| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 26, 2002 as doi:10.1096/fj.01-0272fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2
* Rational Drug Design Program, Biomedicum, and Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital,
Juvantia Pharma Ltd., Turku, Finland; and
Fraser Laboratories, Department of Medicine, Royal Victoria Hospital, Montreal, PQ, Canada
2Correspondence: Transplantation Laboratory, PO Box 21 (Haartmaninkatu 3), FIN 00014 University of Helsinki, Finland. E-mail: pekka.hayry{at}helsinki.fi
SPECIFIC AIM
The somatostatin analogs octreotide and lanreotide, selective to receptor subtypes 2 and 5, failed clinical efficacy for the prevention of restenosis after percutaneous transluminal angioplasty, possibly because a wrong subset of receptors was targeted. As in rat vascular wall subtypes 1 and 4 are expressed three- to fourfold more prominently than subtypes 2 and 5, and subtype 1 is the nearly exclusive subtype in atherosclerotic human vessels, we have tested whether it was possible to prevent fibrointimal dysplasia via targeting to subtype 1,4, only.
PRINCIPAL FINDINGS
Effect of mode and dose of administration on denudation injury
As reported inconsistencies of somatostatin effects on the vessel wall might have been related to different doses and modes of administration, we compared the effects of nonselective somatostatin (SST) -14 and CH275, a somatostatin receptor (SSTR) 1,4-selective agonist, on rat carotid injury response using day 14 as end point, a large dosing range, continuous infusion (osmotic minipump), and daily i.p. injections. Infusions were initiated on day -1 and the first injection was given during the operation.
When given as an infusion, there was no effect of SST14 (P=0.3889) or CH275 (P=0.8909) on intimal area of the left carotid 14 days after endothelial denudation (Fig. 1
). When peptides were injected daily beginning at operation, SST14 (y=11.203–0.012x; P=0.0303) and CH275 (y=13.28–0.022x; P=0.0002, ANOVA) both significantly inhibited the increase of intimal area; at the highest dose (500 µg-1·kg-1·day-1), CH275 virtually eliminated the vascular response to injury (Fig. 1)
. The response to octreotide, a SSTR2,5-selective agonist, was investigated using daily injections only. Octreotide was least effective of the three peptides when the dose-response slopes were compared (y=10.48–0.009x; P=0.098).
|
The same histological specimens were used to quantitate other parameters related to vascular injury in rats. Administrated via daily injections, SST14 significantly reduced not only the intima area but also the number of intima cells (P=0.001). CH275, however, reduced more parameters and more significantly. CH275 also reduced the number of intima cells (P=0.0002), intima/media area ratio (P=0.0009), the adventitial cell number (P=0.0063), and number of replicating (BrdU-positive) cells in the intima (P=0.0037). Octreotide reduced significantly the number of intima cells (P=0.01). When infused continuously, none of the compounds generated a statistically significant dose response to any parameter tested (not shown).
We compared the intimal area of controls to the area obtained with the highest drug dose injected. Controls, 12 ± 1.1 (SE) x104 pixels; SST,14 5.2 ± 2.3 (P=0.025); CH275, 2.1 ± 1.3 (P=0.014); octreotide, 4.8 ± 2.5 (P=0.076, Mann-Whitney).
Serum levels and half-lives of the peptides
As SST14 and C275 protected vascular injury only when administered daily beginning at the operation, we investigated drug levels obtained with different doses to exclude a possible feedback loop after administrating CH275. For control, we used the morning (nonfasting) SST level in 10 nondenuded normal male Wistar rats, which was 60 ± 11 pmol/L.
The plasma levels and half-lives after injection were established only for CH275 and SST14 since we did not have access to an assay system for measuring octreotide. 200 µg/kg of SST14 or CH275 was administered as a single injection; serum specimens were obtained at different intervals, fractionated with HPLC, and RIAs were performed. Peak concentrations of 360 and 280 pmol/L were observed for SST14 and CH275, respectively (after subtraction of the background, 60 pmol/L). Estimated in vivo half-lives were 20 min for SST14 and 90 min for CH275.
