ENDOKRYNOLOGIA POLSKA

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1 E P ENDOKRYNOLOGIA POLSKA POLISH JOURNAL OF ENDOCRINOLOGY ORIGINAL PAPERS

2 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 3/2003 ISSN X HLA antigens and organ specific antimicrosomal antibodies in patients with autoimmune polyglandular syndromes (APS) Elwira Gromniak 1, Andrzej Mikłaszewicz 2, Anhelli Syrenicz 1, Grzegorz Kulig, 1 Stanisław Czekalski 3, Krystyna Pilarska 1 1 Department of Endocrinology, Hypertension and Metabolic Diseases, Pomeranian Medical University, Szczecin, Poland. 2 HLA Laboratory of Department of Microbiology and Immunology, Pomeranian Medical University, Szczecin, Poland. 3 Department of Nephrology, Medical University, Poznań, Poland. Summary The incidence of various HLA class I and II antigens was estimated in a group of 30 patients with autoimmune polyglandular syndromes (APS) in relation to their prevalence in the control population. Moreover, the presence of organ-specific antimicrosomal antibodies (MAB): thyroid, adrenal, pituitary and parietal cell, was evaluated. In the group of APS patients, incidence rates of the following antigens were higher: HLA class I (A1 and B8) and class II (DR3 and DR4). No predictive value of presence of HLA antigens for organ-specific antimicrosomal antibodies was found. Key words: Polyendocrinopathies - Autoimmune - HLA Antigens - Autoantibodies. E. Gromniak Department of Endocrinology, Hypertension and Metabolic Diseases, Pomeranian Medical University, ul. Arkońska 4, Szczecin, Poland. Antygeny HLA i swoiste narządowo przeciwciała antymikrosomalne u chorych z autoimmmunologicznym zespołem wielogruczołowym (AZW) E. Gromniak, A. Mikłaszewicz, A. Syrenicz, G. Kulig, S. Czekalski, K. Pilarska Klinika Endokrynologii, Nadciśnienia Tętniczego i Chorób Przemiany Materii PAM, Szczecin, Polska. Streszczenie W grupie 30 chorych z autoimmunologicznymi zespołami wielogruczołowymi (AZW) oceniano częstość występowania wybranych antygenów zgodności tkankowej HLA klasy I i II w porównaniu do grupy kontrolnej oraz obecność swoistych narządowo przeciwciał antymikrosomalnych (MAB): tarczycowych, nadnerczowych, przysadkowych i żołądkowych. W badanej grupie chorych wykazano częstsze występowanie antygenów HLA klasy I (A1 i B8) oraz klasy II (DR3 i DR4). Nie stwierdzono korelacji miedzy częstszym występowaniem niektórych antygenów z układu HLA, a obecnością swoistych narządowo przeciwciał antymikrosomalnych. Słowa kluczowe: Poliendokrynopatie - Autoimmunologia - Antygeny HLA - Autoprzeciwciała. 269

3 HLA in APS Gromniak E. Introduction Autoimmune polyglandular syndromes (APS) are recognized in patients with at least two autoimmune diseases [1]; moreover, the presence of one autoimmune disease increases the risk of development of other autoimmune disorder of 25% [2]. The following disorders may be included in the APS: chronic lymphocytic thyroiditis with hypothyroidism, Graves disease, infiltrative ophtalmopathy, type 1 diabetes mellitus, vitiligo, autoimmune Addison s disease, pernicious anaemia (atrophic gastritis), acquired primary hypogonadism, alopecia, hypoparathyroidism, myasthenia gravis, hypophysitis, thrombocytopenia, chronic mucocutaneous candidiasis, chronic active hepatitis and primary biliary cirrhosis. With regard to the presence of a combination of various autoimmune disorders, four types of APS have been distinguished: types I, II, III and a mixed type [1]. Characteristic features and diagnostic criteria of the above mentioned APS types are shown in detail in Table I. Development of clinical features of an autoimmune disease should raise the suspicion of coexisting symptoms or immunological markers of damage to other endocrine or non-endocrine organs [3]. The organ- specific antimicrosomal antibodies are immunological markers of tissue damage. In some autoimmune diseases these antibodies are bound to antigens that are thoroughly described. The thyroid microsomal antigen has been identified as peroxidase (TPO) [4,5]; in the Addison s disease these antigens have been recognized as : 17ά- hydroxylase in children [6] and 21-hydroxylase (21-OH) in adults [7, 8, 9]. The H + K + ATP-ase proved to be the antigen in patients with type A chronic autoimmune gastritis [10]. The main antigens in type 1 diabetes include: GABA-synthesizing enzyme glutamic acid decarboxylase [11] and carboxypeptidase-h [12]. The cytosolic