Search

Background and Purpose— Recent symptoms stand as a major determinant of stroke risk in patients with carotid stenosis, likely reflective of atherosclerotic plaque destabilization. In view of emerging links between vascular and adipose biology, we hypothesized that human perivascular adipose characteristics associate with carotid disease symptom status. Methods— Clinical history, carotid plaques, blood, and subcutaneous and perivascular adipose tissues were prospectively collected from patients undergoing carotid endarterectomy. Nine adipose-associated biological mediators were assayed and compared in patients with symptomatic (n=15) versus asymptomatic (n=19) disease. Bonferroni correction was performed for multiple testing ( α /9=0.006). Results— Symptomatic patients had 1.9-fold higher perivascular adiponectin levels ( P =0.005). Other circulating, subcutaneous, and perivascular biomarkers, as well as microscopic plaque characteristics, did not differ between symptomatic and asymptomatic patients. Conclusions— Symptomatic and asymptomatic carotid endarterectomy patients display a tissue-specific difference in perivascular adipose adiponectin. This difference, which was not seen in plasma or subcutaneous compartments, supports a potential local paracrine relationship with vascular disease processes that may be related to stroke mechanisms.

Background Ascending thoracic aortic aneurysm ( ATAA ) is driven by angiotensin II (Ang II ) and contributes to the development of left ventricular ( LV ) remodeling through aortoventricular coupling. We previously showed that locally available leptin augments Ang II ‐induced abdominal aortic aneurysms in apolipoprotein E–deficient mice. We hypothesized that locally synthesized leptin mediates Ang II ‐induced ATAA . Methods and Results Following demonstration of leptin synthesis in samples of human ATAA associated with different etiologies, we modeled in situ leptin expression in apolipoprotein E–deficient mice by applying exogenous leptin on the surface of the ascending aorta. This treatment resulted in local aortic stiffening and dilation, LV hypertrophy, and thickening of aortic/mitral valve leaflets. Similar results were obtained in an Ang II ‐infusion ATAA mouse model. To test the dependence of Ang II ‐induced aortic and LV remodeling on leptin activity, a leptin antagonist was applied to the ascending aorta in Ang II ‐infused mice. Locally applied single low‐dose leptin antagonist moderated Ang II ‐induced ascending aortic dilation and protected mice from ATAA rupture. Furthermore, LV hypertrophy was attenuated and thickening of aortic valve leaflets was moderated. Last, analysis of human aortic valve stenosis leaflets revealed de novo leptin synthesis, whereas exogenous leptin stimulated proliferation and promoted mineralization of human valve interstitial cells in culture. Conclusions Ang II ‐induced ATAA is mediated by locally synthesized leptin. Aortoventricular hemodynamic coupling drives LV hypertrophy and promotes early aortic valve lesions, possibly mediated by valvular in situ leptin synthesis. Clinical implementation of local leptin antagonist therapy may attenuate Ang II ‐induced ATAA and moderate related LV hypertrophy and pre–aortic valve stenosis lesions. Clinical Trial Registration URL : https://www.clinicaltrials.gov/ . Unique identifier: NCT 00449306.

Calcific aortic valve disease (CAVD) encompasses the range of disease from initial alterations in the cell biology of the leaflets to end-stage calcification resulting in left ventricular outflow obstruction. The first detectable macroscopic changes in the leaflets, seen as calcification, or focal leaflet thickening with normal valve function, is termed aortic valve sclerosis, but it is likely that the initiating events in the disease process occur much earlier. Disease progression is characterized by a process of thickening of the valve leaflets and the formation of calcium nodules—often including the formation of actual bone—and new blood vessels, which are concentrated near the aortic surface. End-stage disease, eg, calcific aortic stenosis, is characterized pathologically by large nodular calcific masses within the aortic cusps that protrude along the aortic surface into the sinuses of Valsalva, interfering with opening of the cusps. There is no disease along the ventricular surface. For decades, this disease was thought to be a passive process in which the valve degenerates with age in association with calcium accumulation. Moreover, although CAVD is more common with age, it is not an inevitable consequence of aging. Instead, CAVD appears to be an actively regulated disease process that cannot be characterized exclusively as senile or degenerative. The National Heart, Lung, and Blood Institute convened a group of scientists from different fields of study, including cardiac imaging, molecular biology, cardiovascular pathology, epidemiology, cell biology, endocrinology, bioengineering, and clinical outcomes, to review the scientific studies from the past decade in the field of CAVD. The purpose was to develop a consensus statement on the current state of translational research related to CAVD. Herein, we summarize recent scientific studies and define future directions for research to diagnose, treat, and potentially prevent this complex disease process. ### Key Structure-Function Correlations Heart valves permit unobstructed, unidirectional forward flow through the circulation. …

