Noncanonical atherosclerosis as the driving force in tricuspid aortic valve associated aneurysms - A trace collection.

Pathogenic mechanisms in degenerative thoracic aortic aneurysms (TAA) are still unclear. There is an ongoing debate about whether TAAs are caused by uniform or distinct processes, which would obviously have a major impact on future treatment strategies. Clearly, the ultimate outcome of TAA subgroups associated with a tricuspid aortic valve (TAV) or a bicuspid aortic valve (BAV) is the same, namely a TAA. Based on results from our own and others' studies, we decided to compare the different TAAs (TAV and BAV) and controls using a broad array of analyses, i.e., metabolomic analyses, gene expression profiling, protein expression analyses, histological characterization, and matrix-assisted laser desorption ionization imaging. Central findings of the present study are the presence of noncanonical atherosclerosis, pathological accumulation of macrophages, and disturbances of lipid metabolism in the aortic media. Moreover, we have also found that lipid metabolism is impaired systemically. Importantly, all of the above-described phenotypes are characteristic for TAV-TAA only, and not for BAV-TAA. In summary, our results suggest different modes of pathogenesis in TAV- and BAV-associated aneurysms. Intimal atherosclerotic changes play a more central role in TAV-TAA formation than previously thought, particularly as the observed alterations do not follow classical patterns. Atherosclerotic alterations are not limited to the intima but also affect and alter the TAV-TAA media. Further studies are needed to i) clarify patho-relevant intima-media interconnections, ii) define the origin of the systemic alteration of lipid metabolism, and iii) to define valid biomarkers for early diagnosis, disease progression, and successful treatments in TAV-TAAs.

Transcription factor Sp2 promotes TGFB-mediated interstitial cell osteogenic differentiation in bicuspid aortic valves through a SMAD-dependent pathway.

Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195-5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-ß pathway. However, the transcriptional regulation of miR-195-5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195-5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195-5p. Cotransfection of SP2 shRNA and a miR-195-5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195-5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195-5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.

Advanced Methodology and Preliminary Measurements of Molecular and Mechanical Properties of Heart Valves under Dynamic Strain.

Mammalian heart valves are soft tissue assemblies with multi-scale material properties. This is because they are constructs comprising both muscle and non-contractile extracellular matrix proteins (such as collagens and proteoglycans) and transition regions where one form of tissue structure becomes another, significantly different form. The leaflets of the mitral and tricuspid valves are connected to chordae tendinae which, in turn, bind through papillary muscles to the cardiac wall of the ventricle. The transition regions between these tissue subsets are complex and diffuse. Their material composition and mechanical properties have not been previously described with both micro and nanoscopic data recorded simultaneously, as reported here. Annotating the mechanical characteristics of these tissue transitions will be of great value in developing novel implants, improving the state of the surgical simulators and advancing robot-assisted surgery. We present here developments in multi-scale methodology that produce data that can relate mechanical properties to molecular structure using scanning X-ray diffraction. We correlate these data to corresponding tissue level (macro and microscopic) stress and strain, with particular emphasis on the transition regions and present analyses to indicate points of possible failure in these tissues.

Dexfenfluramine and Pergolide Cause Heart Valve Disease via Valve Metabolic Reprogramming and Ongoing Matrix Remodeling.

Several clinical reports indicate that the use of amphetaminic anorectic drugs or ergot derivatives could cause valvular heart disease (VHD). We sought to investigate whether valvular lesions develop in response to long-term oral administration of these drugs and to identify drug-targeted biological processes that may lead to VHD. Treatment of New Zealand White rabbits with pergolide, dexfenfluramine, or high-dose serotonin for 16 weeks induced valvular alterations characterized by extracellular matrix remodeling. Transcriptome profiling of tricuspid valves using RNA sequencing revealed distinct patterns of differentially expressed genes (DEGs) that clustered according to the different treatments. Genes that were affected by the three treatments were functionally enriched for reduced cell metabolism processes. The two drugs yielded more changes in gene expression than serotonin and shared most of the DEGs. These DEGs were mostly enriched for decreased biosynthetic processes, increased cell-matrix interaction, and cell response to growth factors, including TGF-ß, which was associated with p38 MAPK activation. Treatment with pergolide specifically affected genes involved in homeostasis, which was corroborated by the activation of the master regulator of cell energy homeostasis, AMPK-α, as well as decreased levels of metabolism-related miR-107. Thus, both pergolide and dexfenfluramine may cause VHD through valve metabolic reprogramming and matrix remodeling.

