Growth Hormone Increases Vascular Cell Adhesion Molecule 1 ...

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The Journal of Clinical Endocrinology & Metabolism 89(2):909 –916 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-030223

Growth Hormone Increases Vascular Cell Adhesion Molecule 1 Expression: in Vivo and in Vitro Evidence TROELS KRARUP HANSEN, SANNE FISKER, ROLF DALL, THOMAS LEDET, JENS OTTO L. JØRGENSEN, AND LARS MELHOLT RASMUSSEN Immunoendocrine Research Unit (T.K.H.), Medical Department M (Endocrinology and Diabetes) (T.K.H., S.F., R.D., J.O.L.J.), and Research Laboratory for Biochemical Pathology, Institute of Pathology (T.L., L.M.R.), University Hospital of Aarhus, Kommunehospitalet, DK-8000 Aarhus C, Denmark between groups 151.8 ␮g/liter (95% confidence interval: 95.0 – 208.7 ␮g/liter); P < 0.0001]. In human umbilical vein endothelial cells, there was no direct stimulatory effect of either GH or IGF-I on the expression of VCAM-1 and E-selectin, but serum from GH-treated healthy subjects significantly increased the expression of VCAM-1 (P < 0.01). Our findings are compatible with the notion that GH may stimulate the expression of VCAM-1 indirectly through modulation of circulating factors. VCAM-1-mediated leukocyte extravasation is implicated in several illnesses including atherosclerosis and multipleorgan failure in sepsis, and we hypothesize that enhanced expression of VCAM-1 may contribute to the detrimental effects of GH in critically ill patients. (J Clin Endocrinol Metab 89: 909 –916, 2004)

We investigated the impact of GH administration on endothelial adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, in vivo and in vitro. Soluble VCAM-1, E-selectin, and C-reactive protein concentrations were measured before and after treatment in 25 healthy subjects and 25 adult GH-deficient (GHD) patients randomized to GH treatment or placebo. Furthermore, we studied the direct effect of GH and IGF-I and serum from GH-treated subjects on basal and TNF␣-stimulated expression of VCAM-1 and E-selectin on cultured human umbilical vein endothelial cells. Baseline levels of VCAM-1, but not E-selectin, were significantly lower in GHD patients than in healthy subjects (362 ⴞ 15 ␮g/liter vs. 516 ⴞ 21 ␮g/liter, P < 0.001) and increased in GHD patients during GH treatment, compared with placebo [net difference

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concentrations are related to and increased risk of developing atherosclerosis and ischemic heart disease (5, 6). Extravasation of white blood cells is an important element in both atherogenesis and the acute inflammatory MOF of sepsis and septic shock (7, 8). The endothelium orchestrates this recruitment of leukocytes. It is initiated by members of both the Ig-like supergene and selectin gene families of adhesions molecules, which are expressed on the luminal surface of vascular endothelial cells (9, 10). These molecules can be induced by a number of cytokines (11, 12), and soluble parts of the proteins are cleaved from the cell membrane and shed into the circulation. Vascular cell adhesion molecule-1 (VCAM-1) and E-selectin belong to the Ig and selectin families, respectively. Endothelial expression of VCAM-1 is increased by exposure to lipopolysaccharides (13), and serum concentrations of soluble VCAM-1 are significantly increased in patients with sepsis and septic shock (14, 15). Blockade of VCAM-1 with monoclonal antibodies reduces the endotoxin-induced organ damage (16, 17), supporting a significant role of this adhesion molecule in the pathogenesis of inflammation-related critical illness. Likewise, soluble Eselectin levels are higher in patients with microbiologically documented sepsis than in other critically ill patients (18), but the reported consequences of E-selectin blockade in sepsis and septic shock have been conflicting (19 –21). In the present study, we examined the effects of GH administration on serum concentrations of soluble VCAM-1 and E-selectin in both GHD patients and normal individuals. We also performed in vitro experiments in human umbilical vein endothelial cells (HUVECs) to investigate direct and

