Extracellular Matrix Elements Research Articles

P-424 Placental extracellular matrix remodelling in pregnancies by oocyte donation shows similarities with preeclamptic placenta: a pilot study

Abstract Study question Do stromal alterations occur during placentation upon heterologous assisted reproductive techniques (ART) and are they associated with a higher risk of developing preeclampsia? Summary answer The remodelling of extracellular matrix molecules, key for vascularization and immunity, is altered in pregnancies from oocyte donation and resembles the miss-regulations observed in preeclampsia. What is known already Preeclampsia represents one of the main causes of maternal and perinatal morbidity, affecting 2-8% of spontaneous pregnancies. Its incidence increases two times upon homologous ART and up to four times upon oocyte donation, however the underlying factors are still unknown. Preeclampsia is characterized by high blood pressure and endothelial cell dysfunction, but its pathogenesis is multifactorial relying on vascular, metabolic and immune alterations. All these elements are tightly influenced by the extracellular matrix (ECM) that, during an uneventful pregnancy, is remodelled to support an efficient placentation and immunotolerance. Despite its key functions, little is known regarding ECM remodelling in preeclampsia. Study design, size, duration This is an observational pilot study conducted over a 12 months period. Four groups were included: 1) pregnant women that underwent homologous ART (n = 7; HOM); 2) pregnant women that underwent oocyte donation (n = 10; OD); 3) women that conceived spontaneously affected by PE (n = 7); and 4) uneventful spontaneous pregnancies (n = 8; control group CTRL). Participants/materials, setting, methods For each patient placental samples have been collected upon delivery and processed to perform molecular and proteomic analysis by real time PCR and immunohistochemistry The statistical differences among the groups have been evaluated by the ANOVA and Mann-Whitney tests. Main results and the role of chance We focused our study on specific ECM elements known to modulate angiogenesis and immunity, two key players in preeclampsia occurrence. Our data indicated first that collagen I, collagen IV and Multimerin-2, important components of the vessel wall that contribute to vessel stabilization and efficiency, are significantly decreased in OD compared to HOM group (coll I P = 0.04; coll IV P = 0.043; Multimerin-2 P = 0.036). Next, we analyzed EMILIN-2 and versican, two ECM components exerting not only angiogenic but also immunomodulatory functions. We found that EMILIN-2 is decreased in OD compared to CTRL placentas (P = 0.043), whereas versican undergoes an opposite regulation, being slightly increased in the OD placentas (P = 0.045). We observed an analogous mis-regulation when comparing PE to the CTRL group, suggesting that these stromal alterations may take part in the establishment of a microenvironment more prone to preeclampsia onset and associate with the higher risk related to OD pregnancies. We also found that some features of ECM remodelling seem to be strongly associated with OD. Precisely, our data denoted that the glycoprotein tenascin-C decreased specifically in OD patients, while was comparable between HOM, PE and CTRL groups (P = 0.042). Limitations, reasons for caution The main limitation of this pilot study is the restricted number of patients. Further investigations on a larger sample and prospective cohort of patients might provide more robust data. Wider implications of the findings Our data highlighted for the first time that the placental stroma undergoes an altered remodelling in OD and PE pregnancies. We envision that a deep characterization of ECM will help better understanding of the mechanisms behind the pathogenesis of preeclampsia with possible implications for prediction and prevention. Trial registration number N/A

Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease

AimsCardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. Main methodsEVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. Key findingsProteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SignificanceThe treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.

Differential preoperative transcriptomic profiles are related to the quality of left ventricle reverse remodelling one year after valve replacement in patients with severe aortic valve stenosis

