Journal of Diabetes and Its Complications 29 (2015) 568–571
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Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Serum angiopoietin-2 is associated with angiopathy in type 2 diabetes mellitus Li Li, Lei Qian ⁎, Zheng-Qing Yu Department of Clinical Laboratory, Binhai Hospital, Jiangsu Province, P.R. China
a r t i c l e
i n f o
Article history: Received 6 November 2014 Received in revised form 9 February 2015 Accepted 13 February 2015 Available online 20 February 2015 Keywords: Angiopoietin-1 Angiopoietin-2 Glycosylated hemoglobin Diabetes mellitus Vascular complications
a b s t r a c t Purpose: The aim of the present study was to investigate the association of serum levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) with angiopathy in type 2 diabetes mellitus (T2DM). Methods: The group studied comprised of 120 patients with T2DM (68 males and 52 females), included macroangiopathy complications, microvascular disease and diabetic without vascular disease. The control group consisted of 50 healthy blood donors. Ang-1, Ang-2, fasting plasma glucose (FBG), fasting insulin (FINS) and HbA1c were assessed. Results: The serum Ang-2 levels of T2DM patients with angiopathy were found to be significantly higher compared to patients without angiopathy. Ang-2 levels were significantly positively correlated with homeostasis model assessment for insulin resistance (HOMA-IR) and glycosylated hemoglobin A1c (HbA1c) (r = 0.577 and 0.504, respectively, P b 0.01). In contrast, there was no significant correlation between Ang-1 levels and HOMA-IR (P N 0.05). In multivariable logistic regression analysis, Ang-2 levels (P = 0.02) were found to be independently associated with patients with T2DM angiopathy. Conclusions: An association between the Ang-2 and T2DM with vascular complications was found. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by chronic hyperglycemia, which mainly results from a deficiency in peripheral insulin effects (insulin resistance). However, morbidity and mortality from diabetes are mainly attributed to the development of both macrovascular and microvascular complications that rapidly leads to premature death (Jaumdally, Goon, Varma, et al., 2010; Rasul, Reiter, Ilhan, et al., 2011). Insulin-producing beta-cells and endothelial cells in the pancreatic islets of Langerhans exchange bidirectional signals necessary for development, differentiation, and the proper function of both endocrine and vascular compartments (Rasul et al., 2011). Beta-cells secreted angiogenic factors, like vascular endothelial growth factor, as well as anti-angiogenic factors, like angiopoietin (Calderari et al., 2012). The angiopoietins are a family of seven secreted glycoprotein ligands, angiopoietin-1 to -7, originally identified as important in blood vessel formation. The best characterizations of these ligands are angiopoietin-1 (Ang-1) and angiopoitein-2 (Ang-2) (Davis, Aldrich, Jones, et al., 1996; Valenzuela, Griffiths, Rojas, et al., 1999). Ang-1, a secreted 70 kDa glycoprotein constitutively expressed by pericytes
Conflict of interest: None. ⁎ Corresponding author. Department of Clinical Laboratory, Binhai Hospital, 248 Fudong Road, Jiangsu Province 224500, P.R. China. E-mail address: [email protected] (L. Qian). http://dx.doi.org/10.1016/j.jdiacomp.2015.02.006 1056-8727/© 2015 Elsevier Inc. All rights reserved.
and vascular smooth muscle cells, is a major agonist for the tyrosine kinase receptor Tie-2. Binding of Ang-1 to Tie-2 promotes vessel integrity, inhibits vascular leakage, and suppresses inflammatory gene expression. On the other hand, Ang-2 is also expressed by endothelial cells and acts as an antagonist for Tie-2. Ang-2 has been reported to completely disrupt protective Tie-2 signaling in numerous studies (Chen, Guo, & Chen, 2013; Thomas & Augustin, 2009). Recent results revealed that Ang-2 is closely related to abnormal vascular endothelial inflammation, insulin resistance and vascular injury. However, whether Ang-1 and Ang-2 levels are relevant to T2DM patients with vascular complications remains unknown. Therefore, the aim of this study was to investigate the association of serum Ang-1 and Ang-2 with T2DM patients with vascular lesions and insulin resistance. 2. Material and methods 2.1. Patient selection A total of 120 patients with T2DM (68 males and 52 females) treated at BinHai County People's hospital were recruited in this study. The average patient age was 62 ± 11 years old. The patients were diagnosed according to WHO criteria (Geneva, 1999). Primary diagnoses (by clinical, ECG, and imaging diagnosis) included macroangiopathy complications (n = 32, including heart disease, cerebrovascular disease, and peripheral arterial disease), microvascular disease (n = 52, including diabetic nephropathy (urinary albumin N 30 mg/24 h)), as
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Table 1 Clinical characteristics and research indexes in the study groups.
