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University of Edinburgh, Department of Medical Sciences, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
Address all correspondence and requests for reprints to: Prof. Brian R. Walker, University of Edinburgh, Endocrinology Unit, Department of Medical Sciences, Western General Hospital, Edinburgh EH4 2XU, United Kingdom. E-mail: . b.walker{at}ed.ac.uk
Abstract
Both GH deficiency and excess are associated with cardiovascular disease. The mechanisms are unclear, but direct effects of GH in the vessel wall may be important. Previous reports suggest that GH enhances endothelium-dependent vasodilatation and alters large artery structure. Here we report a detailed assessment of large artery and microvascular structure and function in patients with contrasting GH levels. We studied six age-matched healthy control men, five men with acromegaly, and seven men with adult-onset GH deficiency before and at the end of 16 wk of GH replacement therapy. We measured arterial wall thickness by ultrasound of the common carotid artery; arterial stiffness by pulse wave analysis at the radial artery; microvascular structure by measurement of flow during maximal dilatation in the forearm and dermal circulation and counting dermal capillaries using video microscopy; and endothelial function in the forearm during brachial artery infusion of vasodilators (acetylcholine and sodium nitroprusside). Cardiac output was measured by Doppler ultrasound in GH-deficient patients and controls. GH-deficient patients tended to have increased arterial wall thickness and arterial stiffness, compared with controls. GH replacement reduced arterial stiffness (radial augmentation index 0.28 ± 0.07 to 0.20 ± 0.12, P = 0.02) and increased the number of dermal capillaries perfused (28.6 ± 5.0 to 30.9 ± 6.5 cm-2, P = 0.03), but a reduction in arterial wall thickness was not statistically significant. With respect to maximum flow in forearm and dermis and endothelial function, GH-deficient patients were not different from controls, and GH therapy had no effect. Moreover, acromegalic patients were not different from controls in any vascular parameters studied. We conclude that the direct vascular effects of GH excess and deficiency in man are of modest magnitude and should not therefore be given the highest priority in considering the risks of cardiovascular events in patients with pituitary disease.
EPIDEMIOLOGICAL STUDIES SUGGEST that occlusive vascular disease contributes to excess mortality in patients with both adult GH deficiency and acromegaly (1, 2, 3). Moreover, higher GH levels within the population are predictive of subsequent cardiovascular disease (4). Indirect mechanisms may be responsible, including hyperinsulinemia and hyperglycemia associated with both acromegaly and central obesity in GH deficiency, and hypertension associated with acromegaly. Alternatively, patients with pituitary disease often receive other hormone replacement therapy, including T4 (5), glucocorticoids (6), and androgens, which may have adverse cardiovascular effects. Against this background, little attention has been given to the direct effects of GH and IGF1 on the blood vessel wall in patients with endocrine disease.
There is extensive literature (7) concerning the adverse effects of GH and IGF1 on peripheral vessels in conditions as diverse as diabetic retinopathy (8), neovascularization in tumors, restenosis after angioplasty (9), and the development of atheroma (10). In vitro, IGF1 is mitogenic to vascular smooth muscle, and up-regulates potentially adverse receptors including angiotensin II type 2 receptors (11) and 
- adrenoceptors (12). In vivo the somatostatin analog, lanreotide, has been used effectively in reducing the chances of restenosis after angioplasty in men with coronary artery disease (9). However, GH also has actions that are of potential cardiovascular benefit. IGF1 is a vasodilator, in part because of enhanced vascular nitric oxide generation (13). In rats IGF1 lowers blood pressure and increases regional blood flow (14, 15, 16). It is likely that there is a balance between adverse and beneficial effects of GH in the blood vessel wall.
A few studies have examined aspects of peripheral vascular structure and function in adult patients with GH deficiency (17, 18, 19, 20, 21). Compared with controls, these patients have increased intima-media thickness in conduit arteries (17), a trend toward impaired flow-mediated vasodilatation (20), and decreased systemic nitric oxide generation (19). Administration of replacement GH normalized these abnormalities (19, 20) and increased forearm vasodilatation during brachial artery infusion of acetylcholine (21). It has, therefore, been proposed that GH replacement improves endothelium- dependent nitric oxide generation and vasodilatation and hence ameliorates endothelial dysfunction that occurs in association with cardiovascular risk factors (22, 23). In acromegaly, peripheral vascular function has not been assessed in detail, although there is some evidence for altered structure of dermal capillaries (24, 25), which may contribute to elevated peripheral vascular resistance and hypertension (26), and increased intima-media thickness of the internal carotid artery has been reported (27, 28).
