Collagen Secretion Research Articles

In vitro bioprinted 3D model enhancing osteoblast-to-osteocyte differentiation

In vitrobone models are pivotal for understanding tissue behavior and cellular responses, particularly in unravelling certain pathologies' mechanisms and assessing the impact of new therapeutic interventions. A desirablein vitrobone model should incorporate primary human cells within a 3D environment that mimics the mechanical properties characteristics of osteoid and faithfully replicate all stages of osteogenic differentiation from osteoblasts to osteocytes. However, to date, no bio-printed model using primary osteoblasts has demonstrated the expression of osteocytic protein markers. This study aimed to develop bio-printedin vitromodel that accurately captures the differentiation process of human primary osteoblasts into osteocytes. Given the considerable impact of hydrogel stiffness and relaxation behavior on osteoblast activity, we employed three distinct cross-linking solutions to fabricate hydrogels. These hydrogels were designed to exhibit either similar elastic behavior with different elastic moduli, or similar elastic moduli with varying relaxation behavior. These hydrogels, composed of gelatin (5% w/v), alginate (1%w/v) and fibrinogen (2%w/v), were designed to be compatible with micro-extrusion bioprinting and proliferative. The modulation of their biomechanical properties, including stiffness and viscoelastic behavior, was achieved by applying various concentrations of cross-linkers targeting both gelatin covalent bonding (transglutaminase) and alginate chains' ionic cross-linking (calcium). Among the conditions tested, the hydrogel with a low elastic modulus of 8 kPa and a viscoelastic behavior over time exhibited promising outcomes regarding osteoblast-to-osteocyte differentiation. The cessation of cell proliferation coincided with a significant increase in alkaline phosphatase activity, the development of dendrites, and the expression of the osteocyte marker PHEX. Within this hydrogel, cells actively influenced their environment, as evidenced by hydrogel contraction and the secretion of collagen I. This bio-printed model, demonstrating primary human osteoblasts expressing an osteocyte-specific protein, marks a significant achievement. We envision its substantial utility in advancing research on bone pathologies, including osteoporosis and bone tumors.

P4HA1: an important target for treating fibrosis related diseases and cancer

Fibrosis is significantly associated with a wide variety of diseases and is involved in their progression. Fibrosis activated under the influence of different combinations of factors is considered a double-edged sword. Although there has been much research on organ fibrosis in recent years, a variety of organ fibrosis diseases and cancers are not well controlled in terms of prevention, treatment, and prognosis. Clinical studies still lack exploration and discovery of effective targets for the pathogenesis of organ fibrosis. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is a protein kinase and the synthesis and secretion of collagen are related to the sustained activation of P4HA1. As further studies are being conducted, the potential role of P4HA1 in the development of fibrosis-associated diseases and cancer is becoming clear. Consequently, we conducted a systematic review and discussion on the role of P4HA1 in the pathogenesis of various fibrosis-related diseases and cancers. We reviewed the possible strategies of P4HA1 in the diagnosis and treatment of fibrosis-related diseases and cancers, and analyzed its potential relevance as a biomarker in the diagnosis and treatment of fibrosis-related diseases and cancer.

Palmitic Acid Induced a Dedifferentiation Profile at the Transcriptome Level: A Collagen Synthesis but no Triglyceride Accumulation in Hepatocyte-Like Cells Derived From Human-Induced Pluripotent Stem Cells Cultivated Inside Organ on a Chip.

Nonalcoholic fatty liver disease (NAFLD) is one of the main causes of critical liver diseases leading to steatosis, steatohepatitis, fibrosis, and ultimately to liver cirrhosis and hepatic carcinoma. In this study, the effect of palmitic acid (PA), one of the most abundant dietary fatty acids, was investigated using an organ-on-a-chip (OoC) technology on hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSCs). After 1 week of hepatic maturation, followed by 1 week of exposure, the transcriptomic analysis showed lower liver transcription factor activity. It also revealed that 318 genes were differentially expressed between the control and 0.5-mM PA conditions. The 0.5-mM PA conditions were characterized by the downregulation of hepatic markers (liver transcription factors, phase I and phase II metabolism genes) of lipidic genes (metabolism and transport). In parallel, the 0.5-mM PA treatment upregulated several extracellular matrix genes (such as collagen genes). The physiopathological staining demonstrated no lipid accumulation in our model and confirmed the secretion of collagen in the 0.5-mM PA conditions. However, the production of albumin, the metabolic biotransformation by the cytochrome P450 enzymes, and the biliary acid concentrations were not altered by the PA treatments. Overall, our data illustrated the response to PA characterized by an early stage of dedifferentiation observed at the transcriptomic levels associated with a modification of the collagenic profile but without lipid accumulation. We believe that our model provides new insight of the onset of palmitic lipotoxicity in the early stage of NAFLD.

