Exploring the Early Molecular Pathogenesis of Osteoarthritis Using Differential Network Analysis of Human Synovial Fluid.

The molecular mechanisms that drive the onset and development of osteoarthritis (OA) remain largely unknown. In this exploratory study, we used a proteomic platform (SOMAscan assay) to measure the relative abundance of more than 6000 proteins in synovial fluid (SF) from knees of human donors with healthy or mildly degenerated tissues, and knees with late-stage OA from patients undergoing knee replacement surgery. Using a linear mixed effects model, we estimated the differential abundance of 6251 proteins between the three groups. We found 583 proteins upregulated in the late-stage OA, including MMP1, collagenase 3 and interleukin-6. Further, we selected 760 proteins (800 aptamers) based on absolute fold changes between the healthy and mild degeneration groups. To those, we applied Gaussian Graphical Models (GGMs) to analyze the conditional dependence of proteins and to identify key proteins and subnetworks involved in early OA pathogenesis. After regularization and stability selection, we identified 102 proteins involved in GGM networks. Notably, network complexity was lost in the protein graph for mild degeneration when compared to controls, suggesting a disruption in the regular protein interplay. Furthermore, among our main findings were several downregulated (in mild degeneration versus healthy) proteins with unique interactions in the healthy group, one of which, SLCO5A1, has not previously been associated with OA. Our results suggest that this protein is important for healthy joint function. Further, our data suggests that SF proteomics, combined with GGMs, can reveal novel insights into the molecular pathogenesis and identification of biomarker candidates for early-stage OA.

TRPV4 mediates IL-1-induced Ca2+ signaling, ERK activation and MMP expression.

Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.

Delta/Notch signaling in glia maintains motor nerve barrier function and synaptic transmission by controlling matrix metalloproteinase expression.

While the role of barrier function in establishing a protective, nutrient-rich, and ionically balanced environment for neurons has been appreciated for some time, little is known about how signaling cues originating in barrier-forming cells participate in maintaining barrier function and influence synaptic activity. We have identified Delta/Notch signaling in subperineurial glia (SPG), a crucial glial type for Drosophila motor axon ensheathment and the blood-brain barrier, to be essential for controlling the expression of matrix metalloproteinase 1 (Mmp1), a major regulator of the extracellular matrix (ECM). Our genetic analysis indicates that Delta/Notch signaling in SPG exerts an inhibitory control on Mmp1 expression. In the absence of this inhibition, abnormally enhanced Mmp1 activity disrupts septate junctions and glial ensheathment of peripheral motor nerves, compromising neurotransmitter release at the neuromuscular junction (NMJ). Temporally controlled and cell type-specific transgenic analysis shows that Delta/Notch signaling inhibits transcription of Mmp1 by inhibiting c-Jun N-terminal kinase (JNK) signaling in SPG. Our results provide a mechanistic insight into the regulation of neuronal health and function via glial-initiated signaling and open a framework for understanding the complex relationship between ECM regulation and the maintenance of barrier function.

Substance P promotes transforming growth factor-ß-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-ß on collagen type I synthesis without affecting that of IL-1ß on the expression of matrix metalloproteinase-1. This effect of SP on TGF-ß-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-ß-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-ß on collagen type I synthesis without affecting that of interleukin-1ß on the expression of matrix metalloproteinase-1.

Novel Insights into the Catalytic Mechanism of Collagenolysis by Zn(II)-Dependent Matrix Metalloproteinase-1.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12-Expressing Arteriovenous Malformation-Like Vessels.

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.

Identification of CD64 as a marker for the destructive potential of synovitis in osteoarthritis.

OBJECTIVES: OA is characterized by cartilage degeneration and persistent pain. The majority of OA patients present with synovitis, which is associated with increased cartilage damage. Activated synovial macrophages are key contributors to joint destruction. Therefore, a marker that reflects the activation of these cells could be a valuable tool to characterize the destructive potential of synovitis and benefit monitoring of OA. Here, we aimed to investigate the use of CD64 (FcγRI) as a marker to characterize the damaging potential of synovitis in OA. METHODS: Synovial biopsies were obtained from end-stage OA patients that underwent joint replacement surgery. CD64 protein expression and localization was evaluated using immunohistochemistry and immunofluorescence and quantified using flow cytometry. qPCR was performed to measure the expression of FCGR1 and OA-related genes in synovial biopsies, and in primary chondrocytes and primary fibroblasts stimulated with OA conditioned medium (OAS-CM). RESULTS: Our data exposed a wide range of CD64 expression in OA synovium and showed positive correlations between FCGR1 and S100A8, S100A9, IL1B, IL6 and MMP1/2/3/9/13 expression. CD64 protein correlated with MMP1, MMP3, MMP9, MMP13 and S100A9. Furthermore, we observed that synovial CD64 protein levels in source tissue for OAS-CM significantly associated with the OAS-CM-induced expression of MMP1, MMP3 and especially ADAMTS4 in cultured fibroblasts, but not chondrocytes. CONCLUSION: Together, these results indicate that synovial CD64 expression is associated with the expression of proteolytic enzymes and inflammatory markers related to structural damage in OA. CD64 therefore holds promise as marker to characterize the damaging potential of synovitis.

