The association between MCP-1, VEGF polymorphisms and their serum levels in patients with diabetic foot ulcer.

The purpose of the present study was to investigate distribution of monocyte chemoattractant protein-1 (MCP-1) -2518A/G and vascular endothelial growth factor (VEGF) -634G/C polymorphisms in type 2 diabetes melitus patients (T2DM) presenting diabetic foot ulcer (DFU). Additionally, we evaluated the effects of these 2 polymorphisms on serum levels of MCP-1 and VEGF in the study population.Patients diagnosed with T2DM without or with DFU were recruited in the study. The distribution of MCP-1 -2518A/G and VEGF -634G/C polymorphisms was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Enzyme-linked immunosorbent assay (ELISA) was applied to detect the protein levels of MCP-1 and VEGF. The comparisons of protein levels in DFU patients were performed by student t test according to their genotypes.The frequencies of GG genotype and G allele of MCP-1 -2518A/G was increased in DFU patients, compared with T2DM patients (odds ratio [OR] = 2.60, 95% confidence interval [CI] = 1.23-5.50, P = .011 and OR = 1.72, 95% CI = 1.18-2.50, P = .005, respectively). Moreover, the increased frequency of GG was significantly associated with up-regulated MCP-1 level in DFU patients (P < .001). Analysis for VEGF -634G/C polymorphisms indicated that the prevalence of CC genotype and C allele of the polymorphisms was decreased in DFU patients, compared with T2DM patients (OR = 0.36, 95% CI = 0.17-0.77, P = .008 and OR = 0.63, 95% CI = 0.43-0.91, P = .015, respectively). DFU patients carrying CC genotype had a higher level of VEGF than those with other genotypes (P = .007).MCP-1 -2518A/G and VEGF -634G/C polymorphisms may involve in occurrence and progress of DFU through regulating transcription activity of the genes.

Introduction

Type 2 diabetes melitus (T2DM) is one of the most prevalent metabolic disorders and its prevalence is increasing in recent years.[ T2DM can lead to various complications. Diabetic foot ulcer (DFU) is one of the common diabetic complications which is a leading cause for hospitalization and amputation among patients diagnosed with T2DM.[ Until now, there are no effective treatments for DFU patients, due to the poor blood circulation at the wound sites.[ Therefore, the factors associated with angiogenesis and vascular functions may involve in occurrence and development of DFU.

Monocyte chemoattractant protein-1 (MCP-1), also named chemokine (C-C motif) ligand 2 (CCL2), is a chemokine which could active monocytes, macrophages, and lymphocytes.[ Abnormal expression of MCP-1 has been observed in various diseases, such as clear-cell renal cell carcinoma, cerebral ischemic stroke, coronary artery disease.[ Hyperglycemia can enhance the production of MCP-1 in vascular endothelial cells and its abnormal expression may contribute to the complications related to angiogenesis and vascular functions among T2DM patients.[ Recently, growing studies have indicated that the polymorphisms of MCP-1 –2518A/G may influence the production of MCP-1.[ But the effects of MCP-1 –2518A/G polymorphism and its association with MCP-1 level had been rarely reported among patients with DFU.

Vascular endothelial growth factor (VEGF), a potent angiogenesis and vascular functions factor, is significantly associated with occurrence and development of diabetic complications.[ A meta-analysis including 6 related studies demonstrated that VEGF polymorphisms could influence individual susceptibility to diabetic retinopathy.[ The genetic association of VEGF polymorphisms with risk of DFU had also been reported in the existing literature. Amoli et al[ reported that VEGF polymorphism at position -2578∗C/A was closely correlated with risk of DFU. In the study of Mohajeri-Tehrani et al,[ the increased level of VEGF hold the capacity to improve blood flow and tissue temperature, thus, promoted wound healing for DFU patients. The expression level of VEGF may be influenced by its genetic variants. Sa-Nguanraksa et al[ had found that VEGF expression levels were different among patients carrying different genotypes of VEGF –634G/C polymorphism. However, the polymorphism of VEGF +405C/G might not influence blood concentration of VEGF in Chinese Han population.[ Thus, we hypothesized that VEGF –634G/C polymorphism might influence individual susceptibility to DFU through its regulation on VEGF production.

The purpose of the study was to investigate the genetic effects of VEGF –634G/C and MCP-1 –2518A/G polymorphisms on risk of DFU in Chinese Han population. The T2DM patients without diabetic complications except DFU were recruited in this study, blood specimens were collected from the study subjects. The detected polymorphisms may be useful for targeted therapy of DFU.

