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Methods in Molecular Biology (Clifton,... 2024The proteoglycan versican plays multiple roles in cancer progression, from promoting cell invasion and proliferation to evasion of immune surveillance....
The proteoglycan versican plays multiple roles in cancer progression, from promoting cell invasion and proliferation to evasion of immune surveillance. Metalloproteinases of the A Disintegrin and Metalloproteinase with Thrombospondin-like motif (ADAMTS) family cleave versican at a specific Glu-Ala bond, thus releasing a bioactive fragment named versikine, whose biological function, still not entirely revealed, seems that of antagonizing the effects of the parental molecule. Here we describe an enzyme-linked immunosorbent assay (ELISA) that specifically detects versikine in media, pure component systems, and biological fluids using neoepitope antibodies. Such antibodies recognize their target proteolytic fragment but not the intact, parental molecule. Versikine fragments are captured by neoepitope antibodies and detected by antibodies directed against its N-terminal globular (G1) domain. The method here described can therefore be used to measure ADAMTS versicanase activity and provides a quantitative alternative to immunoblotting.
Topics: Humans; Versicans; Metalloendopeptidases; Peptide Hydrolases; Neoplasms; Enzyme-Linked Immunosorbent Assay
PubMed: 38038934
DOI: 10.1007/978-1-0716-3589-6_8 -
Diabetes, Metabolic Syndrome and... 2023Diabetic kidney disease (DKD) is one of the major microvascular complications of diabetes. DKD is associated with oxidative stress and inflammation. Versican (VCAN), a...
INTRODUCTION
Diabetic kidney disease (DKD) is one of the major microvascular complications of diabetes. DKD is associated with oxidative stress and inflammation. Versican (VCAN), a chondroitin sulphate proteoglycan, has been proven to participate in oxidative stress and inflammation. This study aimed to explore the overall and sex-based relationship between serum VCAN levels and albuminuria in patients with type 2 diabetes mellitus (T2DM).
METHODS
428 patients with T2DM and 84 healthy individuals were enrolled. Patients with diabetes were separated into normal albuminuria, microalbuminuria, and macroalbuminuria groups, according to their urinary albumin/creatinine ratio (UACR). Serum VCAN levels were tested using an enzyme-linked immunosorbent assay.
RESULTS
Compared with males, female patients were older, and had higher total cholesterol and high-density lipoprotein cholesterol, but lower body mass index, diastolic blood pressure, glycated hemoglobin A1, alanine aminotransferase, urinary albumin (UA), and serum creatinine (SCr) ( < 0.05). The VCAN levels in male patients with T2DM were significantly higher than those in the healthy individuals. Male patients with T2DM with albuminuria (micro and macro) had higher levels of VCAN than in patients with normal albuminuria; the highest level was seen in patients with macroalbuminuria ( < 0.05). In male patients with T2DM, serum VCAN correlated positively with systolic blood pressure, blood urea nitrogen, UA, SCr, and UACR, but correlated negatively with the estimated glomerular filtration rate. The area under the receiver operating characteristic curve of serum VCAN to diagnose albuminuria was 0.702, with a corresponding cut-off value of 0.399 ng/mL ( < 0.001). However, the association between serum VCAN and UACR was not observed in female patients with T2DM.
CONCLUSION
Serum VCAN levels correlated positively with the severity of albuminuria in male patients with T2DM.
PubMed: 38028986
DOI: 10.2147/DMSO.S434287 -
Frontiers in Molecular Neuroscience 2023Intracerebral hemorrhage (ICH) is the predominant type of hemorrhagic stroke with high mortality and disability. In other neurological conditions, the deposition of...
BACKGROUND
Intracerebral hemorrhage (ICH) is the predominant type of hemorrhagic stroke with high mortality and disability. In other neurological conditions, the deposition of extracellular matrix (ECM) molecules is a prominent obstacle for regenerative processes and an enhancer of neuroinflammation. Whether ECM molecules alter in composition after ICH, and which ECM members may inhibit repair, remain largely unknown in hemorrhagic stroke.