“Janus” PEEK implant with sandwich Mg-containing coating for infected tissue repair

Having good antibacterial properties and promoting soft and hard tissue repair are the keys to successful implantation of intraosseous transcutaneous. Polyether ether ketone (PEEK) is a class of FDA-approved polymer implants. However, the surface of PEEK is bioinert, which is easy to cause postoperative infection and poor tissue integration. In this study, polypyrrole (Ppy) was polymerized on sulfonated PEEK, Mg3(PO4)2 nanosheets were grown in situ on one side, and polycaprolactone (PCL) was then spun on the surface to form a Janus-like surface on PEEK. The Ppy/Mg3(PO4)2/PCL composite coating could inhibit bacterial adhesion, and the excellent photothermal properties of Ppy/Mg3(PO4)2/PCL and Ppy coatings further promote the removal of bacteria due to the accumulated heat. After the infection was eliminated, the Janus-like surface of modified PEEK switched macrophages to anti-proinflammatory response and promoted both soft and hard tissue repair. The Ppy modified sulfonated PEEK could promote collagen secretion in the soft tissue, while the PCL films on Ppy/Mg3(PO4)2/PCL was densified by temperature response under near-infrared light treatment to close the exposed interface of Mg3(PO4)2 nanosheets that was more conducive to bone repair. In summary, PEEK with Janus-like surface consisting of Ppy/Mg3(PO4)2/PCL and Ppy has multiple biological functions of sequential antibacterial and soft and hard tissue repair, and is a promising candidate material for intraosseous transcutaneous implants.

TMEM39A and TMEM131 facilitate bulk transport of ECM proteins through large COPII vesicle formation

The growth of Caenorhabditis elegans involves multiple molting processes, during which old cuticles are shed, and new cuticles are rapidly formed. This process requires the regulated bulk secretion of cuticle components. The transmembrane protein-39 (TMEM-39) mutant exhibits distinct dumpy and ruptured phenotypes characterized by notably thin cuticles. TMEM-39 primarily co-localizes with the coat protein II complex (COPII) in large vesicles rather than small COPII vesicles. These TMEM-39-associated large vesicles (TMEM-39-LVs) form robustly during the molting period and co-localize with various extracellular matrix (ECM) components, including BLI-1 collagen, BLI-3 dual oxidase, and carboxypeptidases. Through immunoprecipitation using TMEM39A-FLAG and proteomics analysis in human sarcoma cells, we identify TMEM39A-associated proteins, including TMEM131. Knockdown of TMEM131 results in reduced TMEM39A-LV formation and collagen secretion in both C. elegans and human sarcoma cells, indicating a cooperative role between TMEM39A and TMEM131 in the secretion of extracellular components through the formation of large COPII vesicles. Given the conservation of TMEM39A and its associated proteins between C. elegans and humans, TMEM39A-LV may represent a fundamental machinery for rapid and extensive secretion across metazoans.

Integrating single-cell sequencing and genetic lineage tracing reveals endothelial plasticity during atrial remodeling

