Technique for Rapidly Forming Networks of Microvessel-Like Structures.

Modeling organ-blood barriers through the inclusion of microvessel networks within in vitro tissue models could lead to more physiologically accurate results, especially since organ-blood barriers are crucial to the normal function, drug transport, and disease states of vascularized organs. Microvessel networks are difficult to form, since they push the practical limits of most fabrication methods, and it is difficult to coax vascular cells to self-assemble into structures larger than capillaries. Here, we present a method for rapidly forming networks of microvessel-like structures using sacrificial alginate structures. Specifically, we encapsulated endothelial cells within short alginate threads, and then embedded them in collagen gel. Following enzymatic degradation of the alginate, the collagen gel contained a network of hollow channels seeded with cells, all surrounding a perfusable central channel. This method uses a 3D-printed coaxial extruder and syringe pumps to generate short threads in a way that is repeatable and easily transferrable to other labs. The cell-laden, sacrificial alginate threads can be frozen after fabrication and thawed before embedding without significant loss of cell viability. The ability to freeze the threads enables future scale-up and ease of use. Within millifluidic devices that restrict access to media, the threads enhance cell survival under static conditions. These results indicate the potential for use of this method in a range of tissue engineering applications.

Technique for rapidly forming networks of microvessel-like structures.

Modelling organ-blood barriers through the inclusion of microvessel networks within in vitro tissue models could lead to more physiologically accurate results, especially since organ-blood barriers are crucial to the normal function, drug transport, and disease states of vascularized organs. Microvessel networks are difficult to form, since they push the practical limit of most fabrication methods, and it is difficult to coax vascular cells to self-assemble into structures larger than capillaries. Here we present a method for rapidly forming networks of microvessel-like structures using sacrificial, alginate structures. Specifically, we encapsulated endothelial cells within short alginate threads, then embedded them in collagen gel. Following enzymatic degradation of the alginate, the collagen gel contained a network of hollow channels seeded with cells, all surrounding a perfusable central channel. This method uses a 3D printed coaxial extruder and syringe pumps to generate short threads in a way that is repeatable and easily transferrable to other labs. The cell-laden, sacrificial alginate threads can be frozen after fabrication and thawed before embedding without significant loss of cell viability. The ability to freeze the threads enables future scale up and ease of use. Within millifluidic devices that restrict access to media, the threads enhance cell survival under static conditions. These results indicate the potential for use of this method in a range of tissue engineering applications.

Isolation and Characterization of Porcine Endocardial Endothelial Cells.

The heart contains diverse endothelial cell types. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of these cells, we reported our protocol for isolating EECs from porcine hearts and for establishing an EEC population through cell sorting. In addition, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs stained positively for classic phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs at 48 h (1310 ± 251 cells vs. 597 ± 130 cells, p = 0.0361) and at 96 h (2873 ± 257 cells vs. 1714 ± 342 cells, p = 0.0002). Yet EECs migrated more slowly than HUVECs to cover a scratch wound at 4 h (5% ± 1% wound closure vs. 25% ± 3% wound closure, p < 0.0001), 8 h (15% ± 4% wound closure vs. 51% ± 12% wound closure, p < 0.0001), and 24 h (70% ± 11% wound closure vs. 90% ± 3% wound closure, p < 0.0001). Finally, the EECs maintained their endothelial phenotype by positive expression of CD31 through more than a dozen passages (three populations of EECs showing 97% ± 1% CD31+ cells in over 14 passages). In contrast, the HUVECs showed significantly reduced CD31 expression over high passages (80% ± 11% CD31+ cells over 14 passages). These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell types when studying or modeling diseases of interest.

Dantrolene inhibits lysophosphatidylcholine-induced valve interstitial cell calcific nodule formation via blockade of the ryanodine receptor.

Calcific aortic valve disease (CAVD), a fibrocalcific thickening of the aortic valve leaflets causing obstruction of the left ventricular outflow tract, affects nearly 10 million people worldwide. For those who reach end-stage CAVD, the only treatment is highly invasive valve replacement. The development of pharmaceutical treatments that can slow or reverse the progression in those affected by CAVD would greatly advance the treatment of this disease. The principal cell type responsible for the fibrocalcific thickening of the valve leaflets in CAVD is valvular interstitial cells (VICs). The cellular processes mediating this calcification are complex, but calcium second messenger signaling, regulated in part by the ryanodine receptor (RyR), has been shown to play a role in a number of other fibrocalcific diseases. We sought to determine if the blockade of calcium signaling in VICs could ameliorate calcification in an in vitro model. We previously found that VICs express RyR isotype 3 and that its modulation could prevent VIC calcific nodule formation in vitro. We sought to expand upon these results by further investigating the effects of calcium signaling blockade on VIC gene expression and behavior using dantrolene, an FDA-approved pan-RyR inhibitor. We found that dantrolene also prevented calcific nodule formation in VICs due to cholesterol-derived lysophosphatidylcholine (LPC). This protective effect corresponded with decreases in intracellular calcium flux, apoptosis, and ACTA2 expression but not reactive oxygen species formation caused by LPC. Interestingly, dantrolene increased the expression of the regulator genes RUNX2 and SOX9, indicating complex gene regulation changes. Further investigation via RNA sequencing revealed that dantrolene induced several cytoprotective genes that are likely also responsible for its attenuation of LPC-induced calcification. These results suggest that RyR3 is a viable therapeutic target for the treatment of CAVD. Further studies of the effects of RyR3 inhibition on CAVD are warranted.

