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Valve remodeling is a process involving extracellular matrix organization and elongation of valve leaflets. Here, through single-cell RNA sequencing of human fetal valves, we identified an elastin-producing valve interstitial cell (VIC) subtype (apolipoprotein E (APOE) + , elastin-VICs) spatially located underneath valve endothelial cells (VECs) sensing unidirectional flow. APOE knockdown in fetal VICs resulted in profound elastogenesis defects. In valves with pulmonary stenosis (PS), we observed elastin fragmentation and decreased expression of APOE along with other genes regulating elastogenesis. Cell-cell interaction analysis revealed that jagged 1 (JAG1) from unidirectional VECs activates elastogenesis in elastin-VICs through NOTCH2. Similar observations were made in VICs cocultured with VECs under unidirectional flow. Notably, a drastic reduction of JAG1-NOTCH2 was also observed in PS valves. Lastly, we found that APOE controls JAG1-induced NOTCH activation and elastogenesis in VICs through the extracellular signal-regulated kinase pathway. Our study suggests important roles of both APOE and NOTCH in regulating elastogenesis during human valve remodeling., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by β-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

The purpose of this study was to determine the impact of elevated temperature exposure in tissue banking on soft tissues. A secondary objective was to determine the relative ability of various assays to detect changes in soft tissues due to temperature deviations. Porcine pulmonary heart valve leaflets exposed to 37 °C were compared with those incubated at 52 and 67 °C for 10, 30 and 100 min. The analytical methods consisted of (1) viability assessment using the resazurin assay, (2) collagen content using the Sircol assay, and (3) permeability assessment using an electrical conductivity assay. Additionally, histology and two photon microscopy were used to reveal mechanisms of cell and tissue damage. Viability, collagen content, and permeability all decreased following heat treatment. In terms of statistical significance with respect to treatment temperature, cell viability was most affected (p < 0.0001), followed by permeability (p < 0.0001), and then collagen content (p = 0.13). After heat treatment, histology indicated increased apoptosis and two photon microscopy revealed a decrease in collagen fiber organization and an increase in elastin density. These results suggest that measures of cell viability would be best for assessing tissues where the cells are alive and that permeability may be best where cell viability is not intentionally maintained.

Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples.

Recent studies have demonstrated remodeling of aortic and mitral valves leaflets under the volume loading and cardiac expansion of pregnancy. Those valves' leaflets enlarge with altered collagen fiber architecture, content, and cross-linking and biphasic changes (decreases, then increases) in extensibility during gestation. This study extends our analyses to right-sided valves, with additional compositional measurements for all valves. Valve leaflets were harvested from nonpregnant heifers and pregnant cows. Leaflet structure was characterized by leaflet dimensions, and ECM composition was determined using standard biochemical assays. Histological studies assessed changes in cellular and ECM components. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Collagen thermal stability and cross-linking were assessed using denaturation and hydrothermal isometric tension tests. Pulmonary and tricuspid leaflet areas increased during pregnancy by 35 and 55%, respectively. Leaflet thickness increased by 20% only in the pulmonary valve and largely in the fibrosa (30% thickening). Collagen crimp length was reduced in both the tricuspid (61%) and pulmonary (42%) valves, with loss of crimped area in the pulmonary valve. Thermomechanics showed decreased collagen thermal stability with surprisingly maintained cross-link maturity. The pulmonary leaflet exhibited the biphasic change in extensibility seen in left side valves, whereas the tricuspid leaflet mechanics remained largely unchanged throughout pregnancy. The tricuspid valve exhibits a remodeling response during pregnancy that is significantly diminished from the other three valves. All valves of the heart remodel in pregnancy in a manner distinct from cardiac pathology, with much similarity valve to valve, but with interesting valve-specific responses in the aortic and tricuspid valves., (Copyright © 2015 the American Physiological Society.)

Non-linear and anisotropic heart valve leaflet tissue mechanics manifest principally from the stratification, orientation, and inhomogeneity of their collagenous microstructures. Disturbance of the native collagen fiber network has clear consequences for valve and leaflet tissue mechanics and presumably, by virtue of their intimate embedment, on the valvular interstitial cell stress-strain state and concomitant phenotype. In the current study, a set of virtual biaxial stretch experiments were conducted on porcine pulmonary valve leaflet tissue photomicrographs via an image-based finite element approach. Stress distribution evolution during diastolic valve closure was predicted at both the tissue and cellular levels. Orthotropic material properties consistent with distinct stages of diastolic loading were applied. Virtual experiments predicted tissue- and cellular-level stress fields, providing insight into how matrix-to-cell stress transfer may be influenced by the inhomogeneous collagen fiber architecture, tissue anisotropic material properties, and the cellular distribution within the leaflet tissue. To the best of the authors' knowledge, this is the first study reporting on the evolution of stress fields at both the tissue and cellular levels in valvular tissue and thus contributes toward refining our collective understanding of valvular tissue micromechanics while providing a computational tool enabling the further study of valvular cell-matrix interactions.

