| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Monash Institute of Reproduction and Development (D.J.P., K.L.J., D.M.d.K.) and the Center for Heart and Chest Research (D.J.M., J.J.S.), Monash University, Clayton, Victoria 3168, Australia; Oxford Brookes University (N.P.G.), Oxford 0X3 0PB, United Kingdom; and Department of Surgery, St. Vincent’s Hospital (H.P.), Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Dr. D. J. Phillips, Monash Institute of Reproduction and Development, Monash Medical Center, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: david.phillips{at}med.monash.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Given that the biological activity of activin is affected not only by its own circulating level, but also by that of follistatin, there is considerable interest in elucidating the factors that might be responsible for the release of these proteins into the circulation. Animal studies have shown that follistatin is released into the circulation in association with the inflammatory response that accompanies surgery (5). However, activin ßA-subunit and follistatin messenger ribonucleic acids and protein are also present in the endothelial cell compartment of blood vessels (6), and recent evidence indicates that a proportion of cell-associated activin is bound to cell surface proteoglycans as an activin-follistatin 288 complex (7). The latter observation is of particular relevance because an increasing body of data indicates that heparin, a drug routinely used in cardiovascular procedures to reduce blood coagulation activity, can displace a variety of endothelial-bound factors, thereby increasing their circulating levels (8). It is thus possible that heparin given during cardiovascular procedures may also release activin A and follistatin into the circulation, particularly as heparin is known to release follistatin in animal models (9).
The aim of this study was therefore to test the proposition that heparin administration during cardiovascular procedures in humans resulted in circulatory release of activin A and follistatin. Specifically, we examined whether these factors were released during cardiovascular procedures in humans due to the administration of heparin per se or whether this was in part related to an effect of the inflammatory response accompanying surgical trauma previously described in animal studies (5).
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Vascular surgery protocol
Thirty-two patients undergoing either abdominal aortic aneurysm (AAA) repair or carotid endarterectomy (CEA) procedures at St. Vincent’s Hospital were enrolled in this protocol. The AAA group (n = 16) consisted of 14 men and 2 women, with a median age of 70 yr (range, 60–92); aortic grafts were performed in 6 of these patients, and aorto-iliac grafts were performed in the other 10 subjects. All patients received Dacron grafts impregnated with gelatin (Gelsoft Plus, Sulzer Vascutek Ltd., Renfrewshire, Scotland, UK). There was no effect of the type of graft on any parameter measured, so data were pooled for both types of procedures. Blood samples were collected from the cubital vein before surgery, during dissection and clamping of the aorta, and at 6, 12, 24, and 72 h after surgery. All patients received 5000 IU heparin at the time of vessel clamping.
The CEA group (n = 16) comprised nine men and seven women, with a median age of 69 yr (range, 58–82). Blood samples were obtained from an ipsilateral jugular venous catheter inserted for the duration of the operation and were collected before and during clamping of the carotid artery and up to 15 min after unclamping of this vessel. In these patients, 5000 IU heparin were administered at the time of vessel clamping.
To determine to what extent any observed activin A and follistatin responses were related to a local or systemic inflammatory reaction associated with surgical trauma, serum interleukin-1ß (IL-1ß) concentrations were also measured in AAA and CEA patients. Three of the 16 AAA patients had IL-1ß levels above the normal range before surgery, and these were therefore not included in subsequent analyses.
Patients in both the AAA and CEA groups received only the one bolus dose of heparin at the time of vessel clamping and had not been given sc heparin at the commencement of the procedure.
Coronary angiography protocol
Subjects (n = 20) were recruited from patients undergoing elective coronary angiography in the Cardiology Unit at Monash Medical Center. The study group comprised 13 men and 7 women, with a median age of 67 yr (range, 45–77), who were administered 2500 IU heparin (10 patients) or placebo consisting of an equal volume of normal saline (10 patients) just before coronary catheterization. Patients were randomized to the 2 groups using sealed envelopes. Blood samples were collected from the cubital vein before heparin or placebo administration and then at 10, 20, 30, 40, 50, 60, 120, and 180 min after these treatments.