n Age (years) Sex (males/females) BMI (kg/m2) SBP (mmHg) DBP (mmHg) TC (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Ang-1 (ng/ml) Ang-2 (ng/ml) FBG (mmol/L) FINS (mU/L) HOMA-IR HbA1c (%)
Controls
T2DM
T2DM With macroangiopathy complications
T2DM with microangiopathy complications
T2DM without angiopathy complications
P-value
50 60 ± 6 30/20 25 ± 5 128 ± 16 77 ± 7 4.8 ± 1.0 1.5 ± 0.5 1.6 ± 0.5 3.2 ± 0.8 19.5 ± 1.7 0.8 ± 0.2 5.1 ± 0.5 8.0 ± 1.3 1.8 ± 0.1 4.8 ± 0.9
120 62 ± 11 68/52 28 ± 6 139 ± 15 78 ± 11 5.7 ± 1.6 2.8 ± 1.6 1.2 ± 0.4 2.9 ± 0.8 19.6 ± 1.8 2.2 ± 0.7 9.4 ± 2.3 18.1 ± 2.7 7.6 ± 0.2 9.5 ± 2.1
32 64 ± 5 17/15 29 ± 5 137 ± 16 75 ± 9 5.5 ± 1.5 2.0 ± 1.2 1.3 ± 0.2 2.5 ± 0.9 19.1 ± 1.7 2.9 ± 0.7 10.5 ± 2.8 18.7 ± 2.4 8.7 ± 0.3 11.3 ± 3.5
52 69 ± 4 28/24 26 ± 4 138 ± 15 76 ± 8 5.9 ± 1.1 2.5 ± 1.6 1.1 ± 0.5 2.3 ± 0.7 20.2 ± 2.0 2.2 ± 0.6 9.2 ± 2.3 15.5 ± 2.1 6.3 ± 0.2 9.7 ± 2.7
36 66 ± 5 23/13 25 ± 6 136 ± 15 75 ± 7 5.6 ± 1.2 2.4 ± 1.5 1.3 ± 0.3 2.6 ± 1.0 19.8 ± 2.1 1.6 ± 0.4 8.5 ± 1.7 20.1 ± 3.8 7.6 ± 0.3 8.9 ± 2.1
— 0.125 0.314 0.001⁎ 0.060 0.213 0.911 0.895 b0.001 0.058 0.420 0.001 b0.001 b0.001 b0.001 b0.001
Data are presented as mean ± SD, BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Ang, angiopoietin; HOMA-IR, homeostasis model assessment for insulin resistance. ⁎ Significant difference between patients with T2DM and controls but not between patient groups.
well as diabetic retinopathy and diabetic peripheral neuropathy) and diabetic without vascular disease (n = 36). According to the number of macrovascular and microvascular injuries respectively, patients with macrovascular or microvascular disease were divided into the following groups respectively: one kind of macrovascular disease group (n = 10) or microvascular disease group (n = 22), two kinds of macrovascular disease group (n = 15) or microvascular disease group (n = 18), and three kinds of macrovascular disease group (n = 7) or microangiopathy group (n = 12). The control group consisted of 30 males and 20 females, and the average age was 60 ± 15 years old. The exclusion criteria included patients with evidence of neoplastic disease, chronic inflammation (except vascular injuries), significant hepatic and renal disease. The study was approved through the local research ethics committee, and informed consent for all subjects was obtained. 2.2. Laboratory analysis
Ang-1 and Ang-2 levels were independently associated with T2DM with vascular complications during hospitalization. P b 0.05 was considered a statistical significance. All analyses were performed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Clinical and laboratory measures A total of 120 patients with type-2 diabetes and 50 normal healthy controls were recruited. These subjects were age and sex ratio comparable. As expected, HbA1c, FINS, HOMA-IR, TC, and triglyceride levels were higher, and HDL cholesterol was lower in the diabetic patients. Serum Ang-2 levels, but not Ang-1 levels, were significantly higher in patients with T2DM than in healthy controls, and there were differences between the diabetic patient subgroups (Table 1). There were no significant relationships between Ang-1 and Ang-2 levels with age, sex, and body mass index.