proteins: 49- and 40-kDa, were found to be autoantigens in the lymhocytic hypophysitis; the 49-kDa protein has been recently recognized as gamma- and alpha- enolase [13, 14, 15]. The presence of antimicrosomal antibodies may indicate morphological or functional damage to other organ or tissue and may precede by many years the clinical symptoms of autoimmune disease confined to that organ [16, 17]. Endocrine organ failure is caused not only by destruction of the cells, but also by inactivation of enzymatic systems that play role in the production of hormones [18, 19]. The aim of the present study was, firstly to estimate the incidence of HLA class I and II antigens in patients with APS in relation to the distribution of HLA antigens in the healthy population and, secondly, to determine the associations of HLA antigens and the presence of thyroid, adrenal, pituitary and parietal cell antimicrosomal antibodies in the sera of the patients. Material and methods The study group enrolled 30 patients with APS. The diagnosis of APS was established basing on past history, thorough physical examination, hormonal and immunological studies, ultrasonography, scintigraphic study and/or fine-needle biopsy, depending on the type of the disease. Table II shows the characteristic features of patients with APS. No cases of type I, II B or III B were found in the studied group of APS patients. The control group enrolled 73 healthy individuals without clinical or immunological features of autoimmune diseases; moreover, none of the controls had relatives suffering from autoimmune diseases. In order to check the distribution of APS HLA class I and II in own study group, the data were compared with the big reference group that enrolled 600 individuals, studied by Matej from Wrocław [20]. HLA antigens were determined in the HLA Laboratory, Department of Microbiology and Immunology, Pomeranian Medical University, Szczecin. HLA class I antigens were determined using a two-step microcytotoxicity test according to Mittal [21]. HLA class II antigens were determined Table I. Classification of APS Type Possible abnormalities Diagnostic criteria I 1. Addison s disease. 2. Hypoparathyroidism. 3. Chronic mucocutaneous candidiasis. At least two of the three conditions must be present. II III Mixed Addison s disease and: A. Autoimmune thyroid disease. B. Type 1 diabetes mellitus. Autoimmune thyroid disease and: A. Type 1 diabetes mellitus. B. Pernicious anaemia. C. Vitiligo and/or alopecia. All disorders in various combinations not included above. Addison s disease with one or both of the associated diseases. Autoimmune thyroid disease (without Addison s disease) with one of the diseases designated A, B, or C. Any combination of the conditions. 270

4 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 3 (54) Table II. Characteristics of the study group of patients with APS APS Type Number of patients Age (yr.) x ± SD Gender Total APS 30 42,2±11,13 (range23-71) Table III. The HLA class I and II antigen typing in patients with APS. F - 22 (73 %) M - 8 (27 %) II A 5 (17 %) 46.6 ± F - 5 (100 %) M - 0 III Total 19 (63 %) 38.9±8.87 F - 14 (74 %) M - 5 (26 %) III A 12 (40 %) 39.4±11.42 F- 10 (83 %) M - 2 (17 %) III C 7 (23 %) 38.4±5.06 F- 4 (57 %) M - 3 (43 %) Mixed 6 (20 % ) 48.7±14.38 F - 3 (50 %) M - 3 (50 %) APS - autoimmune polyglandular syndrome, F - female, M - male APS Type Antigens in locus HLA - A Class I Antigens in locus HLA - B Antigens in locus HLA - C Class II Antigens in locus HLA -DR 1. II 10 5, 18 1, 5 3, 7 2. II 1, 2 8, 18 * 4 3. II 1, 3 8 * 3, II 1, 3 7, 15 * 6, 2 5. II 2, , 7 2, 3 6. IIIA 1, 2 8, 5 * 9, 3 7. IIIA 1, 10 8, , 3 8. IIIA 2 13 * 1, 4 9. IIIA 1, 2 35 * 6, IIIA 1, 9 8, 40 * IIIA 2, 10 27, IIIA 1, 2 8, IIIA 2, 9 13, 12 * 4, IIIA 11, 28 7, 12 * 4, IIIA 1, 28 8, 40 3, 7 6, IIIC 1, 2 8, 73 * 3, IIIC 2, 3 7, 21 * IIIC 2 15, 27 2, 3 2, IIIC 1, 2 21 * IIIC 3, 11 35, 16 4 ** 21. IIIC 1, 28 5, 8 * 2, IIIC 2, 3 8, , IIIC 2, 11 35, 5 4 1, IIIC 2, 3 13, , M 1, 2 8, 73 * M 2, - 35, M 2, 10 16, 5 * 3, M 2, 9 8, M 11, 30 13, 35 4, M 2, 9 8, 16 4, 7 * - antigens were not typed due to lack of reference sera M - Mixed type APS ** - typing was not performed 271

