Acellular Endocardium as a Novel Biomaterial for the Intima of Tissue-Engineered Small-Caliber Vascular Grafts.

We aimed to investigate whether acellular endocardium can be used as a useful biomaterial for the intima of engineered small-caliber vascular grafts. Fresh endocardium was harvested from the swine left atrium and was decellularized by digestion with the decellularization solution of Triton X-100 and SDS containing DNase I and RNase A. Surface morphological characteristics and Young's modulus were evaluated. To analyze the effect of mechanical characteristics on cell adhesion, the decellularized endocardium was stiffened with 2.5% glutaraldehyde. Small-caliber vascular grafts were constructed using decellularized endocardium treated with or without glutaraldehyde as the intima. CD34+ cells were seeded onto the luminal surface of the vascular grafts and linked to bioreactors that simulate a pulsatile blood stream. Acellular endocardium had distinct surface morphological characteristics, which were quite different from those of other materials. The compliance of acellular endocardium was higher than that of other materials tested by Young's modulus. CD34+ cells formed a monolayer structure and adhered to the inner face of the acellular endocardium. The glutaraldehyde treatment stiffened the acellular endocardium but had little impact on the surface morphological characteristics or static adhesiveness of the cells. Data from the bioreactor study showed that the detachment of the cells from the surface of glutaraldehyde-treated acellular endocardium increased dramatically when the pressure was equal or higher than 40 mm Hg, while the cells on the untreated acellular endocardium remained well and formed confluent monolayers and tight junctions under the same pressure. Acellular endocardium has distinct structures and mechanical characteristics that are beneficial for CD34+ cell adhesion and retention under dynamic fluid perfusion. Thus, it can be used as a useful biomaterial for the construction of the intima of engineered small-caliber vascular grafts.

A 1-Year-Old with Mycobacterium tuberculosis Endocarditis with Mass Spectrometry Analysis of Cardiac Vegetation Composition.

In this study, we report the first case of Mycobacterium tuberculosis endocarditis in an immunocompetent child born in the United States. Mass spectrometry of the vegetation identified coagulation, humoral immune proteins, neutrophil granule proteins, and histones. Few neutrophils on histopathology suggest that neutrophil extracellular traps may contribute to tuberculous endocardiac mass formation.

Urinary bladder matrix promotes site appropriate tissue formation following right ventricle outflow tract repair.

The current prevalence and severity of heart defects requiring functional replacement of cardiac tissue pose a serious clinical challenge. Biologic scaffolds are an attractive tissue engineering approach to cardiac repair because they avoid sensitization associated with homograft materials and theoretically possess the potential for growth in similar patterns as surrounding native tissue. Both urinary bladder matrix (UBM) and cardiac ECM (C-ECM) have been previously investigated as scaffolds for cardiac repair with modest success, but have not been compared directly. In other tissue locations, bone marrow derived cells have been shown to play a role in the remodeling process, but this has not been investigated for UBM in the cardiac location, and has never been studied for C-ECM. The objectives of the present study were to compare the effectiveness of an organ-specific C-ECM patch with a commonly used ECM scaffold for myocardial tissue repair of the right ventricle outflow tract (RVOT), and to examine the role of bone marrow derived cells in the remodeling response. A chimeric rat model in which all bone marrow cells express green fluorescent protein (GFP) was generated and used to show the ability of ECM scaffolds derived from the heart and bladder to support cardiac function and cellular growth in the RVOT. The results from this study suggest that urinary bladder matrix may provide a more appropriate substrate for myocardial repair than cardiac derived matrices, as shown by differences in the remodeling responses following implantation, as well as the presence of site appropriate cells and the formation of immature, myocardial tissue.

Combined analysis of allograft inflammatory factor-1, interleukin-18, and Toll-like receptor expression and association with allograft rejection and coronary vasculopathy.

