Relationship of hydraulic impedance and elasticity in the pulmonary artery of maturing newborn pigs.

The current study determined the dynamic stress-strain elastic moduli (E(Y)) and characteristic impedances (Z(0(2-7Hz))) of the main pulmonary artery in open-chest, anesthetized newborn pigs at 2 days, 2 weeks, and 3 months of age. E(Y) and Z(0(2-7Hz)) were compared to those values derived from the Womersley and Moens-Korteweg equations (denoted E(W-MK) and Z(0W-MK), respectively) to test the validity of these equations in describing the elasticity of the intact newborn pulmonary artery. E(Y) was defined as the ratio of stress to strain. The current study hypothesized that: (1) E(Y) and E(W-MK) are numerically similar, and therefore the Womersley and Moens-Korteweg equations accurately describe the viscoelastic properties of the main pulmonary artery of the newborn pig, and (2) that both E(Y) and Z(0) are elevated at birth and undergo a steady decline with maturation. E(Y) was not significantly different from E(W-MK), while Z(0(2-7Hz)) was nearly identical to Z(0W-MK) in all groups. The elastic modulus peaked (P < 0.001) in 2-week-old pigs compared with both younger and older animals, while Z(0(2-7Hz)) decreased with increasing age (48 h = 1237 +/- 251 [SEM] dyn s cm(-5), 2-week = 433 +/- 95 dyn s cm(-5), 3 month = 162 +/- 17 dyn s cm(-5), P < 0.001). These experiments validate the Womersley and Moens-Korteweg equations as accurately describing the elastic properties of the intact newborn pig pulmonary artery. These data also demonstrate that a diminution in Z(0) may occur even with increased wall stiffness, as observed in our 2-week-old pigs.

Characterization of the pulmonary arterial response to endothelin-1 and bosentan in neonatal pigs.

BACKGROUND: This study determined the pulmonary vascular responses to intravenous (IV) administration of endothelin-1 (ET-1) before and after an IV bolus of bosentan (Ro 47-0203), an endothelin receptor antagonist, in anesthetized open-chest 48-hour-old and 2-week-old Yorkshire pigs. METHODS: Eighteen 48-hour-old and 25 2-week-old pigs were randomly allocated to receive either (1) 400 ng x kg(-1) x min(-1) of ET-1 or (2) 5 mg/kg or 10 mg/kg of Ro 47-0203 followed by 400 ng x kg(-1) x min(-1) of ET-1 over a 10-minute interval. Pulmonary vascular resistance (PVR, dyne sec/cm(-5)), elastic modulus (E(Yo), dyne/cm2), and characteristic impedance (Zo) were determined (+/- SEM). RESULTS: In 48-hour-old pigs, ET-1 decreased pulmonary artery pressure (PAP, dyne/cm2; 21,317 +/- 1,833 versus 17,757 +/- 1,823; p = 0.003). In 2-week-old pigs, ET-1 elevated PAP (19,009 +/- 1,834 versus 21,935 +/- 2,104; p = 0.003) and PVR (1,624 +/- 254 versus 2,302 +/- 416; p = 0.001), whereas bosentan abolished the ET-1 induced pulmonary and systemic vasoconstriction. Neither agent altered E(Y) or Z(o). CONCLUSIONS: ET-1 caused a pulmonary depressor response in 48-hour-old pigs and a constrictor response in 2-week-old pigs, whereas bosentan inhibited the ET-1 induced pulmonary arteriolar vasoconstriction in 2-week-old pigs. The response to ET-1 changes from dilation in 48-hour-old pigs (neonates) to constriction in 2-week-old pigs (infants) suggests a maturational dependent alteration in ET receptors during the first 2 weeks of life. These data suggest that bosentan may have potential clinical application in the treatment of newborn pulmonary hypertensive episodes as it ablated ET-1 induced pulmonary vasoconstriction, while maintaining systemic pressure.

Interstitial cellular and matrix restoration of cardiac valves after cryopreservation.

