Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Tissue response, macrophage phenotype, and intrinsic calcification induced by cardiovascular biomaterials: Can clinical regenerative potential be predicted in a rat subcutaneous implant model?

The host immune response to an implanted biomaterial, particularly the phenotype of infiltrating macrophages, is a key determinant of biocompatibility and downstream remodeling outcome. The present study used a subcutaneous rat model to compare the tissue response, including macrophage phenotype, remodeling potential, and calcification propensity of a biologic scaffold composed of glutaraldehyde-fixed bovine pericardium (GF-BP), the standard of care for heart valve replacement, with those of an electrospun polycarbonate-based supramolecular polymer scaffold (ePC-UPy), urinary bladder extracellular matrix (UBM-ECM), and a polypropylene mesh (PP). The ePC-UPy and UBM-ECM materials induced infiltration of mononuclear cells throughout the thickness of the scaffold within 2 days and neovascularization at 14 days. GF-BP and PP elicited a balance of pro-inflammatory (M1-like) and anti-inflammatory (M2-like) macrophages, while UBM-ECM and ePC-UPy supported a dominant M2-like macrophage phenotype at all timepoints. Relative to GF-BP, ePC-UPy was markedly less susceptible to calcification for the 180 day duration of the study. UBM-ECM induced an archetypical constructive remodeling response dominated by M2-like macrophages and the PP caused a typical foreign body reaction dominated by M1-like macrophages. The results of this study highlight the divergent macrophage and host remodeling response to biomaterials with distinct physical and chemical properties and suggest that the rat subcutaneous implantation model can be used to predict in vivo biocompatibility and regenerative potential for clinical application of cardiovascular biomaterials.

Increased utilization of bioprosthetic aortic valve technology:Trends, drivers, controversies and future directions.

Introduction: Bioprosthetic valves (BPV) implanted surgically or by transcatheter valve implantation (TAVI) comprise an overwhelming majority of substitute aortic valves implanted worldwide.Areas Covered: Prominent drivers of this trend are: 1) BPV patients have generally better outcomes than those with a mechanical valve, and remain largely free of anticoagulation and its consequences; 2) BPV durability has improved over the years; and 3) the expanding use of TAVI and valve-in-valve (VIV) procedures permitting interventional management of structural valve degeneration (SVD). Nevertheless, key controversies exist: 1) optimal anticoagulation regimens for surgical and TAVI BPVs; 2) the incidence, mechanisms and mitigation strategies for SVD; 3) the use of VIV for treatment of SVD, and 4) valve selection recommendations for difficult cohorts, (e.g. patients 50-70 years, patients <50, childbearing age women). This communication reviews trends in and drivers of BPV utilization, current controversies, and future directions affecting BPV use.Expert Opinion: Long-term data are needed in several areas related to aortic BPV use, including anticoagulation/antiplatelet therapy, especially following TAVI. TAVI and especially VIV durability and optimal use warrant will benefit greatly from long-term data. Certain populations may benefit from such high-quality data on multi-year outcomes, particularly younger patients.

Biology and Biomechanics of the Heart Valve Extracellular Matrix.

Heart valves are dynamic structures that, in the average human, open and close over 100,000 times per day, and 3 × 109 times per lifetime to maintain unidirectional blood flow. Efficient, coordinated movement of the valve structures during the cardiac cycle is mediated by the intricate and sophisticated network of extracellular matrix (ECM) components that provide the necessary biomechanical properties to meet these mechanical demands. Organized in layers that accommodate passive functional movements of the valve leaflets, heart valve ECM is synthesized during embryonic development, and remodeled and maintained by resident cells throughout life. The failure of ECM organization compromises biomechanical function, and may lead to obstruction or leaking, which if left untreated can lead to heart failure. At present, effective treatment for heart valve dysfunction is limited and frequently ends with surgical repair or replacement, which comes with insuperable complications for many high-risk patients including aged and pediatric populations. Therefore, there is a critical need to fully appreciate the pathobiology of biomechanical valve failure in order to develop better, alternative therapies. To date, the majority of studies have focused on delineating valve disease mechanisms at the cellular level, namely the interstitial and endothelial lineages. However, less focus has been on the ECM, shown previously in other systems, to be a promising mechanism-inspired therapeutic target. Here, we highlight and review the biology and biomechanical contributions of key components of the heart valve ECM. Furthermore, we discuss how human diseases, including connective tissue disorders lead to aberrations in the abundance, organization and quality of these matrix proteins, resulting in instability of the valve infrastructure and gross functional impairment.

