Matrix Biology Plus最新文献

Background

Non-muscle invasive bladder cancer (NMIBC) patients are affected by a high risk of recurrence. The topography of collagen fibers represents a hallmark of the neoplastic extracellular microenvironment.

Objective

Assess the topographic change associated with different stages of bladder cancer (from neoplastic lesions to bona fide tumor) and whether those changes favour the development of NMIBC.

Design, Setting, and Participants

Seventy-one clinical samples of urothelial carcinoma at different stages were used. Topographic changes preceding tumor onset and progression were evaluated in the rat bladder cancer model induced by nitrosamine (BBN), a bladder-specific carcinogen. The preclinical model of actinic cystitis was also used in combination with BBN. Validated hematoxylin-eosin sections were used to assess the topography of collagen fibrils associated with pre-tumoral steps, NMIBC, and MIBC.

Findings

Linearization of collagen fibers was higher in Cis and Ta vs. dysplastic urothelium, further increased in T1 and greatest in T2 tumors. In the BBN preclinical model, an increase in the linearization of collagen fibers was established since the beginning of inflammation, such as the onset of atypia of a non-univocal nature and dysplasia, and further increased in the presence of the tumor. Linearization of collagen fibers in the model of actinic cystitis was associated with earlier onset of BBN-induced tumor.

Conclusions

The topographic modification of the extracellular microenvironment occurs during the inflammatory processes preceding and favoring the onset of bladder cancer. The topographic reconfiguration of the stroma could represent a marker for identifying and treating the non-neoplastic tissue susceptible to tumor recurrence.

Fish oils rank among the world’s most popular nutritional supplements and are purported to have numerous health benefits. Previous work suggested that fish oils increase collagen production; however, the effect of fish oils on musculoskeletal health is poorly understood. Further, the divergent effects of omega-3 (Ω3FA) and saturated fatty acids (SFA) remains poorly understood. We tested the effects of Ω3FA and SFAs on in vitro-engineered human ligament (EHL) function. EHLs were treated with bovine serum albumin (BSA)-conjugated eicosapentaenoic acid (EPA, 20:5(n-3)), palmitic acid (PA, 16:0), or a BSA control for 6 days. EPA did not significantly alter, whereas PA significantly decreased EHL function and collagen content. To determine whether this was an in vitro artifact, mice were fed a control or high-lard diet for 14 weeks and musculoskeletal mass, insulin sensitivity, and the collagen content, and mechanics of tendon and bone were determined. Body weight was 40 % higher on a HFD, but muscle, tendon, and bone mass did not keep up with body weight resulting in relative losses in muscle mass, tendon, and bone collagen, as well as mechanical properties. Importantly, we show that PA acutely decreases collagen synthesis in vitro to a similar extent as the decrease in collagen content with chronic treatment. These data suggest that Ω3FAs have a limited effect on EHLs, whereas SFA exert a negative effect on collagen synthesis resulting in smaller and weaker musculoskeletal tissues both in vitro and in vivo.

Extracellular matrix (ECM) fabricated using human induced pluripotent stem cells (hiPSCs)-derived cardiac fibroblasts (hiPSC-CFs) could serve as a completely biological scaffold for an engineered cardiac patch, leveraging the unlimited source and outstanding reproducibility of hiPSC-CFs. Additionally, hiPSC-CF-derived ECM (hiPSC-CF-ECM) holds the potential to enhance maturation of exogenous cardiomyocytes, such as hiPSC-derived cardiomyocytes (hiPSC-CMs), by providing a microenvironment rich in cardiac-specific biochemical and signaling cues. However, achieving sufficient robustness of hiPSC-CF-ECM is challenging. This study aims to achieve appropriate ECM deposition, scaffold thickness, and mechanical strength of an aligned hiPSC-CF-ECM by optimizing the culture period, ranging from 2 to 10 weeks, of hiPSC-CFs grown on micro-grated substrates, which can direct the alignment of both hiPSC-CFs and their secreted ECM. The hiPSC-CFs demonstrated a production rate of 13.5 µg ECM per day per 20,000 cells seeded. An anisotropic nanofibrous hiPSC-CF-ECM scaffold with a thickness of 20.0 ± 2.1 µm was achieved after 6 weeks of culture, followed by decellularization. Compositional analysis through liquid chromatography-mass spectrometry (LC-MS) revealed the presence of cardiac-specific fibrillar collagens, non-fibrillar collagens, and matricellular proteins. Uniaxial tensile stretching of the hiPSC-CF-ECM scaffold indicated robust tensile resilience. Finally, hiPSCs-CMs cultured on the hiPSC-CF-ECM exhibited alignment following the guidance of ECM nanofibers and demonstrated mature organization of key structural proteins. The culture duration of the anisotropic hiPSC-CF-ECM was successfully refined to achieve a robust scaffold containing structural proteins that resembles cardiac microenvironment. This completely biological, anisotropic, and cardiac-specific ECM holds great potential for cardiac patch engineering.

