Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

The chemokine receptor Cxcr3 is not essential for acute cardiac allograft rejection in mice and rats.

Chemokine receptors have gained attention as potential targets for novel therapeutic strategies. We investigated the mechanisms of allograft rejection in chemokine receptor Cxcr3-deficient mice using a model of acute heart allograft rejection in the strain combination BALB/c to C57BL/6. Allograft survival was minimally prolonged in Cxcr3-deficient mice compared to wild-type (wt) animals (8 vs. 7 days) and treatment with a subtherapeutic dose of cyclosporine A (CsA) led to similar survival in Cxcr3-deficient and wt recipients (13 vs. 12 days). At rejection grafts were histologically indistinguishable. Microarray analysis revealed that besides Cxcr3 only few genes were differentially expressed in grafts or in spleens from transplanted or untransplanted animals. Transcript analysis by quantitative RT-PCR of selected cytokines, chemokines, or chemokine receptors or serum levels of selected cytokines and chemokines showed similar levels between the two groups. Furthermore, in a rat heart allograft transplantation model treatment with a small molecule CXCR3 antagonist did not prolong survival despite full blockade of Cxcr3 in vivo. In summary, Cxcr3 deficiency or pharmacologic blockade does not diminish graft infiltration, tempo and severity of rejection. Thus, Cxcr3 does not appear to play a pivotal role in the allograft rejection models described here.

Macrophage labeling by SPIO as an early marker of allograft chronic rejection in a rat model of kidney transplantation.

Anatomical and functional information (renography, perfusion) was obtained by MRI in a life-supporting transplantation model, in which Lewis rats received kidneys from Fisher 344 donors. Renography and perfusion analyses were carried out with Gd-DOTA and small particles of iron oxide (SPIO), respectively. Starting 12 weeks posttransplantation, images from grafts of untreated recipients exhibited distinctive signal attenuation in the cortex. Animals treated with cyclosporin (Sandimmune Neoral; Novartis Pharma, Basel, Switzerland) to prevent acute rejection showed a signal attenuation in the cortex at 33 weeks posttransplantation, while kidneys from rats treated additionally with everolimus (Certican; Novartis), a rapamycin derivative, had no changes in anatomical appearance. A significant negative correlation was found between the MRI cortical signal intensity and the histologically determined iron content in macrophages located in the cortex. Renography revealed a significantly reduced functionality of the kidneys of untreated controls 33 weeks after transplantation, while no significant changes in perfusion were observed in any group of rats. These results suggest the feasibility, by labeling macrophages with SPIO, of detecting signs of graft rejection significantly earlier than when changes in function occur. Monitoring early changes associated with chronic rejection can have an impact in preclinical studies by shortening the duration of the experimental period and by facilitating the investigation of novel immunomodulatory therapies for transplantation.

Comparative efficacy of mycophenolate sodium (MPS) and mycophenolate mofetil (MMF) with and without cyclosporine in rat transplantation models.

BACKGROUND: ERL is the enteric-coated sodium salt of mycophenolic acid, presently in clinical development. The drug substance mycophenolate sodium (MPS) was evaluated in rat transplantation models and compared with mycophenolate mofetil (MMF) for therapeutic window and synergy with cyclosporine (CsA). METHODS: Allotransplantation was performed in the Dark Agouti-to-Lewis (DA-to-Lewis; kidney, heart, and aorta) and Brown Norway-to-Lewis (BN-to-Lewis; kidney) strain combinations, and hamster heart xenotransplantation was performed in athymic and euthymic Lewis rats. The compounds were administered daily orally, starting the day of transplantation. RESULTS: In kidney and heart transplantation the minimal efficacious dose of CsA was 5.0 mg/kg/d. For MPS this dose was 10 mg/kg/d in BN-to-Lewis kidney transplantation, 20 mg/kg/d in DA-to-Lewis heart transplantation, and 10 mg/kg/d in hamster-to-athymic rat heart transplantation. At these doses the first signs of adverse effects were evident, indicating a narrow therapeutic window. No window was established for MMF in these models or for MPS in DA-to-Lewis kidney transplantation. There was no potential synergy between CsA and MPS or MMF regarding efficacy, but fewer side effects were noted in efficacious combinations, in particular for MPS. In aorta transplantation, MPS and MMF dose-dependently inhibited intima thickening. The combination of 20 mg/kg/d MPS and 10 mg/kg/d CsA gave long-term survival of hamster-to-rat xenografts. CONCLUSIONS: Despite the overall comparable efficacy and narrow therapeutic window of MPS and MMF when given alone, MPS apparently is better tolerated than MMF in some of the transplant models. The combination of these agents with CsA allows fine-tuning between optimal immunosuppression and adverse side effects.