In cardiac transplantation settings, the initial myocardial ischemia and reperfusion may cause myocyte tissue injury and the release of allograft inflammatory factor-1 (AIF-1). This in part may trigger the innate immune response through the modulation of Toll-like receptor-2 (TLR-2) and AIF-1 expression and function, causing the release of proinflammatory cytokines. The goal was to demonstrate these markers in the peripheral blood and biopsy specimen from recipients with cardiac allograft rejection and coronary vasculopathy (CV). Peripheral blood and endomyocardial specimens were tested by reverse transcriptase-polymerase chain reaction and immunohistochemistry stains for identification of TLR-2, -4, interleukin-18, and AIF-1 markers and analyzed against clinical rejection grades for rejection. The differences for mRNA transcript levels were determined by one-way analysis of variance. The mRNA expression levels were significantly varied for TLR-2 in monocytes with different rejection grades (P < 0.0001). The mean +/- SEM level of mRNA expression for 3A grade rejection was 64.21 +/- 3.8; grade 1A, 38.4 +/- 3.5; and for Grade 0 was 38.46 +/- 2.8. The TLR-4 mRNA expression was increased but the specificity was not statistically significant. The TLR-2 immunoreactivity was strongly detected in infiltrating mononuclear cells and cardiac myocytes in Grade 3A rejection. AIF-1 expression was increased significantly in the group with 3A rejection and Grade III CV as compared with Grade 0 or 1A. Interleukin-18 receptors were strongly detected in Grade 3A rejection and CV. The expression profiles of AIF-1, TLR-2, and interleukin-18 were correlated with biopsy-proven allograft rejection in both peripheral blood and local tissue, suggesting a potential for diagnostic biomarkers for early detection of allograft rejection.

Dopamine receptor subtypes in the native human heart.

The present study first time detected D1-D5 dopamine receptor subtypes in the native human heart simultaneously, found presence of D1, D2, D4, and D5 in cardiac tissues, and revealed distribution features of dopamine receptor subtypes in the epicardium, myocardium, and endocardium. Samples from four native hearts coming from young brain-dead donors, which for technical reason were not used for transplants, were studied. Dopamine receptors were revealed by immunochemistry technique and immunoblot analysis. Morphometrical quantification of the density of each receptor subtype was performed by an image analyzer. Our results demonstrate that only four subtypes of dopamine receptors can be found in cardiac tissues: D1, D2, D4, and D5. These dopamine receptors have been detected in endocardium, myocardium, and epicardium. D1 receptors were stored primarily in the epicardial layer. Dopamine receptors are distributed in the wall of both atria and ventricles, and its transmural gradient can be described in the wall of the human heart. Sections of atria and ventricles exposed to antidopamine receptor antibodies showed fluorescent-positive reaction in the epicardium, myocardium and endocardium. D4 receptor immune reactivity was remarkably less intense than D2 receptor immune reactivity. All the subtypes of dopamine receptors are in close relationships with all cardiac structures. Our findings provide a favorable basis for researching the role of dopamine receptors in controlling functions of the human heart and in the pathogenesis of cardiovascular diseases.

Down regulation of immuno-detectable cardiac connexin-43 in BALB/c mice following acute fasting.

Acute starvation effects for connexin-43 protein expression, in the heart, had not been previously explored. Hence we examined acute fasting on the myocardial immuno-histochemical expression of connexin-43 in 3 groups of 8-week old female BALB/c mice. Groups consisted of control mice (n=5), fasting for 24 h (N=5) and 48 h (N=3). Under light microscopy all control fed cases revealed the presence of some immuno-detectable staining for connexin-43 that is either present or weakly observed in some or all of the regions of interest, that include the cross-sectional left ventricular sub-endocardium, mid-myocardium and papillary muscle. Whereas mice that underwent 24 or 48 h of acute starvation, connexin-43 expression was either difficult to detect visually (N=3) or was completely absent (N=5) at 40x magnification using a light microscope. In starved mice with no membrane staining for connexin-43 we observed an increase in the intracellular accumulation of cytoplasmic connexin-43 expression.

