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Growth Hormone Treatment Improves Markers of Systemic Nitric Oxide Bioavailability via Insulin-Like Growth Factor-I

Universitätsklinikum (T.T., F.F., J.B.), Medizinische Klinik I (Kardiologie), 97080 Würzburg, Germany; Universitätsklinikum (T.T., M.J.), Interdisziplinäres Zentrum für Klinische Forschung, Nachwuchsgruppe Cardiac Wounding and Healing, 97080 Würzburg, Germany; and Medizinische Hochschule Hannover (I.K., D.T., D.O.S.), Institut für Klinische Pharmakologie, D-30625 Hannover, Germany

Address all correspondence and requests for reprints to: Dr. med. Thomas Thum, Medizinische Klinik und Poliklinik I, Kardiologie, Universitätsklinikum, Bayerische Julius-Maximilians-Universität, Josef-Schneider Str. 2, 97080 Würzburg, Germany. E-mail: thum_t{at}klinik.uni-wuerzburg.de.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context and Objective: Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I.

Design, Patients, and Results: Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment, we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P < 0.05). Likewise, plasma nitrate and nitrite levels were increased, whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly, IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P < 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly, blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability.

Conclusions: GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus, GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GH-DEFICIENT PATIENTS display increased peripheral vascular resistance and have a higher risk of death due to cardiovascular diseases (1, 2, 3). Treatment of GH-deficient patients with recombinant human GH reduces peripheral resistance, enhances cardiac output and reduces plasma cholesterol concentrations (2, 3). This obvious relation between GH and cardiovascular biology may be explained at least in part by subsequent activation of IGF-I and the endothelial nitric oxide synthase (eNOS) (2). In contrast, patients with low IGF-I levels have an increased risk of ischemic heart disease, whereas GH therapy may restore physiological nitric oxide (NO) levels, thus contributing to a beneficial cardiovascular outcome (4).

Vascular lesions are partly repaired by endogenous endothelial progenitor cells (EPCs) via NO-dependent mechanisms (5, 6). Enhanced eNOS activity within bone marrow is correlated with increased levels of circulating EPCs (7). EPCs are reduced in patients with coronary artery disease (8, 9) or progressive heart failure (10). EPC levels also serve as a strong predictor for future cardiovascular events, including myocardial infarction and death (11). We recently have shown a stimulatory effect of GH on circulating EPC levels (12), whereas the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) impaired EPCs in patients with coronary artery disease (8). This supports the assumption that systemic NO may influence number of circulating EPCs.

Here we hypothesized that the effect of GH supplementation on EPC in middle-aged healthy volunteers was mediated by improved NO bioavailability. Additional animal and cell culture experiments were performed to clarify whether enhanced NO bioavailability was mediated directly via GH or GH-mediated IGF-I increase.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Experimental subjects

The ethical committee of the Hannover Medical School approved the study. The clinical trial has been registered at www.clinicaltrials.gov (NCT00470002). Written informed consent was obtained from the volunteers. The clinical study was designed as a single-center, nonrandomized, uncontrolled pilot study. Sixteen middle-aged healthy volunteers were enrolled. Inclusion and exclusion criteria are given in Table 1. Treatment dose was 0.4 mg recombinant human GH (Genotropin; Pharmacia and Upjohn, Karlsruhe, Germany) per day, lasting from d 1 to d 10. Each injection was performed at 4 p.m. by study assistance.

Measurement of clinical parameters

Nitrate and nitrite. Nitrate and nitrite in plasma and urine were measured by gas chromatography-tandem mass spectrometry (GC-MS/MS) with use of stable isotope-labeled internal standards as described previously (13). Urinary nitrate and nitrite concentrations were related to urine creatinine levels. To ensure that the urinary nitrate and nitrite levels were not influenced by high dietary nitrate/nitrite intake, volunteers were instructed to avoid food containing high amounts of nitrate/nitrite.

