Hypothermic Perfusion Preservation of Pancreas for Islet Grafts: Validation Using a Split Lobe Porcine Model.

The demand for high-quality islets for transplantation in type I diabetics will increase as the current clinical trials transition into standard of care. The mode of preservation of donor pancreata is critical to this mission since islets are very sensitive to ischemic injury. Hypothermic perfusion preservation (HPP) is being investigated for extended pancreas preservation in light of the beneficial effects reported for other organs. The present pilot study aimed to establish the potency of porcine islets isolated from pancreata after 24 h of HPP at 4-8°C. The study design included a split-lobe pancreas model that permitted paired comparisons of islets isolated from 24-h HPP splenic lobes with nonperfused, fresh control duodenal/connecting lobes stored at 4°C for <3 h. Prior to transplantation, islet viability was assessed in vitro using the ratio of oxygen consumption rate to DNA (OCR/DNA) assay and correlated with subsequent in vivo function by transplantation in diabetic immunodeficient mice. The OCR/DNA (mean ± SD) measured after 7 days of culture and immediately prior to transplantation for islets from the 24-h HPP group was 269 ± 19 nmol/min/mg DNA, which was higher but not statistically different to the mean of 236 ± 43 for the counterpart control group. All four nude mice transplanted with islets from the 24-h HPP group showed diabetes reversal, compared with five of six transplants from the control group. In conclusion, islets isolated from adult porcine pancreata after 24-h HPP exhibited high viability as measured by OCR/DNA and were able to consistently reverse diabetes in a nude mouse bioassay.

Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.

BACKGROUND: A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. METHODS: Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment. RESULTS: As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). CONCLUSIONS: An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.

Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: correlation of freezing visualization with functional recovery.

Cryopreservation is a well-established technique for long-term storage of viable cells and tissues. However, in recent years, application of established cryobiological principles to the preservation of multicellular tissues and organs has demanded considerable attention to ways of circumventing the deleterious effects of ice and thermal stresses in bulky tissues. As part of a multidisciplinary research program designed to study the interactions of thermo-physical events with tissue preservation, we report here on the implementation of a slow cooling (3 degrees C/min) and slow warming (62 degrees C/min) regimen towards scale-up of vitreous preservation of large tissue samples. Specifically, the correlation of thermo-physical events during vitrification of carotid artery segments with function recovery is reported using marginal thermal conditions for achieving vitrification in bulky samples. Moreover, the outcome is compared with a similar study reported previously using a 3-fold higher rate of rewarming (186+/-13 degrees C/min). Tissue vitrification using an 8.4M cryoprotectant cocktail solution (VS55) was achieved in 1 ml samples by imposing a low (2.6+/-0.1 degrees C/min) cooling rate, between -40 degrees C and -100 degrees C, and a low rewarming rate (62+/-4 degrees C/min) between -100 degrees C and -40 degrees C. Following cryoprotectant removal, the artery segments were cut into 3-4mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore and sodium nitroprusside). In addition, non-specific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function, normalized to untreated control samples, in response to the agonists norepinephrine and phenylepinephrine was significantly better in the slowly rewarmed group of carotid segments (74+/-9% and 62+/-11%, respectively) than for the more rapidly warmed group 31+/-7% and 45+/-15%, respectively). However, EC(50) sensitivities were not significantly different between the groups. Thermo-physical events such as ice formation and fractures were monitored throughout the cooling and warming phases using cryomacroscopy with the aid of a purpose-built borescope device. This technique allowed a direct observation of the visual impact of ice formation on specific zones along the blood vessel segment where, in most cases, no ice formation or fractures were observed in the vicinity of the artery segments. However, in specific instances when some ice crystallization was observed to impact the artery segment, the subsequent testing of function revealed a total loss of contractility. The successful vitrification of blood vessel segments using marginal conditions of slow cooling and rewarming, provide essential information for the development of scale-up protocols that is necessary when clinically relevant size samples need to be cryopreserved in an essentially ice-free state. This information can further be integrated into computer simulations of heat transfer and thermo-mechanical stress, where the slowest cooling rate anywhere in the simulated domain must exceed the critical values identified in the current study.

Twenty-four hour hypothermic machine perfusion preservation of porcine pancreas facilitates processing for islet isolation.

