Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complications Arleta Kulwas a, Ewelina Drela a,⁎, Wiesław Jundziłł b, Barbara Góralczyk a, Barbara Ruszkowska-Ciastek a, Danuta Rość a a b
Department of Pathophysiology Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland Department of Vascular Surgery and Angiology, University Hospital of A. Jurasz, Bydgoszcz, Poland
a r t i c l e
i n f o
Article history: Received 6 October 2014 Received in revised form 27 March 2015 Accepted 28 March 2015 Available online xxxx Keywords: Angiogenesis Diabetic foot syndrome VEGF-A CEPCs FGF-2
a b s t r a c t Introduction: Data about angiogenic factors in diabetic foot syndrome (DFS) are insufficient. Therefore, in the present study we focus on circulating endothelial progenitor cells (EPCs) and two major angiogenic factors: vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) in patients with DFS. Materials and methods: We included 75 subjects: 45 patients with type 2 diabetes and 30 controls. The study group was divided into 2 subgroups: 23 patients with diabetic foot and 22 patients without diabetic complications. The concentration of VEGF-A, soluble VEGF receptor 2 (sVEGF-R2) and FGF-2 were measured in plasma samples. The number of circulating EPCs was determined in peripheral venous blood. The number of endothelial progenitor cells was measured with FACSCalibur flow cytometer using monoclonal antibodies directed against antigens specific for EPCs. Results: In our study we observed significant higher levels of VEGF-A and FGF-2 and lower sVEGF-R2 concentration in patients with T2DM compared to healthy subjects. The conducted analysis showed decreased levels of VEGF-A and elevated levels of FGF-2 in patients with DM complicated DFS compared to diabetic patients without DFS. Increased circulating EPCs number was reported in patients with DFS, and the difference was almost statistically significant. Conclusions: The high concentration of VEGF-A and FGF-2, and a positive correlation between them indicate their participation in the process of angiogenesis in T2DM. Decreased sVEGF-R2 may result from inactivation of VEGF-A during complexes formation. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Diabetes is one of the major socioeconomic problems, and it is considered a global epidemic of the XXI century. According to the International Diabetes Federation in 2012 more than 371 million people suffer from diabetes, and it is postulated that in 2030 the number of patients with diabetes rises to 552 million (IDF Diabetes Atlas, 2012; Waniczek et al., 2013). Long lasting (prolonged) hyperglycemia leads to development of diabetic complications, like retinopathy, nephropathy or diabetic foot syndrome (DFS). It is considered that DFS is a significant cause of morbidity and mortality and is not typical for late diabetic complication, but also appears in patients with newly diagnosed diabetes. Normal wound healing is Grant: This study was supported by grant from National Centre of Science (Poland) based on the decision no. DEC-2011/01/N/NZ5/00293. Conflicts of interest: None. ⁎ Corresponding author at: Department of Pathophysiology, Collegium Medicum in Bydgoszcz, Skłodowskiej-Curie 9, 85–094 Bydgoszcz, Poland. Tel.: +48 52 585 3591; fax: +48 52 585 3595. E-mail address: [email protected] (E. Drela).
very important for patients with DFS because of development of diabetic foot ulcer. Impaired tissue regeneration, resulting from insufficient angiogenesis, contributes to non-healing ulcers. Diabetic non-healing ulcers account for more than 60% of all non-traumatic lower limb amputations (Jaiswal, Gambhir, Agrawal, & Harish, 2010). The pathogenesis of each diabetic complication is undoubtedly multifactorial. Nevertheless, impaired angiogenesis is one potential component that might be common for many diabetic complications. Angiogenesis is a multi-stage process involving the endothelium, growth factors and their inhibitors, cytokines, endothelial progenitor cells (EPCs) and enzymes (Jiang & Brey, 2011; Kajdaniuk, Marek, Borgiel-Marek, & Kos-Kudła, 2011; Kota et al., 2012). The process of proper vascular network formation includes four phases: initiation, progression, differentiation and stabilization with maturation. Every phase is controlled by balance between stimulatory and inhibitory factors (Kota et al., 2012). VEGF-A and FGF-2 (basic fibroblast growth factor) are two crucial angiogenic factors. First of them (VEGF-A) activates angiogenesis and is responsible for endothelial cells survival, migration and proliferation (Kajdaniuk et al., 2011). VEGF transmits signal through receptors:
http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013 1056-8727/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
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A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
Flt-1 (VEGF-R1), Flk-1 (VEGF-R2) and Flt-4 (VEGF-R3) that are localized on cell surface. Further receptors are soluble forms of receptor: sVEGF-R1 and sVEGF-R2. Soluble receptors bind VEGF and reduce its biological activity. Soluble receptors for VEGF are known as angiogenesis inhibitor (Kajdaniuk et al., 2011; Shibuya, 2006). Purified FGF-2 stimulates proteases synthesis (metalloproteases and urokinase-type plasminogen activator) that contributes to basement membrane degradation. In that way, cells can migrate into the new blood vessel. FGF-2 plays a critical role in the extracellular matrix (ECM) components synthesis that leads to the maturation of new blood vessels (Mizia-Malarz, Sobol, & Woś, 2008; Presta, Andres, Leali, Dell’Era, & Ronca, 2009; Qazi, Maddula, & Ambati, 2009). Thus, VEGF-A predominate in early angiogenic steps and FGF-2 regulates the later stages of angiogenesis. Co-operation between these factors is needed for property, well-functioning vessels development. The discovery of endothelial progenitor cells has brought new insight into angiogenesis process. These cells exist in the bone marrow (BM) and are heterogenic group of cells. Highly immature EPCs (pre-EPCs) express early hematopoietic markers as CD117 and CD133. Early EPCs (CD133, CD34 and VEGF-R2 positive) may facilitate angiogenesis by secreting pro-angiogenic factors (mainly VEGF). The late EPCs express endothelial markers such as CD31, VEGF-R2, but they are CD133 negative (Lombardo et al., 2012; Qazi et al., 2009; van Ark et al., 2012). EPCs also express receptors for FGF. In order to various stimuli (e. g. ischemia), EPCs are recruited to the peripheral circulation (Lombardo et al., 2012). Recently experimental study has shown that EPCs applied to diabetic wounds increased local VEGF and FGF expression that improved vascularization and process of wound healing (Asai, Takenaka, Ii, et al., 2013). From the angiogenesis point of view, diabetes is a paradoxical disease. An excessive angiogenesis is observed in retinopathy or nephropathy, while in diabetic foot syndrome angiogenic response is impaired. Data about angiogenic factors in DFS are insufficient. Therefore, in the present study we focus on circulating EPCs and two major angiogenic factors: VEGF-A and FGF-2 in patients with DFS.