Determination of IGF-I and IGFBP-3 levels. IGF-I levels were measured using a chemiluminescence immune assay and quantification was performed using Nichols Advantage Specialty system (Nichols Institute Diagnostics, San Clemente, CA) as described previously (12). IGFBP-3 measurement was performed using a RIA assay (Mediagnost, Reutlingen, Germany). As described by the manufacturer, a highly affine, polyclonal antibody specific to IGFBP-3 was used. There was no known cross-reactivity with either IGFBP-1 or IGFBP-2.

Determination of 8-iso-prostaglandin F2 (8-iso-PGF₂). 8-Iso-PGF₂ levels were quantified by a previously described fully validated GC-MS/MS method. Urinary excretion rates of 8-iso-PGF₂ were corrected for creatinine, which was determined spectrophotometrically by the alkaline picric acid reaction with an automatic analyzer (Beckmann 6641, Galway, Ireland). Samples were spiked with [²H₄]8-iso-PGF₂ (Cayman, Ann Arbor, MI) as an internal standard and acidified to pH 3.5. Analytes were solid-phase extracted on octadecylsilica cartridges and eluted from the columns with ethyl acetate. After solvent evaporation, the pentafluorobenzyl esters were prepared, subsequently separated by thin-layer chromatography, further converted to their trimethylsilyl ether derivatives, and analyzed by GC-MS/MS in the selected reaction mode exactly as described elsewhere (14).

Determination of cGMP levels. To quantify urinary cGMP levels, an ELISA (Sunrise; Tecan, Mannedorf, Switzerland) was used. A known quantity of peroxidase-labeled cGMP was added to the probe. A cGMP-specific antibody was immobilized by a secondary antibody to a microtiter plate. After binding of the free cGMP to the plate, the remaining free cGMP was washed off and the bound cGMP/peroxidase-labeled cGMP could be detected by adding 3,3', 5,5'-tetramethylbenzene. The reaction product was quantified by photometry at 450 nm in an ELISA reader. In case of the mouse study, cGMP was measured using a cGMP enzyme immunoassay (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions.

Determination of ADMA and dimethylamine (DMA) levels. In case of the human studies, measurement of ADMA was performed by a combined gas chromatograph/mass spectrometer-based method (8). ADMA plasma levels of mice were determined by a commercial available ADMA-ELISA kit (DLD Diagnostika GmbH, Hamburg, Germany). DMA levels were analyzed as described previously (15).

Determination of basic blood parameters and VEGF. Basic blood parameters were accessed by standard laboratory measurements (Hannover Medical School, Hannover, Germany) and VEGF levels were measured by a commercial ELISA kit (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s recommendation.

Determination of endothelial progenitor cells

We isolated and quantified both CD133⁺/VEGFR2⁺ EPCs and endothelial colony-forming units (CFU). CFU assay was essentially done as described (9, 12). Briefly, peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and 1 x 10⁶ cells were plated on fibronectin-coated 12-well plates in EndoCult medium (StemCell Technologies, St. Katharinen, Germany). To exclude mature endothelial cells, nonadherent cells were collected after 48 h and plated in replicate fibronectin-coated 24-well plates. Colonies were evaluated and quantified 3 d later. A colony was defined as a central core of round cells with more elongated sprouting cells at the periphery and is referred to as early outgrowth CFU-endothelial cell. The endothelial lineage of these cells has been confirmed previously by immunocytochemical staining for von Willebrand factor, vascular endothelial growth factor (VEGF) receptor 2, and CD3 (9). CD133⁺/VEGFR2⁺ EPCs were measured in eight patients before and after GH supplementation and analyzed by fluorescence-activated cell sorter analysis as described (12).

Human endothelial cell culture experiments and expression of dimethylarginine dimethylaminohydrolase (DDAH)-1/2 in response to IGF-I

Human umbilical vein endothelial cells (HUVECs) and human coronary arterial endothelial cells (HCAECs) were cultured until subconfluence as described (16) and then treated with IGF-I (100 nM) for 24 h. Timing and concentrations were based on a previous study (12). Untreated cells served as controls. Total RNA isolation, RT-PCR experiments, and data analysis were performed as described (12). PCR experiments were done within the log-linear range of amplification. DDAH1/2 primer sequence information were obtained from Monsalve et al. (17).