Serum levels obtained by pump infusion were determined for SST14 and CH275. When the peptides were administered with minipumps, serum levels of 
110 pmol/L were obtained 10 h after installation of the pump and remained at this level throughout the 7 day lifetime of the pump.
To exclude a possible feedback loop of CH275 administration, we took advantage of the fact that CH275 did not copurify under the conditions recommended by the manufacturer of the RIA kit. This made it possible to measure serum levels only of SST14 after administration of CH275. There was no induction of endogenous SST14 production in the test animal after a single injection of 200 µg/kg of CH275; levels remained below the control values.
Dose responses to SST14 and CH275 were done using the 20 min measurements postinjection, HPLC separation of the peptides, and RIA. High (<1200 pmol/L) but short-lasting peaks (not shown) were obtained at the highest dose, 500 µg/kg.
Weight gain
The highest peptide doses used were > 10-fold higher than recommended (for octreotide and lanreotide) in humans, so we followed the weight of individual rats used in this study. There was some weight loss postoperatively in CH275-, SST14-, and untreated rats, but rats resumed their growth rate < 4 days after the operation and, by day 14, weighed the same as preoperatively. No difference in weight was seen between the vehicle-treated, CH275-treated, and SST14-treated rats.
Effects of the peptides on the replication and migration of vascular smooth muscle cells in vitro
None of the peptides had any effect on 5% FBS-stimulated in vitro replication as measured by [3H]-thymidine incorporation or on 60 ng/ml PDGF-induced migration as measured in Transwell chambers of primary rat smooth muscle cells. Nor did the peptides affect replication of an established rat smooth muscle cell line, A10 (data not shown).
Effects of the peptides on outgrowth of cells from vascular tissue explants
We first investigated the outgrowth of cells from denuded carotids (and denuded thoracic aortas). The specimens for in vitro culture were obtained before or at different times after denudation from a minimum of three rats per time point. Uniform 1 x 1 mm punch explants were prepared and cultured in 96-well tissue culture plates by placing each at the center of a well. Explant cultures were examined after 24 h for the presence of outgrowing (‘sprouting’) cells, and the result is given as the proportion (%) of positive wells. In both types of vessels the proportion of wells with sprouts increased from < 5% predenudation to 50–75% on days 2–14 postdenudation; thereafter, the proportion of wells with sprouts returned to predenudation levels
Sufficient amounts of material for in vitro drug testing using the sprouting assay were difficult to obtain from denuded carotids because of their small size; we used thoracic aortas instead. The thoracic aortas were denuded in vivo and the animals received 500 µg·kg-1·day-1 of any one of the three peptides for 2 days postdenudation. Animals were subsequently killed, the aortas were removed, and punch explants were obtained and plated in culture. Vehicle only (PBS) or 10 µM of the same peptide administered in vivo was added to the wells at the beginning of culture and harvested 48 h later. The proportion (%) of wells with sprouts was ascertained by visual inspection at 24 and 48 h. The longest distance the sprouting cells had migrated was calculated using an ocular microscopic grid scale (graticule); cell replication was measured by [3H]-TdR incorporation upon termination of the cultures.
The proportion of sprouting punch explants from vehicle-treated rats on day 1 of culture was 54.0 ± 6.0%. Conversely, the proportion of sprouting explants from rats receiving CH275 in vivo and in vitro was 17.2 ± 6.7% (P=0.034 vs. control); from rats receiving CH275 only in vivo, it was 15.5 ± 3.7% (P=0.027). The proportion of sprouting explants from rats receiving octreotide in vivo and in vitro was 28.2 ± 6.1% (P=0.127 vs. control), whereas for those receiving the drug in vivo alone it was 19.6 ± 6.9% (P=0.046). Finally, the proportion of sprouting explants from rats receiving SST14 in vivo and in vitro was 22.6 ± 5.3% (P=0.066 vs. control); from rats receiving SST14 only in vivo, it was 28.2 ± 13.1% (P=0.136).