Tolerability profile of sodium mycophenolate (ERL080) and mycophenolate mofetil with and without cyclosporine (Neoral) in the rat.

Mycophenolic acid sodium salt (ERL080) is currently in Phase III clinical trials for the prophylaxis of kidney transplant rejection upon coadministration with Neoral (cyclosporin A microemulsion). To assess the relative side effect profile of ERL080 and MMF as drug substances in Lewis rats, a rat strain commonly used in transplantation experiments, a comparative 4-week tolerability study was performed. Escalating doses of ERL080 and MMF were administered orally at 10-30 mg/kg/d (i.e., doses within or above the immunosuppressive range in rats), either in single compound treatment or in combination with cyclosporine (CsA) at a daily oral dose of 7.5 mg/kg. The compounds were well tolerated as documented by body weight monitoring, hematologic parameters, and weight and histology of organs. Major abnormalities observed were a dose-dependent reduction in thymus weight associated with immunosuppression, in some cases villous atrophy in the jejunum, a reduction in white blood cell counts and lymphocyte counts (mean value in distinct treatment groups not exceeding 40-50%), a decrease in red blood cell counts and hemoglobin concentration (at maximum 25-30%), and an increase in platelet counts (in some groups up to doubling). At a given dose, these adverse effects were slightly more pronounced for MMF than for ERL080, and for groups under CsA coadministration compared to both compounds given alone. No significant potentiation effect of CsA on the changes induced by ERL080 or MMF was observed. Moreover, there were no new toxic entities evident upon CsA microemulsion coadministration.

Endothelial dysfunction and denudation in rat aortic allografts.

Clinical evidence suggests that early endothelial cell (EC) dysfunction may predict the development of graft vascular disease. We wished to assess the early functional and morphological changes in the graft endothelium in a commonly used animal model of graft vascular disease, the rat aortic interposition allograft model. To assess graft EC function, regulation of vascular tone by ECs was monitored in aortic rings from grafts harvested at various times after transplantation (Tx). EC morphology was assessed by silver staining, which was followed by en face inspection of the luminal side of the grafts. Acetylcholine-induced EC-dependent vasorelaxation was reduced in allografts at post-Tx days 7 and 14, whereas in syngeneic grafts EC-dependent relaxation was unaffected at any time after Tx. In separate grafts collected at the same time points, massive leukocyte adhesion at post-Tx day 7 and EC denudation at days 14 and 28 were evident in allografts but not in syngeneic grafts. At post-Tx day 56 (a time at which vessel wall remodeling is pronounced in this model), an intact EC layer covered the grafts. EC dysfunction and morphological changes were prevented by immunosuppression of recipient rats with cyclosporine. Our study shows that Tx-induced EC dysfunction in rat aortic allografts can be observed within 1 week of Tx in rat aortic allografts and that this is occurring concomitantly with enhanced leukocyte adhesion to the graft ECs. These changes occur before any other morphological or functional changes described thus far in this model and appear to be immune-driven. Taken together, these results show that Tx-induced early EC dysfunction, as described in patients, may be studied in the model of rat aortic Tx.

Transplantation-induced endothelial dysfunction as studied in rat aorta allografts.

