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Acute Effects of Growth Hormone on Vascular Function in Human Subjects

Department of Internal Medicine and Cardiovascular Sciences, University Federico II, 80131 Naples, Italy

Address all correspondence and requests for reprints to: Luigi Saccà, M.D., Department of Internal Medicine, Via Pansini 5, 80131 Naples, Italy. E-mail: sacca{at}unina.it.

	Abstract

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GH is involved in the long-term regulation of peripheral vascular resistance and vascular reactivity. We determined whether GH plays a role in the acute regulation of vascular function in humans. The acute vascular effects of GH were studied in eight healthy subjects according to a double-blind, placebo-controlled design. Forearm blood flow (FBF), vascular resistance, and nitric oxide (NO) production were monitored during a 4-h infusion of GH into the brachial artery at a rate chosen to raise local GH to stress levels (40 ng/ml). During GH infusion, FBF rose 75% (P < 0.05), whereas forearm vascular resistance decreased comparably (P < 0.05). These changes were paralleled by augmented forearm release of NO (P < 0.02). GH heightened the response of FBF to the endothelium-dependent vasodilator acetylcholine (Ach; P < 0.02). With the highest Ach dose, FBF reached 30.4 ± 4.2 and 16.9 ± 3.1 ml/dl·min in the GH and placebo studies, respectively (P < 0.005). The slopes of the dose-response curves also differed markedly (0.45 ± 0.07 and 0.25 ± 0.05 ml/dl·min/µg in the GH and placebo studies, respectively; P < 0.01). GH caused an upward shift of the FBF response to the endothelium-independent vasodilator sodium nitroprusside (P < 0.01), but did not affect the slope of the dose-response curve. GH infusion did not cause any appreciable increment in the venous IGF-I concentration in the test arm. In conclusion, GH acutely lowers peripheral vascular resistance and stimulates endothelial function. These effects are mediated by activation of the NO pathway and appear to be independent of IGF-I.

	Introduction

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BESIDES CONTROLLING LINEAR growth and metabolism, GH and IGF-I also play roles in cardiovascular physiology (1). One of the most consistent findings is the altered peripheral vascular resistance (PVR) in pituitary diseases associated with abnormal GH secretion. In particular, PVR is decreased in patients with chronic GH excess, whereas it is markedly augmented in patients with chronic lack of GH (2, 3).

A retrospective clinical study of patients with hypopituitarism showed that the chronic lack of GH is associated with doubled cardiovascular mortality, mainly due to myocardial infarction and stroke (4). GH-deficient patients present with a cluster of vascular risk factors, including abnormal lipid profile, abdominal obesity, insulin resistance, and increased intima media thickness of the large arteries (5). In addition, vascular reactivity is impaired in patients with chronic GH deficiency (6). Overall, the data suggest that an intact GH activity serves an antiatherogenic function.

The mechanisms underlying the vascular effects of GH are poorly understood. It is not even known whether GH affects vascular reactivity in humans under physiological circumstances. In essence, our understanding of the vascular effects of GH is deduced from studies of patients with pituitary disorders. Thus, it remains unclear whether GH acts directly or through the intervention of indirect mechanisms activated by chronic pituitary disease. Another basic question not yet addressed is whether GH per se is able to induce acute changes in vascular function independent of the time-requiring activation of IGF-I production.

The present investigation was undertaken to determine whether GH exerts an acute action on vascular reactivity and the nitric oxide (NO) pathway in healthy human subjects. The model chosen was based on direct infusion of GH into the brachial artery and measurement of the forearm blood flow (FBF) responses to vasoactive agents by strain gauge plethysmography. With this approach, only the forearm vessels are exposed to elevated GH levels, and the intervention of systemic mechanisms is precluded.

