and AEAbs IgA) showed statistical significance (Table 2). In patients with poor glycemic control and increased vascular risk (*n* = 25), who have HbA1c values >7.5%, the levels of MMP-2 (32.85 ± 1.56 vs. 39.34 ± 2.39; *p* = 0.022) and AEAbs IgA (0.45 ± 0.04 vs. 0.67 ± 0.09; *p* = 0.049) were significantly increased compared to those with better control and HbA1c values ≤7.5% (*n* = 34; Figures 1C and 2B). In the same subgroups of patients, MMP-9, unlike MMP-2, showed significantly decreased levels at HbA1c values >7.5% compared with HbA1c values ≤7.5% (41.89 ± 3.31 vs. 32.51 ± 3.26; *p* = 0.05; Figure 1D).

At the cut-off HbA1c value of 7.0%, statistical significance showed a set of two markers (MMP-9 and AEAbs IgA). MMP-9 showed significantly decreased levels at HbA1c values >7.0% compared with HbA1c values ≤7.0% (43.12 ± 3.45 vs. 32.19 ± 3.05; *p* = 0.023). The levels of AEAbs IgA were significantly increased at HbA1c values >7.0% compared with HbA1c values ≤7.0% (0.44 ± 0.04 vs. 0.66 ± 0.09; *p* = 0.031; Table 2). Only one marker (MMP-9) showed significantly decreased levels at cut off HbA1c values of 6.0% (50.79 ± 5.64 vs. 34.15 ± 2.40; *p* = 0.003) and 6.5% (46.42 ± 4.32 vs. 33.36 ± 2.65; *p* = 0.009). None of the markers showed statistical significance at a cut-off value of HbA1c of 8.0% (Table 2). The relationship between the levels of test markers and HbA1 as a continuous variable is shown in Figure 4.

**Figure 4.** Scatterplots showing the relationship between the levels of (**A**) MMP-2, (**B**) MMP-9, (**C**) AEAbs IgA, (**D**) AEAbs IgG, (**E**) AEAbs IgM, (**F**) CIV-DP, (**G**) ACIVAbs IgM, and HbA1 as a continuous variable.

## *3.3. Correlations of Investigated Immunological Markers*

There were significant correlations of the examined markers for vascular risk in the T2D group, which are presented in Table 3.


**Table 3.** Pearson's correlation coefficients and statistical significance between the variables in the T2D group.

\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; MMP-2: matrix metalloproteinase-2; AEAbs: anti-elastin antibodies; ACIVAbs: anti-collagen IV antibodies; CIV-DP: CIV-derived peptides; BP: blood pressure; BMI: body mass index.

#### **4. Discussion**

HbA1c is major tool for assessing glycemic control and has strong predictive value for diabetes complications [24]. Given the broad informativeness of the test, it is imperative to know how it can be optimally applied to the management and assessment of overall vascular risk (micro- and macrovascular) in patients with diabetes [25]. Systematic review and meta-analysis of multiple databases suggest that in people with diabetes, the target levels for HbA1c to minimize vascular complications should range from 6.0 to 8.0% [26]. The UK Prospective Diabetes Study (UKPDS) [27,28] and the Kumamoto Study [29] confirmed that intensive glycemic control significantly decreased rates of microvascular complications in patients with T2D [24]. The Diabetes Control and Complications Trial (DCCT) [30], a prospective randomized controlled trial of intensive (mean HbA1c about 7%) versus standard (mean HbA1c about 9%) glycemic control in patients with T1D, showed that better glycemic control is associated with 50–76% reductions in rates of development and progression of microvascular complications. Epidemiologic analyses of the DCCT and UKPDS also suggest that further lowering of A1C from 7 to 6% is associated with further reduction in the risk of microvascular complications, although the absolute risk reductions become much smaller [24]. The Analysis of the Action in Diabetes and Vascular disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study has shown that microvascular event risk begins above an HbA1c of 6.5%. For macrovascular event risk, inflection of the curve was seen at around 7%, and the risk increased at higher HbA1c levels [31]. In a cohort of patients with T2D in the UK, Currie and colleagues report that the ideal HbA1c level is 7.5% and that the recommendations should target such a value. Their analyses demonstrate that an HbA1c of approximately 7.5% was associated with the lowest all-cause mortality and the lowest progression to large-vessel disease events. An increase or decrease from this mean HbA1c value was associated with a heightened risk of adverse outcomes [32]. The data in this study are consistent with our results, which give the highest indication of vascular change (set of three assessment markers—MMP-2, MMP-9, and AEAbs IgA) at a cut-off HbA1c value of 7.5% (Table 2). Therefore, vascular damage from preceding long-term hyperglycemia begins to dominate at an HbA1c value greater than 7.5%, which is the likely cut-off point to predict increased vascular risk.