Venous blood samples were obtained from all subjects upon hospital admission. All samples were collected in vacuum blood collection tubes with a clot activator and immediately centrifuged at 1000 × g and 4 °C for 20 min. Plasma was aliquoted and stored at −70 °C until analysis. Serum Ang-1 and Ang-2 levels were measured by the enzyme-linked immunosorbent assay (ELISA) using commercial kits and reagents (R&D Systems, Minneapolis, MN, USA). HbA1c was measured by liquid chromatography (G8-90SL, Tosoh, Japan). Fasting plasma glucose (FBG) and fasting insulin (FINS) were measured by routine techniques. The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated by the following formula: HOMA-IR = (FBG × FINS)/22.5.
In our T2DM cohort, 84 patients had suffered vascular complications. In T2DM patients with increased microvascular or macrovascular disease, Ang-2 concentration also increased. Ang-2 levels were also significantly different (P b 0.05). The concentration of concurrent Ang-1 levels was not significantly different related to different microangiopathy or macroangiopathy numbers (P N 0.05) (Tables 2, 3).
2.3. Statistical analysis
3.3. Correlations
The values are expressed as mean ± S.D. Comparisons of means between two groups were performed using Student's t-test upon test of normality and equality of variances. Correlations within each group were sought using Spearman's or Pearson's method. Multivariable logistic regression analyses were performed to determine whether
Serum Ang-2 levels positively correlated with HbA1c and HOMA-IR (r = 0.504 and 0.577 respectively, P b 0.01; Fig. 1). However, serum Ang-1 levels did not significantly correlate with HbA1c and HOMA-IR (P N 0.05). BMI, as well as systolic and diastolic blood pressure did not significantly correlate with Ang-2 levels.
Table 2 Results of the different numbers of microvascular injuries in T2DM.
Table 3 Results of the different numbers of macrovascular injuries in T2DM.
3.2. Relationship of Ang-2 and the number of microvascular or macrovascular injuries in T2DM
Numbers of microvascular
Ang-1 (μg/L)
Ang-2 (μg/L)
Numbers of macrovascular
Ang-1 (μg/L)
Ang-2 (μg/L)
1 2 3
20.2 ± 1.9 20.3 ± 2.0 20.1 ± 2.1
2.1 ± 0.3 2.5 ± 0.7 2.9 ± 0.9
1 2 3
19.0 ± 1.4 19.2. ± 1.8 19.1 ± 1.9
2.5 ± 0.5 2.9 ± 0.8 3.4 ± 0.7
570
L. Li et al. / Journal of Diabetes and Its Complications 29 (2015) 568–571
Fig. 1. Serum Ang-2 levels (A) and (B) Ang-2 were positively correlated with HbA1c and HOMA-IR respectively.