Differential proteome profile, biological pathways, and network relationships of osteogenic proteins in calcified human aortic valves.

Calcific aortic valve disease (CAVD) is the most common heart valve disease requiring intervention. Most research on CAVD has focused on inflammation, ossification, and cellular phenotype transformation. To gain a broader picture into the wide range of cellular and molecular mechanisms involved in this disease, we compared the total protein profiles between calcified and non-calcified areas from 5 human valves resected during surgery. The 1413 positively identified proteins were filtered down to 248 proteins present in both calcified and non-calcified segments of at least 3 of the 5 valves, which were then analyzed using Ingenuity Pathway Analysis. Concurrently, the top 40 differentially abundant proteins were grouped according to their biological functions and shown in interactive networks. Finally, the abundance of selected osteogenic proteins (osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK) was quantified using ELISA and/or immunohistochemistry. The top pathways identified were complement system, acute phase response signaling, metabolism, LXR/RXR and FXR/RXR activation, actin cytoskeleton, mineral binding, nucleic acid interaction, structural extracellular matrix (ECM), and angiogenesis. There was a greater abundance of osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK in the calcified regions than the non-calcified ones. The osteogenic proteins also formed key connections between the biological signaling pathways in the network model. In conclusion, this proteomic analysis demonstrated the involvement of multiple signaling pathways in CAVD. The interconnectedness of these pathways provides new insights for the treatment of this disease.

The Immune and Inflammatory Basis of Acquired Pediatric Cardiac Disease.

Children with acquired heart disease face significant health challenges, including a lifetime of strict medical management, multiple cardiac surgeries, and a high mortality risk. Though the presentation of these conditions is diverse, a unifying factor is the role of immune and inflammatory responses in their development and/or progression. For example, infectious agents have been linked to pediatric cardiovascular disease, leading to a large health burden that disproportionately affects low-income areas. Other implicated mechanisms include antibody targeting of cardiac proteins, infection of cardiac cells, and inflammation-mediated damage to cardiac structures. These changes can alter blood flow patterns, change extracellular matrix composition, and induce cardiac remodeling. Therefore, understanding the relationship between the immune system and cardiovascular disease can inform targeted diagnostic and treatment approaches. In this review, we discuss the current understanding of pediatric immune-associated cardiac diseases, challenges in the field, and areas of research with potential for clinical benefit.

Congenital Heart Disease: An Immunological Perspective.

Congenital heart disease (CHD) poses a significant global health and economic burden-despite advances in treating CHD reducing the mortality risk, globally CHD accounts for approximately 300,000 deaths yearly. Children with CHD experience both acute and chronic cardiac complications, and though treatment options have improved, some remain extremely invasive. A challenge in addressing these morbidity and mortality risks is that little is known regarding the cause of many CHDs and current evidence suggests a multifactorial etiology. Some studies implicate an immune contribution to CHD development; however, the role of the immune system is not well-understood. Defining the role of the immune and inflammatory responses in CHD therefore holds promise in elucidating mechanisms underlying these disorders and improving upon current diagnostic and treatment options. In this review, we address the current knowledge coinciding CHDs with immune and inflammatory associations, emphasizing conditions where this understanding would provide clinical benefit, and challenges in studying these mechanisms.

Tumor necrosis factor alpha and interleukin 1 beta suppress myofibroblast activation via nuclear factor kappa B signaling in 3D-cultured mitral valve interstitial cells.