Pulmonary conduit valves are used as one of the surgical treatment methods of congenital heart diseases. We have been designing a sophisticated pulmonary conduit valve for the right ventricular outflow tract reconstruction in pediatric patients. In this study, two types of polyester grafts with or without bulging structures for the conduit valves were used and evaluated from the hemodynamic point of view focusing on the application of these conduit valves in the grown-up congenital heart failure patients. We examined valvular function in the originally developed pulmonary mock circulatory system, which consisted of a pneumatic driven right ventricular model, a pulmonary valve chamber, and an elastic pulmonary compliance model with peripheral vascular resistance units. Prior to the measurement, a bileaflet valve was sutured in each conduit. Each conduit valve was installed in the mock right ventricular outflow portion, and its leaflet motion was obtained by using a high-speed camera synchronously with pressure and flow waveforms. As a result, we could obtain hemodynamic changes in two different types of conduits for pulmonary valves, and it was indicated that the presence of the Valsalva shape might be effective for promoting valvular response in the low cardiac output condition.

Objective: To conduct a test of noninferiority for CardioCel (Admedus, Brisbane, Australia), a chemically engineered bovine pericardium over autologous pericardium treated intraoperatively with glutaraldehyde in a chronic juvenile sheep model of pulmonary valve (PV) and mitral valve (MV) reconstruction., Methods: We replaced the posterior leaflet of the MV and of 1 PV cusp with patches in ewes aged 10 months. There were 2 groups: CardioCel (n = 6) and control (n = 4). All valves were competent. Echocardiography was performed before euthanasia. The collected data were function, macroscopy, histology, and calcium contents. The primary end points were thickening and calcium content., Results: All animals survived until sacrifice after 7 months. The valves had normal echo. The macroscopic aspect of the valves was excellent. Examination of the slides for both groups revealed a continuous endothelium on both sides of the patch and a layer of new collagen developed on both sides between patch and endothelium and interstitial cells and smooth muscle cell in these layers. The patch had not thickened but the 2 layers of new collagen for the PV showed a median thickening of 37% in the CardioCel group and 111% in the control group (P = .01), and for the MV a thickening of 108% and 251%, respectively, was seen (P = .01). The median calcium content in the PV was 0.24 μg/mg (range, 0.19-0.30) in the CardioCel group versus 0.34 μg/mg (range, 0.24-0.62) in controls (P = .20). In the MV it was 0.46 μg/mg (range, 0.30-1.0) in the CardioCel group and 0.47 μg/mg (range, 0.29-1.9) in controls (P = 1.0)., Conclusions: In this growing lamb model the CardioCel patch allowed accurate valve repair at both systemic and pulmonary pressure. The mechanical properties of CardioCel after 7 months were preserved with a more controlled healing than the treated autologous pericardium and without calcification., (Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Mathematical models can provide valuable information to assess and evaluate the mechanical behavior and remodeling of native tissue. A relevant example when studying collagen remodeling is the Ross procedure because it involves placing the pulmonary autograft in the more demanding aortic valve mechanical environment. The objective of this study was therefore to assess and evaluate the mechanical differences between the aortic valve and pulmonary valve and the remodeling that may occur in the pulmonary valve when placed in the aortic position. The results from biaxial tensile tests of pairs of human aortic and pulmonary valves were compared and used to determine the parameters of a structurally based constitutive model. Finite element analyzes were then performed to simulate the mechanical response of both valves to the aortic diastolic load. Additionally, remodeling laws were applied to assess the remodeling of the pulmonary valve leaflet to the new environment. The pulmonary valve showed to be more extensible and less anisotropic than the aortic valve. When exposed to aortic pressure, the pulmonary leaflet appeared to remodel by increasing its thickness and reorganizing its collagen fibers, rotating them toward the circumferential direction., (© 2013 Elsevier Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: The Melody transcatheter pulmonary valve (TPV) was approved for implantation in obstructed right ventricular outflow tract conduits in 2010 after a multicenter trial demonstrating improvements in conduit obstruction, regurgitation, and right ventricular pressure. A recognized risk and contraindication to TPV implantation is the demonstration of coronary artery (CA) compression during balloon angioplasty or stent placement in the overlying conduit. This study is the first to characterize the risk of CA compression in this population., Methods and Results: From 2007 to 2012, 404 patients underwent 407 catheterizations for potential TPV implantation (median age, 18 years) at 4 centers. Three hundred forty-three patients (85%) underwent valve implantation. Twenty-one patients (5%) had evidence of CA compression with simultaneous right ventricular outflow tract angioplasty and CA angiography. Sixty-eight patients (17%) had abnormal CA anatomy. Fifteen of 21 (71%) patients with CA compression had abnormal CA anatomy. Eight patients with tetralogy of Fallot and 7 patients with transposition of the great arteries demonstrated compression. Of the 34 patients with tetralogy of Fallot and abnormal CA, 7 (21%) demonstrated CA compression., Conclusions: CA compression following TPV implantation can be catastrophic. CA compression was observed in 5% of patients during test balloon angioplasty. No patients in this study developed clinically apparent CA compression after TPV implantation. CA compression was significantly associated with the presence of abnormal CA anatomy, especially in patients with tetralogy of Fallot or transposition of the great arteries. Preimplantation coronary angiography with simultaneous test angioplasty is an important step to evaluate for the presence of CA compression during TPV implantation.