3.4. Association between serum Ang-2 and vascular complications In multivariable logistic regression analysis, Ang-2 levels (95% CI, 1.002 to 1.020, P = 0.02) were independently associated with T2DM patients with vascular complications, The Ang-2 was positively correlated with microangiopathy and macroangiopathy numbers respectively (r = 0.485,0.467 respectively, P b 0.05) following adjustment for age, gender, BMI, hypertension, diabetes mellitus, and blood fat. 4. Discussion Diabetes is associated with a range of vascular diseases, including atherosclerosis, myocardial infarction and heart failure resulting from endothelial dysfunction (Szarvas, Jäger, Tötsch, et al., 2008). Angiopoietins can promote angiogenesis, remodeling, maturation and maintenance through using a coiled-coil domain in the N-terminus and a fibrinogen-like domain in the C-terminus. In addition, some angiopoietins are potent regulators of lipid, glucose, and energy metabolism (Kitazawa, Nagano, Masumoto, et al., 2011). Ang-1 and Ang-2, two of the most widely studied biomarkers of endothelial activation/dysfunction in infectious diseases, have a high degree of homology. Studies have shown that Ang-1 stabilizes new vessels, leading to vascular maturation by promoting interactions between endothelial cells and covering mural cells (Singh, Nicholas, Brindle, Victor, et al., 2010). Ang-2 is a glycoprotein exclusively expressed by endothelial cells that acts as an antagonist for Tie-2. Ang-2 has been reported to completely disrupt protective Tie-2 signaling in numerous studies (Maisonpierre, Suri, Jones, et al., 1997; Thomas & Augustin, 2009). Ang-2 can inhibit Tie2 phosphorylation, damage blood vessels, promote
blood vessel structure relaxation, relieve inhibition effects on perivascular cells, and extracellular matrix to the endothelium, causing vascular endothelial injury and pathological angiogenesis. Patients with diabetes are at risk for microvascular and macrovascular atherosclerotic disease (Felmeden, Spencer, Belgore, et al., 2003). Ang-1 and Ang-2 are well-known to be associated with several forms of cardiovascular (Chen, Guo, Cui, et al., 2012; Chen, Yu, Sun, et al., 2010; Iribarren, Phelps, Darbinian, et al., 2011; Patel, Lim, Varughese, et al., 2008) and inflammatory diseases (Ong, McClintock, Kallet, et al., 2010; Ricciuto, Santos, Hawkes, et al., 2011). Previous studies revealed that serum Ang-2 levels, but not serum Ang-1 levels, are elevated in patients with diabetes. In agreement with the previous reports, our data complement and extend this work. This study found that serum Ang-2 levels were positively associated with the number of concurrent microvascular and macrovascular lesions in patients with T2DM. Tabata et al. reported that Ang-2 mRNA is mainly expressed in visceral adipose tissue (Tabata, Kadomatsu, Fukuhara, et al., 2009), linking obesity and adipose tissue inflammation with insulin resistance. This study found that serum Ang-2 levels were positively correlated with HbA1c and HOMA-IR, indicating that Ang-2 is closely associated with glucose metabolism disorders and vascular lesions in patients with T2DM. Our results suggest that long-term hyperglycemia increases Ang-2 expression via stimulating endothelial cells. In addition, insulin resistance and insufficient insulin signaling in patients with T2DM may also lead to the increased expression of Ang-2. The high concentrations of glycation end-products in diabetic patients can up-regulate Ang-2 mRNA expression by stimulating endothelial cell (Okamoto, Yamagishi, Inagaki, et al., 2002). Conversely, Ang-2 can stimulate endothelial cell proliferation and induce vascular inflammation, accelerating the generation and development of vascular disease in diabetes (Maisonpierre et al., 1997; Thomas & Augustin, 2009). In our study, we found that Ang-2 levels gradually increased in the following order: T2DM patients without vascular disease group, microangiopathy group, and macroangiopathy group. Importantly, Ang-2 is significantly higher among diabetic subjects with macrovascular disease when compared to those without macrovascular complications. In the multivariable logistic regression analysis, Ang-2 levels were independently associated with T2DM patients with vascular complications. This indicated that the extent of vascular damage may be closely linked to circulating Ang-2 levels. Several hypotheses can be made on the role of Ang-2 levels in diabetic vasculopathy. First, Ang-2 alone may exert pro-angiogenic activity. Second, high Ang-2 levels may result in local inflammation, as evidenced by leukocyte attachment to the wall of post-capillary venules, and increased blood vessel permeability (Oike & Tabata, 2009). In contrast, inflammation stimulates Weibel-Palade body exocytosis and Ang-2 release, allowing Ang-2 to preferentially bind the Tie-2 receptor and promote proinflammatory and pro-thrombotic pathways, as well as microvascular leak (Kadomatsu, Tabata, & Oike, 2011; Page & Liles, 2013).
5. Conclusion The results of this study show that Ang-2 levels, but not Ang-1 levels, are significantly elevated in patients with type 2 diabetes. Plasma Ang-2 levels were associated with glucose metabolism disorders, insulin resistance, and vascular lesions, and were especially closely associated with the degree and extent of vascular lesions in diabetic patients. These observations provide further insights into the pathophysiology of vascular disease in diabetes mellitus.
