Marker-Independent In Situ Quantitative Assessment of Residual Cryoprotectants in Cardiac Tissues.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA's characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

Porcine pulmonary valve decellularization with NaOH-based vs detergent process: preliminary in vitro and in vivo assessments.

BACKGROUND: Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%). METHODS: Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2). RESULTS: Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm2 in the leaflets). In one animal, at three months we documented minimal calcification in the area of sinus leaflet and in one, microthrombi formation on the leaflet surface at 1 month. The endoluminal side of the valves showed partial reendothelialization. CONCLUSIONS: NaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis. Graphical abstract of the study.

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts.

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37-42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg-1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg-1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg-1 (Regan et al. in Transfusion 50:2670-2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.

Calcium and phosphorus concentrations in native and decellularized semilunar valve tissues.

BACKGROUND AND AIM OF THE STUDY: Native, allograft, xenograft and bioprosthetic semilunar valves are all susceptible to calcific degeneration. However, intrinsic differences in baseline calcium and phosphorus tissue concentrations within mammalian normal valve structural components (e.g., cusps, sinus, vessel wall) additionally subdivided by tripartite regions (e.g., right-, left- and non-coronary leaflets) have never been systematically measured and reported. It was originally hypothesized that variations in normative tissue concentrations of calcium and phosphorus may correspond to subsequent clinical patterns of acquired dystrophic calcification; decellularization was also expected to reduce the tissue concentrations of these elements. METHODS: Native semilunar valves were freshly harvested from 12 juvenile sheep. Half of the valves were decellularized (six aortic and six pulmonary), while the other valves were flash-frozen at -80 degrees C within minutes of euthanasia as native valves. Elemental calcium and phosphorus concentrations were measured in the great vessels, sinus walls and cusps using inductively coupled plasma optical emission spectrometry (ICP-OES), and analyzed with non-parametric statistical tests. RESULTS: Calcium concentrations (microg/mg tissue; median (range) were similar in aortic native cusps (0.37 (0.21)), sinus walls (0.37 (0.09)) and aorta (0.37 (0.08)) (p = 0.8298). Pulmonary calcium concentrations were similar in cusps, but 10-25% higher in the native sinus (p = 0.0018) and pulmonary artery (p < 0.0001) compared to analogous aortic structures. All cusps had higher phosphorus concentrations than their respective conduit tissues. No tripartite regional variations were observed. Decellularization did not reduce the calcium content of cusps, but removed 50-55% of vessel and sinus wall calcium. However, up to 85% of phosphorus was removed from all valve tissues (p < 0.001). CONCLUSION: There were no significant differences in normal tissue concentrations of calcium between aortic valve functional structures, and no semilunar tripartite regional differences in either semilunar valve complex. Thus, the distribution of baseline tissue calcium content of healthy young valves is not inherently predictive of selective or asymmetric anatomical patterns of valve degenerative calcification. Native semilunar cusps contain the highest phosphorus concentrations. Decellularization reduces all elemental concentrations except for cuspal calcium.

Development and characterization of acellular porcine pulmonary valve scaffolds for tissue engineering.

Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Immunohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrodynamic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of α-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

Networked-based characterization of extracellular matrix proteins from adult mouse pulmonary and aortic valves.

A precise mixture of extracellular matrix (ECM) secreted by valvular cells forms a scaffold that lends the heart valve the exact mechanical and tensile strength needed for accurate hemodynamic performance. ECM proteins are a key component of valvular endothelial cell (VEC)-valvular interstitial cell (VIC) communication essential for maintenance of the valve structure. This study reports the healthy adult pulmonary and aortic valve proteomes characterized by LC-MS/MS, resulting in 2710 proteins expressed by 1513 genes, including over 300 abundant ECM proteins. Surprisingly, this study defines a distinct proteome for each semilunar valve. Protein-protein networking (PPN) was used as a tool to direct selection of proteomic candidates for biological investigation. Local PPN for nidogen 1 (Nid1), biglycan (Bgn), elastin microfibril interface-located protein 1 (Emilin-1), and milk fat globule-EGF factor 8 protein (Mfge8) were enriched with proteins essential to valve function and produced biological functions highly relevant to valve biology. Immunofluorescent investigations demonstrated that these proteins are functionally distributed within the pulmonary and aortic valve structure, indicative of important contribution to valve function. This study yields new insight into protein expression contributing to valvular maintenance and health and provides a platform for unbiased assessment of protein alterations during disease processes.

Heart valve collagens: cross-species comparison using immunohistological methods.