	Subjects and Methods

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Subjects

The study was performed on eight healthy volunteers (six men and two women; 22 ± 1 yr of age; body mass index, 23 ± 1 kg/m²) according to a double-blind, placebo-controlled design. Each subject underwent two studies in random order, separated by a 4-wk interval. In one study the hemodynamic effects of GH infusion into the brachial artery were monitored for 240 min. Then the responses of FBF to vasoactive test substances were measured while keeping the infusion of GH ongoing. In the control study the protocol was the same, except that placebo was infused instead of GH. The dose of GH was 0.02 µg/kg·min. This was the theoretical dose necessary to raise GH concentration in the forearm circulation to the levels observed during stress (e.g. intense physical activity and hypoglycemia) (7, 8). Using a local infusion approach, the systemic GH concentration is expected to be marginally, or not at all, affected for two reasons. 1) FBF represents less than 1% of cardiac output. Thus, minimal amounts of GH are required to raise its concentration in the forearm. 2) Systemic accumulation of GH is minimized by its short half-life (20 min). GH (Humatrope) was provided by Eli Lilly \|[amp ]\| Co. (Florence, Italy). Written informed consent was obtained from each subject, and the study was approved by the ethics committee of University Federico II.

Experimental procedures

All subjects were studied in the morning after a 12- to 15-h overnight fast in a quiet room kept at 22–24 C, as previously described (6, 9). Briefly, a plastic cannula (20 gauge) was inserted into the brachial artery of the nondominant arm under local anesthesia and used for the infusion of test substances, the monitoring of arterial blood pressure and heart rate, and arterial blood sampling. In the same arm, a second plastic cannula was introduced into a large antecubital vein to obtain venous blood samples. FBF was measured by strain gauge plethysmography, with a calibrated mercury-in-SILASTIC (Dow Corning Corp., Midland, MI) strain gauge applied around the forearm and connected to a plethysmograph (Hokanson 045 EC4, P.M.S. Instruments, Berks, UK) associated with a McLab computer.

Each subject underwent the following stepwise infusions into the brachial artery: 1) acetylcholine (Ach) at rates of 15, 30, 45, and 60 µg/liter forearm·min to assess endothelial-mediated vasodilation; 2) sodium nitroprusside (NP), a direct NO donor, at rates of 1, 3, and 9 µg/liter·min to assess nonendothelial-mediated vasodilation; and 3) L-N-monomethylarginine, a competitive analog of L-arginine, at the rate of 1 mg/liter·min. Each dose of the test substances was infused for 5.5 min, and FBF was measured during the last 1.5 min of infusion. A 30-min wash-out period was allowed between each substance infusion. Blood samples were simultaneously taken from the arterial and venous cannulas in the basal state and during GH infusion to measure nitrate/nitrite concentrations. FBF was measured simultaneously in both arms (experimental and control) to ensure that no systemic effects occurred during the infusion of test agents. Each FBF value represents the mean of six consecutive measurements performed at 8-sec intervals.

To assess the maximal vasodilating capacity (peak flow), the hyperemic response to ischemia was studied. FBF was measured after 5 min of ischemia induced by inflating a sphygmomanometer cuff around the upper arm. Due to the continuous infusion of GH in the test arm, the peak flow was determined only in the control arm.

Analytical methods

GH, IGF-I, and insulin concentrations were measured by RIA. The nitrite concentration was measured in plasma samples in triplicate. Before assay, plasma was ultrafiltered through a 10-kDa molecular mass cut-off filter (Centricon 10, Millipore Corp., Bedford, MA). Total plasma nitrite and nitrate were measured using a colorimetric kit (Cayman Chemical Co., Ann Arbor, MI). Nitrate was converted to nitrite by nitrate reductase, and then nitrite was assayed by the standard Griess diazo reaction. The data are referred to as the nitrite concentration, but they reflect the sum of nitrate and nitrite concentrations.

Calculations

Forearm vascular resistance (FVR) was measured as the ratio of mean arterial blood pressure to FBF. The net forearm balance of nitrite was calculated by multiplying the plasma arterial-venous concentration difference by the plasma flow. A negative balance indicates substrate release, whereas a positive balance indicates uptake. The data for vascular reactivity were analyzed by two-way ANOVA for repeated measures (version 11.0, SPSS, Inc., Chicago, IL). Comparison between the slopes of the dose-response curves was performed by paired t test. Vascular reactivity data are expressed as absolute values of FBF. Results are presented as the mean ± SEM.

	Results

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The study was well tolerated by all subjects, without systemic reactions. In particular, systolic blood pressure (122 ± 4 mm Hg), diastolic blood pressure (57 ± 3), and heart rate (61 ± 3) did not show any change from their basal values.