Introduction: Recent symptoms stand as a major determinant of stroke risk in carotid stenosis patients, likely reflective of atherosclerotic plaque destabilization. Hypothesis: In view of emerging links between vascular and adipose biology, we hypothesized that human perivascular adipose characteristics associate with carotid disease symptom status. Methods: Clinical history, carotid plaques, blood, and subcutaneous and perivascular adipose tissues were prospectively collected from consented patients undergoing carotid endarterectomy (CEA). Nine adipose associated biologic mediators (adiponectin, IL-1 beta, IL-6, IL-8, leptin, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, resistin, and tumor necrosis factor) protein levels were quantified, and compared in patients with symptomatic (n=15) versus asymptomatic (n=19) disease. Bonferroni correction was performed for multiple testing (alpha/9=0.006). Results: Symptomatic patients had 1.9-fold higher perivascular adiponectin levels (median [interquartile range] of symptomatic versus asymptomatic patients was 478.4 ng/ml [315.4-603.9 ng/ml] versus 251.9 ng/ml [212.7-365.4ng/ml], p=0.005). Other circulating, subcutaneous, and perivascular biomarkers, as well as microscopic plaque characteristics, did not differ between symptomatic and asymptomatic patients. Conclusions: Symptomatic and asymptomatic CEA patients display a tissue-specific difference in perivascular adipose adiponectin. This biomarker signature, which was not seen in plasma or subcutaneous compartments, supports a potential local paracrine relationship to vascular disease processes which may relate to stroke mechanisms. Future study of these adipose based paracrine effects may point to novel therapeutic targets and inform patient selection for preventative interventions.

In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-β 2 , individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF- and TGFβ 2 –induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGFβ 2 , a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGFβ 2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGFβ 2 but were resistant to endothelial-inducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves.

Background— Development and validation of novel imaging modalities to assess the composition of human atherosclerotic plaques will improve the understanding of atheroma evolution and could facilitate evaluation of therapeutic strategies for plaque modification. Surface MRI can characterize tissue content of carotid but not deeper arteries. This study evaluated the usefulness of intravascular MRI (IVMRI) to discern the composition of human iliac arteries in vivo. Methods and Results— Initial studies validated IVMRI against histopathology of human atherosclerotic arteries ex vivo. A 0.030-inch-diameter IVMRI detector coil was advanced into isolated human aortoiliac arteries and coupled to a 1.5-T scanner. Information from combined T1-, moderate T2-, and proton-density–weighted images differentiated lipid, fibrous, and calcified components with favorable sensitivity and specificity and allowed accurate quantification of plaque size. The validated approach was then applied to image iliac arteries of 25 human subjects in vivo, and results were compared with those of intravascular ultrasound (IVUS). IVMRI readily visualized inner and outer plaque boundaries in all arteries, even those with extensive calcification that precluded IVUS interpretation. It also revealed the expected heterogeneity of atherosclerotic plaque content that was noted during ex vivo validation. Again, IVUS did not disclose this heterogeneity. The level of interobserver and intraobserver agreement in the interpretation of plaque composition was high for IVMRI but poor for IVUS. Conclusions— IVMRI can reliably identify plaque composition and size in arteries deep within the body. Identification of plaque components by IVMRI in vivo has important implications for the understanding and modification of human atherosclerosis.

Background— An estimated 275 000 patients undergo heart valve replacement each year. However, existing solutions for valve replacement are complicated by the morbidity associated with lifelong anticoagulation of mechanical valves and the limited durability of bioprostheses. Recent advances in tissue engineering and our understanding of stem cell biology may provide a lifelong solution to these problems. Methods and Results— Mesenchymal stem cells were isolated from ovine bone marrow and characterized by their morphology and antigen expression through immunocytochemistry, flow cytometry, and capacity to differentiate into multiple cell lineages. A biodegradable scaffold was developed and characterized by its tensile strength and stiffness as a function of time in cell-conditioned medium. Autologous semilunar heart valves were then created in vitro using mesenchymal stem cells and the biodegradable scaffold and were implanted into the pulmonary position of sheep on cardiopulmonary bypass. The valves were evaluated by echocardiography at implantation and after 4 months in vivo. Valves were explanted at 4 and 8 months and examined by histology and immunohistochemistry. Valves displayed a maximum instantaneous gradient of 17.2±1.33 mm Hg, a mean gradient of 9.7±1.3 mm Hg, an effective orifice area of 1.35±0.17 cm 2 , and trivial or mild regurgitation at implantation. Gradients changed little over 4 months of follow-up. Histology showed disposition of extracellular matrix and distribution of cell phenotypes in the engineered valves reminiscent of that in native pulmonary valves. Conclusions— Stem-cell tissue-engineered heart valves can be created from mesenchymal stem cells in combination with a biodegradable scaffold and function satisfactorily in vivo for periods of >4 months. Furthermore, such valves undergo extensive remodeling in vivo to resemble native heart valves.