Introduction: Congenital cardiac valve disease is common, affecting ∼1% of the population, with substantial morbidity and mortality, but suboptimal treatment options. Characterization of the specific matrix and valve cell phenotypic abnormalities in these valves could lend insight into disease pathogenesis and potentially pave the way for novel therapies., Methods: Thirty-five human aortic and pulmonic valves were categorized based on gross and microscopic assessment into control valves (n=21); dysplastic valves, all except one also displaying hemodynamic changes (HEMO/DYSP, n=6); and hemodynamically altered valves (HEMO, n=8). Immunohistochemistry was performed on valve sections and flow cytometry on valvular interstitial cells., Results: While both hemodynamically altered aortic and pulmonic valves demonstrated increased collagen turnover and cell activation, prolyl 4-hydroxylase and hyaluronan increased in hemodynamically altered aortic valves but decreased in hemodynamically altered pulmonic valves relative to control valves (P<.001). HEMO/DYSP aortic valves demonstrated decreased collagen and elastic fiber synthesis and turnover compared to both hemodynamically altered aortic valves and control aortic valves (each P<.006). Valvular interstitial cells from both hemodynamically altered and HEMO/DYSP pulmonic valves showed altered cell phenotype compared to control valves (each P<.032), especially increased non-muscle myosin. Furthermore, valvular interstitial cells from hemodynamically altered pulmonic valves and HEMO/DYSP aortic and pulmonic valves each demonstrated greater size and complexity compared to control valves (each P<.05)., Conclusions: Dysplastic semilunar valves displayed alterations in collagen and elastic fiber turnover that were distinct from valves similarly exposed to altered hemodynamics as well as to control valves. These results demonstrate that dysplastic valves are not simply valves with gross changes or loss of leaflet layers, but contain complex matrix and cell phenotype changes that, with future study, could potentially be targets for novel nonsurgical treatments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Decellularization of pulmonary valve substitutes is believed to eliminate immunogenicity and improve conduit durability. This study focused on a detailed histopathological and immunohistochemical analysis of explanted Matrix P plus valves, following their early obstruction in pediatric patients., Methods: Occurrence of fibrosis, scar formation, neovascularization, and inflammatory infiltrates were determined in longitudinal sections of four valve specimens explanted after 12-15 months. Valves were immunohistochemically analyzed for presence of different subtypes of inflammatory cells. The expression of smooth muscle actin and connective tissue growth factor was determined., Results: We observed a foreign body-type reaction accompanied by severe fibrosis and massive neointima formation around decellularized porcine valve wall, whereas the equine pericardial patch remained separated from porcine layer and acellular. Re-cellularization of decellularized matrix was low, and neovascularization was observed only in the neointima and scar tissue. Inflammatory infiltrates, composed mainly of T cells, B cells, and plasma cells, as well as the presence of dendritic cells, macrophages, and mast cells were detected in the tissue surrounding the porcine matrix. In the fibrous tissue, overexpression of connective tissue growth factor was observed. The leaflets remained functional, with normal endothelialization and no degenerative changes. Control pre-implant samples of Matrix P plus valve revealed incomplete decellularization of porcine matrix, which may have contributed to increased immunogenicity of these conduits., Conclusions: Early obstruction of decellularized Matrix P plus valve is associated with massive inflammatory reaction and exaggerated fibrotic scaring around porcine conduit wall. Detailed studies will be necessary to determine factors that contribute to remnant immunogenicity of decellularized grafts., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: Aortic homografts were compared with pulmonary homografts in the setting of right ventricular outflow tract reconstruction in adolescent sheep. Furthermore, clinically available stentless porcine and bovine xenografts were studied as an alternative to homografts., Methods: In 51 adolescent sheep cryopreserved aortic and pulmonary (ovine) homografts, as well as 6 different types of clinically available stentless bioprostheses (Prima Plus, Toronto SPV, Toronto BiLinx, Freestyle, Pericarbon Stentless, and Contegra) were implanted in the pulmonary position. After 5 to 6 months, the valves were explanted and studied for structural valve degeneration by means of radiographic analysis, histology, and calcium content determination., Results: Pulmonary homografts calcified significantly less than aortic homografts in the wall portion. Leaflet calcification was mild, hardly detectable on radiographic analysis, and comparable between aortic and pulmonary homografts. Stentless porcine xenografts showed severe calcification in the aortic wall portion, irrespective of the antimineralization treatment. Leaflet calcification was mild and in the range of that seen in homografts. Pannus formation was present but never induced leaflet retraction or cusp immobilization. Calcification was absent in the stentless Pericarbon valve implants, but all valves showed extensive pannus overgrowth, leaflet retraction, and cusp immobilization. The Contegra valves showed wall calcification, but the leaflets were completely free of calcification and pannus., Conclusions: For right ventricular outflow tract reconstruction, the pulmonary homograft remains the first choice. All xenografts result in either calcific degeneration or cusp immobilization., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

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.