Acknowledgments We thank all our colleagues for excellent technical assistance.
L. Li et al. / Journal of Diabetes and Its Complications 29 (2015) 568–571
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Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Serum angiopoietin-2 is associated with angiopathy in type 2 diabetes mellitus Li Li, Lei Qian ⁎, Zheng-Qing Yu Department of Clinical Laboratory, Binhai Hospital, Jiangsu Province, P.R. China
a r t i c l e
i n f o
Article history: Received 6 November 2014 Received in revised form 9 February 2015 Accepted 13 February 2015 Available online 20 February 2015 Keywords: Angiopoietin-1 Angiopoietin-2 Glycosylated hemoglobin Diabetes mellitus Vascular complications
a b s t r a c t Purpose: The aim of the present study was to investigate the association of serum levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) with angiopathy in type 2 diabetes mellitus (T2DM). Methods: The group studied comprised of 120 patients with T2DM (68 males and 52 females), included macroangiopathy complications, microvascular disease and diabetic without vascular disease. The control group consisted of 50 healthy blood donors. Ang-1, Ang-2, fasting plasma glucose (FBG), fasting insulin (FINS) and HbA1c were assessed. Results: The serum Ang-2 levels of T2DM patients with angiopathy were found to be significantly higher compared to patients without angiopathy. Ang-2 levels were significantly positively correlated with homeostasis model assessment for insulin resistance (HOMA-IR) and glycosylated hemoglobin A1c (HbA1c) (r = 0.577 and 0.504, respectively, P b 0.01). In contrast, there was no significant correlation between Ang-1 levels and HOMA-IR (P N 0.05). In multivariable logistic regression analysis, Ang-2 levels (P = 0.02) were found to be independently associated with patients with T2DM angiopathy. Conclusions: An association between the Ang-2 and T2DM with vascular complications was found. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by chronic hyperglycemia, which mainly results from a deficiency in peripheral insulin effects (insulin resistance). However, morbidity and mortality from diabetes are mainly attributed to the development of both macrovascular and microvascular complications that rapidly leads to premature death (Jaumdally, Goon, Varma, et al., 2010; Rasul, Reiter, Ilhan, et al., 2011). Insulin-producing beta-cells and endothelial cells in the pancreatic islets of Langerhans exchange bidirectional signals necessary for development, differentiation, and the proper function of both endocrine and vascular compartments (Rasul et al., 2011). Beta-cells secreted angiogenic factors, like vascular endothelial growth factor, as well as anti-angiogenic factors, like angiopoietin (Calderari et al., 2012). The angiopoietins are a family of seven secreted glycoprotein ligands, angiopoietin-1 to -7, originally identified as important in blood vessel formation. The best characterizations of these ligands are angiopoietin-1 (Ang-1) and angiopoitein-2 (Ang-2) (Davis, Aldrich, Jones, et al., 1996; Valenzuela, Griffiths, Rojas, et al., 1999). Ang-1, a secreted 70 kDa glycoprotein constitutively expressed by pericytes
Conflict of interest: None. ⁎ Corresponding author. Department of Clinical Laboratory, Binhai Hospital, 248 Fudong Road, Jiangsu Province 224500, P.R. China. E-mail address: [email protected] (L. Qian). http://dx.doi.org/10.1016/j.jdiacomp.2015.02.006 1056-8727/© 2015 Elsevier Inc. All rights reserved.
and vascular smooth muscle cells, is a major agonist for the tyrosine kinase receptor Tie-2. Binding of Ang-1 to Tie-2 promotes vessel integrity, inhibits vascular leakage, and suppresses inflammatory gene expression. On the other hand, Ang-2 is also expressed by endothelial cells and acts as an antagonist for Tie-2. Ang-2 has been reported to completely disrupt protective Tie-2 signaling in numerous studies (Chen, Guo, & Chen, 2013; Thomas & Augustin, 2009). Recent results revealed that Ang-2 is closely related to abnormal vascular endothelial inflammation, insulin resistance and vascular injury. However, whether Ang-1 and Ang-2 levels are relevant to T2DM patients with vascular complications remains unknown. Therefore, the aim of this study was to investigate the association of serum Ang-1 and Ang-2 with T2DM patients with vascular lesions and insulin resistance. 2. Material and methods 2.1. Patient selection A total of 120 patients with T2DM (68 males and 52 females) treated at BinHai County People's hospital were recruited in this study. The average patient age was 62 ± 11 years old. The patients were diagnosed according to WHO criteria (Geneva, 1999). Primary diagnoses (by clinical, ECG, and imaging diagnosis) included macroangiopathy complications (n = 32, including heart disease, cerebrovascular disease, and peripheral arterial disease), microvascular disease (n = 52, including diabetic nephropathy (urinary albumin N 30 mg/24 h)), as
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Table 1 Clinical characteristics and research indexes in the study groups.
