Layer Of Extracellular Matrix Research Articles

Microscopic anatomy of the integument in the ribbon worm Micrura bella (Stimpson, 1857) (Pilidiophora, Nemertea).

The integument of ribbon worms in the order Heteronemertea is distinct from the integuments in the other taxa of nemerteans due to the presence of a special subepidermal glandular layer, the cutis. Among heteronemerteans, the ultrastructure of the cutis has been studied only in the Lineus ruber species complex. In the current study, ultrastructural (transmission electron microscopy) and histochemical studies of the epidermis and the cutis of Micrura bella from the basal Lineage A of the family Lineidae were performed. The epidermis consisted of ciliated and serous gland cells and is separated from the cutis by a layer of the subepidermal extracellular matrix; the basal lamina was not detected. The cutis comprised musculature, two types of mucous and four types of granular gland cells, and pigment cells with four types of granules. In the cutis of juvenile worms, type II granular gland cells and type II mucous cells were not observed. The integument of the caudal cirrus consisted of ciliated and serous gland cells and two intraepidermal lateral nerve cords; the cutis was absent. The compositions of the integument glands of M. bella and the L. ruber species complex are similar, except for the presence of type IV granular gland cells with narrow rod-shaped and lamellated granules exhibiting an alternating dark and light transverse layers and type II mucous cells found only in M. bella.

Biot Theory-Based Finite Element Modeling of Continuous Ultrasound Propagation Through Microscale Articular Cartilage.

Low-intensity ultrasound has shown promise in promoting the healing and regeneration of articular cartilage degraded by osteoarthritis. In this study, a two-dimensional finite element method (FEM) model was developed for solving the Biot theory equations governing the propagation of continuous ultrasound through the cartilage. Specifically, we computed the ultrasound-induced dilatations and displacements in the microscale cartilage that is represented as consisting of four zones, namely, the chondrocyte cell and its nucleus, the pericellular matrix (PCM) that forms a layer around the chondrocyte, and the extracellular matrix (ECM). The chondrocyte-PCM complex, referred to as the chondron, is embedded in the ECM. We model multiple cartilage configurations where in the ECM layer contains chondrons along the depth, as well as laterally. The top surface of the ECM layer is subjected to specified amplitude and frequency of continuous ultrasound. The resulting wave propagation is modeled by numerically solving the two-dimensional Biot equations for seven frequencies in the 0.5 MHz-5 MHz range. It is seen that ultrasound is attenuated in the ECM and the attenuation increases monotonically with frequency. In contrast, manyfold augmentation of the ultrasound amplitude is observed inside the cytoplasm and the nucleus of the chondrocyte. Chondrocytes act as a major sink of ultrasound energy, thereby reducing the depthwise propagation of ultrasound fluctuations. Regions of high dilatations and displacements were found at the ECM-PCM interface, PCM-chondrocyte interface, as well as in the cytoplasm and nucleus of the chondrocyte. We observe that the ultrasound field around a chondron interacts with that around a neighboring chondron located at the same depth in the ECM layer. The qualitative and quantitative insights gained from our study may be relevant to designing ultrasound-based therapies for osteoarthritis.

Surrogate modeling of articular cartilage degradation to understand the synergistic role of MMP-1 and MMP-9: a case study.

