Biochemical and immunomodulatory insights of extracellular matrix from decellularized human whole cervix: recellularization and in vivo ECM remodelling interplay.

Extracellular matrix (ECM) rich whole organ bio-scaffolds, preserving structural integrity and essential growth factors, has potential towards regeneration and reconstruction. Women with cervical anomalies or trauma can benefit from clinical cervicovaginal repair using constructs rich in site specific ECM. In this study, complete human cervix decellularization was achieved using a modified perfusion-based stir bench top decellularization method. This was followed by physico-chemical processes including perfusion of ionic agents, enzymatic treatment and washing using detergent solutions for a duration of 10-12 days. Histopathological analysis, as well as DNA quantification confirmed the efficacy of the decellularization process. Tissue ultrastructure integrity was preserved and the same was validated via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies. Biochemical analysis and structural characterizations like FTIR, Raman spectroscopy of decellularised tissues demonstrated preservation of important proteins, crucial growth factors, collagen, and glycosaminoglycans (GAGs ). In vitro studies, using THP-1and HUVEC cells, demonstrated macrophage polarisation from M1 to M2 and vascular functional genes enhancement, respectively, when treated with decellularized cervical matrix. human decellularised cervical matrix (hdCM) () grafts were recellularized with site specific human cervical epithelial cells and HUVEC, showing non-cytotoxic cell viability and enhanced proliferation. Futhermore, hdCM scaffolds showed immunomodulatory effects in vivo on small rodent model via upregulation of M2 macrophage genes as compared to decellularised rat cervical matrix scaffolds (DRC). hdCM scaffolds underwent neo-vascularization followed by ECM remodelling with enhanced tissue integration.

Endothelial progenitor cells control remodeling of uterine spiral arteries for the establishment of utero-placental circulation.

Placental ischemia, resulting from inadequate remodeling of uterine spiral arteries, is a factor in the development of preeclampsia. However, the effect of endothelial progenitor cells that play a role in the vascular injury-repair program is largely unexplored during remodeling. Here, we observe that preeclampsia-afflicted uterine spiral arteries transition to a synthetic phenotype in vascular smooth muscle cells and characterize the regulatory axis in endothelial progenitor cells during remodeling in human decidua basalis. Excessive sEng, secreted by AMP-activated protein kinase (AMPK)-deficient endothelial progenitor cells through the inhibition of HO-1, damages residual endothelium and leads to the accumulation of extracellular matrix produced by vascular smooth muscle cells during remodeling, which is further confirmed by animal models. Collectively, our findings suggest that the impaired functionality of endothelial progenitor cells contributes to the narrowing of remodeled uterine spiral arteries, leading to reduced utero-placental perfusion. This mechanism holds promise in elucidating the pathogenesis of preeclampsia.

Inhibition of neuraminidase-1 sialidase activity by interfering peptides impairs insulin receptor activity in vitro and glucose homeostasis in vivo.

Neuraminidases also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Mammalian Neuraminidase-1 (NEU-1) participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low density lipoprotein receptor and toll like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, non-alcoholic steatohepatitis and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during eight weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway.

BACKGROUND: Osteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear. METHODS: In this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1ß) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1ß-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays. RESULTS: Downregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: This study demonstrated that CHMP5 repressed IL-1ß-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

Unraveling the genetic collagen connection: clinical and therapeutic insights on genetic connective tissue disorders.

Hereditary connective tissue disorders include more than 200 conditions affecting different organs and tissues, compromising the biological role of the extracellular matrix through interference in the synthesis, development, or secretion of collagen and/or its associated proteins. The clinical phenotype includes multiple signs and symptoms, usually nonspecific but of interest to rheumatologists because of musculoskeletal involvement. The patient´s journey to diagnosis is long, and physicians should include these disorders in their differential diagnoses of diseases with systemic involvement. In this review, insights for the diagnosis and treatment of osteogenesis imperfecta, hypermobility spectrum disorder/Ehlers-Danlos syndrome, Marfan, Loeys-Dietz, and Stickler syndromes are presented.

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration.

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism.SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.

Integrated bioinformatics analysis reveals upregulated extracellular matrix hub genes in pancreatic cancer: Implications for diagnosis, prognosis, immune infiltration, and therapeutic strategies.

