Papers by Jonathan Butcher
Development
During valvulogenesis, cytoskeletal, secretory and transcriptional events drive endocardial cushi... more During valvulogenesis, cytoskeletal, secretory and transcriptional events drive endocardial cushion growth and remodeling into thin fibrous leaflets. Genetic disorders play an important role in understanding valve malformations but only account for a minority of clinical cases. Mechanical forces are ever present, but how they coordinate molecular and cellular decisions remains unclear. In this study, we used osmotic pressure to interrogate how compressive and tensile stresses influence valve growth and shape maturation. We found that compressive stress drives a growth phenotype, whereas tensile stress increases compaction. We identified a mechanically activated switch between valve growth and maturation, by which compression induces cushion growth via BMP-pSMAD1/5, while tension induces maturation via pSer-19-mediated MLC2 contractility. The compressive stress acts through BMP signaling to increase cell proliferation and decrease cell contractility, and MEK-ERK is essential for both...
Advanced Healthcare Materials, 2022
3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indisp... more 3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indispensable for the successful construction of tissue engineered heart valves (TEHVs). In this study, novel tri‐layered and gel‐like nanofibrous scaffolds, consisting of poly(lactic‐co‐glycolic) acid (PLGA) and poly(aspartic acid) (PASP), are fabricated by a combination of positive/negative conjugate electrospinning and bioactive hydrogel post‐processing. The nanofibrous PLGA‐PASP scaffolds present tri‐layered structures, resulting in anisotropic mechanical properties that are comparable with native heart valve leaflets. Biological tests show that nanofibrous PLGA‐PASP scaffolds with high PASP ratios significantly promote the proliferation and collagen and glycosaminoglycans (GAGs) secretions of human aortic valvular interstitial cells (HAVICs), compared to PLGA scaffolds. Importantly, the nanofibrous PLGA‐PASP scaffolds are found to effectively inhibit the osteogenic differentiation of HAVI...
Structural Heart, 2021
Objective: The Notch and Wnt pathways participate in calcific aortic valve disease (CAVD), but wi... more Objective: The Notch and Wnt pathways participate in calcific aortic valve disease (CAVD), but with conflicting findings to date. We hypothesize that Notch and Wnt pathway engagement during CAVD initiation are cell-type specific. Methods: Primary porcine aortic valvular endothelial (PAVEC) and/or interstitial (PAVIC) cells were cultured in a 3D spring mechanical constraint system or in 2D culture and challenged with osteogenic media (OGM) to induce calcification. DAPT and IMR-1 were used as Notch1 inhibitors, and lentiviral transduction was used to create constitutively-active b-catenin (B-cat+) PAVIC and doxycycline-inducible Notch1 intracellular domain (NICD+) PAVEC and PAVIC. Calcification was evaluated with alizarin red staining. Results: PAVIC treatment with Notch inhibitors DAPT and IMR-1 resulted in opposite calcific responses, and this correlated with active-form b-catenin signaling. Further interrogation using B-cat+ PAVIC showed increased calcification compared to wild-type (WT) cells. Treatment of B-cat+ PAVIC with DAPT, but not IMR-1, was able to reduce OGM-induced calcification to different extents in B-cat+ and WT cells. NICD overexpression was investigated in PAVIC and PAVEC. Culture of NICD+ PAVIC in 3D decreased calcification compared to controls, while NICD+ PAVIC and PAVEC co-culture increased calcification. Gene expression data in 2D NICD+ VEC found no difference in EndMT SNAI1/2 expression, and an increase in osteogenic genes Runx2, BMP2, SPARC, and SPP1 vs. WT controls. Conclusions: We demonstrate a divergence in Notch1 regulation of calcification with PAVIC DAPT and IMR-1 treatment with and without constitutively active b-catenin, and with NICD+ PAVIC monoculture vs. NICD+ PAVIC and PAVEC co-culture. Given our previous findings demonstrating early upregulation and later downregulation of Notch1 in VEC with TNFa-stimulus, our results are consistent with the hypothesis that Notch1 signaling has a dynamic and cell-specific influence over CAVD progression. Nuances in Notch1 and Wnt signaling could be vital to development of a CAVD therapeutic.