Fabrication of graded TiN coatings on nitinol occluders and effects on in vivo nickel release.

A nano-structured TiN/Ti coating with a total thickness of 0.9 mum was deposited on nitinol cardiac occluders using the filtered multi-arc vacuum ion plating technique at less than 300 degrees C. The coating was composed of laminated TiN/Ti layers with thickness of about 100 nm. The cardiac occluders made of a nitinol mesh with and without a graded nano-structured titanium nitride (TiN) coating were implanted into the hearts of rams. The nickel concentration of the whole blood of the animals were measured one week, one month, three months, and six months after implantation and compared to that before operation. The nickel concentration in the neo-endocardium covered occluders was also measured using graphite furnace atomic absorption spectrophotometry. After one week, the nickel content in the blood increased by a factor of three compared to the level before operation and decreased afterwards returning to the normal level after six months when endothelialization was complete. Statistical analyses showed that the TiN coating could mitigate nickel release into blood (P<0.01). For example, the nickel concentration released from the control increased from about 2.65+/-1.20 microg/kg, the normal concentration, to 7.30+/-1.00 microg/kg but just from 2.56+/-1.16 microg/kg to 4.68+/-1.29 microg/kg from the TiN coated occluder after 7 days. The nickel concentration in the neo-endocardium covered and TiN coated occluders reached 17.0+/-8.05 microg/kg in two months after implantation. In comparison, it was 31.0+/-5.72 microg/kg for the occluder without the TiN coating. While normal concentration of nickel in endocardium is also 2.6+/-1.09 microg/kg. Our results demonstrate that the graded TiN coating can significantly reduce nickel release into the endocardium (P<0.01) under in vivo conditions.

Sympathetic nerve sprouting after orthotopic heart transplantation.

BACKGROUND: Although many studies have documented sympathetic re-innervation in transplanted hearts (allografts) using chemical, imaging, and electrophysiologic methods, little histopathologic proof of this process exists. METHODS AND RESULTS: We used immunohistochemical techniques with antibodies to S-100 protein, to growth-associated protein 43 (GAP43), and to tyrosine hydroxylase (TH) to detect nerves in the left ventricles in allografts from 29 consecutive recipients. Reasons for transplantation included ischemic heart disease (IHD, n=16), non-ischemic dilated cardiomyopathy (DCM, n=12), and both (n=1). We assessed nerve densities (nerves/mm2) with respect to time after transplantation in the endocardium; in the mid-myocardium; and around intramyocardial blood vessels, scars, foci of rejection, and Quilty lesions. Six normal hearts were used for comparison. As in normal hearts, all 29 allografts had epicardial nerve trunks that extended into the mid-myocardium around blood vessels. Although the total number of nerves (S100-positive) progressively decreased over time, GAP43-positive nerves around the blood vessels increased with time (p <0.005). We also observed abundant TH-positive nerves. The density of S100-positive nerves around blood vessels was greater in those undergoing transplantation for IHD (113 +/- 88) than in those with prior DCM (54 +/- 49, p <0.05). Nerve density in each area varied greatly. CONCLUSIONS: Heterogeneous sympathetic nerve sprouting and re-innervation occurred around blood vessels in the allografts. The magnitude of nerve sprouting increased with time and varied greatly from patient to patient. Patients with IHD had greater nerve sprouting and re-innervation than did those with DCM.

Instantaneous quantitative video intensity heterogeneity: evaluation with low mechanical index contrast echocardiography.