BACKGROUND AND AIM OF THE STUDY: Tissue engineering is an emerging strategy for the development of replacement heart valves where the properties of native tissues are to be replicated. The complexity of the distribution of various collagens in the aortic, mitral, and pulmonary valve leaflets of porcine, bovine, and ovine origin, has been examined. METHODS: Immunohistological and transmission electron microscopy analyses using monoclonal antibodies to types I, III, IV, V and VI collagens were performed. RESULTS: The results indicated that each collagen type has its own distinct distribution, with minimal variation between heart valve anatomic sites and species. Of particular interest was type VI collagen, which had an asymmetric distribution that was principally localized along the outflow surface of the valve. CONCLUSION: Successful tissue engineering constructs of heart valves may need to replicate the complex distribution of different collagens found in heart valve tissues.

Hyaluronan and its receptor CD44 in the heart of newborn and adult rats.

The extracellular matrix is a dynamic space and a prerequisite for the function of cardiomyocytes. We have previously reported on the distribution of the glycosaminoglycan hyaluronan (HYA) and its cellular receptor CD44 in the vascular system. In newborn rats, HYA and its receptor were colocalized, but in the adult animals, no such colocalization was observed. Furthermore, the distribution of both HYA and CD44 differed between newborn and adult animals. In this study, the distribution of HYA and its receptor CD44 is explored in the heart. Hearts from newborn and adult rats were stained for visualization of HYA and CD44 using histochemistry and immunohistochemistry. HYA stained the interstitium of the myocardium heterogeneously. Strong staining was seen in the heart valves of both newborn and adult animals. CD44 staining was sparse in hearts from both newborn and adult animals. There are no major changes in the distribution of HYA in the myocardium during the postnatal development in contrast to the blood vessels. Thus, the structure of the interstitium does not change after birth when the heart starts to grow mainly through cardiomyocyte hypertrophy rather than hyperplasia. The abundance of HYA in the heart valves is probably related to their unique physiological properties to withstand repetitive mechanical stress.

Cyclic aortic pressure affects the biological properties of porcine pulmonary valve leaflets.

BACKGROUND AND AIM OF THE STUDY: Native pulmonary valve leaflets (PVL) are exposed to lower pressures compared to aortic valve leaflets. Knowledge of the biology of PVL exposed to aortic pressures is limited. Hence, the study's aim was to investigate the biological properties of PVL subjected to normal aortic pressures. METHODS: Porcine PVL were exposed to mean pulsatile pressures of 30 mmHg or 100 mmHg for 48 h in vitro. Subsequently, PVL were subjected to a mean pulsatile pressure of 30 mmHg for 48 h, followed by increased pressure (100 mmHg) for additional 48 h. Leaflets were evaluated by measuring collagen, DNA and sGAG contents in pressure-exposed and control PVL. Cusp morphology and cell phenotype were examined using hematoxylin and eosin staining (H and E) and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry, respectively. RESULTS: PVL exposed to 30 mmHg showed no significant difference (p > 0.05) in collagen, DNA or sGAG contents compared to statically incubated PVL. However, PVL exposed to 100 mmHg showed a significant increase (p < 0.05) in both collagen and sGAG contents. Collagen content was also significantly increased (p < 0.05) in PVL exposed to varying pressures for 96 h compared to PVL exposed to 30 mmHg. The morphology of PVL exposed to cyclic pressures was comparable to that of both fresh and static leaflets, while alpha-SMA expression was decreased in PVL exposed to cyclic pressures when compared to fresh PVL. CONCLUSION: PVL have the ability to withstand elevated mechanical conditions by increasing the total collagen and sGAG content of the leaflets. The structural integrity of the PVL is unaltered by changes in extracellular matrix composition. However, pulsatile pressures on the PVL did not preserve the native cell phenotype.

Smoothelin-positive cells in human and porcine semilunar valves.

Our aim was to further characterize the interstitial cell phenotypes of normal porcine and human semilunar valves, information necessary for the design of bioengineered valves and for the understanding of valve disease processes such as aortic valve sclerosis. Existence of fibroblasts, myofibroblasts, and smooth muscle-like cells within semilunar heart valves has been established. However, the nature of the smooth muscle cell population has been controversial. We used immunochemical and western blotting methods to determine the status of smoothelin and smooth muscle alpha-actin in the valve. Our examination of valve interstitial cells confirmed the presence of terminally differentiated, contractile smooth muscle cells in situ. They were arranged in small bundles of 5-35 cells within the ventricularis or as individual cells scattered throughout the valvular layers in vivo, and were present in cells explanted from the valves in vitro. Colocalization of these proteins in semilunar heart valves was achieved with double-labeling experiments. Protein extraction, followed by coimmunoprecipitation, electrophoresis, and western blotting confirmed the immunochemical analysis and suggested that smooth muscle alpha-actin and smoothelin interact, as has been previously postulated. The presence of contractile smooth muscle within the valve may be an important factor in understanding valve pathology and in the design of tissue engineering efforts.