Abstract —Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C ( MyBP-C ) gene usually have a better prognosis than individuals bearing β -cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (αMHC 403/+ or a cardiac MyBP-C mutation (MyBP-C t/+ ) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, αMHC 403/+ mice demonstrated considerably more LV hypertrophy than MyBP-C t/+ mice. In older heterozygous mice, hypertrophy continued to be more severe in the αMHC 403/+ mice than in the MyBP-C t/+ mice. Consistent with this finding, hearts from 50-week-old αMHC 403/+ mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C t/+ hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C t/+ mice are not as likely to have inducible ventricular tachycardia as αMHC 403/+ mice. In addition, cardiac function of αMHC 403/+ mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C t/+ mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

Abstract —Advanced atherosclerosis is often associated with dystrophic calcification, which may contribute to plaque rupture and thrombosis. In this work, the localization and association of the noncollagenous bone matrix proteins osteonectin, osteopontin, and osteocalcin with calcification, lipoproteins, thrombus/hemorrhage (T/H), and matrix metalloproteinases (MMPs) in human carotid arteries from endarterectomy samples have been determined. According to the recent American Heart Association classification, 6 of the advanced lesions studied were type V (fibroatheroma) and 16 type VI (complicated). Osteonectin, osteocalcin, and osteopontin were identified by monoclonal antibodies IIIA 3 A 8 , G12, and MPIIIB10 1 and antiserum LF-123. Apolipoprotein (apo) AI, B, and E; lipoprotein(a); fibrinogen; fibrin; fragment D/D-dimer; MMP-2 (gelatinase A); and MMP-3 (stromelysin-1) were identified with previously characterized antibodies. Calcium phosphate deposits (von Kossa’s stain) were present in 82% of samples (3 type V and 15 type VI). Osteonectin was localized in endothelial cells, SMCs, and macrophages and was associated with calcium deposits in 33% of type V and 88% of type VI lesions. Osteopontin was distributed similarly to osteonectin and was associated with calcium deposits in 50% of type V and 94% of type VI lesions. Osteocalcin was localized in large calcified areas only (in 17% of type V and 38% of type VI lesions). ApoB colocalized with cholesterol crystals and calcium deposits. Lipoprotein(a) was localized in the intima, subintima, and plaque shoulder. Fibrin (T/H) colocalized with bone matrix proteins in 33% of type V and 69% of type VI lesions. MMP-3 was cytoplasmic in most cells and colocalized with calcium and fibrin deposits. MMP-2 was less often associated with calcification. The results of this study show that osteonectin, osteopontin, and osteocalcin colocalized with calcium deposits with apoB, fibrin, and MMP-3 in advanced, symptomatic carotid lesions. These data suggest that the occurrence of T/H might contribute to dystrophic arterial calcification in the progression and complications of atherosclerosis.

Abstract We created a mouse that lacks a functional α-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous α-tropomyosin “knockout” mice die between embryonic day 9.5 and 13.5 and lack α-tropomyosin mRNA. Heterozygous α-tropomyosin knockout mice have ≈50% as much cardiac α-tropomyosin mRNA as wild-type littermates but similar α-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous α-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the α-tropomyosin gene produces little or no change in cardiac function or structure, whereas total α-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibrillar tropomyosin despite the reduction in α-tropomyosin mRNA.

Background Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. Methods and Results Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87±0.29 μg/mg tissue compared with control calcium level, 236.00±6.10 μg/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22±2.94 μg/mg tissue; control calcium level, 32.50±11.50 μg/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. Conclusions Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Atherosclerotic plaque rupture may occur when regions of weakened extracellular matrix are subjected to increased mechanical stresses. Since collagen is a major determinant of extracellular matrix strength, enzymes that degrade collagen may play an important role in destabilizing the atherosclerotic lesion. To test the hypothesis that matrix metalloproteinase 1 (interstitial collagenase, or MMP-1), which initiates degradation of fibrillar collagens, colocalizes with increased stress in the fibrous cap of the atherosclerotic lesion, 12 unruptured human coronary lesions were studied. Finite-element analysis was used to determine the distribution of stress in the lesion, with estimates of material properties from previous measurements of human tissues. A computerized image analysis system was used to determine the distribution of immunoreactive MMP-1 within the fibrous tissue of the lesion. There was a significant correlation between immunoreactive MMP-1 and circumferential tensile stress in the fibrous cap within a given lesion (median Spearman rank correlation coefficient, .36; interquartile range, −.02 to .81; P

Background The rate of progression and potential long-term consequences of isolated foci of moderate acute rejection (FMR) on endomyocardial biopsy (EMB) have not been defined; therefore, whether FMR necessitates augmented immunosuppression remains controversial. Methods and Results At our institution, recipients with EMBs having FMR, defined as one or two isolated foci of cellular infiltrates with associated myocyte damage (International Society for Heart and Lung Transplantation [ISHLT] grade 2 and a subset of grade 3A), do not routinely receive intensified immunosuppression. Accordingly, to determine the outcome of untreated FMR, we reviewed 4398 EMBs (mean, 4.4 samples each) obtained after orthotopic heart transplantation in 208 consecutive recipients maintained on triple immunosuppressive therapy. The incidence of progression versus resolution of FMR, the time interval after transplantation when FMR was detected, and the relation of untreated FMR to recipient survival were analyzed. FMR categorized as one (n=312) or two (n=89) foci was present in 401 EMBs (9% of total) obtained 10 days to 7.5 years after transplantation from 149 recipients (72%). EMBs with FMR resolved without treatment in 341 of 401 (85%), and only 60 of 401 (15%) progressed to higher grade rejection. EMBs that progressed occurred 7.5±7.9 months (mean±SD) after transplantation compared with 14.0±16.5 months after transplantation for those that resolved ( P Conclusions First, untreated FMR consisting of either one or two foci has a low rate of progression. Second, progression of FMR decreases with increasing postoperative interval and becomes rare after 2 years. Last, FMR progression did not identify recipients with decreased survival.