BACKGROUND: Instantaneous video intensity of myocardium has been poorly characterized. Myocardial video intensity is usually displayed in the fitted curve from the exponential equation, y = a(1 - e (-bt)). However, information from the fitted curve will be as accurate as the original video intensity data from the perfusion image. Therefore, we sought to characterize the intramyocardial instantaneous video intensity from low mechanical index (MI) contrast echo imaging for variation. METHOD: Low-MI imaging using a nonlinear cancellation technique was performed on 10 subjects with normal myocardium. Quantitative video intensity was analyzed in five segments in the epicardium and subendocardium, as well as in systole and diastole. RESULTS: Video intensity varied between the epicardium and endocardium in each of the region that was analyzed, with the greatest variation in the inferior region (P < 0.0001). Diastolic and systolic differences were also present. CONCLUSION: Instantaneous video intensity is heterogeneous within the myocardium. Differences can result from attenuation, myocardial fiber structure, and even isotropic effects of the contrast agent, and should be taken into account when data are fitted into an exponential function.

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells.

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in fresh-frozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje's cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin-LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.

Simultaneous multisite endocardial mapping of sustained and non-sustained atrial fibrillation in humans.

The aim of this study was to investigate the differences between sustained and non-sustained forms of human atrial fibrillation (AF) using multielectrode endocardial recordings. Methods. Sixty-four pole basket catheters were deployed in the right atrium (RA) of 3 groups of patients: 1) patients with persistent AF (> 48 hours); 2) induced sustained AF (> 15 minutes); and 3) induced non-sustained AF (< 15 minutes). Beat to beat AF intervals (FF) were evaluated for each bipole. On the basis of signal characteristics and direction of wavefront propagation, the degree of spatial and temporal organization of AF was assessed. Results. Persistent AF showed the shortest FF intervals (161 ms) and lowest overall degree of AF organization, induced non-sustained AF the longest FF intervals (192 ms) and highest degree of organization. FF intervals of induced sustained AF were only slightly longer (169 ms) compared to persistent AF. Within each AF group, the lateral wall showed the highest degree of organization, the septal region the lowest. Conclusion. In humans, FF interval and overall degree of AF organization were found to increase significantly from sustained to non-sustained AF. Persistent and induced sustained AF, however, only slightly differed in these parameters.

Prichard's structures of the fossa ovalis are not histogenetically related to cardiac myxoma.

AIMS: Cardiac myxomas are neoplasms of unknown histogenesis. They are thought to arise from hypothetical subendothelial vasoformative reserve cells or from primitive cells which reside in the fossa ovalis and surrounding endocardium. In 1951 Prichard described a kind of microscopic endocardial structure with a predilection for the interatrial septum, which were suggested to be related to cardiac myxomas. To confirm the existence of Prichard's structures and to clarify their role in the genesis of cardiac myxomas, we examined histologically the fossa ovalis and we performed an immunohistochemical study of the endocardial abnormalities that were found. METHODS AND RESULTS: A prospective histological study of 100 interatrial septa and an immunohistochemical study of three out of the 12 endocardial abnormalities that were detected, as well as of four conventional cardiac myxomas were accomplished. Antibodies were used to vimentin, CD31, CD34, alpha-smooth muscle actin, S100 protein, thrombomodulin, calretinin and c-kit (CD117), a tyrosine kinase growth factor receptor for stem cell factor usually expressed by embryonic/fetal endothelium. Structures similar to the ones described by Prichard were found in 12% of septa, most of them in the left side of the fossa ovalis. The hearts with these structures were from patients 10 years older than the ones without them (72 +/- 10 versus 62 +/- 16 years, P=0.006). Immunohistochemically the cells comprising Prichard's structures were positive for vimentin, CD31, CD34 and thrombomodulin, and negative for alpha-smooth muscle actin, S100 protein, calretinin and c-kit. Therefore these cells seem to be mature endothelial cells, but not primitive multipotential mesenchymal cells. Furthermore, these cells were not found in the atrial tissue from the bases of any of the conventional cardiac myxomas. CONCLUSIONS: Our study suggests that there is no apparent relation between Prichard's structures and cardiac myxomas, and that Prichard's minute endocardial deformities are age-related phenomena.