A characteristic feature of arthritic diseases is cartilage extracellular matrix (ECM) degradation, often orchestrated by the overexpression of matrix metalloproteinases (MMPs) and other proteases. The interplay between fibril level degradation and the tissue-level aggregate response to biomechanical loading was explored in this work by a computational multiscale cartilaginous model. We considered the relative abundance of collagenases (MMP-1) and gelatinases (MMP-9) in surrogate models, where the diffusion (spatial distribution) of these enzymes and the subsequent, co-localized fibrillar damage were spatially randomized with Latin Hypercube Sampling. The computational model was constructed by incorporating the results from prior molecular dynamics simulations (tensile test) of microfibril degradation into a hyper-elastoplastic fibril-reinforced cartilage model. Including MMPs-mediated collagen fibril-level degradation in computational models may help understand the ECM pathomechanics at the tissue level. The mechanics of cartilage tissue and fibril show variations in mechanical integrity depending on the different combinations of MMPs-1 and 9 with a concentration ratio of 1:1, 3:1, and 1:3 in simulated indentation tests. The fibril yield (local failure) was initiated at 20.2 ± 3.0 (%) and at 23.0 ± 2.8 (%) of bulk strain for col 1:gel 3 and col 3: gel 1, respectively. The reduction in failure stress (global response) was 39.8% for col 1:gel 3, 37.5% for col 1:gel 1, and 36.7% for col 3:gel 1 compared with the failure stress of the degradation free tissue. These findings indicate that cartilage's global and local mechanisms of failure largely depend on the relative abundance of the two key enzymes-collagenase (MMP-1) and gelatinase (MMP-9) and the spatial characteristics of diffusion across the layers of the cartilage ECM.

The combination of a 3D-Printed porous Ti-6Al-4V alloy scaffold and stem cell sheet technology for the construction of biomimetic engineered bone at an ectopic site.

Cell sheet technology has been widely used in bone tissue engineering and regenerative medicine. However, controlling the shape and volume of large pieces of engineered bone tissue remains impossible without additional suitable scaffolds. Three-dimensional (3D) printed titanium (Ti) alloy scaffolds are mostly used as implant materials for repairing bone defects, but the unsatisfactory bioactivities of traditional Ti-based scaffolds severely limit their clinical applications. Herein, we hypothesize that the combination of bone marrow mesenchymal stem cell (BMSC) sheet technology and 3D porous Ti–6Al–4V (PT) alloy scaffolds could be used to fabricate biomimetic engineered bone. First, various concentrations of BMSCs were directly cocultured with PT scaffolds to obtain complexes of osteoblastic cell sheets and scaffolds. Then, as an experimental control, an osteoblastic BMSC sheet was prepared by continuous culturing under osteogenic conditions for 2 weeks without passaging and used to wrap the scaffolds. The BMSC sheet was composed of several layers of extracellular matrix (ECM) and a mass of BMSCs. The BMSCs exhibited excellent adherent, proliferative and osteogenic potential when cocultured with PT scaffolds, which may be attributed to the ability of the 3D microstructure of scaffolds to facilitate the biological behaviors of cells, as confirmed by the in vitro results. Moreover, the presence of BMSCs and ECM increased the angiogenic potential of PT scaffolds by the secretion of VEGF. Micro-CT and histological analysis confirmed the in vivo formation of biomimetic engineered bone when the complex of cocultured BMSCs and PT scaffolds and the scaffolds wrapped by prepared BMSC sheets were implanted subcutaneously into nude mice. Therefore, the combination of BMSC sheet technology and 3D-printed PT scaffolds could be used to construct customized biomimetic engineered bone, offering a novel and promising strategy for the precise repair of bone defects.

2D and 3D liver models

2D and 3D liver models

Dual‐Phase Aligned Composite Scaffolds Loaded with Tendon‐Derived Stem Cells for Achilles Tendon Repair

AbstractTendon injury is one of the most common musculoskeletal disorders, resulting in a significant decrease in the quality of life of patients with tendon injury. With the in‐depth understanding of the anatomical structure and pathological changes that occur during tendon repair, tissue engineering methods using cells and biomaterials are widely studied. However, simulation of the aligned tissue morphology present in natural tendons remains challenging. The design of a well‐aligned scaffold would be appealing to promote better tendon regeneration. Here, a dual‐aligned composite scaffold comprising an outer layer of polycaprolactone/silk fibroin (PCL/SF) copolymer electrospun membrane and an inner layer of tendon extracellular matrix (TECM) loaded with tendon‐derived stem cells (TDSCs) is developed to promote rabbit Achilles tendon repair. As resident, tissue‐specific cells, TDSCs possess stronger tenogenic differentiation abilities. The PCL/SF electrospun membrane with a dense structure provides mechanical support and TECM with a loose porous structure provides bioactive constituents and a microenvironment for tendon repair. As shown here, the engineered scaffold facilitates TDSC proliferation and migration, favors tenogenesis‐associated gene expression, promotes tendon repair with native‐like hierarchically organized collagen fibers, and enhances the mechanical properties, indicating its potential value in the tendon tissue engineering field.

