Layer Of Extracellular Matrix Research Articles

Basement Membranes, Brittlestar Tendons, and Their Mechanical Adaptability.

Basement membranes (BMs) are thin layers of extracellular matrix that separate epithelia, endothelia, muscle cells, and nerve cells from adjacent interstitial connective tissue. BMs are ubiquitous in almost all multicellular animals, and their composition is highly conserved across the Metazoa. There is increasing interest in the mechanical functioning of BMs, including the involvement of altered BM stiffness in development and pathology, particularly cancer metastasis, which can be facilitated by BM destabilization. Such BM weakening has been assumed to occur primarily through enzymatic degradation by matrix metalloproteinases. However, emerging evidence indicates that non-enzymatic mechanisms may also contribute. In brittlestars (Echinodermata, Ophiuroidea), the tendons linking the musculature to the endoskeleton consist of extensions of muscle cell BMs. During the process of brittlestar autotomy, in which arms are detached for the purpose of self-defense, muscles break away from the endoskeleton as a consequence of the rapid destabilization and rupture of their BM-derived tendons. This contribution provides a broad overview of current knowledge of the structural organization and biomechanics of non-echinoderm BMs, compares this with the equivalent information on brittlestar tendons, and discusses the possible relationship between the weakening phenomena exhibited by BMs and brittlestar tendons, and the potential translational value of the latter as a model system of BM destabilization.

Collagen fibers within skeletal muscle extracellular matrix dynamically change their alignment in a stretch-dependent fashion

Collagen fiber architecture within the skeletal muscle extracellular matrix (ECM) contributes to muscle passive mechanical properties. There is evidence that collagen fibers change their alignment during muscle lengthening (stretching). Thus, the goal of this study was to characterize the dynamic changes of collagen fiber alignment in the muscle ECM during muscle stretching, and to assess the relationship between dynamic collagen re-alignment and muscle passive mechanics. We hypothesized that collagen fibers increase their alignment as the muscle is stretched, and that increased collagen alignment in stretched muscle is correlated to higher elastic stiffness. Extensor digitorum longus (EDL), soleus, and diaphragm muscles were collected from wildtype (WT) and D2.mdx (MDX) mice of 48-52 weeks of age. Muscles underwent a series of twitches to determine the optimum length (Lo) for isometric contraction, and then were decellularized to isolate the muscle ECM. Decellularized muscles (DCMs) were then mechanically tested using a series of stress-relaxation tests at 5% increments from 85% to 115% length relative to Lo. Elastic stiffness was measured as the slope of the tangent line on the stress-strain curve following stress-relaxation. Following mechanical testing, DCMs were imaged using second harmonic generation microscopy to visualize collagen alignment across the range of lengths used in mechanical testing. Collagen alignment was reported as an alignment index on a scale of 0 (perfect unalignment) to 1 (perfect alignment). We found that MDX diaphragm DCMs were less stiff than WT (WT: 910.2±586.7, MDX: 238±168.7, p=0.0041), but there were no significant differences in stiffness between WT and MDX EDL and soleus DCMs. We also observed a significant effect of stretch on collagen alignment in all DCMs tested (Diaphragm: p=0.0071, EDL: p=0.0004, soleus: p<0.0001). Interestingly, the collagen fibers of the epimysium, the outermost layer of the muscle ECM, were more aligned in WT diaphragm DCMs than in MDX (WT 85%: 0.672±.14, MDX 85%: 0.344±0.16, p=0.0027; WT 115%: 0.783±0.04, MDX 115%: 0.480±0.19, p=0.0036). Finally, we found a significant positive correlation between the alignment and elastic stiffness at 110% Lo across all DCMs (p=0.0038, R2=0.1713). These data indicate that collagen fibers dynamically re-align as muscle is stretched. Additionally, increased collagen fiber alignment is associated with higher passive stiffness in the decellularized muscle ECM. Overall, we find that dynamic architectural changes in the muscle ECM contribute to passive mechanical behavior during a muscle stretch. This work was supported by a grant from the NIH NIAMS (R01-AR079545). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of the extracellular matrix in the reduction of lateral force transmission in muscle bundles: A finite element analysis