Activation of the calcium-sensing receptor in human valvular interstitial cells promotes calcification.

INTRODUCTION AND AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in western countries. It has been reported that activation of the calcium-sensing receptor(CaSR) expressed by vascular smooth muscle cells prevents vascular calcification. However, to date, the CaSR's expression and function in cardiac valves have not been studied. The present study sought to evaluate the presence of the CaSR within human valvular interstitial cells (hVICs), assess the CaSR's functionality, and ascertain its involvement in hVIC calcification. METHODS AND RESULTS: Data from Western blot, flow cytometry and immunocytochemistry experiments demonstrated that primary hVICs express the CaSR. The receptor was functional, since the incubation of hVICs with the calcimimetic R-568 significantly increased Ca2+-induced ERK1/2 phosphorylation, and exposure to the calcilytic NPS2143 reduced ERK1/2 activation. A reduction in endogenous CaSR expression by hVICs (using siRNA) was associated with significantly lower levels of Ca2+-induced mineralization (quantified using Alizarin Red staining). Similar data were obtained after the pharmacological inhibition of CaSR activity by the calcilytic NPS2143. In contrast, overexpression of a functional CaSR amplified Ca2+-induced calcification. Pharmacological activation of the CaSR with the calcimimetic R-568 showed similar effects. CaSR's procalcific properties are associated with increased osteogenic transition (as characterized by elevated mRNA expression of bone morphogenetic protein 2 and osterix), and reduced the expression of the calcification inhibitor osteopontin. Histological analysis of 12 human aortic tricuspid valves showed that CaSR expression was greater in calcified areas than in non-calcified areas. These data were confirmed by Western blots. CONCLUSIONS: To the best of our knowledge, this study is the first to have demonstrated that hVICs express a functional CaSR. Taken as a whole, our data suggest that activation of the CaSR expressed by hVICs might be a key promoter of CAVD progression.

Polyamines and microbiota in bicuspid and tricuspid aortic valve aortopathy.

Polyamines are small aliphatic cationic molecules synthesized via a highly regulated pathway and involved in general molecular and cellular phenomena. Both mammalian cells and microorganisms synthesize polyamines, and both sources may contribute to the presence of polyamines in the circulation. The dominant location for microorganisms within the body is the gut. Accordingly, the gut microbiota probably synthesizes most of the polyamines in the circulation in addition to those produced by the mammalian host cells. Polyamines are mandatory for cellular growth and proliferation. Established evidence suggests that the polyamine spermidine prolongs lifespan and improves cardiovascular health in animal models and humans through both local mechanisms, involving improved cardiomyocyte function, and systemic mechanisms, including increased NO bioavailability and reduced systemic inflammation. Higher levels of polyamines have been detected in non-dilated aorta of patients affected by bicuspid aortic valve congenital malformation, an aortopathy associated with an increased risk for thoracic ascending aorta aneurysm. In this review, we discuss metabolism of polyamines and their potential effects on vascular smooth muscle and endothelial cell function in vascular pathology of the thoracic ascending aorta associated with bicuspid or tricuspid aortic valve.

Strong Signs for a Weak Wall in Tricuspid Aortic Valve Associated Aneurysms and a Role for Osteopontin in Bicuspid Aortic Valve Associated Aneurysms.