Evidence for increased pyridinoline concentration in fibrotic tissues in diffuse systemic sclerosis.

Systemic sclerosis is a generalized disease characterized mainly by the accumulation of collagen in the skin and internal organs. The aim of our study was to determine the amount of collagen cross-link pyridinoline (Pyd) in a variety of fibrotic tissues (skin, fascia, endocardium, bladder) from an autopsy patient with diffuse systemic sclerosis, and to compare these with normal tissues from the same patient. Mean concentrations of Pyd in the fibrotic skin samples (66 mmol/mol collagen) were more than two-times greater than those in the uninvolved normal samples (27 mmol/mol collagen). The increase of Pyd in the endocardium, fascia, and bladder was also markedly higher (1.41 x, 1.26 x and 2.64 x higher than normal samples). The increased deposition of collagen in systemic sclerosis is accompanied by a significantly increased amount of Pyd in the collagen of fibrotic tissues.

Heat shock proteins, HSP25 and HSP70, and apoptosis in developing endocardial cushion of the mouse heart.

Formation of the atrioventricular channels and valves from the endocardial cushion occurs through growth and remodeling of the initial endocardial cushion. This process requires balanced coordination of proliferation and apoptosis by still unknown factors. To detect a possible role for the heat shock proteins 25 and 70 (HSP25 and HSP70) as apoptosis-associated proteins and differentiation factors in the development of the endocardial cushion, we analyzed their temporal and regional occurrence during cell proliferation and apoptosis in E11-E17 embryos. The distribution and timing of these events and factors were consistent with the hypothesis that HSP25 is related to myocardial development whereas HSP70 is related to differentiation of the endocardial cushion by cell proliferation and apoptosis.

Comparative histopathology of endomyocardial biopsies in chagasic and non-chagasic heart transplant recipients.

BACKGROUND: Heart transplantation has been an option for the treatment of chagasic (C) cardiomyopathy despite difficulties concerning the control of rejection and reactivation. The parasite-host interaction under the influence of immunosuppressive therapy may affect the immunological response to the graft in a pattern different from that in non-chagasic (NC) patients. The aim of this study was to compare the major histopathological features in heart transplantation in C and NC patients. METHODS: We studied 293 endomyocardial biopsies from two groups of heart transplanted patients, including 18 C and 15 NC. Both groups had identical surgical and clinical procedure except immunosuppressive therapy was lower in C patients. The histopathological parameters evaluated were the Quilty effect, rejection, C myocarditis reactivation, fibrosis, hypertrophy, and ischemia. In addition, lymphocytic cellular infiltration of myocarditis due to rejection or reactivation was immunophenotyped in the biopsies of both groups with rejection grades 3 to 4, in biopsies with signs of reactivation, and in fragments of the receptor heart with chronic C myocarditis. A search for Trypanosoma cruzi was performed in all biopsies in the C group in which lymphocyte immunophenotyping was done. We used immunofluorescence and confocal microscopy. RESULTS: The Quilty effect was present in 23% of the biopsies, involving 69.7% of the patients without a significant difference between groups (p = 0.509). Rejection was frequently observed in biopsies with the Quilty effect and the effect often recurred in the same patient. Rejection grades 3 to 4 was more frequent in the C group (p = 0.023). There were 5 episodes of Chagas' disease reactivation with myocarditis in 2 cases. The mean numbers of CD8+ and CD4+ T cells, and the CD4+-to-CD8+ ratio were similar for rejection in both groups (p > 0.05), while the CD4+-to-CD8+ ratio was significantly lower in chronic C myocarditis compared to rejection in the C group (p = 0.043). There was no significant difference in ischemic damage or interstitial fibrosis in the groups but there was a higher frequency of hypertrophy in the NC group (p = 0.007). CONCLUSIONS: The histopathological features of heart transplantation in C patients did not differ from that in NC patients in regard to the Quilty effect, development of myocardial fibrosis and ischemia. However, the higher involvement of the C group for rejection grades 3 to 4 suggested higher susceptibility to this event. The similarity of the lymphocytic cellular composition for rejection in both groups indicates that C patients respond to immunological stimulus in a similar pattern as NC patients.