Procurement of donor pancreata for islet isolation and transplantation is not yet widely practiced due to concerns about the impact of postmortem ischemia on functional islet yields. Perfusion/preservation technology may help to circumvent ischemic injury as applied in this study of porcine pancreata prior to islet isolation. Pancreata harvested from adult pigs were assigned to 1 of 3 preservation treatment groups: G1, fresh controls, processed immediately with minimum cold ischemia (<1 hour); G2, static cold storage, flushed with cold UW-Viaspan and stored at 2 degrees -4 degrees C for 24 hours; and G3, hypothermic machine perfusion (HMP) on a pulsatile LifePort machine Organ Recovery Systems, Inc., Des Plaines, Ill, United States with KPS1 solution at 4-7 degrees C and low pressure (10 mm Hg) for 24 hours. Islet isolation was then accomplished using conventional methods. Product release criteria were used to assess islet yield and function. Islet yield was markedly different between the treatment groups. There was a statistically significant increased yield in the HMP group over static cold storage in UW-Viaspan (P < .05). Functionally, the islets from each experimental group were equivalent and not significantly different from fresh controls (G1). Dithizone staining of islets showed consistently more uniform digestion of pancreata from G3 compared with G1 and G2, with greater separation of the tissue and fewer entrapped islets. HMP for 24 hours was well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appeared to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreata subjected to 24 hours of static cold storage.

Vitrification of Carotid Artery Segments: An Integrated Study of Thermophysical Events and Functional Recovery Toward Scale-Up for Clinical Applications.

In recent years, ice-free cryopreservation by vitrification has been demonstrated to provide superior preservation of tissues compared with conventional freezing methods. To date, this has been accomplished almost exclusively for small model systems, whereas cryopreservation of large tissue samples-of a clinically useful size-continues to be hampered by thermomechanical effects that compromise the structure and function of the tissue. Reduction of mechanical stress is an integral condition of successful cryopreservation of large specimens. The current study focuses on the impact of sample size on both the physical events, observed by cryomacroscopy, and on the outcome on tissue function. To this end, the current study sought to address the question of functional recovery of vitrified carotid artery segments, processed as either artery rings (3-4 mm long) or segments (25 mm long) as selected models; the latter model represents a significant increase in sample size for evaluating the effects of vitrification. Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1-ml samples by imposing either a high (50-70 °C/min) or a low (2-3 °C/min) cooling rate, between -40°C and -100°C, and a high rewarming rate between -100°C and -40°C. Following cryoprotectant removal, the artery segments were cut into 3 to 4-mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore, and sodium nitroprusside). In addition, nonspecific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function in response to the agonists norepinephrine and phenylepinephrine was maintained at the same level (350%) for the segments as for the rings, when compared with noncryopreserved control samples. Relaxation in response to the antagonists calcium ionophore and sodium nitroprusside was maintained at between 75% and 100% of control levels, irrespective of cooling rate or sample size. No evidence of macroscopic crystallization or fractures was observed by cryomacroscopy at the above rates in any of the samples. In conclusion, this study verifies that the rate of cooling and warming can be reduced from our baseline vitrification technique such that the function of larger tissue samples is not significantly different from that of smaller blood vessel rings. This represents a step toward the goal of achieving vitreous cryopreservation of large tissue samples without the destructive effect of thermal stresses.

Preservation of embryonic kidneys for transplantation.

BACKGROUND: Long-term storage of embryonic kidneys is crucial for the organization of transplantation and organ banking. In this study, we investigated the effects of controlled-rate freezing and ice-free vitrification on metanephroi (MN) viability. METHODS: Metanephroi isolated from 15-day (E15) timed pregnant Lewis rats were either: (i) frozen, using a DMSO/FCS/RPMI solution and a controlled freezing rate of -0.3 degrees C/min, from -10 degrees to -40 degrees C; or (ii) cryopreserved in an ice-free state by rapid cooling to -100 degrees C in cryoprotectant (VS55), followed by vitrification to -120 degrees C. After cryopreservation, the metanephroi were stored at -135 degrees C for 48 hours. After storage the MN were rewarmed, resuspended in culture media, and their viability was assessed using the AlamarBlue assay and histology (light microscopy, TEM, and cryosubstitution). RESULTS: There was statistically no difference in embryonic kidney metabolic activity of either of the cryopreserved MN groups relative to the control untreated group. However, cryosubstitution demonstrated the presence of significant ice formation during controlled-rate freezing, yet in contrast the amount of ice was significantly reduced by vitrification. This was confirmed by TEM, where vacuolation of the cytoplasm of controlled-rate frozen metanephroi was observed, whereas vitrified metanephroi had little cytoplasmic disruption. However, vitrified metanephroi showed mitochondrial and nuclear injury at the cellular level. CONCLUSIONS: There is a need for long-term storage of organs to make MN transplantation a reality. This study demonstrates that standard freezing methods are unsuitable for this purpose. Vitrification yielded more promising results, but further development is required.