Central processes in the pathogenesis of TAV- (tricuspid aortic valve) and BAV- (bicuspid aortic valve) associated ascending thoracic aortic aneurysm (ATAA) development are still unknown. To gain new insights, we have collected aortic tissue and isolated smooth muscle cells of aneurysmal tissue and subjected them to in situ and in vitro analyses. We analyzed aortic tissue from 78 patients (31 controls, 28 TAV-ATAAs, and 19 BAV-ATAAs) and established 30 primary smooth muscle cell cultures. Analyses included histochemistry, immuno-, auto-fluorescence-based image analyses, and cellular analyses including smooth muscle cell contraction studies. With regard to TAV associated aneurysms, we observed a strong impairment of the vascular wall, which appears on different levels-structure and dimension of the layers (reduced media thickness, increased intima thickness, atherosclerotic changes, degeneration of aortic media, decrease of collagen, and increase of elastic fiber free area) as well as on the cellular level (accumulation of fibroblasts/myofibroblasts, and increase in the number of smooth muscle cells with a reduced alpha smooth muscle actin (α-SM actin) content per cell). The pathological changes in the aortic wall of BAV patients were much less pronounced-apart from an increased expression of osteopontin (OPN) in the vascular wall which stem from smooth muscle cells, we observed a trend towards increased calcification of the aortic wall (increase significantly associated with age). These observations provide strong evidence for different pathological processes and different disease mechanisms to occur in BAV- and TAV-associated aneurysms.

Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves.

AIMS: Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. METHODS AND RESULTS: We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. CONCLUSION: Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

Cellular Mechanisms of Aortic Valve Calcification.

Comparative in vitro study examined the osteogenic potential of interstitial cells of aortic valve obtained from the patients with aortic stenosis and from control recipients of orthotopic heart transplantation with intact aortic valve. The osteogenic inductors augmented mineralization of aortic valve interstitial cells (AVIC) in patients with aortic stenosis in comparison with the control level. Native AVIC culture of aortic stenosis patients demonstrated overexpression of osteopontin gene (OPN) and underexpression of osteoprotegerin gene (OPG) in comparison with control levels. In both groups, AVIC differentiation was associated with overexpression of RUNX2 and SPRY1 genes. In AVIC of aortic stenosis patients, expression of BMP2 gene was significantly greater than the control level. The study revealed an enhanced sensitivity of AVIC to osteogenic inductors in aortic stenosis patients, which indicates probable implication of OPN, OPG, and BMP2 genes in pathogenesis of aortic valve calcification.

Downregulated MicroRNA-195 in the Bicuspid Aortic Valve Promotes Calcification of Valve Interstitial Cells via Targeting SMAD7.

BACKGROUND/AIMS: Aortic stenosis caused by leaflet calcification in the bicuspid aortic valve (BAV) is more accelerative than that in the tricuspid aortic valve (TAV). MicroRNA-195 (miR-195) is downregulated more in stenotic than in insufficient BAVs, but its expression in BAVs compared with TAVs is unclear. We aimed to investigate the roles of miR-195 and its calcification-related target SMAD7 in stenotic BAVs compared with those in TAVs. METHODS: Twenty-one stenotic BAV and 29 TAV samples were collected from surgical patients and examined for the expression of miR-195 and SMAD7 by RT-PCR. The samples were also assessed by western blotting and immunohistochemistry for the functional protein alteration associated with calcification. Dual-luciferase assay was performed to determine the putative target of miR-195 before the effects of miR-195 expression on osteogenic progression was demonstrated in cultured porcine valve interstitial cells (VICs). RESULTS: Compared with TAV, the expression of miR-195 was remarkably lower in the BAV leaflet with higher expression of SMAD7, which was then validated as a direct target of miR-195. Their negative correlation was then confirmed in cultured VICs. Under an osteogenic environment, the cellular calcification was promoted in miR-195-repressed VICs expressing higher BMP-2 and Runx2 and higher activity of MMP-2 compared with the controls. Finally, higher MMP-2 and MMP-9 expression and more collagen distribution were observed in BAV than TAV samples. CONCLUSIONS: miR-195 is downregulated more in stenotic BAV than TAV in this study. The downregulation of miR-195 is associated with valvular calcification via targeting SMAD7, which promotes the remodeling of the extracellular matrix.

Stenotic Bicuspid and Tricuspid Aortic Valves　- Micro-Computed Tomography and Biological Indices of Calcification.