ROWTH HORMONE IS a potent anabolic hormone with widespread metabolic actions. Mounting evidence suggest that the GH/IGF-I axis may have important immunomodulatory effects, and it was recently documented that patients with GH deficiency have increased levels of inflammatory markers (1). In critically ill patients, severe catabolism with increased protein turnover and negative nitrogen balance is a major problem (2), and treatment with GH has been proposed as a feasible approach to reverse these changes (3). However, a recent randomized placebocontrolled multicenter study demonstrated that high-dose GH therapy in fact caused a significant increase in mortality and morbidity, compared with placebo (4). Apparently, GH possesses hitherto unknown proinflammatory properties, and elucidation of the involved pathways may increase our general knowledge of the pathophysiology of inflammation, sepsis, and multiple-organ failure (MOF). Likewise, GH and IGF-I have been suggested to be involved in the pathogenesis behind cardiovascular diseases. Detrimental effects of GH has, for example, been suggested to play a role in both acromegaly and diabetes (5). However, GH’s role is complex because GH-deficient (GHD) patients suffer from an increased incidence of cardiovascular disease and low IGF-I Abbreviations: CRP, C-reactive protein; GHD, GH-deficient; HRP, horseradish-peroxidase; HUVEC, human umbilical vein endothelial cell;MOF, multiple-organ failure; VCAM-1, vascular cell adhesion molecule-1. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

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indirect actions of GH and IGF-I on the expression of VCAM-1 and E-selectin. Subjects and Methods Subjects Blood samples were obtained from 25 patients (19 males, 6 females) with known pituitary pathology (10 nonsecreting adenoma, two prolactinoma, three Cushings disease, five craniopharyngioma, and five other). All patients had GH deficiency determined as peak GH less than 5 ␮g/liter at two different provocative tests. Patients with other hormonal deficiencies had been on stable substitution with the relevant hormones for at least 1 yr before participation. The patients were randomized to treatment with either GH [target dose 2 IU/m2 (0.66 mg/ m2)] once daily or placebo for 4 months. Body mass index was 29.9 ⫾ 1.5 kg/m2 in the GH group and 27.0 kg/m2 in the placebo group. During the initial 6 wk, the dose of GH (or placebo) was gradually increased to reach target dose. The mean dose given was 3.7 ⫾ 0.8 IU/d, but due to side effects (edema, myalgia and arthralgia), GH dose was reduced in seven patients during the treatment period to a mean daily dose of 3.3 ⫾ 1.0 IU/d. Serum samples were collected at baseline and after 4 months. Furthermore, samples from 25 healthy unmatched lean subjects (18 males, seven females) randomized to treatment with either high-dose GH (0.1 or 0.2 IU kg⫺1 [0.033 or 0.066 mg kg⫺1] once daily) or placebo in a double-blinded manner for 4 wk were included. Serum samples were obtained on d 0 and 28. GH or placebo was administered as daily sc self-injections in the evening. To minimize side effects, only 50% of the target dose was given during the first week. GH (Norditropin) and placebo preparations were supplied by Novo Nordisk (Copenhagen, Denmark). All blood samples were collected after an overnight fast. The local ethical committee and the Danish National Board of Health approved the study, and informed consent was obtained from each subject before entering the study.

Analyses Serum VCAM-1, E-selectin, IGF-I, and C-reactive protein (CRP) concentrations were measured in all patients and healthy subjects at baseline and after treatment with GH or placebo. Serum VCAM-1 and Eselectin were measured by commercially available ELISA kits, as described by the manufacturer (R&D Systems, Minneapolis, MN, catalog no. BBE3, VCAM-1) and BBE2B (E-selectin). Serum IGF-I was measured with an in-house time-resolved immunofluorometric assay, as previously described (22), and serum concentrations of CRP were analyzed at the Department of Clinical Biochemistry, Aarhus University Hospital, using ultrasensitive latex-enhanced immunotechniques (Cobas Integra 700, Hoffmann-La Roche Ltd., Basel, Switzerland).