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Spain´s Ministerio de Economía y Competitividad [(PI21/00084, CIBERCV (CB16/11/00264), co-funded by Fondo Europeo de Desarrollo Regional (FEDER)] Instituto de Investigación Sanitaria Marqués de Valdecilla, IDIVAL (INNVAL21/24; PREVAL22/09) Background Aortic valve stenosis (AS) promotes a progressive remodelling of the left ventricle (LV) characterised by cardiomyocyte hypertrophy and increased deposition of extracellular matrix elements. These adaptive plastic changes may evolve towards heart failure over time. The extent of LV remodelling and its persistence after valve replacement are significant prognostic factors for the long-term outcome of AS patients. The biological processes that determine the reverse remodelling rate after surgery of AS patients are unknown. Purpose To ascertain preoperative LV transcriptomic determinants of favourable reverse remodelling one year after surgery in AS patients. To this end, a genome-wide transcriptomic analysis was performed in LV biopsies from AS patients and surgical controls. Methods and Results The study was performed in AS (n=24) and control patients (n=12) (age and sex matched) followed up for one year after surgery. RNA-seq was performed on LV biopsies obtained during surgery. AS patients were stratified according to the quality of the reverse remodelling at one year in good (LV mass index ≤51 g/m2.7, n=12) or poor (LV mass index >51 g/m2.7, n=12) performers. RNA-seq libraries were prepared and sequenced using an Illumina Next Seq Unit. Resulting reads were mapped against GRCh38.99 version of the human transcriptome. The results from the principal component analysis revealed a distinctive expression gene pattern for the AS condition, accounting for 60.1 % of the variance, with no difference for the sex condition (8.7 %). Differential gene expression was considered significant when yielding a Benjamini-Hochberg adjusted p-value < 0.05. Compared to controls, 339 and 443 genes were differentially expressed in AS with good and poor reverse remodelling, respectively, with 509 overlapping. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes in patients with poor reverse remodelling compared to control patients, showed a significant enrichment (NES > 1.5) in genes associated with angiogenesis, epithelial-mesenchymal transition (EMT), and heart growth. The down-regulated genes (NES < 0) were associated with transcription processes, such as histone modification and RNA processing-related genes, highlighting the significance of epigenetic mechanisms in AS patients exhibiting poor reverse remodelling. In contrast, such transcriptomic profile patterns were not observed in AS with good-reverse remodelling. Conclusion Aortic stenosis patients with good or poor reverse remodelling one year after aortic valve replacement exhibited distinct preoperative LV transcriptome profile. Gene ontology enrichment analysis revealed angiogenesis, EMT, and epigenetic processes as critical biological functions associated with poor reverse remodelling in AS patients.

Hydrogel formats to model potential drug interactions occurring at the subcutaneous injection site

We have previously developed an in vitro instrument, termed subcutaneous injection site simulator (SCISSOR), that can be used to monitor release properties of an active pharmaceutical ingredient (API) and formulation components of a medicine designed for SC injection. Initial studies to validate the SCISSOR instrument applications used a simple hyaluronic acid (HA) hydrogel to monitor early release events. We now report a type of cross-linked HA that can, when combined with HA, provide a hydrogel (HA-XR) with optical clarity and rheological properties that remain stable for at least 6 days. Incorporation of 0.05–0.1 mg/mL of collagens isolated from human fibroblasts (Col F), bovine type I collagen (Col I), chicken collagen type II (Col II), or chondroitin sulphate (CS) produced HA or HA-XR hydrogel formats with optical clarity and rheological properties comparable to HA or HA-XR alone. HA + Col F hydrogel had a much greater effect on release rates of 70 kDa compared to 4 kDa dextran, while Col F incorporated into the HA-XR hydrogel accentuated differences in release rates of prandial and basal forms of insulin as well as decreased the release rate of denosumab. A hydrogel format of HA + Col I was used to examine the complex events for bevacizumab release under conditions where a target ligand (vascular endothelial growth factor) can interact with extracellular matrix (ECM). Together, these data have demonstrated the feasibility of using a cross-linked HA format to examine API release over multiple days and incorporation of specific ECM elements to prepare more biomimetic hydrogels that allow for tractable examination of their potential impact of API release.