BACKGROUND: Pancreatic cancer (PC) stands out as one of the most formidable malignancies and exhibits an exceptionally unfavorable clinical prognosis due to the absence of well-defined diagnostic indicators and its tendency to develop resistance to therapeutic interventions. The primary objective of this present study was to identify extracellular matrix (ECM)-related hub genes (HGs) and their corresponding molecular signatures, with the intent of potentially utilizing them as biomarkers for diagnostic, prognostic, and therapeutic applications. METHODS: Three microarray datasets were sourced from the NCBI database to acquire upregulated differentially expressed genes (DEGs), while MatrisomeDB was employed for filtering ECM-related genes. Subsequently, a protein-protein interaction (PPI) network was established using the STRING database. The created network was visually inspected through Cytoscape, and HGs were identified using the CytoHubba plugin tool. Furthermore, enrichment analysis, expression pattern analysis, clinicopathological correlation, survival analysis, immune cell infiltration analysis, and examination of chemical compounds were carried out using Enrichr, GEPIA2, ULCAN, Kaplan Meier plotter, TIMER2.0, and CTD web platforms, respectively. The diagnostic and prognostic significance of HGs was evaluated through the ROC curve analysis. RESULTS: Ten genes associated with ECM functions were identified as HGs among 131 DEGs obtained from microarray datasets. Notably, the expression of these HGs exhibited significantly (p < 0.05) higher in PC, demonstrating a clear association with tumor advancement. Remarkably, higher expression levels of these HGs were inversely correlated with the likelihood of patient survival. Moreover, ROC curve analysis revealed that identified HGs are promising biomarkers for both diagnostic (AUC > 0.75) and prognostic (AUC > 0.64) purposes. Furthermore, we observed a positive correlation between immune cell infiltration and the expression of most HGs. Lastly, our study identified nine compounds with significant interaction profiles that could potentially act as effective chemical agents targeting the identified HGs. CONCLUSION: Taken together, our findings suggest that COL1A1, KRT19, MMP1, COL11A1, SDC1, ITGA2, COL1A2, POSTN, FN1, and COL5A1 hold promise as innovative biomarkers for both the diagnosis and prognosis of PC, and they present as prospective targets for therapeutic interventions aimed at impeding the progression PC.

Shaping Oncogenic Microenvironments: Contribution of Fibronectin.

The extracellular matrix (ECM) is a complex network of proteins and glycans, dynamically remodeled and specifically tailored to the structure/function of each organ. The malignant transformation of cancer cells is determined by both cell intrinsic properties, such as mutations, and extrinsic variables, such as the mixture of surrounding cells in the tumor microenvironment and the biophysics of the ECM. During cancer progression, the ECM undergoes extensive remodeling, characterized by disruption of the basal lamina, vascular endothelial cell invasion, and development of fibrosis in and around the tumor cells resulting in increased tissue stiffness. This enhanced rigidity leads to aberrant mechanotransduction and further malignant transformation potentiating the de-differentiation, proliferation and invasion of tumor cells. Interestingly, this fibrotic microenvironment is primarily secreted and assembled by non-cancerous cells. Among them, the cancer-associated fibroblasts (CAFs) play a central role. CAFs massively produce fibronectin together with type I collagen. This review delves into the primary interactions and signaling pathways through which fibronectin can support tumorigenesis and metastasis, aiming to provide critical molecular insights for better therapy response prediction.

Effects of aging on the biomechanical properties of the lung extracellular matrix: dependence on tissular stretch.