Experimental animals

The animal studies conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (publication no. 85-23, revised 1996).

To test whether GH directly or via increase of IGF-I alters markers for systemic NO bioavailability, we treated male mice (6–8 months old, Harlan-Winkelmann, Borchen, Germany) ip with placebo (150 µl PBS; n = 7), GH (Sigma-Aldrich, Munich, Germany; 2.5 µg GH per gram per day dissolved in 150 µl PBS once per day for 2 or 7 d; each n = 5) or mouse IGF-I (Sigma-Aldrich; 1.5 µg mouse IGF-I per gram body weight dissolved in 150 µl PBS three times per day for 2 d; n = 5) (18, 19). We also systemically blocked the IGF-I receptor using a diaryl urea compound [N-(2-methoxy-5-chlorophenyl)-N'-(2-methylquinolin-4-yl)-urea; IGF-IR Inhibitor II, catalog no. 407248, Calbiochem, Darmstadt, Germany; 100 mg/kg every second day for 7 d; n = 4], which recently was described to be a selective and highly potent inhibitor of the IGF-I receptor by blocking autophosphorylation (in both human cell lines and mice in vivo studies; see Ref. 20). After treatment we determined plasma cGMP and ADMA as markers for systemic NO availability. eNOS protein expression was investigated by Western blotting experiments of total protein lysates of aortic tissue of treated mice as described (7).

Statistical methods

Data are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA or paired t test followed by multiple comparisons using Fisher’s protected least significant difference test. To analyze relationships between variables, simple and multivariate regression analyses were performed. Statistical analysis was performed using StatView 5.0 statistic program (Abacus Concepts, Berkley, CA). Statistical significance was assumed at P < 0.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Clinical study

Alterations of basic cardiovascular parameters. All volunteers completed the study in accordance with the study protocol, and no serious adverse events were observed. Diastolic blood pressure was moderately but significantly reduced (86.7 ± 1.7 vs. 83.3 ± 1.67 mm Hg, P < 0.05), whereas systolic blood pressure remained basically unchanged (133.3 ± 3.7 vs. 131.0 ± 2.9 mm Hg, P = 0.2). No change of heart rate (63.2 ± 1.9 vs. 65.3 ± 2.1 beats per minute, P = 0.2) could be detected.

GH-mediated changes in basic blood parameters. A 10-d supplementation of healthy middle-aged volunteers with GH significantly raised IGF-I (126 ± 8.0 vs. 231.3 ± 9.59 ng/ml, P < 0.0001) as well as IGFBP-3 plasma levels (2.49 ± 0.30 vs. 3.12 ± 0.12 mg/liter, P < 0.005) (Fig. 1, A and B). No significant changes were found in total cholesterol, HDL-cholesterol, LDL-cholesterol, or triglyceride plasma levels. Likewise, hemoglobin, erythrocyte, leukocyte, and homocysteine levels remained unaffected (Table 2).

View larger version (20K): [in this window] [in a new window]   FIG. 1. IGF-I (A), IGFBP-3 (B), urinary cGMP/creatinine levels (C), and correlations of IGF-I and urinary cGMP/creatinine (D) as well as plasma NOx levels with diastolic blood pressure (E) in healthy middle-aged volunteers before and after a 10-d treatment with recombinant human GH (daily 0.4 mg).

2

2

View larger version (12K): [in this window] [in a new window]   FIG. 2. Simple regression analysis between IGF-I plasma levels and endothelial CFUs (A) or circulating CD133+/VEGFR2+ cells (B) in healthy middle-aged volunteers. C, Correlation between plasma VEGF levels and endothelial CFUs.