In the present study, we aimed to extend these observations to examine both endothelial function and structural changes in large and small vessels in healthy controls and patients with contrasting GH levels, including those with adult-onset GH deficiency before and after GH replacement therapy, and patients with acromegaly.
Patients and Methods
Participants
Local ethical committee approval and written informed consent were obtained. Patients attending our endocrinology clinic and healthy volunteer subjects were invited to participate if they were male; aged 20–70 yr; and not diagnosed with hypertension, hyperlipidemia, or diabetes mellitus. Patients with acromegaly were diagnosed by measurement of GH during a glucose tolerance test and had received their primary treatment in the form of transsphenoidal surgery with or without radiotherapy. They had either active acromegaly (n = 3; random plasma GH 6.9–34.9 mU/liter) and were studied before commencing octreotide long-acting-release therapy, or they had been cured by transsphenoidal surgery and were studied within 3 months of surgery (n = 2; random plasma GH 1.6 and 2.0 mU/liter). Acromegalic patients had a known duration of disease of 2–33 yr (mean 18.2 yr). Patients with GH deficiency were diagnosed with either an insulin tolerance test or a clonidine stimulation test, had basal plasma IGF1 concentrations less than 150 ng/ml, and were included only if they were deficient in at least two other pituitary hormones for which replacement therapy was unchanged in the previous 3 months and during the study.
GH therapy
Patients with GH deficiency were studied before and after 16 wk of administration of daily sc human GH (Genotropin, Pharmacia and Upjohn, Milton Keynes, UK). Doses were introduced from a starting dose of 0.8 IU/d for 2 wk to 1.2 IU/d for 2 wk and then 1.6 IU/day for 2 wk, after which doses were titrated by measurement of serum IGF1 concentrations every 6 wk to obtain values in the range of 150–350 ng/ml. Efficacy of therapy was also examined by standardized disease-specific quality-of-life questionnaire [Adult Growth Hormone Deficiency Assessment (29)] before and in the fourth month of GH replacement therapy.
Protocol
Participants took their normal hormone replacement therapy and attended our Clinical Research Center at 0900 h and acclimatized to a temperature-controlled environment (23–25 C) for 10 min. The following measurements were made in sequence by a single observer. Further details of several of these measurements and their validation have been published previously (28).
1. Blood pressure was recorded in the right arm using an automated sphygmomanometer (Omron HEM 705CP). The mean of two recordings was used for analysis unless they differed by more than 5 mm Hg when a third recording was made, and the mean of two closest matches was used.
2. Pulse wave analysis was performed in the right radial artery (Sphygmocor, PWV Medical, Sydney, Australia) to assess the arterial wave form and infer differences in conduit artery stiffness (30).
3. Dermal capillary density was recorded on the dorsum of the middle phalanx of the right index finger by intravital videomicroscopy. The skin was prepared with a coating of clear nail varnish. Dermal capillaries were visualized using a microscope (Leitz, Leica Corp. UK, Milton Keynes, UK) under illumination with a mercury filament lamp (Leitz). Six adjacent fields of 0.25 mm2 were recorded via a television camera (Phillips LDH0703, KRP Power Source, Newbury, UK) onto videotape for 1 min at baseline and again after 10 min of venous occlusion, achieved by inflating a cuff (Peni-cuff; Hokanson, Belleview, WA) to 40 mm Hg around the base of the finger. Calibration was checked periodically with a graticule (Graticules Ltd., Tonbridge, UK).
4. Ultrasound images of the heart were recorded using an Acuson xl750 (Mountain View, CA). This measurement was not made in acromegalic patients. From the apical view, continuous-wave Doppler flows through the mitral and aortic valve were recorded during six cardiac cycles and the mean calculated to overcome effects of respiration. Cardiac output was calculated as (aortic velocity time integral) x (aortic root area) x (heart rate).
5. Common carotid artery intima-media thickness was measured using a 7-mHz linear phase array probe (Acuson). The posterior wall of the artery was imaged 1 cm proximal to the carotid bifurcation. Measurements were averaged from left and right.