We conducted a prospective longitudinal study to determine the clinical significance of endothelial adhesion molecule expression in endomyocardial biopsies from human cardiac allografts. Ten to 18 (mean 13) consecutive allograft biopsies were obtained from 20 serial human transplant recipients over a one-year period. A total of 267 biopsies was examined. The expression of endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, as well as the presence of CD3+ T cell infiltrates was assessed by immunocytochemical staining of frozen sections. Separate specimens taken at the same time were analyzed histologically for ischemic injury or rejection. ICAM-1--and, to a lesser extent VCAM-1--was expressed at low levels in normal biopsies. E-selectin was only expressed in 15% of histologically normal biopsy specimens. Ischemic injury noted in the immediate posttransplant period was associated with increased expression of all three adhesion molecules. VCAM-1 expression increased both with the degree of CD3+ T cell infiltrates (P < 0.001) and with the degree of rejection (P < 0.05). ICAM-1 increased over constitutive levels in association with diffuse CD3+ infiltrates (P < 0.001) and with rejection (P < 0.05). E-selectin was increased on occasional vessels in association with CD3+ infiltrates (P < 0.001), but was not associated with active rejection. Increases in E-selectin were most likely to occur in biopsies just prior to rejection episodes (odds ratio 3.3), and were least likely to occur in biopsies following rejection (odds ratio 0.3). ICAM-1, but not VCAM-1, was also elevated in prerejection specimens. VCAM-1 and ICAM-1 declined in postrejection specimens. These data suggest a dynamic pattern in the expression of endothelial cell adhesion molecules during the course of cardiac allograft rejection. This study also suggests that endothelial E-selectin expression may be a useful clinical marker of impending rejection. Finally, inducible VCAM-1 expression may be a helpful adjunct in the diagnosis of ongoing acute rejection, and decreases in its expression may be indicative of successful antirejection therapy.

BACKGROUND Calcification is a frequent cause of the clinical failures of glutaraldehyde-pretreated bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-cross-linked porcine aortic valves. 2-Amino oleic acid (AOA) has been shown in previous in vivo studies to be a promising anticalcification agent. Our objective was to investigate the mechanism of calcification inhibition mediated by AOA pretreatment of porcine aortic valve bioprostheses. METHODS AND RESULTS BPHV tissues were treated with an AOA solution for 72 hours before experimentation. The diffusion of AOA across both cusp and aortic wall was evaluated. The lag time for AOA to diffuse across the aortic wall was prolonged compared with that of the cusp. An extraction study was performed to determine the stability of AOA binding; the results indicated that the binding was relatively stable regardless of solvent extraction conditions. The interaction between ionic calcium and AOA on treated tissue also was investigated by evaluating the patterns of calcium diffusion across both treated and untreated tissues. The results showed that AOA significantly reduced the diffusion of calcium. AOA inhibition of aortic valve calcification (calcium level, 5.5 +/- 3.0 mg/g of tissue compared with control; calcium level, 91.2 +/- 19.5 mg/g of tissue) but not aortic wall (calcium level, 158.7 +/- 10.3 mg/g of tissue compared with control; calcium level, 157.5 +/- 7.9 mg/g of tissue) was demonstrated on representative specimens from valves implanted in left ventricular apicoaortic shunts explanted after 150 days. CONCLUSIONS AOA covalently binds to glutaraldehyde-pretreated bioprosthetic heart valve tissue, presumably as the result of an aldehyde-amino reaction. Covalently bound AOA diminishes Ca2+ diffusion compared with non-AOA-pretreated bioprosthetic tissues. This may explain in part the anticalcification mechanism of AOA. Furthermore, AOA inhibits calcification of porcine BPHV cusps in the circulation.