Structural Heart, 2021
Objective: Bioprinting has strengthened the ability to create complex materials to replace dysfun... more Objective: Bioprinting has strengthened the ability to create complex materials to replace dysfunctional tissue and has been used to replicate the geometry of heart valves. Additionally, embedded printing has expanded the range of bioprintable materials. However, bioprinting microstructure of leaflets remains difficult with traditional printing methods. Extrusion printers can be programmed to move in non-planar directions to bioprint embedded non-planar tissues (BENT) by following surfaces of tissues to reproduce complex structures such as valve leaflets. Methods: Valve leaflet models were created in three diameters (12, 17, and 22mm). Briefly, the BENT method uses leaflet surfaces projected onto the X-Y plane and extruded to the final leaflet thickness (300, 600, and 900µm). These models were imported to Cura and sliced into printable paths. The files were translated to the surfaces using point matching to assign new Z-values each point. This created a 3D print file where paths follow non-planar movement so that layers form the full leaflet surface before moving to the next layer. A 12mm valve was printed in alginate (Figure 1) and BENT print files of three sizes were created to compare extruder changes and total layers using one, two, or three materials. Conclusions: Initial results show that non-planar printing can reduce the number of layers and extruder changes when using multiple materials to print a wide range of heart valve leaflet sizes. Future work will move to optimize printer motion for improving print time and study implications of this method on cell viability, print fidelity, and material mechanics. Results:Layers and extruder changes for leaflets printed with one, two, or three materials 22mm Valve 17mm Valve 12mm Valve Layers Extruder Changes Layers Extruder Changes Layers Extruder Changes 1. Extruder Planar 90 0 68 0 44 0 Non-Planar 9 0 6 0 3 0 2. Extruders Planar 90 80 68 62 46 29 Non-Planar 9 1 6 1 3 1 3. Extruders Planar 90 160 68 124 45 58 Non-Planar 9 2 6 2 3 2
Scientific Reports, 2020
Heart valve disease is a common manifestation of cardiovascular disease and is a significant caus... more Heart valve disease is a common manifestation of cardiovascular disease and is a significant cause of cardiovascular morbidity and mortality worldwide. The pulmonary valve (PV) is of primary concern because of its involvement in common congenital heart defects, and the PV is usually the site for prosthetic replacement following a Ross operation. Although effects of age on valve matrix components and mechanical properties for aortic and mitral valves have been studied, very little is known about the age-related alterations that occur in the PV. In this study, we isolated PV leaflets from porcine hearts in different age groups (~ 4–6 months, denoted as young versus ~ 2 years, denoted as adult) and studied the effects of age on PV leaflet thickness, extracellular matrix components, and mechanical properties. We also conducted proteomics and RNA sequencing to investigate the global changes of PV leaflets and passage zero PV interstitial cells in their protein and gene levels. We found t...
