Clinical immunology
Angiogenic activity of human serum – dependence on sex

Introduction
Angiogenesis, the growth of new blood vessels from the preexistent ones, is required for a variety of normal proliferative processes. Human serum contains substantial amounts of various angiostimulatory and angioinhibitory factors. Recently, we have reported age-dependence of human serum angiogenic activity [1]. Sera of older people were less effective in inducing neovascular response in mice cutaneous test, and contained lower concentration of important proangiogenic cytokine, vascular endothelial growth factor (VEGF), in comparison to the sera of those below the age of 50. In this paper we concentrate on sex differences in in vivo angiogenic activity of human serum. We also describe sex differences in the level of VEGF and in the activity of endogenous angiogenesis inhibitor, antithrombin-III (AT-III), in sera collected from men and women of various ages.

Material and Methods
Sera were collected from 60 people (30 men and 30 women), 20-80 years old, without immunological, inflammatory and neoplastic disorders. Informed consent was obtained from each person and the study was approved by local Ethics Committee.
Serum – (SIA) – induced cutaneous angiogenesis assay
Cutaneous angiogenesis assay was performed according to Sidky and Auerbach method [2] with own modifications [3, 4]. Studies have been performed in 2-month old, female inbred Balb/c mice. Mice have been of local laboratory breed, weighing ca 20 γ each. The sera of healthy subjects were injected intradermally (0.05 ml per one injection, 3-6 injections per mouse) into regionally shaved, anaesthetized with chloral hydrate (POCH, Poland) groups of 3 or more mice. In order to facilitate the localization of injection sites later on, all injected samples were coloured with 0.1% of trypan blue. After 72 hours mice were killed with lethal dose of Morbital (Biowet, Poland). All newly formed blood vessels were identified and counted in dissection microscope in 1/3 central area of microscopic field, at 6 × magnification. Identification was based on the fact that newly-formed blood vessels differ from background vasculature by their small size, tortuosity and divarications. Mean number of newly-formed blood vessels was calculated from a dozen or so separate readings and designated as “angiogenic activity” of tested sample.
Experiments were approved and supervised by the Local Ethics Committee.
Measurement of VEGF concentration
Cytokine levels in examined sera were determined using sandwich ELISA kits (R&D Systems, USA) for human VEGF, according to the producer instructions. Optical density was measured at 450 nm using spectrophotometric reader Elx800 (Biotek Instruments, Inc., USA). Cytokine concentration was expressed as pg/ml.
Measurement of AT-III activity
AT-III levels in plasma were assessed by the kinetic method using commercially available reagents (Dade Behring Marburg, Germany) according to manufacturer’s instructions. Results were expressed as % of reference normal value.
Statistical analysis
Statistical evaluation of the results was done by: Pearson’s correlation test, ANOVA, Mann-Whitney and Kruskal-Wallis tests, Bonferroni Multiple Comparison Test (GraphPad Prism software).

Results
The results of angiogenic activity of sera from men and women in age groups 20-49 and 50-80 are shown on Fig. 1. Young men sera presented higher activity than sera of both women groups, and this activity decreased with age- as low as to the values characteristic for women (Table 1). In men, we have found negative correlation between the age and serum VEGF concentration (Fig. 2). In women, no such relationship was observed (Fig. 3). VEGF content was the highest in young men group and significantly decreased with age (Fig. 4). AT-III serum activity was higher in men than in women (Fig. 5)Positive correlation between the age and AT-III activity was seen in men only (Figs. 6 and 7).