Hansen et al. • GH Stimulates VCAM-1 Expression

Cell cultures of HUVECs HUVECs obtained from collagenase-digested umbilical veins were cultured in DMEM (glucose concentration: 5.5 mm), containing 10% fetal calf serum, 2 ␮g/ml ciprofloxacin, 100 ␮g/ml ampicillin, 25 ␮g/ml endothelial cell growth supplement, 15 U/ml heparin, and 2 mm glutamine in gelatin-coated plates (0.65 ␮g/cm2), maintained at 37 C in an atmosphere of 5% CO2, 95% atmospheric air (12). The cells were subcultured after detaching with trypsin solution and replating. Experiments were performed in 96-well plates, in which cells were exposed to GH or IGF-I at concentrations of 5 or 50 ␮g/liter for 6 or 24 h. Experiments were conducted with or without concomitant addition of TNF␣ at a concentration of 0.1 ng/ml. In a separate series of experiments, cells were incubated with sera from the 25 healthy subjects in a dilution of 1/10. Each individual serum sample was assessed in quadruplicate for both VCAM-1 and E-selectin. Experiments with the addition of hormones were done in 10% fetal calf serum.

ELISA procedures for cellular content of VCAM-1 and E-selectin A modified ELISA procedure was used to measure the cellular Eselectin and VCAM-1 content (23). Cells were grown as indicated above and washed once with 150 ␮l PBS, fixed in 150 ␮l of 100% methanol for 10 min, air dried, and stored at 4 C. Dried cells were rehydrated and blocked in 150 ␮l PBS, 0.1% Tween 20, 0.5% BSA (P⫹T⫹A) for 30 min and washed twice in P⫹T. The wells were then incubated for 2 h at room temperature with either a monoclonal antibody against human Eselectin (BBA-16, R&D Systems) diluted 1/500 in P⫹T⫹A or a polyclonal goat antibody against human VCAM-1 (BBA-19, R&D Systems) diluted 1/500 in P⫹T⫹A. After two washes in P⫹T, wells were incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies diluted in P⫹T⫹A: Rabbit antimouse Ig-HRP (NA9310, Amersham Life Sciences, Arlington Heights, IL) 1/4000 for E-selectin measurements and rabbit antigoat-Ig-HRP (P0160, Dako A/S, Copenhagen, Denmark) 1/4000 for VCAM-1 analysis. After 1 h of incubation at room temperature, wells were washed five times in P⫹T, and they were subsequently stained using 100 ␮l TMB-reagent (Dako, S 1600) as substrate for the bound HRP. After 5 min of incubation, the reaction was stopped by adding 100 ␮l of 3 m H2SO4. Absorbance was read at 540 nm in an ELISA reader.

Statistical methods Statistical calculations were done with SPSS for Windows version 11.0 (SPSS, Chicago, IL). The paired-samples t test or one-way ANOVA was used to evaluate the differences within or between groups. The unpaired t test was used for between-groups comparisons of changes during GH

FIG. 1. Baseline serum concentrations of VCAM-1, E-selectin, and CRP in healthy subjects and GHD patients. Results are expressed as mean ⫾ SE, and P values refer to between-group comparisons.

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treatment vs. changes during placebo (⌬-values). Pearson product moment correlation or Spearman correlation were used to examine the relationship among different variables at baseline and after treatment. P ⬍ 0.05 was considered significant. All results are expressed as mean ⫾ se.