5 HLA in APS Gromniak E. using an isolated lymphocyte test according to Matej [22], with a modification by Mikłaszewicz [23]. Determination of thyroid, adrenal, pituitary and parietal cell antimicrosomal antibodies were performed using a solid-phase radioimmunoassay (RIA) in plastic tubes coated with microsomal proteins obtained from human autopsy tissues according to Kosowicz and Łącka [24, 25, 26], with a modification by Gryczyńska [27, 28] and Sawicka [29]. The achieved results underwent statistical analysis using chi-square test with Yates correction and ANOVA test. Results Among HLA class I antigens, locus A contained A2 and A1 antigens most frequently that were found in 20 and 13 cases, respectively (i.e. 65% and 43%); locus B contained most frequently B8 and B35 antigens, in 13 and 6 cases, respectively (i.e. 43% and 20%); in locus C antigens Cw4 and Cw7 proved to be the most prevalent, however due to the lack of reference sera the studies have been performed only in some of the patients. Among HLA class II antigens, DR3 and DR4 antigens turned out to be the most frequent that were detected in 12 and 11 cases, respectively (i.e. 41% and 38%). The detailed distribution of HLA class I and II antigens in the studied material is shown in Table III. Table IV shows the comparison between incidence of HLA class I and II in APS patients and controls. Patients with APS significantly more frequently revealed HLA class I (A1 and B8) antigens, and HLA class II (DR3 and DR4) antigens. Comparison of incidence of HLA class I and II antigens between the own control group and Wrocław group showed no significant differences. The incidence of antimicrosomal antibodies in APS patients is shown in Table V. The whole studied group most frequently revealed thyroid and parietal cell antibodies, namely in 57% and 53%, respectively. Pituitary and adrenal antibodies were found less frequently, namely in 10% and 7%, respectively. When analyzing patients with each APS type separately, thyroid antibodies were found most frequently in type IIIA and IIA, namely in 92% and 80%, respectively; adrenal antibodies most frequently in type IIA - in 20%; pituitary antibodies - in type IIIA and mixed type - with the same frequency - 17% each; parietal cell antibodies in type IIIA and mixed type, namely in 67% and 50%, respectively. Table VI shows comparison between the incidence of antimicrosomal antibodies and most frequent HLA class I and II antigens (A1; B8; DR3 and DR4) in APS patients. However, incidence of antimicrosomal antibodies in APS patients with HLA-A1; B8; DR3 and DR4 antigens were not higher when compared to patients without these antibodies. Only borderline statistical significance was found for thyroid antibodies in patients with A1 antigen (77% of cases), parietal cell antibodies in patients with DR4 antigen (80% of cases), while in patients without that antigens the results were 44% and 39%, respectively. Discussion Genetic predisposition plays a significant role in the pathogenesis of autoimmune diseases. State-of-theart hypothesis suggests indirect association between HLA antigens and autoimmune diseases. In other words, a specific HLA gene is only a marker of other genes that determine susceptibility to the specific disease and thus determine the course of the specific immune reactions. Such a situation is possible due to linkage disequilibrium frequently present in HLA system [30]. Until recently only few studies on HLA in autoimmune polyglandular syndromes have been done. Type I APS did not reveal increased incidence of any of HLA class I or II antigens that would be a characteristic feature for that type and could be found more frequently in these patients than in healthy individuals [31]. Few studies on the remaining types of APS described higher incidence of HLA-DR3 and DR4 antigens in type II [32]. Higher number of publications describe results on incidence of HLA class I and II antigens in patients with isolated autoimmune diseases of endocrine glands or autoimmune diseases confined to other organs. A lot of studies were performed in Graves disease in which antigens B8 and DR3 in Caucasian patients, Bw35 in Japanese patients and Bw46 in Hong-Kong patients were found significantly more frequently [33, 34, 35, 36, 37]. The presence of HLA antigens in Hashimoto lymphocytic thyroiditis raised an interest. Higher incidences of antigens A1 and A8, DR4 and DR5 were found in that disease; in Hashitoxicosis DR3 and DR5 [38, 39, 40, 41, 42, 43, 44] prevailed. Antigens A1 and B8 and DR3 or DR5 were found more frequently in the atrophic type of Hashimoto thyroiditis [45, 46, 47, 48, 