Discussion
The results obtained in this study reveal differences between some parameters of angiogenic activity of sera collected from healthy men and healthy women. Malamitsi et al. [5] reported controversial results concerning VEGF – they observed higher serum VEGF levels in females than in males. However, their study group included fetuses, neonates, children, and pregnant women, what may explain controversy. The same authors [6] in other paper reported no difference between males and females in respect to the serum levels of basic fibroblast growth factor (bFGF), other important proangiogenic cytokine. Bruserud et al. in their study performed on young athletes and elderly individuals discovered, that serum levels of some pro-angiogenic factors (bFGF, angiogenin and leptin) and of one antiangiogenic (endostatin) were higher in elderly individuals [7].
There is some controversy concerning the effect of testosterone on VEGF production and vascular reactivity. Ray et al. [8] reported deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production in vitro. Shao et al. , however, reported in rats with prostatic hypertrophy that testosterone increased microvessel density and multiplication of vascular endothelial cells [9]. According to Liu et al. [10] testosterone can stimulate prostatic growth probably by upregulating the protein expression of VEGF and FLK-1. Jones et al. reported positive influence of testosterone upon vascular reactivity [11]. In the present study we observed the highest serum angiogenic activity and VEGF level in the group of men below 50 years old, presumably being good testosterone producers. So, it may partly explain decrease of serum angiogenic activity and VEGF content in older men. We also observed in men another age-dependence: positive correlation with antithrombin level.
Antithrombin is a member of the serpin family of proteins and functions as an inhibitor of thrombin. The cleaved and latent forms of antithrombin also inhibit angiogenesis and tumor growth in vivo, acting selectively upon endothelial cells [12]. It was described, that antiangiogenic antithrombin blocks the heparan sulfate-dependent binding of proangiogenic growth factors to their endothelial cell receptors [13]. Then, the other explanation for decreasing angiogenic activity in elderly men might be the increase of AT-III activity in their sera.

References
1. Skopiński P, Skopińska-Różewska E, Jung L et al. (2009): Age dependence of angiogenic activity of human serum. Centr Eur J Immunol 34: 53-56.
2. Sidky YA, Auerbach R (1975): Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of graft-versus-host reaction. J Exp Med 141: 1084-1092.
3. Skopiński P, Szaflik J, Duda-Król B et al. (2004): Suppression of angiogenic activity of sera from diabetic patients with non-proliferative retinopathy by compounds of herbal origin and sulindac sulfone. Int J Mol Med 14: 707-711.
4. Skopiński P, Barcz E, Szaflik J et al. (2006): Angiogenic activity and IL-12p40 concentration in healthy people and diabetic patients sera. Central Eur J Immunol 31: 18-22.
5. Malamitsi-Puchner A, Tziotis J, Tsonou A et al. (2000): Changes in serum levels of VEGF in males and females throughout life. J Soc Gynecol Investig 7: 309-312.
6. Malamitsi-Puchner A, Tziotis J, Tsonou A et al. (2000): bFGF: serum levels in the female. Growth Factors 17: 215-220.
7. Bruserud O, Grovan F, Lindas R et al. (2005): Serum levels of angioregulatory mediators in healthy individuals depend on age and physical activity: studies of angiogenin, bFGF, leptin and endostatin. Scand J Clin Lab Invest 65: 505-511.
8. Ray R, Herring CM, Markel TA et al. (2008): Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production. Am J Physiol Regul Integr Comp Physiol 294: R1498-1503.
9. Shao JC, Wang Y, Zhang SW et al. (2005): Angiogenesis and regulatory factors in rats with BPH induced by testosterone. Zhonghua Nan KeXue 11: 413-418.
10. Liu X, Dong O, Lu Y et al. (2002): Effects of testosterone on VEGF and FLK-1 protein expression in the ventral prostate of rat. Hua Xi Yike Da Xue Xue Bao 33: 226-228.
11. Jones RD, Hugh Jones T, Channer KS (2004): The influence of testosterone upon vascular reactivity. Eur J Endocrinol 151: 29-37.
12. O’Reilly MS (2007): Antiangiogenic antithrombin. Semin Thromb Hemost 33: 660-666.
13. Zhang W, Swanson R, Xiong Y et al. (2006): Antiangiogenic antithrombin blocks the heparin sulfate-dependent binding of proangiogenic growth factors to their endothelial cell receptors: evidence for differential binding of antiangiogenic and anticoagulant forms of antithrombin to proangiogenic heparin sulfate domains. J Biol Chem 281: 37302-37310.