Cell Extracellular Matrix Research Articles

Non-cell-autonomous regulation of interneuron specification mediated by extracellular vesicles.

Disruption in neurogenesis and neuronal migration can influence the assembly of cortical circuits, affecting the excitatory-inhibitory balance and resulting in neurodevelopmental and neuropsychiatric disorders. Using ventral cerebral organoids and dorsoventral cerebral assembloids with mutations in the extracellular matrix gene LGALS3BP, we show that extracellular vesicles released into the extracellular environment regulate the molecular differentiation of neurons, resulting in alterations in migratory dynamics. To investigate how extracellular vesicles affect neuronal specification and migration dynamics, we collected extracellular vesicles from ventral cerebral organoids carrying a mutation in LGALS3BP, previously identified in individuals with cortical malformations and neuropsychiatric disorders. These results revealed differences in protein composition and changes in dorsoventral patterning. Proteins associated with cell fate decision, neuronal migration, and extracellular matrix composition were altered in mutant extracellular vesicles. Moreover, we show that treatment with extracellular vesicles changes the transcriptomic profile in neural progenitor cells. Our results indicate that neuronal molecular differentiation can be influenced by extracellular vesicles.

Large Polyacrylamide Hydrogels for Large-Batch Cell Culture and Mechanobiological Studies.

The rigidity of a cell's substrate or extracellular matrix plays a vital role in regulating cell and tissue functions. Polyacrylamide (PAAm) hydrogels are one of the most widely used cell culture substrates that provide a physiologically relevant range of stiffness. However, it is still arduous and time-consuming to prepare PAAm substrates in large batches for high-yield or multiscale cell cultures. In this communication, a simple method to prepare PAAm hydrogels with less time cost and easily accessible materials is presented. The hydrogel is mechanically uniform and supports cell culture in a large batch. It is further shown that the stiffness of the hydrogel covers a large range of Young's modulus and is sensed by cells, regulating various cell features including changes in cell morphology, proliferation, and contractility. This method improves the reproducibility of mechanobiology studies and can be easily applied for mechanobiology research requiring large numbers of cells or experimental groups.

Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue.

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.

Osteogenesis promotion by injectable methacryloylated gelatin containing psoralen and its bacteriostatic properties.

The treatment of periodontitis focuses on controlling the progression of inflammation, reducing plaque accumulation, and promoting bone tissue reconstruction. Among them, the reconstruction of irregular bone resorption caused by periodontitis is a long-standing challenge. At present, the local drug treatment of periodontitis is mainly anti-inflammatory and antibacterial drugs. In this study, psoralen (Pso), a Chinese herbal medicine with anti-inflammatory, antibacterial, and osteogenic effects, was selected for the local treatment of periodontitis. Meanwhile, an injectable methacrylate gelatin (GelMA) platform loading with Pso was constructed. Pso-GelMA had the properties of fluidity, light cohesion, self-healing, and slow release, which could be better used in the deep and narrow structure of the periodontal pocket, and greatly increased the effectiveness of local drug delivery. The pore size of Gelma hydrogel did not change after loading Pso by SEM. In vitro, Pso-GelMA effectively upregulated the expression of osteogenic genes and proteins, increased alkaline phosphatase activity, promoted the mineralisation of rat bone marrow mesenchymal stem cells (BMSCs) extracellular matrix, and had significant antibacterial effects on Staphylococcus aureus and Fusobacterium nucleatum. Therefore, Pso-GelMA has immense promise in the adjuvant treatment of periodontitis.

Identification of the hub genes associated with prostate cancer tumorigenesis.

