Assessment of the Relationship Between CD34 Antigen and Vitamin D Level and Insulin Resistance in Patients With Type 2 Diabetes

This case-control study included 30 people with type 2 diabetes and 30 healthy individuals referred to Mashhad University Hospitals in 2020.

People with type 2 diabetes who were >25 years of age, had a duration of diabetes of 1–5 years, and had no known nephropathy or liver disease, heart problems, hypothyroidism, hyperthyroidism, stroke, or cancer were eligible. Participants had to have not taken vitamin supplements or medications and not had a fever for at least 1 week for any reason, including viral infection, and pregnant women were also excluded. The control group included people determined by two blood glucose tests to have no diabetes who were matched with the diabetes group in terms of age and sex.

Data collected included participants’ age, sex, duration of diabetes, fasting blood glucose (FBG), A1C, blood pressure, serum creatinine, serum uric acid, lipid profile (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides), presence of other diseases, history of drug use, and BMI. These data were obtained from participants’ medical records.

The vitamin D kit used was the 25(OH)-Vitamin D (TR) ELISA Kit (PishtazTEB Diagnostics). The CD34 kit used was the human cluster of differentiation 34 (CD34) ELISA kit (ZellBio). The procedures used in this study were based on the brochures from each kit manufacturer.

We used the sandwich ELISA method, in which an antigen is placed between two particular antibodies. In this method, one antibody is used to trap the antigen on ELISA wells, and the second antibody is labeled with an enzyme acting as a marker. It should be noted that, in this method, the antigen must have at least two different antigenic regions to bind to both antibodies.

Appropriate tables and statistical indicators (such as mean and SD) have been used to report data. First, normal distribution was examined by the Shapiro-Wilk test. When abnormal data were observed, the Mann-Whitney U test was used, and for confirmation of normality, appropriate parametric methods such as the Student t test were used. A χ² test was used to analyze the data with a nominal scale. Pearson correlation coefficient and a linear model were used to investigate the relationship between variables. Analyses were performed using SPSS v.26 statistical software, and statistical significance was set at P <0.05.

The Ethical Committee of the Mashhad Medical Sciences Branch, Islamic Azad University, Mashhad, Iran, approved the study protocol (registration no. IR.IAU.MSHD.REC.1399. 132).

Patients provided written informed consent before participating in the study.

In this case-control study, the group with type 2 diabetes and the healthy control group each included 30 individuals, of whom 47% were male and 53% female.

The mean age was 48.53 ± 10.36 years in the control group and 52.90 ± 10.38 years in the case group (P = 0.18). The mean BMI was 25.99 ± 3.77 kg/m² in the control group and 29.55 ± 4.80 kg/m² in the case group (P = 0.002). The mean systolic blood pressure was 130.67 ± 16.17 mmHg in the control group and 138.77 ± 12.47 mmHg in the case group (P = 0.034), and the mean diastolic blood pressure was 89.00 ± 14.70 mmHg in the control group and 91.67 ± 13.41 mmHg in the case group (P = 0.466).

The results of the distribution of blood indices in the two groups are shown in Table 1. As can be seen, the mean of FBG, A1C, and insulin showed a statistically significant difference between the two groups (P <0.05), but the mean of serum creatinine, serum uric acid, and lipid profile (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides), had no statistically significant differences between the two groups (P >0.05).

The mean vitamin D level was 47.47 ± 22.54 ng/mL in the control group and 29.51 ± 9.98 ng/mL in the group with diabetes. The distribution of vitamin D in Figure 1 showed a significant difference between the two groups in terms of vitamin D level (P = 0.0001).

The mean insulin resistance calculated by the HOMA-IR formula was 0.57 ± 0.84 in the control group and 3.86 ± 3.56 in the case group, which showed a significant difference between the two groups (P = 0.001) (Figure 2).

The mean CD34 level was 4.80 ± 0.78 ng/mL in the control group and 2.35 ± 1.04 ng/mL in the case group, which was a significant difference between the two groups (P = 0.0001) (Figure 3).

Table 2 shows the relationships between CD34 and other variables. CD34 had a significant inverse relationship with BMI, A1C, FBG (P = 0.0001), insulin resistance (P = 0.005), and insulin level (P = 0.009). CD34 had a significant direct relationship with vitamin D (P = 0.002).

