Aortic valve disease in diabetes: Molecular mechanisms and novel therapies.

Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non-diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic-hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes-induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.

Transforming growth factor-ß1 promotes fibrosis but attenuates calcification of valvular tissue applied as a three-dimensional calcific aortic valve disease model.

Calcific aortic valve disease (CAVD) is characterized by valvular fibrosis and calcification and driven by differentiating valvular interstitial cells (VICs). Expression data from patient biopsies suggest that transforming growth factor (TGF)-ß1 is implicated in CAVD pathogenesis. However, CAVD models using isolated VICs failed to deliver clear evidence on the role of TGF-ß1. Thus, employing cultures of aortic valve leaflets, we investigated effects of TGF-ß1 in a tissue-based three-dimensional (3-D) CAVD model. We found that TGF-ß1 induced phosphorylation of Mothers against decapentaplegic homolog (SMAD) 3 and expression of SMAD7, indicating effective downstream signal transduction in valvular tissue. Thus, TGF-ß1 increased VIC contents of rough endoplasmic reticulum, Golgi, and secretory vesicles as well as tissue levels of RNA and protein. In addition, TGF-ß1 raised expression of proliferation marker cyclin D1, attenuated VIC apoptosis, and upregulated VIC density. Moreover, TGF-ß1 intensified myofibroblastic VIC differentiation as evidenced by increased α-smooth muscle actin and collagen type I along with diminished vimentin expression. In contrast, TGF-ß1 attenuated phosphorylation of SMAD1/5/8 and upregulation of ß-catenin while inhibiting osteoblastic VIC differentiation as revealed by downregulation of osteocalcin expression, alkaline phosphatase activity, and extracellular matrix incorporation of hydroxyapatite. Collectively, these effects resulted in blocking of valvular tissue calcification and associated disintegration of collagen fibers. Instead, TGF-ß1 induced development of fibrosis. Overall, in a tissue-based 3-D CAVD model, TGF-ß1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs, thus triggering dominant fibrosis. Simultaneously, by inhibiting SMAD1/5/8 activation and canonical Wnt/ß-catenin signaling, TGF-ß1 attenuates osteoblastic VIC differentiation, thus blocking valvular tissue calcification. These findings question a general phase-independent CAVD-promoting role of TGF-ß1.NEW & NOTEWORTHY Employing aortic valve leaflets as a tissue-based three-dimensional disease model, our study investigates the role of transforming growth factor (TGF)-ß1 in calcific aortic valve disease pathogenesis. We find that, by activating Mothers against decapentaplegic homolog 3, TGF-ß1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of valvular interstitial cells, thus triggering dominant fibrosis. Simultaneously, by inhibiting activation of Mothers against decapentaplegic homolog 1/5/8 and canonical Wnt/ß-catenin signaling, TGF-ß1 attenuates apoptosis and osteoblastic differentiation of valvular interstitial cells, thus blocking valvular tissue calcification. These findings question a general phase-independent calcific aortic valve disease-promoting role of TGF-ß1.

Activated platelets promote an osteogenic programme and the progression of calcific aortic valve stenosis.

AIMS: Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. METHODS AND RESULTS: Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR-/- apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. CONCLUSIONS: These findings suggest a novel role for platelets in the progression of CAVS.

Aortic Valve Stenosis and Mitochondrial Dysfunctions: Clinical and Molecular Perspectives.

Calcific aortic stenosis is a disorder that impacts the physiology of heart valves. Fibrocalcific events progress in conjunction with thickening of the valve leaflets. Over the years, these events promote stenosis and obstruction of blood flow. Known and common risk factors are congenital defects, aging and metabolic syndromes linked to high plasma levels of lipoproteins. Inflammation and oxidative stress are the main molecular mediators of the evolution of aortic stenosis in patients and these mediators regulate both the degradation and remodeling processes. Mitochondrial dysfunction and dysregulation of autophagy also contribute to the disease. A better understanding of these cellular impairments might help to develop new ways to treat patients since, at the moment, there is no effective medical treatment to diminish neither the advancement of valve stenosis nor the left ventricular function impairments, and the current approaches are surgical treatment or transcatheter aortic valve replacement with prosthesis.

