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(a) Total cell count of varied ratio of support cell to EC for EC:CF and EC:MSC co-cultures. (b) The number of doublings in varied co-culture conditions for the three support cell:EC ratios. Note that for both EC:CF and EC:MSC co-cultures that the number of doublings in the 3:1 support cell:EC ratio is significantly less than the other two ratios for each co-culture type (p < 0.001).
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A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal st...
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... second set of experiments in this manuscript focused on determining any changes in the response of ECs to alterations in the ratios of support cells to ECs. In analyzing EC specific proliferation in the various groups in this study following 10 days of culture in our system via flow cytometry, it became evident that ECs proliferated best and maintained a higher percentage of the total cells in the CF co-culture conditions (Figs. 6a and 6b). When ECs were cultured with the MSCs as a support cell there was a significant decrease in the percent of ECs remaining in the constructs after 10 days (Fig. 6c). One possible explanation for these results could be that the MSCs are proliferating at a greater rate than the ECs and inhibiting EC growth via contact inhibition. Indeed, it has been previously demonstrated that VEGF induces MSC prolifera- tion, 25 and since the constructs for this study were all cultured in 1:1 EC to support cell medium, there would be VEGF present from the EC medium. This seems to agree with the complete data set, as although the total cell count was higher when ECs were cultured with MSCs (Fig. 5a), the EC proliferation was much lower in the cultures that had higher initial support cell numbers (Fig. 6b). Higher ratios of MSC:EC in culture have also led to EC apoptosis via contact-mediated release of reactive oxygen species, 24 which could also negatively impact EC number in co-culture with ...
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... to the mixed media studies detailed above, the total cell number after 10 days in culture was calculated using a DNA assay (Fig. 5a). Because the initial number of cells seeded in the constructs varied by condition even though the EC number was held constant (Table 1), the total number of doublings for each condition was also calculated based on the initial seeding number (Fig. 5b). When compared to the 3:1 condition for both support cell types, the 1:1 and 1:3 ratio of support cells to ECs resulted in a statistically greater number of doublings (p < 0.001). Within the MSC support cell condition, the 1:3 ratio of Support Cell to EC resulted in a significantly greater number of doublings than the 3:1 and 1:1 con- ditions (p < 0.001 and p = 0.027, respectively. While within the CF support cell type, the 1:3 and 1:1 condition both lead to a significantly greater number of doublings compared to the 3:1 condition (p = 0.003 and p = 0.035, ...
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... to the mixed media studies detailed above, the total cell number after 10 days in culture was calculated using a DNA assay (Fig. 5a). Because the initial number of cells seeded in the constructs varied by condition even though the EC number was held constant (Table 1), the total number of doublings for each condition was also calculated based on the initial seeding number (Fig. 5b). When compared to the 3:1 condition for both support cell types, the 1:1 and 1:3 ratio of support cells to ECs resulted in a statistically greater number of doublings (p < 0.001). Within the MSC support cell condition, the 1:3 ratio of Support Cell to EC resulted in a significantly greater number of doublings than the 3:1 and 1:1 con- ditions (p < 0.001 and p = 0.027, respectively. While within the CF support cell type, the 1:3 and 1:1 condition both lead to a significantly greater number of doublings compared to the 3:1 condition (p = 0.003 and p = 0.035, ...
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Rationale
Myoendothelial junctions (MEJs) within the fenestrae of the internal elastic lamina (IEL) are critical sites that allow for endothelial cell (EC) - vascular smooth muscle cell (VSMC) contact and communication. Vascular Notch signaling is a critical determinant of normal vasculogenesis and remodeling, and it regulates cell phenotype via co...
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... The interactions between CFs and other non-myocytes have been examined in previous studies. When tested in the co-culture model, CFs assisted the proliferation of ECs and their sprouting (30). IL-1β expression in CFs, under a disease condition of myocarditis, has been shown to recruit leukocytes and induce the inflammatory process (31). ...
Heart disease is one of the major life-threatening diseases with high mortality and incidence worldwide. Several model systems, such as primary cells and animals, have been used to understand heart diseases and establish appropriate treatments. However, they have limitations in accuracy and reproducibility in recapitulating disease pathophysiology and evaluating drug responses. In recent years, three-dimensional (3D) cardiac tissue models produced using tissue engineering technology and human cells have outperformed conventional models. In particular, the integration of cell reprogramming techniques with bioengineering platforms (e.g., microfluidics, scaffolds, bioprinting, and biophysical stimuli) has facilitated the development of heart-on-a-chip, cardiac spheroid/organoid, and engineered heart tissue (EHT) to recapitulate the structural and functional features of the native human heart. These cardiac models have improved heart disease modeling and toxicological evaluation. In this review, we summarize the cell types for the fabrication of cardiac tissue models, introduce diverse 3D human cardiac tissue models, and discuss the strategies to enhance their complexity and maturity. Finally, recent studies in the modeling of various heart diseases are reviewed.