Differential expression of RANTES chemokine, TGF-beta, and leukocyte phenotype in acute cellular rejection and quilty B lesions.

BACKGROUND: Because of the complexity of the trabeculated endocardial surface and tangential histologic sectioning, the differentiation of acute cellular rejection (ACR) from Quilty B lesions (QB) in endomyocardial biopsies (EMBs) is problematic. We hypothesized that the phenotype chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) expression of infiltrating cells and the pattern of expression of transforming growth factor-beta (TGF-beta) may distinguish ACR from QB. In previous studies, the number of RANTES-positive cells and the expression of TGF-beta correlated with the severity of rejection. METHODS: We used immunohistochemical techniques to stain sections of human EMBs with only QB (n = 14) or with only ACR (International Society for Heart and Lung Transplantation Grades 1A and 1B, n = 7; Grades 3A and 3B, n = 7) for B (CD20) and T-lymphocytes (CD3), macrophages (CD68), RANTES, and TGF-beta expression. We graded the percentage of positive cells from 0 to 4 (1 = 1% to 25%; 2 = 26% to 50%; 3 = 51% to 75%, and 4 = 76% to 100%). RESULTS: When ACR was compared with QB, we found no difference in the proportion of myocardial B cells (0.9 +/- 0.3 vs 1.1 +/- 0.3, p = 0.17); however, we found a lesser proportion of T cells (1.8 +/- 0.5 vs. 2.8 +/- 0.9, p <0.01) but more macrophages (2.9 +/- 0.5 vs. 1.1 +/- 0.6, p < 0.0001) in ACR than in QB. We also found more RANTES-positive leukocytes in ACR vs. QB (2.8 +/- 1.3 vs. 1.9 +/- 0.9, p = 0.03). In QB, many endocardial vessels stained for TGF-beta (2.9 +/- 1.6). Myocardial vessels and injured myocytes in both ACR and QB expressed TGF-beta. CONCLUSIONS: In ACR, although T-lymphocytes are numerous, more than 50% of infiltrating cells are macrophages and more than 50% express RANTES. In QB lesions, more than 50% of infiltrating cells are T-lymphocytes and less that 50% of leukocytes will express RANTES. B cells are present in both ACR and QB, but on average comprise only 25% of the cells present. Thus, a relatively simple immunohistochemical analysis of endomyocardial biopsies may be useful in distinguishing ACR from QB.

Identification and localization of TBX5 transcription factor during human cardiac morphogenesis.

Mutations in the TBX5 transcription factor gene cause human cardiac malformation in Holt-Oram syndrome. To identify and localize TBX5 during cardiac morphogenesis, we performed immunohistochemical studies of TBX5 protein cardiac expression during human embryogenesis. Specific antibody to human TBX5 was generated in rabbits with a TBX5 synthetic peptide and affinity purification of antiserum. Anti-TBX5 was used in immunohistochemical analyses of human cardiac tissue. In embryonic and adult heart, TBX5 is expressed throughout the epicardium and in cardiomyocyte nuclei in myocardium of all four cardiac chambers. Endocardial expression of TBX5 is only present in left ventricle. Asymmetric left-sided transmyocardial gradients of TBX5 protein expression were observed in embryonic but not adult hearts. Human cardiac expression of TBX5 protein correlates with the cardiac manifestations of Holt-Oram syndrome. TBX5 transmyocardial protein gradients may contribute to normal patterning of the human heart during embryogenesis.

Immunohistochemical localization of modified C-reactive protein antigen in normal vascular tissue.

BACKGROUND: The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. METHODS: Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. RESULTS: Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. SIGNIFICANCE: This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.