Materials and methods

Study subjects

All the participants were collected from The First Affiliated Hospital of Shihezi University Medical College. The volunteers recruited in the present study should meet the following inclusion criterion: the Chinese Han adults population, without blood relationship; from the same geographical region; diagnosed with T2DM according to World Health Organization (WTO) criteria; without diabetic complications, except DFU. Exclusion criterion: immune diseases, cerebrovascular diseases, or other serious diseases. The patients who met the included criterion and had no excluded symptoms would be included in current study. According to the presence of DFU, the patients were divided into T2DM and DFU groups. The 2 study groups were matched in age and sex.

After an overnight fast, 6 mL peripheral blood was obtained from all the individuals using EDTA tubes. Then the blood specimens were stored in –80 °C until use.

The present study was supported by the ethic committee of The First Affiliated Hospital of Shihezi University Medical College. All the patients signed the written informed contents before blood collection. The study procedures were in accordance with the Declaration of Helsinki.

DNA extraction and genotyping

QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) was applied for DNA extraction following the instruction of the manufacturer's. In the current study, the genotyping of VEGF –634G/C and MCP-1 –2518A/G were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). PCR primers were designed by Primer Premier 5.0 software and the sequences were listed in Table 1 (MCP-1,[VEGF[). DNA products were amplified in a 25 μL reaction system containing 12.5 μL PCR mastermix (2×), 1 μL DNA template, 0.5 μL specific primers, 0.5 μL MgCl2, 10 μL free nuclease water. PCR programs were carried out according to the following settings: 95 °C for 1 minute, followed by 35 cycles of 95 °C for 45 second, 62 °C (for VEGF –634C/G) or 55 °C (for MCP-1 –2518A/G) for 40 seconds, 72 °C for 40 seconds, then an extra extension at 72 °C for 10 minutes.

Table 1

Primers sequences and restriction enzymes used in the present study.

PCR products were analyzed by 1% agarose gel electrophoresis (AGE) and then submitted to enzymes digestion using the specific restriction enzymes. The enzyme-digested fragments were separated by 2% AGE.

MCP-1 and VEGF level evaluation

Enzyme-linked immunosorbent assay (ELISA) kits were applied to detect the protein levels of MCP-1 and VEGF in collected blood specimens. The MCP-1 levels were detected using human MCP-1 ELISA kits (R&D Systems, Minneapolis, MN),[ while human VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN)[ was used for evaluation of VEGF level following the instructions of the manufacturer.

Statistical analysis

Statistical analysis was performed using SPSS 18.0 software. Odds ratio (OR) with corresponding 95% confidence interval (95% CI) calculated by χ2 test was applied to evaluate the different frequencies of genotypes and alleles between T2DM and DFU patients. The comparisons of VEGF and MCP-1 levels in DFU cases were performed using student t test according to their genotypes. P value <.05 was considered significant difference.

Results

Baseline characteristics of the study population

One hundred eight T2DM patients without DFU (male: 58, female: 50) were enrolled in the present study as T2DM group, and their average age was 57.06 ± 10.96 years old. The DFU groups included 71 men and 50 women and their mean age was 56.02 ± 9.83 years old. Analysis results indicated that the T2DM group and DFU group were age–sex matched (P > .05 for both). The other clinical characteristics of the study population were listed in Table 2. There were no significant differences between the 2 groups (P > .05 for all).

Table 2

Baseline characteristics of the study population.

Genotypes of MCP-1 –2518A/G

RFLP-PCR was applied to analyze the distributions of MCP-1 –2518A/G polymorphism. The frequencies of AA, AG, and GG were separately 24.07%, 48.15%, and 27.78% in T2DM group. Meanwhile, their frequencies were 14.88%, 40.50%, and 44.63% in DFU group, respectively. In addition, chi-square analysis indicated that compared with AA genotype, GG genotype was significantly correlated with DFU susceptibility (OR = 2.60, 95% CI = 1.23–5.50, P = .011). However, there was no association between AG and DFU occurrence.

Allele distribution analysis demonstrated that the allele occurrence was 48.15% (A) and 51.85% (G) in T2DM group. In DFU group, A allele frequency was 35.12% and the frequency of G allele was 64.88%, respectively. Moreover, compared with A allele, G allele was significantly associated with risk of DFU (OR = 1.72, 95% CI = 1.18–2.50, P = .005) (Table 3).

Table 3

Frequencies of alleles and genotypes of MCP-1 and VEGF polymorphisms in study groups.

Analysis for VEGF –634C/G polymorphism

There were 3 genotypes for VEGF –634C/G polymorphism, including CC, CG, and GG. The prevalence of these 3 genotypes was 35.18% (CC), 50.00% (CG), and 14.81% (GG) in T2DM group; while for DFU group, the frequencies were 23.97% (CC), 47.93% (CG), and 28.10% (GG), respectively. The distribution of CC was significantly decreased in DFU group, compared with GG (P = .008). CC genotype might be a protective factor for occurrence of DFU (OR = 0.36, 95% CI = 0.17–0.77).

