Engineering the aortic valve extracellular matrix through stages of development, aging, and disease.

For such a thin tissue, the aortic valve possesses an exquisitely complex, multi-layered extracellular matrix (ECM), and disruptions to this structure constitute one of the earliest hallmarks of fibrocalcific aortic valve disease (CAVD). The native valve structure provides a challenging target for engineers to mimic, but the development of advanced, ECM-based scaffolds may enable mechanistic and therapeutic discoveries that are not feasible in other culture or in vivo platforms. This review first discusses the ECM changes that occur during heart valve development, normal aging, onset of early-stage disease, and progression to late-stage disease. We then provide an overview of the bottom-up tissue engineering strategies that have been used to mimic the valvular ECM, and opportunities for advancement in these areas.

Optimization of polycaprolactone fibrous scaffold for heart valve tissue engineering.

Pore size is generally small in nanofibrous scaffolds prepared by electrospinning polymeric solutions. Increase of scaffold thickness leads to decrease in pore size, causing impediment to cell infiltration into the scaffolds during tissue engineering. In contrast, comparatively larger pore size can be realized in microfibrous scaffolds prepared from polymeric solutions at higher concentrations. Further, microfibrous scaffolds are conducive to infiltration of reparative M2 phenotype macrophages during in vivo/in situ tissue engineering. However, rise of mechanical properties of a fibrous scaffold with the increase of polymer concentration may limit the functionality of a scaffold-based, tissue-engineered heart valve. In this study, we developed microfibrous scaffolds from 14%, 16% and 18% (wt/v) polycaprolactone (PCL) polymer solutions prepared with chloroform solvent. Porcine valvular interstitial cells were cultured in the scaffolds for 14 d to investigate the effect of microfibers prepared with different PCL concentrations on the seeded cells. Further, fresh microfibrous scaffolds were implanted subcutaneously in a rat model for two months to investigate the effect of microfibers on infiltrated cells. Cell proliferation, and its morphologies, gene expression and deposition of different extracellular matrix proteins in the in vitro study were characterized. During the in vivo study, we characterized cell infiltration, and myofibroblast and M1/M2 phenotypes expression of the infiltrated cells. Among different PCL concentrations, microfibrous scaffolds from 14% solution were suitable for heart valve tissue engineering for their sufficient pore size and low but adequate tensile properties, which promoted cell adhesion to and proliferation in the scaffolds, and effective gene expression and extracellular matrix deposition by the cells in vitro. They also encouraged the cells in vivo for their infiltration and effective gene expression, including M2 phenotype expression.

The Transcriptional Signature of Growth in Human Fetal Aortic Valve Development.

BACKGROUND: In the second trimester of human fetal development, a tenfold increase in fetal size occurs while cardiac valves grow and retain their function. Patterns of transcription in normally growing human aortic valves are unknown. METHODS: Discarded human aortic valve samples were collected from the second trimester, 6 from early (14, 15, 17 weeks) and 6 from late (20, 21, 22 weeks) gestation. Network analysis of RNA sequencing data identified subnetworks of significantly increasing and decreasing transcripts. Subsequent cluster analysis identified patterns of transcription through the time course. Pathway enrichment analysis determined the predominant biological processes at each interval. RESULTS: We observed phasic transcription over the time course, including an early decrease in cell proliferation and developmental genes (14 to 15 weeks). Pattern specification, shear stress, and adaptive immune genes were induced early. Cell adhesion genes were increased from 14 to 20 weeks. A phase involving cell differentiation and apoptosis (17 to 20 weeks) was followed by downregulation of endothelial-to-mesenchymal transformation genes and then by increased extracellular matrix organization and stabilization (20 to 22 weeks). CONCLUSIONS: We present a unique data set, comprehensively characterizing human valve development after valve primordia are formed, focusing on key processes displayed by normal aortic valves undergoing significant growth. We build a time course of genes and processes in second trimester fetal valve growth and observe the sequential regulation of gene clusters over time. Critical valve growth genes are potential targets for therapeutic intervention in congenital heart disease and have implications for regenerative medicine and tissue engineering.

Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T type 2 (Tnnt2) Cre lineage.