BACKGROUND: Clinical evidence indicates that vascular endothelial cell (EC) dysfunction occurs early after transplantation (Tx) and initiates chronic graft vasculopathy. This study explored this phenomenon in rat aorta Tx using the stringent Dark Agouti (DA)-to-Lewis (LEW) and the weak Fischer 344 (F344)-to-LEW strain combinations. METHODS: Donor abdominal aortae were orthotopically grafted into LEW rats. At post-Tx days 7, 14, 28, and 56, grafts were collected to assess changes in EC morphology (en face silver staining) and EC function, i.e., vasodilatory response to acetylcholine (ACH) after phenylephrine (PHE) precontraction; changes in vascular smooth muscle cell (VSMC) alpha-actin (western blotting), degree of apoptosis (caspase-3 activity), and morphology. RESULTS: In DA allografts, VSMC and EC dysfunctions developed concomitantly and were completed at 14 days post-Tx, most likely due to the EC and alpha-actin-positive VSMC loss. Meanwhile, allografts revealed markedly increased caspase-3 activity. Neointima formation, restricted to the edges of allografts at day 28, covered the entire allografts by day 56 post-Tx. In F344-allografts, VSMC function was maintained up to day 14 post-Tx, whereas ACH-induced relaxation was reduced by 50% at day 7 and abolished at day 14. EC denudation was not seen up to 56 days post-Tx, despite prominent leukocyte adhesion. Neointima formation was not detected at day 28 post-Tx but appeared along the entire allografts at day 56 post-Tx. CONCLUSIONS: These results confirm that Tx-induced EC dysfunction precedes the development of vasculopathy in rat aorta allografts and suggest that this early phenomenon can be best studied in the F344-to-LEW strain combination.

Complete loss of functional smooth muscle cells precedes vascular remodeling in rat aorta allografts.

BACKGROUND: The functional consequences of vascular remodeling in rat aorta allografts were studied at different times after transplantation (Tx). METHODS: At days 1, 3, 7, 14, 28, and 56 after Tx, rat aorta allografts (Dark Agouti [DA]-to-Lewis) were mounted as isolated organs, and their contractile properties tested with phenylephrine, KCl, or endothelin-1. Controls were native DA-aortae and DA-syngeneic grafts. Changes in alpha smooth muscle actin and morphology were assessed by immunoblotting and histology. RESULTS: PostTx syngeneic grafts presented similar functional and morphological properties to native aortae. In allografts, no morphological changes was detected at day 7 after Tx, but phenylephrine-induced vasoconstriction was reduced by 60%. Signs of medial smooth muscle cell (SMC) loss and adventitial inflammation were observed at day 14 after Tx, without neointima formation. A complete loss of contractile property was observed at day 28 after Tx in association with a 75% decrease in alpha-SMC actin, severe adventitial inflammation, and reduced medial cellularity. At this time, neointima was restricted to both edges of allografts. At day 56 after Tx, allografts were also not functional and exhibited neointima on their entire length. All these changes were prevented by treating recipients with cyclosporine (7.5 mg/kg/day). CONCLUSION: These results indicate that, after Tx, the contractile property of rat aorta allografts is altered before manifest vascular remodeling. Because this can be prevented by cyclosporine, it most likely reflects an acute rejection of SMC. These results also show that vascular graft dysfunction can be used to monitor the development of rejection in the rat aorta allograft model.

Side effects of brequinar and brequinar analogues, in combination with cyclosporine, in the rat.

Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.

Decreased lumen size after balloon injury despite inhibition of neointimal thickening and antivasospastic treatment.