Accumulation of elements in the arteries and cardiac valves of Thai with aging.

To elucidate whether the extent of element accumulation in the arteries and cardiac valves with aging was different between different races, the authors investigated the accumulation of elements in the arteries and cardiac valves of the Thai with aging and the relationships among elements in the cardiac valves. After ordinary dissection at Chiang Mai University was finished, 16 arteries and 4 cardiac valves were resected and element contents were determined by inductively coupled plasma-atomic emission spectrometry. In the 16 arteries, the average content of calcium was the highest in the site of the abdominal aorta ramifying into the common iliac arteries, and it decreased in the order internal iliac, coronary, abdominal aorta, common iliac, external iliac, superior mesenteric, inferior mesenteric, thoracic aorta, brachial, radial, common carotid, subclavian, ulnar, axillary, renal, and internal thoracic arteries. The average contents of phosphorus and magnesium in respective arteries were parallel with the average contents of calcium, except for the coronary artery. In comparison with the arteries of the Japanese, the trend of calcium accumulation in the arteries of the Thai was almost similar to that in the arteries of the Japanese, except for the coronary artery and thoracic aorta. The calcium accumulation in the coronary artery was much higher in the Thai than in the Japanese, whereas that in the thoracic aorta was lower in the Thai than in the Japanese. Regarding elements in the cardiac valves, the calcium content increased remarkably in the seventies in the aortic valve and in the nineties in the pulmonary valve, but it hardly increased in both the mitral and tricuspid valves with aging. The average content of calcium was the highest in the aortic valve, and it decreased in the order pulmonary, tricuspid, and mitral valves. Regarding the relationship among elements in the aortic valves, it was found that there were extremely significant direct correlations among the contents of calcium, phosphorus, and magnesium, whereas there were significant direct correlations between zinc and either calcium or phosphorus contents. Although significant correlations were found between sulfur and the other element contents in the aortic valves fo the Japanese, no significant correlations were found between them in the aortic valves of the Thai. In the mitral valves, extremely or very significant direct correlations were found among the contents of calcium, phosphorus, magnesium, and sulfur, with some exceptions that there were no significant correlations between phosphorus and either magnesium or sulfur contents. in addition, no significant correlation was found in the calcium content between the aortic valve and coronary artery in the same individuals.

Construction of autologous human heart valves based on an acellular allograft matrix.

OBJECTIVE: Tissue engineered heart valves based on polymeric or xenogeneic matrices have several disadvantages, such as instability of biodegradable polymeric scaffolds, unknown transfer of animal related infectious diseases, and xenogeneic rejection patterns. To overcome these limitations we developed tissue engineered heart valves based on human matrices reseeded with autologous cells. METHODS AND RESULTS: Aortic (n=5) and pulmonary (n=6) human allografts were harvested from cadavers (6.2+/-3.1 hours after death) under sterile conditions. Homografts stored in Earle's Medium 199 enriched with 100 IU/mL Penicillin-Streptomycin for 2 to 28 days (mean 7.3+/-10.2 days) showed partially preserved cellular viability (MTT assay) and morphological integrity of the extracellular matrix (H-E staining). For decellularization, valves were treated with Trypsin/EDTA resulting in cell-free scaffolds (DNA-assay) with preserved extracellular matrix (confocal microscopy). Primary human venous endothelial cells (HEC) were cultivated and labeled with carboxy-fluorescein diacetate-succinimidyl ester in vitro. After recellularization under fluid conditions, EC were detected on the luminal surfaces of the matrix. They appeared as a monolayer of positively labeled cells for PECAM-1, VE-cadherin and Flk-1. Reseeded EC on the acellular allograft scaffold exhibited high metabolic activity (MTT assay). CONCLUSIONS: Earle's Medium 199 enriched with low concentration of antibiotics represents an excellent medium for long time preservation of extracellular matrix. After complete acellularization with Trypsin/EDTA, recellularization under shear stress conditions of the allogeneic scaffold results in the formation of a viable confluent HEC monolayer. These results represent a promising step toward the construction of autologous heart valves based on acellular human allograft matrix.

Antimineralization treatments in stentless porcine bioprostheses: an experimental study.

