Jan-Hung Chen

Calcification by Valve Interstitial Cells Is Regulated by the Stiffness of the Extracellular Matrix

Objective—Extensive remodeling of the valve ECM in calcific aortic valve sclerosis alters its mechanical properties, but little is known about the impact of matrix mechanics on the cells within the valve interstitium. In this study, the influence of matrix stiffness in modulating calcification by valve interstitial cells (VICs), and their differentiation to pathological phenotypes was assessed. Methods and Results—Primary porcine aortic VICs were cultured in standard media or calcifying media on constrained type I fibrillar collagen gels. Matrix stiffness was altered by changing only the thickness of the gels. Calcification did not occur in standard media, regardless of matrix stiffness. However, when VICs were grown in calcifying media on relatively compliant matrices with stiffness similar to that of normal tissue, they readily formed calcified aggregates of viable cells that expressed osteoblast-related transcripts and proteins. In contrast, VICs cultured in calcifying media on stiffer matrices (similar to stenotic tissue) differentiated to myofibroblasts and formed calcified aggregates that contained apoptotic cells. Actin depolymerization reduced aggregation on stiff, but not compliant, matrices. TGF-&bgr;1 potentiated aggregate formation on stiff matrices by enhancing &agr;-smooth muscle actin expression and cellular contractility, but not on compliant matrices attributable to downregulation of TGF-&bgr; receptor I. Cell contraction by VICs inhibited Akt activation and enhanced apoptosis-dependent calcification on stiff matrices. Conclusions—Differentiation of VICs to pathological phenotypes in response to biochemical cues is modulated by matrix stiffness. Although osteogenic or myofibrogenic differentiation of VICs can result in calcification, the processes are distinct.

Boning up on Wolff's Law: Mechanical regulation of the cells that make and maintain bone

Bone tissue forms and is remodeled in response to the mechanical forces that it experiences, a phenomenon described by Wolffs Law. Mechanically induced formation and adaptation of bone tissue is mediated by bone cells that sense and respond to local mechanical cues. In this review, the forces experienced by bone cells, the mechanotransduction pathways involved, and the responses elicited are considered. Particular attention is given to two cell types that have emerged as key players in bone mechanobiology: osteocytes, the putative primary mechanosensors in intact bone; and osteoprogenitors, the cells responsible for bone formation and recently implicated in ectopic calcification of cardiovascular tissues. Mechanoregulation of bone involves a complex interplay between these cells, their microenvironments, and other cell types. Thus, dissection of the role of mechanics in regulating bone cell fate and function, and translation of that knowledge to improved therapies, requires identification of relevant cues, multifactorial experimental approaches, and advanced model systems that mimic the mechanobiological environment.

Identification and Characterization of Aortic Valve Mesenchymal Progenitor Cells with Robust Osteogenic Calcification Potential

Advanced valvular lesions often contain ectopic mesenchymal tissues, which may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. The identity, frequency, and differentiation potential of the putative progenitor subpopulation are unknown. The objectives of this study were to determine whether valve interstitial cells (VICs) contain a subpopulation of multipotent mesenchymal progenitor cells, to measure the frequencies of the mesenchymal progenitors and osteoprogenitors, and to characterize the osteoprogenitor subpopulation because of its potential role in calcific aortic valve disease. The multilineage potential of freshly isolated and subcultured porcine aortic VICs was tested in vitro. Progenitor frequencies and self-renewal capacity were determined by limiting dilution and colony-forming unit assays. VICs were inducible to osteogenic, adipogenic, chondrogenic, and myofibrogenic lineages. Osteogenic differentiation was also observed in situ in sclerotic porcine leaflets. Primary VICs had strikingly high frequencies of mesenchymal progenitors (48.0 +/- 5.7%) and osteoprogenitors (44.1 +/- 12.0%). High frequencies were maintained for up to six population doublings, but decreased after nine population doublings to 28.2 +/- 9.9% and 5.8 +/- 1.3%, for mesenchymal progenitors and osteoprogenitors, respectively. We further identified the putative osteoprogenitor subpopulation as morphologically distinct cells that occur at high frequency, self-renew, and elaborate bone matrix from single cells. These findings demonstrate that the aortic valve is rich in a mesenchyma l progenitor cell population that has strong potential to contribute to valve calcification.

Cell–Matrix Interactions in the Pathobiology of Calcific Aortic Valve Disease: Critical Roles for Matricellular, Matricrine, and Matrix Mechanics Cues

The hallmarks of calcific aortic valve disease (CAVD) are the significant changes that occur in the organization, composition, and mechanical properties of the extracellular matrix (ECM), ultimately resulting in stiffened stenotic leaflets that obstruct flow and compromise cardiac function. Increasing evidence suggests that ECM maladaptations are not simply a result of valve cell dysfunction; they also contribute to CAVD progression by altering cellular and molecular signaling. In this review, we summarize the ECM changes that occur in CAVD. We also discuss examples of how the ECM influences cellular processes by signaling through adhesion receptors (matricellular signaling), by regulating the presentation and availability of growth factors and cytokines to cells (matricrine signaling), and by transducing externally applied forces and resisting cell-generated tractional forces (mechanical signaling) to regulate a wide range of pathological processes, including differentiation, fibrosis, calcification, and angiogenesis. Finally, we suggest areas for future research that should lead to new insights into bidirectional cell–ECM interactions in the aortic valve, their contributions to homeostasis and pathobiology, and possible targets to slow or prevent the progression of CAVD.

β-Catenin Mediates Mechanically Regulated, Transforming Growth Factor-β1–Induced Myofibroblast Differentiation of Aortic Valve Interstitial Cells

Objective—In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-&bgr;1 (TGF-&bgr;1) and Wnt/&bgr;-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of &bgr;-catenin and extracellular matrix stiffness in TGF-&bgr;1-induced myofibroblast differentiation of valve interstitial cells (VICs). Methods and Results—TGF-&bgr;1 induced rapid &bgr;-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-&bgr; receptor I kinase. Degrading &bgr;-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-&bgr;1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing &bgr;-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-&bgr;1 and Wnt3A caused greater myofibroblast differentiation than TGF-&bgr;1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-&bgr;1-induced &bgr;-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, &bgr;-catenin, TGF-&bgr;1, and phosphorylated Smad2/3 in the fibrosa. Conclusion—Myofibroblast differentiation of VICs involves matrix stiffness–dependent crosstalk between TGF-&bgr;1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.

The elastic properties of valve interstitial cells undergoing pathological differentiation

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.

A system for automated counting of fetal and maternal red blood cells in clinical KB test

The Kleihauer-Betke test (KBT) is a widely used method for measuring fetal-maternal hemorrhage (FMH) in maternal care. In hospitals, KBT is performed by a certified technologist to count a minimum of 2,000 fetal and maternal red blood cells (RBCs) on a blood smear. Manual counting is inherently inconsistent and subjective. This paper presents a system for automated counting and distinguishing fetal and maternal RBCs on clinical KB slides. A custom-adapted hardware platform is used for KB slide scanning and image capturing. Spatial-color pixel classification with spectral clustering is proposed to separate overlapping cells. Optimal clustering number and total cell number are obtained through maximizing cluster validity index. To accurately identify fetal RBCs from maternal RBCs, multiple features including cell size, shape, gradient and saturation difference are used in supervised learning to generate feature vectors, to tackle cell color, shape and contrast variations across clinical KB slides. The results show that the automated system is capable of completing the counting of over 60,000 cells (vs. 2,000 by technologists) within 5 minutes (vs. 15 minutes by technologists). The counting results are highly accurate and correlate strongly with those from benchmarking flow cytometry measurement.