BACKGROUND: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood. RESULTS: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0. CONCLUSIONS: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc.

A role for primary cilia in aortic valve development and disease.

BACKGROUND: Bicuspid aortic valve (BAV) disease is the most common congenital heart defect, affecting 0.5-1.2% of the population and causing significant morbidity and mortality. Only a few genes have been identified in pedigrees, and no single gene model explains BAV inheritance, thus supporting a complex genetic network of interacting genes. However, patients with rare syndromic diseases that stem from alterations in the structure and function of primary cilia ("ciliopathies") exhibit BAV as a frequent cardiovascular finding, suggesting primary cilia may factor broadly in disease etiology. RESULTS: Our data are the first to demonstrate that primary cilia are expressed on aortic valve mesenchymal cells during embryonic development and are lost as these cells differentiate into collagen-secreting fibroblastic-like cells. The function of primary cilia was tested by genetically ablating the critical ciliogenic gene Ift88. Loss of Ift88 resulted in abrogation of primary cilia and increased fibrogenic extracellular matrix (ECM) production. Consequentially, stratification of ECM boundaries normally present in the aortic valve were lost and a highly penetrant BAV phenotype was evident at birth. CONCLUSIONS: Our data support cilia as a novel cellular mechanism for restraining ECM production during aortic valve development and broadly implicate these structures in the etiology of BAV disease in humans. Developmental Dynamics 246:625-634, 2017. © 2017 Wiley Periodicals, Inc.

Growing potential of small aortic valve with aortic coarctation or interrupted aortic arch after bilateral pulmonary artery banding.

OBJECTIVES: Bilateral pulmonary artery banding (bPAB) is utilized for some patients with a ventricular septal defect (VSD) and aortic coarctation (CoA) or interrupted aortic arch (IAA). We evaluated aortic valve (AoV) diameter and patient outcomes following bPAB. METHODS: Between August 2010 and September 2015, 10 consecutive patients with VSD and patent ductus arteriosus-dependent CoA or IAA underwent bPAB because of an AoV diameter of approximately <50% of the normal value (n = 6), severe subaortic stenosis and poor patient condition (n = 1, respectively), or low birthweight (n = 2). RESULTS: Second-stage operations were conventional total repair in five and Damus-Kaye-Stansel anastomosis, aortic arch reconstruction and right ventricle-pulmonary artery shunt (modified Norwood) type repair in five. After modified Norwood-type repair, four patients were Yasui-type repair candidates and one was a Fontan candidate. For all patients, the mean AoV diameter increased from 3.7 ± 0.7 mm before bPAB to 4.6 ± 0.8 mm before the second-stage operation. In five patients with CoA or IAA type A, the AoV diameter significantly increased from 3.5 ± 0.3 mm to 4.5 ± 0.5 mm during the term between bPAB and the second-stage operation, with an AoV Z-score increase from -5.82 ± 0.92 to -4.28 ± 0.86. IAA type B showed a slight increase in the AoV diameter. CONCLUSIONS: Initial palliation with bPAB enables AoV diameter growth in some patients, improving the likelihood of conventional total repair adaptation rate, particularly for CoA or IAA type A.

The fate of the neoaortic valve and root after the modified Ross-Konno procedure.

OBJECTIVES: In children with aortic valve disease associated with annular hypoplasia or complex multilevel left ventricular outflow tract obstruction, the Ross procedure, combined with a modified Konno-type aortoventriculoplasty, is advocated. We aim to examine the fate of the neoaortic apparatus and assess neoaortic valve function after the modified Ross-Konno procedure. METHODS: Forty-three patients, with a median age of 6 years, underwent the modified Ross-Konno procedure with a myectomy but without the use of a ventricular septal patch. Serial postoperative echocardiograms (n = 187) were analyzed, and regression models adjusted for repeated measures were used to model the longitudinal growth of the neoaortic annulus and root. RESULTS: There were 2 operative deaths (5%) and 1 late mortality. At 8 years, survival was 93% and freedom from autograft, homograft, and all-cause reoperation was 100%, 81%, and 72%, respectively. The median postprocedure diameter and z score were 14 mm (7-21 mm) and +1.3 (-3.0 to +6.1) for the neoaortic annulus and 21 mm (9-30 mm) and +1.6 (-1.3 to +4.1) for the neoaortic root, respectively. Serial echocardiograms showed a progressive increase in annular (+0.56 mm/year, P < .001) and root (+0.89 mm/year, P < .001) diameters but little change in annular (-0.07/year, P = .08) and root (-0.002/year, P = .96) z scores. Autograft regurgitation developed in 9 patients; however, the degree and progression of regurgitation over time were not significant (P = .22). CONCLUSIONS: After the modified Ross-Konno procedure, the neoaortic annulus and root increased in size proportionately to somatic growth. Autograft regurgitation, usually mild and stable, developed in few patients, and none required autograft reoperation. Our findings support the use of the modified Ross-Konno as the procedure of choice in children with aortic valve disease and complex left ventricular outflow tract obstruction.