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.

Myocardial and interstitial matrix metalloproteinase activity after acute myocardial infarction in pigs.

A structural event during the evolution of a myocardial infarction (MI) is left ventricular (LV) remodeling. The mechanisms that contribute to early changes in LV myocardial remodeling in the post-MI period remain poorly understood. Matrix metalloproteinases (MMPs) contribute to tissue remodeling in several disease states. Whether and to what degree MMP activation occurs within the myocardial interstitium after acute MI remains to be determined. Adult pigs (n = 15) were instrumented to measure regional myocardial function and interstitial MMP levels within regions served by the circumflex and left anterior descending arteries. Regional function was measured by sonomicrometry, and interstitial MMP levels were determined by selective microdialysis and zymography as well as by MMP interstitial fluorogenic activity. Measurements were performed at baseline and sequentially for up to 3 h after ligation of the obtuse marginals of the circumflex artery. Regional fractional shortening fell by over 50% in the MI region but remained unchanged in the remote region after coronary occlusion. Release of soluble MMPs, as revealed by zymographic activity of myocardial interstitial samples, increased by 2 h post-MI. The increased zymographic activity after MI was consistent with MMP-9. Myocardial interstitial MMP fluorogenic activity became detectable within the ischemic region as early as 10 min after coronary occlusion and significantly increased after 1 h post-MI. MMP fluorogenic activity remained unchanged from baseline values in the remote region. The present study demonstrated that myocardial MMP activation can occur within the MI region in the absence of reperfusion. These unique results suggest that MMP release and activation occurs within the ischemic myocardial interstitium in the early post-MI period.

Long-term angiotensin-converting enzyme and angiotensin I--receptor inhibition in pacing-induced heart failure: effects on myocardial interstitial bradykinin levels.

BACKGROUND: We examined whether and to what degree long-term angiotensin-converting enzyme (ACE) inhibition, angiotensin type 1 (AT(1))-receptor blockade, or combined inhibition in developing congestive heart failure (CHF) alter myocardial interstitial bradykinin (BF) levels. METHODS AND RESULTS: Pigs (27-30 kg) underwent rapid pacing-induced CHF (240 bpm, 3 weeks; n = 10); pacing CHF with concomitant ACE inhibition (benezaprilat, 3.75 mg/day; n = 10); pacing CHF and concomitant AT(1)-receptor blockade (valsartan, 60 mg/day; n = 10); pacing CHF and combined inhibition (benezaprilat/valsartan, 1.87/60 mg/day, respectively; n = 10); or served as controls (no pacing, no treatment; n = 10). Steady-state myocardial interstitial BK levels were quantitated by microdialysis. Cardiac output decreased to 1.95 +/- 0.18 L/min in pacing CHF compared with control (3.78 +/- 0.38; P < .05). Cardiac output increased from untreated CHF values with concomitant ACE inhibition (3.91 +/- 0.27 L/min), AT(1)-receptor blockade (3.30 +/- 0.41 L/min), or combined ACE/AT(1)-receptor inhibition (4.13 +/- 0.32 L/min; all P < .05 v CHF). With pacing CHF, myocardial interstitial BK levels were reduced by approximately 50% from control values and were normalized in the ACE inhibition and combined inhibition groups. CONCLUSIONS: Long-term ACE inhibition increases myocardial interstitial BK levels with CHF; addition of AT(1)-receptor blockade does not seem to abrogate these effects.

Myocardial bradykinin following acute angiotensin-converting enzyme inhibition, AT1 receptor blockade, or combined inhibition in congestive heart failure.