Background: Tetralogy of Fallot (TOF) is the most commonly observed conotruncal congenital heart defect. Treatment of these patients has evolved dramatically in the last few decades, yet a genetic explanation is lacking for the failure of cardiac development for the majority of children with TOF. Our goal was to perform genome wide analyses and characterize expression patterns in cardiovascular tissue (right ventricle, pulmonary valve and pulmonary artery) obtained at the time of reconstructive surgery from 19 children with tetralogy of Fallot., Methods: We employed genome wide gene expression microarrays to characterize cardiovascular tissue (right ventricle, pulmonary valve and pulmonary artery) obtained at the time of reconstructive surgery from 19 children with TOF (16 idiopathic and three with 22q11.2 deletions) and compared gene expression patterns to normally developing subjects., Results: We detected a signal from approximately 26,000 probes reflecting expression from about half of all genes, ranging from 35% to 49% of array probes in the three tissues. More than 1,000 genes had a 2-fold change in expression in the right ventricle (RV) of children with TOF as compared to the RV from matched control infants. Most of these genes were involved in compensatory functions (e.g., hypertrophy, cardiac fibrosis and cardiac dilation). However, two canonical pathways involved in spatial and temporal cell differentiation (WNT, p = 0.017 and Notch, p = 0.003) appeared to be generally suppressed., Conclusions: The suppression of developmental networks may represent a remnant of a broad malfunction of regulatory pathways leading to inaccurate boundary formation and improper structural development in the embryonic heart. We suggest that small tissue specific genomic and/or epigenetic fluctuations could be cumulative, leading to regulatory network disruption and failure of proper cardiac development.

During the fetal-to-neonatal transition, transvalvular pressures (TVPs) on the aortic and pulmonary valves change dramatically-but differently for each valve. We have examined changes in the molecular stability and crosslinking of collagen during this transition. Aortic and pulmonary valves were harvested from fetal and neonatal cattle. Using differential scanning calorimetry (DSC), denaturation of valvular collagen was examined and, using HPLC, the types and quantities of enzymatic crosslinks were examined. No difference in hydrothermal stability was found between the collagens in the fetal aortic and pulmonary valves; this was expected since the TVP is approximately the same across both valves before birth. Only in the neonatal samples was the collagen from aortic valves (higher TVP) less stable than that from pulmonary valves (lower TVP). Surprisingly, the enthalpy of denaturation did not differ either between valve type or with age, suggesting an entropic mechanism of altered molecular stability. A significant difference in immature-to-mature crosslink ratio was found between neonatal aortic and pulmonary valves: a difference absent in fetal valves. This ratio-indicative of remodeling rate-parallels (and may be a function of) the changing in vivo load. This study highlights the relationship between in vivo load and both (i) molecular stability and (ii) collagen remodeling in heart valves.

Objectives: Our aim was to determine whether aortic valve interstitial cells (AVICs) and pulmonary valve interstitial cells (PVICs) differ in expression of Toll-like receptor (TLR)2 and TLR4, response to TLR agonists, and osteogenic phenotypic changes., Background: Calcific stenosis occurs frequently in aortic valves but rarely in pulmonary valves. Studies have implicated AVICs in the inflammation associated with calcification and progression to stenosis. We previously reported that human AVICs express functional TLR2 and TLR4 and that stimulation of these receptors induces pro-osteogenic factor expression., Methods: Human aortic and pulmonary valve leaflets from the same heart were collected and interstitial cells isolated., Results: Aortic valves express more TLR2 and TLR4, in both tissue and isolated interstitial cells, than pulmonary valves. After stimulation with TLR2 and TLR4 agonists, AVICs express higher levels of pro-inflammatory and pro-osteogenic mediators (bone morphogenetic protein [BMP]-2, runt-related transcription factor 2) and greater osteogenic phenotypic changes (alkaline phosphatase [ALP] activity, calcified nodule formation) than PVICs. Silencing TLR2 and TLR4 in AVICs reduced BMP-2 expression and ALP activity to PVIC levels. ALP activity in AVICs induced by TLR2 and TLR4 agonists was abolished by BMP antagonism with Noggin and mimicked by stimulation with recombinant BMP-2. AVICs isolated from stenotic valves had greater expression of TLR2 and TLR4 and a greater BMP-2 response than AVICs from normal valves., Conclusions: Greater expression of TLR2 and TLR4 and greater pro-inflammatory and pro-osteogenic responses to TLR2 and TLR4 agonists in AVICs than PVICs are associated with osteogenic phenotypic changes. These innate immune receptors may play a critical role in aortic valve calcification and stenosis.

Objective: To test the hypothesis that valve allograft (VA) calcification results from an ossification process in which bone-regulatory proteins are expressed., Methods: 15 VA that were explanted at the time of surgery for dysfunction were studied. VA were analysed and compared with normal aortic valves (n = 20)., Results: All the VA (5 aortic, 10 pulmonary) exhibited heavy calcification and important fibrosis. Immunohistochemistry studies showed that the bone-specific transcription factor Cbfa-1 was expressed by stromal cells. Bone alkaline phosphatase was expressed in calcified regions. Immunostaining for alpha smooth muscle (alpha-SM) actin was increased in VA compared with normal valves and in 6 of the 15 valves formed cellular clusters close to the calcified nodules. In VA osteopontin and osteonectin were expressed by stromal cells, whereas osteocalcin was closely associated with the calcified regions. Furthermore, analysis of the bone-regulatory proteins that control bone resorption showed that receptor activator of nuclear factor kappaB ligand (RANKL), receptor activator of nuclear factor kappaB (RANK) and osteoprotegerin (OPG) were differentially expressed in calcified VA and normal valves. Normal valve leaflets expressed OPG, whereas OPG expression was absent or faint in calcified VA. RANKL and RANK were not detected in normal valves, whereas calcified VA expressed RANKL and RANK., Conclusion: These data suggest that calcification of VA results from an ossification process, which relies on tight control of bone-regulatory protein expression. The expression pattern of the RANKL/RANK/OPG system suggests that it may have a regulatory role not only in osteoclastogenesis but also in the calcification of human VA.

