Dissecting Calcific Aortic Valve Disease—The Role, Etiology, and Drivers of Valvular Fibrosis

Calcific aortic valve disease (CAVD) is a highly prevalent and progressive disorder that ultimately causes gradual narrowing of the left ventricular outflow orifice with ensuing devastating hemodynamic effects on the heart. Calcific mineral accumulation is the hallmark pathology defining this process; however, fibrotic extracellular matrix (ECM) remodeling that leads to extensive deposition of fibrous connective tissue and distortion of the valvular microarchitecture similarly has major biomechanical and functional consequences for heart valve function. Significant advances have been made to unravel the complex mechanisms that govern these active, cell-mediated processes, yet the interplay between fibrosis and calcification and the individual contribution to progressive extracellular matrix stiffening require further clarification. Specifically, we discuss (1) the valvular biomechanics and layered ECM composition, (2) patterns in the cellular contribution, temporal onset, and risk factors for valvular fibrosis, (3) imaging valvular fibrosis, (4) biomechanical implications of valvular fibrosis, and (5) molecular mechanisms promoting fibrotic tissue remodeling and the possibility of reverse remodeling. This review explores our current understanding of the cellular and molecular drivers of fibrogenesis and the pathophysiological role of fibrosis in CAVD

Elastogenesis Correlates With Pigment Production in Murine Aortic Valve Leaflets

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues. Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression. Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species

Elastogenesis Correlates With Pigment Production in Murine Aortic Valve Leaflets

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues.Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression.Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species

High resolution monitoring of valvular interstitial cell driven pathomechanisms in procalcific environment using label-free impedance spectroscopy

IntroductionFibro-calcific aortic valve disease has high prevalence and is associated with significant mortality. Fibrotic extracellular matrix (ECM) remodeling and calcific mineral deposition change the valvular microarchitecture and deteriorate valvular function. Valvular interstitial cells (VICs) in profibrotic or procalcifying environment are frequently used in vitro models. However, remodeling processes take several days to weeks to develop, even in vitro. Continuous monitoring by real-time impedance spectroscopy (EIS) may reveal new insights into this process.MethodsVIC-driven ECM remodeling stimulated by procalcifying (PM) or profibrotic medium (FM) was monitored by label-free EIS. Collagen secretion, matrix mineralization, viability, mitochondrial damage, myofibroblastic gene expression and cytoskeletal alterations were analyzed.Results and DiscussionEIS profiles of VICs in control medium (CM) and FM were comparable. PM reproducibly induced a specific, biphasic EIS profile. Phase 1 showed an initial impedance drop, which moderately correlated with decreasing collagen secretion (r = 0.67, p = 0.22), accompanied by mitochondrial membrane hyperpolarization and cell death. Phase 2 EIS signal increase was positively correlated with augmented ECM mineralization (r = 0.97, p = 0.008). VICs in PM decreased myofibroblastic gene expression (p < 0.001) and stress fiber assembly compared to CM. EIS revealed sex-specific differences. Male VICs showed higher proliferation and in PM EIS decrease in phase 1 was significantly pronounced compared to female VICs (male minimum: 7.4 ± 4.2%, female minimum: 26.5 ± 4.4%, p < 0.01). VICs in PM reproduced disease characteristics in vitro remarkably fast with significant impact of donor sex. PM suppressed myofibroblastogenesis and favored ECM mineralization. In summary, EIS represents an efficient, easy-to-use, high-content screening tool enabling patient-specific, subgroup- and temporal resolution

Standardization of Human Calcific Aortic Valve Disease in vitro Modeling Reveals Passage-Dependent Calcification

Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as in vitro models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD in vitro research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need

Regular Exercise or Changing Diet Does Not Influence Aortic Valve Disease Progression in LDLR Deficient Mice

BACKGROUND: The development and progression of calcific aortic valve disease (CAVD) shares a number of similarities with atherosclerosis. Recently we could demonstrate that regular exercise training (ET) as primary prevention prevents aortic valve disease in LDL-receptor deficient (LDLR(-/-)) mice. We aimed to investigate the impact of exercise training on the progression of CAVD in LDLR(-/-) mice in the setting of secondary prevention METHODS AND RESULTS: Sixty-four LDLR(-/-) mice were fed with high cholesterol diet to induce aortic valve sclerosis. Thereafter the animals were divided into 3 groups: group 1 continuing on high cholesterol diet, group 2 continuing with cholesterol diet plus 1 h ET per day, group 3 continuing with normal mouse chow. After another 16 weeks the animal were sacrificed. Histological analysis of the aortic valve thickness demonstrated no significant difference between the three groups (control 98.3±4.5 µm, ET 88.2±6.6 µm, change in diet 87.5±4.0). Immunohistochemical staining for endothelial cells revealed a disrupted endothelial cell layer to the same extend in all groups. Furthermore no difference between the groups was evident with respect to the expression of inflammatory, fibroblastic and osteoblastic markers. CONCLUSION: Based on the present study we have to conclude that once the development of a CAVD is initiated, exercise training or a change in diet does not have the potential to attenuate the progress of the CAVD

Photon shot-noise limited transient absorption soft X-ray spectroscopy at the European XFEL

Femtosecond transient soft X-ray Absorption Spectroscopy (XAS) is a very promising technique that can be employed at X-ray Free Electron Lasers (FELs) to investigate out-of-equilibrium dynamics for material and energy research. Here we present a dedicated setup for soft X-rays available at the Spectroscopy & Coherent Scattering (SCS) instrument at the European X-ray Free Electron Laser (EuXFEL). It consists of a beam-splitting off-axis zone plate (BOZ) used in transmission to create three copies of the incoming beam, which are used to measure the transmitted intensity through the excited and unexcited sample, as well as to monitor the incoming intensity. Since these three intensity signals are detected shot-by-shot and simultaneously, this setup allows normalized shot-by-shot analysis of the transmission. For photon detection, the DSSC imaging detector, which is capable of recording up to 800 images at 4.5 MHz frame rate during the FEL burst, is employed and allows approaching the photon shot-noise limit. We review the setup and its capabilities, as well as the online and offline analysis tools provided to users.Comment: 11 figure