BACKGROUND: The present study examined the effects of acute angiotensin-converting enzyme inhibition (ACEI), AT(1) receptor blockade (AT(1) block), or combined treatment on in vitro and in vivo bradykinin (BK) levels. METHODS: BK levels were measured in isolated porcine myocyte preparations (n = 13) in the presence of exogenous BK (10(-8) M); with an ACEI (benezaprilat; 0.1 mM) and BK; an AT(1) block (valsartan; 10(-5) M) and BK; and combined treatment and BK. In a second study, myocardial microdialysis was used to measure porcine interstitial BK levels in both normal (n = 14) and pacing-induced congestive heart failure (CHF) (240 beats/min, 3 weeks, n = 16) under the following conditions: baseline, following ACEI (benezaprilat, 0.0625 mg/kg) or AT(1) block (valsartan, 0.1 mg/kg), and a combined treatment (benezaprilat, 0.0625 mg/kg; valsartan, 0.1 mg/kg). RESULTS: In the left ventricular myocyte study, BK levels increased over 93% with all treatments compared to untreated values (P < 0.05). In the in vivo study, basal interstitial BK values were lower in the CHF group than in controls (2.64 +/- 0.57 vs 5.91 +/- 1.4 nM, respectively, P < 0.05). Following acute infusion of the ACEI, BK levels in the CHF state increased from baseline (57% +/- 22; P < 0.05). Following combined ACEI/AT(1) block, BK levels increased from baseline in both control (42% +/- 11) and CHF groups (60% +/- 22; P < 0.05 for both). CONCLUSION: These findings suggest that ACEI, or combined ACEI/AT(1) block increased BK at the level of the myocyte and potentiated BK levels in the CHF myocardial interstitium.

Temporal endothelin dynamics of the myocardial interstitium and systemic circulation in cardiopulmonary bypass.

OBJECTIVE: Increased systemic levels of the bioactive peptide endothelin 1 during and after cardioplegic arrest and cardiopulmonary bypass have been well documented. However, endothelin 1 is synthesized locally, and therefore myocardial endothelin 1 production during and after cardiopulmonary bypass remains unknown. METHODS: Pigs (n = 11) were instrumented for cardiopulmonary bypass, and cardioplegic arrest was initiated. Myocardial interstitial and systemic arterial levels of endothelin 1 were measured before cardiopulmonary bypass, throughout bypass and cardioplegic arrest (90 minutes), and up to 90 minutes after separation from bypass. Myocardial interstitial endothelin 1 was determined by microdialysis and radioimmunoassay. RESULTS: Baseline myocardial endothelin 1 levels were higher than systemic endothelin 1 levels (25.6 +/- 6.7 vs 8.3 +/- 1.1 fmol/mL, P <.05). With the onset of bypass, myocardial endothelin 1 increased by 327% +/- 92% from baseline (P <.05), which preceded the increase in systemic endothelin 1 levels. CONCLUSION: Myocardial compartmentalization of endothelin 1 exists in vivo. Cardiopulmonary bypass and cardioplegic arrest induce temporal differences in endothelin 1 levels within the myocardial interstitium and systemic circulation, which, in turn, may influence left ventricular function in the postbypass period.

ET-1 in the myocardial interstitium: relation to myocyte ECE activity and expression.

Increased plasma levels of endothelin-1 (ET-1) have been identified in congestive heart failure (CHF), but local myocardial interstitial ET-1 levels and the relation to determinants of ET-1 synthesis remain to be defined. Accordingly, myocardial interstitial ET-1 levels and myocyte endothelin-converting enzyme (ECE)-1 activity and expression with the development of CHF were examined. Pigs were instrumented with a microdialysis system to measure myocardial interstitial ET-1 levels with pacing CHF (240 beats/min, 3 wk; n = 9) and in controls (n = 14). Plasma ET-1 was increased with CHF (15 +/- 1 vs. 9 +/- 1 fmol/ml, P < 0.05) as was total myocardial ET-1 content (90 +/- 15 vs. 35 +/- 5 fmol/g, P < 0.05). Paradoxically, myocardial interstitial ET-1 was decreased in CHF (32 +/- 4 vs. 21 +/- 2 fmol/ml, P < 0.05), which indicated increased ET-1 uptake by the left ventricular (LV) myocardium with CHF. In isolated LV myocyte preparations, ECE-1 activity was increased by twofold with CHF (P < 0.05). In LV myocytes, both ECE-1a and ECE-1c mRNAs were detected, and ECE-1a expression was upregulated fivefold in CHF myocytes (P < 0.05). In conclusion, this study demonstrated compartmentalization of ET-1 in the myocardial interstitium and enhanced ET-1 uptake with CHF. Thus a local ET-1 system exists at the level of the myocyte, and determinants of ET-1 biosynthesis are selectively regulated within this myocardial compartment in CHF.

Effects of gene deletion of the tissue inhibitor of the matrix metalloproteinase-type 1 (TIMP-1) on left ventricular geometry and function in mice.