Ultrastructural Pathology of Atherosclerosis, Calcific Aortic Valve Disease, and Bioprosthetic Heart Valve Degeneration: Commonalities and Differences.

Atherosclerosis, calcific aortic valve disease (CAVD), and bioprosthetic heart valve degeneration (alternatively termed structural valve deterioration, SVD) represent three diseases affecting distinct components of the circulatory system and their substitutes, yet sharing multiple risk factors and commonly leading to the extraskeletal calcification. Whereas the histopathology of the mentioned disorders is well-described, their ultrastructural pathology is largely obscure due to the lack of appropriate investigation techniques. Employing an original method for sample preparation and the electron microscopy visualisation of calcified cardiovascular tissues, here we revisited the ultrastructural features of lipid retention, macrophage infiltration, intraplaque/intraleaflet haemorrhage, and calcification which are common or unique for the indicated types of cardiovascular disease. Atherosclerotic plaques were notable for the massive accumulation of lipids in the extracellular matrix (ECM), abundant macrophage content, and pronounced neovascularisation associated with blood leakage and calcium deposition. In contrast, CAVD and SVD generally did not require vasculo- or angiogenesis to occur, instead relying on fatigue-induced ECM degradation and the concurrent migration of immune cells. Unlike native tissues, bioprosthetic heart valves contained numerous specialised macrophages and were not capable of the regeneration that underscores ECM integrity as a pivotal factor for SVD prevention. While atherosclerosis, CAVD, and SVD show similar pathogenesis patterns, these disorders demonstrate considerable ultrastructural differences.

Evaluation of Supercritical CO2-Assisted Protocols in a Model of Ovine Aortic Root Decellularization.

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO2-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO2 medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young's modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.

End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles.

Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.

Microstructural alterations owing to handling of bovine pericardium to manufacture bioprosthetic heart valves: A potential risk for cusp dehiscence.

INTRODUCTION: Cross-linking and anti-calcification of prosthetic heart valves have been continuously improved to prevent degeneration and calcification. However, non-calcific structural deteriorations such as cuspal dehiscences along the stent still require further analysis. MATERIAL AND METHOD: Based upon the previous analysis of an explanted valve after 7 years, a fresh commercial aortic valve was embedded in poly(methyl methacrylate) (PMMA) and cut into slices to ensure the detailed observation of the assembly and material structures. A pericardial patch embossed to provide the adequate shape of the cusps was investigated after paraffin embedding and appropriate staining. The microstructural damages that occurred during manufacturing process were identified and evaluated by light microscopy, polarized microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The wavy collagen bundles, the key structure of the pericardium patch, were damaged to a great extent at suture sites along the stent and in the compressed areas around the stent post. The fixation of the embossed pericardium patch along the plots of the stent aggravated the microstructural modifications. The damages mainly appeared as the elimination of collagen bundle waviness and delamination between the bundles. CONCLUSION: Considering the modes of failure of the explant, the damages to the collagen bundles may identify the vulnerable sites that play an important role in the cusp dehiscence of heart valve implants. Such information is important to the manufacturers. Recommendations to prevent in vivo cusp dehiscence can therefore be formulated.

Decellularized aortic conduits: could their cryopreservation affect post-implantation outcomes? A morpho-functional study on porcine homografts.

Decellularized porcine aortic valve conduits (AVCs) implanted in a Vietnamese Pig (VP) experimental animal model were matched against decellularized and then cryopreserved AVCs to assess the effect of cryopreservation on graft hemodynamic performance and propensity to in vivo repopulation by host's cells. VPs (n = 12) underwent right ventricular outflow tract substitution using AVC allografts and were studied for 15-month follow-up. VPs were randomized into two groups, receiving AVCs treated with decellularization alone (D; n = 6) or decellularization/cryopreservation (DC; n = 6), respectively. Serial echocardiography was carried out to follow up hemodynamic function. All explanted AVCs were processed for light and electron microscopy. No signs of dilatation, progressive stenosis, regurgitation, and macroscopic calcification were echocardiographically observed in both D and DC groups. Explanted D grafts exhibited near-normal features, whereas the presence of calcification, inflammatory infiltrates, and disarray of elastic lamellae occurred in some DC grafts. In the unaltered regions of AVCs from both groups, almost complete re-endothelialization was observed for both valve cusps and aorta walls. In addition, side-by-side repopulation by recipient's fibroblasts, myofibroblasts, and smooth muscle cells was paralleled by ongoing tissue remodeling, as revealed by the ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features. Incipient neo-vascularization and re-innervation of medial and adventitial tunicae of grafted aortic walls were also detected for both D and DC groups. Cryopreservation did not affect post-implantation AVC hemodynamic behavior and was topically propensive to cell repopulation and tissue renewal, although graft deterioration including calcification was present in several areas. Thus, these preliminary data provide essential information on feasibility of decellularization and cryopreservation coupling in the perspective of treatment optimization and subsequent clinical trials using similarly treated human allografts as innovative heart valve substitutes.