METHODS
The collagenase-induced ICH mouse model and an autopsied human ICH specimen were investigated for expression of ECM members by immunofluorescence microscopy. Confocal image z-stacks were analyzed with Imaris 3D to assess the association of immune cells and ECM molecules. Sections from a mouse model of multiple sclerosis were used as disease and staining controls. Tissue culture was employed to examine the roles of ECM members on oligodendrocyte precursor cells (OPCs).
RESULTS
Among the lectican chondroitin sulfate proteoglycan (CSPG) members, neurocan but not aggrecan, versican-V1 and versican-V2 was prominently expressed in perihematomal tissue and lesion core compared to the contralateral area in murine ICH. Fibrinogen, fibronectin and heparan sulfate proteoglycan (HSPG) were also elevated after murine ICH while thrombospondin and tenascin-C was not. Confocal microscopy with Imaris 3D rendering co-localized neurocan, fibrinogen, fibronectin and HSPG molecules to Iba1 microglia/macrophages or GFAP astrocytes. Marked differentiation from the multiple sclerosis model was observed, the latter with high versican-V1 and negligible neurocan. In culture, purified neurocan inhibited adhesion and process outgrowth of OPCs, which are early steps in myelination . The prominent expression of neurocan in murine ICH was corroborated in human ICH sections.
CONCLUSION
ICH caused distinct alterations in ECM molecules. Among CSPG members, neurocan was selectively upregulated in both murine and human ICH. In tissue culture, neurocan impeded the properties of oligodendrocyte lineage cells. Alterations to the ECM in ICH may adversely affect reparative outcomes after stroke.
PubMed: 38025264
DOI: 10.3389/fnmol.2023.1251432 -
Respiratory Research Nov 2023Lung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable...
BACKGROUND
Lung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable and even if the risk is proportionally small, many patients could be affected. However, there is still no data on lung extracellular matrix (ECM) composition in severe COVID-19 and whether it is different from other aetiologies of ARDS.
METHODS
We have quantified different ECM elements and TGF-β expression in lung tissue of 28 fatal COVID-19 cases and compared to 27 patients that died of other causes of ARDS, divided according to MV duration (up to six days or seven days or more). In COVID-19 cases, ECM elements were correlated with lung transcriptomics and cytokines profile.
RESULTS
We observed that COVID-19 cases presented significant increased deposition of collagen, fibronectin, versican, and TGF-β, and decreased decorin density when compared to non-COVID-19 cases of similar MV duration. TGF-β was precociously increased in COVID-19 patients with MV duration up to six days. Lung collagen was higher in women with COVID-19, with a transition of upregulated genes related to fibrillogenesis to collagen production and ECM disassembly along the MV course.
CONCLUSIONS
Fatal COVID-19 is associated with an early TGF-β expression lung environment after the MV onset, followed by a disordered ECM assembly. This uncontrolled process resulted in a prominent collagen deposition when compared to other causes of ARDS. Our data provides pathological substrates to better understand the high prevalence of pulmonary abnormalities in patients surviving COVID-19.
Topics: Humans; Female; Pulmonary Fibrosis; COVID-19; Extracellular Matrix; Collagen; Lung; Transforming Growth Factor beta; Respiratory Distress Syndrome
PubMed: 37964271
DOI: 10.1186/s12931-023-02555-7 -
BioRxiv : the Preprint Server For... Mar 2024Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur...
Integrated single-nuclei and spatial transcriptomic analysis reveals propagation of early acute vein harvest and distension injury signaling pathways following arterial implantation.
BACKGROUND
Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury.
METHODS
Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing (snRNA-seq) and spatial transcriptomics (ST) analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-cartoid vein bypass implantation in a canine model (n=4).
