Premature development of microvascular and macrovascular disease is the most frequent complication of diabetes. It is responsible for diabetic retinopathy, nephropathy, and neuropathy (1). Moreover, diabetes leads to reduced collateralization in ischemic tissues, which causes impaired wound healing, exacerbation of peripheral limb ischemia, and a three- to fourfold increase in cardiac mortality in comparison with nondiabetic patients (2,3).
The pathophysiological mechanisms responsible for impaired angiogenic activity in diabetes remain unknown. Although angiogenesis impairment has been attributed to alterations in the gene regulatory network, which can be involved in the physiological revascularization process, the role of angiogenin in this process has not been clarified (2,4).
The aim of this study therefore was to compare angiogenin serum levels in healthy and type 2 diabetic age-matched individuals. The cutaneous angiogenesis in vivo (serum-induced angiogenesis [SIA]) test in mice was performed in parallel to determine the functional differences between the groups examined.
RESEARCH DESIGN AND METHODS
A total of 43 patients with type 2 diabetes and 43 age-matched healthy control subjects volunteered for the study. Patients suffering from acute and chronic infections and neoplastic diseases were excluded. Diabetes was defined according to American Diabetes Association criteria (5). All patients had blood glucose levels >7 mmol/l. Serum angiogenin levels were measured in duplicate with the ELISA Quantikine kit (R&D System, Minneapolis, MN). The SIA test was performed on Balb/c mice according to the method of Sidky and Auerbach (6) with own modifications (7–9).
The results were analyzed using Statistica software, Version 7 (StatSoft Polska). The level of significance was set at P < 0.05, and two-sided tests were performed as the standard.
RESULTS
The diabetic patients were characterized by significantly higher levels of triglycerides and serum creatinine and lower levels of LDL cholesterol, while BMI and total as well as HDL cholesterol concentrations were not significantly different from those of healthy individuals (Table 1).
Serum angiogenin levels were significantly lower in type 2 diabetic patients in relation to control subjects (P = 0.000002). At the same time, sera from diabetic patients induced significantly fewer newly formed blood vessels on the inner surface of skin from Balb/c mice (P = 0.03).
Serum angiogenin levels as well as SIA values were significantly lower in the patients with late complications (retinopathy and nephropathy) in relation to those without complicated disease (P = 0.04 for angiogenin and P = 0.02 for SIA).
There were no differences, however, in angiogenin and SIA values between the patients receiving statins (P = 0.93 for angiogenin and P = 0.74 for SIA) or ACE inhibitors (P = 0.70 for angiogenin and P = 0.51 for SIA) and those not treated with either. The multivariate linear stepwise regression analysis confirmed the previous results revealing that none of the parameters examined, including medicines, exerted an effect on angiogenin or SIA values (P = 0.70). The angiogenin and SIA values were not correlated with the A1C concentrations (P = 0.4).
CONCLUSIONS
Angiogenin, a 14-kDa protein, is implicated in immunological and inflammatory angiogenesis (10). The level of angiogenin in human plasma is strictly regulated (11); the protein is a normal constituent of blood, and its level usually remains unchanged. However, in some pathological conditions such as peripheral vascular disease, inflammatory bowel disease, rheumatoid arthritis, obesity, proliferative diabetic retinopathy, and proliferative vitreopathy, it can intensify the induction of new blood vessel formation (10).
We discovered that the serum angiogenin level is markedly decreased in type 2 diabetic patients in comparison with age-matched healthy subjects. The lower angiogenic potential of sera from type 2 diabetic patients was confirmed by the in vivo SIA test. The more severe disease, i.e., complicated with retinopathy or nephropathy, was associated with lower values of both angiogenin and SIA. On the other hand, the differences in values of both indicators of angiogenesis were not related, in our analysis, to any of the other clinical parameters examined in the diabetic patients. Since the number of diabetic patients was limited in our study and almost all patients had poorly controlled A1C levels, we can assume that they constituted a very homogenous group. The angiogenin and SIA values were in a rather narrow range. Therefore, they most likely reached a point when any treatment ceased to play a role. The most important finding was that neither statins nor angiotensin inhibitors influenced the values of both angiogenesis indicators.
Our data are compatible with the known phenomenon of limited collateral vessel development in coronary heart disease, which is associated with a pronounced myocardial ischemia in diabetic patients (2). In experimental in vitro and in vivo models of retinal angiogenesis assays, Stitt et al. (1) discovered that sera of type 2 diabetic patients have strong anti-angiogenic effects. The poorer the glycemic control of patients, the more evident the inhibition of angiogenesis. The authors documented that advanced glycation end products and their receptors may be mediators in retinal angiogenesis inhibition. Adding to that, Chou et al. (12) suggest a possible explanation for impaired collateral formation in cardiac tissue by reduction in vascular endothelial growth factor in ventricles from diabetic patients compared with those in control subjects. Similar results were obtained by Chung et al. (13). Their report indicates a 48% reduction of vascular endothelial growth factor in internal mammary artery of diabetic patients undergoing coronary artery bypass graft surgery. Our results imply, additionally, that angiogenesis inhibition may be realized through a reduction in angiogenin level. Whether such a relation really exists is a matter for further investigation.
Basic parameters in diabetic patients and healthy control subjects
| Parameter . | Type 2 diabetic patients . | Healthy control subjects . | P value . |
|---|---|---|---|
| n | 43 | 43 | |
| Age (years) | 64.73 ± 11.47 | 65.16 ± 11.91 | 0.78 |
| BMI (kg/m2) | 32.09 ± 5.91 | 30.11 ± 4.51 | 0.13 |
| Systolic blood pressure (mmHg) | 140.81 ± 15.80 | 137.83 ± 11.91 | 0.4 |
| Diastolic blood pressure (mmHg) | 78.10 ± 7.71 | 81.01 ± 4,80 | 0.1 |
| Diabetes duration (years) | 9.63 ± 5.60 | ||
| A1C | 8.23 ± 1.72 | ||
| Total cholesterol (mmol/l) | 224.36 ± 45.20 | 240.45 ± 39.79 | 0.1 |
| LDL cholesterol (mmol/l) | 135.59 ± 42.61 | 156.52 ± 32.96 | 0.02 |
| HDL cholesterol (mmol/l) | 51.42 ± 17.74 | 55.12 ± 12.10 | 0.3 |
| Triglycerides (mmol/l) | 187.25 ± 89.69 | 135.6 ± 54.0 | 0.004 |
| Serum creatinine (mg/100 ml) | 1.01 ± 0.185 | 0.86 ± 0.23 | 0.04 |
| Retinopathy (%) | 71.87 | ||
| Nephropathy (%) | 35.71 | ||
| ACE inhibitor treatment (%) | 86.49 | ||
| Statin treatment (%) | 48.65 | ||
| Angiogenin serum level (pg/ml) | 319.72 ± 107.04 | 550.54 ± 187.99 | 0.0000015 |
| Cutaneous angiogenesis in vivo in serum-induced angiogenesis test* | 38.17 ± 11.80 | 52.51 ± 20.22 | 0.03 |
| Parameter . | Type 2 diabetic patients . | Healthy control subjects . | P value . |
|---|---|---|---|
| n | 43 | 43 | |
| Age (years) | 64.73 ± 11.47 | 65.16 ± 11.91 | 0.78 |
| BMI (kg/m2) | 32.09 ± 5.91 | 30.11 ± 4.51 | 0.13 |
| Systolic blood pressure (mmHg) | 140.81 ± 15.80 | 137.83 ± 11.91 | 0.4 |
| Diastolic blood pressure (mmHg) | 78.10 ± 7.71 | 81.01 ± 4,80 | 0.1 |
| Diabetes duration (years) | 9.63 ± 5.60 | ||
| A1C | 8.23 ± 1.72 | ||
| Total cholesterol (mmol/l) | 224.36 ± 45.20 | 240.45 ± 39.79 | 0.1 |
| LDL cholesterol (mmol/l) | 135.59 ± 42.61 | 156.52 ± 32.96 | 0.02 |
| HDL cholesterol (mmol/l) | 51.42 ± 17.74 | 55.12 ± 12.10 | 0.3 |
| Triglycerides (mmol/l) | 187.25 ± 89.69 | 135.6 ± 54.0 | 0.004 |
| Serum creatinine (mg/100 ml) | 1.01 ± 0.185 | 0.86 ± 0.23 | 0.04 |
| Retinopathy (%) | 71.87 | ||
| Nephropathy (%) | 35.71 | ||
| ACE inhibitor treatment (%) | 86.49 | ||
| Statin treatment (%) | 48.65 | ||
| Angiogenin serum level (pg/ml) | 319.72 ± 107.04 | 550.54 ± 187.99 | 0.0000015 |
| Cutaneous angiogenesis in vivo in serum-induced angiogenesis test* | 38.17 ± 11.80 | 52.51 ± 20.22 | 0.03 |
Data are arithmetic means ± SD or percentage of patients, unless otherwise indicated.
Mean±SD newly formed blood vessels on inner skin surface of Balb/c mice.
References
Published ahead of print at http://care.diabetesjournals.org on 18 September 2007. DOI: 10.2337/dc07-0629.
Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/dc07-0629.
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C Section 1734 solely to indicate this fact.