Preclinical Assessment of Cardiac Valve Substitutes: Current Status and Considerations for Engineered Tissue Heart Valves.

Tissue engineered heart valve (TEHV) technology may overcome deficiencies of existing available heart valve substitutes. The pathway by which TEHVs will undergo development and regulatory approval has several challenges. In this communication, we review: (1) the regulatory framework for regulation of medical devices in general and substitute heart valves in particular; (2) the special challenges of preclinical testing using animal models for TEHV, emphasizing the International Standards Organization (ISO) guidelines in document 5840; and (3) considerations that suggest a translational roadmap to move TEHV forward from pre-clinical to clinical studies and clinical implementation.

Functional remodeling of an electrospun polydimethylsiloxane-based polyether urethane external vein graft support device in an ovine model.

Saphenous vein graft (SVG) failure rates are unacceptably high, and external mechanical support may improve patency. We studied the histologic remodeling of a conformal, electrospun, polydimethylsiloxane-based polyether urethane external support device for SVGs and evaluated graft structural evolution in adult sheep to 2 years. All sheep (N = 19) survived to their intended timepoints, and angiography showed device-treated SVG geometric stability over time (30, 90, 180, 365, or 730 days), with an aggregated graft patency rate of 92%. There was minimal inflammation associated with the device material at all timepoints. By 180 days, treated SVG remodeling was characterized by minimal/nonprogressive intimal hyperplasia; polymer fragmentation and integration; as well as the development of a neointima, and a confluent endothelium. By 1-year, the graft developed a media-like layer by remodeling the neointima, and elastic fibers formed well-defined structures that subtended the neo-medial layer of the remodeled SVG. Immunohistochemistry showed that this neo-media was populated with smooth muscle cells, and the intima was lined with endothelial cells. These data suggest that treated SVGs were structurally remodeled by 180 days, and developed arterial-like features by 1 year, which continued to mature to 2 years. Device-treated SVGs remodeled into arterial-like conduits with stable long-term performance as arterial grafts in adult sheep.

Repeated generalized seizures can produce calcified cardiac lesions in DBA/1 mice.

Studies suggest that cardiorespiratory dysfunction likely contributes to sudden unexpected death in epilepsy (SUDEP). Seizures result in autonomic and respiratory dysfunction, leading to sympathetic hyperactivity and respiratory distress, including apnea. While the heart is vulnerable to catecholamine surges and hypoxia, it remains unknown if repetitive generalized seizures lead to cardiac damage. DBA/1 mice exhibit seizure-induced respiratory arrest (S-IRA) following generalized audiogenic seizures (AGS), which can be resuscitated using a rodent ventilator. In the current study, we induced different numbers of S-IRA episodes in DBA/1 mice and determined the association of repeated S-IRA induction with cardiac damage using histology. After repetitive induction of 18 S-IRA, calcified lesions, as revealed by calcium (Ca2+)-specific alizarin red staining, were observed in the ventricular myocardium in 61.5% of DBA/1 mice, which was higher compared to mice with 5 S-IRA and 1 S-IRA as well as age-matched untested control mice. The incidence of lesions in mice with 9 S-IRA was only higher than that of control mice. Only 1-2, small lesions were observed in mice with 5 S-IRA and 1 S-IRA and in control mice. Larger lesions (>2500 µm2) were observed in mice with 9 and 18 S-IRA. The incidence of larger lesions was higher in mice with 18 S-IRA (53.8%) as compared to mice with 5 S-IRA and 1 S-IRA as well as with control mice, and the incidence of larger lesions in mice with 9 S-IRA was only higher than that of control mice. Repeated induction of S-IRA in DBA/1 mice can result in calcified necrotic lesions in the ventricles of the heart, and their incidence and size are dependent on the total number of S-IRA.

After 50 Years of Heart Transplants: What Does the Next 50 Years Hold for Cardiovascular Medicine? A Perspective From the International Society for Applied Cardiovascular Biology.