Abstract Background Atrial fibrillation (AF), as the most burdensome cardiac arrhythmia, could lead to the significant mortality and morbidity. Atrial fibrosis is a well-recognized pathophysiological factor of the development and progression of AF, which also hampers its treatment. However, the underlying mechanisms of this intricate process remain largely unknown. Purpose This study sought to reveal the cellular composition of AF substrate and investigate the vital contributors to atrial structural remodeling. Methods Left atrial appendages from three patients with AF undergoing surgical ablation and three individuals with sinus rhythm undergoing heart transplantation were subjected to 10X single-cell RNA sequencing (scRNA-seq). Another five public scRNA-seq or snRNA-seq datasets of potentially remodeled right or left appendages were retrieved across three publications. The vital contributors of atrial structural remodeling and their characteristics were identified by intercellular communication analysis and trajectory inference. Transverse aortic constriction (TAC) was used to induce atrial remodeling in Cdh5-CreERT2;RFP mice, followed by scRNA-seq of endothelial cells (ECs). Human primary atrial ECs and fibroblasts were isolated for cell-cell interaction experiments. ECs-specific mouse models of Transforming growth factor beta 1 (TGFB1) (knockout and overexpression) were generated. Electrophysiological and histology analyses were performed to determine the consequences of ECs-derived TGFB1 gain and loss of function in the atrium. Results The comprehensive analysis of different scRNA-seq datasets revealed that ECs played an important regulatory role in the homeostasis of atrial substrate and displayed remarkable mesenchymal activation during atrial remodeling, which was further confirmed by TAC mice. Immunofluorescence staining and high-content screening suggested that the complete transition from ECs to mesenchymal cells slightly contributed to atrial remodeling. However, TGFB1 secreted from mesenchymal activated ECs significantly promoted activation, proliferation, and collagen secretion of fibroblasts. We discovered that overexpression of TGFB1 in ECs of mice significantly increased atrial fibrosis, and thus prolonging AF duration. Besides, knockout of TGFB1 in ECs of mice underwent TAC significantly attenuated the atrial fibrosis and decreased the rate of AF. Conclusion Our work demonstrates that mesenchymal ECs-derived Tgfb1 plays an important role in atrial remodeling by regulating the fibroblast function. EC-specific interventions were suggested to facilitate the development of novel strategies for mitigating AF substrate remodeling.

7587 The Estrogen Receptors and ARE/Nrf2 are Involved in Protecting Human Dermal Fibroblasts from Damage Caused by Mitochondrial Disfunction

Abstract Disclosure: A. Darawshe: None. A. Trachtenberg: None. Y. Sharoni: None. Skin ageing is influenced by several factors including environmental exposure and hormonal changes. Reactive oxygen species (ROS), which mediate many of the effects of these factors, can be formed by extrinsic factors, such as sun exposure, or can result from mitochondrial dysfunction as occurs during ageing. Several studies have shown the protective role of estrogens on skin health. The aim of the current study was to examine the damage to dermal fibroblasts by mitochondrially generated ROS, and to study the mechanism of the protective effects of estradiol. Rotenone, a complex I inhibitor, was used to cause mitochondrial dysfunction in human dermal fibroblasts and its effects on mitochondrial and cytosolic ROS levels, mitochondrial respiration, cell death, apoptosis, matrix metalloproteinase-1 (MMP1) and pro-collagen secretion were determined as markers of skin damage. Rotenone caused substantial reduction of respiration, followed by increased mitochondrial and cytosolic ROS which resulted in apoptotic cell death, increased MMP1 secretion and decreased collagen secretion. Pretreatment with estradiol recovered more than 50% of the respiratory activity, reduced mitochondrial and cytosolic ROS levels and MMP1 secretion and increased cell number and collagen secretion. These effects can be partially explained by a cooperative effect of estradiol and rotenone on Antioxidant Response Element (ARE/Nrf2) transcriptional activity, which leads to upregulation of antioxidant proteins such as NQO1 and Trxr1. To determine if estrogen receptors (ER) are involved in the protective effects, we used the ER inhibitor, Fulvestrant. This inhibitor, which completely inhibited ER Response Element reporter activity, partially prevented the protective effects of estradiol. The protective effects of estradiol were Similarly reduced by ML385 and Ochratoxin A, that are inhibitors of the ARE/Nrf2 transcription system. Incubating the cells with Fulvestrant and Ochratoxin A, which inhibit both pathways, completely blocked the protective effect of estradiol. These results suggest that the estrogen receptors and the ARE/Nrf2 signaling pathways are involved in the protective effects of estradiol against the damage caused by rotenone-induced mitochondria generated oxidative stress. Presentation: 6/3/2024

Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells.