Results Serum concentrations of IGF-I

Baseline concentrations of IGF-I were significantly lower among GHD patients than healthy subjects (112 ⫾ 59 ␮g/liter vs. 320 ⫾ 19 ␮g/liter, P ⬍ 0.0001). During treatment of GHD patients, IGF-I levels remained stable in the placebo group (110 ⫾ 17 ␮g/liter vs. 103 ⫾ 18 ␮g/liter, NS), but increased significantly in the GH group (115 ⫾ 18 ␮g/liter vs. 300 ⫾ 27

␮g/liter, P ⬍ 0.0001) (Table 1). Likewise, IGF-I concentrations were unaltered by placebo treatment in healthy subjects (279 ⫾ 17 ␮g/liter vs. 299 ⫾ 19 ␮g/liter, NS), and increased equally in both GH treatment groups (374 ⫾ 41 ␮g/liter vs. 769 ⫾ 79 ␮g/liter, P ⬍ 0.0001; and 301 ⫾ 22 ␮g/liter vs. 766 ⫾ 100 ␮g/liter, P ⬍ 0.001, respectively). Data from the two GH treatment groups in healthy subject were subsequently analyzed together (Table 1). Serum concentrations of soluble VCAM-1, E-selectin, and CRP in GHD patients and healthy subjects

As seen in Fig. 1, baseline concentrations of VCAM-1 were significantly lower in GHD patients, compared with healthy

TABLE 1. Absolute changes in VCAM-1, E-selectin, and CRP levels from baseline during GH treatment of healthy subjects and GHD patients Healthy subjects (change from baseline)

VCAM-1 (␮g/liter) E-selectin (␮g/liter) CRP (mg/liter) IGF-I (␮g/liter)

Placebo (n ⫽ 10)

GH (n ⫽ 15)

Net difference between groups (95% CI)

17.8 ⫺5.3 ⫺0.24 19.8

50.1 6.5 ⫺0.55 419.1

32.3 (⫺45.1 to 109.8) 11.8 (2.5 to 21) ⫺0.31 (⫺1.77 to 1.14) 399.3 (274.3 to 524.2)

GHD patients (change from baseline) P

Placebo (n ⫽ 13)

GH (n ⫽ 12)

Net difference between groups (95% CI)

P

NS ⬍0.05 NS ⬍0.0001

⫺54.4 ⫺4.6 0.63 ⫺6.3

97.4 2.5 ⫺1.58 184.8

151.8 (95.0 to 208.7) 7.2 (⫺4.5 to 18.9) ⫺2.2 (⫺4.91 to 0.50) 191.1 (143.9 to 238.1)

⬍0.0001 NS 0.10 ⬍0.0001

P values refer to changes during GH treatment vs. changes during placebo treatment. NS, Not significant.

FIG. 2. Changes in serum concentrations of soluble VCAM-1 during treatment with GH or placebo in healthy subjects (A) or GHD patients (B). Open circles represent mean values ⫾ SE before and after treatment. *, P ⬍ 0.01, and **, P ⬍ 0.001, compared with baseline for within-group comparisons. The indicated P values refer to between-group comparisons of changes during GH treatment vs. changes during placebo (⌬-values). The bottom panels depict correlations between the observed changes in VCAM-1 and IGF-I in healthy subjects (C) and GHD patients (D).

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individuals (362 ⫾ 15 ␮g/liter vs. 516 ⫾ 21 ␮g/liter, P ⬍ 0.001). No differences were observed in the concentrations of E-selectin (60.2 ⫾ 4.8 ␮g/liter vs. 55.4 ⫾ 5.4 ␮g/liter, NS), whereas CRP concentrations were significantly higher at baseline in GHD patients (3.51 ⫾ 0.67 mg/liter vs. 1.57 ⫾ 0.38 mg/liter, P ⫽ 0.015) (Fig. 1). There were no significant correlations between baseline levels of VCAM-1, E-selectin, CRP, and IGF-I in either GHD patients or healthy subjects. In GHD patients GH treatment was associated with a highly significant increase in VCAM-1 concentrations, compared with placebo, and there was a strong positive correlation between the observed changes in VCAM-1 and IGF-I (Table 1, Fig. 2). GH treatment did not significantly increase serum concentrations of VCAM-1 in healthy subjects, and there was no correlation between the changes in VCAM-1 and IGF-I (Table 1, Fig. 2). E-selectin concentrations increased during GH treatment in healthy subjects, but there was no significant correlation between the changes in Eselectin and IGF-I (Table 1, Fig. 3). In GHD patients E-selectin levels were unaffected by GH treatment (Table 1, Fig. 3). There were no significant changes in CRP levels during GH treatment in either healthy subjects or GHD patients, al-