Additionally, the frequency of C allele was separately 60.18% and 47.93%, while the prevalence of G allele was respectively 39.72% and 52.07% in T2DM and DFU group. Chi-square analysis demonstrated that compared with G allele, the frequency of C allele significantly reduced the risk of DFU (OR = 0.63, 95% CI = 0.43–0.91, P = .015) (Table 3).

Serum levels of MCP-1 and VEGF

ELISA was applied to evaluate the serum concentrations of MCP-1 and VEGF in the included patients. Analysis results indicated that MCP-1 expressed was elevated in DFU group, compared with T2DM group (23.88 ± 3.72 pg/mL vs 16.63 ± 2.87 pg/mL, P < .001) (Fig. 1A).

Figure 1

Protein levels of MCP-1 and VEGF in collected blood specimens. A: Comparison of MCP-1 level between T2DM and DFU patients. The DFU patients showed an increased level of MCP-1, compared with T2DM patients. ∗∗∗: P < .001. B: The comparison analysis for VEGF level in the study population. Down-regulated level of VEGF was detected in DFU patients, compared with T2DM patients. ∗∗∗: indicated P value <.001. DFU = diabetic foot ulcers, MCP-1 = monocyte chemoattractant protein-1, T2DM = type 2 diabetes melitus, VEGF = vascular endothelial growth factor.

VEGF concentration analysis suggested the expression level of VEGF was obviously down-regulated in patients diagnosed with DFU, compared with those in T2DM group (71.06 ± 8.80 pg/mL vs 107.77 ± 10.98 pg/mL, P < .001) (Fig. 1B).

Association between MCP-1 and VEGF level and their variants

In the present study, we analyzed the effects of MCP-1 –2518A/G polymorphism on MCP-1 production among DFU patients. Analysis results indicated that the expression level of MCP-1 was higher in GG group than that in AA group (P < .001). There were no significant differences between AA group and AG group (P > .05) (Fig. 2A).

Figure 2

Association between gene polymorphisms and protein level in DFU patients. A: Relationship between MCP-1 –2518A/G polymorphism and MCP-1 level. DFU patients carrying GG genotype showed a higher level of MCP-1 than those carrying AA genotype. There was no difference between AA and AG genotypes. ∗∗∗: P < .001. B: The effects of VEGF –634C/G polymorphism on VEGF level in DFU patients. CC genotype was significantly associated with up-regulated level of VEGF, compared with GG genotype. No significant difference was detected between patients carrying GG and GC genotypes. ∗∗: indicated P value <.01. DFU = diabetic foot ulcers, MCP-1 = monocyte chemoattractant protein-1, T2DM = type 2 diabetes melitus, VEGF = vascular endothelial growth factor.

In addition, the comparison of VEGF expression was also performed in DFU patients according to their genotypes of VEGF–634C/G polymorphism. The results suggested that the DFU patients carrying CC genotype had a higher level of VEGF than those with GG genotype (P = .007). Moreover, there were no significant differences between DFU patients with CG genotype and GG genotype (P > .05) (Fig. 2B).

Discussion

DFU is a main reason for amputation and death among diabetic patients, moreover, the treatments for DFU lead to a heavy economic burden to the patients and their family.[ Early detection and timely treatment may significantly improve life quality and outcomes of the patients.[ There are 2 major reasons contributing to the occurrence of DFU: diabetic neuropathy and peripheral vascular diseases.[ Therefore, the expression level and polymorphisms of MCP-1 and VEGF which were related to angiogenesis and vascular functions were investigated in the study. The present study may be helpful for diagnosis and prevention of DFU in T2DM patients.

MCP-1 protein which is encoded by MCP-1 gene is involved in various processes, such as inflammation, wound healing, fibrosis, and formation of vessels.[ Accumulating evidences have demonstrated that MCP-1 was involved in various diabetic complications.[ A study carried out by Jeon et al[ had indicated that MCP-1 –2518A/G polymorphism was correlated with risk of proliferative diabetic retinopathy in a Korean population with T2DM. In the study of Raina et al,[ genotypes of MCP-1–2518A/G polymorphism may influence the susceptibility of end stage renal disease caused by T2DM based on north-west Indian population of Punjab. In the current study, we compared the distribution of MCP-1 –2518A/G polymorphism between T2DM patients and DFU patients. PCR-RFLP results indicated that the prevalence of GG genotype was significantly different between test group and control group. The results indicated that GG genotype may increase the risk of DFU for T2DM patients. In addition, we investigated the serum level of MCP-1 in collected specimens and the results suggested that compared with T2DM patients, the DFU patients showed a high level of MCP-1. The abnormal expression of MCP-1 may involve in the progress of DFU. Kasiewicz et al[ had proved that down-regulated of MCP-1 could break the signal way of chronic inflammation within diabetic wound healing in an in vitro co-culture model of DFU. The results were accorded with our findings.