BACKGROUND Acute rejection may be suspected in heart transplant recipients in the setting of new onset of clinical symptoms or alterations in cardiac function. Immediate diagnosis may be obtained by performing a frozen section on endomyocardial biopsy (EMB) specimens. However, little is known about the indications for, and the diagnostic reliability of, this procedure. METHODS AND RESULTS EMBs with frozen section (n = 98) from 65 of 214 consecutive orthotopic heart transplant recipients were reviewed and divided into early (< or = 45 days; n = 47) and late (> 45 days; n = 51) posttransplant periods. Frozen section diagnoses (means = 1.5 EMB samples) were compared with corresponding permanent section diagnoses (means = 4.4 EMB samples), and clinical indications were analyzed. Comparison of frozen and permanent section interpretation revealed concordant pathological processes-rejection (n = 31) versus no rejection (n = 37) versus ischemic injury (n = 20)-in 88 of 98 (90%) cases. Discordant pathological processes on frozen versus permanent section in 10 of 98 (10%) cases could be attributed to ischemic injury (n = 5), sampling (n = 4), and infection (n = 1). In the 92 cases with defined clinical indications, the indication and number of EMBs positive for rejection early and late after transplantation were arrhythmia: 2 of 12 early, 4 of 10 late; congestive heart failure: 1 of 2 early, 5 of 12 late; fever: 0 of 2 early, 1 of 4 late; echo abnormality: 0 of 5 early, 0 of 1 late; syncope: 1 of 5 early, 0 of 1 late; hypotension: 1 of 3 early, 1 of 2 late; noncompliance: 0 of 0 early, 4 of 5 late; more than one of the above: 3 of 7 early, 2 of 5 late; other: 1 of 7 early, 1 of 9 late; total: 9 of 43 early, 18 of 49 late. CONCLUSIONS Frozen section on EMB specimens accurately reflected the permanent section diagnosis in 90% of cases. No specific clinical indication predicted EMB rejection positivity with high sensitivity in either the early or late posttransplant periods.

BACKGROUND The intracardiac conversion rate of angiotensin (Ang) I to Ang II and the expression of angiotensin converting enzyme (ACE) mRNA are amplified in rat hearts with left ventricular hypertrophy (LVH). To examine whether the accelerated intracardiac Ang II generation in LVH is related to an induction of cardiac ACE, we studied localization and function of cardiac ACE in hypertrophied rat hearts using specific ACE inhibitors. METHODS AND RESULTS Cardiac ACE was localized and quantified in hearts from male Wistar rats with LVH due to chronic experimental aortic stenosis and from control rats. With the ACE inhibitor 125I-351A, a derivative of lisinopril, as a radioligand on coronal sections of LVH and control hearts, in vitro autoradiography demonstrated ACE binding in aorta, coronary arteries, atria, and ventricles of both groups. Quantitative analyses revealed that ACE density (counts per minute per cross-sectional area of tissue) was twofold higher within the myocardium of hypertrophied left ventricles compared with controls (p < 0.005). Quantitative morphometry demonstrated a modest increase in the fractional volume of myocytes as well as capillary volume without an increase in the fractional volume of endothelial cells in left ventricular tissue from aortic stenosis rats. These data suggest that an increase in endothelial cell volume per se cannot alone account for the observed doubling of ACE density and support an upregulation of ACE production in hypertrophied tissue. The role of cardiac ACE in intracardiac conversion of Ang I to Ang II and its specific inhibition was studied in isolated, isovolumic beating, buffer-perfused LVH and control hearts. Biochemical conversion rates as well as functional changes in response to 3 x 10(-7) M Ang I were examined in the absence or presence of the ACE inhibitor enalaprilat (4 x 10(-6) M). After a brief stabilization period, groups of LVH and control hearts were subjected to the following infusion protocols: 15 minutes of vehicle followed by 30 minutes of Ang I plus vehicle, 15 minutes of enalaprilat followed by 30 minutes of Ang I plus enalaprilat (enal/Ang I), or 45 minutes of vehicle only to allow comparison with a time control. Intracardiac Ang I-to-Ang II conversion rate was fourfold higher in LVH than in control hearts (p < 0.05). Infusion of enalaprilat reduced the intracardiac Ang I-to-Ang II conversion rate in LVH hearts by 70% (p < 0.05 versus Ang I). At similar levels of constant coronary flow per gram, Ang I increased coronary perfusion pressure by 23 +/- 5 mm Hg (p < 0.01 versus vehicle) in LVH hearts and by 36 +/- 10 mm Hg (p < 0.005 versus vehicle) in control hearts. When enalaprilat was infused with Ang I, the increase in perfusion pressure was limited to 5 +/- 5 mm Hg (NS versus vehicle) in LVH hearts and 12 +/- 3 mm Hg (p < 0.05 versus vehicle) in control hearts and was significantly lower than in hearts infused with Ang I only (p < 0.05 in LVH and p < 0.05 in control hearts, respectively). Systolic function was not affected by either infusion protocol. In contrast, Ang I infusion was associated with diastolic dysfunction. In LVH hearts, left ventricular end-diastolic pressure (LVEDP) increased from 10 +/- 1 mm Hg at baseline to 25 +/- 2 mm Hg at the end of the Ang I infusion (p < 0.001 versus vehicle), which was inhibited by infusion of enalaprilat. In control hearts, there was a lesser increase in LVEDP from 10 +/- 1 mm Hg to 15 +/- 1 mm Hg in response to Ang I (p < 0.05 versus LVH). Control hearts treated with enalaprilat with Ang I displayed no increase in LVEDP: CONCLUSIONS These observations indicate that ACE protein is increased within the myocardium of LVH hearts, extending recent findings of increased cardiac ACE activity and mRNA levels in this model of pressure-overload LVH in the rat. Blockade of the enzyme by an ACE inhibitor decreases intracardiac Ang I-to-Ang II conversion rate and prevents the functional changes of Ang I-to-Ang II activation