The peptide hormone kisspeptin attenuates liver steatosis, metabolic dysfunction-associated steatohepatitis (MASH), and fibrosis in mouse models by signaling via the kisspeptin 1 receptor (KISS1R). However, whether kisspeptin impacts fibrogenesis in the human liver is not known. We investigated the impact of a potent kisspeptin analog (KPA) on fibrogenesis using human precision-cut liver slices (hPCLS) from fibrotic livers from male patients, in human hepatic stellate cells (HSCs), LX-2, and in primary mouse HSCs. In hPCLS, 48 h and 72 h of KPA (3 nM, 100 nM) treatment decreased collagen secretion and lowered the expression of fibrogenic and inflammatory markers. Immunohistochemical studies revealed that KISS1R is expressed and localized to HSCs in MASH/fibrotic livers. In HSCs, KPA treatment reduced transforming growth factor b (TGFβ)-the induced expression of fibrogenic and inflammatory markers, in addition to decreasing TGFβ-induced collagen secretion, cell migration, proliferation, and colony formation. Mechanistically, KISS1R signaling downregulated TGFβ signaling by decreasing SMAD2/3 phosphorylation via the activation of protein phosphatases, PP2A, which dephosphorylates SMAD 2/3. This study revealed for the first time that kisspeptin reverses human hepatic fibrogenesis, thus identifying it as a new therapeutic target to treat hepatic fibrosis.

Transcriptomic profiling of lung fibroblasts in silicosis: Regulatory roles of Nrf2 agonists in a mouse model

Silicosis is an occupational disease caused by long-term inhalation of free silica, resulting in a significant global health burden. Its pathogenesis remains unclear, and there is no effective treatment. Proliferative and activated myofibroblasts play a key role in the development of silicosis. Traditional studies have focused on fibroblast proliferation and collagen secretion, neglecting their functional heterogeneity. With the advancement of omics research, more pathogenic fibroblast subgroups and their functions have been identified. In this study, we applied transcriptomics to analyze gene changes in primary lung fibroblasts during silicosis development using a mouse model. Our results indicate that DEGs are enriched in collagen secretion, ECM synthesis, leukocyte migration, and chemotaxis functions. Altered core genes are associated with immune cell recruitment and cell migration. Nrf2 agonists, known for anti-inflammatory and antioxidant properties, have shown potential therapeutic effects in fibrotic diseases. However, their effects on fibroblasts in silicosis are not fully understood. We used four common Nrf2 agonists to study gene expression changes in lung fibroblasts at the transcriptome level, combined with histopathological and biochemical methods, to investigate their effects on silicosis in mice. Results show that Nrf2 agonists can exert anti-silicosis fibrosis functions by downregulating genes like Fos and Egr1, involved in cell differentiation, proliferation, and inflammation. In conclusion, this study suggests that inflammation-related co-functions of fibroblasts may be a potential mechanism in silicosis pathogenesis. Targeting Nrf2 may be a promising strategy to alleviate oxidative stress and inflammation in silicosis.

Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment.

Cardiac fibrosis is a prevalent pathological process observed in the progression of numerous cardiovascular diseases and is associated with an increased risk of sudden cardiac death. Although the BRD4 inhibitor JQ1 has powerful antifibrosis properties, its clinical application is extremely limited due to its side effects. There remains an unmet need for effective, safe, and low-cost treatments. Here, we present a multifunctional biomimetic nanoparticle drug delivery system (PM&EM nanoparticles) assembled by platelet membranes and erythrocyte membranes for targeted JQ1 delivery in treating cardiac fibrosis. The platelet membrane endows PM&EM nanoparticles with the ability to target cardiac myofibroblasts and collagen, while the participation of the erythrocyte membrane enhances the long-term circulation ability of the formulated nanoparticles. In addition, PM&EM nanoparticles can deliver sufficient JQ1 with controllable release, achieving excellent antifibrosis effects. Based on these advantages, it is demonstrated in both pressures overloaded induced mouse cardiac fibrosis model and MI-induced mouse cardiac fibrosis that injection of the fusion membrane biomimetic nanodrug carrier system effectively reduced fibroblast activation, collagen secretion, and improved cardiac fibrosis. Moreover, it significantly mitigated the toxic and side effects of long-term JQ1 treatment on the liver, kidney, and intestinal tract. Mechanically, bioinformatics prediction and experimental validation revealed that PM&EM/JQ1 NPs reduced liver and kidney damage via alleviated oxidative stress and mitigated cardiac fibrosis via the activation of oxidative phosphorylation activation. These results highlight the potential value of integrating native platelet and erythrocyte membranes as a multifunctional biomimetic drug delivery system for treating cardiac fibrosis and preventing drug side effects.