BACKGROUND: Valve calcification is well estimated by ex-vivo micro-computed tomography (micro-CT). The objective of this study was to investigate the associations between micro-CT findings and biological indices of calcification in aortic stenosis (AS), as well as differences between bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV).Methods and Results:Aortic valves and plasma were obtained from patients undergoing valve surgery. Valves were dissected and underwent micro-CT, genetic analyses, and calcium content assessment. Plasma levels of calcification markers were measured. Forty-two patients with isolated severe AS, including 22 with BAV, were studied. BAV patients had a lower median CT value (140.0 [130.0-152.0] vs. 157.0 [147.0-176.0], P=0.002) and high-density calcification (HDC) fraction (9.3 [5.7-23.3] % vs. 21.3 [14.3-31.2] %, P=0.01), as compared with TAV. Calcification fraction (CF) correlated with AS severity (measured as maximal transvalvular pressure gradient [r=0.34, P=0.03], maximal flow velocity [r=0.38, P=0.02], and indexed aortic valve area [r=-0.37, P=0.02]). For TAV patients only, mRNA expression of integrin-binding sialoprotein correlated with CF (r=0.45, P=0.048), and the receptor activator of the nuclear factor κ-B ligand transcript correlated with HDC corrugation (r=0.54, P=0.01). CONCLUSIONS: TAV patients with AS present more mineralized calcifications in micro-CT than BAV subjects. The relative volume of calcifications increases with the AS severity. In TAV patients, upregulated expression of genes involved in osteoblastogenesis in AS correlates with leaflet mineralization in micro-CT.

RNA expression profile of calcified bicuspid, tricuspid, and normal human aortic valves by RNA sequencing.

The molecular mechanisms leading to premature development of aortic valve stenosis (AS) in individuals with a bicuspid aortic valve are unknown. The objective of this study was to identify genes differentially expressed between calcified bicuspid aortic valves (BAVc) and tricuspid valves with (TAVc) and without (TAVn) AS using RNA sequencing (RNA-Seq). We collected 10 human BAVc and nine TAVc from men who underwent primary aortic valve replacement. Eight TAVn were obtained from men who underwent heart transplantation. mRNA levels were measured by RNA-Seq and compared between valve groups. Two genes were upregulated, and none were downregulated in BAVc compared with TAVc, suggesting a similar gene expression response to AS in individuals with bicuspid and tricuspid valves. There were 462 genes upregulated and 282 downregulated in BAVc compared with TAVn. In TAVc compared with TAVn, 329 genes were up- and 170 were downregulated. A total of 273 upregulated and 147 downregulated genes were concordantly altered between BAVc vs. TAVn and TAVc vs. TAVn, which represent 56 and 84% of significant genes in the first and second comparisons, respectively. This indicates that extra genes and pathways were altered in BAVc. Shared pathways between calcified (BAVc and TAVc) and normal (TAVn) aortic valves were also more extensively altered in BAVc. The top pathway enriched for genes differentially expressed in calcified compared with normal valves was fibrosis, which support the remodeling process as a therapeutic target. These findings are relevant to understand the molecular basis of AS in patients with bicuspid and tricuspid valves.

Load-dependent extracellular matrix organization in atrioventricular heart valves: differences and similarities.

The extracellular matrix of the atrioventricular (AV) valves' leaflets has a key role in the ability of these valves to properly remodel in response to constantly varying physiological loads. While the loading on mitral and tricuspid valves is significantly different, no information is available on how collagen fibers change their orientation in response to these loads. This study delineates the effect of physiological loading on AV valves' leaflets microstructures using Second Harmonic Generation (SHG) microscopy. Fresh natural porcine tricuspid and mitral valves' leaflets (n = 12/valve type) were cut and prepared for the experiments. Histology and immunohistochemistry were performed to compare the microstructural differences between the valves. The specimens were imaged live during the relaxed, loading, and unloading phases using SHG microscopy. The images were analyzed with Fourier decomposition to mathematically seek changes in collagen fiber orientation. Despite the similarities in both AV valves as seen in the histology and immunohistochemistry data, the microstructural arrangement, especially the collagen fiber distribution and orientation in the stress-free condition, were found to be different. Uniaxial loading was dependent on the arrangement of the fibers in their relaxed mode, which led the fibers to reorient in-line with the load throughout the depth of the mitral leaflet but only to reorient in-line with the load in deeper layers of the tricuspid leaflet. Biaxial loading arranged the fibers in between the two principal axes of the stresses independently from their relaxed states. Unlike previous findings, this study concludes that the AV valves' three-dimensional extracellular fiber arrangement is significantly different in their stress-free and uniaxially loaded states; however, fiber rearrangement in response to the biaxial loading remains similar.