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.

Background and Aim of the Study: The generation of a functional, non-immunogenic, non-thrombogenic construct based on autologous cells seeded onto an acellular extracellular matrix is the major goal in heart valve tissue engineering. The study aim was to identify culturing conditions required to achieve a stable endothelial cell (EC) layer under physiological flow conditions, a prerequisite for the requested characteristics., Methods: Eleven detergent-decellularized ovine pulmonary valves (PVs) were statically reseeded in special bioreactors with ovine venous ECs (1.2x10(7) cells per valve). The dynamic culture was started with 0.1 l/min in eight bioreactors. In four bioreactors the initial flow rate was slow, and increased by 0.1 l/min twice each day until maximal flow was 0.5 l/min and pulsation rate (PR) was 20 beats/min; in four other bioreactors the flow was increased by 0.7 l/min/day and reached 2.0 l/min with a PR of 50 beats/min. The mean system pressure was maintained at 25 +/- 5 mmHg during the whole dynamic cultivation in both groups. Three statically reseeded valves served as baseline. After achieving maximal appointed flow, the valves were investigated morphologically (hematoxylin and eosin staining, electron microscopy, von Willebrand factor, endothelial nitric oxide synthase immunostaining) and for metabolic activity (MTS assay)., Results: After reseeding, the endothelium appeared on the luminal surface of the PV as a non-confluent monolayer. Moderate pulsatile circulation induced complete confluence of EC monolayers on both cusp sides and the pulmonary wall. A high flow rate led to a partial loss of cells on the wall surface with large defects, and to complete cell wash-off from cusps. Cusp and wall metabolic activity was significantly higher after culture under moderate flow (p < 0.001) than in other groups, and was absent from cusps in high-flow bioreactors., Conclusion: Moderate pulsatile flow with small increments stimulates EC proliferation on the ovine decellularized valve scaffold. A rapid increase in bioreactor flow to physiological levels leads to significant damage of the reseeded endothelium and complete loss of cusp cellularity. This effect may be responsible for the in-vivo failure of static reseeded tissue-engineered valves exposed to physiological hemodynamic forces.

Objective: This study was performed to evaluate the possibility of creating a glutaraldehyde-free porcine xenograft to improve long-term durability., Methods: A decellularized porcine pulmonary valve was implanted into the right ventricular outflow tract of 7 juvenile sheep. Valves were explanted after 3 months (n = 4) and 6 months (n = 3). Evaluation was performed by gross examination, radiography, histology (hematoxylin-eosin and Sirius red staining), and immunohistochemistry. Quantitative determination of calcium content was investigated by atomic absorption spectrometry., Results: All animals showed fast recovery without complications. At explantation, all decellularized valves showed smooth and pliable leaflets without evidence of thrombosis. The valve wall was also smooth and pliable without hardness. Light microscopy showed a monolayer of host endothelial cells covering the inner surface of the heart valves and repopulation of host fibroblasts into the deeper layers. Sirius red staining enabled visualization of the production of new collagen. Radiographic results showed an absence of calcification, confirmed by the low calcium levels (1.08 +/- 0.28 microg/g and 0.73 +/- 0.31 microg/g at 3 and 6 months, respectively) revealed by atomic absorption spectrometry., Conclusions: The results with the juvenile sheep model showed that decellularized heart valves are recellularized in vivo. Host endothelial cells form a monolayer on the inner surface of the valve matrix. Furthermore, host fibroblasts repopulate the valve matrix and produce collagen; thus, a remodeling potential can be expected.

A biologically engineered xenogenic heart valve based on the concept of acellularization offers promise as a means of overcoming the limitations of current prosthetic valves. We evaluated the process of repopulation by recipient cells on an acellularized xenograft treated with a saline (NaCl)-sodium dodecyl sulfate (SDS) solution. Porcine pulmonary valved conduits acellularized with NaCl-SDS solution were implanted in the right ventricular outflow tract of goats under cardiopulmonary bypass. The goats were sacrificed at 1 week and at 1, 3, 6, and 12 months after implantation. Echocardiographic evaluations of the valves were performed before sacrifice. Visual inspections and histopathologic examinations of the explanted xenogenic valved conduits were performed. Immunohistochemical staining was used to identify the composition of the recellularized cells. Fibroblasts and myofibroblasts were stained by vimentin and smooth muscle actin, and endothelial cells were stained by factor VII related antigen and CD31. Echocardiographic examinations of implanted acellularized xenogenic pulmonary valves revealed satisfactory function except for mild regurgitation and stenosis. On gross examination, all leaflets were well preserved, and no calcification was observed. Microscopic analysis of the acellularized leaflets showed progressive cellular ingrowth over time. The recellularization started from the leaflet base and progressed toward the free margin. Microthrombi were detected on unrecellularized portions. Inflammatory response was detected in the early phase, although it gradually subsided. At 6 months after implantation, cellular ingrowth by fibroblasts, myofibroblasts, and endothelial cells progressed up to 50% of the leaflet's height and progressively increased to 70% after 12 months. Extracellular matrices were regenerated by repopulating cells on the recellularized portion. This study suggests that acellularized xenogenic porcine valved conduits are biocompatible heart valve prostheses that are repopulated with autologous cells and extracellular matrices over a reasonable span of time and preserve the functional integrity without degeneration or calcification of leaflets.