2. Materials and methods This study was conducted according to the tenets of the Declaration of Helsinki and was approved by the Bioethics Commission of the Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń (no. KB/367/2009). Patients and volunteers were informed about the purpose of the study and the procedures involved. They all gave written informed consent. We included 75 subjects and divided them into two groups: 45 patients with type 2 diabetes and 30 controls. The study group consisted of 22 women and 23 men. The study group was divided into 2 subgroups: 23 patients with diabetic foot and 22 patients without diabetic complications. The control group consisted of 30 healthy volunteers selected with regard to the age and gender (Table 1). All patients were being treated with appropriate therapy in the Diabetic Foot Clinic, University Hospital and the Department of Vascular Surgery and Angiology, University Hospital of A. Jurasz, Bydgoszcz. The criteria for inclusion in the study were glycated hemoglobin value above 7% providing poor diabetes control and the presence of vascular complication with diagnosed diabetic foot syndrome. Classification according to Wagner was done to determine the deep foot lesions during the first visit. T2DM was diagnosed using blood glucose cut-off values as defined by WHO. The patients were treated with insulin, metformin and other appropriate therapy. Hypertension was defined as a systolic pressure ≥ 140 mm Hg or diastolic pressure ≥ 90 mm Hg. Patients with hypertension-received angiotensin-converting enzyme (ACE) alone or associated with other anti-hypertensive agents. The patients who had an operation within the last month, had the end-stage renal failure, chronic liver disease or
Table 1 Clinical characteristics of the patients. Characteristic
Patients with DF
Patients without DF
Gender (M/F) Age (mean) DM duration (years) DFS duration (months) BMI Normal Overweight Obesity Hypertension Smoking Retinopathy Nephropathy Neuropathy
16/7 67 12.4 17
7/15 63 5.3 –
7 10 6 18 7 12 7 12
12 8 2 16 6 – – –
DF – diabetic foot, DM – diabetes mellitus.
any other severe medical conditions requiring active treatment were excluded from this study. The comparative analysis of the examined group (only patients with DFS) included the influence of factors as: gender, smoking (n = 7), retinopathy (n = 12), nephropathy (n = 7), neuropathy (n = 12) and hypertension (n = 18) on measured parameters. 2.1. Immunoassays methods The material for the research of angiogenic factors was blood samples drawn after 12 hours of fasting. Plasma was obtained from the whole blood collected into tubes containing ethylenediaminetetraacetic (EDTA) and centrifuged at 3000 rpm for 15 minutes. All the samples were stored at −80 °C until the analysis, but no longer than for 6 months. VEGF concentration was determined in the plasma samples considering the known release of it by platelets. The concentration of VEGF-R2 and FGF-2 was also measured in plasma samples. The growth factors and receptors were measured using a human Elisa kit: Quantikine Human Immunoassay produced by R&D Systems, Minneapolis, MN, USA. 2.2. Quantification of circulating EPCs by flow cytometry To determine the number of circulating EPCs, 3 ml of peripheral venous blood was collected in EDTA tubes and processed within 5 h after collection. The number of endothelial progenitor cells was measured with FACSCalibur flow cytometer (Becton Dickinson, San Diego, USA) using monoclonal antibodies directed against antigens specific for endothelial progenitor cells (EPCs). Acquired data were analyzed by using CellQuest software (Becton Dickinson). The selection of antigens allowed to assess the population of early and late circulating EPCs. The following monoclonal antibodies were used in this study: fluorescein isothiocyanate (FITC)-conjugated anti-CD31, PerCP-Cy5.5conjugated anti-CD45, as well as APC-conjugated anti-CD34 antibody (all BD Biosciences, Pharmingen, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). Endothelial progenitor cells (EPCs) were defined as negative for the hematopoietic marker CD 45 and positive for the endothelial progenitor marker CD 133 and positive for the endothelial cell markers CD 31 and CD 34. At least 100,000 events were collected before analysis. TruCount tubes (BD Biosciences, San Jose, CA, USA) containing a calibrated number of fluorescent beads and `lyse-no-wash` procedures were used in the present study. To evaluate immunophenotyping of circulating endothelial progenitor cells, 50 μl of whole blood (taken at the EDTA-K2) were incubated in TruCOUNT tube (Becton Dickinson) in the dark for 15 minutes with antibodies: anti-CD45, anti-CD31, anti-CD34,
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Table 2 Levels of circulating EPCs, VEGF, sVEGF-R2 and FGF-2 in patients with T2DM and healthy volunteers. Parameter
Circulating EPCs/μl VEGF-A [pg/ml] sVEGF-R2 [pg/ml] FGF-2 [pg/ml]
Study groupN = 45
Control groupN = 30
p value
Me
Q1; Q3
Me
Q1; Q3
0.40 48.7 9419.0 5.65
0.1; 1.22 19.08; 90.81 8449.5; 10677,0 5.02; 7.34
0.41 15.06 10738.75 4.92
0.2; 0.92 7.98; 27.84 9670.5; 11766.0 4.6; 5.44
p p p p
= = = =
0.978 0.0002 0.007 0.002
Me – median, Q1 – lower quartile, Q3 – upper quartile, p – value p. VEGF-A – vascular endothelial growth factor A, sVEGF-R2 – soluble receptor 2 of vascular endothelial growth factor, FGF-2 - fibroblast growth factor 2, circulating EPCs – circulating endothelial progenitor cells, DF – diabetic foot, T2DM – type 2 diabetes mellitus.
Table 3 Circulating EPCs and levels of angiogenic parameters and in patients with DF and without DF. Parameter
Circulating EPCs/μl VEGF-A [pg/ml] sVEGF-R2 [pg/ml] FGF-2 [pg/ml]
Patients with DF N = 23
Patients without DF N = 22
p value
Me
Q1; Q3
Me
Q1; Q3
0.41 36.0 8930.5 6.39
0.2; 1.22 13.74; 73.95 8360.0; 10356.0 5.54; 8.42
0.31 79.16 10099.0 4.97
0.1; 1.53 29.56; 131.55 8765.25; 10841.5 4.45; 6.02
p p p p
= = = =
0.0581 0.04 0.26 0.0002
Me – median, Q1 – lower quartile, Q3 – upper quartile, p – value p. VEGF-A – vascular endothelial growth factor A, sVEGF-R2 – soluble receptor 2 of vascular endothelial growth factor, FGF-2 - fibroblast growth factor 2, circulating EPCs – circulating endothelial progenitor cells, DF – diabetic foot.
anti–CD133 (20 μl each). The material was subjected to 10 minutes lysed with a solution of Lysing Solution, without subsequent rinsing (method "lyse no-wash"). During each analysis, the fluorescence of 100,000 cells was measured. The statistical analysis was carried out using Statistica 10,0 (StatStoft Inc, USA). The Shapiro–Wilk test was used to assess the normality of distributions. The median (Me), lower quartile (Q1) and upper quartile (Q3) were used for variables that were not normally distributed. The significance of differences between two groups was analyzed using Mann–Whitney U test (nonparametric data). For comparison between three groups, ANOVA and post hoc tests were used. p-value of b 0.05 was considered statistically significant.
