Structural Extracellular Matrix Proteins Research Articles

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor β1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFβ1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.

Involvement of kinesins in skeletal dysplasia - a review.

Skeletal dysplasias are group of rare genetic diseases resulting from mutations in genes encoding structural proteins of the cartilage extracellular matrix (ECM), signaling molecules, transcription factors, epigenetic modifiers, and several intracellular proteins. Cell division, organelle maintenance, and intracellular transport are all orchestrated by the cytoskeleton associated proteins, and intracellular processes effected through microtubule-associated movement are important for the function of skeletal cells. Amongst microtubule associated motor proteins, kinesins in particular have been shown to play a key role in cell cycle dynamics, including chromosome segregation, mitotic spindle formation and ciliogenesis, in addition to cargo trafficking, receptor recycling and endocytosis. Recent studies highlight the fundamental role of kinesins in embryonic development and morphogenesis and have shown that mutations in kinesin genes lead to several skeletal dysplasias. However, many questions concerning the specific functions of kinesins and their adaptor molecules as well as specific molecular mechanisms in which the kinesin proteins are involved during skeletal development remain unanswered. Here we present a review of the skeletal dysplasias resulting from defects in kinesins and discuss the involvement of kinesin proteins in the molecular mechanisms that are active during skeletal development.

Proteomic analysis of the extracellular matrix of human atherosclerotic plaques shows marked changes between plaque types

Cardiovascular disease is the leading cause of death, with atherosclerosis the major underlying cause. While often asymptomatic for decades, atherosclerotic plaque destabilization and rupture can arise suddenly and cause acute arterial occlusion or peripheral embolization resulting in myocardial infarction, stroke and lower limb ischaemia. As extracellular matrix (ECM) remodelling is associated with plaque instability, we hypothesized that the ECM composition would differ between plaques. We analyzed atherosclerotic plaques obtained from 21 patients who underwent carotid surgery following recent symptomatic carotid artery stenosis. Plaques were solubilized using a new efficient, single-step approach. Solubilized proteins were digested to peptides, and analyzed by liquid chromatography-mass spectrometry using data-independent acquisition. Identification and quantification of 4498 plaque proteins was achieved, including 354 ECM proteins, with unprecedented coverage and high reproducibility. Multidimensional scaling analysis and hierarchical clustering indicate two distinct clusters, which correlate with macroscopic plaque morphology (soft/unstable versus hard/stable), ultrasound classification (echolucent versus echogenic) and the presence of hemorrhage/ulceration. We identified 714 proteins with differential abundances between these groups. Soft/unstable plaques were enriched in proteins involved in inflammation, ECM remodelling, and protein degradation (e.g. matrix metalloproteinases, cathepsins). In contrast, hard/stable plaques contained higher levels of ECM structural proteins (e.g. collagens, versican, nidogens, biglycan, lumican, proteoglycan 4, mineralization proteins). These data indicate that a single-step proteomics method can provide unique mechanistic insights into ECM remodelling and inflammatory mechanisms within plaques that correlate with clinical parameters, and help rationalize plaque destabilization. These data also provide an approach towards identifying biomarkers for individualized risk profiling of atherosclerosis.

Transcriptional Profiling of Muscle in Females With Distal Radius Fracture and Functional Sarcopenia.

Skeletal muscle and bone interact with each other in mechanical and biochemical ways. This study aimed to investigate the molecular mechanisms of interaction between muscle and bone by analyzing the transcriptional profiles of total RNA from the muscle tissue of females with distal radius fractures. A total of 30 female participants (mean age 71.1 ± 8.9 years) with distal radius fractures were recruited. Participants were categorized into 2 groups: the NORM group consisted of participants with T score of the areal bone mineral density (aBMD) of the femoral neck higher than -1.0, handgrip strength greater than 18kg, and gait speed faster than 1.0 m/s (n = 10). Otherwise, participants with T score of the aBMD of the femoral neck equal to or less than -1.0, handgrip strength lower than 18kg, and gait speed slower than 1.0 m/s (n = 20) were categorized into EXP group. Pronator quadratus muscle samples were obtained from all participants. Total RNA was extracted from frozen muscle samples and sequenced. The gene ontology analysis demonstrated that the potential interactions between attached muscle function and the density of the associated bone would be linked with collagen biosynthetic activity and maintenance of extracellular matrix structures. The analysis of the pathway, network, and protein class exhibited that integrin signaling, inflammatory reactions, matrix metalloproteinase (MMP) activity, and extracellular matrix protein structure had possible associations with the molecular background of muscle-bone interaction. Through integrin signaling, MMP activity, inflammatory reactions, and collagen biosynthesis, muscle and bone may mutually interact with one another.