OBJECTIVES: We previously characterized the porcine aortic leaflet interstitial cell phenotype as having both synthetic and contractile characteristics; that is, it is a myofibroblast. In this study we hypothesized (1) that the cryopreservation of aortic valves causes a significant reduction in cell density, (2) that it simultaneously causes alterations in representative components of extracellular matrix, and (3) that both of these processes are reversible. METHODS: Seventy-two leaflets from 24 porcine aortic valves were studied. Whole valves were subjected to variable lengths of preharvest ischemia (group 1), ischemia followed by processing analogous to clinical methods (group 2), and ischemia followed processing with an organ culture type of resuscitation (group 3). Vital dye exclusion by cells enzymatically dispersed from leaflets was used to quantify viability. Electron and light microscopy, immunohistochemical assay, and a silicone rubber substratum contractility assay were used both in dispersed cell preparations and in leaflet cross sections to examine structural, ultrastructural, and functional changes across the 3 groups through a range of preharvest ischemic times. RESULTS: Results indicated that harvest ischemic periods between 2 and 24 hours after donor death were not responsible for cell number reductions. During this interval overt dissolution of chondroitin sulfate simultaneous with a relative sparing of fibronectin was evidenced by immunohistochemical staining. Although not reduced in number, ischemic interstitial cells did show significant ultrastructural evidence of injury and suppressed monoclonal binding to vimentin and alpha-smooth muscle actin. After cryopreservation, viable cell numbers were always markedly reduced at all ischemic intervals and damage to both soluble extracellular matrix components and cell ultrastructure was increased. At all time and processing points, however, some retention of matrix secretory and cellular contractile capabilities was observed among the surviving cells. After the extended periods of preharvest ischemia (2-24 hours) followed by processing, a restitution of functioning cells was accomplished by means of whole-leaflet incubation in 15% fetal bovine serum. CONCLUSIONS: After application of the described methods, new cells within restored intact leaflets as well as in single-cell preparations demonstrated normal ultrastructure and contractile and synthetic functions (normal phenotypic expression). If functioning leaflet interstitial cells can contribute to homograft durability, bioengineering methods for pretransplantation cell repopulation could be refined with these techniques and applied to clinical valve transplantation.

Sympathetic blockade blunts hypercapnic pulmonary arterial vasoconstriction in newborn piglets.

OBJECTIVE: Hypercapnia has been implicated in the pathophysiology of pulmonary hypertensive disease in newborns. However, little has been done to determine how its vasoconstrictive actions are mediated. The purpose of this study is to define the role of the sympathetic nervous system in modulating the response of the newborn pulmonary circulation to hypercapnia. Specifically, we studied the effect of sympathetic blockade on mean and pulsatile pulmonary arterial hemodynamics in 48-h-old, intact, open-chest Yorkshire piglets during hypercapnic ventilation. METHODS: All animals were anesthetized and then instrumented for high fidelity measurement of pulmonary artery pressure (PAP), flow (PAF), aortic pressure and radius of the main pulmonary artery (Rmn). Baseline data were acquired in all animals. Control animals (n = 7) were subjected to 30 s intervals of hypercapnia (inspired CO2 fraction (FiCO2) = 0.20). Experimental animals (n = 7) were pre-treated with an intravenous bolus of the adrenergic blocking agent guanethidine (20 mg/kg) before being subjected to the hypercapnic stress. Characteristic impedance (Zo) and input mean impedance (Zm) were determined through application of a Fourier analysis of the PAP and PAF waveforms. The modulus of elasticity (Ey) was calculated from Zo and Rmn. Pulmonary vascular resistance (PVR) was calculated as (PAP - LAP/PAF). RESULTS: Control animals underwent significant increases in PVR (4860 +/- 341 dyne cm s(-5) versus 8090 +/- 387 dyne cm s(-5), P < 0.01) and Zm (7215 +/- 495 dyne cm s(-5) versus 10228 +/- 993 dyne cm s(-5), P < 0.01) when exposed to hypercapnia. Pre-treatment with guanethidine attenuated this response (PVR, 5552 +/- 368 dyne cm s(-5) versus 7105 +/- 611 dyne cm s(-5), P = 0.31 and ZM, 7922 +/- 446 dyne cm s(-5) versus 9745 +/- 600 dyne cm s(-5), P = 0.31). Characteristic impedance, modulus of elasticity and the radius of the main pulmonary artery were unchanged in both groups. CONCLUSIONS: These data indicate that vasoconstriction secondary to hypercapnia in the neonatal pulmonary arterial circulation occurs at the level of the distal arteriolar bed, rather than the more proximal pulmonary arteries. In addition, this response is partially modulated by the sympathetic nervous system and may therefore respond clinically to manipulation of sympathetic input to the pulmonary arterial circulation.