Background and objectiveAging is associated with a reduction in muscle performance, but muscle weakness is characterized by a much greater loss of force loss compared to mass loss. The aim of this work is to assess the contribution of the extracellular matrix (ECM) to the lateral transmission of force in humans and the loss of transmitted force due to age-related modifications. MethodsFinite element models of muscle bundles are developed for young and elderly human subjects, by considering a few fibers connected through an ECM layer. Bundles of young and elderly subjects are assumed to differ in terms of ECM thickness, as observed experimentally. A three-element-based Hill model is adopted to describe the active behavior of muscle fibers, while the ECM is modeled assuming an isotropic hyperelastic neo-Hookean constitutive formulation. Numerical analyses are carried out by mimicking, at the scale of a bundle, two experimental protocols from the literature. ResultsWhen comparing numerical results obtained for bundles of young and elderly subjects, a greater reduction in the total transmitted force is observed in the latter. The loss of transmitted force is 22 % for the elderly subjects, while it is limited to 7.5 % for the young subjects. The result for the elderly subjects is in line with literature studies on animal models, showing a reduction in the range of 20–34 %. This can be explained by an alteration in the mechanism of lateral force transmission due to the lower shear stiffness of the ECM in elderly subjects, related to its higher thickness. ConclusionsComputational modeling allows to evaluate at the bundle level how the age-related increase of the ECM amount between fibers affects the lateral transmission of force. The results suggest that the observed increase in ECM thickness in aging alone can explain the reduction of the total transmitted force, due to the impaired lateral transmission of force of each fiber.

Colony phase variation switch modulates antimicrobial tolerance and biofilm formation in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.IMPORTANCEAs a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Alignment, cross linking, and beyond: a collagen architect's guide to the skeletal muscle extracellular matrix.

The muscle extracellular matrix (ECM) forms a complex network of collagens, proteoglycans, and other proteins that produce a favorable environment for muscle regeneration, protect the sarcolemma from contraction-induced damage, and provide a pathway for the lateral transmission of contractile force. In each of these functions, the structure and organization of the muscle ECM play an important role. Many aspects of collagen architecture, including collagen alignment, cross linking, and packing density affect the regenerative capacity, passive mechanical properties, and contractile force transmission pathways of skeletal muscle. The balance between fortifying the muscle ECM and maintaining ECM turnover and compliance is highly dependent on the integrated organization, or architecture, of the muscle matrix, especially related to collagen. While muscle ECM remodeling patterns in response to exercise and disease are similar, in that collagen synthesis can increase in both cases, one outcome leads to a stronger muscle and the other leads to fibrosis. In this review, we provide a comprehensive analysis of the architectural features of each layer of muscle ECM: epimysium, perimysium, and endomysium. Further, we detail the importance of muscle ECM architecture to biomechanical function in the context of exercise or fibrosis, including disease, injury, and aging. We describe how collagen architecture is linked to active and passive muscle biomechanics and which architectural features are acutely dynamic and adapt over time. Future studies should investigate the significance of collagen architecture in muscle stiffness, ECM turnover, and lateral force transmission in the context of health and fibrosis.