n Age (years) Sex (males/females) BMI (kg/m2) SBP (mmHg) DBP (mmHg) TC (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Ang-1 (ng/ml) Ang-2 (ng/ml) FBG (mmol/L) FINS (mU/L) HOMA-IR HbA1c (%)
Controls
T2DM
T2DM With macroangiopathy complications
T2DM with microangiopathy complications
T2DM without angiopathy complications
P-value
50 60 ± 6 30/20 25 ± 5 128 ± 16 77 ± 7 4.8 ± 1.0 1.5 ± 0.5 1.6 ± 0.5 3.2 ± 0.8 19.5 ± 1.7 0.8 ± 0.2 5.1 ± 0.5 8.0 ± 1.3 1.8 ± 0.1 4.8 ± 0.9
120 62 ± 11 68/52 28 ± 6 139 ± 15 78 ± 11 5.7 ± 1.6 2.8 ± 1.6 1.2 ± 0.4 2.9 ± 0.8 19.6 ± 1.8 2.2 ± 0.7 9.4 ± 2.3 18.1 ± 2.7 7.6 ± 0.2 9.5 ± 2.1
32 64 ± 5 17/15 29 ± 5 137 ± 16 75 ± 9 5.5 ± 1.5 2.0 ± 1.2 1.3 ± 0.2 2.5 ± 0.9 19.1 ± 1.7 2.9 ± 0.7 10.5 ± 2.8 18.7 ± 2.4 8.7 ± 0.3 11.3 ± 3.5
52 69 ± 4 28/24 26 ± 4 138 ± 15 76 ± 8 5.9 ± 1.1 2.5 ± 1.6 1.1 ± 0.5 2.3 ± 0.7 20.2 ± 2.0 2.2 ± 0.6 9.2 ± 2.3 15.5 ± 2.1 6.3 ± 0.2 9.7 ± 2.7
36 66 ± 5 23/13 25 ± 6 136 ± 15 75 ± 7 5.6 ± 1.2 2.4 ± 1.5 1.3 ± 0.3 2.6 ± 1.0 19.8 ± 2.1 1.6 ± 0.4 8.5 ± 1.7 20.1 ± 3.8 7.6 ± 0.3 8.9 ± 2.1
— 0.125 0.314 0.001⁎ 0.060 0.213 0.911 0.895 b0.001 0.058 0.420 0.001 b0.001 b0.001 b0.001 b0.001
Data are presented as mean ± SD, BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Ang, angiopoietin; HOMA-IR, homeostasis model assessment for insulin resistance. ⁎ Significant difference between patients with T2DM and controls but not between patient groups.
well as diabetic retinopathy and diabetic peripheral neuropathy) and diabetic without vascular disease (n = 36). According to the number of macrovascular and microvascular injuries respectively, patients with macrovascular or microvascular disease were divided into the following groups respectively: one kind of macrovascular disease group (n = 10) or microvascular disease group (n = 22), two kinds of macrovascular disease group (n = 15) or microvascular disease group (n = 18), and three kinds of macrovascular disease group (n = 7) or microangiopathy group (n = 12). The control group consisted of 30 males and 20 females, and the average age was 60 ± 15 years old. The exclusion criteria included patients with evidence of neoplastic disease, chronic inflammation (except vascular injuries), significant hepatic and renal disease. The study was approved through the local research ethics committee, and informed consent for all subjects was obtained. 2.2. Laboratory analysis
Ang-1 and Ang-2 levels were independently associated with T2DM with vascular complications during hospitalization. P b 0.05 was considered a statistical significance. All analyses were performed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Clinical and laboratory measures A total of 120 patients with type-2 diabetes and 50 normal healthy controls were recruited. These subjects were age and sex ratio comparable. As expected, HbA1c, FINS, HOMA-IR, TC, and triglyceride levels were higher, and HDL cholesterol was lower in the diabetic patients. Serum Ang-2 levels, but not Ang-1 levels, were significantly higher in patients with T2DM than in healthy controls, and there were differences between the diabetic patient subgroups (Table 1). There were no significant relationships between Ang-1 and Ang-2 levels with age, sex, and body mass index.