3. Results In our study we observed significant higher levels of VEGF-A (p = 0.0002) and FGF-2 (p = 0.002) and lower sVEGF-R2 concentration (p = 0.007) in patients with type 2 diabetes compared to healthy subjects. There was no difference in circulating EPCs number in these groups (p = 0.9) (Table 2). The conducted analysis of the angiogenesis parameters including patients with and without DFS showed decreased levels of VEGF-A (p = 0.04) and elevated levels of FGF-2 (p = 0.0002) in patients with diabetes mellitus complicated DFS. The differences were statistically significant. Increased circulating EPCs number was reported in patients with DFS, and the difference was almost statistically significant. There were no essential differences in the concentration of the soluble form of the receptor for VEGF-A (Table 3). No significant difference was seen as regards clinical characteristics. We observed the only significantly lower FGF-2 concentrations (p = 0.05) in patients with DFS and associated retinopathy (Table 4). The analysis of correlation was made between the concentrations of VEGF-A, sVEGF-R2, FGF-2, the number of circulating EPCs and gender, age, BMI, WHR, diabetes duration, DFS duration in the group with diabetes complicated DFS. There was a negative correlation between BMI and FGF-2 (R = −0.48, p = 0.02) and between duration of diabetes and the number of circulating EPCs (R = −0.43, p = 0.04). There was no relationship between other parameters.
There was a positive correlation between VEGF-A and FGF-2 (R = 0.42, p = 0.04) when analyzing these parameters in a group of 45 patients with type 2 diabetes. 4. Discussion In this study, we evaluated the cell population of CD31+, CD133+, CD34+ and CD45 – (we did not distinguish circulating EPCs for early and late). The inclusion of CD133 marker into the identification panel may indicate the presence of the pre-EPCs (so-called early EPCs) cells that have angiogenic properties. In our study, we observed no statistically
Table 4 Circulating EPCs, angiogenic factors and clinical parameters in patients with diabetic foot. Clinical parameter
Circulating EPCs [/μl]
VEGF-A [pg/ml]
sVEGF-R2 [pg/ml]
FGF-2 [pg/ml]
Gender Female Male
Me = 0.61 Me = 0.41
Me = 41.08 Me = 31.52
Me = 8649.5 Me = 9077.5
Me = 6.39 Me = 6.65
Smoking Yes No
Me = 0.31 Me = 0.65
Me = 48.72 Me = 31.44
Me = 9511.0 Me = 8790.0
Me = 7.77 Me = 6.33
Hypertension Yes No
Me = 0.41 Me = 1.22
Me = 38.54 Me = 22.26
Me = 8790.0 Me = 11619.5
Me = 6.1 Me = 7.02
Retinopathy Yes No
Me = 0.41 Me = 0.40
Me = 35.92 Me = 41.08
Me = 8790.0 Me = 9017.0
Me = 5.75⁎ Me = 7.45
Nephropathy Yes No
Me = 0.31 Me = 0.46
Me = 27.04 Me = 38.54
Me = 9138.0 Me = 8790.0
Me = 7.45 Me = 6.12
Neuropathy Yes No
Me = 0.46 Me = 0.41
Me = 44.9 Me = 25.45
Me = 9034.2 Me = 8449.5
Me = 5.75 Me = 7.13
Me – median. “Hypertension yes” – means: patients with hypertension. “Hypertension no” – means: patients without hypertension, etc. ⁎ p b 0.05 statistically significant.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
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A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
significant difference in the number of circulating EPCs in patients with diabetes and healthy subjects. For further research the angiogenic variables were tested among the subgroups (patients with DF and without DF). The analysis of the subgroups showed increased number of circulating EPCs in patients with DFS compared to patients with uncomplicated diabetes (p value of borderline statistical significance). Evaluation of the available literature concerning the circulating EPCs in patients with T2DM complicated DFS and without DF complications shows different results. Lombardo et al. indicate an increased number of pre-EPCs and decreased number of late EPCs in patients with T2DM compared to the healthy subjects (Lombardo et al., 2012). In 2002, van Ark et al. also reported the reduced number of EPCs in patients with diabetes compared to the healthy (van Ark et al., 2012). Tepper et al. showed that EPCs from patients with type 2 diabetes were characterized by incorrect proliferation and adhesion. Further studies confirmed the abnormal function and accelerated apoptosis of EPCs in patients with diabetes (Chen et al., 2009; Cheng, Dong, Wang, Kang, & Xu, 2009; Jung, Rafnsson, Shemyakin, Bohm, & Pernow, 2010; Tepper et al., 2002). Studies conducted by Li et al. indicate that advanced glycation end products (AGEs) impair the functioning of late EPCs. The researchers suggest that this may lead to the development of vascular complications in diabetes (Li et al., 2012). In our study, patients with diabetes had higher VEGF-A concentration and lower sVEGF-R2 concentration compared to the healthy subjects. Both factors are involved in angiogenesis, with the difference that VEGF-A is a proangiogenic factor, and soluble receptor is considered as a natural VEGF-A inhibitor (Shibuya, 2006). Unfortunately, data in the available literature about systemic VEGF-A concentration in diabetic patients are not clear. Tecilazich et al. did not observe significant differences in VEGF-A levels in the healthy controls and in the three groups of patients with diabetes mellitus: DM-not at risk of DFU development, and DM-at risk of DFU and DM-DFU (Tecilazich et al., 2013). Different results were obtained by Doupis et al. (serum VEGF-A level) and Nandy et al. (plasma VEGF-A level). In both studies, higher level of VEGF-A in diabetic patients compared to controls (healthy subjects) was observed (Doupis et al., 2011; Nandy & Mukhopadhyay, 2010). In addition, Choksy et al. demonstrated an intense expression of VEGF in distal ischemic tissues (foot), especially in the necrotic area and ulcers (Choksy, Pockley, Wajeh, & Chan, 2004). We suggest that increased VEGF-A level in our patients may result from tissue hypoxia. Elevated level of VEGF-A in diabetic patients may also be explained by the effect of chronic inflammation that occurs in T2DM. IL-6 is a pro-inflammatory cytokine. Increased levels of this cytokine may lead to the VEGF-A secretion by platelets and leukocytes (van den Over, Raterman, Nurmohamed, & Simsek, 2010). The