6. Forearm blood flow was then assessed by venous occlusion plethysmography. This technique has been described in detail elsewhere (31, 32). Briefly, subjects lay supine with their arms inclined at approximately 30 degrees to improve venous drainage. Wrist cuffs were applied and, during the recording periods, were inflated to 220 mm Hg to exclude the hand circulation from the measurements. Upper-arm congesting cuffs were inflated to 40 mm Hg and blood flow was recorded for 10 sec followed by a 5-sec refilling period. Multiple 10-sec measurements were made over a 3-min period and the slopes of the final five recordings averaged to determine forearm blood flow. To correct for the effects of nonspecific variations in blood flow during the protocol, changes in forearm blood flow during drug infusions are presented in the figures as the ratio of flow in the infused arm to that in the control arm, expressed as percentage change in this ratio from baseline. For drug infusions, a 27 standard wire gauge needle (Cooper’s Needle Works, Birmingham, UK), connected to a 16G epidural catheter (Portex Ltd., Hythe, Kent, UK), was introduced into the brachial artery of the nondominant arm under local anesthesia of 1% lidocaine (Astra USA, Inc. Pharmaceuticals Ltd., Kings Langley, UK). Patency was maintained by infusion of physiologic saline (0.9%, Baxter Healthcare Ltd., Thetford, Norfolk, UK) or drug solutions (see below) at a rate of 1.0 ml/min-1. After intraarterial saline had been infused for 20 min, basal forearm blood flow was measured in both arms simultaneously. Then acetylcholine (Miochol, IOLAB, Bracknell, UK) or sodium nitroprusside (Nipride, Roche Pharmaceutical Products Ltd., Welwyn Garden City, UK) was infused in physiologic saline at cumulative doses of 3.74, 7.5, and 15 µg/min-1 and 0.3, 3, and 10 µg/min-1, respectively, for 6 min at each dose. Saline was then infused to provide a 12-min washout period, and a cumulative dose response was obtained with the other drug (sodium nitroprusside or acetylcholine). Forearm blood flow measurements were obtained during the final 3 min of each dose of drug or saline infusion. The order of administration of acetylcholine and sodium nitroprusside was randomized.
7. At the end of the intraarterial infusion, maximum forearm blood flow was measured in the noninfused arm by inflating an upper arm cuff above systolic pressure for 12 min and measuring forearm flow as above during the first 30 sec after release of the cuff.
8. Subjects then had body composition measured using a dual-energy x-ray absorptiometry scanner (Hologic, Inc., Bedford, MA).
Statistics
Data are mean ± SD. Differences among groups (controls, acromegalic patients, and GH-deficient patients before replacement therapy) were sought by single-way ANOVA for single measurements and repeated-measures ANOVA for dose-response curves. Measurements before and after GH therapy were compared by paired t tests for single measurements or repeated-measures ANOVA for dose-response curves. Within the acromegalic group, cardiovascular measurements were correlated with random GH levels by simple regression to identify the influence of the current activity of the disease.
Results
Participants
Characteristics of participants are shown in Table 1
. The groups were well matched for body mass index and age. Of patients with adult-onset GH deficiency, primary diagnoses included six with nonfunctioning pituitary macroadenoma and one idiopathic hypopituitarism. These patients received a mean GH replacement dose of 1.14 ± 0.28 IU/d resulting in IGF1 plasma concentrations of 304 ± 3 ng/ml and an improvement in Adult Growth Hormone Deficiency Assessment score from 13.6 ± 7.7 to 9.0 ± 7.6. GH-deficient patients had higher percent body fat at baseline, and GH therapy increased lean body mass (Table 2).
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Systemic hemodynamic measurements including blood pressure and cardiac output were not different among groups at baseline, not altered by GH therapy, and did not correlate with GH levels in acromegalic patients. More direct measurements reflecting large artery and microvascular structure are presented in Table 2
. These can be considered as indices of large artery structure and function and indices of microvascular structure and function.