Assays
Serum or plasma derived from the blood samples (collected into
ethylenediamine tetraacetate-coated tubes) was measured in duplicate
for total activin A or follistatin concentrations using previously
validated immunoassays (10, 11). In both assays there was no effect of
heparin on the detection of activin A. In both assays we tested heparin
at concentrations that far exceeded the levels measured in the
circulation of patients given 2500 IU heparin (Fig. 1
). The activin A assay uses human
recombinant activin A as the reference preparation, has a detection
limit of 10 pg/mL, and has within- and between-assay coefficients of
variation of less than 12%. The follistatin assay uses human
recombinant follistatin 288 (National Hormone and Pituitary Program,
Torrance, CA) as the reference preparation, has a detection limit of 2
ng/mL, and has within- and between-assay coefficients of variation of
7% and 9% respectively.
|
Statistical analysis
Peak and baseline concentrations within groups were compared with paired Student’s t test. The fold increases in activin and follistatin were compared between groups using one-way ANOVA, followed by the Neuman-Keuls post-hoc test. Disappearance rates for activin, follistatin, and heparin in the coronary angiography study were estimated by fitting curves to a monoexponential equation. Half-life values were compared using unpaired t tests. Where appropriate, data were transformed before analysis to correct for heterogeneity of variance. The data are presented as the mean ± SEM, and a P value of 0.05 was regarded as significant.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
In both AAA and CEA procedures, serum total activin A and
follistatin increased rapidly and robustly (Fig. 2). Peak concentrations were reached
5–15 min after the clamping of the artery and administration of 5000
IU heparin and were significantly higher than baseline in both groups
(P < 0.0002). Despite the differing sampling regimens,
the activin A and follistatin response profiles were qualitatively
similar in the two procedures. However, the duration of the response
was longer in the AAA procedure; 1 h after clamping, both activin
A and follistatin were still approximately 50% of peak values, whereas
in the CEA patients, concentrations had returned to preclamp levels
within 1 h. The fold increase in activin A levels was
significantly (P < 0.001) higher in the CEA group than
in the AAA group (8.4 ± 0.7 vs. 5.0 ± 0.4,
respectively), whereas the fold increase in follistatin was similar
between the two groups (5.4 ± 0.9 vs. 5.3 ±
0.7).
|
). In the CEA patients, there was a
small, but significant, increase in IL-1ß levels 30 min after
clamping of the carotid (P < 0.05). In both groups,
the IL-1ß response lagged behind and had a much lower magnitude than
that observed for activin A and follistatin. Coronary angiography protocol
In patients undergoing coronary angiography, injection of 2500 IU
heparin elicited an almost immediate 10-fold increase in plasma activin
A and a 6-fold increase in follistatin levels. By contrast, no
significant (P > 0.05) change in plasma activin A and
follistatin levels was observed in the placebo group (Fig. 3). The response profile of plasma
activin A and follistatin levels in the heparin-treated angiography
group was qualitatively similar to that observed in the AAA and CEA
patients. However, the fold increase in activin A (9.6 ± 1.0) was
significantly higher (P < 0.001) than the response in
the AAA group, but was not different from that in the CEA patients. The
fold increase in follistatin levels (5.5 ± 0.8) in the
angiography patients was not significantly different from that in the
CEA and AAA patient groups. In terms of the duration of the response,
angiography patients had a similar response duration as the AAA group,
in which both activin A and follistatin were still approximately 50%
of peak values 1 h after heparin administration.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Activin and follistatin are normally present in the circulation in a complex (4, 13). In addition, recent studies have suggested that activin is complexed through follistatin 288 to heparin sulfate proteoglycans on the cell membrane (7), and through this mechanism may be fated for intracellular degradation through a lysosomal pathway. However, the presence of the activin-follistatin 288 complex on the vascular endothelium raises the possibility that this complex may be displaced by heparin. Indeed, such a phenomenon is observed with a range of endothelial-bound factors, including basic fibroblast growth factor (14), platelet-derived growth factor, vascular endothelial growth factor, hepatocyte growth factor (8, 15, 16), and tissue factor pathway inhibitor (17).
The close temporal relation between the administration of heparin and the subsequent increase in the circulating levels of activin and follistatin during abdominal aortic aneurysm and carotid endarterectomy surgery was consistent with the idea that heparin caused the release of these proteins from vascular endothelium. However, as previous studies by us and others have suggested that inflammation can elevate circulating activin and follistatin levels (18), it was possible that the initiation of an inflammatory response by tissue trauma accompanying aortic and carotid surgery also contributed to our findings. Two indirect lines of evidence suggest, however, that an inflammatory component was unlikely to be a major contributor to the rises in activin and follistatin levels observed during vascular surgery. First, the response of the inflammatory marker IL-1ß was relatively minor, suggesting that the robust release of activin and follistatin was not related to local trauma. Second, our previous animal studies indicate that, at least with follistatin, the peak response occurs some 12–24 h after a surgical intervention (5), much later than the almost immediate response detected in the present studies.
To more directly determine whether the increase in circulating activin and follistatin levels observed in vascular surgery patients was related to heparin administration, we sought to establish whether the response could be replicated by administering heparin to patients undergoing elective coronary angiography, a procedure associated with relatively minor vascular trauma. The similarity of activin and follistatin responses between the coronary surgery and angiography protocols suggested that the majority of the release of activin and follistatin observed during aortic and carotid surgery could be accounted for by the use of heparin during these procedures.