Structural changes in perineuronal nets and their perforating GABAergic synapses precede motor coordination recovery post stroke

BackgroundStroke remains one of the leading causes of long-term disability worldwide, and the development of effective restorative therapies is hindered by an incomplete understanding of intrinsic brain recovery mechanisms. Growing evidence indicates that the brain extracellular matrix (ECM) has major implications for neuroplasticity. Here we explored how perineuronal nets (PNNs), the facet-like ECM layers surrounding fast-spiking interneurons, contribute to neurological recovery after focal cerebral ischemia in mice with and without induced stroke tolerance.MethodsWe investigated the structural remodeling of PNNs after stroke using 3D superresolution stimulated emission depletion (STED) and structured illumination (SR-SIM) microscopy. Superresolution imaging allowed for the precise reconstruction of PNN morphology using graphs, which are mathematical constructs designed for topological analysis. Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (tMCAO). PNN-associated synapses and contacts with microglia/macrophages were quantified using high-resolution confocal microscopy.ResultsPNNs undergo transient structural changes after stroke allowing for the dynamic reorganization of GABAergic input to motor cortical L5 interneurons. The coherent remodeling of PNNs and their perforating inhibitory synapses precedes the recovery of motor coordination after stroke and depends on the severity of the ischemic injury. Morphological alterations in PNNs correlate with the increased surface of contact between activated microglia/macrophages and PNN-coated neurons.ConclusionsOur data indicate a novel mechanism of post stroke neuroplasticity involving the tripartite interaction between PNNs, synapses, and microglia/macrophages. We propose that prolonging PNN loosening during the post-acute period can extend the opening neuroplasticity window into the chronic stroke phase.Graphical

Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice.

Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1's role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar extracellular matrix (ECM) of murine AoV leaflets. To determine whether disruption of Prox1 localization affects heart valve development, we generated mice (NFATc1enCre Prox1 gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and NFATc1enCre Prox1 gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in NFATc1enCre Prox1 gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome (Fbn1C1039G/+). The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including Flt1, Efnb2, Egfl7, and Cx37. Ectopic Prox1 colocalized with induced Flt1, Efnb2, and Cx37 expression in NFATc1enCre Prox1 gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs. Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.

Exploring the Therapeutic Potential of Elastase Inhibition in Age-Related Macular Degeneration in Mouse and Human.

Abnormal turnover of the extracellular matrix (ECM) protein elastin has been linked to AMD pathology. Elastin is a critical component of Bruch's membrane (BrM), an ECM layer that separates the retinal pigment epithelium (RPE) from the underlying choriocapillaris. Reduced integrity of BrM's elastin layer corresponds to areas of choroidal neovascularization (CNV) in wet AMD. Serum levels of elastin-derived peptides and anti-elastin antibodies are significantly elevated in AMD patients along with the prevalence of polymorphisms of genes regulating elastin turnover. Despite these results indicating significant associations between abnormal elastin turnover and AMD, very little is known about its exact role in AMD pathogenesis. Here we report on results that suggest that elastase enzymes could play a direct role in the pathogenesis of AMD. We found significantly increased elastase activity in the retinas and RPE cells of AMD mouse models, and AMD patient-iPSC-derived RPE cells. A1AT, a protease inhibitor that inactivates elastase, reduced CNV lesion sizes in mouse models. A1AT completely inhibited elastase-induced VEGFA expression and secretion, and restored RPE monolayer integrity in ARPE-19 monolayers. A1AT also mitigated RPE thickening, an early AMD phenotype, in HTRA1 overexpressing mice, HTRA1 being a serine protease with elastase activity. Finally, in an exploratory study, examining archival records from large patient data sets, we identified an association between A1AT use, age and AMD risk. Our results suggest that repurposing A1AT may have therapeutic potential in modifying the progression to AMD.

Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13

Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model.