Intra-arterial GH infusion raised the venous GH concentration in the ipsilateral forearm from 4.6 ± 2 to 40.2 ± 5 ng/ml. The systemic GH concentration, as measured in the control arm, was marginally affected by local GH infusion (4.6 ± 2, 7.2 ± 2, and 9.2 ± 1 ng/ml at baseline, 240 min, and the end of the infusion period, respectively; P = NS). In the placebo study the forearm venous GH concentration was 4.5 ± 2 ng/ml at baseline and remained unchanged until the end of the study period. The systemic insulin concentration decreased slightly throughout the experimental period in both studies (from 13 ± 4 to 9 ± 2 µU/ml and from 12 ± 3 to 7 ± 2 µU/ml in the GH and placebo studies, respectively).

To obtain an index of IGF-I production by the forearm tissues exposed to high GH levels, IGF-I was measured in the venous blood from the test arm. In the GH study the IGF-I concentration was 234 ± 53 ng/ml basally and remained unchanged until the end of the study (239 ± 43). Similarly, the IGF-I concentration was stable in the placebo study (226 ± 32 and 245 ± 24 ng/ml at baseline and at the end of study, respectively).

In the test arm FBF was comparable in the two studies at baseline (Table 1). During intra-arterial GH infusion, FBF remained stable during the first 2 h. Thereafter, it started to rise and reached values significantly higher than those in the placebo study at 210 and 240 min. As arterial blood pressure remained unchanged (data not shown), the acceleration of FBF reflected a progressive decline in FVR to levels significantly lower than those in the placebo study.

View larger version (16K): [in this window] [in a new window]   Figure 1. Forearm blood flow response to infusion of Ach into brachial artery in placebo- and GH-treated subjects (n = 8). GH or placebo was infused for 4 h, and then vascular reactivity was tested while keeping the infusion ongoing. Data (mean ± SEM) were analyzed by ANOVA for repeated measures. P = 0.017 for the effect of GH treatment; P = 0.004 for the interaction between GH and Ach.

View larger version (15K): [in this window] [in a new window]   Figure 2. Forearm blood flow response to infusion of NP in placebo- and GH-treated subjects (n = 8). GH or placebo was infused for 4 h, and then vascular reactivity was tested while keeping the infusion ongoing. Data (mean ± SEM) were analyzed by ANOVA for repeated measures. P = 0.007 for the effect of NO; P = 0.13 for the interaction between GH and NO.

In the control arm the peak blood flow in response to 5-min ischemia was virtually identical in the two studies (38 ± 4 and 39 ± 5 ml/dl·min in the GH and placebo studies, respectively).

	Discussion

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The new findings of the present study can be summarized as follows. 1) Acute increments in GH to levels commonly observed during stress lower vascular resistance and raise blood perfusion in the forearm tissues (mainly skeletal muscle). 2) GH induces marked activation of the endothelium-dependent component of vasodilation. 3) The vascular effects of GH are linked to the activity of the NO pathway and are not associated with increments in IGF-I levels in the forearm circulation. 4) Given the local intra-arterial mode of GH administration, the observed effects are accountable by an action of GH on the forearm.

In recent years increasing interest has focused on the relationship between the GH/IGF-I axis and vascular biology. Many key concepts have been generated by clinical models of chronic abnormalities of GH secretion or activity, such as pituitary disease and congestive heart failure. In these conditions it is not easy to dissect the vascular action of GH per se from that of the accompanying complications. Moreover, no study has addressed the question of whether GH exerts an acute action on vascular reactivity and the related NO pathway. In a previous metabolic study the incidental observation was made that GH acutely accelerates FBF (10). However, no other hemodynamic parameter was measured, and the study was uncontrolled. Thus, it could not be excluded that the changes in FBF represented a reflex or time-dependent phenomenon.

The present study demonstrates that acute increments in GH levels induce vasodilation and enhance endothelial sensitivity to vasodilating agents. These effects are associated with increased NO production by the endothelium. In addition, inhibition of NO synthesis by an L-arginine analog abrogates the vasodilating response to GH. Our study does not clarify exactly how GH affects endothelial function and what signaling is activated. It is pertinent, however, to recall that in recent studies GH was able to up-regulate endothelial NO synthase in vascular tissues (11).