Venous blood samples were obtained from all subjects upon hospital admission. All samples were collected in vacuum blood collection tubes with a clot activator and immediately centrifuged at 1000 × g and 4 °C for 20 min. Plasma was aliquoted and stored at −70 °C until analysis. Serum Ang-1 and Ang-2 levels were measured by the enzyme-linked immunosorbent assay (ELISA) using commercial kits and reagents (R&D Systems, Minneapolis, MN, USA). HbA1c was measured by liquid chromatography (G8-90SL, Tosoh, Japan). Fasting plasma glucose (FBG) and fasting insulin (FINS) were measured by routine techniques. The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated by the following formula: HOMA-IR = (FBG × FINS)/22.5.
In our T2DM cohort, 84 patients had suffered vascular complications. In T2DM patients with increased microvascular or macrovascular disease, Ang-2 concentration also increased. Ang-2 levels were also significantly different (P b 0.05). The concentration of concurrent Ang-1 levels was not significantly different related to different microangiopathy or macroangiopathy numbers (P N 0.05) (Tables 2, 3).
2.3. Statistical analysis
3.3. Correlations
The values are expressed as mean ± S.D. Comparisons of means between two groups were performed using Student's t-test upon test of normality and equality of variances. Correlations within each group were sought using Spearman's or Pearson's method. Multivariable logistic regression analyses were performed to determine whether
Serum Ang-2 levels positively correlated with HbA1c and HOMA-IR (r = 0.504 and 0.577 respectively, P b 0.01; Fig. 1). However, serum Ang-1 levels did not significantly correlate with HbA1c and HOMA-IR (P N 0.05). BMI, as well as systolic and diastolic blood pressure did not significantly correlate with Ang-2 levels.
Table 2 Results of the different numbers of microvascular injuries in T2DM.
Table 3 Results of the different numbers of macrovascular injuries in T2DM.
3.2. Relationship of Ang-2 and the number of microvascular or macrovascular injuries in T2DM
Numbers of microvascular
Ang-1 (μg/L)
Ang-2 (μg/L)
Numbers of macrovascular
Ang-1 (μg/L)
Ang-2 (μg/L)
1 2 3
20.2 ± 1.9 20.3 ± 2.0 20.1 ± 2.1
2.1 ± 0.3 2.5 ± 0.7 2.9 ± 0.9
1 2 3
19.0 ± 1.4 19.2. ± 1.8 19.1 ± 1.9
2.5 ± 0.5 2.9 ± 0.8 3.4 ± 0.7
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Fig. 1. Serum Ang-2 levels (A) and (B) Ang-2 were positively correlated with HbA1c and HOMA-IR respectively.