Moreover, the results of the linear model to investigate the relationship between CD34 or vitamin D with different variables showed that CD34 and vitamin D were significantly associated with type 2 diabetes (P <0.05).

Accelerated atherogenesis is an important complication of diabetes, which increases the risk of atherothrombotic events compared with people without diabetes. Despite various treatments and preventive measures, there is an urgent need to identify new strategies for managing the residual risk of cardiovascular events.

In people with type 2 diabetes, progenitor cells modify their proliferation, adhesion, and incorporation into vascular structures, and in patients at high risk for CVD, EPCs may be reduced. CD34 is a standard marker for stem cells, including vascular endothelial components, heart cells, and smooth muscle cells (10,19).

On the other hand, the primary physiological function of vitamin D is to regulate the metabolism of calcium and bones. Vitamin D deficiency is prevalent in autoimmune diseases and can affect the function of the vascular endothelium and lead to the onset or progression of atherosclerosis (20). Recent studies have shown a direct link between vitamin D and EPCs.

Because the relationships between the above factors in diabetes were unclear, we designed a study to examine the relationship between serum CD34 and vitamin D levels with insulin resistance in people with type 2 diabetes by comparing their results with those of a control group.

In this study, the mean age and sex proportions in the control group were not significantly different from those in the case group, but mean BMI and systolic blood pressure in the control group were significantly lower than in the case group. Also, there were statistically significant differences in mean FBG, A1C, and insulin between the two groups, but mean serum creatinine, serum uric acid, and lipid profile were not statistically different between the two groups. With regard to vitamin D, there was a significant difference between the two groups, such that the level of vitamin D was lower in people with diabetes than in the control group. In addition, a significant increase in insulin resistance was observed in the case group, and mean CD34 level in the control group was significantly higher than in the case group. We showed a significant inverse relationship between CD34 and BMI, A1C, FBG, insulin resistance, and insulin level. Interestingly, CD34 had a significant direct relationship with vitamin D. We also showed that vitamin D had a significant inverse relationship with A1C, FBG, and BMI. Consequently, our results showed that CD34 and vitamin D are significantly associated with type 2 diabetes.

In a study conducted in Slovenia by Kresnik et al. (21), cell grafts from 100 adult patients scheduled for autologous CD34+ cell transplantation were investigated. The number of CD34+ cells and their viability were determined by flow cytometry. The researchers found that CD34+ cell levels significantly decreased with increasing age. Also, they concluded that there was a tendency for an inverse relationship between diabetes and CD34+ cell count; however, this relationship was not significant.

A 2006 Italian study by Fadini et al. (22) examined the relationship between CD34 and metabolic syndrome in patients with cardiovascular risk. In this study, different types of CD34 were studied in 200 patients at different levels of cardiovascular risk. Results indicated a close negative correlation between CD34 and cardiovascular risk, as well as a synergistic detrimental effect of clustered metabolic components.

Another study by Fadini et al. (13) examined the role of EPCs in peripheral vascular disease in patients with type 2 diabetes. By analyzing flow cytometry, the researchers evaluated circulating progenitor cells (CPCs) and EPCs in 51 patients in a case group and 17 patients in a control group. They found that CPC and EPC levels in people with diabetes were decreased by 33 and 40%, respectively, compared with the control group.

A study conducted in California by Vrtovec et al. (17) examined the effectiveness of CD34 cell therapy in nonischemic cardiomyopathy in 45 patients, including 12 people with type 2 diabetes, 17 people without diabetes but with insulin resistance based on HOMA-IR, and 16 people without insulin resistance or diabetes. All of the patients had heart disease. All patients were treated with granulocyte-colony stimulating factor ampoules 5 mg/kg for 5 days, and then CD34 cell levels were measured. The patients were then treated with CD34 cells injected into the left ventricle. They were evaluated for CD34, blood glucose, insulin resistance, and response to cell therapy 1, 3, and 6 months after cell therapy. In both groups without diabetes, the level of the N-terminal pro-brain natriuretic peptide marker, which indicates heart damage, decreased significantly after 3 months, but a reduction was not observed in the group with diabetes. This study indicates that diabetes alone affects the function of CD34 cells, but insulin resistance without diabetes has no effect on the function of CD34 cells. This study found no significant relationship between insulin resistance and diabetes and CD34, which contradicts the findings of our study. More research is therefore needed to clarify the role of insulin resistance.