BACKGROUND The coronary arteries of transplanted hearts frequently develop accelerated diffuse arteriosclerosis. The effects of this disease on resistance vessel function are unknown. METHODS AND RESULTS To investigate the integrity of endothelium-dependent small-vessel vasodilation in transplanted hearts, coronary blood flow (CBF) responses to the endothelium-dependent dilator acetylcholine (10(-8) to 10(-6) M) and the essentially endothelium-independent dilator adenosine (10(-6) to 10(-4) M) were assessed in 40 studies of 29 transplant patients 1-3 years after transplantation and in seven nontransplanted controls. CBF was measured at constant arterial pressure with a Doppler catheter in the left anterior descending coronary artery. Controls, year 1 transplant patients, and year 2 transplant patients had similar increases in CBF in response to acetylcholine (232 +/- 40%, 200 +/- 41%, and 201 +/- 54%, respectively; p = NS), whereas year 3 transplant patients had increased CBF of only 100 +/- 39% (p less than 0.05 versus controls). An index of the proportion of CBF reserve attributable to endothelium-dependent dilation was obtained by normalizing each patient's peak acetylcholine flow response by the peak adenosine flow response. In patients receiving both acetylcholine and adenosine, endothelium-dependent flow responses declined over time [57 +/- 9% in controls, 56 +/- 10% for year 1, 47 +/- 12% for year 2, and 29 +/- 9% for year 3 (p less than 0.05 versus controls)]. An increased mean cyclosporine level (range, 99-261 ng/ml) (r = 0.67, p = 0.004) and increased transplant recipient age (range, 20-63 years) (r = 0.51, p = 0.004) predicted a preserved endothelium-dependent microvascular response. CONCLUSIONS Thus, microvascular endothelium-dependent dilation deteriorates over time in the transplanted heart, which may reflect underlying graft arteriosclerosis and contribute to ischemic damage of the myocardium.

Calcification (CALC) is the most frequent cause of failure in bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated porcine aortic valve or bovine pericardium. Site specific controlled administration of ethanehydroxy diphosphonate (EHDP), using numerous carriers, has been successful in inhibiting CALC of BHV tissue in an accelerated rat subdermal model, without adverse effects on serum calcium, bone development, or overall somatic growth. The current study was designed to evaluate refillable reservoir devices fabricated from polyurethane (Biomer Woburn, MA) with regard to their transport properties relative to EHDP. The refillable reservoirs were evaluated for EHDP release in vitro at 22 degrees C (under perfect sink conditions) into both a physiologic receptor phase (N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, pH = 7.4) with (1.5 mM) or without Ca2+ present. Transport parameters for EHDP diffusion through the polyurethane membranes used to fabricate the reservoirs were significantly different for the two receptor phases evaluated. The mean diffusion coefficients (D) for EHDP through the polyurethane membrane into each receptor phase were not significantly different. However, an approximately 3.5-fold reduction was observed in the mean value of the partition coefficient (K) for EHDP when EHDP was evaluated for release into a receptor phase that contained 1.5 mM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravascular ultrasound may be useful for studying the natural history of atherosclerotic lesions of different morphologies and for guiding interventional strategies. This study was designed to test the hypothesis that tissue appearance by intravascular ultrasound is related to the biomechanical properties of atheroma components. Forty-three atheroma caps were obtained from the abdominal aortas of 22 patients at autopsy and studied with an ultrasensitive, servo-controlled spectrometer. By measuring the static strain caused by increasing levels of compressive stress from 30 to 90 mm Hg, the uniaxial unconfined compression stiffness (ratio of stress to strain) was determined. After mechanical testing, specimens were imaged with a 6F, 20-MHz intravascular ultrasound transducer, and images were interpreted by an investigator who was unaware of the mechanical measurements. Specimens were classified as nonfibrous (n = 14), fibrous (n = 18), or calcified (n = 11) based on intravascular ultrasound appearance. The static stiffnesses of the nonfibrous, fibrous, and calcified ultrasound classes were 41.2 +/- 18.8 kPa, 81.7 +/- 33.2 kPa, and 354.5 +/- 245.4 kPa, respectively (p = 0.0002 by analysis of variance). The times to reach static equilibrium (creep time) for the nonfibrous, fibrous, and calcified classes were 79.6 +/- 26.5 minutes, 50.2 +/- 20.0 minutes, and 19.4 +/- 8.1 minutes, respectively (p = 0.0007). Intravascular ultrasound appearance was most significantly related to biomechanical behavior when calcium deposits were noted; the differences in biomechanical behavior between nonfibrous and fibrous tissue appearances were less apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND Although thrombosis associated with a fissured atherosclerotic plaque is believed to be the most common cause of acute coronary syndromes, the underlying factors that trigger plaque rupture are currently unknown. However, the mechanical behavior of the plaque is probably of critical importance. METHODS AND RESULTS To test the hypothesis that the mechanical properties of a plaque are dependent on its composition and, in particular, that the stiffness of fibrous caps changes within the range of frequencies carried by a physiological pressure wave, the stress-strain relation was studied in 27 fibrous caps and related to the underlying histological structure of the fibrous cap. Fibrous caps were obtained during 14 autopsies from the abdominal aorta and were classified by histological examination as cellular (n = 7), hypocellular (n = 9), or calcified (n = 11). Hypocellular fibrous caps were 1-2 times stiffer than cellular caps (p less than 0.005), and calcified caps were 4-5 times stiffer than cellular caps (p less than 0.005). All 27 fibrous caps demonstrated an increase in stiffness with increasing frequencies of stress ranging from 0.05 to 10 Hz; the increase in stiffness was similar in all three histological classes. CONCLUSIONS We conclude that the stiffness of fibrous caps from human atherosclerotic plaques is related to the underlying histological structure and that the stiffness increases with frequency in the range of physiological heart rates. The protective benefit of beta-adrenergic receptor blocking agents in coronary artery disease may, in part, be related to the frequency dependence of atherosclerotic plaque stiffness.