RESULTS
Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways ( < 0.05) involved in the activation of endothelial cells (ECs), fibroblasts (FBs), and vascular smooth muscle cells (VSMCs), namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and extracellular matrix (ECM) remodeling throughout the vein wall. Subsequent snRNA-seq analysis supported these findings and further unveiled distinct EC and FB subpopulations with significant upregulation ( < 0.00001) of markers related to endothelial injury response and cellular activation of ECs, FBs, and VSMCs. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury-response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including (versican), (fibrillin-1), and (vascular endothelial growth factor C), in addition to novel genes of interest such as (GLIS family zinc finger 3) and (ephrin-A3). These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as and . By integrating the ST and snRNA-seq datasets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and FBs were notably enriched in the intima and media of distended veins. Lastly, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal transitioning ECs, protomyofibroblasts, and VSMCs in upregulating signaling pathways associated with cellular proliferation (MDK, PDGF, VEGF), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension.
CONCLUSIONS
Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.
PubMed: 37961724
DOI: 10.1101/2023.10.31.564995 -
Experimental Dermatology Jan 2024Dermal papilla cells (DPCs) undergo premature ageing in androgenetic alopecia and senescent alopecia. As critical components of hair follicle reconstruction, DPCs are...
Dermal papilla cells (DPCs) undergo premature ageing in androgenetic alopecia and senescent alopecia. As critical components of hair follicle reconstruction, DPCs are also prone to senescence in vitro, resulting in a diminished hair follicle inductivity capacity. Dermal sheath cup cells (DSCCs), a specific subset of hair follicle mesenchymal stem cells, intimately linked to the function of DPCs. The primary objective of this research is to investigate the anti-ageing effect of exosomes derived from DSCCs (Exo ) on DPCs. Exosomes were utilized to treat H O -induced DPCs or long-generation DPCs(P10). Our findings demonstrate that Exo promote the proliferation, viability and migration of senescent DPCs while inhibiting cell apoptosis. The expression of senescence marker SA-β-Gal were significantly downregulated in senescent DPCs. When treated with Exo , expression of inducibility related markers alkaline phosphatase and Versican were significantly upregulated. Additionally, Exo activated the Wnt/β-catenin signalling in vitro. In patch assay, Exo significantly promoted hair follicle reconstruction in senescent DPCs. In summary, our work highlights that Exo may restore the biological functions and improve the hair follicle induction ability of senescent DPCs. Therefore, Exo may represent a new strategy for intervening in the ageing process of DPCs, contributing to the prevention of senile alopecia.
Topics: Humans; Hair Follicle; Dermis; Exosomes; Cells, Cultured; Alopecia; Aging; Regeneration; Cell Proliferation
PubMed: 37950506
DOI: 10.1111/exd.14948 -
Investigative Ophthalmology & Visual... Oct 2023Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is...
PURPOSE
Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we characterized the LSCN to identify key components of the HA-specific LSCN.
METHODS
The cornea and limbal rim were dissected from mouse corneas, subjected to mRNA extraction, and sequenced using a NextSeq 500 (Illumina) and data processed using CLC Genomics Workbench 20 (Qiagen) and the STRING database to identify key components of the LSCN. Their expression was confirmed by real-time PCR, Western blotting, and immunohistochemistry. Furthermore, the differential expression of key compounds in different corneal cell types were determined with single-cell RNA sequencing.
RESULTS
We identified that the hyaladherins inter-alpha-inhibitor (IαI), TSG-6 and versican are highly expressed in the limbus. Specifically, HA/HC complexes are present in the LSCN, in the stroma underlying the limbal epithelium, and surrounding the limbal vasculature. For IαI, heavy chains 5 and 2 (HC5 and HC2) were found to be the most highly expressed HCs in the mouse and human limbus and were associate with HA-forming HA/HC-specific matrices.
CONCLUSIONS
The LSCN contains HA/HC complexes, which have been previously correlated with stem cell niches. The identification of HA/HC complexes in the LSCN could serve as a new therapeutic avenue for treating corneal pathology. Additionally, HA/HC complexes could be used as a substrate for culturing LESCs before LESC transplantation.
Topics: Humans; Animals; Mice; Stem Cell Niche; Blotting, Western; Cornea; Databases, Factual; Epithelium; Hyaluronic Acid
PubMed: 37906057
DOI: 10.1167/iovs.64.13.48 -
Circulation Mar 2024The adult mammalian heart is incapable of regeneration, whereas a transient regenerative capacity is maintained in the neonatal heart, primarily through the...