Assessing trans-endothelial transport of nanoparticles for delivery to abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are localized, rupture-prone expansions of the abdominal aorta wall. In this condition, structural extracellular matrix (ECM) proteins of the aorta wall, elastic fibers and collagen fibers, that impart elasticity and stiffness respectively, are slowly degraded by overexpressed matrix metalloproteinases (MMPs) following an injury stimulus. We are seeking to deliver therapeutics to the AAA wall using polymer nanoparticles (NPs) that are capable of stimulating on-site matrix regeneration and repair. This study aimed to determine how NP shape and size impacts endocytosis and transmigration past the endothelial cell (EC) layer from circulation into the medial layer of the AAA wall. First, rod-shaped NPs were shown to be created based mechanical stretching of PLGA NPs while embedded in a PVA film with longer rod-shaped NPs created based of the degree in which the PVA films are stretched. Live/dead assay reveals that our PLGA NPs are safe and do not cause cell death. Immunofluorescence staining reveal cytokine activation causes endothelial dysfunction in ECs by increasing expression of inflammatory marker Integrin αVβ3 and decreasing expression of adhesion protein vascular endothelial (VE)-cadherin. We showed this disruption enable greater EC uptake and translocation of NPs. Fluorescence studies demonstrate high endothelial transmigration and endocytosis with rod-shaped NPs in cytokine activated ECs compared to healthy control cells, arguing for the benefits of using higher aspect ratio (AR) NPs for accumulation at the aneurysm site. We also demonstrated that the mechanisms of NP transmigration across an activated EC layer depend on NP AR. These results show the potential of using shape as a modality for enhancing permeation of NPs into the aneurysm wall. These studies are also significance to understanding the mechanisms that are likely engaged by NPs for penetrating the endothelial lining of aneurysmal wall segments.

Characterisation of Matrix-Bound Nanovesicles (MBVs) Isolated from Decellularised Bovine Pericardium: New Frontiers in Regenerative Medicine.

Matrix-bound nanovesicles (MBVs) are a recently discovered type of extracellular vesicles (EVs), and they are characterised by a strong adhesion to extracellular matrix structural proteins (ECM) and ECM-derived biomaterials. MBVs contain a highly bioactive and tissue-specific cargo that recapitulates the biological activity of the source ECM. The rich content of MBVs has shown to be capable of potent cell signalling and of modulating the immune system, thus the raising interest for their application in regenerative medicine. Given the tissue-specificity and the youthfulness of research on MBVs, until now they have only been isolated from a few ECM sources. Therefore, the objective of this research was to isolate and identify the presence of MBVs in decellularised bovine pericardium ECM and to characterise their protein content, which is expected to play a major role in their biological potential. The results showed that nanovesicles, corresponding to the definition of recently described MBVs, could be isolated from decellularised bovine pericardium ECM. Moreover, these MBVs were composed of numerous proteins and cytokines, thus preserving a highly potential biological effect. Overall, this research shows that bovine pericardium MBVs show a rich and tissue-specific biological potential.

Human extracellular matrix (ECM)-like collagen and its bioactivity.

Collagen, the most abundant structural protein in the human extracellular matrix (ECM), provides essential support for tissues and guides tissue development. Despite its widespread use in tissue engineering, there remains uncertainty regarding the optimal selection of collagen sources. Animal-derived sources pose challenges such as immunogenicity, while the recombinant system is hindered by diminished bioactivity. In this study, we hypothesized that human ECM-like collagen (hCol) could offer an alternative for tissue engineering. In this study, a facile platform was provided for generating hCol derived from mesenchymal stem cells with a hierarchical structure and biochemical properties resembling native collagen. Our results further demonstrated that hCol could facilitate basal biological behaviors of human adipose-derived stem cells, including viability, proliferation, migration and adipocyte-like phenotype. Additionally, it could promote cutaneous wound closure. Due to its high similarity to native collagen and good bioactivity, hCol holds promise as a prospective candidate for in vitro and in vivo applications in tissue engineering.

A Noninvasive Circulating Signature of Combined Right Ventricular Pressure and Volume Overload in Tetralogy of Fallot/Pulmonary Atresia/Major Aortopulmonary Collateral Arteries.