Hansen et al. • GH Stimulates VCAM-1 Expression

though CRP concentrations tended to decrease in GHD patients (P ⫽ 0.10; Table 1). There were no significant correlations between baseline or posttreatment levels of VCAM-1, E-selectin, and CRP in either GHD patients or healthy subjects, and there were no significant correlations between the observed changes in VCAM-1, E-selectin, and CRP concentrations. In vitro effects of GH and IGF-I on VCAM-1 and E-selectin expression in human endothelial cells

When GH or IGF-I was added at concentrations of 5 or 50 ␮g/liter to cultured HUVECs for 6 or 24 h, no alterations were seen in cellular expression of VCAM-1 as depicted in Fig. 4. We have previously demonstrated that the expression of VCAM-1 is up-regulated by incubation with TNF␣ (10, 24), which was confirmed in the present study. However, when cells were incubated with GH and IGF-I in conjunction with TNF␣ for 6 or 24 h, no further increase in VCAM-1 expression was observed (Fig. 4). Likewise, no differences were seen after GH or IGF-I incubation either with or without concomitant TNF␣ stimulation when cellular E-selectin content was considered (Fig. 5).

FIG. 3. Changes in serum concentrations of soluble E-selectin during treatment with GH or placebo in healthy subjects (A) or GHD patients (B). Open circles represent mean values ⫾ SE before and after treatment; *, P ⬍ 0.05, compared with baseline for within-group comparisons. The indicated P values refer to between-group comparisons of changes during GH treatment vs. changes during placebo (⌬-values). The bottom panels depict correlations between the observed changes in E-selectin and IGF-I in healthy subjects (C) and GHD patients (D).

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FIG. 4. Effects of GH and IGF-I on VCAM-1 expression in human endothelial cells cultured in vitro. Cells were incubated for 6 or 24 h in the presence of GH or IGF-I (5 and 50 ␮g/liter) with or without the addition of TNF␣ for the last 6 h. Results are expressed as mean ⫾ SE of six cultures.

FIG. 5. Effects of GH and IGF-I on E-selectin expression in human endothelial cells cultured in vitro. Cells were incubated for 6 or 24 h in the presence of GH or IGF-I (5 and 50 ␮g/liter) with or without the addition of TNF␣ for the last 6 h. Results are expressed as mean ⫾ SE of six cultures.

In vitro effects of serum from GH-treated healthy adults on VCAM-1 and E-selectin expression in human endothelial cells

To investigate a possible indirect effect of GH treatment mediated through one or more circulating factors, HUVECs were incubated with serum from each healthy subjecttreated with GH or placebo. The average expression of VCAM-1 was significantly increased when endothelial cells were incubated with sera from GH-treated subjects, compared with sera from placebo-treated subjects (Fig. 6A). By contrast, there were no significant differences between the two treatment groups on the effects of on cellular E-selectin content (Fig. 6B).

Discussion

The present study is the first to demonstrate decreased levels of soluble VCAM-1 in GHD patients. It should be emphasized, though, that our patients and the healthy controls were not matched for known cardiovascular risk factors, such as body mass index and smoking habits. The GHD patients were slightly overweight, which is known to be associated with increased levels of VCAM-1 (25). In our study, this would, however, tend to give higher VCAM-1 levels in GHD and not lower, as observed. The finding of decreased VCAM-1 values in GHD is strongly supported by the normalization of VCAM-1 after 4 months of GH treatment in combination with the correlation between VCAM-1

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Hansen et al. • GH Stimulates VCAM-1 Expression