Current consensus-based guidelines do not fix one exact cut-point of HbA1c, beyond which vascular risk increases. All recommend individualizing HbA1c targets on the basis of patient characteristics. The ADA recommends the reasonable target of HbA1c for many non-pregnant adults to be less than 7%. An HbA1c of <7.0% may be targeted in the majority of patients, acknowledging the individual needs [12]. When a patient has just been diagnosed and is free from significant cardiovascular disease, the aim should be a range from 6.0–6.5%. By contrast, in an elderly patient with long-standing and/or complicated disease, relaxing the target to 7.5–8.0% may be wiser, given that the vascular benefits in terms of life expectancy are less relevant [24]. The American Association of Clinical Endocrinologists and American College of Endocrinology (AACE/ACE) guidelines recommend a target of 6.5% (if it can be achieved safely). The National Institute for Health and Care Excellence (NICE) guideline specifies 6.5% or 7%, depending on the patient's treatment regimen. The Clinical Systems Improvement (ICSI) guideline recommends a target range less than 7% to less than 8% based on patient factors. According to recommendations of the American College of Physicians (ACP), for most patients with T2D, targets levels of HbA1c should be between 7.0% and 8.0% [6]. Our HbA1c cut-point of 7.5%, evaluated to predict higher vascular risk, falls in the middle of this recommended range.

When we compared the levels of MMP-2 and MMP-9 between the control group and the T2D group, we observed higher levels in patients with T2D (Figure 1A,B). As to our findings that MMP-2 and MMP-9 levels were significantly higher in T2D patients, similar findings have been reported by other researchers [33,34]. However, an impression in our study makes the observed opposite levels of the two gelatinases at HbA1c values greater than 7.5% (Figure 1C,D). In patients with poor glycemic control and increased vascular risk, who have HbA1c values >7.5%, MMP-2 levels were significantly increased compared to those with better control and HbA1c values ≤7.5% (Figure 1C). In the same subgroups of patients, MMP-9, unlike MMP-2, showed significantly decreased levels

at HbA1c values >7.5% compared with HbA1c values ≤7.5% (Figure 1D). Similar results have been reported by Derosa and colleagues in children and adolescents with T1D. They reported that MMP-2 levels were significantly higher in patients with microangiopathic complications compared with control subjects and patients without complications. MMP-9 levels were significantly lower in patients with microangiopathic complications compared with control subjects and patients without complications. Based on these results, the authors postulated that MMP-2 may be a good index of the severity and stability of microangiopathy, and MMP-9 is a marker of macroangiopathy in diabetes [35]. We also found positive correlations between MMP-2 and AEAbs IgG and between MMP-2 and ACIVAbs IgM in the T2D group (Table 3). A possible explanation for this result is that MMP-2 may be involved in the process of elastin and collagen IV destruction and the development of vascular complications.