The first successful heart transplant 50 years ago by Dr.Christiaan Barnard in Cape Town, South Africa revolutionized cardiovascular medicine and research. Following this procedure, numerous other advances have reduced many contributors to cardiovascular morbidity and mortality; yet, cardiovascular disease remains the leading cause of death globally. Various unmet needs in cardiovascular medicine affect developing and underserved communities, where access to state-of-the-art advances remain out of reach. Addressing the remaining challenges in cardiovascular medicine in both developed and developing nations will require collaborative efforts from basic science researchers, engineers, industry, and clinicians. In this perspective, we discuss the advancements made in cardiovascular medicine since Dr. Barnard's groundbreaking procedure and ongoing research efforts to address these medical issues. Particular focus is given to the mission of the International Society for Applied Cardiovascular Biology (ISACB), which was founded in Cape Town during the 20th celebration of the first heart transplant in order to promote collaborative and translational research in the field of cardiovascular medicine.

Morphology and mechanisms of a novel absorbable polymeric conduit in the pulmonary circulation of sheep.

BACKGROUND: Right ventricular outflow tract (RVOT) conduits used in children with congenital heart disease often degenerate rapidly or develop other complications, and they do not grow with the patient. This leads to multiple surgeries until adult-sized conduits can be implanted. We report experimental in vivo experience with an entirely synthetic absorbable graft, designed to be replaced by tissue in-vivo by host cells, in a process termed Endogenous Tissue Restoration (ETR), and to grow commensurate with somatic growth. METHODS: We characterized the structure, mechanical properties, biocompatibility, and in vivo remodelling of a bioabsorbable polyester based on the self-complementary ureido-pyrimidinone (UPy) quadruple hydrogen-bonding motif. Electrospinning was used to process the polymer into a tubular graft with a highly porous wall structure, which was implanted as a pulmonary artery interposition graft in 9 adult sheep with a maximum follow-up of 1 year, followed by pathologic and mechanical analysis. RESULTS: All grafts were patent by transthoracic echocardiography. Eight were intact at post-mortem examination. One graft had aneurysmal dilation. Graft polymer resorption in vivo was consistent among specimens. Histologic examination revealed progressive tissue replacement of graft polymer, ongoing at one year, with remodeling to a structure that had some key features of native vascular wall. Burst pressures for all explants at 8 weeks and beyond were higher than those of native pulmonary artery (PA) and largely determined by newly formed tissue. CONCLUSIONS: Preclinical studies of a new, absorbable polymeric graft for PA replacement showed remodelling by endogenous cells up to one-year follow-up. Our results show that ETR leads to progressive and substantial replacement of an off-the-shelf synthetic bioabsorbable conduit by functional host tissue to one year in sheep. Thus, further development of this novel concept is warranted.

Early animal model evaluation of an implantable contrast agent to enhance magnetic resonance imaging of arterial bypass vein grafts.

Background Non-invasive monitoring of autologous vein graft (VG) bypass grafts is largely limited to detecting late luminal narrowing. Although magnetic resonance imaging (MRI) delineates vein graft intima, media, and adventitia, which may detect early failure, the scan time required to achieve sufficient resolution is at present impractical. Purpose To study VG visualization enhancement in vivo and delineate whether a covalently attached MRI contrast agent would enable quicker longitudinal imaging of the VG wall. Material and Methods Sixteen 12-week-old male C57BL/6J mice underwent carotid interposition vein grafting. The inferior vena cava of nine donor mice was treated with a gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA)-based contrast agent, with control VGs labeled with a vehicle. T1-weighted (T1W) MRI was performed serially at postoperative weeks 1, 4, 12, and 20. A portion of animals was sacrificed for histopathology following each imaging time point. Results MRI signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were significantly higher for treated VGs in the first three time points (1.73 × higher SNR, P = 0.0006, and 5.83 × higher CNR at the first time point, P = 0.0006). However, MRI signal enhancement decreased consistently in the study period, to 1.29 × higher SNR and 2.64 × higher CNR, by the final time point. There were no apparent differences in graft morphometric analyses in Masson's trichrome-stained sections. Conclusion A MRI contrast agent that binds covalently to the VG wall provides significant increase in T1W MRI signal with no observed adverse effects in a mouse model. Further optimization of the contrast agent to enhance its durability is required.

Nanoparticles targeting extra domain B of fibronectin-specific to the atherosclerotic lesion types III, IV, and V-enhance plaque detection and cargo delivery.