BACKGROUND AND AIM OF THE STUDY: Photo-oxidation treatment of porcine stentless bioprostheses (Photofix) was compared with glutaraldehyde fixation, with either AOA (Freestyle valve) or Tween-80 (Edwards Prima Plus valve). METHODS: Six valves of each type were implanted in juvenile sheep, in the pulmonary position. Valves were explanted after three or six months and examined macroscopically, by X-radiography, and by light and transmission electron microscopy. Calcium content was determined by atomic absorption spectrometry. RESULTS: The cusps of all valves were free of calcification, and had normal histology and function. Calcium contents (median +/- IQR) were 0.63+/-0.45, 0.73+/-1.46 and 0.46+/-1.42 microg/mg for the Photofix, Freestyle and Prima Plus valves, respectively (p = NS). Calcium contents of the aortic wall portions were 0.71+/-1.27 (Photofix), 10.78+/-77.22 (Freestyle) and 28.70+/-66.53 (Prima Plus) (p <0.05 for Photofix versus Freestyle or Prima Plus). CONCLUSION: Photo-oxidation of a porcine stentless valve prevents calcification not only in the cusps, but also in the aortic wall portion.

Differences in accumulation of elements in human cardiac valves.

To elucidate changes of human cardiac valves with aging, the authors determined age-related changes of element contents in the four human cardiac valves by inductively coupled plasma-atomic emission spectrometry and attempted to examine the relationships in the element contents among the four cardiac valves. The subjects consisted of 10 men and 15 women, ranging in age from 65 to 102 yr. The accumulation of calcium and phosphorus was the highest in the aortic valve, and decreased in the order mitral, pulmonary, and tricuspid valves. The contents of calcium and phosphorus in the aortic valves corresponded to about 12 and 19 times the amounts of those in the tricuspid valves, in which the contents were very low. The contents of calcium and phosphorus in the aortic valves were about 2.5-fold the amounts of those in the mitral valves. An examination was attempted to determine whether or not there were relationships in element contents among the four cardiac valves. As for the aortic and mitral valves, there were no relationships in the contents of calcium and phosphorus between them, but there were relationships in the contents of sulfur and magnesium between them. Three out of 24 cases contained high contents of calcium and phosphorus in both the mitral and aortic valves, whereas 16 out of 24 cases contained high contents of calcium and phosphorus in the aortic valves alone, without the high contents in the mitral valves. Likewise, there were no relationships in the element contents, such as calcium, phosphorus, sulfur, and magnesium, between the mitral and pulmonary valves or between the mitral and tricuspid valves. It is suggested that the accumulation of calcium and phosphorus in the cardiac valve occurs independent of the other cardiac valves.

Tissue-engineered valved conduits in the pulmonary circulation.

OBJECTIVE: Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue-engineering techniques. METHODS: Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (+/-3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. RESULTS: Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. CONCLUSION: This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue-engineered structure continues for at least 6 months.

Accumulation of oxidized LDL in human semilunar valves correlates with coronary atherosclerosis.

OBJECTIVE: Recent data indicate that oxidized low-density lipoprotein (ox-LDL) has several proatherogenic effects, e.g. induction of macrophage chemoattractants, adhesion molecules, cytokines, type-1 plasminogen activator inhibitor and platelet-derived growth factor A-chain by smooth muscle cells. Therefore, ox-LDL has been utilized as a marker of oxidative modification of proteins in atherosclerosis. Because heart valves consist of smooth muscle cells, fibroblasts and endothelial cells, and because valvular disease and coronary atherosclerosis could result from similar biological processes, we investigated ox-LDL accumulation in isolated aortic and pulmonary valves and coronary arteries from patients with angiographically proven coronary heart disease (CHD, n = 19), patients with idiopathic congestive heart failure (IDCM = idiopathic dilated cardiomyopathy, n = 20), and transplant donors. METHODS: Masson-Goldner staining and immunohistochemistry utilizing anti ox-LDL and CD68 were performed on paraffin sections of freshly isolated semilunar valves. Data were analyzed by digital image planimetry and by visual scoring of staining intensity. RESULTS: Ox-LDL immunoreactivity was identified in the vascular aspect of the attachment line, in the deep valve stroma, and in the ventricular and vascular endothelium of the semilunar valves, colocalizing with macrophages. Valvular ox-LDL area was significantly increased in CHD-patients (P < 0.03) and IDCM-patients (P < 0.04) compared with controls. More ox-LDL was accumulating in the pulmonary valves than in the aortic valves (P = 0.04) as assessed by area and staining intensity. Valvular ox-LDL area in pulmonary valve and aortic valve was significantly correlated with ox-LDL accumulation in the intimal layer (P < 0.001) and medial layer (P < 0.001) of coronary arteries from the same patients. CONCLUSION: The data suggest that the biological process leading to ox-LDL accumulation in coronary atherosclerosis also involves heart valves. Therefore, accumulation of the oxidative stress marker ox-LDL in heart valves illustrates atherosclerosis as an additional mechanisms accelerating valvular degeneration in these patients.