The role of N omega-nitro-L-arginine in modulation of pulmonary vascular tone in the maturing newborn pig.

Current therapeutic modalities for treatment of newborn pulmonary hypertensive crisis include but are not limited to the administration of nitric oxide (endothelium-derived relaxing factor). However, few data are available on the role of endogenously produced endothelium-derived relaxing factor in the modulation of pulmonary vascular tone in the neonate. In the current study, we investigated the acute effects of N omega-nitro-L-arginine (a potent competitive inhibitor of endothelium-derived relaxing factor synthase) on the pulmonary vasculature of anesthesized open-chest 48-hour-old (n = 8) and 2-week-old (n = 7) Yorkshire pigs. After baseline data were acquired, all animals received a 10 mg/kg per minute infusion of N omega-nitro-L-arginine for 10 minutes. To discern distal and proximal pulmonary arterial vessel changes, input mean and characteristic impedance were respectively determined. Pulmonary vascular resistance was also calculated (units determined in dyne.sec.cm-5 plus or minus the standard error of the mean). Results showed N omega-nitro-L-arginine infusion did not significantly alter baseline pulmonary arterial pressure (22,370 +/- 1473 dyne/cm2), pulmonary vascular resistance (5171 +/- 805 dyne.sec.cm-5), input impedance (6343 +/- 806 dyne.sec.cm-5), or characteristic impedance (2073 +/- 418 dyne.sec.cm-5) in 48-hour-old pigs. In 2-week-old pigs, infusion of N omega-nitro-L-arginine elevated pulmonary arterial pressure (18,162 +/- 1415 dyne/cm2 versus 23,838 +/- 1810 dyne/cm2, p = 0.015), pulmonary vascular resistance (810 +/- 137 dyne.sec.cm-5 versus 1519 dyne.sec.cm-5, p = 0.030), and input impedance (2302 +/- 251 dyne.sec.cm-5 versus 2900 +/- 255 dyne.sec.cm-5, p = 0.018). Characteristic impedance was not altered in 2-week-old pigs. These data indicate that N omega-nitro-L-arginine infusion resulted in pulmonary arteriolar vasoconstriction in 2-week-old pigs, but not in 48-hour-old pigs. This finding suggests that endothelium-derived relaxing factor does not modulate basal pulmonary arteriolar tone during the early newborn period, but does play a significant role in 2-week-old pigs. These data also suggest that the functional role for endothelium-derived relaxing factor is confined to the distal arteriolar pulmonary bed and does not extend to the larger proximal arterial vessels.

Adenine nucleotide depletion in cryopreserved human cardiac valves: the "stunned" leaflet interstitial cell population.

Preparation protocols for human cardiac valves are intended to minimize cytotoxicity because it has been thought that viable leaflet interstitial cells may enhance homograft durability. Preimplantation factors influencing the status of these cells at the time of transplantation include ischemia, disinfection, and cryopreservation freezing programs. In these experiments, adenine nucleotide quantitation was undertaken to assess metabolic consequences of preparation; preharvest ischemia served as an independent variable to examine the relationship between time of procurement (postmortem) and high-energy phosphate status of the cryopreserved leaflets at thaw. Nucleotides were measured using high-performance liquid chromatography performed on extracts of semilunar cusps from 25 cryopreserved human valves with documented ischemic times. Results indicate total adenine nucleotides (TAN; [ATP] + [ADP] + [AMP], in nmol TAN/mg leaflet protein) are higher (P < 0.05) after < 2 h of harvest ischemia (1.16 +/- 0.36) than with ischemic times of 3-6 h (undetected), 7-12 h (0.18 +/- 0.07), and 13-20 h (0.06 +/- 0.06). Depletion of ATP was similar, with many leaflets devoid of detectable levels. Net utilization of leaflet energy stores demonstrates time dependency when assayed after completed processing. However, relatively elevated catabolites, even with brief ischemia, and infrequently identified ATP, ADP, and AMP, suggest a consumption so accelerated that the following cryopreservation it is virtually independent of procurement-associated ischemia. We conclude resumption of a functional cell population obligates significant de novo phosphoanhydride boned reformation or a repopulation of dead/dying interstitial cells from a subset surviving the apparently severe rigors of valve preparation.

Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblast.

The cellular properties of semilunar cardiac valve leaflets may be more complex than previously assumed. In particular, the cells of the leaflet matrix which are likely critical to proper cusp function are a poorly described population to date. We hypothesized that, similar to the matrix cells of atrioventricular valves, aortic valve leaflet interstitial cells (AoLIC's) possess characteristics of both fibroblasts (matrix secretion) and smooth muscle cells (contraction). Porcine AoLIC's were structurally examined for contractile and stress fiber protein assemblies using transmission electron microscopy and immunocytochemistry. Contractile function in response to vasoactive stimuli was directly assessed using AoLIC's cultured on flame-polymerized silicone, with cell contraction identified by the appearance of wrinkles in the substratum after challenge with each agent. The structural analyses showed cellular microfilaments were often organized into various contractile arrangements including polygonal networks, and that AoLIC's are rich in smooth muscle-specific alpha-actin. Incomplete basal laminae often associated with myofibroblasts were observed. Contraction experiments indicated a responsivity of similar latency, but variable peak and duration to 10(-7) M L-epinephrine, 3.2 x 10(-7) M angiotensin II, 110 microM carbachol, 50 mM KCl, 3.2 x 10(-7) M bradykinin, 110 microM isoproterenol, and 5 x 10(-7) M endothelin I. Soluble and insoluble matrix secretion was confirmed with FITC-conjugated monoclonal antibodies to chondroitin sulfate, fibronectin, and prolyl-4-hydroxylase. These data show that the AoLIC's are best designated as myofibroblasts. The unusual features of the myofibroblast may be central to lifelong aortic leaflet durability.

Pulmonary hydraulic impedance responses to hypoxia and hypercapnia in newborn pigs.

The purpose of this study was to determine the cumulative effects of brief intervals of hypoxia and hypercapnia on the pulsatile characteristics of the pulmonary arterial circulation of 48-h-old compared with 2-wk-old open-chest Yorkshire pigs while using two different anesthetic regimens: 1) azaperone and ketamine (4 and 12 mg/kg im, respectively) and 2) thiopental sodium (25 mg/kg i.v.). Animals 48 h old were randomly allocated to undergo mild hypoxia (inspired O2 fraction = 0.15), severe hypoxia (inspired O2 fraction = 0.05), or hypercapnia (inspired CO2 fraction = 0.20), whereas animals 2 wk old underwent severe hypoxia or hypercapnia. With use of Fourier analysis, characteristic impedance (Zo), mean input impedance (Zm), impedance moduli, and phase angles were determined. In 48-h-old pigs anesthetized with azaperone-ketamine, neither mild nor severe hypoxia altered Zo, Zm, or pulmonary vascular resistance (PVR), whereas hypercapnia increased Zo by 22% (P < 0.001), which persisted despite a return to normocapnia. In 48-h-old animals anesthetized with thiopental, baseline control Zo and Zm were lower than those in same-age pigs anesthetized with azaperone-ketamine. In thiopental-anesthetized 48-h-old pigs, both severe hypoxia and hypercapnia increased Zm and PVR but Zo was unaltered. In 2-wk-old pigs anesthetized with thiopental, severe hypoxia but not hypercapnia elevated Zm and PVR, whereas Zo was not changed with either stress. Results indicate age- and anesthetic-dependent responses of Zo, Zm, and PVR to severe hypoxia and hypercapnia. The persistent elevation in Zo caused by hypercapnia indicates a prolonged decrease in arterial compliance or a reduction in effective proximal pulmonary arterial radius.

Pulmonary hydraulic impedance response to cromakalim (BRL 34915) in newborn pigs.

Cromakalim (BRL 34915), a novel vasodilator, is used clinically to manage systemic hypertension. Its effects on the intact newborn pulmonary circulation remain unclear. The purpose of this study was to determine the response of both the mean and pulsatile pulmonary hemodynamic parameters to intravenous infusion of BRL 34915 (0.1 mg/kg/min for 10 min) in 48-hr-old open-chest Yorkshire pigs. Animals were divided into two groups: Group I pigs had a high mean baseline pulmonary artery pressure (PAP) (> 17 mm Hg), while Group II pigs had a low to normal baseline mean PAP (< 17 mm Hg). Following instrumentation for high-fidelity recordings of PAP and flow, baseline data were acquired. Employing Fourier analysis, characteristic impedance (Zo), input mean impedance (Zm), impedance harmonic moduli (units in dyn sec cm-5), and phase angles (radians) were determined. Pulmonary vascular resistance (PVR) was calculated and aortic pressure (AP, mm Hg) was also measured. All values = mean +/- SEM. Group I animals ("high tone") underwent a decrease in PAP (17.9 +/- 1.6 mm Hg vs 13.6 +/- 1.9 mm Hg, P = 0.008) and PVR (4310 +/- 816 dyn sec cm-5 vs 3713 +/- 687 dyn sec cm-5, P = 0.04). Group II animals ("low tone") responded with an increase in PAP (11.5 +/- 0.4 mm Hg vs 16.6 +/- 1.8 mm Hg, P = 0.029). AP decreased in Group I (40 +/- 3.8 mm Hg vs 21.5 +/- 2.8 mm Hg, P = 0.008) and Group II (51.2 +/- 10.8 mm Hg vs 31.2 +/- 10.9 mm Hg, P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of adenosine deaminase and nucleoside transport. Utility in a model of homograft cardiac valve preimplantation processing.

Human cardiac valves are increasingly used in the reconstruction of ventricular outflow tracts and offer performance advantages over porcine and mechanical prostheses; the durability of these replacements has been associated with leaflet interstitial cell viability and a presumed sustained function after implantation. Preimplantation tissue preparation entails sequential steps that are potentially cytotoxic and may therefore affect functional cell survival at thaw. We defined the metabolic consequences of each interval using semilunar cusps from 118 porcine valves to model a homograft preparation with 40 minutes of fixed cadaveric (harvest) ischemia. Fifty-eight valves served as controls and were first processed according to standard cryopreservation protocol; nucleosides were extracted at the end of each step to differentiate independent contributions to high-energy phosphate depletion. Sixty simultaneously harvested leaflets were administered the nucleoside transport inhibitor p-nitrobenzy-thionosine (NBMPR) and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) at procurement, to attempt adenosine salvage and restitution of processing-incurred adenine nucleotide losses. High-performance liquid chromatography was used to compare adenosine triphosphate, diphosphate, and monophosphate and diffusible nucleopurines of the control and EHNA/NBMPR-treated groups. Control results indicate that disruption of the adenosine triphosphate-diphosphate cycle occurs independently with antibiotic disinfection and cryopreservation. However, throughout all preparation steps, adenine nucleotides were maintained at harvest (baseline) concentrations in the EHNA/NBMPR valves. This suggests that salvage therapy may protect a significant number of cells from net high-energy phosphate catabolism. If, with further study, the durability of transplanted valves is concluded to benefit from retained leaflet interstitial cell viability, such enhancement of metabolic tolerance to the obligatory processing may facilitate functional recovery.

Preimplantation alteration of adenine nucleotides in cryopreserved heart valves.

To assess the initial metabolic phase of cellular injury from cardiac valve processing, high-energy phosphate concentrations were analyzed in valve leaflets subsequent to critical processing steps. Using a porcine model, valves were processed in a manner identical to human homografts, with 58 randomly assigned to five groups representing distinct preparation phases. Group I (controls) sustained 40 minutes of warm ischemia concluded by liquid nitrogen immersion. Remaining groups similarly endured 40 minutes of ischemia, but were subsequently prepared according to stepwise design: II, warm ischemia + 24 hours of 4 degrees C ischemia; III, warm ischemia + 24 hours of 4 degrees C antibiotic disinfection; IV, warm ischemia + 24 hours at 4 degrees C (without antibiotics) + cryopreservation (-1 degrees C/min cryoprotected freezing); and V, warm ischemia+disinfection+cryopreservation. At each regimen's conclusion leaflet extracts were assayed by high-performance liquid chromatography for high-energy adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate) and catabolites. A 47% and 86% decrease in cellular adenosine triphosphate level was observed in group III and group V leaflets, respectively. The level of total adenine nucleotides was maintained up to cryopreservation; thereafter a 74% decrease was noted. Catabolite analysis confirmed incomplete degradation of adenine nucleotides indicating cellular metabolic resilience throughout standard homograft preparation in valves previously exposed to 40 minutes of warm ischemia.

Human cryopreserved homografts: electron microscopic analysis of cellular injury.

Twenty-five human cryopreserved valves with harvest-related warm ischemic times (WITs) ranging from 0 to 20 hours were studied using transmission electron microscopy to characterize the effects of harvesting and preservation on leaflet matrix cells. The valves were divided into seven groups on the basis of WIT and processed using standard transmission electron microscopic methods. Each cell (528 micrographs) was graded for reversible and irreversible cellular injury and subjected to a Cochran-Mantel-Haenszel trend analysis. Our results demonstrated a progression in cellular injury with increasing WIT. During the first 12 hours of warm ischemia, reversible cellular injury predominated (0.0%, 30.0%, 51.2%, 31.3%, 35.1%, 45.1%, and 40.0% at WITs of 0, 1, 2, 8, 12, 16, and 20 hours, respectively). A positive correlation (p < 0.0001) between increasing WIT and reversible cellular injury through the first 12 hours was observed. Minimal morphologic evidence of irreversible injury was noted in valves harvested with less than 12 hours of warm ischemia; however, after 12 hours there was a marked increase (0.0%, 0.0%, 4.7%, 2.4%, 2.7%, 31.4%, and 40.0% at WITs of 0, 1, 2, 8, 12, 16, and 20 hours, respectively) in irreversible cellular injury (p < 0.001 between 12 and 20 hours WIT). These data demonstrate a progression in cellular injury with increasing WIT. There was virtually no morphologic injury in valves with harvest-related WITs less than 2 hours and minimal irreversible cellular injury observed in valves exposed to 12 hours or less of warm ischemia. If cellular viability is critical to homograft durability then harvest-related warm ischemia may need to be restricted to 12 hours.

High energy phosphate depletion in leaflet matrix cells during processing of cryopreserved cardiac valves.

Preimplantation preparation of cardiac valves includes three major steps: (1) harvesting with accompanying ischemia (warm time from cessation of donor heart beat), (2) antibiotic disinfection, and (3) controlled-rate cryopreservation. To define the interdependent injury effects of these manipulations on leaflet matrix cells and specifically the potential for prolonged harvest-related ischemia to predispose greater injury by the subsequent steps, 96 semilunar valves were harvested from pigs in a manner analogous to human heart valve retrievals and randomly allocated to study groups as follows: 48 control valves were exposed to increasing harvested-related ischemic times, (2, 6, 12, 24 hr) and immersed in liquid nitrogen to arrest metabolic activity (i.e., prior to cryopreservation) and conclude the ischemia; another 48 were similarly harvested, subjected to identical ischemic times, then disinfected in 4 degrees C RPMI medium with standard antibiotics for 24 hr and dimethylsulfoxide cryopreserved at -1 degrees C/min to -170 degrees C (i.e., formal cryopreservation protocol). At thawing, each valve was extracted in 12% trichloroacetic acid and assayed by high performance liquid chromatography for components of the adenine nucleotide pool including ATP, lower energy nucleotides (total adenine nucleotides, [TAN] = [ATP] + [ADP] + [AMP]), adenosine, and the diffusible purines. Results are reported as nanomoles metabolite/milligram of leaflet cell protein (Lowry) and reflect a maintenance of total high energy phosphates in the control groups (5.41 +/- 0.29 nmole TAN at 2 hr; 8.34 +/- 0.67 nmole TAN at 24 hr), which fell significantly in all cryopreserved groups (1.27 +/- 0.33 nmole TAN at 2 hr; 0.34 +/- 0.22 nmole TAN at 24 hr).(ABSTRACT TRUNCATED AT 250 WORDS)