Extra domain B of fibronectin (FN-EDB) is upregulated in the extracellular matrix during tissue remodeling and has been postulated as a potential biomarker for atherosclerosis, yet no systematic test for FN-EDB in plaques has been reported. We hypothesized that FN-EDB expression would intensify in advanced plaques. Furthermore, engineering of FN-EDB-targeted nanoparticles (NPs) could enable imaging/diagnosis and local delivery of payloads to plaques. Methods: The amount of FN-EDB in human atherosclerotic and normal arteries (ages: 40 to 85 years) was assessed by histological staining and quantification using an FN-EDB-specific aptide (APTFN-EDB). FN-EDB-specific NPs that could serve as MRI beacons were constructed by immobilizing APTFN-EDB on the NP surface containing DTPA[Gd]. MRI visualized APTFN-EDB-[Gd]NPs administered to atherosclerotic apolipoprotein E-deficient mice in the brachiocephalic arteries. Analysis of the ascending-to-descending thoracic aortas and the aortic roots of the mice permitted quantitation of Gd, FN-EDB, and APTFN-EDB-[Gd]NPs. Cyanine, a model small molecule drug, was used to study the biodistribution and pharmacokinetics of APTFN-EDB-NPs to evaluate their utility for drug delivery. Results: Atherosclerotic tissues had significantly greater FN-EDB-positive areas than normal arteries (P < 0.001). This signal pertained particularly to Type III (P < 0.01), IV (P < 0.01), and V lesions (P < 0.001) rather than Type I and II lesions (AHA classification). FN-EDB expression was positively correlated with macrophage accumulation and neoangiogenesis. Quantitative analysis of T1-weighted images of atherosclerotic mice revealed substantial APTFN-EDB-[Gd]NPs accumulation in plaques compared to control NPs, conventional MRI contrast agent (Gd-DTPA) or accumulation in wild-type C57BL/6J mice. Additionally, the APTFN-EDB-NPs significantly prolonged the blood-circulation time (t1/2: ~ 6 h) of a model drug and increased its accumulation in plaques (6.9-fold higher accumulation vs. free drug). Conclusions: Our findings demonstrate augmented FN-EDB expression in Type III, IV, and V atheromata and that APTFN-EDB-NPs could serve as a platform for identifying and/or delivering agents locally to a subset of atherosclerotic plaques.

Morphology, Clinicopathologic Correlations, and Mechanisms in Heart Valve Health and Disease.

The clinical and pathological features of the most frequent intrinsic structural diseases that affect the heart valves are well established, but heart valve disease mechanisms are poorly understood, and effective treatment options are evolving. Major advances in the understanding of the structure, function and biology of native valves and the pathobiology, biomaterials and biomedical engineering, and the clinical management of valvular heart disease have occurred over the past several decades. This communication reviews contemporary considerations relative to the pathology of valvular heart disease, including (1) clinical significance and epidemiology of valvular heart disease; (2) functional and dynamic valvular macro-, micro- and ultrastructure; (3) causes, morphology and mechanisms of human valvular heart disease; and (4) pathologic considerations in valve replacement, repair and, potentially, regeneration of the heart valves.

Reproducible in vitro model for dystrophic calcification of cardiac valvular interstitial cells: insights into the mechanisms of calcific aortic valvular disease.

Calcific aortic valvular disease (CAVD) is the most prevalent valvular pathology in the United States. Development of a pharmacologic agent to slow, halt, or reverse calcification has proven to be unsuccessful as still much remains unknown about the mechanisms of disease initiation. Although in vitro models of some features of CAVD exist, their utility is limited by the inconsistency of the size and time course of the calcified cell aggregates. In this study, we introduce and verify a highly reproducible in vitro method for studying dystrophic calcification of cardiac valvular interstitial cells, considered to be a key mechanism of clinical CAVD. By utilizing micro-contact printing, we were able to consistently reproduce cell aggregation, myofibroblastic markers, programmed cell death, and calcium accumulation within aggregates of 50-400 µm in diameter on substrates with moduli from 9.6 to 76.8 kPa. This method is highly repeatable, with 70% of aggregates staining positive for Alizarin Red S after one week in culture. Dense mineralized calcium-positive nanoparticles were found within the valvular interstitial cell aggregates as shown by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The area of micro-contact printed aggregates staining positive for caspase 3/7 activity increased from 5.9 ± 0.9% to 12.6 ± 4.5% over one week in culture. Z-VAD-FMK reduced aggregates staining positive for Alizarin Red S by 60%. The state of cell stress is hypothesized to play a role in the disease progression; traction force microscopy indicates high substrate stresses along the aggregate periphery which can be modulated by altering the size of the aggregates and the modulus of the substrate. Micro-contact printing is advantageous over the currently used in vitro model as it allows the independent study of how cytokines, substrate modulus, and pharmacologic agents affect calcification. This controlled method for aggregate creation has the potential to be used as an in vitro assay for the screening of promising therapeutics to mitigate CAVD.