In studies that attempt to measure intracellular calcium [( Ca2+]i) in the intact heart with the calcium indicator indo 1-AM, a fundamental assumption is that the signals report changes in myocyte [Ca2+]i. We studied isolated perfused rabbit hearts loaded with the calcium probe indo 1-AM and recorded surface fluorescence of the left ventricle during continuous excitation at 360 nm. In cells containing indo 1, an increase in [Ca2+]i is associated with an increase in fluorescence intensity at 400 nm, a decrease in intensity at 500 nm, and an increase in the 400:500 nm ratio. Beat-to-beat fluorescence transients were recorded from the surface of the heart coincident with contraction, indicating that a component of the fluorescence signals is derived from beating myocytes. To evaluate the potential contribution of endothelial cells, we compared the response to increases in [Ca2+]o or bradykinin (10(-5) M). In response to an increase of the [Ca2+] in the perfusate from 0.6 to 3.0 mM, left ventricular developed pressure and +dP/dt increased with a simultaneous increase in the [Ca2+]i-sensitive 400:500 nm ratio. Perfusion with the endothelial cell agonist bradykinin caused no change in left ventricular isovolumic peak systolic pressure or left ventricular dP/dt, whereas bradykinin evoked an immediate elevation in both the diastolic and systolic levels of the [Ca2+]i-sensitive 400:500 nm ratio. In additional experiments with indo 1-loaded isolated beating myocytes, superfusion with bradykinin had no effect on either the fluorescence [Ca2+]i transients or contractility. In contrast, superfusion of indo 1-loaded cultured endothelial cells with bradykinin caused the elevation of [Ca2+]i within seconds. Fluorescence microscopy of unstained frozen tissue sections from indo 1-loaded hearts also suggested the presence of more intense microvascular endothelial cell indo 1 fluorescence relative to that observed in myocytes. These experiments provide evidence that a component of [Ca2+]i-sensitive fluorescence of whole hearts loaded with indo 1 is contributed by nonmyocyte sources, including endothelial cells. These results also raise the caution that the abrupt rise of [Ca2+]i that has been observed during the initial phase of ischemia in whole hearts loaded with indo 1 may be partly derived from endothelial cells rather than myocytes.

Background —Previous tissue engineering approaches to create heart valves have been limited by the structural immaturity and mechanical properties of the valve constructs. This study used an in vitro pulse duplicator system to provide a biomimetic environment during tissue formation to yield more mature implantable heart valves derived from autologous tissue. Methods and Results —Trileaflet heart valves were fabricated from novel bioabsorbable polymers and sequentially seeded with autologous ovine myofibroblasts and endothelial cells. The constructs were grown for 14 days in a pulse duplicator in vitro system under gradually increasing flow and pressure conditions. By use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and the valve constructs were implanted into 6 lambs (weight 19±2.8 kg). All animals had uneventful postoperative courses, and the valves were explanted at 1 day and at 4, 6, 8, 16, and 20 weeks. Echocardiography demonstrated mobile functioning leaflets without stenosis, thrombus, or aneurysm up to 20 weeks. Histology (16 and 20 weeks) showed uniform layered cuspal tissue with endothelium. Environmental scanning electron microscopy revealed a confluent smooth valvular surface. Mechanical properties were comparable to those of native tissue at 20 weeks. Complete degradation of the polymers occurred by 8 weeks. Extracellular matrix content (collagen, glycosaminoglycans, and elastin) and DNA content increased to levels of native tissue and higher at 20 weeks. Conclusions —This study demonstrates in vitro generation of implantable complete living heart valves based on a biomimetic flow culture system. These autologous tissue-engineered valves functioned up to 5 months and resembled normal heart valves in microstructure, mechanical properties, and extracellular matrix formation.

