Unmasking of molecular players: proteomic profiling of vitreous humor in pathologic myopia.

BACKGROUND: This study aimed to identify the differentially expressed proteins in the vitreous humor (VH) of eyes with and without pathologic myopia (PM), providing insights into the molecular pathogenesis. METHODS: A cross-sectional, observational study was conducted. VH samples were collected from patients undergoing vitrectomy for idiopathic epiretinal membrane (ERM), macular hole (MH), or myopic retinoschisis (MRS). Label-free quantitative proteomic analysis identified differential protein expression, with validation using ELISA. RESULTS: The proteomic profiling revealed significantly higher expressions of tubulin alpha 1a (TUBA1A) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) in PM groups (MH-PM, MRS-PM) compared to controls (MH, ERM). Conversely, xylosyltransferase 1 (XYLT1), versican core protein (VCAN), and testican-2 (SPOCK2) expressions were lower in PM. ELISA validation confirmed these findings. CONCLUSIONS: Our study provides novel insights into the molecular mechanisms of PM. The differentially expressed proteins EEF1A1, TUBA1A, XYLT1, VCAN, and SPOCK2 may play crucial roles in chorioretinal cell apoptosis, scleral extracellular matrix (ECM) synthesis, and scleral remodeling in PM. These proteins represent potential new targets for therapeutic intervention in PM, highlighting the importance of further investigations to elucidate their functions and underlying mechanisms in disease pathogenesis.

Hydrogen Ion Capturing Hydrogel Microspheres for Reversing Inflammaging.

Inflammaging is deeply involved in aging-related diseases and can be destructive during aging. The maintenance of pH balance in the extracellular microenvironment can alleviate inflammaging and repair aging-related tissue damage. In this study, the hydrogen ion capturing hydrogel microsphere (GMNP) composed of mineralized transforming growth factor-ß (TGF-ß) and catalase (CAT) nanoparticles is developed via biomimetic mineralization and microfluidic technology for blocking the NLRP3 cascade axis in inflammaging. This GMNP can neutralize the acidic microenvironment by capturing excess hydrogen ions through the calcium carbonate mineralization layer. Then, the subsequent release of encapsulated TGF-ß and CAT can eliminate both endogenous and exogenous stimulus of NLRP3, thus suppressing the excessive activation of inflammaging. In vitro, GMNP can suppress the excessive activation of the TXNIP/NLRP3/IL-1ß cascade axis and enhance extracellular matrix (ECM) synthesis in nucleus pulposus cells. In vivo, GMNP becomes a sustainable and stable niche with microspheres as the core to inhibit inflammaging and promote the regeneration of degenerated intervertebral discs. Therefore, this hydrogen ion-capturing hydrogel microsphere effectively reverses inflammaging by interfering with the excessive activation of NLRP3 in the degenerated tissues.

SPARC promotes fibroblast proliferation, migration, and collagen production in keloids by inactivation of p53.

BACKGROUND: Keloid, an aggressive fibroproliferative disease of the skin, is usually caused by infectious skin diseases, burns, and trauma. OBJECTIVE: This study aimed to assess the effect of SPARC on the keloid pathogenesis. METHODS: In normal skin and keloid scar tissues, changes in SPARC expression were analysed by qRT-PCR, western blotting, and immunohistochemistry. Keloid fibroblasts were isolated from human keloid tissue. GSEA was performed to investigate the signalling pathways related to SPARC. Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine, transwell assay, and scratching assays were used to assess fibroblast proliferation and migration. Changes in α-SMA, fibronectin, collagen I, and collagen III levels were examined in fibroblasts by western blotting. RESULTS: SPARC expression was upregulated in keloid scar tissues. In fibroblasts, cell proliferation, migration, collagen production, and extracellular matrix (ECM) synthesis were promoted by SPARC overexpression, whereas SPARC knockdown resulted a converse result. GSEA showed that SPARC regulates the p53 pathway. In keloid scar tissues, there was a negative correlation between SPARC and p53 expression. p53 expression was decreased by SPARC overexpression, whereas SPARC knockdown increased p53 expression. Furthermore, the effects of SPARC on the fibroblast phenotype were reversed by p53 overexpression. CONCLUSIONS: Fibroblast proliferation, migration, and ECM synthesis were promoted by SPARC overexpression, which was achieved by regulating the p53 pathway. Our findings provide new therapeutic targets for keloids.

NFκB inhibition to lift the mechano-competence of mesenchymal stromal cell-derived neocartilage toward articular chondrocyte levels.