MMP-2 and MMP-9 play an important role in the development of microvascular and macrovascular complications in T2D patients. The recent results advocate that due to diabetes, the overexpression of MMP-2 and MMP-9 in the retina inhibits cell proliferation and differentiation and accelerates apoptosis, a phenomenon that precedes the development of histopathology characteristic of DR [36]. During the first stage of DR, increased retinal MMP-2 and MMP-9 enhance the permeability of the blood–retinal barrier via proteolytic degradation of tight junction protein occludin and disruption of the overall tight junction complex [37]. In addition, MMP-2 and MMP-9 facilitate apoptosis of retinal capillary cells and pericytes, which disrupts the normal vascular structure and leads to the formation of microaneurysms and hemorrhages [38]. During the advanced stage, MMP-2 and MMP-9 dissolve the vascular basement membrane and create the conditions for the formation of new vessels [39]. MMP-2 and MMP-9 are also implicated in the pathogenesis of diabetic macular edema and fibrovascular proliferation with tractional retinal detachment, which are the most common causes of vision loss in patients with DR [40–42]. The impact and contribution of MMP-2 and MMP-9 to the onset and progression of DN may be most critical in the earlier phases of the disease process, at a time in which enhanced matrix turnover, release of pro-fibrotic growth factors, and altered cell motility may damage the glomerular apparatus and tubular architecture [43]. In these phases of DN, MMP-9 can predict microalbuminuria several years before its appearance and can be prognostic marker for the renal involvement. In the late period of diabetes, decreased activity of MMP-2 and MMP-9 is observed with increased activity of tissue inhibitor of metalloproteinases-1 (TIMP-1). These leads to excessive deposition of type IV collagen and fibronectin in the BM and a decrease in effective filtration surface area [44]. In the advanced stage of chronic kidney disease, the activity of MMP-2 and MMP-9 is decreased and, in this late period, the fibrosis is difficult to reverse [45]. On the other hand, MMP-2 and MMP-9 are involved in the process of atherogenesis and development of arterial lesions in T2D [46,47]. They are synthesized in atheromatous plaques and are present at elevated levels in rupture-prone regions of arterial blood vessels. MMP-2 and MMP-9 were both correlated with plaque instability and there was a correlation between increased MMP-9 expression and cap rupture [48]. Plasma levels and zymographic activities of MMP-2 and MMP-9 are increased in T2D patients with peripheral arterial disease in comparison with healthy control subjects, and MMP-9 may be a useful marker for development of macrovascular complications in T2D [34].

EL fibers are essential structural elements of the vascular wall, especially of the arteries. They are considered the most resilient element of vascular ECM. The EL half-life is in the order of 40 years. [49]. Elastases are endopeptidases that cleave EL, resulting in the formation of EDPs. Elastases include serine- and cysteine- proteinases and four MMPs—MMP-2, MMP-9, MMP-7 (matrilysin), and MMP-12 (macrophage elastase) [50]. T2D is associated with an increase in the expression and activity of MMP-2 and MMP-9, and an increase generation of EDPs [51,52]. EDPs have immunogenic properties and favor the formation of specific AEAbs from IgM, IgG and IgA classes [53]. The presence of AEAbs can lead to the formation of circulating immune complexes and complement activation and K-cell-mediated antibody dependent cytotoxicity, which may further contribute to the destruction of EL in the arterial wall. This process can be maintained by specific T- and B-lymphocytes at sites of arterial damage [21,54,55]. When we compared the serum levels of AEAbs IgM, AEAbs IgG, and AEAbs