Extracellular matrix remodeling and cell phenotypic changes in dysplastic and hemodynamically altered semilunar human cardiac valves.

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.

Environmental regulation of valvulogenesis: implications for tissue engineering.

Ongoing research efforts aim at improving the creation of tissue-engineered heart valves for in vivo systemic application. Hence, in vitro studies concentrate on optimising culture protocols incorporating biological as well as biophysical stimuli for tissue development. Important lessons can be drawn from valvulogenesis to mimic natural valve development in vitro. Here, we review the up-to-date status of valvulogenesis, focussing on the biomolecular and biophysical regulation of semilunar valve development. In addition, we discuss potential benefits of incorporating concepts derived from valvulogenesis, as well as alternative approaches, in tissue-engineering protocols, to improve in vitro valve development. The combined efforts from clinicians, cell biologists and engineers are required to implement and evaluate these approaches to achieve optimised protocols for heart-valve tissue engineering.

Aortic valve surgery in congenital heart disease: a single-center experience.

The optimal treatment of congenital aortic valve lesions is a controversial issue. This study was performed to evaluate the outcome after surgical treatment of aortic valve lesions in congenital aortic valve disease. Between the years of 2000 and 2008, 61 patients (mean age: 12.6 +/- 9.6 years, range: 1 day to 40 years) underwent aortic valve surgery for congenital aortic valve disease. Twenty-four patients had undergone previous cardiovascular operations. Indications for surgery were aortic regurgitation in 14.7% (n = 9), aortic stenoses in 26.2% (n = 16), and mixed disease in 59.1% (n = 36). The Ross procedure was performed in 37.7% (n = 23), aortic valve replacement with biological or mechanical prostheses in 29.5% (n = 18). Concomitant procedures were performed in 91.8% (n = 56) due to associated congenital cardiac defects. The overall mortality rate was 5%. Six patients needed reoperation. Implantation of permanent pacemakers occurred in six patients for permanent atrioventricular block. At the latest clinical evaluation, all survivors are in New York Heart Association class I-II and are living normal lives. Aortic valve surgeries in patients with congenital heart disease have had low mortality and morbidity rates in our series. Surgical technique as well as timing should be tailored for each patient. Aortic valve replacement should be delayed until the implantation of an adult-sized prosthesis is possible.

Wnt signaling in heart valve development and osteogenic gene induction.

Wnt signaling mediated by beta-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of beta-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.

Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells.

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-beta (TGF-beta). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-beta-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-beta. A positive role for Notch1 was revealed in three experiments: (1) TGF-beta induced Notch1 mRNA in valve ECs, (2) a gamma-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.

Left heart growth, function, and reintervention after balloon aortic valvuloplasty for neonatal aortic stenosis.