Mitral valve disease is a major cause of cardiovascular morbidity throughout the world. Many different mitral valve pathologies feature fibrotic remodeling, often accompanied by an inflammatory state. Mitral valve fibrosis is mediated by valvular interstitial cells (VICs), which reside in the valve leaflets and often differentiate into myofibroblast-like cells during disease conditions. In this study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) on mitral VICs, since these pro-inflammatory cytokines have been shown to exert pleiotropic effects on various cell types in other fibrotic disorders. Using biomimetic three-dimensional culture systems, we demonstrated that TNF-α and IL-1ß suppress myofibroblast differentiation in mitral VICs, as evidenced by gene and protein expression of alpha smooth muscle actin and smooth muscle 22 alpha. Addition of TNF-α and IL-1ß also inhibited mitral VIC-mediated contraction of collagen gels. Furthermore, inhibition of NF-κB, which is downstream of TNF-α and IL-1ß, reversed these effects. These results reveal targetable pathways for potential development of pharmaceutical treatments for alleviating fibrosis during mitral valve disease. STATEMENT OF SIGNIFICANCE: Mitral valve disease is a common cardiovascular condition that is often accompanied by fibrotic tissue remodeling. Valvular interstitial cells (VICs), the fibroblast-like cells that reside in heart valve leaflets, are thought to drive fibrosis during valve disease by differentiating into activated myofibroblasts. However, the signaling pathways that regulate this process in the mitral valve are not fully understood. In the present study, we cultured mitral VICs in collagen and poly(ethylene glycol) scaffolds designed to mimic the heart valve microenvironment and treated the cell-seeded scaffolds with cytokines. Using these 3D culture models, we found that the pro-inflammatory cytokines TNF-α and IL-1ß downregulate myofibroblast and fibrosis markers in mitral VICs via the canonical NF-κB signaling pathway.

Poly(ethylene glycol)-Based Coatings for Bioprosthetic Valve Tissues: Toward Restoration of Physiological Behavior.

Bioprosthetic valves (BPVs) have a limited lifespan in the body necessitating repeated surgeries to replace the failed implant. Early failure of these implants has been linked to various surface properties of the valve. Surface properties of BPVs are significantly different from physiological valves because of the fixation process used when processing the xenograft tissue. To improve the longevity of BPVs, efforts need to be taken to improve the surface properties and shield the implant from the bodily interactions that degrade it. Toward this goal, we evaluated the use of hydrogel coatings to attach to the BPV tissue and impart surface properties that are close to physiological. Hydrogels are well characterized for their biocompatibility and highly tunable surface characteristics. Using a previously published coating method, we deposited hydrogel coatings of poly(ethylene glycol)diacrylate (PEGDA) and poly(ethylene glycol)diacrylamide (PEGDAA) atop BPV samples. Coated samples were evaluated against the physiological tissue and uncoated glutaraldehyde-fixed tissue for deposition of hydrogel, surface adherence, mechanical properties, and fixation properties. Results showed both PEGDA- and PEGDAA-deposited coatings were nearly continuous across the valve leaflet surface. Further, the PEGDA- and PEGDAA-coated samples showed restoration of physiological levels of protein adhesion and mechanical stiffness. Interestingly, the coating process rather than the coating itself altered the material behavior yet did not alter the cross-linking from fixation. These results show that the PEG-based coatings for BPVs can successfully alter surface properties of BPVs and help promote physiological characteristics without interfering with the necessary fixation.

Interfacial Coating Method for Amine-Rich Surfaces using Poly(ethylene glycol) Diacrylate Applied to Bioprosthetic Valve Tissue Models.

Bioprosthetic heart valve implants are beset by calcification and failure due to the interactions between the body and the transplant. Hydrogels can be used as biological blank slates that may help to shield implants from these interactions; however, traditional light-based hydrogel polymerization is impeded by tissue opacity and topography. Therefore, new methods must be created to bind hydrogel to implant tissues. To address these complications, a two-step surface-coating method for bioprosthetic valves was developed. A previously developed bioprosthetic valve model (VM) was used to investigate and optimize the coating method. Generally, this coating is achieved by first reacting surface amine groups with an NHS-PEG-acrylate while also allowing glucose to absorb into the bulk. Then, glucose oxidase, poly(ethylene glycol) diacrylate (PEGDA), and iron ions are added to the system to initiate free-radical polymerization that bonds the PEGDA hydrogel to the acrylates sites on the surface. Results showed a thin (∼8 µm), continuous coating on VM samples that is capable of repelling protein adhesion (2% surface fouling versus 20% on uncoated samples) and does not significantly affect the surface mechanical properties. Based on this success, the coating method was translated to glutaraldehyde-fixed valve tissue samples. Results showed noncontinuous but evident coating on the surface, which was further improved by adjusting the coating solution. These results demonstrate the feasibility of the proposed two-step surface coating method for modifying the surface of bioprosthetic valve replacements.

Monitoring Oxygen Levels within Large, Tissue-Engineered Constructs Using Porphyin-Hydrogel Microparticles.