A number of subtle differences were, however, evident in the profiles of activin and follistatin responses during coronary angiography, aortic surgery, and carotid endartectomy. First, there were apparent differences in the fold increase in activin A between the AAA group and the CEA and angiography groups. The most plausible explanation for this difference was the intensity of the sampling regimens used. Thus, the initial, rapid release of these factors was detected in the coronary angiography and carotid endarterectomy protocols, but not in the AAA group, in which samples were collected less frequently. Second, the pattern of decline in circulating activin and follistatin levels differed with the various protocols, with the release of activin A and follistatin being of shorter duration in the CEA patients than in either the angiography or AAA group. The basis for this differential response to the various procedures is not clear at present, but may potentially relate to the nature of the carotid endarterectomy procedure itself, the ability of different vessel beds to release activin A and follistatin, or differences in the sites of blood sampling.
In the coronary angiography protocol, the calculated circulating half-life of heparin was less than that of activin, which, in turn, was less than that of follistatin. This finding differs from another heparin-releasable factor, tissue factor pathway inhibitor, which seems to have similar disappearance kinetics as heparin (17). One possible explanation for the disparate circulating half-lives was that heparin became dissociated from the activin-follistatin complex, followed by dissociation of the activin-follistatin components. The differing half- lives of activin and follistatin might thus reflect alternative fates of these two factors. If activin dissociated from follistatin, it could become accessible for binding to the activin-receptor complex and thereby initiate activin-specific intracellular signaling pathways. However, it should be pointed out that in biochemical studies, the activin-follistatin complex is remarkably stable and is not subject to significant dissociation (19). Another explanation for the differences in the disappearance rates of activin and follistatin could be that heparin released follistatin not bound to activin from cell membranes in addition to follistatin bound to activin. Conceivably, the former has a longer circulating half-life than the proportion bound to activin, and this accounts for the overall longer circulation time of follistatin. We are currently exploring these intriguing issues in various experimental models.
The findings in the present study have potential significance to cardiovascular medicine, as activin is a known mitogen for vascular smooth muscle cells and is up-regulated during vessel injury and in restenotic lesions (20). This is of relevance, because recent findings from Medalion and colleagues (14) have demonstrated that basic fibroblast growth factor, another endothelial-bound factor released by heparin administration, is preferentially sequestered at sites of vessel injury, where it may stimulate mitogenic processes. At present we do not have any information about whether activin and follistatin are also preferentially targeted to injured sites, but a mechanism similar to that shown for basic fibroblast growth factor is conceivable, particularly as activin accumulates at the site of atheromatous plaques (21). In addition, further investigations are needed to determine whether low molecular weight variants of heparin and other anticoagulants, such as direct thrombin inhibitors, also cause the release of activin and follistatin into the circulation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Recipient of a Research Fellowship from the Cardiac Society of
Australia and New Zealand.
Received December 15, 1999.
Revised December 25, 1999.
Accepted March 1, 2000.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  T. F. Lerch, S. Shimasaki, T. K. Woodruff, and T. S. Jardetzky Structural and Biophysical Coupling of Heparin and Activin Binding to Follistatin Isoform Functions J. Biol. Chem., May 25, 2007; 282(21): 15930 - 15939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Anderson Candidate-based proteomics in the search for biomarkers of cardiovascular disease J. Physiol., February 15, 2005; 563(1): 23 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Smith, A. Yndestad, B. Halvorsen, T. Ueland, T. Waehre, K. Otterdal, H. Scholz, K. Endresen, L. Gullestad, S. S. Froland, et al. Potential anti-inflammatory role of activin A in acute coronary syndromes J. Am. Coll. Cardiol., July 21, 2004; 44(2): 369 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Borawski, B. Naumnik, and M. Mysliwiec Activin A/Follistatin System: Another Link to Heparin-induced Osteoporosis? Clinical and Applied Thrombosis/Hemostasis, April 1, 2004; 10(2): 191 - 192. [PDF]
|
|
|
|
 |
|
 D. M. Bonucchi, G. Cappelli, and A. Albertazzi Heparin and dialysis: reasons to make a change? Nephrol. Dial. Transplant., March 1, 2002; 17(3): 531 - 532. [Full Text] [PDF]
|
|
|
|
 |
|
 T. Eldar-Geva, I.M. Spitz, N.P. Groome, E.J. Margalioth, and R. Homburg Follistatin and activin A serum concentrations in obese and non-obese patients with polycystic ovary syndrome Hum. Reprod., December 1, 2001; 16(12): 2552 - 2556. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
,
heparin alone; 
, heparin plus standard.