Introduction: Aging induces functional and structural changes in the lung, characterized by a decline in elasticity and diminished pulmonary remodeling and regenerative capacity. Emerging evidence suggests that most biomechanical alterations in the lung result from changes in the composition of the lung extracellular matrix (ECM), potentially modulating the behavior of pulmonary cells and increasing the susceptibility to chronic lung diseases. Therefore, it is crucial to investigate the mechanical properties of the aged lung. This study aims to assess the mechanical alterations in the lung ECM due to aging at both residual (RV) and functional (FV) lung volumes and to evaluate their effects on the survival and proliferation of mesenchymal stromal cells (MSCs). Methods: The lungs from young (4-6-month-old) and aged (20-24-month-old) mice were inflated with optimal cutting temperature compound to reach FV or non-inflated (RV). ECM proteins laminin, collagen I and fibronectin were quantified by immunofluorescence and the mechanical properties of the decellularized lung sections were assessed using atomic force microscopy. To investigate whether changes in ECM composition by aging and/or mechanical properties at RV and FV volumes affects MSCs, their viability and proliferation were evaluated after 72 h. Results: Laminin presence was significantly reduced in aged mice compared to young mice, while fibronectin and collagen I were significantly increased in aged mice. In RV conditions, the acellular lungs from aged mice were significantly softer than from young mice. By contrast, in FV conditions, the aged lung ECM becomes stiffer than that of in young mice, revealing that strain hardening significantly depends on aging. Results after MSCs recellularization showed similar viability and proliferation rate in all conditions. Discussion: This data strongly suggests that biomechanical measurements, especially in aging models, should be carried out in physiomimetic conditions rather than following the conventional non-inflated lung (RV) approach. The use of decellularized lung scaffolds from aged and/or other lung disease murine/human models at physiomimetic conditions will help to better understand the potential role of mechanotransduction on the susceptibility and progression of chronic lung diseases, lung regeneration and cancer.

Development of a Cissus quadrangularis-Doped Extracellular Matrix and a Hyaluronic Acid-Incorporated Scaffold for Periodontal Regeneration: An In Vitro Study.

PURPOSE: The study aimed to analyze whether adding Cissus quadrangularis (CQ) extract and the extracellular matrix of ovine tendon (TENDON) increases the regenerative potential of mesenchymal stem cells produced in hyaluronic acid (HA) scaffolds for tenogenesis. MATERIALS AND METHODS: Fifty grams of powdered CQ was mixed with 250 mL of ethanol to prepare the extract. Two grams of hyaluronic acid powder was added to 100 mL of distilled water to make the HA solution. The ovine tendon was decellularized using a mixture of 10% phosphate-buffered saline (PBS), sodium dodecyl sulfate (SDS), and Triton-X. The hydrogel samples were prepared by mixing the extracellular matrix of tendon, HA, and CQ, after which they were divided into study groups such as HA, HA + CQ, HA + TENDON, and HA + CQ + TENDON. Scanning electron microscopy (SEM) analysis, swelling analysis, differentiation analysis, compression test, compatibility assay, and tenogenesis assay were later conducted. RESULTS: The morphology of the samples was analyzed using SEM. Low levels of swelling of the hydrogels were observed. Cells were found to be viable and showed good differentiation and tenogenesis. Optimal compression levels were observed, and the properties of the prepared hydrogels were satisfactory. CONCLUSION: The results suggest that the addition of CQ considerably increases the tenogenic potential of the extracellular matrix/HA scaffold. Hence, it can be used as a regenerative material for periodontal tissue regeneration.

Interactions between Schwann cell and extracellular matrix in peripheral nerve regeneration.

Peripheral nerve injuries, caused by various reasons, often lead to severe sensory, motor, and autonomic dysfunction or permanent disability, posing a challenging problem in regenerative medicine. Autologous nerve transplantation has been the gold standard in traditional treatments but faces numerous limitations and risk factors, such as donor area denervation, increased surgical complications, and diameter or nerve bundle mismatches. The extracellular matrix (ECM) is a complex molecular network synthesized and released into the extracellular space by cells residing in tissues or organs. Its main components include collagen, proteoglycans/glycosaminoglycans, elastin, laminin, fibronectin, etc., providing structural and biochemical support to surrounding cells, crucial for cell survival and growth. Schwann cells, as the primary glial cells in the peripheral nervous system, play various important roles. Schwann cell transplantation is considered the gold standard in cell therapy for peripheral nerve injuries, making ECM derived from Schwann cells one of the most suitable biomaterials for peripheral nerve repair. To better understand the mechanisms of Schwann cells and the ECM in peripheral nerve regeneration and their optimal application, this review provides an overview of their roles in peripheral nerve regeneration.

An Extracellular Matrix Overlay Model for Bioluminescence Microscopy to Measure Single-Cell Heterogeneous Responses to Antiandrogens in Prostate Cancer Cells.