BACKGROUND: Fully functional regeneration of skeletal defects by multipotent progenitor cells requires that differentiating cells gain the specific mechano-competence needed in the target tissue. Using cartilage neogenesis as an example, we asked whether proper phenotypic differentiation of mesenchymal stromal cells (MSC) into chondrocytes in vitro will install the adequate biological mechano-competence of native articular chondrocytes (AC). METHODS: The mechano-competence of human MSC- and AC-derived neocartilage was compared during differentiation for up to 35 days. The neocartilage layer was subjected to physiologic dynamic loading in a custom-designed bioreactor and assayed for mechano-sensitive gene and pathway activation, extracellular matrix (ECM) synthesis by radiolabel incorporation, nitric oxide (NO) and prostaglandin E2 (PGE2) production. Input from different pathways was tested by application of agonists or antagonists. RESULTS: MSC and AC formed neocartilage of similar proteoglycan content with a hardness close to native tissue. Mechano-stimulation on day 21 and 35 induced a similar upregulation of mechano-response genes, ERK phosphorylation, NO production and PGE2 release in both groups, indicating an overall similar transduction of external mechanical signals. However, while AC maintained or enhanced proteoglycan synthesis after loading dependent on tissue maturity, ECM synthesis was always significantly disturbed by loading in MSC-derived neocartilage. This was accompanied by significantly higher COX2 and BMP2 background expression, > 100-fold higher PGE2 production and a weaker SOX9 stimulation in response to loading in MSC-derived neocartilage. Anabolic BMP-pathway activity was not rate limiting for ECM synthesis after loading in both groups. However, NFκB activation mimicked the negative loading effects and enhanced PGE2 production while inhibition of catabolic NFκB signaling rescued the load-induced negative effects on ECM synthesis in MSC-derived neocartilage. CONCLUSIONS: MSC-derived chondrocytes showed a higher vulnerability to be disturbed by loading despite proper differentiation and did not acquire an AC-like mechano-competence to cope with the mechanical stress of a physiologic loading protocol. Managing catabolic NFκB influences was one important adaptation to install a mechano-resistance closer to AC-derived neocartilage. This new knowledge asks for a more functional adaptation of MSC chondrogenesis, novel pharmacologic co-treatment strategies for MSC-based clinical cartilage repair strategies and may aid a more rational design of physical rehabilitation therapy after AC- versus MSC-based surgical cartilage intervention.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p.

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

The inclusion of leukocytes into platelet rich plasma reduces scaffold stability and hinders extracellular matrix remodelling.

BACKGROUND: Scaffolds should have controllable degradation rate and allow cells to produce their own extracellular matrix. Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of PRPs. The aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF) may alter the process of fibrinolysis. The effect of different combinations of cellular phenotypes with PRGF and L-PRP clots on both the fibrinolysis and matrix deposition process was also determined. METHODS: PRGF and L-PRP clots were incubated for 14 days and D-dimer and type I collagen were determined in their conditioned media to evaluate clots' stability. For remodelling assays, gingival fibroblasts, alveolar osteoblasts and human umbilical vein endothelial cells (HUVEC) were seeded onto the two types of clots for 14 days. D-dimer, type I collagen, and laminin α4 were measured by ELISA kits in their conditioned media. Morphological and histological analysis were also performed. Cell proliferation was additionally determined RESULTS: PRGF clots preserved their stability as shown by the low levels of both D-dimer and collagen type I compared to those obtained for L-PRP clots. The inclusion of both gingival fibroblasts and alveolar osteoblasts stimulated a higher fibrinolysis in the PRGF clots. In contrast to this, the degradation rates of both PRGF and L-PRP clots remained unchanged after culturing with the endothelial cells. In all cases, type I collagen and laminin α4 levels were in line with the degree of clots' degradation. In all phenotypes, cell proliferation was significantly higher in PRGF than in L-PRP clots. CONCLUSION: The inclusion of leukocytes in PRGF scaffolds reduced their stability, decreased cell number and slowed down cell remodelling.

PLCγ1 inhibition-driven autophagy of IL-1ß-treated chondrocyte confers cartilage protection against osteoarthritis, involving AMPK, Erk and Akt.

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1ß for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.

Lysyl oxidase inhibits TNF-α induced rat nucleus pulposus cell apoptosis via regulating Fas/FasL pathway and the p53 pathways.