Recreating cellular barriers in human microphysiological systems in-vitro.

Within cellular barriers, cells are separated by basement membranes (BMs), nanometer-thick extracellular matrix layers. In existing in-vitro cellular-barrier models, cell-to-cell signaling can be preserved by culturing different cells in individual chambers separated by a semipermeable membrane. Their structure does not always replicate the BM thickness nor diffusion through it. Here, a porous polymeric nanofilm made of poly(D-L-lactic acid) (PDLLA) is proposed to recreate the BM in a microfluidic blood-brain-barrier model. Nanofilms showed an average thickness of [Formula: see text] and a maximum pore diameter of 1.6 μm. Human umbilical vein endothelial cells (HUVECs) were cultured on PDLLA. After 7 days, viability was >95% and cell morphology did not show relevant differences with HUVECs grown on control substrates. A protocol for suspending the nanofilm between 2 microfluidic chambers was identified and showed no leakage and good sealing. Clinical Relevance- Preclinical models of cellular barriers are a key step towards a deeper understanding of their roles in pathogenesis of various diseases: a physiologically relevant microfluidic model of the blood brain barrier (BBB) allows high-throughput investigations of BBB contribution in neurodegenerative diseases and cruelty-free screenings of drugs targeting the brain.

Proximal deposition of collagen IV by fibroblasts contributes to basement membrane formation by colon epithelial cells in vitro.

The basement membrane (BM) underlying epithelial tissue is a thin layer of extracellular matrix that governs tissue integrity and function. Epithelial BMs are generally assembled using BM components secreted from two origins: epithelium and stroma. Although de novo BM formation involves self-assembly processes of large proteins, it remains unclear how stroma-derived macromolecules are transported and assembled, specifically in the BM region. In this study, we established an in vitro co-culture model of BM formation in which DLD-1 human colon epithelial cells were cultured on top of collagen I gel containing human embryonic OUMS-36T-2 fibroblasts as stromal cells. A distinct feature of our system is represented by OUMS-36T-2 cells which are almost exclusively responsible for synthesis of collagen IV, a major BM component. Exploiting this advantage, we found that collagen IV incorporation was significantly impaired in culture conditions where OUMS-36T-2 cells were not allowed to directly contact DLD-1 cells. Soluble collagen IV, once diluted in the culture medium, did not accumulate in the BM region efficiently. Live imaging of fluorescently tagged collagen IV revealed that OUMS-36T-2 cells deposited collagen IV aggregates directly onto the basal surface of DLD-1 cells. Collectively, these results indicate a novel mode of collagen IV deposition in which fibroblasts proximal to epithelial cells exclusively contribute to collagen IV assembly during BM formation.

Abstract 6030: Reconstructed Pancreas: A tumor-specific 3D culture platform for accurate evaluation of therapeutic agents in pancreatic cancer