... Combined, these two aspects also have a significant impact on the maturation of iPSC-CMs. Indeed, different studies demonstrated that iPSC-CM maturation is improved by the presence of other cell types and 3D structure provided by the ECM [137,345,[384][385][386][387][388]. Although, to date, these advanced 3D multicellular models have not been applied to MD patients' iPSCs, they do stand a greater ability to recapitulate DAC phenotypes more faithfully and with better resolution. ...
The ultimate goal of precision disease modeling is to artificially recreate the disease of affected people in a highly controllable and adaptable external environment. This field has rapidly advanced which is evident from the application of patient-specific pluripotent stem-cell-derived precision therapies in numerous clinical trials aimed at a diverse set of diseases such as macular degeneration, heart disease, spinal cord injury, graft-versus-host disease, and muscular dystrophy. Despite the existence of semi-adequate treatments for tempering skeletal muscle degeneration in dystrophic patients, nonischemic cardiomyopathy remains one of the primary causes of death. Therefore, cardiovascular cells derived from muscular dystrophy patients' induced pluripotent stem cells are well suited to mimic dystrophin-associated cardiomyopathy and hold great promise for the development of future fully effective therapies. The purpose of this article is to convey the realities of employing precision disease models of dystrophin-associated cardiomyopathy. This is achieved by discussing, as suggested in the title echoing William Shakespeare's words, the settlements (or "leagues") made by researchers to manage the constraints ("betwixt mine eye and heart") distancing them from achieving a perfect precision disease model.
... [43,44] TGFβ1 and vascular endothelial growth factor (VEGF), released by both anti-inflammatory macrophages and cardiac fibroblasts, increase connective tissue growth factor (CTGF) secretion by fibroblasts. [4,[45][46][47][48][49] CTGF is also downregulated by IL-1 to yield lower activity in the inflammatory phase. [50] CTGF mAb treatment in MI mice at day 3 improved ejection fraction and survival at day 7. Long term treatment with CTGF blocking antibody limited fibrosis and cardiac hypertrophy. ...
Cardiac wound healing after myocardial infarction (MI) evolves from pro-inflammatory to anti-inflammatory to reparative responses, and the cardiac fibroblast is a central player during the entire transition. The fibroblast mirrors changes seen in the left ventricle infarct by undergoing a continuum of polarization phenotypes that follow pro-inflammatory, anti-inflammatory, and pro-scar producing profiles. The development of each phenotype transition is contingent upon the MI environment into which the fibroblast enters. In this mini-review, we summarize our current knowledge regarding cardiac fibroblast activation during MI and highlight key areas where gaps remain.
... 114 Furthermore, these new methods need endothelial cells, smooth muscle cells, myofibroblasts, mesenchymal stem cells and cardiac fibroblasts, which are important for contractile force improvements and producing angiogenic factors essential for a stable vessel maturation in vitro. 115 In addition, nanotechnologies have been created to check and record the cardiac microtissue-generated forces in real time, using mini-engineered heart tissue grown into microgauges. 117 However, engineered cardiac tissue is not fully developed for efficient drug screening analysis; the major hurdle to the application of these technologies is the inability to assess cell variability as well as chemical, mechanical, and electrical stimuli. ...
Duchenne and Becker muscular dystrophies are the most common muscle diseases and are both currently incurable. They are caused by mutations in the dystrophin gene, which lead to the absence or reduction/truncation of the encoded protein, with progressive muscle degeneration that clinically manifests in muscle weakness, cardiac and respiratory involvement and early death. The limits of animal models to exactly reproduce human muscle disease and to predict clinically relevant treatment effects has prompted the development of more accurate in vitro skeletal muscle models. However, the challenge of effectively obtaining mature skeletal muscle cells or satellite stem cells as primary cultures has hampered the development of in vitro models. Here, we discuss the recently developed technologies that enable the differentiation of skeletal muscle from human induced pluripotent stem cells (iPSCs) of Duchenne and Becker patients. These systems recapitulate key disease features including inflammation and scarce regenerative myogenic capacity that are partially rescued by genetic and pharmacological therapies and can provide a useful platform to study and realize future therapeutic treatments. Implementation of this model also takes advantage of the developing genome editing field, which is a promising approach not only for correcting dystrophin, but also for modulating the underlying mechanisms of skeletal muscle development, regeneration and disease. These data prove the possibility of creating an accurate Duchenne and Becker in vitro model starting from iPSCs, to be used for pathogenetic studies and for drug screening to identify strategies capable of stopping or reversing muscular dystrophinopathies and other muscle diseases.