A major barrier to the creation of engineered organs is the limited diffusion of oxygen through biological tissues. Advances in biofabrication bring us increasingly closer to complex vascular networks capable of supplying oxygen to large cellularized scaffolds. However, technologies for monitoring oxygen levels in engineered tissues do not accommodate imaging depths of more than a few dozen micrometers. Here, we report the creation of fluorescent porphyrin-hydrogel microparticles that can be used at depths of 2 mm into artificial tissues. By combining an oxygen-responsive porphyrin dye with a reference dye, the microparticles generate a ratiometric signal that is photostable, unaffected by attenuation from biological material, and responsive to physiological change in oxygen concentration. These microparticles can measure long-distance oxygen gradients within 3D, cellularized constructs and accurately report cellular oxygen consumption rates. Furthermore, they are compatible with a number of hydrogel polymerization chemistries and cell types, including primary human cells. We believe this technology will significantly advance efforts to visualize oxygen gradients in cellularized constructs and inform efforts to tissue engineer solid organs.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Left-Ventricular Assist Device Impact on Aortic Valve Mechanics, Proteomics and Ultrastructure.

BACKGROUND: Aortic regurgitation is a prevalent, detrimental complication of left ventricular assist devices (LVADs). The altered hemodynamics of LVADs results in aortic valves (AVs) having distinct mechanical stimulation. Our hypothesis was that the altered AV hemodynamics modulates the valve cells and matrix, resulting in changes in valvular mechanical properties that then can lead to regurgitation. METHODS: AVs were collected from 16 LVAD and 6 non-LVAD patients at time of heart transplant. Standard demographic and preoperative data were collected and comparisons between the two groups were calculated using standard statistical methods. Samples were analyzed using biaxial mechanical tensile testing, mass spectrometry-based proteomics, and transmission electron microscopy to assess ultrastructure. RESULTS: The maximum circumferential leaflet strain in LVAD patients was less than in non-LVAD patients (0.35 ± 0.10MPa versus 0.52 ± 0.18 MPa, p = 0.03) with a trend of reduced radial strain (p = 0.06) and a tendency for the radial strain to decrease with increasing LVAD duration (p = 0.063). Numerous proteins associated with actin and myosin, immune signaling and oxidative stress, and transforming growth factor ß were increased in LVAD patients. Ultrastructural analysis showed a trend of increased fiber diameter in LVAD patients (46.2 ± 7.2 nm versus 45.1 ± 6.9 nm, p = 0.10), but no difference in fiber density. CONCLUSIONS: AVs in LVAD patients showed decreased compliance and increased expression of numerous proteins related to valve activation and injury compared to non-LVAD patients. Further knowledge of AV changes leading to regurgitation in LVAD patients and the pathways by which they occur may provide an opportunity for interventions to prevent and/or reverse this detrimental complication.

Extracellular matrix remodeling in wound healing of critical size defects in the mitral valve leaflet.

The details of valvular leaflet healing following valvuloplasty and leaflet perforation from endocarditis are poorly understood. In this study, the synthesis and turnover of valvular extracellular matrix due to healing of a critical sized wound was investigated. Twenty-nine sheep were randomized to either CTRL (n = 11) or HOLE (n = 18), in which a 2.8-4.8 mm diameter hole was punched in the posterior mitral leaflet. After 12 weeks, posterior leaflets were harvested and histologically stained to localize extracellular matrix components. Immunohistochemistry was also performed to assess matrix components and markers of matrix turnover. A semi-quantitative grading scale was used to quantify differences between HOLE and CTRL. After 12 weeks, the hole diameter was reduced by 71.3 ± 1.4 % (p < 0.001). Areas of remodeling surrounding the hole contained more activated cells, greater expression of proteoglycans, and markers of matrix turnover (prolyl 4-hydroxylase, metalloproteases, and lysyl oxidase, each p ≤ 0.025), along with fibrin accumulation. Two distinct remodeling regions were evident surrounding the hole, one directly bordering the hole rich in versican and hyaluronan and a second adjacent region with abundant collagen and elastic fiber turnover. The remodeling also caused reduced delineation between valve layers (p = 0.002), more diffuse staining of matrix components and markers of matrix turnover (p < 0.001), and disruption of the collagenous fibrosa. In conclusion, acute valve injury elicited distinct, heterogeneous alterations in valvular matrix composition and structure, resulting in partial wound closure. Because these changes could also affect leaflet mechanics and valve function, it will be important to determine their impact on healing wounds.

Therapeutic Transdifferentiation: A Novel Approach for Ischemic Syndromes.

The technological development of induced pluripotent stem cells (iPSCs) has overcome many of the limitations of adult and embryonic stem cells. We have found that activation of innate immunity signaling is necessary for this process, as it facilitates epigenetic plasticity in cells by a process called transflammation. More recently, we have discovered that transflammation also facilitates the transdifferentiation of cells directly from one somatic cell type to another. This insight may lead to a promising therapeutic pathway that avoids reverting cells all the way back to pluripotency before achieving a cell type of interest. While there is much therapeutic promise to transflammation and transdifferentiation, there is also evidence that transdifferentiation plays a role in some pathological conditions, including atherosclerosis. Ultimately, better understanding of transflammation will facilitate the development of regenerative therapies.