Abstract As it is rarely diagnosed early, the prognosis for patients with pancreatic cancer is grim. The average 5-year survival rate is 10% and only 3% for patients with metastatic disease. Partially because of the late diagnosis and partially because of its biology, treatment options for patients with pancreatic cancer are limited and new interventions are desperately needed to gain the upper hand on this disease. One of the challenges faced by the drug developers looking for agents against pancreatic cancer is capturing the elements of the tumor microenvironment, which provides both a physical barrier and stroma-driven resistance to therapeutic agents. We developed a tumor-specific Reconstructed Pancreas (r-Pancreas) model, which recapitulates the cellular and extracellular elements of the pancreatic tumor microenvironment. In the r-Pancreas model, tumor and stromal cells are embedded in a dense extracellular matrix (ECM) characteristic of pancreatic tumors. Pancreatic tumor cell lines, MiaPaca-2, Panc-1, and Bx-PC3 robustly proliferate and survive for 14 days providing an extended therapeutic window to test anti-tumor compounds in the context of pancreas-specific ECM. Mimicking clinical response pancreatic tumor cells grown in r-Pancreas are sensitive to gemcitabine (IC50=0.4-0.6µM) but are resistant to 5-fluorouracil (IC50=not reached). These cell lines were also 5-10 times less sensitive to gemcitabine when cultured in Matrigel (IC50=3-6µM), soft ECM rich in collagen IV/laminin neither of which is abundant in the pancreas. Incorporating a collagen-rich capsule into the r-Pancreas model further expands its physiological relevance as human pancreatic tumors have been shown to have an outer layer of stiff ECM, which provides a physical barrier that impedes drug access to the tumor. Finally, establishing a co-culture of pancreatic tumor cell lines with primary pancreatic stromal cells creates a complete model enabling testing of novel therapeutic agents within the pancreatic tumor microenvironment. Taken together these data suggest that to be clinically useful in vitro tumor models not only need to allow for 3-dimensional cell growth but also have to incorporate tumor-specific elements of the microenvironment. Citation Format: Eleanore J. Kirshner, Aayushi Ahlawat, Matthew Ryou. Reconstructed Pancreas: A tumor-specific 3D culture platform for accurate evaluation of therapeutic agents in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6030.

Abstract 3456: Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems

Abstract Due to the lack of early symptoms and valid screening methods, most patients with the most prevalent histotype of ovarian cancer, high-grade serous ovarian cancer (HGSOC), are diagnosed at late stage, at which point metastasis has already occurred. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells, representing different stages of disease evolution, in both two-dimensional (2D) and three-dimensional (3D) organotypic assays. Five HGSOC cell lines, collected at different stages of disease progression were used throughout this study; PEO1, PEO4, and PEO6 cells were obtained from a first patient, whereas PEO14 and PEO23 cells were isolated from a second patient. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of each cell line, while invasion was assessed by adding a layer of extracellular matrix to the BC assays. Finally, 2D adhesion assays were performed on fibronectin coated plates. On the other hand, 3D organotypic models composed of a mixture of collagen I and fibroblasts, topped with a monolayer of mesothelial cells, were constructed to further study the metastatic potential of all five HGSOC cell lines; HGSOC were plated on top of the mesothelial cells, and their adhesive behavior and capacity to displace mesothelial cells was followed by fluorescence microscopy. In 2D studies, the migratory and invasive capacities of the HGSOC cells decreased along disease evolution in both cell line series. Moreover, a distinct migration pattern was observed; cell lines representing early-stage disease had a tendency to migrate as individual cells, while cells representing late stage disease migrated in clusters. Also in 2D conditions, adhesion rates were found to be similar between all cell lines. Noteworthy, these results obtained in 2D conditions were somewhat contradicted by the results obtained in the 3D organotypic models, as both late-state disease PEO6 and PEO23 cells adhered more rapidly to the mesothelial cell monolayer than their early-stage counterparts. Furthermore, cell lines previously found to be tumorigenic in vivo such as PEO4, PEO6, and PEO14, demonstrated to trigger higher rates of mesothelial cell displacement in the 3D organotypic model system. This mesothelial cell displacement was also observed after exposure of mesothelial cells to conditioned media derived from PEO4, PEO6, or PEO14 cultures. In conclusion, 2D in vitro assays demonstrated lower migration and invasion capacities at late- rather than early-stage disease, while the opposite was observed in the organotypic models. Mesothelial cell displacement was found to be associated with tumorigenicity in vivo and to be independent of physical interactions between cancer cells and mesothelial cells. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3456.