FIG. 6. Expression of VCAM-1 (A) and E-selectin (B) in cultured human endothelial cells after incubation for 24 h with serum from healthy subjects at baseline (black columns) and after treatment with placebo or GH for 4 wk (gray columns). Columns indicate the average value (⫾ SE) of individual sera analyzed in quadruple. The indicated P values refer to between-group comparisons of changes during GH treatment vs. changes during placebo (⌬-values).

and IGF-I changes. No differences were observed in GHtreated healthy subjects, maybe because GH is only capable of increasing VCAM-1 to a certain threshold, beyond which there is no further effect of adding GH. The finding of low but GH-sensitive VCAM-1 values in GHD is surprising because the cardiovascular mortality is increased in these patients (26, 27), and it has been suggested that GH replacement therapy may reduce the risk of cardiovascular events in GHD (28). Moreover, CRP levels are increased in GHD patients (1), indicating chronic low-grade inflammation and furthermore increased levels of endothelial dysfunction markers have also been observed. In this context and with the knowledge that VCAM-1 is a marker of endothelial dysfunction and cardiovascular diseases, increased VCAM-1 levels were to be expected in GHD patients. Remarkably, our data show the opposite, indicating that GH may play a special role in the regulation of VCAM-1, leading to lower levels in the case of GH deficiency. In contrast to VCAM-1, we did not find differences concerning serum levels of E-selectin in GHD. In a recent study, E-selectin levels were found to be higher in GHD patients than healthy controls (28). The baseline E-selectin concentrations among GHD patients in our study was in the same range as found in the study by Elhadd et al. (28) but not significantly higher than in our healthy subjects, which could be attributable to the smaller sample size in our study. Eselectin was increased slightly in healthy subjects treated with GH, compared with placebo, whereas there was no significant difference between the two treatment groups in GHD patients. It has previously been reported in an uncontrolled study that GH treatment reduces soluble E-selectin levels in GHD patients (29), but in that particular study, baseline concentrations of E-selectin were considerably higher than in our study (mean concentration 72.4 ⫾ 11 ␮g/liter, n ⫽ 11). Another aspect in which E-selectin and VCAM-1 results differ in our study concerns the fact that we were not able to detect any effects of GH treatment on serum E-selectin in GHD but observed a small effect in healthy

persons. VCAM-1 and E-selectin are often viewed as very similar markers of endothelial dysfunction. It has, however, been shown, that in some clinical settings, they do not always change in parallel. The time-dependent pattern of VCAM-1 and E-selectin is for example very different after infusion of TNF␣ (30). Interestingly, it has also been shown that there are large differences in the regulation of the two molecules in vitro. Although both molecules are induced by proinflammatory peptides, the treatment with, for example, statins leads to highly different expression patterns due to different half-lives of the molecules on the cell surface (12). The present observations support the concept that the regulation of VCAM-1 and E-selectin differs and seem to indicate that GH or IGF-I may exert regulatory effects on the turnover of the molecules. The expression of adhesion molecules on the endothelial surface can be mimicked in cultured endothelial cells as in our in vitro set-up. We observed no direct stimulatory effect of either GH or IGF-I on the expression of VCAM-1 and E-selectin after incubation for both 6 and 24 h The level of expression can be strongly up-regulated by several proinflammatory factors including TNF␣ (12, 31). Because it is possible to influence the cellular susceptibility to TNF␣, we furthermore tested whether GH or IGF-I added for 6 or 24 h would influence TNF␣-induced expression of the adhesion molecules. We could, however, not demonstrate any effects of GH and IGF-I. A recent paper has dealt with this issue (32). Similar to our data, these authors could not demonstrate any direct effect of IGF-I on expression of VCAM-1 and E-selectin. However, in contrast to our findings, they found that IGF-I added to endothelial cells for 24 h indirectly enhanced TNF␣-induced expression of VCAM-1 and E-selectin. The experiments were done in human endothelial cells, as in the present study, but a considerably higher TNF concentration was used (5 ng/ml vs. 0.1 ng/ml; normal plasma concentration 1–10 pg/ml). In our in vitro experiments, we found no direct stimulatory effect of GH or IGF-I on either basal or TNF␣-induced