Prostate cancer (PCa) displays diverse intra-tumoral traits, impacting its progression and treatment outcomes. This study aimed to refine PCa cell culture conditions for dynamic monitoring of androgen receptor (AR) activity at the single-cell level. We introduced an extracellular matrix-Matrigel (ECM-M) culture model, enhancing cellular tracking during bioluminescence single-cell imaging while improving cell viability. ECM-M notably tripled the traceability of poorly adherent PCa cells, facilitating robust single-cell tracking, without impeding substrate permeability or AR response. This model effectively monitored AR modulation by antiandrogens across various PCa cell lines. Single-cell imaging unveiled heterogeneous antiandrogen responses within populations, correlating non-responsive cell proportions with drug IC50 values. Integrating ECM-M culture with the PSEBC-TSTA biosensor enabled precise characterization of ARi responsiveness within diverse cell populations. Our ECM-M model stands as a promising tool to assess heterogeneous single-cell treatment responses in cancer, offering insights to link drug responses to intracellular signaling dynamics. This approach enhances our comprehension of the nuanced and dynamic nature of PCa treatment responses.

Cell Senescence-Independent Changes of Human Skin Fibroblasts with Age.

Skin ageing is defined, in part, by collagen depletion and fragmentation that leads to a loss of mechanical tension. This is currently believed to reflect, in part, the accumulation of senescent cells. We compared the expression of genes and proteins for components of the extracellular matrix (ECM) as well as their regulators and found that in vitro senescent cells produced more matrix metalloproteinases (MMPs) than proliferating cells from adult and neonatal donors. This was consistent with previous reports of senescent cells contributing to increased matrix degradation with age; however, cells from adult donors proved significantly less capable of producing new collagen than neonatal or senescent cells, and they showed significantly lower myofibroblast activation as determined by the marker α-SMA. Functionally, adult cells also showed slower migration than neonatal cells. We concluded that the increased collagen degradation of aged fibroblasts might reflect senescence, the reduced collagen production likely reflects senescence-independent processes.

Recent Advancements in Hydrogel Biomedical Research in Italy.

Hydrogels have emerged as versatile biomaterials with remarkable applications in biomedicine and tissue engineering. Here, we present an overview of recent and ongoing research in Italy, focusing on extracellular matrix-derived, natural, and synthetic hydrogels specifically applied to biomedicine and tissue engineering. The analyzed studies highlight the versatile nature and wide range of applicability of hydrogel-based studies. Attention is also given to the integration of hydrogels within bioreactor systems, specialized devices used in biological studies to culture cells under controlled conditions, enhancing their potential for regenerative medicine, drug discovery, and drug delivery. Despite the abundance of literature on this subject, a comprehensive overview of Italian contributions to the field of hydrogels-based biomedical research is still missing and is thus our focus for this review. Consolidating a diverse range of studies, the Italian scientific community presents a complete landscape for hydrogel use, shaping the future directions of biomaterials research. This review aspires to serve as a guide and map for Italian researchers interested in the development and use of hydrogels in biomedicine.

Differential Development of the Chordae Tendineae and Anterior Leaflet of the Bovine Mitral Valve.

There is increasing evidence that some adult mitral valve pathologies may have developmental origins involving errors in cell signaling and protein deposition during valvulogenesis. While early and late gestational stages are well-documented in zebrafish, chicks, and small mammalian models, longitudinal studies in large mammals with a similar gestational period to humans are lacking. Further, the mechanism of chordae tendineae formation and multiplication remains unclear. The current study presents a comprehensive examination of mitral anterior leaflet and chordae tendineae development in a bovine model (a large mammal with the same gestational period as humans). Remarkably distinct from small mammals, bovine development displayed early branched chordae, with increasing attachments only until birth, while the anterior leaflet grew both during gestation and postnatally. Chordae also exhibited accelerated collagen deposition, maturation, and crimp development during gestation. These findings suggest that the bovine anterior leaflet and chordae tendineae possess unique processes of development despite being a continuous collagenous structure and could provide greater insight into human valve development.

Unlocking the Therapeutic Potential of Marine Collagen: A Scientific Exploration for Delaying Skin Aging.