Heart Valve Biomechanics and Underlying Mechanobiology.

Heart valves control unidirectional blood flow within the heart during the cardiac cycle. They have a remarkable ability to withstand the demanding mechanical environment of the heart, achieving lifetime durability by processes involving the ongoing remodeling of the extracellular matrix. The focus of this review is on heart valve functional physiology, with insights into the link between disease-induced alterations in valve geometry, tissue stress, and the subsequent cell mechanobiological responses and tissue remodeling. We begin with an overview of the fundamentals of heart valve physiology and the characteristics and functions of valve interstitial cells (VICs). We then provide an overview of current experimental and computational approaches that connect VIC mechanobiological response to organ- and tissue-level deformations and improve our understanding of the underlying functional physiology of heart valves. We conclude with a summary of future trends and offer an outlook for the future of heart valve mechanobiology, specifically, multiscale modeling approaches, and the potential directions and possible challenges of research development. © 2016 American Physiological Society. Compr Physiol 6:1743-1780, 2016.

Heart valve health, disease, replacement, and repair: a 25-year cardiovascular pathology perspective.

The past several decades have witnessed major advances in the understanding of the structure, function, and biology of native valves and the pathobiology and clinical management of valvular heart disease. These improvements have enabled earlier and more precise diagnosis, assessment of the proper timing of surgical and interventional procedures, improved prosthetic and biologic valve replacements and repairs, recognition of postoperative complications and their management, and the introduction of minimally invasive approaches that have enabled definitive and durable treatment for patients who were previously considered inoperable. This review summarizes the current state of our understanding of the mechanisms of heart valve health and disease arrived at through innovative research on the cell and molecular biology of valves, clinical and pathological features of the most frequent intrinsic structural diseases that affect the valves, and the status and pathological considerations in the technological advances in valvular surgery and interventions. The contributions of many cardiovascular pathologists and other scientists, engineers, and clinicians are emphasized, and potentially fruitful areas for research are highlighted.

Local Application of Leptin Antagonist Attenuates Angiotensin II-Induced Ascending Aortic Aneurysm and Cardiac Remodeling.

BACKGROUND: Ascending thoracic aortic aneurysm (ATAA) is driven by angiotensin II (AngII) and contributes to the development of left ventricular (LV) remodeling through aortoventricular coupling. We previously showed that locally available leptin augments AngII-induced abdominal aortic aneurysms in apolipoprotein E-deficient mice. We hypothesized that locally synthesized leptin mediates AngII-induced ATAA. METHODS AND RESULTS: Following demonstration of leptin synthesis in samples of human ATAA associated with different etiologies, we modeled in situ leptin expression in apolipoprotein E-deficient mice by applying exogenous leptin on the surface of the ascending aorta. This treatment resulted in local aortic stiffening and dilation, LV hypertrophy, and thickening of aortic/mitral valve leaflets. Similar results were obtained in an AngII-infusion ATAA mouse model. To test the dependence of AngII-induced aortic and LV remodeling on leptin activity, a leptin antagonist was applied to the ascending aorta in AngII-infused mice. Locally applied single low-dose leptin antagonist moderated AngII-induced ascending aortic dilation and protected mice from ATAA rupture. Furthermore, LV hypertrophy was attenuated and thickening of aortic valve leaflets was moderated. Last, analysis of human aortic valve stenosis leaflets revealed de novo leptin synthesis, whereas exogenous leptin stimulated proliferation and promoted mineralization of human valve interstitial cells in culture. CONCLUSIONS: AngII-induced ATAA is mediated by locally synthesized leptin. Aortoventricular hemodynamic coupling drives LV hypertrophy and promotes early aortic valve lesions, possibly mediated by valvular in situ leptin synthesis. Clinical implementation of local leptin antagonist therapy may attenuate AngII-induced ATAA and moderate related LV hypertrophy and pre-aortic valve stenosis lesions. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/. Unique identifier: NCT00449306.