Abstract 4305: Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice

Abstract Cancer is a complex disease that involves both malignant cells and the surrounding microenvironment. The tumor microenvironment (TME) is comprised of various components, including cancer-associated fibroblasts, immune cells, blood vessels, and extracellular matrix elements, all of which interact with cancer cells. It is increasingly recognized that the components of the TME are a critical determinant of therapeutic response and resistance, and as such targeting the TME has emerged as a promising strategy in cancer therapy. Syngeneic models are necessary to study the TME, and more specifically the tumor infiltrating immune cell populations, as these models allow us to monitor various aspects of the immune response. Another important aspect to consider is tumor heterogeneity, which can cause issues during analysis due to immune cell populations not being evenly distributed throughout the tumor. The decision of whether to analyze whole tumors or halved tumors when monitoring changes in immune cell populations is an important consideration. When considering multiple endpoints from a single tumor sample, halved tumors are oftentimes considered as we remain mindful of the 3Rs (replacement, reduction and refinement) in animal research. As such, it is important to understand the effects of splitting samples and how it can affect the data that is generated. To determine the effects of analyzing whole and halved tumor samples on immune cell subsets, studies were designed to subcutaneously implant either CT26.WT or 4T1-Luc2-1A4 cells into female BALB/cAnNHsd mice and harvest tumor samples after the completion of treatment. Mice were treated with isotype control (Clone MPC-11) or anti-mCTLA-4 (Clone 9D9). At the time of sample collection, tumors were kept whole intact or halved and analyzed via flow cytometry to quantify lymphoid and myeloid immune cell subsets within tumor samples. The data revealed differences in immune cell subset populations for both isotype vs. anti-CTLA-4 treated tumors and whole vs. halved tumors. Changes in the lymphoid cell compartment were observed for whole vs. halved tumors of ± ~25-100% and ± ~0-40% for isotype treated and of ± ~0-30% and ± ~25-2600% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively. Changes in the myeloid cell compartment of ± ~5-45% and ± ~10-65% for isotype treated and of ± ~15-100% and ± ~0-70% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively, were also observed. Additionally, differences were observed between the two tumor halves, not just between whole and halved tumors. Taken together, the data support that splitting tumor samples can negatively impact the analysis of the immune cell populations. Tumors can be split if required to perform multiple analyses, however, this should be done with caution, as it can result in trend differences and increases variability within the dataset. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Sheri Barnes, David Draper, Scott Wise. Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4305.

Gelatin from specific freshwater and saltwater fish extracted using six different methods: Component interactions, structural characteristics, and functional properties

Dry-salting, wet-salting, trypsin, pepsin, acid, and heat methods were used to extract the gelatin from a few saltwater (snakehead) and freshwater (shark) fish skins, and their structural and functional properties and components were then investigated. The results show that enzyme techniques produce fish skin gelatin with a robust triple-helix organization and a sponge-like mesh structure, exhibiting good water absorption(10.67%), fat binding(10.63%), and emulsification properties(89.37 m2/g). The gelatin fiber length and height of shark gelatin extracted by wet salt method were the highest with 617.77 ± 44.04 nm and 17.30 ± 1.45 nm, respectively. Shark skin gelatin produced via heat methods and blackfish skin produced via trypsin exhibits impressive thermal stability, its Tm value was 77.5 °C and 75.7 °C respectively. heatly extracted snakehead and shark gelatin has a high gel strength with 3.06 ± 0.04 N and 3.14 ± 0.04 N. A more in-depth component analysis demonstrates that blackfish skin and shark skin gelatins contain 14 and 9 different forms of collagen proteins, respectively, according to a more thorough component study. Different varieties of gelatin also have other extracellular matrix elements. Notably, trypsin-derived gelatin from shark and blackfish skin has been shown to include biglycan and member 3 (NESH)-binding protein b. The co-assembly of type II improved the gel strength and thermal stability of the gelatin and type XI collagen. Biglycan and decorin can promote the growth of dense network structures. These findings will significantly contribute to the customized applications of fish gelatin and the mechanistic clarification of synthetic biology.

COVID-19 induces more pronounced extracellular matrix deposition than other causes of ARDS

BackgroundLung 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.MethodsWe 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.ResultsWe 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.ConclusionsFatal 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.

Matrix Metalloproteinases and the Pathogenesis of Recurrent Corneal Erosions and Epithelial Basement Membrane Dystrophy.