... MI day 3 fibroblasts showed a pro-angiogenic profile. Cardiac fibroblasts secrete several factors that regulate angiogenesis, including VEGF [40,60,71,72]. The positive regulation of angiogenesis at MI day 3 coincides with the peak of vessel formation and endothelial cell proliferation in the mouse, indicating that fibroblasts are a significant in vivo contributor to endothelial cell activation [63]. ...
Cardiac fibroblasts are the major producers of extracellular matrix (ECM) to form infarct scar. We hypothesized that fibroblasts undergo a spectrum of phenotype states over the course of myocardial infarction (MI) from early onset to scar formation. Fibroblasts were isolated from the infarct region of C57BL/6J male mice (3–6 months old, n = 60) at days 0 (no MI control) and 1, 3, or 7 after MI. Whole transcriptome analysis was performed by RNA-sequencing. Of the genes sequenced, 3371 were differentially expressed after MI. Enrichment analysis revealed that MI day 1 fibroblasts displayed pro-inflammatory, leukocyte-recruiting, pro-survival, and anti-migratory phenotype through Tnfrsf9 and CD137 signaling. MI day 3 fibroblasts had a proliferative, pro-fibrotic, and pro-angiogenic profile with elevated Il4ra signaling. MI day 7 fibroblasts showed an anti-angiogenic homeostatic-like myofibroblast profile and with a step-wise increase in Acta2 expression. MI day 7 fibroblasts relied on Pik3r3 signaling to mediate Tgfb1 effects and Fgfr2 to regulate PI3K signaling. In vitro, the day 3 MI fibroblast secretome stimulated angiogenesis, while day 7 MI fibroblast secretome repressed angiogenesis through Thbs1 signaling. Our results reveal novel mechanisms for fibroblasts in expressing pro-inflammatory molecules and regulating angiogenesis following MI.
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The online version of this article (10.1007/s00395-019-0715-4) contains supplementary material, which is available to authorized users.
... FBs have been also regarded as an indispensable and significant component within the heart (8) and proven to be involved in to maintain the integrity of microvessels and prevent from leaking. Cardiac fibroblasts (CFs) are considered to play a pivotal role in normal cardiac homeostasis and pathological fibrosis after MI, which also have been presented to promote ECs proliferation and sprout formation (52,53). Compared to the CFs, EmFs are more likely to get available. ...
Heart failure (HF) is the terminal state of cardiovascular disease (CVD), leading numerous patients to death every year. Cardiac tissue engineering is a multidisciplinary field of creating functional cardiac patches in vitro to promote cardiac function after transplantation onto damaged zone, giving the hope for patients with end-stage HF. However, the limited thickness of cardiac patches results in the graft failure of survival and function due to insufficient blood supply. To date, prevascularized cardiac tissue, with the use of circulatory scaffolds, holds the promise to be inosculated and perfused with host vasculature to eventually promote cardiac pumping function. Circulatory scaffolds play its role to provide oxygen and nutrients and take metabolic wastes away, and achieve anastomosis with host vasculature in vivo. Of worth note, hearton- a-chip based on circulatory scaffolds now has been considered as a valuable unit to broaden the research for building cardiac tissue. In this review, we will present recent different strategies to engineer circulatory scaffolds for building cardiac tissue with microvasculature, followed by its current state and future direction.
... Fibroblasts are proven to secrete growth factors that support endothelial cell form lumen in three-dimensional co-cultures. 44,45 In the presence of broblasts, HUVEC spreading is robust and forms intracellular lumen within four to ve days. As shown in Fig. 4, 3D-cultured HUVEC development was monitored using a camera in a confocal microscope. ...
Exposure to ambient fine particulate matter (FPM) has been thought to be associated with cardiovascular disease. However, the pathogenesis remains largely unknown. Animal models have been widely used in toxicological research, but species difference makes it impossible to directly translate discoveries from animals to humans. In this study, we developed a 3D functional human microvascular network in a microfluidic device. The established model enables endothelial cells to form vessel-like microtissues and have physiological functions which are closer to cells in human blood vessels. The perfusable microvasculature allows the delivery of nutrients, and oxygen, as well as flow-induced mechanical stimuli into the luminal space of the endothelium. The microflow effectively mimic the blood flow in human vessels. FPMs were introduced into this physiologically human vessel-like microenvironment following the fluid flow. The vascular toxicity was evaluated based on this organotypic 3D microvessel model. Our results demonstrated that intravascular accumulation of FPM could enhance ROS generation which may further cause endothelial dysfunction by oxidative stress. This is expressed in disorder of NO expression and IL-6 up-regulation. These are expected to enhance endothelial inflammation which might in turn accelerate coagulation that is associated with thrombosis. Human organotypic 3D microvessel models provide a possible bridge for how the research outcomes translate to humans. These models could partly simulate the physiological responses of human vessels to FPM stimulation. This simple and versatile platform can be used for a wide range of applications in vascular physiology studies of particulate matter in the context of cardiovascular disease.