BACKGROUND: Transcatheter balloon aortic valvuloplasty (BAVP) has become the first-line treatment for critical aortic stenosis (AS) in neonates. However, little is known about the growth and function of left heart structures or about patterns of reintervention on the left heart after neonatal BAVP. METHODS AND RESULTS: Between 1985 and 2002, 113 patients underwent neonatal BAVP at < or =60 days of age. There were 16 early deaths (14%), with a significant decrease from 1985 to 1993 (22%) to 1994 to 2002 (4%), and 6 patients had successful early conversion to a univentricular circulation. In the short term, the mean relative gradient reduction was 54+/-26%, and significant aortic regurgitation (AR) developed in 15% of patients. The 91 early survivors with a biventricular circulation were followed up for 6.3+/-5.3 years, during which time there was a steady increase in the frequency of significant AR. Freedom from moderate or severe AR was 65% at 5 years. In almost all patients with a baseline aortic annulus z score less than -1, the annulus diameter increased to within the normal range within 1 to 2 years. Similarly, left ventricular (LV) end-diastolic dimension z scores, which ranged from -5 to 7.5 before BAVP, normalized within 1 to 2 years in nearly all patients with a predilation z score less than -1. Among early survivors with a biventricular circulation, reintervention-free survival on the LV outflow tract was 65% at 1 year and 48% at 5 years, with younger age, higher pre- and post-BAVP gradients, and a larger balloon-annulus diameter ratio associated with decreased reintervention-free survival (P<0.01). Seventeen surgical interventions were performed on the aortic valve in 15 patients, including replacement in 7. Survival free from aortic valve replacement was 84% at 5 years. CONCLUSIONS: BAVP for AS during the first 60 days of life results in short-term relief of AS in the majority of patients. Among early survivors, initially small left heart structures may be associated with worse subacute outcomes but typically normalize within 1 year. Reintervention for residual/recurrent AS or iatrogenic AR is relatively common, particularly during the first year after BAVP, but aortic valve replacement during early childhood is seldom necessary.

Angiogenic activation of valvular endothelial cells in aortic valve stenosis.

Here, we demonstrate the angiogenic response of valvular endothelial cells to aortic valve (AV) stenosis using a new ex vivo model of aortic leaflets. Histological analysis revealed neovascularization within the cusps of stenotic but not of non-stenotic aortic valves. Correspondingly, the number of capillary-like outgrowth in 3D collagen gel was significantly higher in stenotic than in non-stenotic valves. Capillary-like sprouting was developed significantly faster in stenotic than in non-stenotic valves. New capillary sprouts from stenotic aortic valves exhibited the endothelial cell markers CD31, CD34 and von-Willebrand factor (vWF) as well as carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), Tie-2 and angiogenesis inhibitor endostatin. Western blot analyses revealed a significant increase of CEACAM1 and endostatin in stenotic aortic valve tissue. Electron microscopic examinations demonstrate that these capillary-like tubes are formed by endothelial cells containing Weibel-Palade bodies. Remarkably, inter-endothelial junctions are established and basement membrane material is partially deposited on the basal side of the endothelial tubes. Our data demonstrate the capillary-like sprout formation from aortic valves and suggest a role of angiogenesis in the pathogenesis of aortic valve stenosis. These data provide new insights into the mechanisms of valvular disorders and open new perspectives for prevention and early treatment of calcified aortic stenosis.

Fate of the aortic root after arterial switch operation.

OBJECTIVE: Concerns have been voiced about possible dilation and insufficiency of the neo-aortic valve after the arterial switch operation (ASO). AIMS: To determine growth of the neo-aortic valve and the aortic anastomosis after ASO and the prevalence of insufficiency or stenosis. PATIENTS AND METHODS: Since 1977, 144 consecutive patients (pts) underwent ASO for transposition of the great arteries (TGA). Median follow-up was 8.65 years (0.1--22.5 years). Simple TGA was present in 97 pts and 47 had TGA with ventricular septal defect (VSD). Detailed echocardiography included measurements of aortic diameter at four levels. The 608 measurements were compared with published normal values. RESULTS: The mean aortic valve z-score was 1.5, without significant change with age (P=0.75). Under 4 months, mean valve z-score was 0.63+/-2.20, between 5 and 12 months 2.56+/-2.30 (P<0.0001). Gradual growth occurs thereafter. The aortic sinus follows an identical growth pattern. The aorta at the anastomosis, is initially smaller than normal (z-score -0.64). After 4 months the z-score is 0.83, followed by continued growth of 0.1 z-score per year. At the last visit, the aortic valve z-score was above 2 in 51 patients, between -2 and 2 in 72 and less than -2 in six patients, none of whom had a flow velocity above 2 m/s. z-score of patients with VSD remained above those without VSD (P<0.0001). Aortic insufficiency was grade 2/4 in three patients, grade 3/4 in one and grade 4/4 in one. No patient developed aortic stenosis. CONCLUSION: After ASO the neo-aortic valve and sinus are larger than normal, representing the natural size difference in the prenatal situation and influence of associated cardiac malformations. In the first year of life, rapid dilatation of the new aorta is observed, followed by growth towards normalization of the valve and sinus size. Stenosis at the anastomosis was not observed. Aortic dilatation by itself is rarely associated with significant insufficiency.