Prostate cancer (PCa) is one of the most common malignant tumors of the male urogenital system; however, the underlying mechanisms remain largely unclear. This study integrated two cohort profile datasets to elucidate the potential hub genes and mechanisms in PCa. Gene expression profiles GSE55945 and GSE6919 were filtered from the Gene Expression Omnibus (GEO) database to obtain 134 differentially expressed genes (DEGs) (14 upregulated and 120 downregulated) in PCa. Gene Ontology and pathway enrichment were performed using the Database for Annotation, Visualization, and Integrated Discovery, showing that these DEGs were mainly involved in biological functions such as cell adhesion, extracellular matrix, migration, focal adhesion, and vascular smooth muscle contraction. The STRING database and Cytoscape tools were used to analyze protein-protein interactions and identify 15 hub candidate genes. Violin plot, boxplot, and prognostic curve analyses were performed using Gene Expression Profiling Interactive Analysis, which identified seven hub genes, including upregulated expressed SPP1 and downregulated expressed MYLK, MYL9, MYH11, CALD1, ACTA2, and CNN1 in PCa compared with normal tissue. Correlation analysis was performed using the OmicStudio tools, which showed that these hub genes were moderately to strongly correlated with each other. Finally, quantitative reverse transcription PCR and western blotting were performed to validate the hub genes, showing that the abnormal expression of the seven hub genes in PCa was consistent with the analysis results of the GEO database. Taken together, MYLK, MYL9, MYH11, CALD1, ACTA2, SPP1, and CNN1 are hub genes significantly associated with PCa occurrence. These genes are abnormally expressed, leading to the formation, proliferation, invasion, and migration of PCa cells and promoting tumor neovascularization. These genes may serve as potential biomarkers and therapeutic targets in patients with PCa.

Oligosaccharide Ligands of Galectin-4 and Its Subunits: Multivalency Scores Highly.

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.

Aloe vera-loaded natural rubber latex dressing as a potential complementary treatment for psoriasis

Psoriasis is a disease that causes keratinocytes to proliferate ten times faster than normal, resulting in chronic inflammation and immune cell infiltration in the skin. Aloe vera (A. vera) creams have been used topically for treating psoriasis because they contain several antioxidant species; however, they have several limitations. Natural rubber latex (NRL) has been used as occlusive dressings to promote wound healing by stimulating cell proliferation, neoangiogenesis, and extracellular matrix formation. In this work, we developed a new A. vera-releasing NRL dressing by a solvent casting method to load A. vera into NRL. FTIR and rheological analyzes revealed no covalent interactions between A. vera and NRL in the dressing. We observed that 58.8 % of the loaded A. vera, present on the surface and inside the dressing, was released after 4 days. Biocompatibility and hemocompatibility were validated in vitro using human dermal fibroblasts and sheep blood, respectively. We observed that ~70 % of the free antioxidant properties of A. vera were preserved, and the total phenolic content was 2.31-fold higher than NRL alone. In summary, we combined the antipsoriatic properties of A. vera with the healing activity of NRL to generate a novel occlusive dressing that may be indicated for the management and/or treatment of psoriasis symptoms simply and economically.

Role of non‑coding RNAs in cartilage endplate (Review).

Cartilage endplate (CEP) degeneration is considered one of the major causes of intervertebral disc degeneration (IDD), which causes non-specific neck and lower back pain. In addition, several non-coding RNAs (ncRNAs), including long ncRNAs, microRNAs and circular RNAs have been shown to be involved in the regulation of various diseases. However, the particular role of ncRNAs in CEP remains unclear. Identifying these ncRNAs and their interactions may prove to be is useful for the understanding of CEP health and disease. These RNA molecules regulate signaling pathways and biological processes that are critical for a healthy CEP. When dysregulated, they can contribute to the development disease. Herein, studies related to ncRNAs interactions and regulatory functions in CEP are reviewed. In addition, a summary of the current knowledge regarding the deregulation of ncRNAs in IDD in relation to their actions on CEP cell functions, including cell proliferation, apoptosis and extracellular matrix synthesis/degradation is presented. The present review provides novel insight into the pathogenesis of IDD and may shed light on future therapeutic approaches.

Development and characterization of a recombinant silk network for 3D culture of immortalized and fresh tumor-derived breast cancer cells.

Traditional cancer models rely on 2D cell cultures or 3D spheroids, which fail to recapitulate cell-extracellular matrix (ECM) interactions, a key element of tumor development. Existing hydrogel-based 3D alternatives lack mechanical support for cell growth and often suffer from low reproducibility. Here we report a novel strategy to make 3D models of breast cancer using a tissue-like, well-defined network environment based on recombinant spider silk, functionalized with a cell adhesion motif from fibronectin (FN-silk). With this approach, the canonical cancer cells SK-BR-3, MCF-7, and MDA-MB-231, maintain their characteristic expression of markers (i.e., ERα, HER2, and PGR) while developing distinct morphology. Transcriptomic analyses demonstrate how culture in the FN-silk networks modulates the biological processes of cell adhesion and migration while affecting physiological events involved in malignancy, such as inflammation, remodeling of the ECM, and resistance to anticancer drugs. Finally, we show that integration in FN-silk networks promotes the viability of cells obtained from the superficial scraping of patients' breast tumors.