The present findings support the hypothesis that GH is one of the many factors governing vascular homeostasis. It should be noted, however, that the changes in forearm hemodynamics were evident 2–3 h after the beginning of GH infusion. This excludes the possibility that GH is involved in the moment to moment regulation of peripheral circulation. Instead, it is reasonable to hypothesize that GH plays a role in the adaptive response to sustained stress. In this condition the vasodilatory effect of GH may be particularly important to prevent an excessive rise in PVR and maintain high rates of blood perfusion in the skeletal muscle, an essential process to meet the increased demand of oxygen and nutrients.

The current data, obtained with an acute approach, are in line with those from previous studies performed in patients with pituitary disease (1, 2) or chronic heart failure (12, 13), which demonstrated the ability of GH to affect vascular function and NO bioavailability. In addition, the chronic lack of GH was shown to cause marked impairment of both the endothelium-mediated and nonendothelium-mediated components of vascular reactivity (6). In the present study GH stimulated the endothelial component, but did not affect the response of vascular smooth muscle cells (VSMCs) to NO. A likely explanation for this difference is that in chronic GH deficiency, the function of VSMCs is also impaired as a consequence of structural alterations, which are corrected by long-term treatment with GH. In line with this interpretation is the observation that in patients with chronic heart failure, a 3-month GH treatment corrects endothelium dysfunction, but leaves unchanged the already normal nonendothelium-dependent component (13).

The current model of local GH infusion implies that the effects of GH are mediated by a direct action on the forearm, rather than indirect, systemic mechanisms. This conclusion is also supported by the following considerations. 1) The local arterial infusion of GH did not result in significant changes in the systemic levels of the hormone. 2) No change in FBF or peak flow was observed in the contralateral arm during GH infusion.

Another interesting question is whether the present findings reflect an effect of GH per se or are mediated by IGF-I. Although the major source of circulating IGF-I is the liver, IGF-I is also produced by a variety of cells, including endothelial cells and VSMCs (14). It is unclear whether these cells respond to GH stimulation by acutely increasing their IGF-I production. It is known, however, that endothelial cells are endowed with high affinity receptors for IGF-I (15) and that IGF-I exerts a vasodilatory action mediated by NO (16, 17). Therefore, IGF-I could well be the mediator of all of the effects reported here. The following arguments, however, make this interpretation unlikely.

During intra-arterial GH infusion, IGF-I levels in the effluent blood from the forearm remained unchanged. One could argue that the time allowed for GH to activate IGF-I synthesis was too short. This seems unlikely because a 6-h systemic infusion of GH at a rate even lower than the current one caused significant increments in systemic IGF-I (18). Thus, the explanation for the lack of increase in IGF-I in our study is that the synthesis of IGF-I by forearm tissues in response to GH is not as rapid and sensitive a process as it is in other body tissues. On purely theoretical grounds, GH might have stimulated forearm IGF-I production to an extent that did not reflect changes in venous IGF-I, but sufficient to promote local biological effects by autocrine/paracrine mechanisms. This possibility, however, appears quite remote given the remarkable stability of IGF-I in venous blood from the forearm during GH infusion. Taken together, the present data suggest that GH per se was responsible for the observed acute vascular effects.

Apart from the physiological implications, the present data may also have clinical relevance. Patients with chronic heart failure show a characteristic impairment of endothelial function, which may be reversed by 3-month treatment with GH (13). The current study raises the question of whether the same goal may be reached by acute or short-term GH administration. Furthermore, recent studies underline the importance of an intact GH/IGF-I activity to the outcome of acute myocardial infarction (19, 20, 21). This has been explained by the growth-stimulating and antiapoptotic effects of IGF-I. Based on the present findings it is possible that vascular mechanisms are also at play.

In conclusion, the present study demonstrates that GH acutely stimulates endothelium-dependent vasodilation and the related NO pathway. These effects may have implications for the adaptive cardiovascular response to stress.

	Footnotes

 
Abbreviations: Ach, Acetylcholine; FBF, forearm blood flow; FVR, forearm vascular resistance; NO, nitric oxide; NP, sodium nitroprusside; PVR, peripheral vascular resistance; VSMC, vascular smooth muscle cell.

Received January 28, 2003.

Accepted March 12, 2003.

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