The role of Bcl11b in vascular smooth muscle cell death

Arterial stiffening is a strong independent risk factor for cardiovascular disease. B-cell leukaemia 11b (BCL11B) is a transcription factor known as an essential regulator in T lymphocytes differentiation and neuronal development. Several genome-wide association studies showed that single nucleotide polymorphisms (SNPs) in the 3’gene desert of Bcl11b are associated with a higher susceptibility for aortic stiffness and blood pressure control. However, its role in smooth muscle cells and mechanism of action remains to be uncovered. Our aims are: (1) to analyse the role of Bcl11b in vascular system; (2) to characterize the signalling pathways related to Bcl11b in cell death. Morphological, cellular and molecular analysis were carried out on the arteries of smooth muscle cell-specific Bcl11b-knockout mice at 3, 7, 28 days after tamoxifen injections as well as on the smooth muscle cells isolated from the aorta of Bcl11b-floxed mice that were cultured and inactivated by Cre recombinase using adenoviral vectors. We analysed mice with Bcl11b deletion in vascular SMC (Bcl11b-SMKO). There is no difference between control and mutant mice at the macroscopic level 3 days after Bcl11b KO induction, however, 7 days after Bcl11b inactivation, 70% of the Bcl11b-SMKO mice showed signs of haemorrhage in the distal part of the thoracic aorta near the abdominal aorta. The histological examination of thoracic aorta at day 7 indicated the presence of “bumpy region” in the mutant aorta, these areas seem to be covered by a thicker layer of extracellular matrix and the presence of IgG positive cells, indicating that cell death via necroptosis is occurring in this tissue. Our preliminary results from smooth muscle cell culture indicate higher cell death rate in the mutant cells compared to control cells. Our data suggest a primary effect of Bcl11b inactivation on cell death, probably through necroptosis. The mechanisms linked to cell death induced by Bcl11b loss is under investigation.

In vivo engraftment into the cornea endothelium using extracellular matrix shrink-wrapped cells

Cell injection is a common clinical approach for therapeutic delivery into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site have generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These μMonolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted μMonolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in extracellular matrix dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness.

Small, but smart: Fine structure of an avicularium in Dendrobeania fruticosa (Bryozoa: Cheilostomata).

Bryozoans are small benthic suspension-feeding colonial animals. Among this phylum, there are representatives showing a lesser or greater degree of polymorphism, and the most common type of polymorphic zooids is the avicularium. Here we present a detailed description of the bird's-head shaped avicularium in Dendrobeania fruticosa. The body cavity of the avicularium demonstrates an acoelomate condition: along the cystid walls, there is neither the layer of extracellular matrix toward the epidermis, nor coelomic lining. However, a layer of extracellular matrix and epithelialized cells lie under the epidermis of the tentacle sheath. Probably, such construction helps the tentacle sheath to acquire some rigidity-it is the only region of the body wall without an ectocyst. We did not find typical funicular strands in the avicularium, but there is a delicate mesh composed of stellate cells with thin and long projections, which sometimes isolate the spaces filled with a heterogeneous matrix. The proximal ends of the adductors, abductors, and polypide retractors are attached to the body wall via typical epidermal tendon cells, which possess numerous bundles of tonofilaments. The distal ends of the abductors and adductors attach to the frontal membrane or upper vestibular membrane, respectively. The inner organic layer of the ectocyst in these regions forms large protrusions, from which numerous thin outgrowths branch off. We suggest them to be a functional analogue of apodemes and apodemal filaments in arthropods. "Apodemal" tendon cells have long and thin projections that line the outgrowths of the ectocyst and surround the distal ends of the muscle cells. At these sites, "apodemal" tendon cells possess numerous tonofilaments. The vestigial polypide includes the tentacle sheath, rudimentary lophophore, cerebral ganglion, and polypide retractors. The sensory part of 5HT-positive cells of the frontal membrane is dendrite-shaped and embedded in the inner organic layer of the ectocyst.