49]. Other authors revealed no correlation between HLA DR antigens and Hashimoto thyroiditis [50, 51]. It is likely that 2 or more loci in the major histocompatibility complex MHC/HLA determine predisposition for autoimmune diseases of the thyroid gland. One of them - DR3 - may show a non-specific effect of magnification of auto-reaction possibility. It is hypothesized that in both healthy and sick individuals the presence of DR3 antigen is associated with high genetic polymorphism which leads to negative reactions in some of them and positive reactions in other individuals [52], this is a phenomenon associated with protective antigens including HLA antigens class I and II B 35; B 7, 27; B 8, 18; DR 2, 3, 4 [53]. 272

6 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 3 (54) Table IV. The comparison of HLA class I and class II antigen incidence rates between the APS group and the control Wrocław group. Antigens in locus HLA - A HLA Antigens Percentage of antigen positive subjects. Class I Antigens in locus HLA - B Antigens in locus HLA - Cw Class II Antigens in locus HLA - DR P C P C P C P C A 1 43* 27 B Cw DR A B Cw DR A B 8 43* 17 Cw DR 3 41* 20 A B Cw DR 4 38* 16 A B Cw DR A B Cw DR A B DR A B DR A B DR A B DR A B A B B B P - patients with APS, C - control group, * statistically significant, p< 0,05 ( test χ2). Table V. The incidence of organ-specific antimicrosomal antibodies in patients with APS. APS Type Antymicrosomal antibodies - number of positiv patients. Thyroidal Adrenal Pituitary Parietal cell Total APS n=30 17 (57%) 2 (7%) 3 (10%) 16 (53%) II A n= 5 4 (80%) 1 (20%) - 2 (40%) III (A+C) n=19 13 (68%) 1 (5%) 2 (11%) 11 (58%) III A n=12 11 (92%) 1 (8%) 2 (17%) 8 (67%) III C n=7 2 (29%) (43%) Mixed n= (17 %) 3 (50%) APS autoimmune polyglandular syndrome. Table VI. The incidence of organ-specific antimicrosomal antibodies in relation to the presence of most frequent HLA class I and II antigens in patients with APS. HLA Antigens (percentage of patients) Class I Class II Antibodies A1 B8 DR3 DR4 with without with without with without with without Thyroidal Adrenal Pituitary Parietal cell * 39 * p>0,05 borderline statistical significance (test χ2) 273

7 HLA in APS Gromniak E. Characteristic patterns of HLA antigens were sought also in other autoimmune diseases. Antigens B8 and DR3 are most common in the Addison disease [54]. Some authors found higher incidence of HLA- A3 [55] and B7 antigens [56] in Addison- Biermer anemia; however, many reports deny the association of pernicious anemia with HLA antigens [57, 58]). Type 1 diabetes mellitus is believed to be associated both with HLA class I -A1 and B8 [59] antigens, and class II - DR3 and DR4 antigens [60]. In our study we evaluated the incidence of individual HLA antigens in patients with APS in comparison to the control group. The distribution of HLA antigens in the own group was identical as in the Wrocław group that enrolled 600 patients, and proved to be characteristic for the Caucasian race [20]. We found significantly higher incidence of two class I antigens: A1 and B8, and two class II antigens: DR3 and DR4 in patients with APS when compared to healthy individuals. The incidence rates of HLA antigens in patients with APS and in patients with isolated autoimmune endocrine disorders, as mentioned above, didn t differ as far as specific HLA class I and II antigens were concerned. Thus, we may assume that any patient with an isolated autoimmune disease is predisposed for development of other autoimmune disorders including both endocrine and non-endocrine ones. On the other hand, it is surprising that in as many as 6 patients with APS (20% of the studied group) no characteristic antigens of HLA class I (A1 and B8) or class II (DR3 and DR4) have been found. Thus, HLA antigens should be treated as weak and nonspecific markers of autoimmune disorders [30] (18). Their presence in healthy individuals reflects rather a familial than individual predisposition to development of autoimmune disorders [40, 61]. Moreover, it seems that any specific HLA gene is only the marker of the presence of other genes that determine the course of immune reactions but they don t play any role in the pathogenesis of the disorders themselves. That hypothesis is consistent with studies by Stenszky [62], who investigated the incidence of B8 antigen in patients with Graves disease divided into three groups according