Pregnancy-induced remodeling of heart valves.

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.

Transcatheter treatment of tricuspid regurgitation by caval valve implantation--experimental evaluation of decellularized tissue valves in central venous position.

BACKGROUND: Caval valve implantation has been suggested for transcatheter treatment of severe tricuspid regurgitation (TR). Combining the interventional technique with the promising surgical experience with decellularized valves, we sought to evaluate the functional and structural outcome of decellularized pericardial tissue valves (dTVs) in the low-pressure venous circulation in a chronic model of TR. METHODS AND RESULTS: Sixteen pericardial tissue valves were heterotopically implanted in the inferior and superior vena cava in a sheep model (54-98 kg; median 74.5 kg, n = 8) of severe TR. The devices were assembled using self-expanding nitinol stents and bovine pericardia decellularized by a detergent-based protocol (group dTV; n = 8). Glutaraldehyde-fixed pericardial tissue valves served as control (GaTV, n = 8). After 6 months, device function and structural maturation were analyzed using echocardiographic, histologic, immunohistologic, and electron microscopic approaches. After implantation, cardiac output increased significantly from 3.7 ± 1.1 l/min to 4.8 ± 1.1 l/min (P < 0.05) and competent valve function was verified by angiography. At 6 months, angiographic and echocardiographic evaluation revealed moderate to severe regurgitation in all GaTV. In contrast, five of the eight dTVs functioned well with only minor regurgitation. In these animals, autopsy revealed preserved valve structure with tender leaflets without signs of thrombosis or calcification. Conversely, GaTV showed severe degeneration with large calcification areas. Microscopic and histologic analysis confirmed endothelial repopulation in both valve types. However, additional interstitial reseeding was observed in decellularized valves. CONCLUSIONS: In the venous circulation in severe TR, decellularized valves show superior functional performance compared to Ga-fixed tissue valves. Macroscopic and microscopic analyses suggest preserved structural integrity and advanced endothelial and interstitial repopulation with evidence of less degradation in dTV. © 2014 Wiley Periodicals, Inc.

A combined proteomic and transcriptomic approach shows diverging molecular mechanisms in thoracic aortic aneurysm development in patients with tricuspid- and bicuspid aortic valve.

Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.

Steady flow hemodynamic and energy loss measurements in normal and simulated calcified tricuspid and bicuspid aortic valves.

The bicuspid aortic valve (BAV), which forms with two leaflets instead of three as in the normal tricuspid aortic valve (TAV), is associated with a spectrum of secondary valvulopathies and aortopathies potentially triggered by hemodynamic abnormalities. While studies have demonstrated an intrinsic degree of stenosis and the existence of a skewed orifice jet in the BAV, the impact of those abnormalities on BAV hemodynamic performance and energy loss has not been examined. This steady-flow study presents the comparative in vitro assessment of the flow field and energy loss in a TAV and type-I BAV under normal and simulated calcified states. Particle-image velocimetry (PIV) measurements were performed to quantify velocity, vorticity, viscous, and Reynolds shear stress fields in normal and simulated calcified porcine TAV and BAV models at six flow rates spanning the systolic phase. The BAV model was created by suturing the two coronary leaflets of a porcine TAV. Calcification was simulated via deposition of glue beads in the base of the leaflets. Valvular performance was characterized in terms of geometric orifice area (GOA), pressure drop, effective orifice area (EOA), energy loss (EL), and energy loss index (ELI). The BAV generated an elliptical orifice and a jet skewed toward the noncoronary leaflet. In contrast, the TAV featured a circular orifice and a jet aligned along the valve long axis. While the BAV exhibited an intrinsic degree of stenosis (18% increase in maximum jet velocity and 7% decrease in EOA relative to the TAV at the maximum flow rate), it generated only a 3% increase in EL and its average ELI (2.10 cm2/m2) remained above the clinical threshold characterizing severe aortic stenosis. The presence of simulated calcific lesions normalized the alignment of the BAV jet and resulted in the loss of jet axisymmetry in the TAV. It also amplified the degree of stenosis in the TAV and BAV, as indicated by the 342% and 404% increase in EL, 70% and 51% reduction in ELI and 48% and 51% decrease in EOA, respectively, relative to the nontreated valve models at the maximum flow rate. This study indicates the ability of the BAV to function as a TAV despite its intrinsic degree of stenosis and suggests the weak dependence of pressure drop on orifice area in calcified valves.