cGMP levels were increased after a 7-d treatment (1479 ± 111 vs. 2343 ± 407 fmol/well; P < 0.05) or after treatment with IGF-I (2182 ± 315 fmol/well; P < 0.05). Aortic eNOS expression was significantly increased by 30% after GH (7 d) treatment (see Fig. 4). In contrast, ADMA levels were decreased after GH treatment for 7 d (0.89 ± 0.03 vs. 0.76 ± 0.03 µmol/liter; P < 0.05) as well as after IGF-I treatment (0.76 ± 0.01 µmol/liter; P < 0.05). In contrast, short GH treatment for only 2 d did not alter cGMP or ADMA plasma levels. Blocking of IGF-I/IGF1 receptor signaling cascade in vivo by a specific small molecule inhibitor of the IGF-I receptor completely abolished the effects of GH treatment on the latter markers of NO bioavailability (see Figs. 3, A and B, and 4). Treatment of human HCAECs with IGF-I for 24 h resulted in a significant increase in DDAH1 and DDAH2 expression (see Fig. 5), whereas HUVECs were only partly responsive to IGF-I treatment.

View larger version (19K): [in this window] [in a new window]   FIG. 4. A, Protein expression of eNOS in aortae of mice treated with placebo, GH (7 d), or GH and an IGF-I receptor (IGF-IR) inhibitor (n = 5–6 per group). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Markers of NO bioavailability in mice treated with GH (2 or 7 d), GH and an IGF-I receptor inhibitor (7 d), or IGF-I alone. cGMP levels (A) and ADMA levels (B) after different treatments are shown. Correlation of circulating EPCs (sca-1+/flk-1+ cells) with plasma levels of cGMP (C) and ADMA (D) is shown.

View larger version (19K): [in this window] [in a new window]   FIG. 5. DDAH1, DDAH2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in cultured HUVECs and HCAECs treated with IGF-I (100 nM, 24 h; each n = 4).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This study provides evidence that IGF-I mediates the increase in systemic NO bioavailability induced by GH supplementation in vivo. In healthy middle-aged volunteers, a short supplementation period of 10 d with GH was sufficient to modulate several markers for increased systemic NO levels, e.g. elevation of the major NO downstream messenger cGMP and decrease of the endogenous NO synthase (NOS) inhibitor ADMA. IGF-I levels were correlated with cGMP levels pointing to a causal relationship. This was proved in an animal model using IGF-I receptor blockade, suggesting that IGF-I is mainly responsible for the regulation of NO bioavailability by GH treatment in vivo.

Evidence for IGF-I to regulate NOS activity stems from various in vitro studies (21, 22).

IGF-I interacts with a tyrosine kinase membrane receptor that activates the phosphoinositide 3-kinase and serine/threonine kinase Akt signaling pathway (21, 22), facilitating eNOS expression and activity (23). Clinical studies have demonstrated that GH-deficient patients display endothelial dysfunction and are at increased cardiovascular risk (1, 2, 3). GH supplementation of GH-deficient patients resulted in significant reductions in total peripheral resistance via increased NO bioavailability (2, 3, 24). Likewise, patients with chronic heart failure are characterized by impaired NO-dependent vasodilation, and GH substitution significantly improved NO production and vascular dysfunction (25). It was assumed that the improved NO bioavailability in response to GH is mediated by IGF-I. However, no direct proof has so far been put forward. We here provide evidence that in vivo IGF-I is the major stimulus for an increase in NO availability after GH supplementation for the following reasons; first, IGF-I and cGMP levels significantly correlated in volunteers after GH treatment; second, short-term treatment of mice with GH for only 2 d was not sufficient to increase IGF-I levels nor altered markers for NO. In contrast, a longer GH treatment period of 7 d significantly raised IGF-I and cGMP levels, increased aortic eNOS protein expression, and decreased the circulating endogenous NOS inhibitor ADMA. Third, direct treatment of animals with recombinant IGF-I improved markers of NO availability. Lastly and most importantly, blocking the IGF-I/IGF-I receptor signaling cascade by a small molecule inhibitor of the IGF-I receptor (20) completely prevented the GH-mediated changes in cGMP and ADMA plasma levels as well as aortic eNOS expression.