ECM-Like Adhesive Hydrogel for the Regeneration of Large Corneal Stromal Defects.

The repair of large-diameter corneal stroma defects is a major clinical problem. Although some studies have attempted to use hydrogels to repair corneal damage, most of these hydrogels can only be used for focal stromal defects that are ≤3.5mm in diameter due to poor hydrogel adhesion. Here, a photocurable adhesive hydrogel that mimics the extracellular matrix (ECM) with regard to composition for repairing 6mm-diameter corneal stromal defects in rabbits is investigated. This ECM-like adhesive can be rapidly cured after light exposure, with high light transmittance and good mechanical properties. More importantly, this hydrogel maintains the viability and adhesion of cornea-derived cells and promotes their migration in vitro in 2D and 3D culture environments. Proteomics analysis confirms that the hydrogel promotes cell proliferation and ECM synthesis. Furthermore, in rabbit corneal stromal defect repair experiments, it is proven by histological and proteomic analysis that this hydrogel can effectively promote corneal stroma repair, reduce scar formation, and increase corneal stromal-neural regeneration at the sixmonths follow-up. This work demonstrates the great application of ECM-like adhesive hydrogels for the regeneration of large-diameter corneal defects.

The role of flavonoids in the regulation of epithelial-mesenchymal transition in cancer: A review on targeting signaling pathways and metastasis.

One of the hallmarks of cancer is metastasis, a process that entails the spread of cancer cells to distant regions in the body, culminating in tumor formation in secondary organs. Importantly, the proinflammatory environment surrounding cancer cells further contributes to cancer cell transformation and extracellular matrix destruction. During metastasis, front-rear polarity and emergence of migratory and invasive features are manifestations of epithelial-mesenchymal transition (EMT). A variety of transcription factors (TFs) are implicated in the execution of EMT, the most prominent belonging to the Snail Family Transcriptional Repressor (SNAI) and Zinc Finger E-Box Binding Homeobox (ZEB) families of TFs. These TFs are regulated by interaction with specific microRNAs (miRNAs), as miR34 and miR200. Among the several secondary metabolites produced in plants, flavonoids constitute a major group of bioactive molecules, with several described effects including antioxidant, antiinflammatory, antidiabetic, antiobesogenic, and anticancer effects. This review scrutinizes the modulatory role of flavonoids on the activity of SNAI/ZEB TFs and on their regulatory miRNAs, miR-34, and miR-200. The modulatory role of flavonoids can attenuate mesenchymal features and stimulate epithelial features, thereby inhibiting and reversing EMT. Moreover, this modulation is concomitant with the attenuation of signaling pathways involved in diverse processes as cell proliferation, cell growth, cell cycle progression, apoptosis inhibition, morphogenesis, cell fate, cell migration, cell polarity, and wound healing. The antimetastatic potential of these versatile compounds is emerging and represents an opportunity for the synthesis of more specific and potent agents.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Exosomal microRNAs from Mesenchymal Stem Cells: Novel Therapeutic Effect in Wound Healing.

Wound healing is a complicated biological process that leads to the regeneration of damaged skin tissue. Determining the methods to promote wound healing has become a hot topic in medical cosmetology and tissue repair research. Mesenchymal stem cells (MSCs) are a group of stem cells with the potential of self-renewal and multi-differentiation. MSCs transplantation has a broad application prospect in wound healing therapy. Many studies have demonstrated that the therapeutic capacity of MSCs is mainly mediated by paracrine actions. Exosomes (EXOs), which are nanosized vesicles carrying a variety of nucleic acids, proteins and lipids, are an important component of paracrine secretion. It has been demonstrated that exosomal microRNAs (EXO-miRNAs) play a key role in the function of exosomes. In this review, we focus on current research on miRNAs from MSC-derived exosomes (MSC-EXO miRNAs) in terms of sorting, releasing and function and their effects on inflammation regulation, epidermal cell function, fibroblast function, and extracellular matrix formation. At last, we discuss the current attempts to improve the treatment of MSC-EXO-miRNAs. Many studies have demonstrated that MSC-EXO miRNAs play a key role in promoting wound healing. They have been shown to regulate inflammation response, enhance epidermal cell proliferation and migration, stimulate fibroblast proliferation and collagen synthesis, and regulate extracellular matrix formation. Besides, there have been a number of strategies developed to promote MSC-EXO and MSC-EXO miRNAs for wound healing treatment. Utilizing the association of exosomes from MSCs with miRNAs may be a promising strategy to promote trauma healing. MSC-EXO miRNAs may provide a new approach to promote wound healing and improve the quality of life for patients with skin injuries.

Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5h to ∼1.5h. Loss of K2 inhibits focal adhesion turnover and β1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

Extrusion-based 3D printing of biodegradable, osteogenic, paramagnetic, and porous FeMn-akermanite bone substitutes.

The development of biodegradable Fe-based bone implants has rapidly progressed in recent years. Most of the challenges encountered in developing such implants have been tackled individually or in combination using additive manufacturing technologies. Yet not all the challenges have been overcome. Herein, we present porous FeMn-akermanite composite scaffolds fabricated by extrusion-based 3D printing to address the unmet clinical needs associated with Fe-based biomaterials for bone regeneration, including low biodegradation rate, MRI-incompatibility, mechanical properties, and limited bioactivity. In this research, we developed inks containing Fe, 35 wt% Mn, and 20 or 30 vol% akermanite powder mixtures. 3D printing was optimized together with the debinding and sintering steps to obtain scaffolds with interconnected porosity of 69%. The Fe-matrix in the composites contained the γ-FeMn phase as well as nesosilicate phases. The former made the composites paramagnetic and, thus, MRI-friendly. The in vitro biodegradation rates of the composites with 20 and 30 vol% akermanite were respectively 0.24 and 0.27mm/y, falling within the ideal range of biodegradation rates for bone substitution. The yield strengths of the porous composites stayed within the range of the values of the trabecular bone, despite in vitro biodegradation for 28 d. All the composite scaffolds favored the adhesion, proliferation, and osteogenic differentiation of preosteoblasts, as revealed by Runx2 assay. Moreover, osteopontin was detected in the extracellular matrix of cells on the scaffolds. Altogether, these results demonstrate the remarkable potential of these composites in fulfilling the requirements of porous biodegradable bone substitutes, motivating future in vivo research. STATEMENT OF SIGNIFICANCE: We developed FeMn-akermanite composite scaffolds by taking advantage of the multi-material capacity of extrusion-based 3D printing. Our results demonstrated that the FeMn-akermanite scaffolds showed an exceptional performance in fulfilling all the requirements for bone substitution in vitro, i.e., a sufficient biodegradation rate, having mechanical properties in the range of trabecular bone even after 4 weeks biodegradation, paramagnetic, cytocompatible and most importantly osteogenic. Our results encourage further research on Fe-based bone implants in in vivo.

In vitro construction of liver organoids with biomimetic lobule structure by a multicellular 3D bioprinting strategy

Liver disease is one of the serious threats to human life and health. Three-dimensional (3D) liver models, which simulate the structure and function of natural liver tissue in vitro, have become a common demand in medical, scientific and pharmaceutical fields nowadays. However, the complex cellular composition and multi-scale spatial arrangement of liver tissue make it extremely challenging to construct liver models in vitro. According to HepaRG preference and printing strategy, the formulation of bioink system with opposite charge is optimized. The sodium alginate-based bioink 1 and dipeptide-based bioink 2 are used to ensure structural integrity and provide flexible designability, respectively. The HepaRG/HUVECs/LX-2-laden liver organoids with biomimetic lobule structure are fabricated by a multicellular 3D droplet-based bioprinting strategy, to mimic the cell heterogeneity, spatial structure and extracellular matrix (ECM) features. The liver organoids can maintain structural integrity and multicellular distribution within the printed lobule-like structure after 7 days of culture. Compared with the 2D monolayer culture, the constructed 3D organoids show high cell viability, ALB secretion and urea synthesis levels. This study provides a droplet-based and layer-by-layer 3D bioprinting strategy for in vitro construction of liver organoids with biomimetic lobule structure, giving meaningful insights in the fields of new drugs, disease modelling, and tissue regeneration.