Cryoprotective Effect and Optimal Concentration of Trehalose on Aortic Valve Homografts.

BACKGROUND AND AIM OF THE STUDY: Cryopreservation allows for the long-term storage of aortic homografts, but a high risk of calcification and degeneration is often observed following their transplantation. The study aim was to investigate the cryoprotective effect of trehalose on aortic valve homografts preserved in liquid nitrogen, and to determine the optimal trehalose concentration for such purpose. METHODS: Aortic valve homografts obtained from New Zealand White rabbits were processed using different protectants. Samples were assigned at random to four groups: a control group treated with dimethyl sulfoxide (DMSO; 0.1 mol/l), and test groups A, B and C, which were treated with 0.1 mol/l trehalose + 0.1 mol/l DMSO, 0.2 mol/l trehalose + 0.1 mol/l DMSO, or 0.3 mol/l trehalose + 0.1 mol/l DMSO, respectively, as protectant. Samples in each group were allocated randomly to three subgroups and cryopreserved for 12, 15, and 18 months, respectively. After thawing, apoptosis of the cryopreserved homograft samples was evaluated using immunohistochemistry, semi-quantitative RT-PCR and Western blotting methods. The viability of the tissue cells was assessed by monitoring glucose utilization capacity. RESULTS: The apoptosis assay showed that, among the four groups and at all time points, the expression of caspase-3 was lowest in test groups A and B and highest in the DMSO group. In comparison, the glucose metabolic rates of test groups A and B were highest, while rates for test group C and the control (DMSO) group ranked second and third. CONCLUSION: When aortic homografts are preserved in liquid nitrogen, the cryoprotective effect of trehalose combined with DMSO was superior to that of DMSO alone. The optimal trehalose concentration to cryoprotect rabbit aortic homografts was between 0.1 and 0.2 mol/l.

[CALCIFYING NANOPARTICLES IN PATHOMORPHOGENESIS OF STRUCTURAL LESIONS OF HEART VALVES].

Over the last 10 years, calcifying nanoparticles (CNP) have attracted attention as structures detected. together with many other nanostructures in biopsies from patients operated for the correction of aortic valve malformations. The results of the present work performed with the use of high-resolution transmission and scanning electron microscopes agree on the whole with the data of other authors. Some new findings include CNP adhesion to collagen fibers and specifically-shaped, shallow invaginations or craters at their surface. The possible pathophysiological mechanisms that promote involvement of CNP in the development of the disease are considered.

Replacement of the st jude medical regent valve in the aortic position with a continuous suture technique in the small aortic root.