Background: Despite surgical advances, children with tetralogy of Fallot/pulmonary atresia/major aortopulmonary collaterals (TOF/PA/MAPCAs) are subject to chronic right ventricular (RV) pressure and volume overload. Current diagnostic tools do not identify adverse myocardial remodeling and cannot predict progression to RV failure. We sought to identify a noninvasive, circulating signature of the systemic response to right heart stress to follow disease progression. Methods: Longitudinal data were collected from patients with TOF/PA/MAPCAs (N = 5) at the time of (1) early RV pressure overload and (2) late RV pressure and volume overload. Plasma protein and microRNA expression were evaluated using high-throughput data-independent mass spectroscopy and Agilent miR Microarray, respectively. Results: At the time of early RV pressure overload, median patient age was 0.34 years (0.02-9.37), with systemic RV pressures, moderate-severe hypertrophy, and preserved systolic function. Late RV pressure and volume overload occurred at a median age of 4.08 years (1.51-10.83), with moderate RV hypertrophy and dilation, and low normal RV function; 277 proteins were significantly dysregulated (log2FC ≥0.6/≤-0.6, FDR≤0.05), predicting downregulation in lipid transport (apolipoproteins), fibrinolytic system, and extracellular matrix structural proteins (talin 1, profilin 1); and upregulation in the respiratory burst. Increasing RV size and decreasing RV function correlated with decreasing structural protein expression. Similarly, miR expression predicted downregulation of extracellular matrix-receptor interactions and upregulation in collagen synthesis. Conclusion: To our knowledge, we show for the first time a noninvasive protein and miR signature reflecting the systemic response to adverse RV myocardial remodeling in TOF/PA/MAPCAs which could be used to follow disease progression.

Identification of PCPE-2 as the endogenous specific inhibitor of human BMP-1/tolloid-like proteinases

BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-β superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.

Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.

Assessment of connective tissue metabolism in patients with head and neck malignancies treated with radiotherapy.

<br><b>Introduction:</b> Despite the use of highly specialized irradiation techniques in the treatment of head and neck tumors, it is still impossible to selectively destroy cancer cells without damaging normal structures, including connective tissue cells.</br> <br><b>Aim:</b> The aim of the study was to analyze the concentration of degradation markers such as collagen type I (carboxyterminal telopeptide of type I collagen; ICTP) and elastin (elastin-derived peptides; EDPs) as well as selected metalloproteinases (MMP-1, MMP-2, MMP-9) in patients with head and neck malignancies undergoing radiotherapy.</br> <br><b>Material and methods:</b> The test group consisted of 56 men, who underwent radical or palliative radiotherapy. The concentrations of ICTP, EDPs, MMP-1, MMP-2, MMP-9 were determined in three blood samples collected from patients prior to radiotherapy, immediately after its completion and 3 months after the therapy.</br> <br><b>Results</b>: Both radical and palliative radiotherapy contribute to a significant increase in the concentration of EDPs. At the time of healing of post-irradiation lesions, the level of EDPs was reduced in both groups. The ICTP concentration was not affected by radiotherapy. No significant differences were observed in the concentration of MMP-1 and MMP-2 before and after radiotherapy. Radical radiotherapy caused a statistically significant late reduction in the concentration of MMP-9. The lowest concentrations of MMP-1, MMP-2, MMP-9 in the serum of patients qualified for palliative radiotherapy were recorded in a samples collected three months post-irradiation.</br> <br><b>Conclusions:</b> The degradation markers of key extracellular matrix structural proteins may be helpful tools in the objective assessment of radiation-induced injuries to the connective tissue.</br&gt.

Extracellular Matrix of Echinoderms.

This review considers available data on the composition of the extracellular matrix (ECM) in echinoderms. The connective tissue in these animals has a rather complex organization. It includes a wide range of structural ECM proteins, as well as various proteases and their inhibitors. Members of almost all major groups of collagens, various glycoproteins, and proteoglycans have been found in echinoderms. There are enzymes for the synthesis of structural proteins and their modification by polysaccharides. However, the ECM of echinoderms substantially differs from that of vertebrates by the lack of elastin, fibronectins, tenascins, and some other glycoproteins and proteoglycans. Echinoderms have a wide variety of proteinases, with serine, cysteine, aspartic, and metal peptidases identified among them. Their active centers have a typical structure and can break down various ECM molecules. Echinoderms are also distinguished by a wide range of proteinase inhibitors. The complex ECM structure and the variety of intermolecular interactions evidently explain the complexity of the mechanisms responsible for variations in the mechanical properties of connective tissue in echinoderms. These mechanisms probably depend not only on the number of cross-links between the molecules, but also on the composition of ECM and the properties of its proteins.