... Several cell populations play its role in neoangiogenesis -endothelial cells, endothelial stem cells, pericytes, endocardial cells [17], telocytes [15,18], partially fibroblasts, vascular smooth muscle cells [19] and cardiomyocytes [20]. ...
Economic and social burden of cardiovascular diseases remains enormous and even still rising. There is not enough mass evidence in scientific journals that could describe the course of the process of neoangiogenesis in relatively “healthy” heart after regular endurance exercise. Even though, in this review, we are showing preliminary evidence that this can be one of really cheap and effective preventive means. We are elucidating some of the cellular signaling pathways how exercise could affect neoangiogenesis and ameliorate performance of the heart. Key roles in this process are mechanical forces (mainly increased velocity of blood flow and shear stress) and subsequent rise of angiogenic biological factors (mainly VEGFA).
... The coculture of ECs and CMs or triculture of ECs, CFs, and CMs is now accepted as a viable method to enhance the CM growth and spatial organization [8]. Iyer et al. fabricated microchannels by photo- polymerization of poly(ethylene glycol) diacrylate, and a mixture of CMs, fibroblasts and ECs were seeded in the microchannels [9]. ...
Challenges remain in engineering cardiac tissues with functional and morphological properties similar to those of native myocardium. In the current study, micropatterned fibrous mats are obtained by deposition of electrospun fibers on lithographic collectors to reproduce the anisotropic structure of myocardium, and carbon nanotubes are included in fibers to provide conductivities at the same level of cardiac muscles. The patterned mats are assembled layer-by-layer into patterned scaffolds for coculture of primary cardiomyocytes (CMs) with cardiac fibroblasts (CFs) and endothelial cells (ECs). CMs are organized along the fibers with clear cardiac strips of sarcomeric α-actinin and belt-like connexin-43, showing strong cellular extraction forces and intercellular communications. Compared with square and rectangle patterns, honeycomb (Hc)-patterned scaffolds shows higher ultimate tensile strength and strain to failure. The finite element analysis indicates no apparent stress concentration under stress application in the two orthogonal directions. The Hc-patterned coculture demonstrates significantly higher CM viabilities, deeper penetrations of cells into scaffolds, stronger expression of troponin I, connexin-43 and sarcomeric α-actinin by CMs and more abundant formations of capillary-like networks by ECs than other scaffolds. CMs on Hc-patterned scaffolds display spontaneous beating rates at 101 ± 12 times/min after coculture for 5 days and remain synchronously beating at 94 ± 8 times/min after 15 days, which is close to those of adult and neonatal rats. The layered and patterned coculture strategy achieves the spatial arrangement of multiple types of cells and vascularization potential, providing a biomimetic strategy for engineering functional cardiac patches in vitro.
... Since hADSCs are much larger than HUVECs, some of the initially seeded hADSCs may not have reached the interior of the scaffolds; those who did, proliferated at a faster rate than HUVECs, and thus the ratio was reversed in due time. Many HUVECs in the PEA-s systems establish cell-cell contacts (Fig. 8), which leads to a lower proliferation due to inhibition by contact, as discussed in Ref. 38. The low magnification reconstruction of bare and SAP-filled PEA-s scaffolds ( Fig. 9) reveals that cells were capable of migrating through the scaffolds pores. ...
The interior of tissue engineering scaffolds must be vascularizable and allow adequate nutrients perfusion in order to ensure the viability of the cells colonizing them. The promotion of rapid vascularization of scaffolds is critical for thick artificial constructs. In the present study co-cultures of human endothelial and adipose tissue-derived stem cells have been performed in poly(ethyl acrylate) scaffolds with two different pore structures: grid-like (PEA-o) or sponge-like (PEA-s), in combination with a self-assembling peptide gel filling the pores, which aims to mimic the physiological niche. After 2 and 7 culture days, cell adhesion, proliferation and migration, the expression of cell surface markers like CD31 and CD90 and the release of VEGF were assessed by means of immunocytochemistry, scanning electronic microscopy, flow cytometry and ELISA analyses. The study demonstrated that PEA-s scaffolds promoted greater cell organization into tubular-like structures than PEA-o scaffolds, and this was enhanced by the presence of the peptide gel. Paracrine signaling from adipose cells significantly improved endothelial cell viability, proving the advantageous combination of this system for obtaining easily vascularizable tissue engineered grafts. This article is protected by copyright. All rights reserved.