Nonlaminar flow is a major predisposing factor to atherosclerosis. Yet little is known regarding hemodynamic gene regulation in disease-prone areas of the vascular tree in vivo. We have determined spatial patterns of expression of endothelial cell receptors in the arterial tree and of reporter gene constructs in transgenic animals. In this study we show that the endothelial cell-specific receptor Tie1 is induced by disturbed flow in atherogenic vascular niches. Specifically, tie1 expression in the adult is upregulated in vascular bifurcations and branching points along the arterial tree. It is often confined to a single ring of endothelial cells functioning as sphincters and hence experiencing the steepest gradient in shear stress. In aortic valves, tie1 is asymmetrically induced only in endothelial cells encountering changes in flow direction. Disturbance of laminar flow by a surgical interposition of a vein into an artery led to induction of tie1, specifically in the region where the differently sized vessels adjoin. In pathological settings, tie1 expression is specifically induced in areas of disturbed flow because of the emergence of aneurysms and, importantly, in endothelial cells precisely overlying atherosclerotic plaques. Hemodynamic features of atherosclerotic lesion-prone regions, recreated in vitro with the aid of a flow chamber with a built-in step, corroborated an upregulated tie1 promoter activity only in cells residing where flow separation and recirculation take place. These defined promoter elements might be harnessed for targeting gene expression to atherosclerotic lesions.

Background and Aims of the Study: Tissue turnover is one of many factors involved in the operational longevity of heart valves. An understanding of how valves remodel their matrix in response to the hemodynamic environment in health and disease is crucial to the design and biological responsiveness of tissue-engineered valve substitutes. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in matrix remodeling in several tissues, and include interstitial collagenase (MMP-1, MMP-13), the gelatinases (MMP-2, MMP-9) and stromelysin (MMP-3)., Methods: Expression of MMPs and their tissue inhibitors (TIMPs) in human aortic, mitral, tricuspid and pulmonary valves from unused donor or transplant recipient hearts was determined by immunohistochemical staining using antibodies against human MMP-1, MMP-2, MMP-3 and MMP-9 and their inhibitors TIMP-1, TIMP-2, TIMP-3. Cell identification was achieved using antibodies against CD31(endothelial cells), smooth muscle alpha-actin (microfilaments) and CD68 (macrophages)., Results: MMP-1 was expressed in all valves, whereas MMP-2 was only expressed in aortic and pulmonary leaflets. MMP-3 and MMP-9 were not expressed. TIMP-1 and TIMP-2 were expressed in all leaflets, whereas TIMP-3 was observed only in tricuspid leaflets., Conclusion: Valves have a specific pattern of expression of MMPs and TIMPs, which appears to vary in different heart valves. The functional implications and central mechanisms responsible require further study. These findings have important implications in understanding the dynamic nature of valve remodeling and in aiding the development of tissue-engineered valves.

Kinins locally synthesized in the cardiovascular tissue are believed to contribute to the regulation of cardiovascular homeostasis by stimulating the endothelial cells to release nitric oxide, prostacyclin, or a hyperpolarizing factor via autocrine-paracrine mechanisms. This study was designed to investigate the action of sevoflurane on bradykinin-induced Ca2+ mobilization in endothelial cells in situ. Utilizing fura-2-loaded rat pulmonary arterial valve leaflets, the effects of sevoflurane were examined on bradykinin-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ. In the presence of extracellular Ca2+ (1.5 mM), bradykinin (3-30 microM) produced an initial phasic and a subsequent tonic increase in [Ca2+]i in a concentration-dependent manner. However, it produced only the phasic increase in [Ca2+]i in the absence of extracellular Ca2+. Sevoflurane (5%, 0.67 mM) inhibited both the phasic and tonic responses to bradykinin. In these experiments, sevoflurane (3-5%) generated sustained increases (approximately 20-40% of the bradykinin-induced maximal increase in [Ca2+]i) in the resting [Ca2+]i level. Sevoflurane still increased [Ca2+]i after depletion of the intracellular Ca stores with ionomycin (0.1 microM ). However, the sevoflurane-induced increase in [Ca2+]i was eliminated by removal of the extracellular Ca and attenuated by NiCl (1-3 mM). In conclusion, in the pulmonary arterial valvular endothelial cells, sevoflurane inhibits both bradykinin-induced Ca2+ release from the intracellular stores and bradykinin-induced plasmalemmal Ca2+ influx. In addition, sevoflurane appears to stimulate the plasmalemmal Ca2+ influx and thereby increase the endothelial [Ca2+]i level. Sevoflurane might influence the pulmonary vascular tone through its direct action on the pulmonary arterial valvular endothelial cells.