Simulation of early calcific aortic valve disease in a 3D platform: A role for myofibroblast differentiation.

PURPOSE: Calcific aortic valve disease (CAVD) is the most prevalent valve disease in the Western world. Recent difficulty in translating experimental results on statins to beneficial clinical effects warrants the need for understanding the role of valvular interstitial cells (VICs) in CAVD. In two-dimensional culture conditions, VICs undergo spontaneous activation similar to pathological differentiation, which intrinsically limits the use of in vitro models to study CAVD. Here, we hypothesized that a three-dimensional (3D) culture system based on naturally derived extracellular matrix polymers, mimicking the microenvironment of native valve tissue, could serve as a physiologically relevant platform to study the osteogenic differentiation of VICs. PRINCIPAL RESULTS: Aortic VICs loaded into 3D hydrogel constructs maintained a quiescent phenotype, similar to healthy human valves. In contrast, osteogenic environment induced an initial myofibroblast differentiation (hallmarked by increased alpha smooth muscle actin [α-SMA] expression), followed by an osteoblastic differentiation, characterized by elevated Runx2 expression, and subsequent calcific nodule formation recapitulating CAVD conditions. Silencing of α-SMA under osteogenic conditions diminished VIC osteoblast-like differentiation and calcification, indicating that a VIC myofibroblast-like phenotype may precede osteogenic differentiation in CAVD. MAJOR CONCLUSIONS: Using a 3D hydrogel model, we simulated events that occur during early CAVD in vivo and provided a platform to investigate mechanisms of CAVD. Differentiation of valvular interstitial cells to myofibroblasts was a key mechanistic step in the process of early mineralization. This novel approach can provide important insight into valve pathobiology and serve as a promising tool for drug screening.

Comparative Histopathological Analysis of Mitral Valves in Barlow Disease and Fibroelastic Deficiency.

Whether Barlow disease (BD) and fibroelastic deficiency (FED), the main causes of mitral valve prolapse (MVP), should be considered 2 distinct diseases remains unknown. Mitral valves from patients who required surgery for severe mitral regurgitation due to degenerative nonsyndromic MVP were analyzed. Intraoperative diagnosis of BD or FED was based on leaflet redundancy and thickness, number of segments involved, and annular dimension. The removed medial scallop of the posterior leaflet and attached chordae were used for histopathological and immunohistological assessment. Histologically, compared to normal controls (n = 3), BD (n = 14), and FED (n = 9) leaflets demonstrated an altered architecture and increased thickness. Leaflet thickness was greater and chordae thickness lower in BD than FED (P < 0.0001). In BD, increased thickness was owing to spongiosa expansion (proteoglycan accumulation) and intimal thickening on fibrosa and atrialis; in FED, local thickening was predominant on the fibrosa side, with accumulation of proteoglycan-like material around the chordae. Collagen accumulation was observed in FED leaflets and chords and decreased in BD. Fragmented elastin fibers were present in BD and FED; elastin decreased in BD but increased in FED leaflets and around chordae. Activated myofibroblasts accumulate in both diseased leaflets and chords, but more abundantly in FED chordae (P < 0.0001), independently of age, suggesting a role of these cells in chordal rupture. There were more CD34-positive cells in BD leaflets and in FED chordae (P < 0.01). In BD leaflets (but not chordae) proliferative Ki67-positive cells were more abundant (P < 0.01) and matrix metalloproteinase 2 levels were increased (P < 0.01) indicating tissue remodeling. Upregulation of transforming growth factor beta and pERK signaling pathways was evident in both diseases but more prominent in FED leaflets (continued on next page)(P < 0.001), with pERK upregulation in FED chordae (P < 0.0001). Most cellular and signaling markers were negligible in control valves. Quantitative immunohistopathological analyses demonstrated distinct changes between BD and FED valves: predominant matrix degradation in BD and increased profibrotic signaling pathways in FED, indicating that BD and FED are 2 different entities. These results may pave the way for genetic studies of MVP and development of preventive drug therapies.