expression of VEGF is observed in megakaryocytes, which then is stored in platelet α granules (Kajdaniuk et al., 2011). Diabetes has been considered as a prothrombotic state. It is associated with the increased number of platelets and their hyperreactivity, which is characterized by excessive activation and aggregation (Ferreiro, Gomez-Hospital, & Angiolillo, 2010; Kim, Bae, & Kim, 2013). Platelet activation contributes to PDGF and VEGF secretion from their granules (Kodiatte et al., 2012). Therefore, it might be assumed that elevated number of activated platelets is an additional source of VEGF in diabetic patients. In patients with diabetes there are other factors responsible for increased VEGF-A level. It may result from active process of angiogenesis that occurs during wound healing. Francesco et al. demonstrated that VEGF concentration was higher in DM patients complicated with DF compared to the healthy subjects. Moreover, the concentration of VEGF underwent reduction during healing process in DM-DFU group (duration of treatment: 12 weeks) (Tecilazich et al., 2013). FGF-2, next to the VEGF, plays an essential role in angiogenesis. FGF-2 has both angiogenic properties (stimulates endothelial cells to VEGF synthesis) and proinflammatory properties (enhances IL-6
expression, which also increases the synthesis of VEGF) (Qazi et al., 2009). In our study, increased levels of FGF-2 was found in patients with diabetes (n = 45; DM compared to the controls) as well as in patients with DF (DM-DF compared to DM). Similar results were obtained by Gui et al. They observed increased level of FGF-2 in diabetic patients compared to the controls (Gui et al., 2013). The interaction between VEGF-A and FGF-2 in patients with and without DF is worth noting. Decreased concentration of VEGF-A is associated with elevated concentrations of FGF-2 in patients with diabetic foot. The opposite situation is observed in patients with uncomplicated diabetes. It may result from a specific release kinetics of these two factors during healing process. The physiological process of wound healing is characterized by specific temporal secretion of VEGF-A and FGF-2. High level of FGF-2 is observed immediately after injury and stabilizes to basal level within 3 days. The maximal expression of VEGF mRNA appears between 3 and 7 days. When the VEGF concentration decreases, FGF-2 levels rise again. VEGF expression decreases when hypoxia is reduced or other angiogenic factor (e.g., FGF-2) takes control of blood vessels formation (Bao, Kodra, Tomic-Canic, et al., 2009). This mechanism indicates that these two factors, at the appropriate concentration and time, are necessary to create vascularization. Our study is in agreement with this scenario: we noted positive correlation between VEGF-A and FGF-2 levels in the whole group of patients with type 2 diabetes. In the available literature, we found no data about sVEGF-R2 concentration in patients with DFS. Research conducted by Wierzbowska et al. shows that sVEGF-R2 inhibits angiogenesis dependent of VEGF (Wierzbowska, RObak, Wrzesień-Kuś, et al., 2003). The results presented in this study may suggest that VEGF gene activation occurred in diabetes in response to the tissue hypoxia. It might also suggest that the activation of pro-angiogenic factors is associated with the simultaneous activation of the inhibitory factor. VEGF released into the circulation forms complexes with a soluble receptor 2 (followed by lowering the concentration of sVEGF-R2). In this way, the development of blood vessels is controlled. With regard to the analyzing of selected clinical parameters and tested parameters, we observed increased FGF-2 level in patients without retinopathy compared to the patients with DF and accompanying retinopapthy. There were no differences in VEGF-A concentration and CEPCs number in patients with DF and accompanying retinopathy, nephropathy, and neuropathy. There is no similar study in this field. Baharivand et al. shows increased levels of VEGF in diabetic patients with proliferative retinopathy compared to patients without active retinopathy. This is in contrast with results reported by Hernandez et al. The authors observed no difference in VEGF level in serum (Baharivand et al., 2012; Hernandez, Lecube, Segura, Sararols, & Simo, 2002). Study conducted by Kim et al. showed no difference in the concentration of VEGF in plasma depending on the severity of nephropathy. In contrast, urine VEGF secretion increased with the stage of renal failure (Kim, Oh, Seo, et al., 2005).
5. Conclusions A study shows that the presence of DF has a significant effect on the systemic concentration of the factors involved in angiogenesis. These results may indicate an active process of blood vessel formation during healing in patients with DF, but a concomitant hyperglycemia significantly impairs this process. The role of EPCs in angiogenesis in patients with DF needs further investigation.
Acknowledgments This study was supported by grant from the National Centre of Science (Poland) based on the decision no. DEC-2011/01/N/NZ5/00293.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Kim, J. H., Bae, H. Y., & Kim, S. Y. (2013). Clinical marker of platelet hyperreactivity in diabetes mellitus. Diabetes and Metabolism Journal, 37, 423–428. Kim, N. H., Oh, J. H., Seo, J. A., Lee, K. W., Kim, S. G., Choi, K. M., et al. (2005). Vascular endothelial growth factor (VEGF) and soluble VEGF receptor FLT-1 in diabetic nephropathy. Kidney International, 67, 167–177. Kodiatte, T. A., Manikyam, U. K., Rao, S. B., Jagadish, T. M., Reddy, M., Lingaiah, H. K. M., et al. (2012). Mean platelet volume in type 2 diabetes mellitus. Journal of Laboratory Physicians, 1, 5–9. Kota, S. K., Meher, L. K., Jammula, S., Kota, S. K., Krishna, S. V., & Modi, K. D. (2012). Abberant angiogenesis: The gateway to diabetic complications. Indian Journal of Endocrinology and Metabolism., 6, 918–930. Li, H., Zhang, X., Guan, X., Cui, X., Wang, Y., Chu, H., & Cheng, M. (2012). Advanced glycation end products impair the migration, adhesion and secretion potentials of late endothelial progenitor cells. Cardiovascular Diabetology, 11, 