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are members of the zinc endopeptidase family. They have the ability to degrade extracellular matrix elements, allowing for the release of binding molecules and cell migration. Although metalloproteinases regulate numerous physiological processes within the cornea, overexpression of metalloproteinase genes and an imbalance between the levels of metalloproteinases and their inhibitors can contribute to the inhibition of repair processes, the development of inflammation and excessive cellular proliferation. The involvement of MMPs in the pathogenesis of dystrophic corneal diseases needs clarification. Our analyses focus on the involvement of individual metalloproteinases in the pathogenesis of recurrent corneal erosions and highlight their impact on the development of corneal epithelial basement membrane dystrophy (EBMD). We hypothesize that abnormalities observed in patients with EBMD may result from the accumulation and activation of metalloproteinases in the basal layers of the corneal epithelium, leading to basement membrane degradation. A barrier formed from degradation materials inhibits the normal migration of epithelial cells to the superficial layers, which contributes to the development of the aforementioned lesions. This hypothesis seems to be lent support by the elevated concentrations of metalloproteinases in the corneal epithelium of these patients found in our previous studies on the relationships between MMPs and recurrent corneal erosions.

A Multilayered Imaging and Microfluidics Approach for Evaluating the Effect of Fibrinolysis in Staphylococcus aureus Biofilm Formation.

The recognition of microbe and extracellular matrix (ECM) is a recurring theme in the humoral innate immune system. Fluid-phase molecules of innate immunity share regulatory roles in ECM. On the other hand, ECM elements have immunological functions. Innate immunity is evolutionary and functionally connected to hemostasis. Staphylococcus aureus (S. aureus) is a major cause of hospital-associated bloodstream infections and the most common cause of several life-threatening conditions such as endocarditis and sepsis through its ability to manipulate hemostasis. Biofilm-related infection and sepsis represent a medical need due to the lack of treatments and the high resistance to antibiotics. We designed a method combining imaging and microfluidics to dissect the role of elements of the ECM and hemostasis in triggering S. aureus biofilm by highlighting an essential role of fibrinogen (FG) in adhesion and formation. Furthermore, we ascertained an important role of the fluid-phase activation of fibrinolysis in inhibiting biofilm of S. aureus and facilitating an antibody-mediated response aimed at pathogen killing. The results define FG as an essential element of hemostasis in the S. aureus biofilm formation and a role of fibrinolysis in its inhibition, while promoting an antibody-mediated response. Understanding host molecular mechanisms influencing biofilm formation and degradation is instrumental for the development of new combined therapeutic approaches to prevent the risk of S. aureus biofilm-associated diseases.

PHEMA-based cryogels as 3D placental scaffolds

The placental barrier regulates the passage of nutrients, waste, drugs, and chemicals between maternal and fetal tissues. Mono and co-culture systems were constituted with human placental choriocarcinoma cells (BeWo) and human umbilical cord vein cells (HUVEC) on biocompatible poly (2-hydroxyethyl methacrylate) (PHEMA)-based cryogels. PHEMA cryogels were supported with extracellular matrix (ECM) elements. According to the Fourier transform infrared spectrometry-Attenuated Total Reflectance (FTIR-ATR) results, it was confirmed that gelatin, folic acid (FA), fibronectin (FN), and collagen type IV (COL Type IV) were successfully incorporated into the structure of cryogels. The mean pore size of the cryogels confirmed by micro-computed tomography analysis was in the range of 15–195 µm. The antidepressant active ingredient Vortioxetine was investigated by 3-(4,5-dimethylthiazol2-yl)− 2,5-diphenyltetrazolium-bromide (MTT) viability analysis in the constituted cryogel co-culture system. The cryogels did not cause any toxic effect on the cells. High doses of Vortioxetine showed reduced cell viability in monocultures, but not the expected reduction in cell viability at the same doses of co-culture. The highest amount of human chorionic gonadotropin hormone was in the PHEMA+COL Type IV+FA group. There were no significant changes in matrix metalloproteinase (MMP) 2 and 9 protein levels due to Vortioxetine dose administration.

IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

Canonical interleukin-2 (IL-2) signaling via the high-affinity CD25-containing IL-2 receptor-Janus kinase (JAK)1,3-signal transducer and activator of transcription 5 (STAT5) pathway is essential for development and maintenance of CD4+CD25HiFoxp3+ regulatory Tcells (Tregs) that support immune homeostasis. Here, we report that IL-2 signaling via an alternative CD25-chemokine receptor pathway promotes the suppressive function of Tregs. Using an antibody against CD25 that biases IL-2 signaling toward this alternative pathway, we establish that this pathway increases the suppressive activity of Tregs and ameliorates murine experimental autoimmune encephalomyelitis (EAE). Furthermore, heparan sulfate, an IL-2-binding element of cell surfaces and extracellular matrix, or an engineered IL-2 immunocytokine can also direct IL-2 signaling toward this alternative pathway. Overall, these data reveal a non-canonical mechanism for IL-2 signaling that promotes suppressive functions of Tregs, further elucidates how IL-2 supports immune homeostasis, and suggests approaches to promote or suppress Treg functions.

Ablation of the carboxy-terminal end of MAMDC2 causes a distinct muscular dystrophy.

The extracellular matrix (ECM) has an important role in the development and maintenance of skeletal muscle, and several muscle diseases are associated with the dysfunction of ECM elements. MAMDC2 is a putative ECM protein and its role in cell proliferation has been investigated in certain cancer types. However, its participation in skeletal muscle physiology has not been previously studied. We describe 17 individuals with an autosomal dominant muscular dystrophy belonging to two unrelated families in which different heterozygous truncating variants in the last exon of MAMDC2 co-segregate correctly with the disease. The radiological aspect of muscle involvement resembles that of COL6 myopathies with fat replacement at the peripheral rim of vastii muscles. In this cohort, a subfascial and peri-tendinous pattern is observed in upper and lower limb muscles. Here we show that MAMDC2 is expressed in adult skeletal muscle and differentiating muscle cells, where it appears to localize to the sarcoplasm and myonuclei. In addition, we show it is secreted by myoblasts and differentiating myotubes into to the extracellular compartment. The last exon encodes a disordered region with a polar residue compositional bias loss of which likely induces a toxic effect of the mutant protein. The precise mechanisms by which the altered MAMDC2 proteins cause disease remains to be determined. MAMDC2 is a skeletal muscle disease-associated protein. Its role in muscle development and ECM-muscle communication remains to be fully elucidated. Screening of the last exon of MAMDC2 should be considered in patients presenting with autosomal dominant muscular dystrophy, particularly in those with a subfascial radiological pattern of muscle involvement.

Genetic exploration of roles of acid-sensing ion channel subtypes in neurosensory mechanotransduction including proprioception.

Although acid-sensing ion channels (ASICs) are proton-gated ion channels responsible for sensing tissue acidosis, accumulating evidence has shown that ASICs are also involved in neurosensory mechanotransduction. However, in contrast to Piezo ion channels, evidence of ASICs as mechanically gated ion channels has not been found using conventional mechanoclamp approaches. Instead, ASICs are involved in the tether model of mechanotransduction, with the channels gated via tethering elements of extracellular matrix and intracellular cytoskeletons. Methods using substrate deformation-driven neurite stretch and micropipette-guided ultrasound were developed to reveal the roles of ASIC3 and ASIC1a, respectively. Here we summarize the evidence supporting the roles of ASICs in neurosensory mechanotransduction in knockout mouse models of ASIC subtypes and provide insight to further probe their roles in proprioception.