to their clinical criteria. The first group enrolled patients with a small goitre without thyroid antibodies and with tendency for remission following anti-thyroid drug treatment. The second group enrolled patients with clinical and biochemical features of Hashitoxicosis ; and the third group enrolled patients with associated ophtalmopathy, familial predisposition to autoimmune disorders and tendency for recurrent thyrotoxicosis. The HLA B8 antigen was found in 8.9%, 20.7% and 86.7% of the patients, respectively. The increasing frequency of the antigen in patients with severe immune disorders justifies the conclusion that its direct association with the given disease is not strong; however, it clearly indicates the severity of the immune disturbances which are most likely associated with the lack of protective In our patients antigens [53] with APS we found the antimicrosomal antibodies occurring in the decreasing order: thyroid, parietal cell, pituitary and adrenal antibodies. Thus, the thyroid antibodies could be used as a valuable marker not only in the isolated autoimmune thyroid diseases, but also as a marker of autoimmune polyglandular syndromes. Kasperlik-Załuska and Czarnocka applied that marker in order to evaluate the autoimmune background of the Addison s disease and secondary adrenal insufficiency; the authors measured thyroid peroxidase antibodies (atpo) in their patients [63, 64, 65]. High incidence of parietal cell antibodies in our patients with APS without clinical diagnosis of Addison-Biermer disease should be also underlined. It may indicate that the presence of immunological markers may precede development of clinical signs that require additional pathological factors to be present [61, 66]. Our study did not reveal any significant correlation between the most frequently present HLA antigens: A1, B8, DR3 or DR4, and presence of thyroid, adrenal, pituitary and parietal cell antimicrosomal antibodies in APS patients. The strongest correlation was found between A1 antigen and thyroid antibodies, and between DR4 antigen and parietal cell antibodies; however, these results showed only borderline statistical significance. The literature data on isolated autoimmune diseases are contradictory; however, majority of the reports describe the lack of correlation between HLA antigens and presence of antimicrosomal antibodies. Dahlberg [34], Łącka [35], Mather [67], Nakao [36] and others [68, 69], showed no such correlation in Graves disease. However, Thorsby [70] and Balázs [71] found correlation between presence of B8 antigen and antimicrosomal or anti-thyreoglobulin antibodies in patients with Graves disease. In autoimmune atrophic thyroiditis Irvine [46] found no correlation between the presence of HLA B8 antigen and thyroid antimicrosomal antibodies. Jansson [72] proved the correlation between the presence of HLA DR4 antigen and thyroid antimicrosomal antibodies in postpartum thyroiditis. In type 1 diabetes mellitus no correlation between islet cell antibodies (ICA) and HLA antigens was found by Barbosa [73] and Ginsberg-Fellner [74] in Caucasians, and by Kobayashi [75] and Kida [76] in Japanese population. The latter author in patients with type 1 diabetes mellitus evaluated not only ICA antibodies, but also antimicrosomal and antithyreoglobulin antibodies, antibodies to adrenal cortex and gastric mucosa; however, he did not find any correlation between these antibodies and HLA antigens either. Roman [77] studied patients with type 1 diabetes mellitus and found a positive correlation between the presence of DR5 antigen and 274

8 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 3 (54) thyroid antimicrosomal antibodies. In Addison s disease Partanen [78] did not find any correlation between HLA antigens and presence of antibodies to 21-hydroxylase, the major autoantigen of adrenal cortex microsomal fraction. In the Addison-Biermer anemia Karmel [79] and Feldt- Rasmussen [61] found no correlation between parietal cell antibodies and HLA antigens. Conclusions 1. Presence of class I HLA-A1 and B8 and class II DR3 and DR4 antigens increases the probability of development not only of the isolated autoimmune disease, but also of the autoimmune polyglandular syndrome. 2. Despite lack of presence of HLA -A1, B8, DR3 or DR4 antigens, a certain group of patients may develop the autoimmune polyglandular syndrome. 