46, http://dx.doi. org/10.1186/1475-2840-11-46. Lombardo, M. F., Iacopino, P., Cuzzola, M., Spiniello, E., Garreffa, C., Farrelli, F., et al. (2012). Type 2 diabetes mellitus impairs the maturation of endothelial progenitor cells and increases the number of circulating endothelial cells in peripheral blood. Cytometry. Part A, 81A, 856–864. Mizia-Malarz, A., Sobol, G., & Woś, H. (2008). Proangiogenic factors: Vascularendothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) - the characteristic and function. Przegla̧d Lekarski, 65, 353–357. Nandy, D., & Mukhopadhyay, D. (2010). Both vascular endothelial growth factor and soluble Flt-1 are increased in type 2 diabetes but not in impaired fasting glucose. Journal of Investigative Medicine : the Official Publication of the American Federation for Clinical Research, 58, 804–806. Presta, M., Andres, G., Leali, D., Dell’Era, P., & Ronca, R. (2009). Inflammatory cells and chemokines sustain FGF-2 induced angiogenesis. European Cytokine Network, 2, 39–50. Qazi, Y., Maddula, S., & Ambati, B. K. (2009). Mediators of ocular angiogenesis. Journal of Genetics, 88, 495–515. Shibuya, M. (2006). Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. Journal of Biochemistry and Molecular Biology, 5, 469–478. Tecilazich, F., Dinh, T., Pradhan-Nabzdyk, L., Leal, E., Tellechea, A., Kafanas, A., et al. (2013). Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers. PLoS One, 8(12), e83314, http://dx.doi.org/10.1371/journal. pone.0083314. Tepper, O. M., Galiano, R. D., Capla, J. M., Kalka, C., Gagne, P. J., Jocobowitz, G. R., et al. (2002). Human endothelial progenitor cells from type 2 diabetic exhibit impaired proliferation, adhesion and incorporation into vascular structures. Circulation, 106, 2781–2786. van Ark, J., Moser, J., Lexis, C. P. H., Bekkema, F., Pop, I., van der Horst, I. C. C., et al. (2012). Type 2 diabetes mellitus is associated with an imbalance in circulating endothelial and smooth muscle progenitor cell number. Diabetologia, 55, 2501–2512. van den Over, I. A. M., Raterman, H. G., Nurmohamed, M. T., & Simsek, S. (2010). Endothelial dysfunction, inflammation and apoptosis in diabetes mellitus. Mediators of Inflammation(2010), 1–15. Waniczek, D., Kozowicz, A., Muc-Wierzgoń, M., Kokot, T., Świętochowska, E., & Nowakowska-Zajdel, E. (2013). Adjunct methods of the standard diabetic foot ulceration therapy. Evidence-Based Complementary and Alternative Medicine, 2013, 243568, http://dx.doi.org/10.1155/2013/243568. Wierzbowska, A., RObak, T., Wrzesień-Kuś, T., Krawczynska, A., Lech-Maranda, E., & Urbanska-Rys, H. (2003). Circulating VEGF-A and its soluble receptors VEGF-R1 and VEGF-R2 in patient with acute leukemia. European Cytokine Network, 14, 149–153.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
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Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complications Arleta Kulwas a, Ewelina Drela a,⁎, Wiesław Jundziłł b, Barbara Góralczyk a, Barbara Ruszkowska-Ciastek a, Danuta Rość a a b
Department of Pathophysiology Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland Department of Vascular Surgery and Angiology, University Hospital of A. Jurasz, Bydgoszcz, Poland
a r t i c l e
i n f o
Article history: Received 6 October 2014 Received in revised form 27 March 2015 Accepted 28 March 2015 Available online xxxx Keywords: Angiogenesis Diabetic foot syndrome VEGF-A CEPCs FGF-2
a b s t r a c t Introduction: Data about angiogenic factors in diabetic foot syndrome (DFS) are insufficient. Therefore, in the present study we focus on circulating endothelial progenitor cells (EPCs) and two major angiogenic factors: vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) in patients with DFS. Materials and methods: We included 75 subjects: 45 patients with type 2 diabetes and 30 controls. The study group was divided into 2 subgroups: 23 patients with diabetic foot and 22 patients without diabetic complications. The concentration of VEGF-A, soluble VEGF receptor 2 (sVEGF-R2) and FGF-2 were measured in plasma samples. The number of circulating EPCs was determined in peripheral venous blood. The number of endothelial progenitor cells was measured with FACSCalibur flow cytometer using monoclonal antibodies directed against antigens specific for EPCs. Results: In our study we observed significant higher levels of VEGF-A and FGF-2 and lower sVEGF-R2 concentration in patients with T2DM compared to healthy subjects. The conducted analysis showed decreased levels of VEGF-A and elevated levels of FGF-2 in patients with DM complicated DFS compared to diabetic patients without DFS. Increased circulating EPCs number was reported in patients with DFS, and the difference was almost statistically significant. Conclusions: The high concentration of VEGF-A and FGF-2, and a positive correlation between them indicate their participation in the process of angiogenesis in T2DM. Decreased sVEGF-R2 may result from inactivation of VEGF-A during complexes formation. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Diabetes is one of the major socioeconomic problems, and it is considered a global epidemic of the XXI century. According to the International Diabetes Federation in 2012 more than 371 million people suffer from diabetes, and it is postulated that in 2030 the number of patients with diabetes rises to 552 million (IDF Diabetes Atlas, 2012; Waniczek et al., 2013). Long lasting (prolonged) hyperglycemia leads to development of diabetic complications, like retinopathy, nephropathy or diabetic foot syndrome (DFS). It is considered that DFS is a significant cause of morbidity and mortality and is not typical for late diabetic complication, but also appears in patients with newly diagnosed diabetes. Normal wound healing is Grant: This study was supported by grant from National Centre of Science (Poland) based on the decision no. DEC-2011/01/N/NZ5/00293. Conflicts of interest: None. ⁎ Corresponding author at: Department of Pathophysiology, Collegium Medicum in Bydgoszcz, Skłodowskiej-Curie 9, 85–094 Bydgoszcz, Poland. Tel.: +48 52 585 3591; fax: +48 52 585 3595. E-mail address: [email protected] (E. Drela).