The aim of the present study was to investigate whether the isoprostane 8-epi-PGF2 alpha differently accumulates in semilunar valves of patients suffering from coronary heart disease (CHD, n = 19) as compared to valves from healthy heart donors (controls, n = 6). Sections from isolated aortic and pulmonary valves were analyzed by semiquantitative immunohistochemistry. The 8-epi-PGF2 alpha-content was determined by using a specific radioimmunoassay. The accumulation of 8-epi-PGF2 alpha in both valves was higher in CHD-patients in comparison to controls (Aortic valves: 36.49 +/- 11.26% vs. 15.78 +/- 3.04%; pulmonary valves: 46.79 +/- 9.80% vs. 14.99 +/- 3.57%). The results from the radioimmunoassay revealed comparable findings in both groups (CHD vs. controls: 395.95 +/- 86.09 vs. 139.50 +/- 47.46 pg/mg protein in the aortic valves and 430.47 +/- 76.30 vs. 147.33 +/- 53.84 pg/mg protein in pulmonary valves). Pulmonary valves seem to be more susceptible to oxidative stress than aortic valves as evidenced by a higher accumulation of 8-epi-PGF2 alpha in CHD patients. Considering the data presented in this study, we suggest that 8-epi-PGF2 alpha is a valuable indicator of oxidative injury in human semilunar valves.

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.

To date, little information is available concerning oxidative injury in human cardiac valves. Therefore, we sought to investigate whether the isoprostane, 8-epi-PGF(2alpha), a novel oxidative stress marker, is localized in aortic and pulmonary valves derived from explanted hearts of patients suffering from idiopathic dilative cardiomyopathy (IDC). By using semiquantitative immunohistochemistry, we demonstrated that 8-epi-PGF(2alpha) is localized in both valves with pulmonary valves accumulating more of this isoprostane compared to aortic valves (36.69+/-12.04% vs. 31.54+/-11.49%, P<.05). These results were confirmed by a radioimmunoassay (RIA) analysis showing a similar, but not significant, difference between the two valves (288.50+/-72.18 pg/mg protein in the pulmonary valves and 267.30+/-58.77 pg/mg protein in aortic valves, P=.09). Considering the data presented in this study, we suggest that 8-epi-PGF(2alpha) is a valuable indicator of oxidative injury in human semilunar valves.

Objective: Allograft valves are frequently used in the repair of congenital cardiac anomalies. The failure rate may differ depending on the type of allograft used. Previous studies have shown that rat aortic valve grafts exhibit synthesis of procollagen, suggesting a capacity for repair and regeneration after implantation. No studies of pulmonary valve grafts in the heterotopic rat implant model have thus far been reported. This study was designed to investigate whether pulmonary valve grafts maintain in vivo viability, as demonstrated by procollagen synthesis, and whether cryopreservation, histocompatibility, or both affect this property., Methods: Cryopreserved and fresh rat pulmonary valves were implanted into the abdominal aorta of syngeneic and allogeneic recipients. The grafts and native valves were excised 3 to 21 days after implantation. Valves were sectioned and immunohistochemically stained for procollagen. Computerized morphometry was used to calculate changes in intima, media, and adventitia as a percentage of cross-sectional area of the graft. Procollagen content was graded by semiquantitative methods., Results: Pulmonary valve grafts had significantly greater collagen density in the intima and adventitia compared with native aortic and pulmonary tissues, but collagen density in the media was similar in all groups. The grafts demonstrated appreciably greater procollagen than the corresponding native valves. These findings were consistent in all grafts (ie, both fresh and cryopreserved, both syngeneic and allogeneic), irrespective of duration of implantation., Conclusions: Procollagen synthesis occurs in pulmonary valve grafts early after implantation, indicating viability of these tissues. This model of pulmonary valve implantation may have wide applicability to questions of allograft biology.

Hypoxia (0% O2) evokes a late-phase, endothelium-dependent contractile response in porcine isolated pulmonary arteries that may be caused by a cyclooxygenase-independent, endothelium-derived contractile factor. The aim of this study was to further analyze the mechanism underlying this hypoxic response. Proximal porcine pulmonary arterial rings were suspended for isometric tension recording in organ chambers. Hypoxia (0% O2) caused a late-phase, endothelium-dependent contractile response that was not inhibited by the endothelin (ET)A-receptor antagonist BQ-123 (10(-6) M), by the ETB-receptor antagonist BQ-788 (10(-7) M), or by their combination. In contrast, ET-1 caused a concentration-dependent contraction of arterial rings that was inhibited by BQ-123 (10(-6) M) and a relaxation that was abolished by BQ-788 (10(-7) M) or by endothelial cell removal. Therefore, the endothelium-dependent contraction to hypoxia is not mediated by ET. Hypoxia caused only relaxation in endothelium-denuded rings. However, when a pulmonary valve leaflet, a rich source of pulmonary endothelial cells, was placed into the lumen of endothelium-denuded rings, hypoxia caused a late-phase contractile response that was similar to that observed in arterial rings with native endothelium. This hypoxic contraction persisted in the presence of indomethacin (10(-5) M) and N-nitro-L-arginine methyl ester (3 x 10(-5) M) to block cyclooxygenase and nitric oxide synthase, respectively. These results suggest that hypoxic contraction of pulmonary arteries is mediated by a diffusible, contractile factor released from hypoxic endothelial cells. This contractile mediator is distinct from ET.