Abstract 4561: 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer

Abstract As it is rarely discovered early, the prognosis for people with pancreatic cancer is poor, with an average 5-year survival rate of 10% and only 3% for those with metastatic illness. Pancreatic cancer patients have few therapeutic options and innovative therapies are sorely needed to improve treatment of the disease. This is mainly due to the late diagnosis and partially due to the biology of the disease. Capturing the components of the tumor microenvironment, which serve as both a physical barrier and a source of stromal-driven resistance to therapeutics, is one of the issues faced by drug developers searching for agents to combat pancreatic cancer. We have created a 3D model of the pancreatic tumor microenvironment, the Reconstructed Pancreas (r-Pancreas), using Mia PaCa-2, PANC-1, and BxPC-3 pancreatic tumor cell lines embedded in an extracellular matrix (ECM) that was formulated to recapitulate the typical ECM of pancreatic tumors. We have shown that gemcitabine was effective against pancreatic tumor cells cultured in r-Pancreas (IC50=0.4-0.8µM), while 5-fluorouracil was ineffective (IC50 not reached), mimicking clinical response. In nude mice, subcutaneous PANC-1 tumors exhibited similar responses to gemcitabine and 5-fluorocurcil. Treatment with gemcitabine resulted in a Day 63 median ΔT/ΔC of 37% while 5-fluorouracil resulted in a Day 63 median delta ΔT/ΔC of 102%. Incorporating a collagen-rich capsule increases the physiological relevance of the r-Pancreas model because it has been shown that human pancreatic tumors have an outer layer of stiff ECM that functions as a physical barrier and prevents drugs from penetrating the tumor. Recently, we showed that, at least in the Mia PaCa-2 and BxPC-3 tumor cell lines, introducing such a collagen capsule dramatically reduced sensitivity to gemcitabine. Disrupting the collagen capsule with MMP-9 restored the sensitivity of the tumor cells to gemcitabine demonstrating that the capsule provides a physical barrier to drug entry. Additionally, creating a co-culture of primary activated pancreatic stromal cells and pancreatic tumor cell lines produces a more complete 3D model that permits the testing of potential therapeutic agents inside the pancreatic tumor microenvironment. Hence, we have created a 3D model where pancreatic tumor cells were co-cultured with human pancreatic fibroblasts. According to our results, the IC50 values for pancreatic tumor cell lines treated with gemcitabine increased by twofold when co-cultured with activated pancreatic fibroblasts compared to control sets of tumor cells alone (IC50=1-3µM). Together, these data imply that, to be clinically relevant, in vitro models of pancreatic cancer must integrate tumor-specific components of the microenvironment, including the collagen capsule and activated fibroblasts. Citation Format: Arnat Balabyev, Justin D. Phillips, Michael Steffey, Eleanore J. Kirshner, Aayushi Ahlawat, Arlette H. Uihlein, Daniel Saims, Scott Wise, Julia Kirshner. 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4561.

Abstract 5272: 3D cell-culture strategy for screening novel agents in Fanconi anemia chemoprevention