Site-specific modification of N-terminal α-amino groups of succinylated collagen

Polymer based protein engineering provides an attractive strategy to endow novel properties to protein and overcome the inherent limitations of both counterparts. The exquisite control of site and density of attached polymers on the proteins is crucial for the bioactivities and properties of the protein-polymer bioconjugates, but is still a challenge. Collagen is the major structural protein in extracellular matrix of animals. Based on the advancements of polymer-based protein engineering, collagen bioconjugates has been widely fabricated and applied as biomaterials. However, the site-specific synthesis of well-defined collagen-polymer bioconjugates is still not achieved. Herein, a versatile strategy for the specific modification of N-terminal α-amino groups in collagen was developed. Firstly, all reactive amino groups of tropocollagen (collagen with telopeptides) were protected by succinic anhydride. Then, the telopeptides were digested to give the active N-terminal α-amino groups, which were subsequently attached with poly(N-isopropylacrylamide) (PNIPAAm) via “grafting from” method based on the atom transfer radical polymerization (ATRP). The site-specific N-terminal PNIPAAm modified succinylated collagen was prepared and its structure, thermal responsive behaviour, and properties was explored.

The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging.

Tissue stem cells (SCs) divide infrequently as a protective mechanism against internal and external stresses associated with aging. Here, we demonstrate that slow- and fast-cycling SCs in the mouse skin epidermis undergo distinct aging processes. Two years of lineage tracing reveals that Dlx1+ slow-cycling clones expand into the fast-cycling SC territory, while the number of Slc1a3+ fast-cycling clones gradually declines. Transcriptome analysis further indicate that the molecular properties of each SC population are altered with age. Mice lacking fibulin 7, an extracellular matrix (ECM) protein, show early impairments resembling epidermal SC aging, such as the loss of fast-cycling clones, delayed wound healing, and increased expression of inflammation- and differentiation-related genes. Fibulin 7 interacts with structural ECM and matricellular proteins, and the overexpression of fibulin 7 in primary keratinocytes results in slower proliferation and suppresses differentiation. These results suggest that fibulin 7 plays a crucial role in maintaining tissue resilience and epidermal SC heterogeneity during skin aging.

Real‐Time Measurement of Cell Mechanics as a Clinically Relevant Readout of an In Vitro Lung Fibrosis Model Established on a Bioinspired Basement Membrane (Adv. Mater. 41/2022)

Biomimetic Membranes In article number 2205083, Ali Doryab, Otmar Schmid, and co-workers describe how a biomimetic basement (BETA) membrane with a thickness of only 1 μm and excellent biocompatibility demonstrates the formation of the nanoscale fibrous network structure of extracellular matrix proteins (green) deposited by the cells (violet). These unique features of the BETA membrane including Young's modulus <10 kPa allow monitoring of cell mechanics in real-time.

Systematic Analysis of Actively Transcribed Core Matrisome Genes Across Tissues and Cell Phenotypes

The extracellular matrix (ECM) is a highly dynamic, well-organized acellular network of tissue-specific biomolecules, that can be divided into structural or core ECM proteins and ECM-associated proteins. The ECM serves as a blueprint for organ development and function and, when structurally altered through mutation, altered expression, or degradation, can lead to debilitating syndromes that often affect one tissue more than another. Cross-referencing the FANTOM5 SSTAR (Semantic catalog of Samples, Transcription initiation And Regulators) and the defined catalog of core matrisome ECM (glyco)proteins, we conducted a comprehensive analysis of 511 different human samples to annotate the context-specific transcription of the individual components of the defined matrisome. Relative log expression normalized SSTAR cap analysis gene expression peak data files were downloaded from the FANTOM5 online database and filtered to exclude all cell lines and diseased tissues. Promoter-level expression values were categorized further into eight core tissue systems and three major ECM categories: proteoglycans, glycoproteins, and collagens. Hierarchical clustering and correlation analyses were conducted to identify complex relationships in promoter-driven gene expression activity. Integration of the core matrisome and curated FANTOM5 SSTAR data creates a unique tool that provides insight into the promoter-level expression of ECM-encoding genes in a tissue- and cell-specific manner. Unbiased clustering of cap analysis gene expression peak data reveals unique ECM signatures within defined tissue systems. Correlation analysis among tissue systems exposes both positive and negative correlation of ECM promoters with varying levels of significance. This tool can be used to provide new insight into the relationships between ECM components and tissues and can inform future research on the ECM in human disease and development. We invite the matrix biology community to continue to explore and discuss this dataset as part of a larger and continuing conversation about the human ECM. An interactive web tool can be found at matrixpromoterome.github.io along with additional resources that can be found at dx.doi.org/10.6084/m9.figshare.19794481 (figures) and https://figshare.com/s/e18ecbc3ae5aaf919b78 (python notebook).

Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells

PurposeSecreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors.MethodsA lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively.ResultsSuppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography.ConclusionsSuppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.

ONOO- -mediated oxidative modification of extracellular matrix aggravates atherosclerosis by affecting biological behaviors of vascular smooth muscle cells.

Atherosclerosis (AS) is a principal contributor to stroke and coronary heart disease in humans characterized by chronic low-grade inflammation. The extracellular matrix (ECM) plays critical roles in regulating the function of arteries. However, the effect of changes in ECM on AS development is rarely studied. In this context, we intend to study the effect of oxidizing agent peroxynitrite (ONOO- )-mediated oxidization of ECM proteins on the biological behaviors of vascular smooth muscle cells(SMCs) and the development of AS. AS mouse models were established, and mouse coronary artery smooth muscle cells (MCASMCs) were cultured in vitro to derive ECM (SMC-ECM), which was obtained by deoxycholate (DOC)-based decellularization. Further, MCASMCs were subjected to the determination of ECM oxidative damage and ECM protein structure. Finally, roles of ONOO- -mediated oxidization of ECM in SMC adhesion and migration and in AS development were explored through Transwell assay, transcriptome sequencing, and gene enrichment analysis. High concentration of ONOO- was found in the serum of AS mice, and ONOO- could stimulate the development of AS. SMC-ECM with intact structure can be obtained in vitro by DOC treatment. Functionally, ONOO- -mediated oxidization destroyed the three-dimensional structure of SMC-ECM proteins, affected SMC adhesion and migration and promoted the absorption efficiency of lipids while reducing the efflux of cholesterol. In addition, the expression of inflammation- and oxidative stress-related genes was significantly increased in ECM subjected to ONOO- -mediated oxidization, thereby contributing to AS progression. ONOO- -mediated oxidative modification of ECM aggravates AS by affecting the biological behavior of SMCs.

Investigating the Effects of IRF6 on Focal Adhesions in Keratinocytes

Focal adhesions are one of the two main types of adhesions between cells and the extra‐cellular matrix (ECM). Focal adhesions are dynamic complexes anchored to the actin cytoskeleton through intracellular adaptor proteins, and to the ECM through heterodimers of α and β integrins, forming a transmembrane link between the intra‐ and extra‐cellular space. β‐1 integrin is the primary β subunit found in keratinocytes, and its corresponding heterodimers show affinity for a wide range of ECM proteins including collagen, fibronectin, and laminin. Collagen is the main structural protein in cutaneous ECM (the dermis), while fibronectin is a glycoprotein found in the ECM that binds integrins and plays a crucial role in wound healing. Interferon regulatory factor 6 (IRF6) is a transcription factor expressed by epithelial cells that regulates keratinocyte differentiation, cell‐cell adhesions, and migration. It is well known that focal adhesions are required for cell migration, however the role of IRF6 in regulating focal adhesions is unknown. Therefore, we hypothesize that IRF6 regulates focal adhesion formation. To determine if IRF6 levels affect attachment to the ECM, we seeded wildtype and IRF6 knockdown keratinocytes on collagen IV and plastic and quantified the number of cells attached to the plate after thirty and sixty minutes. No differences were found between wild‐type and IRF6 knockdown keratinocytes on plastic. However, less IRF6 knockdown keratinocytes were attached on collagen IV compared to wildtypes. To explore how IRF6 affects focal adhesions, we plated IRF6 knockdown and wild‐type keratinocytes on different types of ECM that were representative of both normal and wounded conditions of the dermis. Immunofluorescent staining for β‐1 integrin and phalloidin was performed, followed by quantitative analysis of focal adhesion length and intensity. Initial observations indicate that IRF6 knockdown keratinocytes consistently have longer focal adhesions compared to the wildtype. Variations of β‐1 fluorescent intensity was also observed in wild‐type cells compared to IRF6 knockdown keratinocytes. These preliminary results suggest a role for IRF6 in regulating focal adhesions, providing a potential mechanism for the underlying defect observed in the migration of IRF6 deficient keratinocytes.