Large artery structure was measured as wall thickness in the common carotid artery. Wall thickness tended to be higher in GH-deficient patients and tended to fall with GH replacement therapy, but these observations did not reach statistical significance. Large artery function is reflected in the height of the notch in the systolic pulse wave created by reflection; a stiffer arterial system generates an earlier and larger reflected wave, measured as a higher augmentation index. GH-deficient patients tended to have a higher augmentation index than healthy controls (P = 0.10), and GH therapy reduced this measure of arterial stiffness (P < 0.05). Neither wall thickness nor augmentation index were altered in acromegaly or correlated with GH levels in these patients.
Microvascular structure was measured in the forearm (principally skeletal muscle) and dermal circulations. Maximum flow reflects vascular resistance in the absence of any tone in the vessel wall and hence is a measure of structural change. This was measured in the forearm following ischemia and the dermis following heating. Neither of these was different among groups or affected by GH therapy. However, the number of capillaries being perfused in the skin at rest was increased by GH therapy (P < 0.05). This did not result from an increase in total capillary number, measured during venous occlusion, and is consistent with a functional rather than structural change in the microcirculation. These variables did not correlate with GH levels in acromegalic patients.
Microvascular function was assessed pharmacologically in the forearm circulation. Basal forearm blood flow and blood pressure were not different among groups. Forearm vasodilatation during brachial artery infusion of the endothelium-dependent vasodilator acetylcholine and the endothelium-independent nitric oxide donor sodium nitroprusside is shown in Fig. 1. There were no differences among groups and no change in responses following GH therapy. Among acromegalic patients, higher GH values were associated with increased vasodilator response to acetylcholine (r = 0.94, P < 0.02 vs. area under the curve) but not sodium nitroprusside (r = 0.67, P = 0.20).
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We describe here a highly detailed in vivo examination of systemic hemodynamics, vascular structure, and endothelial function in patients with contrasting baseline GH secretion and in GH-deficient patients after GH therapy. Selection criteria and matching among groups excluded confounding effects such as hypertension. GH therapy had anticipated effects on IGF1, body composition, and quality-of-life score. The influence of acute or chronic variations in GH levels on peripheral vascular structure and function was more subtle than predicted by existing literature. We found no vascular abnormalities in patients with long-standing acromegaly, although these patients tended to be younger than the controls and two patients were studied within 3 months of curative transsphenoidal surgery. In GH-deficient patients, who were well matched to controls, we found evidence of an improvement in arterial stiffness and increased microvascular perfusion in the dermal circulation following GH administration. However, these effects were not associated with measurable changes in systemic hemodynamic indices, we did not find differences in endothelial function, and changes in large artery structure were of borderline significance.
Many of these measurements have not been made previously in patients with these diagnoses. However, our results contrast with some previous observations. In acromegaly, Schiavon et al. (25) performed capillaroscopy in the nailfold in 25 patients and 26 controls and reported greater tortuosity and fewer capillaries in acromegalic patients, including the small subgroup without hypertension or hyperglycemia. However, although the nailfold is an ideal site for studying capillary loops to measure flow, it is not an ideal site for measuring numbers of vessels. A similar study by Bach et al. (24) failed to demonstrate a relationship between severity of acromegaly and characteristics of nailfold capillaries. We measured capillary number end-on on the dorsum of the middle phalanx. Our technique has been used widely, including demonstrating rarefaction of capillaries in young men with an inherited predisposition to essential hypertension (28). If capillaries are rarefied, then this is important if it is associated with impaired maximum blood flow in the circulation. We also assessed maximal blood flow during heating in the skin, which we have previously found to be impaired in hypertension (28), and again found no difference in acromegaly, suggesting that a change in dermal capillary structure, if it occurs, is not hemodynamically important.
The current study also extended these previous observations in acromegalic patients to examine other aspects of vascular structure and function. In light of the increased risk of atheromatous disease in acromegaly (33), it is perhaps surprising that we did not detect evidence of vascular endothelial dysfunction or structural change. This may in part reflect the selection of patients who were without associated cardiovascular risk factors such as hypertension and diabetes mellitus, were included even if they were recently cured of their acromegaly, and tended to be younger than controls (albeit not statistically significant). Nonetheless, the only cardiovascular variable that we found to be correlated with current levels of GH, and might therefore have been confounded by inclusion of patients with recently achieved biochemical cure, was the vasodilator response to acetylcholine, which was in fact greater in the patients with highest GH, consistent with improved endothelial function. In one previous report of increased carotid artery intima-media thickness, the acromegalic group were unselected and so were hypertensive (27). Larger studies will be required to establish mediators of vascular disease in this group, but the current data suggest that the effects of acromegaly on the vessel wall are likely to be indirect.