In the study, we evaluated the relationship between MCP-1 level and genotypes of MCP-1–2518A/G polymorphism in DFU patients. Analysis results indicated that patients carrying GG genotype showed a higher expression level than those with AA genotype. There was no significant difference between AA genotype and AG genotype. The results can be explained that polymorphisms in the location of -2518 may influence the transcriptional activity of the gene, thus, regulate its expression. The results were supported by the previous researches. A study carried out by Pham et al[ had demonstrated that G allele in MCP-1 –2518A/G polymorphism was preferentially transcribed and the donors with G allele exhibited an up-regulated level of MCP-1, compared with those carrying C allele. In a word, MCP-1 –2518A/G polymorphism may take part in occurrence and progress of DFU through regulating the gene expression.

In addition, we investigated the distribution of VEGF –634C/G polymorphism in the study populations. Results demonstrated that the prevalence of CC genotype was significantly decreased in DFU patients, compared with T2DM patients. The results implied that T2DM patients with CC genotype of VEGF –634C/G polymorphism may be susceptible to DFU. VEGF–634C/G was a common polymorphism in the 5′-untranslated region of the gene. The polymorphism of the locations had been reported to be associated with risk of osteonecrosis of femoral head, therapeutic effects of 5-FU based chemo/radiotherapy in patients with esophageal squamous cell carcinoma, as well as diabetic complications.[ These results supported the findings in the present study. In addition, low expression of VEGF was detected in DFU patients, compared with T2DM patients. The increased level of VEGF in the local wounding may contribute to wound healing, which may be a potential therapeutic target for DFU.[ Moreover, we found that the expression of VEGF was imbalance between patients carrying different genotypes of VEGF –634C/G polymorphism. Analysis results indicated that DFU patients with CC genotype showed a higher level of VEGF than those carrying GG. The expression of VEGF was not significantly different between GG and GC genotypes. The effects of VEGF –634C/G polymorphism on VEGF expression was approved by Awata et al.[ In their article, VEGF serum level was proved to be higher in healthy individuals with CC genotype of VEGF –634C/G polymorphisms than that in those carrying the other genotypes. However, some studies hold the opposite opinions. A study based on Parkinson disease population indicated that there was not association between VEGF polymorphisms and serum level of VEGF.[ The study carried out by Ungerback et al[ had indicated that there were not significant association between VEGF –634C/G polymorphism and expression of VEGF in colorectal cancer patients based on a Swedish population. The differences might be attributed to the different study populations and the divergences in study diseases. The issue was needed to be verified in the following researches.

In current study, we found that the production of VEGF and MCP-1 was significantly associated with genetic variants in their coding genes. The present study might be helpful in early prevention and diagnosis of DFU in T2DM patients. Moreover, to detect polymorphisms of VEGF and MCP-1, as well as their protein production, might provide guidance for treatment of DFU. However, there were still several limitations in current study. Firstly, the sample size was relatively small that might reduce the reliability of our results. Second, all the patients were collected from the same hospital. The results might be not suitable for other populations due to the regional differences. Additionally, the molecular mechanisms underlying the regulatory function of MCP-1 and VEGF polymorphisms on their protein production remained poorly known. In order to improve our conclusions, further well-designed studies with a larger sample size will be required.

In conclusion, the distributions of MCP-1 –2518A/G and VEGF –634C/G polymorphisms are significantly different between T2DM and DFU patients. Moreover, the genotypes of the 2 studied polymorphisms may influence serum levels of MCP-1 and VEGF in DFU patients. The detected polymorphisms of the genes may play important roles in the occurrence and progress of DFU through their regulatory function on transcription activity of the genes.

Author contributions

Conceptualization: Xiaolei Li.

Data curation: Xiaolei Li.

Formal analysis: Xiaolei Li.

Funding acquisition: Xiaolei Li.

Investigation: Xiaolei Li.

Methodology: Xiaolei Li.

Project administration: Xiaolei Li.

Resources: Xiaolei Li.

Software: Xiaolei Li.

Supervision: Xiaolei Li.

Validation: Xiaolei Li.

Visualization: Xiaolei Li.

Writing – original draft: Xiaolei Li.

Writing – review and editing: Xiaolei Li.