Skeletal muscle cardiac assistance as a treatment modality for heart failure is considered a high-risk procedure subject to strict patient selection. The aim here is to develop minimally invasive techniques to improve surgical outcomes and increase clinical indications. Ten goats (45-55 kg) were studied. In six, the latissimus dorsi muscle (LDM) was harvested via an open technique on one side vs a minimally invasive technique on the other using video assistance through two 3 cm incisions. Surgical maneuvers and length of procedures were noted. Animals were recovered, observed daily for local complications, and killed after 1 week for comparative anatomic and histopathologic studies. In four other goats, minimally invasive aortomyoplasty or cardiomyoplasty was performed using video assistance (2 aortomyoplasty, 2 cardiomyoplasty). In this experimental series, there were no surgical complications. The minimally invasive LDM harvest required a mean of 81 min (range 55-116 mn) with no gross evidence of muscle damage. The technique of LDM harvesting was standardized and is reproducible. Aortic and cardiac wraping were also achieved through three ports and a left minithoracotomy of 4 cm, using the right or left LDM. A scarf technique for the descending aortomyoplasty using the left LDM, and an anterior wrapping for cardiomyoplasty using the left or right LDM was technically feasible with video assistance. This study suggests future clinical applicability.

In vitro (10 MHz) proton magnetic resonance (MR) was used to study myocardial infarction 30 hours after ligation of the left coronary artery in rats. The location of large anterolateral myocardial wall infarcts was confirmed when the rats were killed. In vitro measurements of control animals (n = 7) showed T1 prolongation in right as compared with left ventricles (559 +/- 41 vs. 522 +/- 35 milliseconds [sd]; P less than .02); this correlated with a higher water content and is believed to be caused by increased extracellular compartment size. Infarcted myocardium (n = 9) had lower T1 relaxation times than control left ventricle anterior walls (480 +/- 37 vs. 522 +/- 35 milliseconds; P less than .01) despite increased water content (78 +/- 1.1% vs. 76.5 +/- 6%; P less than .01) and a mild increase in T2 values. We conclude that the MR characteristics of myocardial infarction in rats differ from those of other models, possibly because of differences in infarct sizes and rates of proteolysis within the damaged tissues.

To determine the biocompatibility and thrombogenicity of nitinol blood clot filters, we inserted 27 nitinol wire devices into the venae cavae of 16 dogs and one sheep and studied the results angiographically and at autopsy after periods of one week to four years. Filter shape, location in the vena cava, wire cleaning procedure and wire surface finish were varied. All 18 cleaned nitinol wire filters remained patent by venogram, although some showed small venographic filling defects caused by adherent organized thrombi. Filters in larger veins tended to have less thrombus. Surface polishing and filter shape had no observable effect on thrombogenicity. Histologic study revealed patchy chronic inflammation at the surface of uncleaned filters but only benign fibrous tissue reaction for the cleaned filters. Neointimal tissue overgrowth was observed where nitinol wire contacted the wall of the vena cava. Two filters implanted for four years had no appreciable weight loss caused by corrosion. Platelet adhesion and plasma coagulation effects of nitinol wire were tested in vitro in human blood and found to be similar to those of stainless steel, which is used in comparable devices. These encouraging results suggest that nitinol may be a promising material for human intravascular prosthetic applications.

By virtue of its location at the interface of flowing blood and vascular tissue, the endothelial cell monolayer is in a unique position for interactions with soluble and cellular elements of the blood on one side and with component cells of the vascular tissue on the other. This brief review outlines humoral and contact-mediated endothelial communication with other cells, particularly the resident cells of the vessel wall. Evidence for gap junctional communication channels between endothelium and vascular cells is summarized and discussed in relation to endothelial ion channel activity. Myoendothelial gap junctional communication is proposed as a mechanism involved in vasorelaxation, either independent of or in concert with secreted endothelium-derived relaxing factor(s).

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.

Beat-to-beat heart rate variability was studied by power spectral analysis in 17 orthotopic cardiac transplant patients. Heart rate power spectra were calculated from eighty-four 256-second recordings and compared with those taken from six normal subjects. The power spectra from the control subjects resolved into discrete peaks at 0.04-0.12 Hz and 0.2-0.3 Hz, whereas those of heart transplant recipients resembled broad-band noise without peaks. Log total power in the 0.02-1.0 Hz range was greater in the control subjects (0.982 +/- 0.084 [0.206], mean +/- SEM [SD]) than in the transplanted subjects (-0.766 +/- 0.059 [0.541]), (p less than 0.0001). Fifty-five electrocardiographic recordings from transplant patients were done within 48 hours of an endomyocardial biopsy. When the power spectra of those patients whose endomyocardial biopsies showed evidence of myocardial rejection were compared with those from patients who were found to be free of rejection, a significant difference was found in log total power (-0.602 +/- 0.090 [0.525] vs. -0.909 +/- 0.136 [0.577], p less than 0.02). We conclude that denervation of the heart significantly reduces heart rate variability and abolishes the discrete spectral peaks seen in untransplanted control subjects and that the development of allograft rejection may significantly increase heart rate variability.