Cardiac fibrosis is characterized by excessive accumulation and deposition of ECM proteins. Cardiac fibrosis is commonly implicated in a variety of cardiovascular diseases, including post-myocardial infarction (MI). We have previously developed a dual-delivery nanogel therapeutic to deliver tissue plasminogen activator (tPA) and Y-27632 (a ROCK inhibitor) to address MI-associated coronary artery occlusion and downregulate cell-contractility mediated fibrotic responses. Initial in vitro studies were conducted on glass substrates. The study presented here employs the use of polyacrylamide (PA) gels and microgel thin films to mimic healthy and fibrotic cardiac tissue mechanics. Soft and stiff polyacrylamide substrates or high and low loss tangent microgel thin films were utilized to examine the influence of cell-substrate interactions on dual-loaded nanogel therapeutic efficacy. In the presence of Y-27632 containing nanogels, a reduction of fibrotic marker expression was noted on traditional PA gels mimicking healthy and fibrotic cardiac tissue mechanics. These findings differed on more physiologically relevant microgel thin films, where early treatment with the ROCK inhibitor intensified the fibrotic related responses.

The pancreatic islet is surrounded by ECM that provides both biochemical and mechanical cues to the islet β-cell to regulate cell survival and insulin secretion. Changes in ECM composition and mechanical properties drive β-cell dysfunction in many pancreatic diseases. While several studies have characterized changes in islet insulin secretion with changes in substrate stiffness, little is known about the mechanotransduction signaling driving altered islet function in response to mechanical cues. We hypothesized that increasing matrix stiffness will lead to insulin secretion dysfunction by opening the mechanosensitive ion channel Piezo1 and disrupting intracellular Ca²⁺ dynamics in mouse and human islets. To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D. Our results indicate that increased scaffold stiffness causes insulin secretion dysfunction mediated by increases in Ca²⁺ influx and altered Ca²⁺ dynamics via opening of the mechanosensitive Piezo1 channel. Additionally, inhibition of Piezo1 rescued glucose-stimulated insulin secretion (GSIS) in islets in stiff scaffolds. Overall, our results emphasize the role mechanical properties of the islet microenvironment plays in regulating function. It also supports further investigation into the modulation of Piezo1 channel activity to restore islet function in diseases like type 2 diabetes (T2D) and pancreatic cancer where fibrosis of the peri-islet ECM leads to increased tissue stiffness and islet dysfunction.

Although the mechanism for activation of latent TGFβ1 and TGFβ3 is understood to involve the binding of the TGFβ propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFβ2 has been unclear because the TGFβ2 LAP does not have the classical integrin binding sequence found in the other two TGFβ isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFβ2, we generated mice in which the TGFβ2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2^C24S). We reasoned that, if covalent interaction with a second molecule is required for latent TGFβ2 activation, mutant mice should display a Tgfb2 null (Tgfb2^−/−)-like phenotype. Tgfb2^C24S mice closely phenocopy Tgfb2^−/− mice with death in utero between E18 and P1 and with congenital heart and kidney defects similar to those described for Tgfb2^−/− mice. The mutant latent TGFβ2 is secreted at levels similar to WT, yet TGFβ signaling monitored as nuclear pSmad2 is suppressed. We conclude that, like latent TGFβ1, latent TGFβ2 activation requires binding to an immobilized matrix or plasma membrane molecule.

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.

Collagen is a key component of the extracellular matrix (ECM). In the remodeling of ECM, a remarkable variation in collagen post-translational modifications (PTMs) occurs. This makes collagen a potential target for understanding extracellular matrix remodeling during pathological conditions. Over the years, scientists have gathered a huge amount of data about collagen PTM during extracellular matrix remodeling. To make such information easily accessible in a consolidated space, we have developed ColPTMScape (https://colptmscape.iitmandi.ac.in/), a dedicated knowledge base for collagen PTMs. The identified site-specific PTMs, quantitated PTM sites, and PTM maps of collagen chains are deliverables to the scientific community, especially to matrix biologists. Through this knowledge base, users can easily gain information related to the difference in the collagen PTMs across different tissues in different organisms.

Macrophages are highly plastic immune cells known to exist on a spectrum of phenotypes including pro-inflammatory (M1) or pro-healing (M2). Macrophages interact with extracellular matrix (ECM) ligands, such as fragments of collagen and laminin. Interaction of macrophages with ECM ligands is mediated through integrin receptors. However, the role of ECM ligands in directing macrophage function through integrins is not yet fully understood. Particularly, α2β1 has been implicated in modulating macrophage function, but complexity in mechanisms employed for integrin-ligation especially with laminin-derived peptides makes it challenging to understand macrophage-ECM interactions. We hypothesize that targeting α2β1 through laminin-derived peptide, IKVAV, will modulate macrophage phenotype. In this work we: i) investigated macrophage response to IKVAV in 2D and in a 3D platform, and ii) identified α2β1′s role as it pertains to macrophage modulation via IKVAV. Soluble IKVAV treatment significantly reduced M1 markers and increased M2 markers via immunocytochemistry and gene expression. While the 3D ECM-mimicking PEG-IKVAV hydrogels did not have significant effects in modulating macrophage phenotype, we found that macrophage modulation via IKVAV is dependent on the concentration of peptide used and duration of exposure. To investigate integrin-ligand interactions for macrophages, α2β1 signaling was modulated by antagonists and agonists. We observed that blocking α2β1 reduces M1 activation. To understand integrin-ligand interactions and leveraging the therapeutic ability of macrophages in designing immunomodulatory solutions, it is critical to elucidate IKVAV’s role in mediating macrophage phenotype.