A comparison of macroscopic lipid content within porcine pulmonary and aortic valves. Implications for bioprosthetic valves.

Lipid droplets have been demonstrated within both explanted porcine bioprostheses and normal porcine aortic valves. Because of the increasing interest in pulmonary valves as an allograft or xenograft aortic valve substitute, we examined the incidence and distribution of such lipid deposits in 50 porcine aortic valves and 50 matched porcine pulmonary valves. All 300 cusps were removed with surgical scissors and, under a dissecting microscope, the ventricularis layer was removed to expose the spongiosal layer. Macroscopic extracellular lipid droplets analyzed by means of a dissecting microscope with an eyepiece grid and stereology point-counting techniques to provide an area-density average spatial probability map for each cusp. Only 8% of porcine aortic valves were free of lipid, with the distribution of the lipids being 52% +/- 14% right coronary cusp, 90% +/- 8% left coronary cusp, and 68% +/- 13% noncoronary cusp. Of the pulmonary valves, 60% were free of lipid, with the incidence of lipids being 26% +/- 12% left cusp, 6% +/- 7% right cusp, and 12% +/- 9% anterior cusp. Subsequently, lipid cluster samples underwent thin-layer chromatography, which showed them to be phospholipids, oleic acid (fatty acid), triglycerides, and unesterified cholesterol. One primary mode of bioprosthetic valve failure is leaflet calcification. The similarity of distribution within the spongiosal layer between leaflet calcification and intrinsic cusp lipids suggests that the observed lipids might act as a nucleation site for calcification. The substantially lower incidence of lipid in pulmonary valves therefore may represent a potential benefit when these valves are considered for use as aortic valve replacements.

Histopathologic studies of innervation of normal and prolapsed human mitral valves.

We evaluated the distribution of the nerves in valve tissue of humans to clarify the relationship between mitral valve prolapse and autonomic nerve dysfunction. We studied 15 autopsy specimens of normal mitral valve, 10 prolapsed mitral valves, five each of normal tricuspid, aortic, and pulmonary valves, and three prolapsed mitral valves obtained at cardiac surgery. Immunohistochemical studies utilized the avidinbiotin peroxidase complex (ABC) method and several nerve-related antigens: 1) S-100 protein, glial fibrillary acidic protein (GFAP), and neurofilament protein (NFP) as markers of glial and Schwann cells of the nervous system; 2) choline acetyltransferase (ChAT) to identify cholinergic nerve endings; 3) neuropeptide Y (NPY), a neuropeptide that is distributed in accordance with sympathetic nerves; and 4) calcitonin gene-related peptide (CGRP), a neuropeptide that is distributed in accordance with afferent nerves. Distribution of adrenergic nerve fibers was also examined by fluorescence method. Morphology of nerve endings of the normal mitral valve was studied by electron microscopy. In normal valves, distributions of S-100 protein, GFAP, and NFP immunoreactivities were clearly visible along the subendocardial site on the coaptation aspect of the base-to-body portion of each valve, regardless of the kind of valve. In contrast, there was only a scanty distribution of these reactivities on the physiologic coaptation area of the tip. In prolapsed mitral valves, there was no distribution of S-100-positive protein or other nerve-related antigens in areas of the valve with myxomatous degeneration. Distribution of CGRP, ChAT, and NPY immunoreactivities, and adrenergic fluorescence, were the same as those of the nerve-related antigens in both normal and prolapsed mitral valves. Electron microscopic study of the atrial aspect of normal mitral valves revealed numerous small axons with aggregations of small clear vesicles, indicating cholinergic features. The results suggest that the subendocardial site on the atrial aspect at the middle portion of the mitral valve is rich in nerve endings, including the afferent nerves, and that mechanical stimuli from this area caused by abnormal coaptation in mitral valve prolapse may produce an improper circuit in autonomic nerve function between the central and mitral valve nervous systems.

Histochemical and ultrastructural changes in the extracellular matrix of the developing chick semilunar heart valves.

The present study analyzes the composition and organization of the extracellular matrix (ECM) and its changes in the course of development of the chick embryo semilunar heart valves. In the present work we have employed chick embryos from stage 29 until hatching, using silver and picrosirius red staining, lectin probes and light and transmission electron microscopy. Our results show that during semilunar valve development a series of elements arise and are organized in the ECM which seem to be more closely related to the maintenance of the structural and biomechanical properties of the valvular leaflets than with morphogenetic processes per se.