Hansen et al. • GH Stimulates VCAM-1 Expression

VCAM-1 expression on HUVECs. By contrast, serum from GH-treated healthy subjects increased VCAM-1 expression in vitro, compared with serum from placebo-treated subjects. This observation is compatible with the hypothesis, that the effects of GH could be mediated indirectly via one or more circulating factors. Our in vitro findings show that VCAM-1 expression can be induced by TNF␣, and proinflammatory cytokines are pertinent candidates for the link between GH and VCAM-1 expression. So far, the reported effects of GH on the synthesis of cytokines have varied, depending on the experimental conditions. In macrophages from hypophysectomized rats, treatment with GH increased endotoxininduced synthesis of TNF␣ (24), whereas treatment with GH decreased plasma concentrations of IL-1, TNF␣, and IL-6 in mice (33) and reduced the cytokine response to endotoxin in calves (34). In vitro treatment of human mononuclear cells with GH inhibited endotoxin-induced production of IL-1 and TNF␣ (35), whereas the proinflammatory cytokine response to endotoxin or surgery in humans was unaffected by highdose GH therapy (36). CRP has been shown to directly increase the expression of adhesion molecules on endothelial cells in vitro (37), and this as well as other acute phase proteins could therefore also represent the link between GH and VCAM-1 expression. We have previously demonstrated that the effects of GH administration on different acute phase proteins are highly diverse and include reductions in CRP and haptoglobin levels and an IGF-I-independent increase in mannose-binding lectin levels (38). It is thus unlikely that the effects of GH are mediated via CRP, whereas the direct effects of mannose-binding lectin on endothelial cells should be further elucidated. A putative physiological role for GH-dependable upregulation of VCAM-1 may relate to situations with leukocyte extravasation, which is an important element in, for example, the atherogenic process. Low levels of VCAM-1 in atherosclerosis-prone GHD patients are therefore, as previously mentioned, unexpected and seem to indicate that VCAM-1 is not causally linked to increased cardiovascular mortality in GHD. Induced VCAM-1 due to increased GHlevels could, however, play a role for the development of arterial disease in acromegaly and diabetes (5). Another setting, in which GH up-regulated VCAM-1 may play a role is in sepsis and MOF. Tissue damage caused by leukocyte infiltration is an important element in the pathophysiology of sepsis and MOF (7, 8), and blockade of VCAM-1 with monoclonal antibodies reduces the endotoxin-induced organ damage (16, 17). Critical ill patients are characterized by reduced GH sensitivity and low IGF-I levels, and even though the present study does not involve samples from GH-treated critically ill patients, our findings raise the possibility that the detrimental effects of high-dose GH therapy in critically ill patients may involve augmented VCAM-1 expression and increased leukocyte-induced tissue damage. In conclusion, we have shown that GH administration increases circulating soluble VCAM-1 concentrations in GHD subjects in vivo, a finding that was supported by our in vitro demonstration of a stimulatory effect of serum from GH-treated subjects on VCAM-1 expression on HUVEC cells. Although this proinflammatory effect of GH on the endothelium merits further investigation, it could be hypothe-

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sized that the detrimental effects of GH in critically ill patients might involve aggravated leukocyte-induced tissue damage through increased VCAM-1 expression. Acknowledgments We thank Novo Nordisk A/S (Copenhagen, Denmark), which generously supplied the GH. Received February 11, 2003. Accepted October 27, 2003. Address all correspondence and requests for reprints to: Troels Krarup Hansen, M.D., Ph.D., Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital, Norrebrogade 42-44, DK-8000 Aarhus C, Denmark. E-mail: [email protected]. This work was supported by grants from the Danish Research Council (Novo Nordisk Center for Research in Growth and Regeneration, Aarhus University, Grant 960082) and the Danish Heart Foundation.

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