BACKGROUND: The aim of this study is to review the strategy of performing aortic valve replacement (AVR) by using the St. Jude Medical (SJM) Regent valve with a continuous suture technique in patients with a small aortic root. METHODS: Forty-six patients with small aortic annulus underwent AVR by using 19 or 21 mm SJM Regent valves. There were 15 males and 31 females. The mean age of the patients was 51.8 ± 12.4 years. The aortic annular diameter was 20.2 ± 0.9 mm. AVR procedures were performed with continuous suture technique using SJM Regent valves under standard cardiopulmonary bypass. Echocardiaographic data were collected before operation, at discharge, and at a follow-up time, respectively. RESULTS: The intraoperative course was uneventful and there was no operative mortality. The implanted SJM Regent valves consisted of 21 mm valves in 15 patients and 19 mm valves in 31 patients. Echocardiography at 5.6 ± 1.3 months after operation showed a significant increase in the mean effective orifice area index (0.97 ± 0.24 cm(2) /m(2) ), decrease in the mean and peak transvavluar pressure gradient (12.5 ± 5.9 and 22.3 ± 9.6 mmHg), and decrease in the mean left ventricular mass index (106 ± 41.3 g/m(2) ). Moderate prosthesis-patient mismatch (PPM) (effective orifice area index between 0.65 and 0.85 cm(2) /m(2) ) was present in three patients and no severe PPM (effective orifice area index <0.65 cm(2) /m(2) ) occurred at discharge and during follow-up. CONCLUSION: Replacement of SJM Regent valve with a continuous suture technique maybe a good option to prevent PPM in the aortic position.

Collagen mineralization in human aortic valve stenosis: a field emission scanning electron microscopy and energy dispersive spectroscopy analysis.

Abstract Calcific aortic stenosis is a slowly progressive disorder characterized by an important extracellular matrix remodeling with fibrosis and massive deposition of minerals (primarily calcium) in the valve leaflet. The main structural components of human aortic valve are the large, thick collagen bundles that withstand the diastolic loading. Collagen has been studied in a number of reports that aim to clarify the mechanisms underlying the structural deterioration of heart valve substitutes, however to date, little is known regarding the morphological interaction between collagen and mineral crystals in the calcifying tissue of native aortic valve. Here, we have analyzed a total of 12 calcified native aortic valves by using scanning electron microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) to depict the morphological appearance of mineralized collagen and to determine the location of calcium phosphate minerals in the collagen matrix of the valve cusp. Our results demonstrate that crystals probably nucleate and grow in the interior of the collagen fibers in the absence of surface events.

Massive accumulation of glycosaminoglycans in the aortic valve of a patient with Hunter syndrome during enzyme replacement therapy.

This report describes the pathologic findings for a patient with Hunter syndrome who underwent aortic valve replacement at 10 years of age, 3 years after the initiation of enzyme replacement therapy. Aortic valve pathology showed mild thickening and fibrosis as well as massive glycosaminoglycan accumulation. This suggests that enzyme replacement therapy has limited efficacy for cardiac valve disease both clinically and pathologically.

Deletion of RBP-J in adult mice leads to the onset of aortic valve degenerative diseases.

Transcription factor RBP-J-mediated Notch signaling has been implicated in several inherited cardiovascular diseases including aortic valve diseases (AVD). But whether Notch signal plays a role in AVD in adults has been unclear. This study aims to test whether the deletion of RBP-J in adult mice would lead to AVD and to investigate the underlying mechanisms. Cre-LoxP-mediated gene deletion was employed to disrupt Notch signal in adult mice. Immunofluorescence and electron microscope observations showed that deletion of RBP-J in adult mice led to early morphological changes of AVD. The size of aortic valve was enlarged. The endothelial homeostasis was perturbed, probably due to the up-regulation of VEGFR2. The endothelial cells exhibited increased proliferation and loose endothelial junctions. The valvular mesenchyme displayed significant fibrosis, consistent with the up-regulation of TGF-ß1 and activation of endothelial-mesenchymal transition. We observed melanin-producing cells in aortic valves. The number of melanin-producing cells increased significantly, and their location changed from the mesenchyme to subendothelial layer of valve cusps in RBP-J deficient mice. These results suggest that RBP-J-mediated Notch signaling in aortic valves may be critically involved in valve homeostasis and valve diseases as well. These findings will be helpful for the understanding of the molecular mechanisms of AVD in adults.

Extracellular matrix in deoxycholic acid decellularized aortic heart valves.