In GH-deficient patients, several groups have reported evidence of premature atherosclerosis or greater intima- media thickness of conduit arteries detected by ultrasonography (17, 20, 34). Our results showed similar trends, although because the intima-media thickness took several months to reduce to normal during GH therapy (20), it is perhaps no surprise that the reduction in wall thickness we observed over the relatively short duration (4 months) of our study of GH administration was not statistically significant. One of the consequences of vascular hypertrophy is increased arterial stiffness, which can be measured by pulse wave analysis (30). In accord with a previous report using carotid ultrasound measurements (18), we found a trend for stiffer arteries in GH-deficient patients and showed that GH therapy improved this parameter. In keeping with several previous reports, this was not associated with any change in blood pressure or cardiac output (35, 36, 37) but nevertheless may be of pathophysiological significance because an augmented pulse wave has been proposed to increase the risk of atherogenesis in large vessels, which is a poorly understood complication of hypopituitarism (1, 2, 3).
Two published reports described apparent improvement in endothelium-dependent vasodilatation with GH therapy in deficient patients (20, 21). One of these examined flow-mediated vasodilatation in the brachial artery and found no baseline difference compared with healthy controls, but enhanced responses after 3 months of GH (20). No control was included for nonendothelium-dependent vasodilatation (e.g. sublingual glyceryl trinitrate). The other measured forearm blood flow during brachial artery infusion (21), as we have done, and found that GH for 3 months enhanced vasodilatation to acetylcholine, but there was a similar magnitude of enhanced response to sodium nitroprusside, suggesting enhanced vascular smooth muscle sensitivity to nitric oxide may be as important as altered endothelial release of vasodilators. We did not reproduce these results. Strikingly, vasodilatation in response to acetylcholine in our patients and controls was of small magnitude, compared with the response to sodium nitroprusside. This suggests that controls of this age in our local population have endothelial dysfunction that may obscure any effect of GH.
In this study, we have also extended these previously published measurements to examine the microvasculature in patients with GH deficiency. This did not show evidence of structural change, but did suggest that GH administration increases the dynamic flow in the dermal capillary bed. The mechanism for this, however, is unclear because it was not associated with a change in total number of capillaries (measured after venous occlusion) or an increase in maximal dermal flow. Also, the hemodynamic significance of a change of this relatively small magnitude is uncertain.
Most studies of patients with pituitary disease involve small numbers and should therefore not be interpreted in isolation. We have not been able to identify systematic differences in selection criteria for patients and controls between our study and those performed by other investigators. However, our controls had higher intima-media thickness and more variable endothelial function (Fig. 1) than expected and were sampled from a Scottish population in which the prevalence of atheromatous disease is remarkably high. So how are we to regard the evidence of peripheral vascular effects of GH, given conflicting evidence from this and a handful of previous small studies? Perhaps the most important inference from the current study is that it tests the benefit of GH therapy, and the magnitude of adverse effects of GH excess, in a group of patients who are typical of those attending most adult endocrinology clinics. Our study supports some of the previously observed differences among patient groups and shows previously unknown effects of GH on arterial stiffness and capillary perfusion. However, none of these changes was of large magnitude. Moreover, we did not find further evidence to support the concept of improved endothelial function with GH administration, and we suggest that other factors may have a more important influence on this variable. Arguably, the benefits or adverse effects of GH on vascular function in man should therefore not be given the highest priority in considering the risks of cardiovascular events in patients with pituitary disease.
Acknowledgments
We are grateful to Dr. Jenny Jones for IGF1 assays.
Footnotes
This work was supported by Pharmacia, Novartis, and the British Heart Foundation.
Received August 1, 2001.
Accepted April 2, 2002.
References
This article has been cited by other articles:
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  G. Li, J.-P. del Rincon, L. A. Jahn, Y. Wu, B. Gaylinn, M. O. Thorner, and Z. Liu Growth Hormone Exerts Acute Vascular Effects Independent of Systemic or Muscle Insulin-like Growth Factor I J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1379 - 1385. [Abstract] [Full Text] [PDF]
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