38P Collablot, a new immunodetection assay for the quantification of collagen VI expression and secretion in cell culture

38P Collablot, a new immunodetection assay for the quantification of collagen VI expression and secretion in cell culture

Topical hADSCs-HA Gel Promotes Skin Regeneration and Angiogenesis in Pressure Ulcers by Paracrine Activating PPARβ/δ Pathway.

Pressure ulcer is common in the bedridden elderly with high mortality and lack of effective treatment. In this study, human-adipose-derived-stem-cells-hyaluronic acid gel (hADSCs-HA gel) was developed and applied topically to treat pressure ulcers, of which efficacy and paracrine mechanisms were investigated through in vivo and in vitro experiments. Pressure ulcers were established on the backs of C57BL/6 mice and treated topically with hADSCs-HA gel, hADSCs, hyaluronic acid, and normal saline respectively. The rate of wound closure was observed continuously during the following 14 days and the wound samples were obtained for Western blot, histopathology, immunohistochemistry, and proteomic analysis. Human dermal fibroblasts (HDFs) and human venous endothelial cells (HUVECs) under normal or hypoxic conditions were treated with conditioned medium of human ADSCs (ADSC-CM), then CCK-8, scratch test, tube formation, and Western blot were conducted to evaluate the paracrine effects of hADSCs and to explore the underlying mechanism. The in vivo data demonstrated that hADSCs-HA gel significantly accelerated the healing of pressure ulcers by enhancing collagen expression, angiogenesis, and skin proliferation. The in vitro data revealed that hADSCs strengthened the proliferation and wound healing capabilities of HDFs and HUVECs, meanwhile promoted collagen secretion and tube formation through paracrine mode. ADSC-CM was also proved to exert protective effects on hypoxic HDFs and HUVECs. Besides, the results of proteomic analysis and Western blot elucidated that lipid metabolism and PPARβ/δ pathway mediated the healing effect of hADSCs-HA gel on pressure ulcers. Our research showed that topical application of hADSCs-HA gel played an important role in dermal regeneration and angiogenesis. Therefore, hADSCs-HA gel exhibited the potential as a novel stem-cell-based therapeutic strategy of treating pressure ulcers in clinical practices.

Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits

BackgroundMesenchymal stem cells (MSCs) have shown a positive effect on Osteoarthritis (OA), but the efficacy is still not significant in clinical. Conventional two-dimensional (2D) monolayer culture method is prone to cause MSCs undergoing replication senescence, which may affect the functions of MSCs. Three-dimensional (3D) culture strategy can sustain cell proliferative capacity and multi-differentiation potential. This study aimed to investigate the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured by 3D hanging drop method on OA.MethodshUC-MSCs were isolated from umbilical cord and cultured by 3D hanging drop method for 48 h. Scanning electron microscopy (SEM) was used to observe gross morphology 2D and 3D hUC-MSCs. Transcriptome comparison of gene expression differences between 2D and 3D hUC-MSCs. GO enrichment analysis, KEGG pathway enrichment analysis and GSEA enrichment analysis were used to analyze the impact of 3D hanging drop culture on the biological functions of hUC-MSCs. Female New Zealand rabbits (n = 12) were divided into 4 groups: Normal group, Model group, 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group. After 8 weeks, the gross and histological appearance of the cartilage was evaluated by safranin O-fast green staining and Mankin scoring system. The expression of type I collagen and type II collagen was detected by immunohistochemistry. The levels of IL-6, IL-7, TNFα, TGFβ1 and IL-10 in the knee joint fluid were tested by ELISA.Results3D hanging drop culture changed cell morphology but did not affect phenotype. The MSCs transcriptome profiles showed that 3D hanging drop culture method enhanced cell-cell contact, improved cell responsiveness to external stimuli and immunomodulatory function. The animal experiment results showed that hUC-MSCs could promote cartilage regeneration compared with Model group. 3D hUC-MSCs treatment group had a higher histological score and significantly increased type II collagen secretion. In addition, 3D hUC-MSCs treatment group increased the expression of anti-inflammatory factors TGFβ1 and IL-10.ConclusionThe above experimental results illustrated that 3D hanging drop culture method could enhance the therapeutic effect of hUC-MSCs, and showed a good clinical application prospect in the treatment of OA.