very important for patients with DFS because of development of diabetic foot ulcer. Impaired tissue regeneration, resulting from insufficient angiogenesis, contributes to non-healing ulcers. Diabetic non-healing ulcers account for more than 60% of all non-traumatic lower limb amputations (Jaiswal, Gambhir, Agrawal, & Harish, 2010). The pathogenesis of each diabetic complication is undoubtedly multifactorial. Nevertheless, impaired angiogenesis is one potential component that might be common for many diabetic complications. Angiogenesis is a multi-stage process involving the endothelium, growth factors and their inhibitors, cytokines, endothelial progenitor cells (EPCs) and enzymes (Jiang & Brey, 2011; Kajdaniuk, Marek, Borgiel-Marek, & Kos-Kudła, 2011; Kota et al., 2012). The process of proper vascular network formation includes four phases: initiation, progression, differentiation and stabilization with maturation. Every phase is controlled by balance between stimulatory and inhibitory factors (Kota et al., 2012). VEGF-A and FGF-2 (basic fibroblast growth factor) are two crucial angiogenic factors. First of them (VEGF-A) activates angiogenesis and is responsible for endothelial cells survival, migration and proliferation (Kajdaniuk et al., 2011). VEGF transmits signal through receptors:
http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013 1056-8727/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
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A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
Flt-1 (VEGF-R1), Flk-1 (VEGF-R2) and Flt-4 (VEGF-R3) that are localized on cell surface. Further receptors are soluble forms of receptor: sVEGF-R1 and sVEGF-R2. Soluble receptors bind VEGF and reduce its biological activity. Soluble receptors for VEGF are known as angiogenesis inhibitor (Kajdaniuk et al., 2011; Shibuya, 2006). Purified FGF-2 stimulates proteases synthesis (metalloproteases and urokinase-type plasminogen activator) that contributes to basement membrane degradation. In that way, cells can migrate into the new blood vessel. FGF-2 plays a critical role in the extracellular matrix (ECM) components synthesis that leads to the maturation of new blood vessels (Mizia-Malarz, Sobol, & Woś, 2008; Presta, Andres, Leali, Dell’Era, & Ronca, 2009; Qazi, Maddula, & Ambati, 2009). Thus, VEGF-A predominate in early angiogenic steps and FGF-2 regulates the later stages of angiogenesis. Co-operation between these factors is needed for property, well-functioning vessels development. The discovery of endothelial progenitor cells has brought new insight into angiogenesis process. These cells exist in the bone marrow (BM) and are heterogenic group of cells. Highly immature EPCs (pre-EPCs) express early hematopoietic markers as CD117 and CD133. Early EPCs (CD133, CD34 and VEGF-R2 positive) may facilitate angiogenesis by secreting pro-angiogenic factors (mainly VEGF). The late EPCs express endothelial markers such as CD31, VEGF-R2, but they are CD133 negative (Lombardo et al., 2012; Qazi et al., 2009; van Ark et al., 2012). EPCs also express receptors for FGF. In order to various stimuli (e. g. ischemia), EPCs are recruited to the peripheral circulation (Lombardo et al., 2012). Recently experimental study has shown that EPCs applied to diabetic wounds increased local VEGF and FGF expression that improved vascularization and process of wound healing (Asai, Takenaka, Ii, et al., 2013). From the angiogenesis point of view, diabetes is a paradoxical disease. An excessive angiogenesis is observed in retinopathy or nephropathy, while in diabetic foot syndrome angiogenic response is impaired. Data about angiogenic factors in DFS are insufficient. Therefore, in the present study we focus on circulating EPCs and two major angiogenic factors: VEGF-A and FGF-2 in patients with DFS.
2. Materials and methods This study was conducted according to the tenets of the Declaration of Helsinki and was approved by the Bioethics Commission of the Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń (no. KB/367/2009). Patients and volunteers were informed about the purpose of the study and the procedures involved. They all gave written informed consent. We included 75 subjects and divided them into two groups: 45 patients with type 2 diabetes and 30 controls. The study group consisted of 22 women and 23 men. The study group was divided into 2 subgroups: 23 patients with diabetic foot and 22 patients without diabetic complications. The control group consisted of 30 healthy volunteers selected with regard to the age and gender (Table 1). All patients were being treated with appropriate therapy in the Diabetic Foot Clinic, University Hospital and the Department of Vascular Surgery and Angiology, University Hospital of A. Jurasz, Bydgoszcz. The criteria for inclusion in the study were glycated hemoglobin value above 7% providing poor diabetes control and the presence of vascular complication with diagnosed diabetic foot syndrome. Classification according to Wagner was done to determine the deep foot lesions during the first visit. T2DM was diagnosed using blood glucose cut-off values as defined by WHO. The patients were treated with insulin, metformin and other appropriate therapy. Hypertension was defined as a systolic pressure ≥ 140 mm Hg or diastolic pressure ≥ 90 mm Hg. Patients with hypertension-received angiotensin-converting enzyme (ACE) alone or associated with other anti-hypertensive agents. The patients who had an operation within the last month, had the end-stage renal failure, chronic liver disease or
Table 1 Clinical characteristics of the patients. Characteristic
Patients with DF
Patients without DF
Gender (M/F) Age (mean) DM duration (years) DFS duration (months) BMI Normal Overweight Obesity Hypertension Smoking Retinopathy Nephropathy Neuropathy
16/7 67 12.4 17
7/15 63 5.3 –
7 10 6 18 7 12 7 12
12 8 2 16 6 – – –
DF – diabetic foot, DM – diabetes mellitus.
any other severe medical conditions requiring active treatment were excluded from this study. The comparative analysis of the examined group (only patients with DFS) included the influence of factors as: gender, smoking (n = 7), retinopathy (n = 12), nephropathy (n = 7), neuropathy (n = 12) and hypertension (n = 18) on measured parameters. 2.1. Immunoassays methods The material for the research of angiogenic factors was blood samples drawn after 12 hours of fasting. Plasma was obtained from the whole blood collected into tubes containing ethylenediaminetetraacetic (EDTA) and centrifuged at 3000 rpm for 15 minutes. All the samples were stored at −80 °C until the analysis, but no longer than for 6 months. VEGF concentration was determined in the plasma samples considering the known release of it by platelets. The concentration of VEGF-R2 and FGF-2 was also measured in plasma samples. The growth factors and receptors were measured using a human Elisa kit: Quantikine Human Immunoassay produced by R&D Systems, Minneapolis, MN, USA. 2.2. Quantification of circulating EPCs by flow cytometry To determine the number of circulating EPCs, 3 ml of peripheral venous blood was collected in EDTA tubes and processed within 5 h after collection. The number of endothelial progenitor cells was measured with FACSCalibur flow cytometer (Becton Dickinson, San Diego, USA) using monoclonal antibodies directed against antigens specific for endothelial progenitor cells (EPCs). Acquired data were analyzed by using CellQuest software (Becton Dickinson). The selection of antigens allowed to assess the population of early and late circulating EPCs. The following monoclonal antibodies were used in this study: fluorescein isothiocyanate (FITC)-conjugated anti-CD31, PerCP-Cy5.5conjugated anti-CD45, as well as APC-conjugated anti-CD34 antibody (all BD Biosciences, Pharmingen, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). Endothelial progenitor cells (EPCs) were defined as negative for the hematopoietic marker CD 45 and positive for the endothelial progenitor marker CD 133 and positive for the endothelial cell markers CD 31 and CD 34. At least 100,000 events were collected before analysis. TruCount tubes (BD Biosciences, San Jose, CA, USA) containing a calibrated number of fluorescent beads and `lyse-no-wash` procedures were used in the present study. To evaluate immunophenotyping of circulating endothelial progenitor cells, 50 μl of whole blood (taken at the EDTA-K2) were incubated in TruCOUNT tube (Becton Dickinson) in the dark for 15 minutes with antibodies: anti-CD45, anti-CD31, anti-CD34,
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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Table 2 Levels of circulating EPCs, VEGF, sVEGF-R2 and FGF-2 in patients with T2DM and healthy volunteers. Parameter
Circulating EPCs/μl VEGF-A [pg/ml] sVEGF-R2 [pg/ml] FGF-2 [pg/ml]
Study groupN = 45
Control groupN = 30
p value
Me
Q1; Q3
Me
Q1; Q3
0.40 48.7 9419.0 5.65
0.1; 1.22 19.08; 90.81 8449.5; 10677,0 5.02; 7.34
0.41 15.06 10738.75 4.92
0.2; 0.92 7.98; 27.84 9670.5; 11766.0 4.6; 5.44
p p p p
= = = =
0.978 0.0002 0.007 0.002
Me – median, Q1 – lower quartile, Q3 – upper quartile, p – value p. VEGF-A – vascular endothelial growth factor A, sVEGF-R2 – soluble receptor 2 of vascular endothelial growth factor, FGF-2 - fibroblast growth factor 2, circulating EPCs – circulating endothelial progenitor cells, DF – diabetic foot, T2DM – type 2 diabetes mellitus.