Extracellular RNA as a potential activator of Neutrophil Extracellular Traps by Candida albicans biofilm

Neutrophils represent the first line of innate host defense. The ability to inhibit the development of infections is associated with the involvement of several fighting strategies. The still poorly understood mechanism is netosis, involving the release of Extracellular Neutrophil Traps (NETs). NETs are complexes of chromosomal DNA and granule content. Such a web-like structure inhibits the spread of invaders. Netosis plays a significant role in combating Candida albicans infections. It has been shown that several factors, composing C. albicans cell surface mediate NETs production. However, the development of difficult to eradicate fungal infection is associated with the formation of the biofilm structure, which partially protects the pathogen cells from contact with the host’s immune system. One of the reasons for the creation of a such protective environment is the production of the extracellular matrix (ECM). The major components of the C. albicans ECM layer are lipids, proteins, carbohydrates but also extracellular nucleic acids, among which we observed a significant RNA content. Considering that the ECM consisting of RNA molecules is one of the first lines of contact between biofilms and neutrophils, our current studies aimed to assess the potential role of extracellular RNA in the triggering of the netosis process by human neutrophils in vitro. We showed that RNA purified from C. albicans biofilm structure and the whole cells have the capability to induction of ROS-dependent netosis pathway. Additionally, cell migration analysis indicate that RNA molecules may also be an effective chemotactic agent. This work was supported by NCN (2019/33/B/NZ6/02284).

Polypide anatomy of hornerid bryozoans (Stenolaemata: Cyclostomatida).

Bryozoans are small colonial coelomates whose colonies are made of individual modules (zooids). Like most coelomate animals, bryozoans have a characteristic body wall composition, including an epidermis, an extracellular matrix (ECM) and a coelothelium, all pressed together. The order Cyclostomatida, however, presents the most striking deviation, in which the ECM and the corresponding coelothelium underlying major parts of the skeletal wall epidermis are detached to form an independent membranous sac. It forms a separate, much smaller compartment, suspended in the zooid body cavity and working as an important element of the cyclostome lophophore protrusion mechanism. The polypide anatomy and ultrastructure of this group is best known from studies of one family, the Crisiidae (Articulata). Here, we examined four species from the phylogenetically and ecologically contrasting family Horneridae (Cancellata) from New Zealand, and provide the first detailed ultrastructural description of the hornerid polypide, including tentacles, mouth region, digestive system and the funiculus. We were able to trace continuity and transitions of cell and ECM layers throughout the whole polypide. In addition, we identified that the funiculus is a lumen-free ECM cord with two associated muscles, disconnected from interzooidal pores. Except for funicular core composition, the polypide anatomy of hornerids agrees well with the general cyclostomate body plan.