Pharmacological targeting of AMPK to restore glucose and fatty acid metabolism homeostasis attenuates transplanted kidney fibrosis

Chronic fibrosis often occurs in transplanted kidneys, leading to progressive functional decline. The underlying mechanisms may involve disruption in the metabolism of renal tubular epithelial cells. The liver kinase B1 (LKB1)-AMPK pathway is a pivotal regulatory hub for glucose and fatty acid metabolism and may play a role in transplanted kidney fibrosis, but it has not been reported. In this study we administered fenofibrate, 2-deoxyglucose, or metformin to modulate metabolism in Brown Norway rat kidney transplants and investigated pathways involved in fibrosis using various assays. We identified an impaired LKB1-AMPK pathway within epithelial cells, resulting in perturbed glucose and fatty acid metabolism, collagen secretion, extracellular matrix remodeling, and epithelial-mesenchymal transition. ACOX1, a pivotal enzyme in the fatty acid peroxisomal β-oxidation pathway, played an important role in transplanted renal fibrosis. Furthermore, several metabolism-targeting drugs, particularly metformin, emerged as potent fibrosis inhibitors. Metformin attenuated fibrosis, improved renal function, and reduced inflammation and macrophage infiltration in the transplanted kidneys. These results provide new perspectives for understanding the complex molecular basis underlying transplanted renal fibrosis and developing novel therapeutic strategies.

Bimetallic clusterzymes-loaded dendritic mesoporous silica particle regulate arthritis microenvironment via ROS scavenging and YAP1 stabilization

Clusterzymes are synthetic enzymes exhibiting substantial catalytic activity and selectivity, which are uniquely driven by single-atom constructs. A dramatic increase in antioxidant capacity, 158 times more than natural trolox, is noted when single-atom copper is incorporated into gold-based clusterzymes to form Au24Cu1. Considering the inflammatory and mildly acidic microenvironment characteristic of osteoarthritis (OA), pH-dependent dendritic mesoporous silica nanoparticles (DMSNs) coupled with PEG have been employed as a delivery system for the spatial-temporal release of clusterzymes within active articular regions, thereby enhancing the duration of effectiveness. Nonetheless, achieving high therapeutic efficacy remains a significant challenge. Herein, we describe the construction of a Clusterzymes-DMSNs-PEG complex (CDP) which remarkably diminishes reactive oxygen species (ROS) and stabilizes the chondroprotective protein YAP by inhibiting the Hippo pathway. In the rabbit ACLT (anterior cruciate ligament transection) model, the CDP complex demonstrated inhibition of matrix metalloproteinase activity, preservation of type II collagen and aggregation protein secretion, thus prolonging the clusterzymes' protective influence on joint cartilage structure. Our research underscores the efficacy of the CDP complex in ROS-scavenging, enabled by the release of clusterzymes in response to an inflammatory and slightly acidic environment, leading to the obstruction of the Hippo pathway and downstream NF-κB signaling pathway. This study illuminates the design, composition, and use of DMSNs and clusterzymes in biomedicine, thus charting a promising course for the development of novel therapeutic strategies in alleviating OA.

ARE/Nrf2 Transcription System Involved in Carotenoid, Polyphenol, and Estradiol Protection from Rotenone-Induced Mitochondrial Oxidative Stress in Dermal Fibroblasts.

Skin aging is associated with the increased production of mitochondrial reactive oxygen species (mtROS) due to mitochondrial dysfunction, and various phytonutrients and estrogens have been shown to improve skin health. Thus, the aim of the current study was to examine damage to dermal fibroblasts by chemically induced mitochondrial dysfunction and to study the mechanism of the protective effects of carotenoids, polyphenols, and estradiol. Rotenone, a Complex I inhibitor, caused mitochondrial dysfunction in human dermal fibroblasts, substantially reducing respiration and ATP levels, followed by increased mitochondrial and cytosolic ROS, which resulted in apoptotic cell death, an increased number of senescent cells, increased matrix metalloproteinase-1 (MMP1) secretion, and decreased collagen secretion. Pre-treatment with carotenoid-rich tomato extracts, rosemary extract, and estradiol reversed these effects. These protective effects can be partially explained by a cooperative activation of antioxidant response element (ARE/Nrf2) transcriptional activity by the protective compounds and rotenone, which led to the upregulation of antioxidant proteins such as NQO1. To determine if ARE/Nrf2 activity is crucial for cell protection, we inhibited it using the Nrf2 inhibitors ML385 and ochratoxin A. This inhibition markedly reduced the protective effects of the test compounds by diminishing their effect to reduce cytosolic ROS. Our study results indicate that phytonutrients and estradiol protect skin cells from damage caused by mtROS, and thus may delay skin cell senescence and improve skin health.