Circulating activated and effector memory T cells are associated with calcification and clonal expansions in bicuspid and tricuspid valves of calcific aortic stenosis.

We sought to delineate further the immunological significance of T lymphocytes infiltrating the valve leaflets in calcific aortic stenosis (CAS) and determine whether there were associated alterations in circulating T cells. Using clonotypic TCR ß-chain length and sequence analysis we confirmed that the repertoire of tricuspid CAS valves contains numerous expanded T cell clones with varying degrees of additional polyclonality, which was greatest in cases with severe calcification. We now report a similar proportion of clonal expansions in the much younger bicuspid valve CAS cases. Peripheral blood flow cytometry revealed elevations in HLA-DR(+) activated CD8 cells and in the CD8(+)CD28(null)CD57(+) memory-effector subset that were significantly greater in both bicuspid and tricuspid CAS cases with more severe valve calcification. Lesser increases of CD4(+)CD28(null) T cells were identified, principally in cases with concurrent atherosclerotic disease. Upon immunostaining the CD8 T cells in all valves were mainly CD28(null), and CD8 T cell percentages were greatest in valves with oligoclonal repertoires. T cell clones identified by their clonotypic sequence as expanded in the valve were also found expanded in the circulating blood CD28(null)CD8(+) T cells and to a lesser degree in the CD8(+)CD28(+) subset, directly supporting the relationship between immunologic events in the blood and the valve. The results suggest that an ongoing systemic adaptive immune response is occurring in cases with bicuspid and tricuspid CAS, involving circulating CD8 T cell activation, clonal expansion, and differentiation to a memory-effector phenotype, with trafficking of T cells in expanded clones between blood and the valve.

Reduced sox9 function promotes heart valve calcification phenotypes in vivo.

RATIONALE: Calcification of heart valve structures is the most common form of valvular disease and is characterized by the appearance of bone-like phenotypes within affected structures. Despite the clinical significance, the underlying etiology of disease onset and progression is largely unknown and valve replacement remains the most effective treatment. The SRY-related transcription factor Sox9 is expressed in developing and mature heart valves, and its function is required for expression of cartilage-associated proteins, similar to its role in chondrogenesis. In addition to cartilage-associated defects, mice with reduced sox9 function develop skeletal bone prematurely; however, the ability of sox9 deficiency to promote ectopic osteogenic phenotypes in heart valves has not been examined. OBJECTIVE: This study aims to determine the role of Sox9 in maintaining connective tissue homeostasis in mature heart valves using in vivo and in vitro approaches. METHODS AND RESULTS: Using histological and molecular analyses, we report that, from 3 months of age, Sox9(fl/+);Col2a1-cre mice develop calcific lesions in heart valve leaflets associated with increased expression of bone-related genes and activation of inflammation and matrix remodeling processes. Consistently, ectopic calcification is also observed following direct knockdown of Sox9 in heart valves in vitro. Furthermore, we show that retinoic acid treatment in mature heart valves is sufficient to promote calcific processes in vitro, which can be attenuated by Sox9 overexpression. CONCLUSIONS: This study provides insight into the molecular mechanisms of heart valve calcification and identifies reduced Sox9 function as a potential genetic basis for calcific valvular disease.

Diverging alternative splicing fingerprints in the transforming growth factor-ß signaling pathway identified in thoracic aortic aneurysms.

Impaired regulation of the transforming growth factor-ß (TGFß) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGFß pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGFß pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGFß pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription-polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGFß pathway.