PLOS ONE, 2020
In patients with ascending aortic (AA) aneurysms, prosthetic graft replacement yields benefit but... more In patients with ascending aortic (AA) aneurysms, prosthetic graft replacement yields benefit but risk for complications in the descending aorta persists. Longitudinal impact of AA grafts on native descending aortic physiology is poorly understood. Transthoracic echocardiograms (echo) in patients undergoing AA elective surgical grafting were analyzed: Descending aortic deformation indices included global circumferential strain (GCS), time to peak (TTP) strain, and fractional area change (FAC). Computed tomography (CT) was used to assess aortic wall thickness and calcification. 46 patients undergoing AA grafting were studied; 65% had congenital or genetically-associated AA (30% bicuspid valve, 22% Marfan, 13% other): After grafting (6.4±7.5 months), native descending aortic distension increased, irrespective of whether assessed based on circumferential strain or area-based methods (both p<0.001). Increased distensibility paralleled altered kinetics, as evidenced by decreased time to peak strain (p = 0.01) and increased velocity (p = 0.002). Augmented distensibility and flow velocity occurred despite similar preand post-graft blood pressure and medications (all p = NS), and was independent of pre-surgical aortic regurgitation or change in left ventricular stroke volume (both p = NS). Magnitude of change in GCS and FAC was 5-10 fold greater among patients with congenital or genetically associated AA vs. degenerative AA (p<0.001), paralleling larger descending aortic size,
Arteriosclerosis, Thrombosis, and Vascular Biology, 2020
Objective: Although often studied independently, little is known about how aortic valve endotheli... more Objective: Although often studied independently, little is known about how aortic valve endothelial cells and valve interstitial cells interact collaborate to maintain tissue homeostasis or drive valve calcific pathogenesis. Inflammatory signaling is a recognized initiator of valve calcification, but the cell-type–specific downstream mechanisms have not been elucidated. In this study, we test how inflammatory signaling via NFκB (nuclear factor κ-light-chain enhancer of activated B cells) activity coordinates unique and shared mechanisms of valve endothelial cells and valve interstitial cells differentiation during calcific progression. Approach and Results: Activated NFκB was present throughout the calcific aortic valve disease (CAVD) process in both endothelial and interstitial cell populations in an established mouse model of hypercholesterolemia-induced CAVD and in human CAVD. NFκB activity induces endothelial to mesenchymal transformation in 3-dimensional cultured aortic valve e...
Frontiers in Cell and Developmental Biology, 2017
Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with hea... more Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This information can then be utilized to develop natural engineering approaches that can emulate this fetal microenvironment and thus make prominent progress in pluripotent stem cell-derived maturity toward a more clinically relevant model for cardiac regeneration.
Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue ... more Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue morphogenesis and remodeling. EMT is induced by soluble transforming growth factor β (TGF-β) family members, and restricted by vascular endothelial growth factor fam- ily members. While many downstream molecular regulators of EMT have been identified, these have been largely evaluated individually without considering potential crosstalk. In this study, we created an ensemble of dynamic mathematical models describing TGF-β induced EMT to better understand the operational hierarchy of this complex molecular pro- gram. We used ordinary differential equations (ODEs) to describe the transcriptional and post-translational regulatory events driving EMT. Model parameters were estimated from multiple data sets using multiobjective optimization, in combination with cross-validation. TGF-β exposure drove the model population toward a mesenchymal phenotype, while an epithelial phenotype was enhanced...
Cancer Research, 2015
Background: Extracellular matrix (ECM) characteristics, including stiffness, geometry, chemistry,... more Background: Extracellular matrix (ECM) characteristics, including stiffness, geometry, chemistry, and spatial interaction with the neighboring cells and soluble factors are key components for cancer cell survival in a pathogenic tissue microenvironment (TME). However, poor performance of 2D in vitro systems and animal models for metastatic cancer types demands physiologically relevant well controlled 3D platforms to quantitatively assess (a) metastatic cancer stem cell survival (b) oncogenic mechanisms and (c) drug resistant phenotypes and (d) new drug efficacy. Advances in the “state of the art” 3D platforms with difference in matrix stiffness and viscoelastic properties are showing promising results in identifying changes in drug resistant phenotypes, cell behavior and gene expression profiles. Preliminary findings by us and others using 3D matrices for human multiple myeloma (MM) reveal promising results on cancer /stromal cell interactions. In this study we have shown that Hyalu...
SAE Technical Paper Series, 2001
This paper presents an analysis of the displacement measurement of the Hybrid III 50 th percentil... more This paper presents an analysis of the displacement measurement of the Hybrid III 50 th percentile male dummy chest in quasistatic and dynamic loading environments. In this dummy, the sternal chest deformation is typically characterized using a sliding chest potentiometer, originally ...
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Papers by Jonathan Butcher