OBJECTIVE: Both calcium antagonists and angiotensin converting enzyme (ACE) inhibitors are known to diminish the development of intimal thickening after a balloon catheter lesion. It was previously shown that narrowing of carotid artery lumen induced by balloon injury was not influenced by treatment, even though the two ACE inhibitors used inhibited neointimal thickening. The aim of the present study was to include a calcium antagonist as well, in order to investigate whether vasospasm contributes to the persistence of lumen narrowing in ACE inhibitor treated rats after a balloon lesion. METHODS: Six groups of 10 rats were subject to balloon lesion of the left carotid artery. They received spirapril (10 mg.kg-1 x d-1) or isradipine (30 or 100 mg.kg-1 x d-1) or both, given throughout the study in the food. Controls received no drug. Neointimal thickening was measured histologically two weeks after injury. The cross sectional carotid lumen area was measured in vivo by nuclear magnetic resonance imaging, both before and two weeks after balloon injury, and also postmortem by histological techniques. RESULTS: Two weeks after injury, the lumen area of the left carotid artery was significantly reduced following balloon injury, as measured by both techniques. Treatment did not modify the stenosis process as assessed by either method for measuring lumen size. Neointimal thickening, however, was inhibited by between 4% (low dose isradipine) and 59% (combined spirapril + high dose isradipine) in the various treatment groups. CONCLUSIONS: Since calcium antagonist treatment was not able to influence the reduction of lumen size, it is unlikely that the narrowing is due to reversible spasm of the carotid artery in the first two weeks after inducing a balloon lesion. Alternatively, chronic vasospasm of neointimal smooth muscle might not be very sensitive to calcium antagonists.

Spirapril and cilazapril inhibit neointimal lesion development but cause no detectable inhibition of lumen narrowing after carotid artery balloon catheter injury in the rat.

Five groups of 12 rats were subject to balloon lesion of the left carotid artery and neointimal thickening was measured histologically 2 weeks after injury. Rat groups received either spirapril (3, 10 or 30 mg/kg/day, administered throughout the study in the food), cilazapril (10 mg/kg/day) or placebo. Spirapril caused a dose-dependent inhibition of the neointimal thickening of the rat carotid artery. The degree of inhibition with 10 mg/kg/d spirapril and cilazapril was similar (-44% and -42% respectively). The carotid lumen area was measured in vivo by nuclear magnetic resonance (NMR) imaging both before and 2 weeks after balloon injury and also postmortem by histological techniques. Two weeks after injury, the lumen area of the left carotid artery was significantly reduced following balloon injury, as measured by both techniques. Treatment did not detectably modify this stenosis process despite the use of two independent methods for assessing lumen size, even though neointimal thickening was strongly attenuated by both angiotensin converting enzyme inhibitors. This dissociation between inhibition of neointimal lesion development and decrease of lumen size provides a new view of the role of angiotensin converting enzyme inhibitors in vascular damage situations. Our results suggest that the focus, particularly in clinical studies, on lumen size, may mean that potentially beneficial effects of these drugs on other parts of the vascular wall be overlooked.

The effects of two synthetic glycoprotein IIb/IIIa antagonists, Ro 43-8857 and L-700,462, on platelet aggregation and bleeding in guinea-pigs and dogs: evidence that Ro 43-8857 is orally active.

In vitro platelet aggregation studies in whole blood were used to define the species-specificity profile of two synthetic GP-IIb/IIIa antagonists, Ro 43-8857 and L-700,462. Aggregation of rhesus monkey platelets was inhibited with a similar potency to human platelets, whereas both compounds were poor antagonists in mini-pig, rabbit or hamster blood. Compared to human platelets, Ro 43-8857 was 2-3-fold less active as an inhibitor of dog and guinea-pig platelet aggregation, whereas L-700,462 was, respectively, 4- and 14-fold less active in these species. In vivo investigations with these two compounds were performed in anesthetized guinea-pigs and conscious dogs, with bleeding times measured on small mesenteric arteries or on the inner jowl respectively. Ex vivo ADP-induced whole blood platelet aggregation was completely inhibited in guinea-pigs by Ro 43-8857 following intravenous administration of 0.1 mg/kg and intraduodenal administration of 3 mg/kg, with a duration of action exceeding 5 hours. Mesenteric bleeding times were prolonged by Ro 43-8857 only at doses causing supra-maximal inhibition of aggregation, suggesting these two effects could be partially dissociated. L-700,462 (3 mg/kg i.v.) was shorter acting than Ro 43-8857 in guinea-pigs (duration approximately 1 hour) and the anti-aggregatory effect was accompanied by mesenteric bleeding time prolongations. In conscious dogs, ex vivo aggregation was inhibited to approximately 80% by Ro 43-8857 (0.3 mg/kg i.v. or 10 mg/kg p.o.) and L-700,462 (1 mg/kg i.v.). However, bleeding time prolongations accompanied these anti-aggregatory effects with both compounds. In conclusion, we have shown clear differences between two synthetic GP-IIb/IIIa antagonists, both in terms of their species-specificity in vitro and in terms of their in vivo profile, and in particular the propensity to promote bleeding from mesenteric arteries in guinea-pigs. However, the ability of Ro 43-8857 to discriminate between anti-aggregatory and bleeding effects was not evident when the bleeding time measurements were performed on the dog jowl. This suggests that the species and/or vessels on which the bleeding time is performed, is also an important consideration when characterizing and comparing anti-platelet compounds, even with drugs acting via the same mechanism. These results are relevant for the future design of in vivo animal experiments to characterize this new class of compounds and in the interpretation of the data obtained to the clinical situation.(ABSTRACT TRUNCATED AT 400 WORDS)