Table 3 Circulating EPCs and levels of angiogenic parameters and in patients with DF and without DF. Parameter
Circulating EPCs/μl VEGF-A [pg/ml] sVEGF-R2 [pg/ml] FGF-2 [pg/ml]
Patients with DF N = 23
Patients without DF N = 22
p value
Me
Q1; Q3
Me
Q1; Q3
0.41 36.0 8930.5 6.39
0.2; 1.22 13.74; 73.95 8360.0; 10356.0 5.54; 8.42
0.31 79.16 10099.0 4.97
0.1; 1.53 29.56; 131.55 8765.25; 10841.5 4.45; 6.02
p p p p
= = = =
0.0581 0.04 0.26 0.0002
Me – median, Q1 – lower quartile, Q3 – upper quartile, p – value p. VEGF-A – vascular endothelial growth factor A, sVEGF-R2 – soluble receptor 2 of vascular endothelial growth factor, FGF-2 - fibroblast growth factor 2, circulating EPCs – circulating endothelial progenitor cells, DF – diabetic foot.
anti–CD133 (20 μl each). The material was subjected to 10 minutes lysed with a solution of Lysing Solution, without subsequent rinsing (method "lyse no-wash"). During each analysis, the fluorescence of 100,000 cells was measured. The statistical analysis was carried out using Statistica 10,0 (StatStoft Inc, USA). The Shapiro–Wilk test was used to assess the normality of distributions. The median (Me), lower quartile (Q1) and upper quartile (Q3) were used for variables that were not normally distributed. The significance of differences between two groups was analyzed using Mann–Whitney U test (nonparametric data). For comparison between three groups, ANOVA and post hoc tests were used. p-value of b 0.05 was considered statistically significant.
3. Results In our study we observed significant higher levels of VEGF-A (p = 0.0002) and FGF-2 (p = 0.002) and lower sVEGF-R2 concentration (p = 0.007) in patients with type 2 diabetes compared to healthy subjects. There was no difference in circulating EPCs number in these groups (p = 0.9) (Table 2). The conducted analysis of the angiogenesis parameters including patients with and without DFS showed decreased levels of VEGF-A (p = 0.04) and elevated levels of FGF-2 (p = 0.0002) in patients with diabetes mellitus complicated DFS. The differences were statistically significant. Increased circulating EPCs number was reported in patients with DFS, and the difference was almost statistically significant. There were no essential differences in the concentration of the soluble form of the receptor for VEGF-A (Table 3). No significant difference was seen as regards clinical characteristics. We observed the only significantly lower FGF-2 concentrations (p = 0.05) in patients with DFS and associated retinopathy (Table 4). The analysis of correlation was made between the concentrations of VEGF-A, sVEGF-R2, FGF-2, the number of circulating EPCs and gender, age, BMI, WHR, diabetes duration, DFS duration in the group with diabetes complicated DFS. There was a negative correlation between BMI and FGF-2 (R = −0.48, p = 0.02) and between duration of diabetes and the number of circulating EPCs (R = −0.43, p = 0.04). There was no relationship between other parameters.
There was a positive correlation between VEGF-A and FGF-2 (R = 0.42, p = 0.04) when analyzing these parameters in a group of 45 patients with type 2 diabetes. 4. Discussion In this study, we evaluated the cell population of CD31+, CD133+, CD34+ and CD45 – (we did not distinguish circulating EPCs for early and late). The inclusion of CD133 marker into the identification panel may indicate the presence of the pre-EPCs (so-called early EPCs) cells that have angiogenic properties. In our study, we observed no statistically
Table 4 Circulating EPCs, angiogenic factors and clinical parameters in patients with diabetic foot. Clinical parameter
Circulating EPCs [/μl]
VEGF-A [pg/ml]
sVEGF-R2 [pg/ml]
FGF-2 [pg/ml]
Gender Female Male
Me = 0.61 Me = 0.41
Me = 41.08 Me = 31.52
Me = 8649.5 Me = 9077.5
Me = 6.39 Me = 6.65
Smoking Yes No
Me = 0.31 Me = 0.65
Me = 48.72 Me = 31.44
Me = 9511.0 Me = 8790.0
Me = 7.77 Me = 6.33
Hypertension Yes No
Me = 0.41 Me = 1.22
Me = 38.54 Me = 22.26
Me = 8790.0 Me = 11619.5
Me = 6.1 Me = 7.02
Retinopathy Yes No
Me = 0.41 Me = 0.40
Me = 35.92 Me = 41.08
Me = 8790.0 Me = 9017.0
Me = 5.75⁎ Me = 7.45
Nephropathy Yes No
Me = 0.31 Me = 0.46
Me = 27.04 Me = 38.54
Me = 9138.0 Me = 8790.0
Me = 7.45 Me = 6.12
Neuropathy Yes No
Me = 0.46 Me = 0.41
Me = 44.9 Me = 25.45
Me = 9034.2 Me = 8449.5
Me = 5.75 Me = 7.13
Me – median. “Hypertension yes” – means: patients with hypertension. “Hypertension no” – means: patients without hypertension, etc. ⁎ p b 0.05 statistically significant.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
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significant difference in the number of circulating EPCs in patients with diabetes and healthy subjects. For further research the angiogenic variables were tested among the subgroups (patients with DF and without DF). The analysis of the subgroups showed increased number of circulating EPCs in patients with DFS compared to patients with uncomplicated diabetes (p value of borderline statistical significance). Evaluation of the available literature concerning the circulating EPCs in patients with T2DM complicated DFS and without DF complications shows different results. Lombardo et al. indicate an increased number of pre-EPCs and decreased number of late EPCs in patients with T2DM compared to the healthy subjects (Lombardo et al., 2012). In 2002, van Ark et al. also reported the reduced number of EPCs in patients with diabetes compared to the healthy (van Ark et al., 2012). Tepper et al. showed that EPCs from patients with type 2 diabetes were characterized by incorrect proliferation and adhesion. Further studies confirmed the abnormal function and accelerated apoptosis of EPCs in patients with diabetes (Chen et al., 2009; Cheng, Dong, Wang, Kang, & Xu, 2009; Jung, Rafnsson, Shemyakin, Bohm, & Pernow, 2010; Tepper et al., 2002). Studies conducted by Li et al. indicate that advanced glycation end products (AGEs) impair the functioning of late EPCs. The researchers suggest that this may lead to the development of vascular complications in diabetes (Li et al., 2012). In our study, patients with diabetes had higher VEGF-A concentration and lower sVEGF-R2 concentration compared to the healthy subjects. Both factors are involved in angiogenesis, with the difference that VEGF-A is a proangiogenic factor, and soluble receptor is considered as a natural VEGF-A inhibitor (Shibuya, 2006). Unfortunately, data in the available literature about systemic VEGF-A concentration in diabetic patients are not clear. Tecilazich et al. did not observe significant differences in VEGF-A