3. The presence of antimicrosomal thyroid, adrenal, pituitary and parietal cell antibodies in patients with APS shows no correlation with the presence of HLA antigens. 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10 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 3/2003 ISSN X Thyroliberin augments the peripheral blood plasma concentration of vascular endothelial growth factor in obese humans # # This study has been presented at the 10 th Meeting of the European Neuroendocrine Association, München, Germany; September 12-14, Jan Komorowski, Joanna Jankiewicz-Wika, Beata Misztal-Dethloff, Dominika Stańczyk-Tomecka, Henryk Stępień Institute of Endocrinology, Medical University of Łódź, Poland Summary Obesity is associated with an excessive growth of the adipose tissue. The fat cells are able to produce several cytokines, among other proangiogenic factors: the vascular endothelial growth factor (VEGF) and leptin. The aim of the study was to evaluate the effect of a single thyroliberin administration on VEGF concentration in peripheral blood circulation. The peripheral blood plasma levels of VEGF (by ELISA) were measured during a standard TRH test (at 0, 30, 60, and 120 min) in regularly menstruating, euthyroid, non-smoking and normoglycaemic 10 obese and 10 lean women. Peripheral blood TSH, PRL, ft 3, ft 4 concentrations in the TRH test were also evaluated by MEIA or IRMA methods and leptin (by RIA). The concentration of VEGF at 60 min of the test as well as mobilization of VEGF secretion ( % over basal values) increased (p<0.05) in obese subjects only. Serum levels of leptin decreased at 120 min of the test in both group of studied subjects (p<0.05). Positive relationships between ft 4 and VEGF values in 0, 30, 60 min and between ft 3 and VEGF in the 30 th min. in obese females were noted. In the control group (lean subjects) we revealed a positive relationship between VEGF and leptin levels in 0, 30, 60, and 120 min. The obtained results show that a single TRH administration may augment vascular endothelial growth factor concentrations in peripheral blood of obese subjects. Key words: TRH, VEGF, leptin, obesity Jan Komorowski, MD, PhD Institute of Endocrinology Medical University of Łódź Łódź, Poland Phone: + (48-42) Fax: + (48-42) komorowski.j@wp.pl Tyreoliberyna zwiększa stężenia śródbłonkowego czynnika wzrostu w osoczu krwi u ludzi otyłych # Jan Komorowski, Joanna Jankiewicz-Wika, Beata Misztal-Dethloff, Dominika Stańczyk-Tomecka, Henryk Stępień Instytut Endokrynologii, Uniwersytetu Medycznego w Łodzi Streszczenie Otyłość pojawia się w wyniku zwiększenia tłuszczowej masy ciała. Komórki tkanki tłuszczowej wytwarzają szereg cytokin, między innymi białkowe czynniki peptydowe o działaniu pobudzającym wzrost naczyń krwionośnych: śródbłonkowy czynnik wzrostu (VEGF) i leptynę. Celem pracy była zbadanie czy jednorazowe podanie tyreoliberyny wpływa na stężenie VEGF we krwi. W osoczu krwi żylnej przeprowadzono pomiary stężeń VEGF (metodą ELISA) w przebiegu standardowego testu z TRH (przed i w 30, 60, 120 min.) u 10 otyłych oraz 10 kobiet z prawidłową masą ciała. Równocześnie oceniano stężenia TSH, PRL, ft 3, ft 4 (metodą MEIA lub IRMA) oraz leptynę (metodą RIA). Tylko u otyłych kobiet ujawniono zwiększenie stężenia oraz mobilizacji VEGF ( %) w 60 minucie testu z TRH (p<0,05). Stężenia leptyny malały w obu badanych grupach w 120 minucie testu (p<0,05). Zaobserwowano dodatnią zależność pomiędzy stężeniami ft 4 i VEGF w 0, 30 i 60 min. oraz pomiędzy ft 3 i VEGF w 30 min. testu tylko w grupie kobiet z otyłością. W grupie kobiet bez otyłości stwierdzono dodatnią zależność pomiędzy stężeniami VEGF i leptyny w 0, 30, 60, 120 min. testu. Uzyskane wyniki wskazują na to, że jednorazowe podanie TRH może zwiększać stężenie śródbłonkowego czynnika wzrostu we krwi u kobiet z otyłością. Słowa kluczowe: VEGF, TRH, leptyna, otyłość 277