Similarities in the mechanism of action of two new vasodilator drugs: pinacidil and BRL 34915.

The present study compares the in vitro and in vivo activities of pinacidil with another new vasodilator drug, BRL 34915, claimed to act via the opening of K+ channels in vascular smooth muscle. In the rabbit aorta, BRL 34915 and pinacidil caused rightward shifts of the KCl concentration-response curve and noncompetitively antagonized angiotensin II contractions, yielding an IC50 of 5 and 10 microM, respectively. In 86Rb-loaded guinea pig portal veins, both BRL 34915 and pinacidil stimulated 86Rb+ efflux over the concentration range 0.3-30 microM. At saturating concentrations, the maximum efflux elicited by pinacidil was only one-third that of BRL 34915. Spontaneous activity measured simultaneously from the same vessels was inhibited by BRL 34915 and pinacidil with an IC50 of 12 and 32 nM, respectively. Effects on mechanical activity were thus observed at drug concentrations 100-fold lower than those required to stimulate 86Rb+ efflux. In anesthetized rats, both compounds rapidly lowered blood pressure, with BRL 34915 being threefold more potent than pinacidil in this respect. Tachycardia was more pronounced after BRL 34915 than after pinacidil. Angiotensin II pressor responses were poorly antagonized by these two vasodilators in rats, in marked contrast to the potent effects of Ca2+ antagonists on these responses at equieffective hypotensive doses. We conclude the pinacidil, like BRL 34915, is a potent antihypertensive that is able to enhance the K+ permeability of vascular smooth muscle. The similarities between these two drugs suggest they have a common mechanism of action. However, the discrepancy between the concentrations of these drugs necessary to stimulate 86Rb efflux, and those at which mechanical effects are seen in the portal vein, do not rule out the possibility that other actions may contribute to their vasodilator activities.

Effects of the vasodilator endralazine given with the antihypertensive agent guanfacine on heart rate and blood pressure of spontaneously hypertensive rats and normotensive dogs.

Endralazine (BQ 22-708, Miretilan) and guanfacine (BS 100-141, Estulic) were tested alone and in combination for their effects on blood pressure and heart rate in conscious spontaneously hypertensive rats and conscious normotensive dogs. During combined administration of endralazine and guanfacine to spontaneously hypertensive rats, guanfacine 1 mg kg-1 i.v. blocked the tachycardia caused by endralazine, 1 mg kg-1 i.v. After oral administration, the tachycardia induced by 0.5 and 1 mg kg-1 of endralazine was inhibited dose-dependently by 0.05, 0.1 and 0.5 mg kg-1 of guanfacine. The antihypertensive efficacy of endralazine was either unchanged or increased by guanfacine at 0.05-1 mg kg-1. In normotensive conscious dogs, 0.2 mg kg-1 i.v. of guanfacine antagonised the tachycardia elicited by 0.3 mg kg-1 i.v. of endralazine. The elimination of the endralazine-induced reflex tachycardia by guanfacine suggests that the combination of both drugs could be useful in antihypertensive therapy.