BACKGROUND
The adult mammalian heart is incapable of regeneration, whereas a transient regenerative capacity is maintained in the neonatal heart, primarily through the proliferation of preexisting cardiomyocytes. Neonatal heart regeneration after myocardial injury is accompanied by an expansion of cardiac fibroblasts and compositional changes in the extracellular matrix. Whether and how these changes influence cardiomyocyte proliferation and heart regeneration remains to be investigated.
METHODS
We used apical resection and myocardial infarction surgical models in neonatal and adult mice to investigate extracellular matrix components involved in heart regeneration after injury. Single-cell RNA sequencing and liquid chromatography-mass spectrometry analyses were used for versican identification. Cardiac fibroblast-specific deletion was achieved using the mouse strains and . Molecular signaling pathways related to the effects of versican were assessed through Western blot, immunostaining, and quantitative reverse transcription polymerase chain reaction. Cardiac fibrosis and heart function were evaluated by Masson trichrome staining and echocardiography, respectively.
RESULTS
Versican, a cardiac fibroblast-derived extracellular matrix component, was upregulated after neonatal myocardial injury and promoted cardiomyocyte proliferation. Conditional knockout of in cardiac fibroblasts decreased cardiomyocyte proliferation and impaired neonatal heart regeneration. In adult mice, intramyocardial injection of versican after myocardial infarction enhanced cardiomyocyte proliferation, reduced fibrosis, and improved cardiac function. Furthermore, versican augmented the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Mechanistically, versican activated integrin β1 and downstream signaling molecules, including ERK1/2 and Akt, thereby promoting cardiomyocyte proliferation and cardiac repair.
CONCLUSIONS
Our study identifies versican as a cardiac fibroblast-derived pro-proliferative proteoglycan and clarifies the role of versican in promoting adult cardiac repair. These findings highlight its potential as a therapeutic factor for ischemic heart diseases.
Topics: Animals; Humans; Mice; Animals, Newborn; Cell Proliferation; Heart; Heart Injuries; Induced Pluripotent Stem Cells; Mammals; Myocardial Infarction; Myocytes, Cardiac; Regeneration; Versicans
PubMed: 37886839
DOI: 10.1161/CIRCULATIONAHA.123.066298 -
American Journal of Translational... 2023Versican (VCAN), a member of the multifunctional glycoprotein family, is involved in various aspects of cancer progression. However, the role of VCAN in diverse cancers...
OBJECTIVE
Versican (VCAN), a member of the multifunctional glycoprotein family, is involved in various aspects of cancer progression. However, the role of VCAN in diverse cancers remains poorly defined. This research aimed to investigate the correlation between VCAN expression and the oncogenic role, as well as visualize its prognostic landscape in pan-cancer.
METHODS
Raw data in regard to VCAN expression in cancer patients were acquired from GEO GeneChip public database in NCBI. Besides, we selected microarray data GSE16088 for analysis. We retrieved the genes associated with osteosarcoma (OS) from the OMIM database and identified their intersection with the core module. VCAN was suggested to be a potential marker gene for OS. Subsequently, we conducted Gene Set Enrichment Analysis (GSEA) to explore gene functional enrichment. Moreover, we performed pan-cancer analysis on VCAN to gain a comprehensive understanding of its implications across various cancer types.
RESULTS
The VCAN expression in the tumor tissue was higher than that in normal tissue. Elevated expression of VCAN was associated with high the tumor stage and poor long-term survival. There was a significant positive correlation between VCAN and cancer fibroblasts in all pan cancers. Moreover, FBN1 was the intersection gene of VCAN-related genes and linker genes. ANTXR1, COL5A2, CSGALNACT2, and SPARC were the target genes of VCAN genes. GSEA analysis showed that VCAN was mainly enriched in the extracellular matrix (ECM) signaling pathway.
CONCLUSION
VCAN can be used as a marker molecule for the early diagnosis of OS and holds significance as a molecule in cases of OS with distant metastasis. The ECM signaling pathway may be a core pathway in OS development and distant metastasis. These findings shed new light on therapeutics of cancers.
PubMed: 37854213
DOI: No ID Found