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Microangiopathy is a main complication of diabetes mellitus (1). Folkman and Klagsbrun (2) recognized that the dominant pathology in many diseases, including diabetic complications, is persistent angiogenesis. Various factors are implicated in the phenomenon of angiogenesis, e.g. heparin-binding endothelial cell growth factors and angiogenin (2). In a previous study, it was demonstrated that angiogenin was significantly elevated in the serum of children and adolescents with type 1 diabetes mellitus, presumably reflecting the angiogenic reaction, consequent to not yet clinically evident microangiopathy (3). In addition to angiogenin, bFGF and VEGF are also important angiogenic factors involved in vascular endothelial cell growth (4,5). bGFG, an 18-kD heparin-binding peptide, was one of the first angiogenic factors to be characterized and isolated. It stimulates the proliferation in vitro of a wide range of mesoderm-derived cells and in particular is a potent mitogen for vascular endothelial cells (6). It has been purified from various tissues, including pituitary, brain, hypothalamus, retina, adrenal gland, corpus luteum, human placenta, and kidney and in view of its wide distribution, bFGF has been proposed to be a significant mediator of angiogenesis (7). VEGF is a homodimeric, 40-45-kD glycosylated heparin-binding glycoprotein, secreted by many mesenchymal and stromal cells (5). At least five transcripts of this factor have been detected, each preceded by a 26-amino acid signal peptide, so it is efficiently secreted (8). VEGF is selectively mitogenic for endothelial cells and appears to play a major role in angiogenesis (5).

This study aimed at investigating the serum levels of these two angiogenic factors, in children and adolescents with type 1 diabetes mellitus, as well as their relationship to parameters representing the severity of the disease, i.e. the duration of diabetes and HbA1C.

METHODS

Subjects. After approval by the Hospital Ethics Committee and informed consent, 40 children and adolescents (youngsters) with type 1 diabetes mellitus (19 male and 21 female) as well as 30 healthy control subjects (16 male and 14 female) were included in the study. Diabetic youngsters were recruited consecutively during the study period (January 1996 to December 1996), without any exclusion, from the youngsters returning to our Diabetes Center for their follow-up examination. Control subjects were personnel offspring and young students, selected with the criterion of being in good health.

Clinical data are presented in Table 1. Pubertal status was assessed through Tanner staging and was found to be in accordance to age in both the type 1 diabetes mellitus and control youngsters.

Table 1 Clinical data of the patients with type 1 diabetes mellitus (n = 40), and the control group (n = 30) (mean ± SD)
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Youngsters with type 1 diabetes mellitus underwent a thorough clinical and laboratory examination to document the existence or not of complications in the eyes, kidneys, heart, vessels, and nervous system. No signs of retinopathy (fundoscopy and fundus photography), neuropathy (autonomic nervous system control), or heart enlargement (echocardiogram) were found. Blood pressure (measured by a mercury sphygmomanometer) in each examined youngster was within normal limits for his or her age. Microalbuminuria was detected only in one 20-y-old girl, with urinary albumin excretion of 116.5 µg/min. (Microalbuminuria is determined by nephelometry in 12-h overnight urine collection and is considered abnormal if ≥20 µg/min × 1.73 m2 in at least two out of three urine collections).

Measurements. Three milliliters of blood were drawn from all youngsters. Blood was collected in pyrogen-free tubes, and serum was immediately separated by centrifugation after clotting and was kept frozen at -20°C until assayed. The analysis was performed by enzyme immunoassays, using commercially available kits (human FGF basic Quantikine™ HS and human VEGF Quantikine™, R&D Systems Minneapolis, MN 55413). The minimum detectable dose for bFGF and VEGF were 0.28 pg/mL and 9.0 pg/mL, respectively. Intra- and interassay coefficients of variation of the assays were 6.0 and 6.1% for bFGF and 5.1 and 6.2% for VEGF, respectively. HbA1C was determined in one drop of blood by MAb, using the Bayer DCA 2000 apparatus (normal limits, 4-5.5%).

Data analysis. Simple correlation and multiple regression procedures were used. Separate analyses were undertaken for the type 1 diabetes mellitus and the control group, as well as the combined dataset.