Background: This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT)., Methods: Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 x 10(-10) mol/L to 1 x 10(-4) mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 x 10(-6) mol/L) and contracting (NG-monomethyl-L-arginine, 1 x 10(-4) mol/L) drugs., Results: Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage (p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction (p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and NG-monomethyl-L-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions (p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions (p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves., Conclusions: Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.

Intracellular Ca2+ signals of intact endothelium from rabbit aortic or pulmonic valves loaded with fura 2 were studied using imaging fluorescence microscopy. Agonists such as ATP or carbachol and inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase such as cyclopiazonic acid (CPA), thapsigargin, and 2', 5'-di(tert-butyl)-1, 4,-benzohydroquinone (BHQ) induced an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) that was maintained above the prestimulated levels as long as extracellular Ca2+ was present, indicating that the maintained [Ca2+]i increase is dependent on the entry of extracellular Ca2+. The voltage-gated channel was not found to contribute to [Ca2+]i increase. SK&F-96365, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), a postulated phospholipase C inhibitor, were shown to effectively block ROC, since they greatly reduced the maintained [Ca2+]i increase caused by ATP, but not that caused by CPA, which was blocked by Ni2+. To further investigate the Ca2+ influx involved in this process, divalent cation entry was measured as Mn2+ quenching of fura 2 fluorescence at 360-nm excitation wavelength. At rest, fluorescence quenching at 360 nm by Mn2+ was observed, which was inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry through the leak pathway. The quenching was enhanced following ATP stimulation, and this enhancement was abolished by pretreatment with NCDC. In contrast, the rate of Mn2+ quenching was unaffected by the application of CPA. These results demonstrate that ATP stimulates divalent cation influx that is not related to the Ca2+ content of ER. Abolition of Ca2+ uptake into ER was postulated to increase the effectiveness of the Ca2+ leak in raising [Ca2+]i.

Cytosolic Ca2+ plays a critical role in the secretion of endothelium-derived factors. A new preparation that allows fluorescence imaging of intracellular free Ca2+ concentration ([Ca2+]i) in endothelial cells of rabbit cardiac valves is described. Electron micrographs of the valves revealed no underlying smooth muscle cells that might influence endothelial cell responses or contribute to [Ca2+]i signaling. The valve leaflets, which were < 100 microns in diameter, were visualized using a specially designed chamber and a long working distance fluorescence objective. The semilunar valves (pulmonary and aortic) responded to endothelium-dependent vasodilators, including acetylcholine, with an increase in [Ca2+]i. Synchronized [Ca2+]i transients were observed in the endothelial monolayer in response to agonist stimulation in K(+)-free solutions. The ability to monitor changes in [Ca2+]i in a native endothelial monolayer provides a more realistic assessment of stimulus-response coupling within individual cells and communication between cells of native endothelium. In addition, this preparation affords an opportunity for comparative studies of endothelium-related pathophysiologies, which can be induced experimentally in animal models.

A method for determining the viability of homograft valves has been developed based on sequential measurements of glucose and pH levels of the culture medium in which cardiac valves have been maintained for short periods at 37 degrees C. Viable valves, as determined by tissue culture, showed a characteristic pattern of glucose utilization and pH reduction that was absent in nonviable valves. Upon explantation of valve leaflet fragments into tissue culture, only fragments from valves that metabolized glucose produced viable fibroblast cultures. The method reported here is rapid, requires no specialized equipment, is nondestructive, and can directly determine the viability of the valve homograft within 24 to 48 hours.

Viability of canine heart valve leaflet fibroblasts was assessed after varying periods of sterilization and storage in antibiotic-nutrient solution. Tissue culture and assessment of protein and collagen synthesis showed that tissue obtained under optimal conditions rarely retains viability beyond 3 weeks in antibiotic-nutrient solution and is severely impaired after 2 weeks. This casts serious doubts on viability in current clinical homograft valve practice.

1. Two distinct hexosamine-containing substances have been obtained from bovine cardiac valvular tissue which was incubated with labelled glucosamine. These were identified as mucopolysaccharides and glycopeptides respectively, both by the elution pattern from a Sephadex G-50 column and by chemical analysis. 2. In the mucopolysaccharide fraction about 80% of both the total hexosamine and total radioactivity were present; the galactosamine/glucosamine ratio of the amounts was about 1.36. 3. The glycopeptide fractions had about 20% of both total hexosamine and total radioactivity; the galactosamine/glucosamine ratio of amounts was about 0.44. In this fraction, over 15% of radioactivity was present in sialic acid. 4. In contrast with the concentrations of the several chemical components, there were remarkable differences in the biosynthetic activities among the four valves; the tricuspid valve had highest specific radioactivity in all components of both substances, followed then in turn by mitral, aortic and pulmonary valves. 5. Glucosamine in mucopolysaccharides had a rapid rate of turnover, followed then in turn by turnover rates of both glucosamine and galactosamine in glycopeptides, and of galactosamine in mucopolysaccharides. The possible significance of these findings is discussed.