Abstract 461: Alcohol Modulates Chemokine- And Hypoxia-induced Endothelial-to-mesenchymal Transition In Vitro And Following Ligation Injury In Vivo

Endothelial-to-mesenchymal transition (EndMT) plays a role in arteriosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol consumption (i.e., 1-3 drinks/d range) protects against endothelial dysfunction and arteriosclerosis, in part by stimulating Notch, but whether it does so by influencing EndMT is unknown. Our aim was to address this question. Human coronary artery endothelial cells (HCAEC) (p5-p14) were treated (7 d) with transforming growth factor β (TGFβ, 10 ng/ml) or exposed to hypoxia (5% O 2 ), in the absence or presence of ‘moderate’ level EtOH (25 mM), or the Notch ligand DLL4 +/- the Notch inhibitor DAPT. EndMT was assessed by determining endothelial marker CD31 and smooth muscle cell marker αSMA expression by immunohistochemistry and qRT-PCR, as well as by cell morphology and extracellular matrix protein Fibronectin production. Both TGFβ and hypoxia induced EndMT of HCAEC as determined by simultaneously decreased CD31 and increased αSMA expression, together with a change in morphology from cobblestone (typical of endothelial cells) to spindle-shaped (typical of mesenchymal cells), and increased fibronectin expression. EtOH inhibited EndMT caused by both TGFβ and hypoxia as indicated by maintenance of CD31 expression and cobblestone morphology, and attenuated αSMA and fibronectin expression in the EtOH treated group vs controls. Similar to EtOH, treatment with DLL4 inhibited TGFβ-induced myogenic αSMA and fibronectin mRNA expression in HCAEC, effects blocked by DAPT. Following carotid ligation in C57Bl/6 mice, ~25% of neointimal and medial cells in the remodeled vessel co-expressed αSMA and CD31 (indicative of myo-endothelial cells), suggesting the involvement of EndMT. In the ‘daily moderate’ alcohol group (2 drinks per day equivalent; peak BAC=15 mM) carotid neointimal hyperplasia was reduced compared to controls concomitant with fewer (<10%) αSMA + /CD31 + myo-endothelial cells suggesting repression of EndMT in that cohort. In conclusion, these data illuminate an inhibitory effect of alcohol on EndMT, potentially mediated via Notch. This effect on endothelial transdifferentiation may underlie, in part, the protective effect of moderate alcohol consumption on CVD.

Redox sensitive thiols identified on Jagged1 regulate endothelial cell cycle, adhesion, and extracellular matrix proteins

Redox sensitive thiols identified on Jagged1 regulate endothelial cell cycle, adhesion, and extracellular matrix proteins

Abstract 112: Pcsk6 Ablation In Vascular Smooth Muscle Cells Promotes Abdominal Aortic Aneurysm Development And Rupture

Background and Aim: We have previously shown that Proprotein Convertase Subtilisin/Kexin type 6 (Pcsk6) is strongly enriched in atherosclerotic plaques and plays a key role in lesion vulnerability and vessel wall healing by regulating vascular smooth muscle cell (VSMC) migration, proliferation, and extracellular matrix remodeling. Considering the broad importance of these processes in vascular pathologies, this study aimed to investigate the expression and function of Pcsk6 in the abdominal aortic aneurysm (AAA). Methods and Results: Bioinformatic analysis of human transcriptome microarray and RNA-sequencing data from human AAA biobanks of the Karolinska Institute and the Technical University of Munich have demonstrated a significantly increased level of Pcsk6 transcript in AAA tissues compared to controls, and even higher levels in ruptured AAAs. Single-cell RNA sequencing data demonstrated a significantly increased level of Pcsk6 transcript in fibroblasts and T cells but decreased in VSMCs. To further clarify if Pcsk6 dysregulation in VSMCs is a pathological reaction or if it has a causal role in AAA development and aortic dissection, we performed pilot animal studies: 1) peri-adventitial porcine pancreatic elastase (PPE) induced AAA model on male VSMC conditional Tagln-Pcsk6 deficient and control mice; 2) AngII infusion model on male Tagln-Pcsk6 deficient and control mice with ApoE-/- background. Interestingly, although no difference in baseline blood pressure, aortic dilatation, and constriction was found between Tagln-Pcsk6 deficient and control mice, ultrasound imaging and histology analyses showed that mice with Pcsk6 deficiency in VSMCs manifested remarkably larger AAA, as well as significantly increased rupture and dissection rate. Mechanistically, RNA sequencing analysis indicated significant inhibition of extracellular matrix components and biosynthesis signaling in aortas from Tagln-Pcsk6 deficient mice. Conclusion: PCSK6 expressed by VSMCs plays a substantial role in AAA progression and aortic dissection, opening new avenues for the therapy of aortic disease. Further mechanistic studies are necessary before this translational aspect can be evaluated.

SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process.

Abstract Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease. CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD