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We previously described a transgenic mouse line (alpha(q)*52) in which cardiac-specific expression of activated G alpha(q)protein (HA alpha(q)*) leads to activation of phospholipase C beta (PLC beta), the immediate downstream target of HA alpha(q)*, with subsequent development of cardiac hypertrophy and dilation. We now describe a second, independent line in the same genetic background (alpha(q)*44h) with lower expression of HA alpha(q)* protein that ultimately results in the same phenotype: dilated cardiomyopathy (DCM) with severely impaired left ventricular systolic function (assessed by M-mode and 2D echocardiography), but with a much delayed disease onset. We asked if PLC activation correlates with the development of the phenotype. At 12-14 months, 65% of alpha(q)*44h mice still had normal cardiac function and ventricular weight/body weight ratios (VW/BW). However, their basal PLC activity, which began to increase in ventricles at 6 months, was threefold higher than in wild-type by 12 months. This increase was even more pronounced than in 2.5-month-old alpha(q)*52 mice, in which a twofold increase was accompanied by a 25% increase in VW/BW. Furthermore, at 12-14 months the increase in PLC activity in alpha(q)*44h mice with and without DCM was comparable. Thus, the delayed time course in alpha(q)*44h mice unmasked a lack of correlation between PLC activation and development of DCM in response to HA alpha(q)* expression, suggesting a role for additional pathways and/or mechanisms. It also revealed a differential temporal regulation of protein kinase C isoform expression. The markedly different ages of disease onset in these two mouse lines provide a model for studying both genetic modifying factors and potential environmental influences in DCM.

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.

Background —Unstable atherosclerotic plaques that cause acute coronary events usually contain abundant macrophages expressing matrix metalloproteinases (MMPs) and tissue factor (TF), molecules that probably contribute to plaque rupture and subsequent thrombus formation. Lipid lowering with HMG-CoA reductase inhibitors reduces acute coronary events. Methods and Results —To test whether lipid lowering with an HMG-CoA reductase inhibitor retards macrophage accumulation in rabbit atheroma, we administered cerivastatin to immature Watanabe heritable hyperlipidemic rabbits (cerivastatin group, n=10, cerivastatin 0.6 mg · kg −1 · d −1 ; control group, n=9, saline 0.6 mL · kg −1 · d −1 ) for 32 weeks and measured macrophage accumulation and expression of MMPs and TF. Serum cholesterol levels after 32 weeks were 809±40 mg/dL (control group) and 481±24 mg/dL (treated group). Cerivastatin diminished accumulation of macrophages in aortic atheroma. Macrophage expression of MMP-1, MMP-3, MMP-9, and TF also decreased with cerivastatin treatment. Cerivastatin reduced the number of macrophages expressing histone mRNA (a sensitive marker of cell proliferation) detected by in situ hybridization but did not alter macrophages bearing a marker of death (TUNEL staining). Cerivastatin treatment (≥0.01 μmol/L) also reduced growth, proteolytic activity due to MMP-9, and TF expression in cultured human monocyte/macrophages. Conclusions —These results suggest that lipid lowering with HMG-CoA reductase inhibitors alters plaque biology by reducing proliferation and activation of macrophages, prominent sources of molecules responsible for plaque instability and thrombogenicity.

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (alphaMHC(403/+)), when treated with calcineurin inhibitors or a K(+)-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Background —Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. Methods and Results —We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 μm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell–seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were Conclusions —Tissue-engineered heart valve scaffolds fabricated from polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP-GlcNAc: Galbeta1-3GalNAcalphaRbeta1-6-N-acetylglucosaminyltransferas e (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc-T with severe increase in cardiac core 2 GlcNAc-T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O-glycan and increases of c-fos gene expression and AP-1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc-T also induced c-fos promoter activation, mitogen activated-protein kinase (MAPK) phosphorylation, Trk receptor glycosylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc-T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.-Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.

Background —Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. Methods and Results —Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1– and MMP-13–positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. Conclusions —This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

Background —biomechanical forces generated by blood flow within the cardiovascular system have been proposed as important modulators of regional endothelial phenotype and function. This process is thought to involve the regulation of vascular gene expression by physiological fluid mechanical stimuli such as fluid shear stresses. Methods and Results —We demonstrate sustained upregulation of a recently identified gene encoding a human prostaglandin transporter ( hPGT ) in cultured human vascular endothelium exposed to a physiological fluid mechanical stimulus in vitro. This biomechanical induction is selective in that steady laminar shear stress is sufficient to upregulate the hPGT gene at the level of transcriptional activation, whereas a comparable level of turbulent shear stress (a nonphysiological stimulus) is not. Various biochemical stimuli, such as bacterial endotoxin and the inflammatory cytokines recombinant human interleukin 1β cytokines (rhIL-1β) and tumor necrosis factor-α (TNF-α), did not significantly induce hPGT . Using a specific antiserum to hPGT, we demonstrate endothelial expression within the arterial vasculature and the microcirculation of highly vascularized tissues such as the heart. Conclusions —Our results identify hPGT as an inducible gene in vascular endothelium and suggest that biomechanical stimuli generated by blood flow in vivo may be important determinants of hPGT expression. Furthermore, this demonstration of regulated endothelial expression of hPGT implicates this molecule in the regional metabolism of prostanoids within the cardiovascular system.

Abstract —Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive α-smooth muscle actin, SM1, and SM2. α-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the α-actin-positive intimal area occupied by smooth muscle myosin–positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially Gq, whose α subunit activates phospholipase Cβ (PLCβ). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the Gqα subunit (HAα*q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HAα*qprotein is expressed at less than 50% of endogenous αq/11, and the transgenic hearts are essentially normal morphologically. Although HAα*qprotein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous αq/11rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous αq/11. We conclude that the pathological changes initiated by early, transient HAα*qexpression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998)Cell93, 215–228]. Because HAα*qacts upstream of calcineurin, we hypothesized that HAα*qmight initiate additional pathways leading to hypertrophy and dilatation. Treating HAα*qmice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.

Aminooleic acid treatment has been demonstrated to prevent porcine valve calcification and to protect valvular hemodynamic function. Initial enthusiasm was tempered by histologic studies of these AOA valves, which showed cuspal hematomas, structural loosening, and surface roughening. This prompted a systematic review of the AOA treatment process. Unsolubilized particles of alpha aminooleic acid present in the treatment solution were identified as the cause of mechanical abrasion of valve cusps during processing. These particles were eliminated with a revamped protocol, which included filtration of the AOA solution before valve preparation.Porcine aortic valve cusps treated with this modified AOA protocol (AOA II) were studied in a rat subdermal implant model of mineralization. A juvenile sheep trial was then used to confirm the antimineralization effects of AOA II on glutaraldehyde-fixed porcine aortic roots in a circulatory model of accelerated calcification.Retrieved AOA II-treated cusps from the subdermal model were markedly less calcified than control cusps (AOA II, 1 +/- 0, 17 +/- 4, 23 +/- 6, and 17 +/- 10 versus control, 189 +/- 14, 251 +/- 16, 250 +/- 14, and 265 +/- 10 mg calcium/mg sample at 4, 8, 12, and 16 weeks, respectively; p0.0001). Morphologic examination of the AOA II cusps of the valves retrieved from the sheep demonstrated freedom from the structural loosening, surface roughening, and hematoma formation that had limited the utility of the original AOA preparation technique. Cusps from AOA II-treated porcine roots had significantly less calcium than control cusps (AOA II, 5.5 +/- 3.0 mg/g; control, 91.2 +/- 19.5 mg/g; p = 0.0004). The aortic walls had similar levels of calcification (AOA II, 156 +/- 73 mg/g; control, 159 +/- 10 mg/g; p = not significant).These data suggest that the modified AOA technique warrants further evaluation as an antimineralization treatment for glutaraldehyde-fixed porcine bioprostheses.