3.4. Association between serum Ang-2 and vascular complications In multivariable logistic regression analysis, Ang-2 levels (95% CI, 1.002 to 1.020, P = 0.02) were independently associated with T2DM patients with vascular complications, The Ang-2 was positively correlated with microangiopathy and macroangiopathy numbers respectively (r = 0.485,0.467 respectively, P b 0.05) following adjustment for age, gender, BMI, hypertension, diabetes mellitus, and blood fat. 4. Discussion Diabetes is associated with a range of vascular diseases, including atherosclerosis, myocardial infarction and heart failure resulting from endothelial dysfunction (Szarvas, Jäger, Tötsch, et al., 2008). Angiopoietins can promote angiogenesis, remodeling, maturation and maintenance through using a coiled-coil domain in the N-terminus and a fibrinogen-like domain in the C-terminus. In addition, some angiopoietins are potent regulators of lipid, glucose, and energy metabolism (Kitazawa, Nagano, Masumoto, et al., 2011). Ang-1 and Ang-2, two of the most widely studied biomarkers of endothelial activation/dysfunction in infectious diseases, have a high degree of homology. Studies have shown that Ang-1 stabilizes new vessels, leading to vascular maturation by promoting interactions between endothelial cells and covering mural cells (Singh, Nicholas, Brindle, Victor, et al., 2010). Ang-2 is a glycoprotein exclusively expressed by endothelial cells that acts as an antagonist for Tie-2. Ang-2 has been reported to completely disrupt protective Tie-2 signaling in numerous studies (Maisonpierre, Suri, Jones, et al., 1997; Thomas & Augustin, 2009). Ang-2 can inhibit Tie2 phosphorylation, damage blood vessels, promote
blood vessel structure relaxation, relieve inhibition effects on perivascular cells, and extracellular matrix to the endothelium, causing vascular endothelial injury and pathological angiogenesis. Patients with diabetes are at risk for microvascular and macrovascular atherosclerotic disease (Felmeden, Spencer, Belgore, et al., 2003). Ang-1 and Ang-2 are well-known to be associated with several forms of cardiovascular (Chen, Guo, Cui, et al., 2012; Chen, Yu, Sun, et al., 2010; Iribarren, Phelps, Darbinian, et al., 2011; Patel, Lim, Varughese, et al., 2008) and inflammatory diseases (Ong, McClintock, Kallet, et al., 2010; Ricciuto, Santos, Hawkes, et al., 2011). Previous studies revealed that serum Ang-2 levels, but not serum Ang-1 levels, are elevated in patients with diabetes. In agreement with the previous reports, our data complement and extend this work. This study found that serum Ang-2 levels were positively associated with the number of concurrent microvascular and macrovascular lesions in patients with T2DM. Tabata et al. reported that Ang-2 mRNA is mainly expressed in visceral adipose tissue (Tabata, Kadomatsu, Fukuhara, et al., 2009), linking obesity and adipose tissue inflammation with insulin resistance. This study found that serum Ang-2 levels were positively correlated with HbA1c and HOMA-IR, indicating that Ang-2 is closely associated with glucose metabolism disorders and vascular lesions in patients with T2DM. Our results suggest that long-term hyperglycemia increases Ang-2 expression via stimulating endothelial cells. In addition, insulin resistance and insufficient insulin signaling in patients with T2DM may also lead to the increased expression of Ang-2. The high concentrations of glycation end-products in diabetic patients can up-regulate Ang-2 mRNA expression by stimulating endothelial cell (Okamoto, Yamagishi, Inagaki, et al., 2002). Conversely, Ang-2 can stimulate endothelial cell proliferation and induce vascular inflammation, accelerating the generation and development of vascular disease in diabetes (Maisonpierre et al., 1997; Thomas & Augustin, 2009). In our study, we found that Ang-2 levels gradually increased in the following order: T2DM patients without vascular disease group, microangiopathy group, and macroangiopathy group. Importantly, Ang-2 is significantly higher among diabetic subjects with macrovascular disease when compared to those without macrovascular complications. In the multivariable logistic regression analysis, Ang-2 levels were independently associated with T2DM patients with vascular complications. This indicated that the extent of vascular damage may be closely linked to circulating Ang-2 levels. Several hypotheses can be made on the role of Ang-2 levels in diabetic vasculopathy. First, Ang-2 alone may exert pro-angiogenic activity. Second, high Ang-2 levels may result in local inflammation, as evidenced by leukocyte attachment to the wall of post-capillary venules, and increased blood vessel permeability (Oike & Tabata, 2009). In contrast, inflammation stimulates Weibel-Palade body exocytosis and Ang-2 release, allowing Ang-2 to preferentially bind the Tie-2 receptor and promote proinflammatory and pro-thrombotic pathways, as well as microvascular leak (Kadomatsu, Tabata, & Oike, 2011; Page & Liles, 2013).
5. Conclusion The results of this study show that Ang-2 levels, but not Ang-1 levels, are significantly elevated in patients with type 2 diabetes. Plasma Ang-2 levels were associated with glucose metabolism disorders, insulin resistance, and vascular lesions, and were especially closely associated with the degree and extent of vascular lesions in diabetic patients. These observations provide further insights into the pathophysiology of vascular disease in diabetes mellitus.
Acknowledgments We thank all our colleagues for excellent technical assistance.
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