11 Effect of TRH on EGF Komorowski J. Introduction Obesity, a result of an excessive growth of the adipose tissue, is a worldwide epidemic and public health disaster leading to diabetes mellitus, coronary heart disease, tumours, and sleep breathing problems. Adipocyte hypertrophy and hyperplasia together with excessive angiogenesis contribute to the growth of the fat mass. The fat cells, apart from their metabolic activities, are able to produce several cytokines among other proangiogenic factors: vascular endothelial growth factor (VEGF) [1] and leptin [2]. These factors, originating from adipocytes, may contribute to the formation of new blood vessels mainly inside the fat pad [2]. Blood levels of VEGF can be seen as surrogate markers of angiogenesis [4, 5]. It was found that leptin, which is involved in the control of satiety and energy expenditure, was also shown to stimulate angiogenesis [6]. In our previous study, we revealed that intravenous administration of 200 µg of thyreoliberin (in bolus) decreased peripheral blood levels of leptin in normal and obese women [7]. The aim of the study was to evaluate the effect of a single thyroliberin administration on the vascular endothelial growth factor concentration in peripheral blood circulation. Material and methods The blood plasma which had been obtained during the standard (0.2 mg) TRH test (at 0, 30, 60, and 120 min; stored in 70 o C) from regularly menstruating, euthyroid (without goiter), non-smoking and normoglycaemic 10 obese women (age: 27.50±2.64 yrs; BMI [body mass index]: 36.61±1.28; WHR [waist to hip ratio]: 0.89±0.03; IRI [insulin]: 13.59±11.1 miu/l (by IRMA; Biosource, Belgium) and 10 lean control group (so-called healthy, euthyroid, without goiter; age: 23.10±0.67 yrs; BMI: 20.44±0.61; WHR: 0.69±0.01; IRI: 8.44±3.51 miu/l) measured for VEGF concentrations by ELISA method (R & D Systems, USA). The results of BMI, WHR and leptin values in obese subjects were statistically higher (p<0.01) comparing to controls. During the same TRH test the peripheral blood serum TSH [by IMx ultrasensitive MEIA; Abbott, USA], PRL [by IRMA; Byk- Sangtec Diagnostica, Germany], ft 3, ft 4 [by MEIA Axsym, Abbott, USA] and leptin (by RIA; Linco, USA) concentrations were evaluated. All the obtained results were expressed as means ±SEM. Comparisons between tested groups were made by one way analysis of variance. When ANOVA revealed a statistically significant difference, comparisons of the individual groups were further evaluated by Mann-Whitney s U test. Differences were considered significant if P<0.05. Correlations between different parameters were calculated by the linear analysis. Results The basal levels of TSH, ft3, ft4 and PRL were in normal range in all obese and lean subjects. The mobilization of secretion of the a. m. hormones into circulations also occurred. The concentration of VEGF at 60 min of the test as well mobilization of VEGF secretion ( % over basal values) increased in obese subjects only (p<0.05) (Fig. 1). Leptin levels (lower in controls: 7.90±0.21 ng/ml vs ±2.02 ng/ml) decreased at 120 min of the TRH test in both groups of women tested (p<0.05). No correlations between leptin or insulin basal concentrations and VEGF levels as well as between PRL or TSH and VEGF release were revealed (p>0.05) in both groups of females studied. Positive relationships between ft 4 and VEGF values in 0 (r=0.8291; p<0.001), 30 (r=0.6915; p<0.001), 60 (r=0.6376; p<0.01) min and between ft 3 and VEGF in 30 (r=0.5171; p<0.05) min in obese females were noted. There was no positive correlation between ft 3 or ft 4 and VEGF during TRH test in lean subjects (p>0.05). In the control group (lean subjects) we revealed a positive relationship between VEGF and leptin levels (p<0.001) in 0 r=0.7607), 30 (r=0.7750), 60 (r=0.7059), and 120 min (r=0.7834) of the TRH test. There were no significant relationships between VEGF and leptin concentrations in obese women (p>0.05). Discussion VEGF is an endothelial specific mitogen that has vascular permeability-enhancing activity and has also been shown to play a key role in vasculogenesis and angiogenesis [8, 9]. VEGF expression has been found in activated macrophages, kerationocytes, renal glomerular visceral epithelium and mesangial cells, hepatocytes, smooth muscle cells, Leydig cells, embryonic fibroblasts and bronchial and chorioid plexus epithelium [9]. Thyroliberin is a tripeptide with a wide distribution in the brain and in other tissues [10]. In human TRH stimulates the release of prolactin and thyrotropin via its direct effect on the pituitary lactotrophs and thyrotrophs. When it is administered intravenously there is an immediate release of pituitary TSH and PRL, with a peak levels of both hormones occurring min after the bolus injection. Moreover, our previous study also revealed that administration of thyroliberin augments human immune system activity in vitro [11] and in vivo [12]. We have noted that leptin levels decreased at the 120 min of TRH administration in both lean and obese subjects. The other authors also showed, in male Wistar rats, that intracerebroventricular administration of TRH lowered the levels of blood circulating leptin, too [13]. At present we found a positive correlation between leptin and VEGF levels in control group only. However, it was also proven by others [6], studying the formation of vascular fenestrations and perme- 278