Purpose: To investigate the ECM-related gene expression and thrombogenicity of endothelial cells (EC) grown on electrospun fibrous scaffolds; to elucidate the role of surface topology versus matrix composition on the modulation of ECMrelated differential gene expression and EC thrombogenicity. Methods: Human aortic endothelial cells (HAECs) were cultured for 24 h on glass cover slips coated with electrospun (PLGA-GelatinElastin–PGE) mats (PGE, 3D), PGE solution (PGE film, 2D) and various defined ECM protein solutions. ECM-related gene expression of HAECs was evaluated for cells cultured on PGE, PGE film, gelatin and collagen IV via a focused PCR microarray, which surveyed 84 genes encoding ECM and adhesion molecules. Both basal and TNF-α induced tissue factor (TF) mRNA expression and protein activity levels were assessed for HAECs cultured on all substrates. Results: HAECs cultured for 24 h on 3D electrospun PGE fibrous scaffolds were able to form confluent monolayers, expressing similar level of VE-cadherin, as they do on their 2D counterparts (PGE film) and other protein substrates. A differential expression “profile” of HAECs cultured on PGE scaffolds was observed as compared to cells on other substrates examined; cells on PGE scaffolds were more quiescent and passivated than those on gelatin. In addition, cells cultured on PGE scaffolds expressed TF mRNA levels similar to those on gelatin and exhibited similar TF activity levels as HAECS cultured on collagen IV. Conclusions: HAECs grown on electrospun PGE scaffolds essentially exhibited “healthy” phenotype and were more quiescent and less thrombogenic than those on gelatin.

Background. The purpose of this study was to determine whether cerebral cortical microvascular responses to platelet-derived vasoactive substances are altered after normothermic cardiopulmonary bypass (CPB), and whether these alterations are modified by moderate hypothermia. Methods. Pigs were placed on normothermic CPB (37°C) for 2 hours and then perfused off CPB with normothermic blood for either 15 minutes (n = 8) or 60 minutes (n = 6). Another group was placed on moderately hypothermic CPB (25°C) for 2 hours and then perfused off CPB at 37°C for 15 minutes (n = 6). Alpha-stat pH management was used. In vitro responses of isolated cortical cerebral arterioles (90 to 170 μ m internal diameter) to platelet-derived vasoactive substances were examined in a pressurized no-flow state with video-microscopy. Microvessels from noninstrumented pigs (n = 14) were used as controls for in vitro studies. Results. Cerebrovascular resistance and internal carotid artery blood flow were similar 15 minutes after CPB in both normothermic and hypothermic groups. However, relaxations of microvessels to adenosine 5′ diphosphate or serotonin were reduced in vessels from both groups. One hour of after CPB cerebral perfusion did not change this pattern of altered vascular reactivity. Hypothermia caused a partial but significant reduction in impairment of responses to adenosine 5′ diphosphate and serotonin. Microvascular relaxation to the endothelium-independent agent sodium nitroprusside and contraction to a thromboxane A 2 analog were similar in all experimental groups, suggesting normal vascular smooth muscle responsiveness. Conclusions. This study demonstrates that normothermic extracorporeal circulation reduces endothelium-dependent relaxation responses to products of platelet activation in the cerebral microcirculation. Moderate hypothermia attenuates the CPB-induced impairment of endothelium-dependent relaxation, but has no effect on baseline cerebral blood flow after rewarming.