levels in the healthy controls and in the three groups of patients with diabetes mellitus: DM-not at risk of DFU development, and DM-at risk of DFU and DM-DFU (Tecilazich et al., 2013). Different results were obtained by Doupis et al. (serum VEGF-A level) and Nandy et al. (plasma VEGF-A level). In both studies, higher level of VEGF-A in diabetic patients compared to controls (healthy subjects) was observed (Doupis et al., 2011; Nandy & Mukhopadhyay, 2010). In addition, Choksy et al. demonstrated an intense expression of VEGF in distal ischemic tissues (foot), especially in the necrotic area and ulcers (Choksy, Pockley, Wajeh, & Chan, 2004). We suggest that increased VEGF-A level in our patients may result from tissue hypoxia. Elevated level of VEGF-A in diabetic patients may also be explained by the effect of chronic inflammation that occurs in T2DM. IL-6 is a pro-inflammatory cytokine. Increased levels of this cytokine may lead to the VEGF-A secretion by platelets and leukocytes (van den Over, Raterman, Nurmohamed, & Simsek, 2010). The expression of VEGF is observed in megakaryocytes, which then is stored in platelet α granules (Kajdaniuk et al., 2011). Diabetes has been considered as a prothrombotic state. It is associated with the increased number of platelets and their hyperreactivity, which is characterized by excessive activation and aggregation (Ferreiro, Gomez-Hospital, & Angiolillo, 2010; Kim, Bae, & Kim, 2013). Platelet activation contributes to PDGF and VEGF secretion from their granules (Kodiatte et al., 2012). Therefore, it might be assumed that elevated number of activated platelets is an additional source of VEGF in diabetic patients. In patients with diabetes there are other factors responsible for increased VEGF-A level. It may result from active process of angiogenesis that occurs during wound healing. Francesco et al. demonstrated that VEGF concentration was higher in DM patients complicated with DF compared to the healthy subjects. Moreover, the concentration of VEGF underwent reduction during healing process in DM-DFU group (duration of treatment: 12 weeks) (Tecilazich et al., 2013). FGF-2, next to the VEGF, plays an essential role in angiogenesis. FGF-2 has both angiogenic properties (stimulates endothelial cells to VEGF synthesis) and proinflammatory properties (enhances IL-6
expression, which also increases the synthesis of VEGF) (Qazi et al., 2009). In our study, increased levels of FGF-2 was found in patients with diabetes (n = 45; DM compared to the controls) as well as in patients with DF (DM-DF compared to DM). Similar results were obtained by Gui et al. They observed increased level of FGF-2 in diabetic patients compared to the controls (Gui et al., 2013). The interaction between VEGF-A and FGF-2 in patients with and without DF is worth noting. Decreased concentration of VEGF-A is associated with elevated concentrations of FGF-2 in patients with diabetic foot. The opposite situation is observed in patients with uncomplicated diabetes. It may result from a specific release kinetics of these two factors during healing process. The physiological process of wound healing is characterized by specific temporal secretion of VEGF-A and FGF-2. High level of FGF-2 is observed immediately after injury and stabilizes to basal level within 3 days. The maximal expression of VEGF mRNA appears between 3 and 7 days. When the VEGF concentration decreases, FGF-2 levels rise again. VEGF expression decreases when hypoxia is reduced or other angiogenic factor (e.g., FGF-2) takes control of blood vessels formation (Bao, Kodra, Tomic-Canic, et al., 2009). This mechanism indicates that these two factors, at the appropriate concentration and time, are necessary to create vascularization. Our study is in agreement with this scenario: we noted positive correlation between VEGF-A and FGF-2 levels in the whole group of patients with type 2 diabetes. In the available literature, we found no data about sVEGF-R2 concentration in patients with DFS. Research conducted by Wierzbowska et al. shows that sVEGF-R2 inhibits angiogenesis dependent of VEGF (Wierzbowska, RObak, Wrzesień-Kuś, et al., 2003). The results presented in this study may suggest that VEGF gene activation occurred in diabetes in response to the tissue hypoxia. It might also suggest that the activation of pro-angiogenic factors is associated with the simultaneous activation of the inhibitory factor. VEGF released into the circulation forms complexes with a soluble receptor 2 (followed by lowering the concentration of sVEGF-R2). In this way, the development of blood vessels is controlled. With regard to the analyzing of selected clinical parameters and tested parameters, we observed increased FGF-2 level in patients without retinopathy compared to the patients with DF and accompanying retinopapthy. There were no differences in VEGF-A concentration and CEPCs number in patients with DF and accompanying retinopathy, nephropathy, and neuropathy. There is no similar study in this field. Baharivand et al. shows increased levels of VEGF in diabetic patients with proliferative retinopathy compared to patients without active retinopathy. This is in contrast with results reported by Hernandez et al. The authors observed no difference in VEGF level in serum (Baharivand et al., 2012; Hernandez, Lecube, Segura, Sararols, & Simo, 2002). Study conducted by Kim et al. showed no difference in the concentration of VEGF in plasma depending on the severity of nephropathy. In contrast, urine VEGF secretion increased with the stage of renal failure (Kim, Oh, Seo, et al., 2005).
5. Conclusions A study shows that the presence of DF has a significant effect on the systemic concentration of the factors involved in angiogenesis. These results may indicate an active process of blood vessel formation during healing in patients with DF, but a concomitant hyperglycemia significantly impairs this process. The role of EPCs in angiogenesis in patients with DF needs further investigation.
Acknowledgments This study was supported by grant from the National Centre of Science (Poland) based on the decision no. DEC-2011/01/N/NZ5/00293.
Please cite this article as: Kulwas, A., et al., Circulating endothelial progenitor cells and angiogenic factors in diabetes complicated diabetic foot and without foot complicatio..., Journal of Diabetes and Its Complications (2015), http://dx.doi.org/10.1016/j.jdiacomp.2015.03.013
A. Kulwas et al. / Journal of Diabetes and Its Complications xxx (2015) xxx–xxx
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