Jesper Hjortnaes1, Claudia Goettsch2, Joshua D Hutcheson2, Gulden Camci-Unal3, Lilian Lax4, Katrin Scherer4, Simon Body5, Frederick J Schoen6, Jolanda Kluin7, Ali Khademhosseini8, Elena Aikawa9. 1. Center of Excellence in Vascular Biology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, The Netherlands. 2. Center for Interdisciplinary Cardiovascular Sciences, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. 3. Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA. 4. Center of Excellence in Vascular Biology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. 5. Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, USA. 6. Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. 7. Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, The Netherlands. 8. Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA. Electronic address: alik@bwh.harvard.edu. 9. Center of Excellence in Vascular Biology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Center for Interdisciplinary Cardiovascular Sciences, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. Electronic address: eaikawa@partners.org.
Abstract
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.
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.
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