IgA in T2D patients with those of non-diabetic subjects, we observed significant increases in AEAbs IgA (Figure 2A), whereas the levels of AEAbs IgM and AEAbs IgG were decreased (Figure 2C,D). This feature of the humoral immune response, with the prevalence of higher serum levels of general or specific IgA Abs in diabetic patients, is a generalized phenomenon documented in a number of studies [56–59]. In one of these, the patients with T1D and T2D with micro- or macrovascular complications have had higher serum IgA concentrations than the corresponding groups of patients without complications. Furthermore, the patients with three kinds of microangiopathy had slightly higher IgA levels than patients with only one kind; those with nephropathy and hypertension had even higher levels. The macroangiopathy groups have shown the highest IgA levels among the T2D subgroups with complications, and the lowest among the T1D subgroups. These data suggest that monitoring IgA may provide early warning of the possible presence simultaneously of micro- or macrovascular complications in T2D [56]. Studies in T2D patients have also shown that poor glycemic control may be associated with an increase in serum IgA Abs [60]. Our results provide compelling evidence for this and show that the levels of AEAbs IgA are influenced by the degree of glycaemic control reflect by measurement of HbA1c (Figure 2B). In patients with poor glycemic control and increased vascular risk, who have HbA1c values >7.5%, AEAb IgA levels were significantly increased compared to those with better control and HbA1c values ≤7.5%. Similar to total IgA [56], an increase in AEAb IgA levels may indicate increased degradation of EL in the vascular wall and may be a specific marker for micro- or macrovascular damage in T2D [21,22]. These findings are also supported by the positive correlations that we found between AEAbs IgA and AEAbs IgM, AEAbs IgG, ACIVAbs IgM, and the CIV-DP. AEAbs also showed significant negative correlations with SBP, DBP, and BMI in the T2D group (Table 3).

CIV represents up to 50% of all BM proteins [61]. Unlike fibrillar COLs of type I, II, and III, CIV forms a network structure and it is found to be crucial for vascular BM assembly and stability [62]. MMP-2 and MMP-9 can cleave most of the major macromolecules of the ECM, including COL types IV, V, VII, and X. Processing of CIV gives rise to the release of fragments that are able to behave as epitopes since they can be bound by circulating Abs [63]. Autoantibodies (autoAbs) against CIV are present in various inflammatory and autoimmune diseases [64]. This is the case of recurrent Goodpasture's disease secondary to an autoreactive IgA Ab [63,65]. Moreover, autoAbs against CIV have been detected in children with T1D and vascular complications [66,67], as well as in hypertensive T2D patients with microangiopathy [19]. When we compared the serum levels of ACIVAbs IgM and CIV-DP, they were significantly lower in the patient group than in the control subjects (Figure 3A,B). A possible explanation for this result is that the levels and activity of MMP-9 in chronic hyperglycemia (HbA1c values >7.5%) are decreased, which leads to excessive deposition of CIV in the vascular BM and to its thickening [68]. Vascular BM thickening is the most characteristic structural abnormality of small blood vessels in DR and DN [69]. In addition, the highest HbA1c values exhibited the highest BM thickness in both the retinal and glomerular capillaries, which was observed in diabetic rats [70]. We also found positive correlations between ACIVAbs IgM and AEAbs IgM, between ACIVAbs IgM and AEAbs IgA, and between the CIV-DP and AEAbs IgA in the T2D group (Table 3).

Strengths of our study, unlike retrospective epidemiological studies, are that it reflects the direct relationship of hyperglycemia with vascular changes through the levels of appropriately selected biomarkers. Also, a design has been used in which the division of biomarkers into groups according to the degree of glycemic control (HbA1c levels) can provide valuable information on the vascular status of patients. A limitation of the study is the relatively small number studied persons, which requires these results to be confirmed in larger studies.

#### **5. Conclusions**

Considering the broad informativeness of the HbA1 test, it can be successfully applied to the management and assessment of overall vascular risk in patients with diabetes. Our results give the highest indication of vascular change (set of three assessment markers, MMP-2, MMP-9, and AEAbs IgA) at a cut-off HbA1c value of 7.5%. This indicates that vascular damage from preceding long-term hyperglycemia begins to dominate at HbA1c values ≥7.5%, which is the likely cut-off point to predict increased vascular risk.

**Author Contributions:** Conceptualization, software, formal analysis, writing—review and editing, visualization, K.K.; methodology, resources, A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was accomplished with the financial support of the Medical University, Pleven.

**Conflicts of Interest:** The authors declare no conflict of interest.