Abstract Fanconi anemia (FA) patients have DNA repair mutations predisposing them to oral cancers so treatment with non-DNA damaging agents is highly desired. As cancer experimental therapeutics advance towards study paradigms that more carefully mimic physiology, e.g. organoids and spheroids. Presently, we are using a 3D cell culture method emphasizing layers of extracellular matrix (ECM) as FA premalignant lesions retain epithelial architecture. We conducted 3D screening studies using 2 layered geometries: 1) nanofibers coated with 1 ng/mL 2:1 Collagen III/IV (C3C4) then 100 pg/mL 2.75:1 Tenascin C/Fibronectin (TNFN) and 2) polystyrene layered with 50 ug/mL C3C4 then 3.5 ug/mL TNFN. We developed these coatings to maximize lipid raft recovery, a drug transport feature lost in 2D culture. The ECM impact on FA cell drug transport was characterized by comparing effective nuclear delivery of a 20nm nanoencapsulated FITC-labeled RNAi compound (s50-TBG-RNAi3UTR), known to require lipid rafts, to the same oligo formulated with Dotap in a FA HNSCC cell line (FA1). Using confocal microscopy and IMARS software to segment delivery at maximal nuclear intensity into nuclear or endolysosomal compartments, we found, for FA1 cells on ECM, nuclear oligo signal colocalization with nuclear compartment ± coefficient of variation was 62 ± 2.2% for the capsule vs 9.8 ± 8.5% for Dotap oligo. On glass, nuclear delivery was 10 ± 52% for s50 capsule vs 25 ± 32% for Dotap. Endolysosomal compartment sequestration was respectively 28 ± 12, 62 ± 10, 44 ± 19 and 51 ± 15%. This illustrates a relevant decrease in variation in FA cell biology from ECM addition. Next, we studied metformin (Met) and pioglitazone (Pio)(agents in current oral cancer prevention studies) combined with G2/M blockade inhibitors, MK1775 (Wee1 kinase inhibitor) and GSK461364 (PLK inhibitor) in FA1s. Inhibiting G2/M blockade, already induced by FA-derived DNA damage, could promote inappropriate cell division, mitotic catastrophe (MOC) and death. In initial 2D FA1 growth studies, increased death was observed with Met plus MK1775. 3D studies, in contrast, showed increased cell death for FA1s treated with Pio plus either MK1775 or s50-TBG-RNAiCK2 (RNAiCK2). Confocal mechanistic studies in cells plated on ECM and treated for 18 hours, indicated Pio + MK1775 inhibited Survivin and ß-catenin upregulation and FA1 cells did proceed into MOC and death. For Pio (PPARg agonist) + RNAiCK2, RXRa was dephosphorylated at S260, enabling its escape from cytosolic sequestration to bind nuclear PPARg, initiating differentiation. Differentiation was indicated by upregulation panKeratin and Transglutaminase-3. We conclude combination therapies with high interest agents in FA-associated oral cancer can be performed in 3D culture systems and might confirm drug mechanisms of action, thus augmenting other standard methods of cancer drug evaluation and screening (e.g. cell proliferation and clonogenicity). Citation Format: Gretchen M. Unger, Beverly R. Wuertz, Mary E. Brown, Sanjana Arji, Janeen H. Trembley, Frank G. Ondrey. 3D cell-culture strategy for screening novel agents in Fanconi anemia chemoprevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5272.

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting.

Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5-/- aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5-/- PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5-/- PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5-/- mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

Microfluidic aspiration-assisted electrical impedance spectroscopy system is a reliable tool for the characterization of oocyte hardening

The fertilization rate in assisted reproduction depends significantly on the quality of the oocyte, especially on the hardening of the zona pellucida, which is a gelatinous layer of extracellular matrix surrounding the oocyte. Current reproduction techniques use elaborated and frequent manual manipulation steps, which are expensive, non-standardized, and observer dependent. However, a technically simple to perform, reliable and robust, indisputable and observer independent method is needed to characterize the biophysical properties of the oocyte, especially the hardening state of the zona pellucida. Therefore, a microfluidic-based cell trap technique is proposed, which offers a simple oocyte handling through hydrodynamic trapping. The zona pellucida aspiration at an aperture was measured observer-independently by electrical impedance spectroscopy for the first time. Impedance spectra were fitted by an appropriate equivalent electrical circuit model. A numerical in combination with an analytical model correlated the impedance changes with the aspiration length changes, from which Young’s modulus of the zona pellucida was calculated. As a proof-of-concept, the setup was applied to three different genetic mouse oocyte models owing a super soft, a normal and a stiff zona pellucida structure, respectively. The presented measurement technique can either be used for human and animal reproductive medicine to assist artificial fertilization, for the development of contraceptives using the zona pellucida as a barrier or, in general, for all kinds of characterization of rheological properties of single cells.

The metastatic capacity of high-grade serous ovarian cancer cells changes along disease progression: inhibition by mifepristone.

Simplistic two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them. High-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells, which were originally harvested from two patients at different stages of disease progression, in the absence or presence of cytostatic doses of mifepristone. 2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behavior is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behavior among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over simplistic or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation of these multicellular structures. Differences in cellular behaviours were observed between 2 and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied.

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche.

The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration

BackgroundOlder adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol.ResultsYoung and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of − 5.4% and − 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration.ConclusionsWater restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

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.