Background The frequency and clinical significance of perioperative ischemic myocardial injury (PIMI) after heart transplantation and the diagnostic features distinguishing PIMI from rejection are not well defined. Methods and Results We evaluated PIMI in the first four weekly endomyocardial biopsies and/or autopsy myocardium from 140 consecutive orthotopic heart transplantation recipients (1984 to 1991) by grading the severity of coagulative myocyte necrosis (CMN) as absent, 0; mild-focal, 1; moderate-multifocal, 2; or severe-confluent, 3, and determining the evolution of morphological features of its healing. CMN (often with contraction bands) was noted in 124 patients (89%); 24 patients (17%) had grade 3 CMN, of which 4 died within 30 days of transplantation. Nevertheless, at 1 year after surgery, survival was similar in patients with and without severe injury. Increased cold ischemic time but neither donor age nor intensity of inotropic support correlated with more severe early ischemic injury. PIMI inflammation was characterized by a predominantly polymorphonuclear/histiocytic infiltrate that contained lymphocytes and plasma cells, expanding the interstitium but not encroaching upon and separable from adjacent viable myocytes. Histological features of PIMI developed and resolved more slowly than those of typical myocardial infarct necrosis in nonimmunosuppressed patients; at 4 weeks, CMN persisted in 20% of patients and residual healing in nearly half. Diagnostic rejection was observed concurrently with PIMI in 54 of 533 biopsies (10%). Conclusions Diagnosed by conventional histological criteria, PIMI is prevalent early after heart transplantation and has a protracted healing phase that can mimic or coexist with rejection. Extensive PIMI has deleterious impact on short-term survival, but the long-term impact of PIMI remains to be established.

Publisher Summary This chapter discusses proteins, its structure, its properties, and its adsorption top surfaces. The importance of proteins in biomaterials science stems primarily from their inherent tendency to deposit on surfaces as a tightly bound adsorbate and the strong influence these deposits have on subsequent cellular interactions with the surfaces. The chapter discusses cells, their surfaces, and their interactions with materials. It also describes tissues. Cells, tissues, and organs are adapted to the organized and regulated activities that makeup body functions. The key concepts of biological structure–function correlation include compartmentalization, differentiation, the basic tissues, organs, regeneration following injury, and multicellular communication. Various techniques are available for observing tissue structure and function. The microscopic study of tissue slices, called histology, is the most important tool used to investigate functional tissue architecture in clinical or laboratory investigation.

Publisher Summary Artificial organs comprise complex medical devices that have active mechanical or biochemical functions such as heart, lung, kidney, liver, pancreas, or neurosensory organs. Artificial organs can be either surgically implanted or extra corporeal (in which blood is temporarily processed outside the patient's body). Although the range of devices that constitute artificial organs is at present limited in clinical use, considerable research and development has involved devices that have active mechanical, biologic, or mass exchange functions. In this logical extension of biomaterials science, often called tissue engineering, the cells are either transplanted or induced in the recipient by the implantation of an appropriate resorbable or a permanent substrate. Congestive heart failure is the only class of heart disease that has continued to increase in incidence. Total artificial hearts (TAHs) and ventricular assist devices (VADs) offer the greatest potential to fill this clinical need by providing permanent cardiac assist or replacement. The initial designs of TAHs focused on mimicking the natural heart. All TAHs must fulfill specific criteria necessary for successful application in man. These areas of design are (1) adequate volume of blood pumping necessary to meet the physiologic needs of the recipient, (2) proper anatomic alignment in relation to recipient structures that transport blood entering and exiting the TAH, (3) lack of interference with other organs and maintenance of the ability to approximate the chest wall structures, and (4) avoidance of any complications caused directly or indirectly by the TAH.

Publisher Summary This chapter describes biomaterials used in implanted medical devices—cardiovascular, dental, ophthalmological, and orthopedic prostheses. The most frequent surgical procedure performed upon humans is the dental extraction. Without question, the most frequently employed prosthesis is the complete or partial dentures. These are available as both fixed and removable devices. Most often they are supplied in the latter form owing to their relative ease of production as well as economy. Materials implanted into the body of a human or an animal must be sterile to avoid subsequent infection that can lead to serious illness or death. The chapter describes various sterilization methods, including their advantages and disadvantages, and the validation of sterilization processes from the point of view of sterility as well as the integrity of the physical and biological properties of the implant. The first sterilization method to be used for implants was autoclaving, which involves exposure to saturated steam under pressure. The first concern when choosing a sterilization method is the documentation that the method is compatible with the implant itself as well as the packaging required to maintain its sterile integrity prior to delivery to the operative site.