Aging is closely associated with collagen degradation, impacting the structure and strength of the muscles, joints, bones, and skin. The continuous aging of the skin is a natural process that is influenced by extrinsic factors such as UV exposure, dietary patterns, smoking habits, and cosmetic supplements. Supplements that contain collagen can act as remedies that help restore vitality and youth to the skin, helping combat aging. Notably, collagen supplements enriched with essential amino acids such as proline and glycine, along with marine fish collagen, have become popular for their safety and effectiveness in mitigating the aging process. To compile the relevant literature on the anti-aging applications of marine collagen, a search and analysis of peer-reviewed papers was conducted using PubMed, Cochrane Library, Web of Science, and Embase, covering publications from 1991 to 2024. From in vitro to in vivo experiments, the reviewed studies elucidate the anti-aging benefits of marine collagen, emphasizing its role in combating skin aging by minimizing oxidative stress, photodamage, and the appearance of wrinkles. Various bioactive marine peptides exhibit diverse anti-aging properties, including free radical scavenging, apoptosis inhibition, lifespan extension in various organisms, and protective effects in aging humans. Furthermore, the topical application of hyaluronic acid is discussed as a mechanism to increase collagen production and skin moisture, contributing to the anti-aging effects of collagen supplementation. The integration of bio-tissue engineering in marine collagen applications is also explored, highlighting its proven utility in skin healing and bone regeneration applications. However, limitations to the scope of its application exist. Thus, by delving into these nuanced considerations, this review contributes to a comprehensive understanding of the potential and challenges associated with marine collagen in the realm of anti-aging applications.

Hemodynamics and Wall Mechanics of Vascular Graft Failure.

Blood vessels are subjected to complex biomechanical loads, primarily from pressure-driven blood flow. Abnormal loading associated with vascular grafts, arising from altered hemodynamics or wall mechanics, can cause acute and progressive vascular failure and end-organ dysfunction. Perturbations to mechanobiological stimuli experienced by vascular cells contribute to remodeling of the vascular wall via activation of mechanosensitive signaling pathways and subsequent changes in gene expression and associated turnover of cells and extracellular matrix. In this review, we outline experimental and computational tools used to quantify metrics of biomechanical loading in vascular grafts and highlight those that show potential in predicting graft failure for diverse disease contexts. We include metrics derived from both fluid and solid mechanics that drive feedback loops between mechanobiological processes and changes in the biomechanical state that govern the natural history of vascular grafts. As illustrative examples, we consider application-specific coronary artery bypass grafts, peripheral vascular grafts, and tissue-engineered vascular grafts for congenital heart surgery as each of these involves unique circulatory environments, loading magnitudes, and graft materials.

RAC1-mediated Integrin alpha-6 expression in E-cadherin-deficient gastric cancer cells promotes interactions with the stroma and peritoneal dissemination.

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer that is prone to peritoneal dissemination, with poor patient prognosis. Although intercellular adhesion loss between cancer cells is a major characteristic of DGCs, the mechanism underlying the alteration in cell-to-extracellular matrix (ECM) adhesion is unclear. We investigated how DGCs progress and cause peritoneal dissemination through interactions between DGC cells and the tumour microenvironment (TME). p53 knockout and KRASG12V-expressing (GAN-KP) cells and Cdh1-deleted GAN-KP (GAN-KPC) cells were orthotopically transplanted into the gastric wall to mimic peritoneal dissemination. The GAN-KPC tumour morphology was similar to that of human DGCs containing abundant stroma. RNA sequencing revealed that pathways related to Rho GTPases and integrin-ECM interactions were specifically increased in GAN-KPC cells compared with GAN-KP cells. Notably, we found that Rac Family Small GTPase 1 (RAC1) induces Integrin Subunit Alpha 6 (Itga6) trafficking, leading to its enrichment on the GC cell membrane. Fibroblasts activate the FAK/AKT pathway in GC cells by mediating extracellular matrix (ECM)-Itga6 interactions, exacerbating the malignant phenotype. In turn, GC cells induce abnormal expression of fibroblast collagen and its transformation into cancer-associated fibroblasts (CAFs), resulting in DGC-like subtypes. These findings indicate that Cdh1 gene loss leads to abnormal expression and changes in the subcellular localization of ITGA6 through RAC1 signalling. The latter, through interactions with CAFs, allows for peritoneal dissemination.