Sigma-1 Receptor Activation Is Protective against TGFβ2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Highlighting In Vitro the Role of Brain-like Endothelial Cells on the Maturation and Metabolism of Brain Pericytes by SWATH Proteomics

Within the neurovascular unit, brain pericytes (BPs) are of major importance for the induction and maintenance of the properties of the blood-brain barrier (BBB) carried by the brain microvessel endothelial cells (ECs). Throughout barriergenesis, ECs take advantage of soluble elements or contact with BPs to maintain BBB integrity and the regulation of their cellular homeostasis. However, very few studies have focused on the role of ECs in the maturation of BPs. The aim of this study is to shed light on the proteome of BPs solocultured (hBP-solo) or cocultured with ECs (hBP-coc) to model the human BBB in a non-contact manner. We first generated protein libraries for each condition and identified 2233 proteins in hBP-solo versus 2492 in hBP-coc and 2035 common proteins. We performed a quantification of the enriched proteins in each condition by sequential window acquisition of all theoretical mass spectra (SWATH) analysis. We found 51 proteins enriched in hBP-solo related to cell proliferation, contractility, adhesion and extracellular matrix element production, a protein pattern related to an immature cell. In contrast, 90 proteins are enriched in hBP-coc associated with a reduction in contractile activities as observed in vivo in 'mature' BPs, and a significant gain in different metabolic functions, particularly related to mitochondrial activities and sterol metabolism. This study highlights that BPs take advantage of ECs during barriergenesis to make a metabolic switch in favor of BBB homeostasis in vitro.

Nanomaterials based on chitosan for skin regeneration: an update.

Nanomaterials based on chitosan for skin regeneration: an update.

Timing Expression of miR203a-3p during OA Disease: Preliminary In Vitro Evidence.

Osteoarthritis (OA) is a degenerative bone disease that involves the microenvironment and macroenvironment of joints. Progressive joint tissue degradation and loss of extracellular matrix elements, together with different grades of inflammation, are important hallmarks of OA disease. Therefore, the identification of specific biomarkers to distinguish the stages of disease becomes a primary necessity in clinical practice. To this aim, we investigated the role of miR203a-3p in OA progression starting from the evidence obtained by osteoblasts isolated from joint tissues of OA patients classified according to different Kellgren and Lawrence (KL) grading (KL ≤ 3 and KL > 3) and hMSCs treated with IL-1β. Through qRT-PCR analysis, it was found that osteoblasts (OBs) derived from the KL ≤ 3 group expressed high levels of miR203a-3p and low levels of ILs compared with those of OBs derived from the KL > 3 group. The stimulation with IL-1β improved the expression of miR203a-3p and the methylation of the IL-6 promoter gene, favoring an increase in relative protein expression. The gain and loss of function studies showed that the transfection with miR203a-3p inhibitor alone or in co-treatments with IL-1β was able to induce the expression of CX-43 and SP-1 and to modulate the expression of TAZ, in OBs derived from OA patients with KL ≤ 3 compared with KL > 3. These events, confirmed also by qRT-PCR analysis, Western blot, and ELISA assay performed on hMSCs stimulated with IL-1β, supported our hypothesis about the role of miR203a-3p in OA progression. The results suggested that during the early stage, miR203a-3p displayed a protective role reducing the inflammatory effects on CX-43, SP-1, and TAZ. During the OA progression the downregulation of miR203a-3p and consequently the upregulation of CX-43/SP-1 and TAZ expression improved the inflammatory response and the reorganization of the cytoskeleton. This role led to the subsequent stage of the disease, where the aberrant inflammatory and fibrotic responses determined the destruction of the joint.

PS-P15-5: EFFECT OF SODIUM RESTRICTION ON LARGE ARTERY STIFFNESS AND DISTENSIBILITY OF SMALL ARTERIES IN HYPERTENSIVE PATIENTS: A RANDOMIZED TRIAL