Alterations in the expression and activity of the matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in tissue remodeling in a number of disease states. One of the better characterized TIMPs, TIMP-1, has been shown to bind to active MMPs and to regulate the MMP activational process. The goal of this study was to determine whether deletion of the TIMP-1 gene in mice, which in turn would remove TIMP-1 expression in LV myocardium, would produce time-dependent effects on LV geometry and function. Age-matched sibling mice (129Sv) deficient in the TIMP-1 gene (TIMP-1 knock-out (TIMP-1 KO), n=10) and wild-type mice (n=10) underwent comparative echocardiographic studies at 1 and 4 months of age. LV catheterization studies were performed at 4 months and the LV harvested for histomorphometric studies. LV end-diastolic volume and mass increased (18+/-4 and 38+/-3%, respectively, P<0.05) at 4 months in the TIMP-1 KO group; a significant increase compared to wild-type controls (P<0.05). At 4 months, LV and end-diastolic wall stress was increased by over two-fold in the TIMP-1 KO compared to wild type (P<0.05). However, LV systolic pressure and ejection performance were unchanged in the two groups of mice. LV myocyte cross-sectional area was unchanged in the TIMP-1 KO mice compared to controls, but myocardial fibrillar collagen content was reduced. Changes in LV geometry occurred in TIMP-1 deficient mice and these results suggest that constitutive TIMP-1 expression participates in the maintenance of normal LV myocardial structure.

Bradykinin degradation and relation to myocyte contractility.

BACKGROUND: Past studies have demonstrated that exogenous bradykinin (BK) causes vasodilation and increases coronary blood flow, effects that may be beneficial in the setting of cardiac disease states. An important pathway for BK degradation is through angiotensin-converting enzyme (ACE), which results in the formation of a degradative peptide, BK((1-7)). The goal of this study was to examine the effects of BK, BK((1-7)), and the potential modulation of BK by ACE inhibition on myocyte contractility. METHODS AND RESULTS: Contractile function was examined in isolated adult porcine (n = 15) left ventricular (LV) myocyte preparations in the presence or absence of BK (10(-8) mol/L), BK((1-7)) (10(-8) mol/L), and with pretreatment by ACE inhibition (benazaprilat). Myocyte velocity of shortening fell by over 15% in the presence of BK and by 8% with BK((1-7)) (P <.05 vs basal). ACE inhibition blunted the negative effect of BK on myocyte velocity of shortening by over 60% (P <.05). Furthermore, robust ACE activity coupled with significant BK degradation was demonstrated in LV-isolated myocyte preparations, and BK proteolysis was influenced by ACE inhibition. CONCLUSION: These results suggest that BK has a direct effect on LV myocyte contractility, and that this effect may be mediated by proteolysis of BK at the level of the LV myocyte sarcolemma.

Effects of angiotensin type-I receptor blockade on pericardial fibrosis.

BACKGROUND: Reoperative cardiac surgical procedures are associated with a significantly greater complication rate than that of the initial procedure. Enhanced collagen synthesis can occur due to increased production of angiotensin II (Ang-II) and subsequent activation of Ang AT(1) receptor. Accordingly, the goal of the current study is to test the hypothesis that increased Ang AT(1) receptor activity following pericardiotomy contributes to pericardial thickening and fibrosis. MATERIALS AND METHODS: Adult pigs were randomly assigned to three protocols: (1) pericardiotomy with 28-day recovery period (n = 5); (2) pericardiotomy with Ang AT(1) receptor blockade instituted throughout the 28-day recovery period using 60 mg/day valsartan (n = 5); and (3) sham controls (n = 6). Pericardium samples were collected and analyzed by biochemical and histomorphometrical methods. Pericardial fibrosis occurred postpericardiotomy as indicated by increased hydroxyproline content from normal value of 50 +/- 3 microg/mg to 75 +/- 4 microg/mg (P < 0. 05). RESULTS: Pericardial thickness was increased postpericardiotomy to 2.7 +/- 0.4 mm compared to normal values of 0.4 +/- 0.05 mm (P < 0.05). Ang AT(1) receptor blockade reduced pericardial thickness by 50% and the relative degree of fibrosis was comparable to that of the normal group. CONCLUSIONS: The results from this pericardial fibrosis animal model suggest that Ang AT(1) receptor activation contributes to the development of pericardial thickening and collagen accumulation in the postoperative period. Thus, Ang AT(1) receptor inhibition may provide a novel therapeutic strategy to prevent pericardial fibrosis that follows cardiac surgical procedures.