Pulmonary autograft replacement in children. The ideal solution?

Fifty-one children, aged 1.8 to 21 years (mean, 11.4) with aortic valve replacement using a pulmonary autograft are reviewed. Twenty-nine were intra-aortic implants and 22 were root replacements. There was one operative death, no late deaths, and two have required reoperation. Actuarial freedom from reoperation was 93% +/- 5.5 at 5.6 years. Freedom from progression of aortic insufficiency (AI) was 81% +/- 9 at 5.6 years in the intra-aortic implants and 86% +/- 10 in the root replacement. Enlargement of the pulmonary autograft was seen echocardiographically in both groups. This enlargement was consistent with somatic growth and not associated with progression of AI. Ten of 19 patients with aortic stenosis had an LV mass index suggestive of LV hypertrophy before operation. At 1 year, 18 of 25 had a normal LV mass index. Thirteen of 16 patients with AI had preoperative abnormal LV mass index. All but four returned to normal by 1 year. Low operative risk, excellent function, resolution of abnormal LV hemodynamics, and enlargement consistent with somatic growth suggest that the pulmonary autograft is the ideal replacement for the malfunctioning aortic valve.

Growth and cell viability of aortic versus pulmonic homografts in the systemic circulation.

Growth characteristics and cell viability of aortic and pulmonic valve homografts in the systemic circulation were compared in a growing sheep model. Seven aortic and seven pulmonic cryopreserved homografts were implanted in the descending aortae of recipient female lambs. Of seven sheep per group, three were killed at 8 months, three at 12 months, and one at 15 months. Tissue cultures were obtained on homograft valve and root wall specimens. Male donor cells were identified by chromosome analysis. Preimplant to explant changes in pulmonic homograft external diameters at the valve annulus and sinotubular junction increased significantly more than in aortic roots. The postoperative dimensions of the distal anastomotic diameter and length of the graft increased significantly more in pulmonic than in aortic roots by time of explant. Leaflet calcification and valvular stenosis did not develop in either pulmonic or aortic homografts over the period of observation. Calcification in the root wall was significantly less in pulmonic than aortic homografts. Growth on tissue culture was obtained from over 70% of homograft specimens. Viable donor cells were demonstrated in none and 43% of aortic and pulmonic leaflets, respectively, and 71% and 57% of aortic and pulmonary arterial homograft walls, respectively. In conclusion, both aortic and pulmonic homograft valves provide freedom from calcification and stenosis in this model. Continuing expansion of pulmonic root diameters under systemic pressure might lead to late aneurysm formation. Although host cell repopulation of grafts may be advantageous, the presence of viable donor cells in leaflet tissue does not seem necessary to prevent calcific degeneration.

Calcification in aging canine aortic valve.

Aging changes of aortic valves are thought to underlie the mechanism of calcification, which leads to calcific aortic stenosis in humans. The study of calcification in the aging valvular connective tissue has been hindered by the lack of a suitable animal model. In search of the model, canine aortic valves demonstrated age changes including calcification remarkably similar to those in humans. The mechanism of calcification was studied in the aortic valves of aged Beagles by electron microscopy. Fibroblasts in the canine aortic valves showed the most prominent age changes. The cells accumulated numerous residual bodies and appeared to disintegrate. The resultant membranous cellular degradation products which sequestered in the extracellular space were the nidi of calcification. It appeared that the membrane of cell debris played an important role in calcification. Canine aortic valve is an ideal model for the study of calcification in relation to aging of the valvular connective tissue.