AIMS: Intervertebral disc degeneration (IVDD) has been regarded as the main cause of low back pain, which affects 80% of adults and still lack effective treatment. In IVDD, nucleus pulposus (NP) cell apoptosis has widely existed. Lysyl oxidase (LOX) has been demonstrated to protect chondrocyte against apoptosis in the TNF-α-treated human chondrocytes. Therefore, in this study, we investigated the anti-apoptosis effect of LOX on TNF-α-treated rat NP cells. MAIN METHODS: Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot analyses were used to detect the expression of LOX in TNF-α-treated rat NP cells. Then, the toxicity of exogenous LOX and its protective effect was evaluated by Cell Counting kit-8 (CCK-8). NP cell apoptosis was evaluated by flow cytometry analysis and TUNEL assay. The regulatory effects of LOX on the expression of extracellular matrix (ECM) molecules in TNF-α-treated rat NP cells were measured by RT-qPCR, western blot, and ELISA analyses. The molecular mechanism of LOX in regulating NP cell apoptosis was investigated by RT-qPCR and western blot analyses. KEY FINDINGS: The expression of LOX in TNF-α-treated rat NP cells was significantly decreased. Exogenous LOX preserved the cell viability, reduced the rate of apoptosis and improved the ECM secretion in TNF-α-treated rat NP cells. Further molecular mechanism investigation showed that LOX inhibited the Fas/FasL and p53 pathways. SIGNIFICANCES: LOX played an anti-apoptotic role in TNF-α-treated rat NP cells which could be a promising reagent in IVDD treatment.

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun-protected and sun-exposed body sites.

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun-protected) and photoaged (sun-exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun-protected and sun-exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.

Programmable Hydrogels for Cell Encapsulation and Neo-Tissue Growth to Enable Personalized Tissue Engineering.

Biomimetic and biodegradable synthetic hydrogels are emerging as a promising platform for cell encapsulation and tissue engineering. Notably, synthetic-based hydrogels offer highly programmable macroscopic properties (e.g., mechanical, swelling and transport properties) and degradation profiles through control over several tunable parameters (e.g., the initial network structure, degradation kinetics and behavior, and polymer properties). One component to success is the ability to maintain structural integrity as the hydrogel transitions to neo-tissue. This seamless transition is complicated by the fact that cellular activity is highly variable among donors. Thus, computational models provide an important tool in tissue engineering due to their unique ability to explore the coupled processes of hydrogel degradation and neo-tissue growth across multiple length scales. In addition, such models provide new opportunities to develop predictive computational tools to overcome the challenges with designing hydrogels for different donors. In this report, programmable properties of synthetic-based hydrogels and their relation to the hydrogel's structural properties and their evolution with degradation are reviewed. This is followed by recent progress on the development of computational models that describe hydrogel degradation with neo-tissue growth when cells are encapsulated in a hydrogel. Finally, the potential for predictive models to enable patient-specific hydrogel designs for personalized tissue engineering is discussed.

Blockade of extracellular heat shock protein 90α by 1G6-D7 attenuates pulmonary fibrosis through inhibiting ERK signaling.

Pulmonary fibrosis is characterized by lung fibroblast activation and ECM deposition and has a poor prognosis. Heat shock protein 90 (Hsp90) participates in organ fibrosis, and extracellular Hsp90α (eHsp90α) promotes fibroblast activation and migration. This study aimed to investigate whether a selective anti-Hsp90α monoclonal antibody, 1G6-D7, could attenuate lung fibrosis and whether 1G6-D7 presents a protective effect by inactivating the profibrotic pathway. Our results showed that eHsp90α was increased in mice with BLM-induced pulmonary fibrosis and that 1G6-D7 attenuated inflammation and collagen deposition in the lung. TGF-ß1 induced eHsp90α secretion, concomitantly promoting HFL-1 activation and ECM synthesis. 1G6-D7-mediated inhibition of eHsp90α significantly blocked these effects, meanwhile inhibiting downstream profibrotic pathways such as ERK, Akt, and P38. Human recombinant (hr)Hsp90α mimicked the effects of TGF-ß1, by activating profibrotic pathways and by upregulating LRP-1. Moreover, ERK inhibition effectively blocked the effect of (hr)Hsp90α. In conclusion, 1G6-D7 significantly protects against BLM-induced pulmonary fibrosis by ameliorating fibroblast activation and ECM production, which may be through blocking ERK signaling. Our results suggest a safer molecular therapy, 1G6-D7, in pulmonary fibrosis.