Objective: Arterial stiffness is characterized by a decrease in arterial distensibility, resulting from the interaction between the extracellular matrix and cellular elements. Large and small arteries can be differently influenced by extrinsic factors, such as excessive sodium intake. This study aim to evaluate the impact of sodium restriction on large artery stiffness and distensibility of small arteries in young adults diagnosed with arterial hypertension. Methods: Randomized controlled clinical trial, including 20 hypertensive patients, aged between 30 and 59 years, followed up at the hypertension outpatient clinic of a tertiary hospital. Subjects were randomized into two groups: group 1 - low-sodium diet (2 g sodium/day); group 2 - usual diet. To analyze the stiffness and distensibility of the arteries, it was measured aortic pulse wave velocity by Complior®, elasticity of small arteries and vascular resistance by HDI®, and also through the measurement of metalloproteinase-9. For nutritional assessment it were considered blood glucose levels, 24-h urinary sodium, body mass index (BMI) and other anthropometric measurements. For nutritional intake we analized the 24-hour dietary recall. Results and Discussion: In comparison with group 2, it was observed a significant (p < 0.05) reduction in office blood pressure (p = 0,037) and pulse wave velocity (p = 0,010) in group 1 subjects. Also, in group 1 it occurred reduction in blood glucose levels, BMI, waist and neck circumference. Sodium intake was significantly reduced in group 1, taking into account 24 hs urinary sodium and evaluated by dietary recall. It is worth mentioning that excessive sodium intake linked to arterial hypertension causes a remodeling process, increased oxidative stress, inflammation and fibrosis of the arteries, contributing to arterial stiffness. Regarding the distensibility of small arteries, no significant differences were found between the two groups, but the intervention period and the number of individuals in the study should be taken into account, since these factors may have influenced the results. Conclusion: Sodium restriction for a short period of time promoted reduction of arterial stiffness, in parallel to reduction in obesity parameters and blood pressure in adults diagnosed with systemic arterial hypertension.

Macrophage-, Dendritic-, Smooth Muscle-, Endothelium-, and Stem Cells-Derived Foam Cells in Atherosclerosis.

Atherosclerosis is an inflammatory disease depending on the buildup, called plaque, of lipoproteins, cholesterol, extracellular matrix elements, and various types of immune and non-immune cells on the artery walls. Plaque development and growth lead to the narrowing of the blood vessel lumen, blocking blood flow, and eventually may lead to plaque burst and a blood clot. The prominent cellular components of atherosclerotic plaque are the foam cells, which, by trying to remove lipoprotein and cholesterol surplus, also participate in plaque development and rupture. Although the common knowledge is that the foam cells derive from macrophages, studies of the last decade clearly showed that macrophages are not the only cells able to form foam cells in atherosclerotic plaque. These findings give a new perspective on atherosclerotic plaque formation and composition and define new targets for anti-foam cell therapies for atherosclerosis prevention. This review gives a concise description of foam cells of different pedigrees and describes the main mechanisms participating in their formation and function.

Plasma and Urine Levels of Glycosaminoglycans in Patients with Systemic Sclerosis and Their Relationship to Selected Interleukins and Marker of Early Kidney Injury.

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by immune system dysfunction, vasculopathy, and progressive fibrosis of the skin and internal organs, resulting from excessive accumulation of extracellular matrix (ECM) elements, including collagen and proteoglycans (PGs). An uncontrolled PG proliferation, caused by disturbances in their metabolism in tissues, is most likely reflected in the quantitative changes of their components, i.e., glycosaminoglycans (GAGs), in body fluids. Therefore, the aim of this study was to quantify the different types of GAGs in the blood and urine of systemic sclerosis patients. Chondroitin/dermatan sulfates (CS/DS) and heparan sulfates/heparin (HS/H) were quantified by hexuronic acid assay and electrophoretic fractionation, while hyaluronic acid (HA) and keratan sulfates were evaluated using ELISA tests. In turn, individual urinary GAGs were determined using the Blyscan™ Sulfated Glycosaminoglycan Assay Kit. Our results showed that the plasma concentrations of CS/DS, HS/H, HA, and KS in systemic sclerosis patients were significantly higher compared with those in healthy subjects. In the case of urine measurements, we have found that in SSc patients, CS/DC concentrations were significantly higher, while HA concentrations were significantly lower compared with the values observed in the urine of healthy subjects. Importantly, the found by us correlations between plasma keratan sulfate levels and both the duration of the disease and the severity of skin lesions, as expressed by the Rodnan scale, seems to suggest this GAG as a potential marker in assessing disease progression and activity. In addition, a level of urinary excretion of all types of GAGs due to their high positive correlation with uACR, may be a valuable complementary test in the diagnosis of early renal dysfunction in the course of SSc.