The alterations in systemic NO availability likely influence EPC biology. EPCs contribute to adult vessel formation, and injection of EPC improved vascular function, blood flow in ischemic tissue, and heart function (26, 27, 28). NO not only plays a major role in the release of EPCs from bone marrow but also improves EPC function and migration capacity (5, 7, 12, 29). Likewise, the VEGF is partly involved during the process of EPC mobilization, recruitment, and overall neovascularization (5). Interestingly in our study, both markers of NO availability and VEGF plasma levels were correlated with the amount of circulating EPCs.

In contrast, the endogenous NOS inhibitor ADMA has negative effects on number, function, and migration capacity of EPCs (8). ADMA is a strong and independent predictor of mortality and cardiovascular outcome and is correlated with endothelial dysfunction in systemic cardiovascular diseases (30, 31, 32). In the vascular system, ADMA is produced by protein arginine methyltransferases and metabolized by DDAH1 and DDAH2 (33, 34). We here have shown reduction of DDAH1 and DDAH2 expression, especially in arterial endothelial cells treated with IGF-I. Supplementation of both healthy volunteers and mice with GH resulted in modest but significant reductions of plasma ADMA levels, probably due to the IGF-I-mediated induction in endothelial DDAH1/2 expression. The overall improved NO bioavailability may contribute to the rise in EPC levels after GH therapy as shown recently (12). A positive effect on circulating EPC levels by GH supplementation could be of advantage in the treatment of cardiovascular diseases with impaired number of EPCs, such as coronary artery disease (8, 9). This may particularly apply for GH-deficient patients due to advanced age or other primary or secondary causes.

Although the correlations between systemic NO markers and EPC levels do not ultimately prove a direct cause/effect relationship, they are suggestive for that. Our findings also indicate that circulating EPC levels may be a useful biosensor for the general NO bioavailability in an individual. Indeed, clinical and animal data have shown that drugs that improve NO bioavailability such as statins also increase circulating EPC levels (7, 29, 35), whereas patients with high amounts of NOS-inhibiting ADMA and low NO bioavailability display diminished circulating EPC numbers (8). Further studies should test the potential diagnostic and prognostic use of EPC number and function as a surrogate marker for overall NO bioavailability.

In conclusion, GH therapy improves markers of NO bioavailability and circulating EPC levels via IGF-I. Our initial pilot results warrant further and larger placebo-controlled studies that examine the potential vasoprotective effects of GH and/or IGF-I as well as clinical outcome of treated patients with endothelial dysfunction and/or atherosclerosis including coronary artery disease.

	Acknowledgments

 
The authors thank Annette Horn and Sabrina Thum (University of Würzburg) as well as Bibiana Beckmann, Maria-Theresia Suchy, Frank-Mathias Gutzki, and Dagmar Becker (Medical School Hannover) for technical assistance. The advice of Professor E.-G. Brabant, M.D. (Department of Endocrinology, Medizinische Hochschule Hannover) is acknowledged.

	Footnotes

 
This work was supported by an institutional grant from Pharmacia (Karlsruhe, Germany; to D.O.S.) and grants from the Interdisziplinäres Zentrum für Klinische Forschung (IZKF)-Nachwuchsgruppe Cardiac Wounding and Healing (E-31; to T.T.).

Disclosure Statement: T.T., F.F., I.K., D.T., M.J., J.B., and D.O.S. have nothing to declare.

First Published Online August 28, 2007

Abbreviations: ADMA, Asymmetric dimethylarginine; CFU, colony-forming unit; DDAH, dimethylarginine dimethylaminohydrolase; DMA, dimethylamine; eNOS, endothelial nitric oxide (NO) synthase; EPC, endothelial progenitor cell; GC-MS/MS, gas chromatography-tandem mass spectrometry; HCAEC, human coronary arterial endothelial cell; HDL, high-density lipoprotein; HUVEC, human umbilical vein endothelial cell; IGFBP, IGF binding protein; 8-iso-PGF₂,8-iso-prostaglandin F2; LDL, low-density lipoprotein; NOx, nitrate and nitrite; VEGF, vascular endothelial growth factor.

Received May 7, 2007.

Accepted August 17, 2007.

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