Publisher Summary This chapter introduces biodegradation issues for a number of classes of materials. Biodegradation is the chemical breakdown of materials by the action of living organisms that leads to changes in physical properties. It is a concept of vast scope, ranging from the decomposition of environmental waste involving microorganisms to the host-induced deterioration of biomaterials in implanted medical devices. The calcification of biomaterial implants is an important pathologic process affecting a variety of tissue-derived biomaterials as well as synthetic polymers in various functional configurations. The pathophysiology has been partially characterized with a number of useful animal models; a key common feature is the involvement of devitalized cells and cellular debris. To understand the biological degradation of implant materials, synergistic pathways should be considered. Degradation products can alter the local pH, stimulating further reaction. The hydrolysis of polymers can generate more hydrophilic species leading to polymer swelling and entry of degrading species into the bulk of the polymer.

Background The relation between episodes of acute rejection and the development of graft coronary arteriosclerosis remains controversial. We examined the hypothesis that acute rejection episodes accelerate graft coronary arteriosclerosis lesion formation in rabbit allografts. Methods and Results A control group (n=5) received cyclosporine 5 mg · kg −1 · d −1 for 6 weeks after heterotopic heart transplantation. In a rejection group (n=5), cyclosporine was omitted for 4 days at 1 and 4 weeks after transplantation. We studied cross sections of grafted hearts at 6 weeks and evaluated myocardial rejection grade, incidence, and severity and cell composition of intimal lesions in multiple coronary artery profiles. Episodic withdrawal of cyclosporine augmented myocardial rejection (International Society for Heart and Lung Transplantation grades 0, 0, 0, 0, and 1A in the control group to grades 1A, 1B, 2, 3A, and 3B in the rejection group). Episodes of acute rejection significantly increased the incidence (7.8±2.7% to 49.7±1.9%) and severity (from grade 0.10±0.04 to 0.79±0.24) of intimal thickening in graft coronary arteries. Most intimal lesions consisted of smooth muscle cells and contained various degrees of T-lymphocyte infiltration but sparse macrophages. Conclusions In this experimental model, episodes of acute rejection precipitated by cyclosporine withdrawal accelerated the development of graft vascular lesion formation. Activation of vascular cells and leukocyte recruitment during acute rejection may thus contribute to the pathogenesis of graft arteriosclerosis.

This communication briefly summarizes a morphologic investigation of explanted cryopreserved heart valves and discusses the findings in the context of ongoing debates regarding modes of failure, cellular viability, durability of the extracellular matrix, and the contribution of immune responses. We studied 20 cryopreserved human heart valve allografts functioning up to 9 years as either orthotopic aortic valves/root replacements or right ventricle-to-pulmonary artery conduits explanted for valve failure, infection, or growth-related conduit or valve stenosis. Implanted valves had progressively severe loss of normal layered structure and were devoid of stainable deep connective tissue cells. Inflammation was minimal. Other late findings included minimal inflammation, mild cuspal hematomas, mural thrombus, and calcification, most prominent in the aortic wall. Transmission electron microscopy of late expiants revealed nonviable cells and their debris, and a collagenous skeleton that was largely intact. We conclude that cryopreserved allograft heart valves have minimal, if any, viable cells, but largely retain the original collagen network; preservation of the autolysis-resistant collagenous skeleton likely provides the structural basis of function. Our results also suggest that immune responsiveness has little, if any, impact on late allograft function or degradation.

Pathologists are likely to encounter substitute heart valves with increasing frequency. Informed evaluation of such valves provides valuable information that contributes to both patient care and our understanding of the pathobiology of host interactions with mechanical devices. This article summarizes the most important considerations underlying such analyses-including valve identification, common morphologic features and modes of failure, technical details of evaluation, and potential pitfalls.