Abstract P316: Role Of Scleraxis In Cardiac Valve Development And Disease

Valvular heart disease is one of the major causes of cardiac-related deaths in the US and effective treatment is currently limited to surgical repair or replacement. Mature heart valves are composed of highly organized and stratified layers of extracellular matrix (ECM) regulated by valve interstitial cells (VICs). This stratification that is initiated during embryogenesis and completed during valve maturation after birth, needs to be maintained throughout life for normal valve function. Myxomatous degeneration is a common disease histologically characterized by imbalance in ECM composition and organization resulting in valve biomechanical failure. Yet, key regulators of ECM homeostasis are not well characterized. Scleraxis (Scx) is a bHLH transcription factor that we previously showed to be critical for heart valve development and its loss of function leads to defective VIC maturation and aberrant ECM organization. However, due to lack of efficient tools, the mechanistic function of Scx in valve development and disease in vivo remains to be understood. Herein, we performed temporal analysis of Scx transcript levels during development of murine homeostatic valves and identified that Scx is predominantly expressed in the VICs between E13.5 and P14. We also assessed Scx levels and ECM abnormalities in valve tissues derived from patients with cardiac valve diseases and identified several distinct populations of VICs with high Scx levels that partially colocalized with elevated collagen fragmentation and proteoglycan (PG) deposition in the neighboring ECM. Scx levels were also elevated in valves of Fbn1 C1039G/+ and osteogenesis imperfecta murine ( OIM ) mice that develop myxomatous valve abnormalities signifying a potential role in pathogenesis. To further study the function of Scx in valve development and disease in vivo , we have generated a conditional Scx-transgenic model (Scx-TG) that will allow for overexpression in targeted cell lineages upon Cre recombination. Additionally, we will employ Scx-Cre mouse model expressing Scx-promoter driven Cre -recombinase to perform Scx-expressing cell lineage analyses and high-throughput sequencing analyses to identify direct gene targets and protein-interaction partners of Scx to better understand its mechanistic role in regulating valve ECM homeostasis. Together these in vivo approaches will provide novel insights into the function of Scx in heart development and disease.

Abstract 2897: The metastatic potential of high-grade serous ovarian cancer cells along disease progression

Abstract Epithelial ovarian cancer (EOC) represents 90% of all ovarian tumors and is considered to be the deadliest gynecological disease. High grade serous ovarian cancer (HGSOC) is the most aggressive form of EOC and represents 70% of all deaths from the disease. Due to a lack of early symptoms and valid screening methods, HGSOC is often diagnosed at late stages, when metastasis has already occurred. HGSOC disseminates from the primary tumor as single cells or clusters that typically adheres to the peritoneal organs and wall. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells representing different stages of disease evolution. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of five HGSOC cell lines; PEO1, PEO4 and PEO6, from a first patient, and PEO14 and PEO23, from a second patient. The cell lines were generated along different stages of the disease (e.g. at presentation, recurrence, and before death), thus representing disease evolution. To study the invasive capacity of each cell line, a layer of extracellular matrix was added to the BC assays. Adhesion assays were performed on fibronectin coated plates. It was found that the migratory and invasive capacities of the HGSOC cell lines decreased along disease evolution in both cellular series. PEO1 and PEO14, two chemo-sensitive cell lines, were found to have the highest migratory and invasive capacities, while their chemoresistant counterparts, PEO4, PEO6 and PEO23, showed a decrease in migration and invasion. Moreover, a distinct migration pattern was found to be conserved in both WH and BC assays. Cell lines representing an early stage disease had a tendency to migrate as individual cells, while cells representing a late stage disease were observed migrating in clusters. Adhesion rates were found to be similar between all cells lines. The results suggest that HGSOC disease progression seems to be associated with reduced migratory and invasive capacities, contradicting the idea that advanced stage EOC cancers have higher metastatic potential. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. The metastatic potential of high-grade serous ovarian cancer cells along disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2897.

Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps.

Neutrophils, the first line of the host’s defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation

Basement membrane (BM) is a thin layer of extracellular matrix that surrounds most animal tissues, serving as a physical barrier while allowing nutrient exchange. Although they have important roles in tissue structural integrity, physical properties of BMs remain largely uncharacterized, which limits our understanding of their mechanical functions. Here, we perform pressure-controlled inflation and deflation to directly measure the nonlinear mechanics of BMs in situ. We show that the BMs behave as a permeable, hyperelastic material whose mechanical properties and permeability can be measured in a model-independent manner. Furthermore, we find that BMs exhibit a remarkable nonlinear stiffening behavior, in contrast to the reconstituted Matrigel. This nonlinear stiffening behavior helps the BMs to avoid the snap-through instability (or structural softening) widely observed during the inflation of most elastomeric balloons and thus maintain sufficient confining stress to the enclosed tissues during their growth.

Sub-Regional Differences of the Human Amniotic Membrane and Their Potential Impact on Tissue Regeneration Application.

For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.