12 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 3 (54) 600 * TRH TEST 100 * 500 O 80 Fig. 1. Peripheral blood VEGF mobilization during TRH intravenous test in normal (c) and obese (o) women [X ±SEM; * - p< 0.05]. VEGF [pg/ml] C T [min] C O ability, that leptin plays a significant role in the stimulation of angiogenesis synergistically with the fibroblast growth factor (bfgf) and VEGF, the two most potent and commonly expressed angiogenic factors. They concluded, that the regulation of vascular fenestrations in adipose tissue seems to be complex and involves several factors, as well as that leptin, but not VEGF, is the key molecule in the maitenance of a. m. process. The present study provides the first evidence that a single thyroliberin administration increases the peripheral blood plasma concentration of the vascular endothelial growth factor in adult obese females. It may be done via nitric oxide release [14]. However, we were not able to find any relationship between VEGF and a. m. pituitary hormones after a single administration of TRH in normal as well as in obese subjects. Other studies [15, 16, 17] have revealed some relations between TSH and VEGF secretion. Sato et al [18] identified VEGF mrna in human thyroid follicles isolated from patients with Graves disease, and found it to be increased after exposure to TSH, Graves IgG, or insulin. Moreover, the significant correlation between serum TSH and VEGF was also noted in patients with Hashimoto s thyroiditis [15] and the results suggested that TSH may promote the growth in some thyroid cancers by stimulating VEGF secretion and angiogenesis [16]. The results obtained by others did not support the hypothesis that the short term TSH stimulation in humans with recombinant TSH may increase peripheral blood VEGF concentrations [17]. The positive relationship between ft 4 and ft 3 and VEGF concentrations in obese women during TRH test indicates that thyroid hormones may be involved not only in energy homeostasis but in specific conditions they can accelerate the development of new blood vessels as a result of VEGF hypersecretion because nitric oxide synthase gene expression is upregulated by thyroid hormones [19]. Previously we also revealed that the high levels of thyroid [20] and pituitary secreted hormones [4] affect systemic levels of VEGF in similar matter. However, these effects may be also partly upregulated by insulin. The study performed in vitro by Mick et al. [21] demonstrated that physiological insulin concentrations stimulated VEGF formation and expression in cultured rodent white adipocytes. In conclusion, the obtained results show that a single TRH administration may augment vascular endothelial growth factor concentrations in peripheral blood of obese subjects. References 1. Claffey KP, Wilkison WO, Spiegelman BM. Vascular endothelial growth factor: regulation by cell differentation and activated second messenger pathways. J Biol Chem 1992; 267: Bouloemie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998; 83: Bouloumie A, Sengenes C, Portolan G, Galitzky J, Lafontan M. Adipocyte produces matrix metalloproteinases 2 and 9. Involvement in adipocyte differentation. Diabetes 2001; 50: Komorowski J, Jankiewicz J, Stępień H: Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bfgf) and soluble interleukin-2 receptor (sil-2r) concentrations in peripheral blood as markers of pituitary tumours. Cytobios 2000; 101: Stępień HM, Kołomecki K, Pasieka Z, Komorowski J, Stępień T, Kuzdak K. Angiogenesis of endocrine gland tumours new molecular targets in diagnostics and therapy. Eur J Endocrinol 2002; 146: Cao R, Brakenhielm E, Wahlestedt C, Thyberg J, Cao Y. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci USA 2001; 98: Komorowski J, Jankiewicz-Wika J, Stępień H. Effects of Gn- RH, TRH, and CRF administration on plasma leptin levels in lean and obese women. Neuropeptides 2000; 34: Neufeld G, Cohen T, Gengrynowitch S, Poltorok Z. Vascular 279