A 2014 Ukrainian study by Berezin (23) examined the relationship between circulating EPCs and insulin resistance in patients with chronic heart failure but without diabetes. The study included 300 patients who were 48–62 years of age and diagnosed with coronary artery disease by angiography. Insulin resistance was determined using the HOMA-IR formula. Also, CD45, CD34, CD14, and CD309 cell populations were measured as markers expressing EPCs. In this study, the EPC count was significantly lower in the group with insulin resistance than in the group without insulin resistance. This study indicates that insulin resistance may be an essential factor in reducing EPC function and EPC number in blood circulation, and insulin resistance and EPCs may have a significant relationship with each other, which is in line with the results of our study.

Previous studies have shown that a reduction in circulating CD34 in the early stages of type 2 diabetes may be apparent in individuals with glucose intolerance and that this decrease persists over time and may be worse in patients with advanced complications (8,24).

Also, Cubbon et al. (25) believe that the effects of insulin resistance on EPCs could be caused indirectly by the effects of insulin (e.g., oxidative stress, inflammation, and increased free fatty acids) or directly in a mechanism related to cells in the bone marrow.

To date, it is not clear by what mechanisms diabetes affects EPCs. Previous studies have suggested that a reduction of EPC effects by nitric oxide (NO) may be the result of altered mobilization from the bone marrow returned to damaged vessels or tissues (26,27), as well as an increase in apoptosis and reduced cell survival (28). These disturbances in EPC levels and functional properties may contribute to the endothelial dysfunction, progression of atherosclerotic disease, and reduced scarring seen in people with diabetes (29). More studies are needed to clarify the complex interplay among insulin resistance, EPCs, and endothelial damage and repair.

Many studies have also shown that vitamin D deficiency is associated with endothelial dysfunction and an increased risk of CVD (30–33). Also, previous studies have shown that vitamin D may have a protective effect on EPCs and that a depletion of EPCs is induced by oxidative stress (34–36).

The vitamin D receptor is present in vascular tissues such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes. Indeed, vitamin D has several beneficial effects in the vascular wall via this receptor, including reduction in thrombogenicity, decrease in vasoconstrictors, inhibition of oxidative stress and atherogenesis, improvement of endothelial repair, reduction in foam cell formation, and vascular dilatation (37). A recent cohort study demonstrated that higher vitamin D concentrations could be associated with a lower risk of CVD-related mortality (38).

Hammer et al. (29) investigated the effect of vitamin D on the functional aspects of EPCs isolated from healthy subjects and from people with diabetes, who are known to have attenuated EPC activity. Their results showed that, in people with diabetes, vitamin D supplementation (in vitro) improved EPCs’ capacity to form colonies and viability. A recent study also showed that vitamin D is independently associated with insulin resistance in people with type 2 diabetes (39).

Today we know that diabetes negatively affects EPCs and that insulin resistance can cause endothelial dysfunction, leading to vascular complications (40). Diabetes leads to multiple areas of vascular damage. EPC count and vitamin D status can be used as biomarkers in people with diabetes to predict vascular complications, and their reduction can worsen the situation for these patients.

One of the main limitations of this study was its relatively low sample size, which was the result of time limitations and the coronavirus disease 2019 pandemic. The obtained results could be confirmed in a study with a similar scheme performed with a larger participant sample. In addition, a lack of financial resources meant that it was not possible to measure more serum markers in this study, which could have provided valuable additional information.

Our findings suggest that vitamin D deficiency is common in type 2 diabetes and associated with endothelial dysfunction and a reduced number of EPCs. The reduced CD34 and vitamin D levels simultaneously associated with an increase in insulin resistance in people with type 2 diabetes may have an impact on the progression of this disease, as controlling these factors may prevent or slow disease progression. Hence, these factors can be helpful in providing a more accurate assessment of diabetes status and to predict the development of its complications, especially CVD.

The authors thank all of their colleagues at Islamic Azad University, Mashhad branch, for their help and support, especially the colleagues at the Innovation Research Center of Shahinfar Medical School, as well as those at Imam Reza Hospital in Mashhad.

No potential conflicts of interest relevant to this article were reported.

V.P. conceptualized the study. V.P. and S.E. wrote the manuscript and collected/analyzed data. V.P. and M.S.Y. reviewed/edited the manuscript and contributed to the discussion. V.P. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.