Mechanical force regulates the paracrine functions of ADSCs to assist skin expansion in rats

BackgroundIn the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion.MethodsRat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses.ResultsEndothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group.ConclusionsThe optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.

Potential skin anti-aging effects of main phenolic compounds, tremulacin and tremuloidin from Salix chaenomeloides leaves on TNF-α-stimulated human dermal fibroblasts

The genus Salix spp. has long been recognized as a healing herb for its use in treating fever, inflammation, and pain relief, as well as a food source for its nutritional value. In this study, we aimed to explore the potential bioactive natural products in the leaves of Salix chaenomeloides, commonly known as Korean pussy willow, for their protective effects against skin damage, including aging. Utilizing LC/MS-guided chemical analysis of the ethanol extract of S. chaenomeloides leaves, with a focus on major compounds, we successfully isolated two main phenolic compounds, tremulacin (1) and tremuloidin (2). Subsequently, we investigated the protective effects of tremulacin (1) and tremuloidin (2) in TNF-α-stimulated human dermal fibroblasts (HDFs). The results revealed that both tremulacin (1) and tremuloidin (2) inhibited TNF-α-stimulation-induced ROS, suppressed matrix metalloproteinase-1 (MMP-1) expression, and enhanced collagen secretion. This implies that both tremulacin (1) and tremuloidin (2) hold promise as preventive agents against photoaging-induced skin aging. Furthermore, we assessed the activity of mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2), and heme oxygenase 1 (HO-1) to elucidate the mechanism of photoaging inhibition by tremuloidin (2), which exhibited superior efficacy. We found that tremuloidin (2) inhibited ERK and p38 phosphorylation and notably suppressed COX-2 expression while significantly upregulating HO-1 expression. These findings suggest potent anti-inflammatory and antioxidant properties of tremuloidin (2), positioning it as a potential candidate for combating photoaging-induced skin aging.

Mechanical properties and biological behavior of refractory TiZrNbTa medium-entropy and TiZrHfNbTa high-entropy alloy nanofilms on AISI 316L for bone implants

To investigate the feasibility of high-entropy alloys as the coating materials for the application of bone implants, TiZrNbTa refractory medium-entropy alloy (RMEA) and TiZrHfNbTa refractory high-entropy alloy (RHEA) nanofilms were prepared on AISI 316L stainless steel by DC magnetron sputtering and post-annealing treatment. Then, the microstructure, mechanical properties, wear resistance, corrosion resistance, biocompatibility and osteogenic activity of the nanofilms were systematically studied. The results showed that both the films exhibited uniform and dense nanostructures. Post-annealing treatment not only improved the adhesion strength, but also formed an oxidized layer. Notably, owing to the enhanced high-entropy and lattice distortion effects, the RHEA nanofilm showed finer grain sizes, higher hardness (14 GPa), H/E, H3/E2 and better wear resistance. The in vitro experiments proved that RHEA nanofilm had the highest Ecorr (−0.127 V), with the Icorr value was two orders of magnitude lower than substrate. Its osteoblast proliferative capacity was 1.5 times higher than substrate (after culturing for 5 days), and the collagen secretion and extracellular matrix mineralization reached an optimal level, showing excellent osteogenic activity. Therefore, RHEA nanofilm coated biomaterials can be a more attractive candidate for bone replacement.

Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells.

Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. The current methods for detecting GTPase activation using fluorescent proteins rely on the interaction between the GTPase and its effector. Consequently, these methods are not applicable to factors, such as Sar1, where the effector also functions as a GTPase-activating protein. Here, we present a novel method, the Small GTPase ActIvitY ANalyzing (SAIYAN) system, for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at the ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion confined the activated Sar1 at the ER exit sites, implying the importance of Sar1 activation through the ERGIC in collagen secretion.