RESULTS

Serum bFGF and VEGF levels (pg/mL) in the type 1 diabetes mellitus and control groups are presented in Table 2. Simple correlation between serum levels of bFGF as well as VEGF and age, diabetes duration, HbA1C, Tanner stage, insulin dose, albumin excretion (log transformed), and blood pressure were not statistically significant. Values of r ranged from 0.01 to 0.19 for bFGF and from 0.02 to 0.23 for VEGF. After adjustment through multiple regression, for each examined growth factor, no statistically significant difference was found between the type 1 diabetes mellitus and the control groups (bFGF, r = 0.071, p = 0.952; VEGF, r = 0.174, p = 0.559). Restricting the multiple regression to youngsters with type 1 diabetes mellitus, neither bFGF nor VEGF serum levels varied with any of the parameters used in the analysis. Similarly no statistically significant results were observed after application of multiple regression in the control group. Finally, a significant correlation between serum levels of bFGF and VEGF was found both in the type 1 diabetes mellitus (r = 0.352, p = 0.025) and the control group (r = 0.556, p = 0.001).

Table 2 Serum bFGF and VEGF levels (pg/ml), in children and adolescents (youngsters) of the type 1 diabetes mellitus and control group (mean ± SE)
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DISCUSSION

The vascular disease that occurs in diabetes affects virtually every organ but is most evident in the eyes, kidneys, and the nervous system (9). Retinopathy, renal failure, and peripheral neuropathy are seldom symptomatic in children, but are responsible for considerable morbidity in young adults with type 1 diabetes mellitus (9). Consequent to microangiopathy, neovascularization is expected in the affected tissues. Indeed, increased angiogenesis has already been demonstrated in diabetic rats by Norrby et al. (10). Based on this finding, we have speculated that the so called "angiogenic factors" might be elevated in type 1 diabetes mellitus patients, even before clinical manifestations of microangiopathy are evident. In this respect we have recently determined, in the same cohort as described here, levels of an indirect angiogenic factor, angiogenin, and have shown that they are significantly increased in type 1 diabetes mellitus subjects compared with healthy control subjects (3).

This study investigated for the first time the role of two other angiogenic factors, bFGF and VEGF, in type 1 diabetes mellitus youngsters. Our results indicate that serum levels of both factors do not vary significantly between the type 1 diabetes mellitus and control groups and do not correlate with age, sex, duration, or metabolic control in the diabetic group. With reference to bFGF, a possible explanation for the low levels detected in this study could rely on the fact that bFGF is not normally found in the adult human circulation (11) due, in part, to the lack of a signal sequence domain in the bFGF precursor protein that is necessary for secretion from the cell via the endoplasmic reticulum (12). However, plasma bFGF-like endothelial cell growth-promoting activity has recently been measured using high sensitivity bioassays in a group of men with type 2 diabetes mellitus and persistent microalbuminuria or overt proteinuria, and bFGF serum levels correlated significantly (p < 0.05) with HbA1C (13).

Moreover, another report showed that serum bFGF immunoreactivity was increased in women with gestational diabetes and correlated with glycemic levels at term (14). Nevertheless, bFGF levels are also detected in normal pregnant women (15). In the present study, a high sensitivity bioassay was equally applied, but our population was on the one hand young and on the other, without diabetic complications.

With respect to the VEGF, to our knowledge, no data exist in the available literature referring to VEGF blood levels in diabetic subjects. However, in our study, with the restrictions of the young age and lack of complications in the type 1 diabetes mellitus patients, serum VEGF levels were unaffected compared with those of control subjects.

As the results of this study are in contrast to our previous report determining angiogenin serum levels, we have speculated about the reasons of this discrepancy. Thus, it might be possible that the increment of serum levels of angiogenin could precede that of bFGF and VEGF, possibly reflecting the angiogenic demands of the tissue at the early stage of the disease examined in this study. However, investigations in patients with advanced disease and probably with more extensive vascular dysfunction may reveal increased serum levels of the other angiogenic factors.

Pepper et al. (16) found a potent synergism between bFGF and VEGF in the induction of angiogenesis in vitro. They suggested that the same synergy exists in vivo as well. Our observation that bFGF and VEGF serum levels correlate in youngsters may indicate that possibly some unknown physiologic factor(s) simultaneously stimulates the release of VEGF and bFGF into the circulation. Whether these two factors act synergistically in vivo cannot be confirmed by this study.

In conclusion, the results of this study show that serum levels of bFGF and VEGF are not significantly elevated in the serum of youngsters with type 1 diabetes mellitus lacking clinically evident complications, and do not correlate with age, sex, duration, and metabolic control of the disease. However, a positive correlation exists between these two factors in the type 1 diabetes mellitus as well as in the control group. Further studies on the angiogenic growth factors involving subjects with long standing disease and overt diabetic complications are required to understand the mechanisms implicating the angiogenic process of diabetes.