BACKGROUND: Only limited information is available regarding the influence of decellularization on the extracellular matrix in heart valves. Within the extracellular matrix proteoglycans (PG) play a central role in the structural organization and physical functioning of valves and in their capability of settling with endothelial and interstitial cells partially myofibroblasts. We have therefore estimated the effects of decellularization using deoxycholic acid on the structure of the extracellular matrix and PG´s in porcine aortic valves. MATERIAL/METHODS: Cupromeronic blue was used, alone or in combination with OsO4/thio-carbo-hydrazide/OsO4 for electron microscopic visualization. For PG and glycosaminoglycan (GAG) investigation a papain digestion was employed in combination with photometric determination using dimethylmethylene blue. RESULTS: The results indicate that deoxycholic acid affects the compartmentation of the PG-associated interstitial network not significantly. Compared to controls the PG-rich network was preserved even after deoxycholic acid treatment for 48 h. In parallel to electron microscopy immune assays (ELISA) showed smooth muscle cell -actin to be reduced to 0.96% ± 0.71 and total soluble protein to 6.68% ± 2.0 (n=3) of untreated controls. Protein loss corresponded well with the observations in electron micrographs of rupture and efflux of cell content. Further signs of lysis were irregular cell contours and loss of the basement membrane. CONCLUSIONS: Efficient cell-lysis without disintegration or loss of integrity of the interstitial PG network can be achieved by treatment of aortic valves with deoxycholic acid for 48h. This protocol might also be suitable for clinical use to optimize conditions for growth and autologous remodelling of valves.

Decellularization of heart valve matrices: search for the ideal balance.

OBJECTIVE: Currently used decellularization procedures have negative effects on extracellular matrix (ECM) integrity. The objective of this study is to evaluate four decellularization methods and their effect on the collagen ultrastructure, mechanical behavior and antigenicity of porcine aortic valves. METHODS: Aortic valves were placed in a trypsin, osmotic, trypsin-osmotic or detergent-osmotic solution. Leaflets were processed for histology and mechanical testing. Matrices were implanted subdermally in rats to evaluate immune reaction and calcification. RESULTS: Trypsin-osmotic methodology effected near-complete decellularization. Trypsin treatment resulted in cell removal only in the spongiosa layer. Osmotic and detergent-osmotic treatments did not remove any cells from the cusps. Mechanical strength was significantly inferior in the trypsin (p50,03) and trypsin-osmotic treated group (p50,04). Trypsin and trypsin-osmotic decellularized matrices evoked a strong CD31 inflammatory cell infiltration. CONCLUSION: Enzymatic-osmotic decellularization appears to be the only effective method to remove cellular components. However, the near cell free scaffolds still evokes a strong CD31 T-cell inflammatory reaction.

New evidence for a critical role of elastin in calcification of native heart valves: immunohistochemical and ultrastructural study with literature review.

AIMS: Calcific aortic stenosis is a progressive disease characterized by massive fibrosis andmineralization of the valve leaflets. The aim of this study was to determine whether the onset of native calcific aortic stenosis is associated primarily with matrix remodelling events, and particularly with elastin degradation. METHODS AND RESULTS: The immunohistochemical expression profile of matrix degradating enzymes and tenascin-C was investigated in both healthy and native calcified aortic valves. Collagen and elastic tissue were studied by light microscopy and electron microscopy. Immunophenotypic analysis of inflammatory cells was carried out by using monoclonal antibodies to macrophages, T and B lymphocytes. Immunoreactivity for tenascin-C and matrix metalloproteinase-12 (MMP-12) was associated with areas of dense mineralization, which were characterized by fibrosis, fragmentation and calcification of elastic fibres a positive reaction was also found around small islands of calcification. MMP-11 was not detected in the diseased valves. Osteopontin and osteonectin were also found at sites of mineralization. All calcified valves examined showed inflammatory cell infiltration. CONCLUSIONS: Our results demonstrate the direct involvement of MMP-12 in native aortic valve stenosis. MMP-mediated degradation of elastic fibres might contribute actively to valve mineralization by inducing calcium deposition onto fragmented elastin.

Complex aortic valve repair following excision of a papillary fibroelastoma.

Complex aortic valve repair after mass lesion resection, in an otherwise normal, thin leafleted valve, is rarely described in the literature. We present a 68-year-old woman who underwent resection of an asymptomatic aortic valve papillary fibroelastoma. Due to extensive involvement of her left coronary cusp, the resection resulted in a significant defect in the leaflet, requiring a complex repair to preserve her otherwise normal aortic valve. We describe the operative findings, repair technique, and associated literature.