The effects of the three peptides on the proportion of sprouting specimens on day 2 of culture were correspondingly similar. By this time, the proportion of sprouting explants from vehicle-treated rats had reached 85.9 ± 4.1%. Treatment with CH275 in vivo and in vitro reduced the proportion of sprouting explants to 43.1 ± 13.6% (P=0.002 vs. control); in vivo treatment alone was slightly less effective (58.1±7.5%; P=0.024 vs. control). Treatment with octreotide was equally effective in reducing the proportion of sprouting specimens whether performed in vivo and in vitro (58.3±10.7%; P=0.028 vs. control) or just in vivo (58.7±10.3%; P=0.029). This was also the case with SST14, which reduced the frequency of sprouting explants to 63.1 ± 12.3% (P=0.068 vs. control) and 62.4 ± 12.6% (P=0.062), respectively.
The longest in vitro migration distance in explant cultures established from vehicle-treated rat aortas was 0.46 ± 0.04 mm. This same distance in explant cultures from rats receiving CH275 in vivo and in vitro was 0.18 ± 0.75 mm (P=0.043 vs. control) and that from rats receiving CH275 only in vivo was 0.28 ± 0.04 mm (P=0.060). Octreotide treatment showed comparable efficacy, with the longest migration distance after combined in vivo and in vitro administration being 0.17 ± 0.05 mm (P=0.037 vs. control) and that after in vivo exposure only being 0.26 ± 0.10 mm (P=0.156). SST14 treatment in vivo and in vitro (0.34±0.06 mm; P=0.340 vs. control) or in vivo alone (0.39±0.06 mm; P=0.536) was without effect.
None of the peptides significantly inhibited cell proliferation in vitro whether in vivo + in vitro, in vivo alone, or in vitro alone; a statistical trend suggesting an anti-proliferative effect was seen in most experiments (data not shown).
Rebound of intimal growth
Compared with vehicle-treated rats, 14 day administration of SST14 at either 200 or 500 mg-1·kg-1·day-1 had no significant effect on intimal area at 28 days. Similarly, the effect of octreotide was nonsignificant. Conversely, CH275 significantly inhibited 28 day intimal area at 200 (P=0.008) and 500 mg·kg-1·day-1 (P=0.011) dose levels. When intima/media area ratios were evaluated, the effect of SST14 remained nonsignificant. However, doses of CH275 (P=0.008 and P=0.020, respectively) and octreotide (P=0.045 and P=0.017, respectively) significantly reduced the intima/media area ratio at 28 days.
CONCLUSIONS AND SIGNIFICANCE
The message of this study is that by targeting exclusively to SSTR1 and 4, equal or better vasculoprotection is obtained than by using the nonselective SST-14 or SSTR2,5-selective octreotide. We earlier demonstrated that SSTR1,3 and 4 are the predominant receptors in rat carotid wall on mRNA level and that there is an acute increase in the expression of SSTR1 after injury, coinciding with the replicative burst of vascular smooth muscle cells, and a slow sustained increase of SSTR3 and 4, coinciding with the migration of the cells into the intima. Curtis et al. have shown that SSTR1 is the predominant SSTR in healthy human coronary and carotid arteries and that atherosclerotic arteries express predominantly SSTR1 on mRNA and protein levels.
The sprouting vs. replication assays described here suggest that the effect of somatostatins may be targeted to cell migration rather than proliferation. The frequency of sprouts escalated rapidly after vascular injury and returned to baseline 1 month postinjury, when the injury response was completed. As somatostatins inhibited the sprouts when administered in vivo and inhibited cell migration (but not the replication of cells) in vitro, we suggest that somatostatin affects cell migration rather than replication and that drug treatment should occur before the precursors of intima cells appear in the vessel wall.
We think our observations are relevant to future development of SST analogs for prophylaxis and treatment of vascular fibroproliferative disease. Targeting with agonist ligands exclusively to SSTR1,4 but bypassing SSTR2 and 5 should make it possible to generate compounds with vasculoprotective properties without GH, glucagon, and insulin release, common side effects of SST-based vasculoprotective drug therapy in humans.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0272fje; to cite this article, use FASEB J. (March 26, 2002)10.1096/fj.01-0272fje; 
This article has been cited by other articles:
|
|
 |
|
  M. Engstrom, J. Tomperi, K. El-Darwish, M. Ahman, J.-M. Savola, and S. Wurster Superagonism at the Human Somatostatin Receptor Subtype 4 J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 332 - 338. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