Brain microvascular endothelial cells and blood-brain barrier dysfunction in psychotic disorders.

Although there is convergent evidence for blood-brain barrier (BBB) dysfunction and peripheral inflammation in schizophrenia (SZ) and bipolar disorder (BD), it is unknown whether BBB deficits are intrinsic to brain microvascular endothelial cells (BMECs) or arise via effects of peripheral inflammatory cytokines. We examined BMEC function using stem cell-based models to identify cellular and molecular deficits associated with BBB dysfunction in SZ and BD. Induced pluripotent stem cells (iPSCs) from 4 SZ, 4 psychotic BD and 4 healthy control (HC) subjects were differentiated into BMEC-"like" cells. Gene expression and protein levels of tight junction proteins were assessed. Transendothelial electrical resistance (TEER) and permeability were assayed to evaluate BBB function. Cytokine levels were measured from conditioned media. BMECs derived from human iPSCs in SZ and BD did not show differences in BBB integrity or permeability compared to HC BMECs. Outlier analysis using TEER revealed a BBB-deficit (n = 3) and non-deficit (n = 5) group in SZ and BD lines. Stratification based on BBB function in SZ and BD patients identified a BBB-deficit subtype with reduced barrier function, tendency for increased permeability to smaller molecules, and decreased claudin-5 (CLDN5) levels. BMECs from the BBB-deficit group show increased matrix metallopeptidase 1 (MMP1) activity, which correlated with reduced CLDN5 and worse BBB function, and was improved by tumor necrosis factor α (TNFα) and MMP1 inhibition. These results show potential deficits in BMEC-like cells in psychotic disorders that result in BBB disruption and further identify TNFα and MMP1 as promising targets for ameliorating BBB deficits.

Screening the active ingredients of plants via molecular docking technology and evaluating their ability to reduce skin photoaging.

The active ingredients of plants were screened by molecular docking technology and the result were verified. According to the verification results of molecular docking, the five active ingredients were combined in equal proportions to form a compound drug. In the HaCaT photoaging model, the effects of the compound drug on antioxidant and senescence-associated secretory phenotype (SASP) factors of the NF-κB and MAPK pathways were studied via SOD and MDA kits, DCFH-DA fluorescent probes and ELISA. In the skin photoaging model, the effects of the compound drug on antioxidants and the SASP factors of the NF-κB and MAPK pathways were studied via SOD, MDA, and CAT kits and ELISA. The results revealed that the compound drug increased SOD activity, decreased the MDA content and intracellular ROS, inhibited IL-6 in the NF-κB pathway, and inhibited MMP-1 and collagen I in the MAPK pathway. The results of HE, Masson and Victoria blue skin staining revealed that the compound drug inhibited abnormal thickening of the epidermis, abnormal breaking and accumulation of collagen fibers and elastic fibers, and maintained their orderly arrangement. Moreover, the results revealed that the compound drug increased SOD, CAT and collagen I, and reduced the MDA content, the SASP factors IL-6 and TNF-α of the NF-κB pathway, and the SASP factors MMP-1 of the MAPK pathway. The above results indicate that the active ingredients of the compound drug screened by molecular docking have the potential to reduce skin photoaging.

Inhibitory effects of bioactive compounds on UVB-induced photodamage in human keratinocytes: modulation of MMP1 and Wnt signaling pathways.

UVB radiation significantly threatens skin health, contributing to wrinkle formation and an elevated risk of skin cancer. This study aimed to explore bioactive compounds with potential UVB-protective properties. Using in silico analysis, we chose compounds to reduce binding energy with matrix metalloproteinase-1 (MMP1). Piperitoside, procyanidin C1, and mulberrofuran E emerged as promising candidates through this computational screening process. We investigated the UVB-protective efficacy of the selected compounds and underlying mechanisms in human immortalized keratinocytes (HaCaT). We also investigated the molecular pathways implicated in their action, focusing on the transforming growth factor (TGF)-ß and wingless-related integration site (Wnt)/ß-catenin signaling pathways. In UVB-exposed HaCaT cells (100 mJ/cm2 for 30 min), piperitoside, procyanidin C1, and mulberrofuran E significantly reduced reactive oxygen species (ROS) and lipid peroxides, coupled with an augmentation of collagen expression. These compounds suppressed MMP1, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) expression, while they concurrently enhanced collagen-1 (COL1A1), ß-catenin (CTNNB1), and superoxide dismutase type-1 (SOD1) expression. Furthermore, Wnt/ß-catenin inhibitors, when administered subsequently, partially counteracted the reduction in MMP1 expression and alleviated inflammatory and oxidative stress markers induced by the bioactive compounds. In conclusion, piperitoside, procyanidin C1, and mulberrofuran E protected against UVB-induced damage in HaCaT cells by inhibiting MMP1 expression and elevating ß-catenin expression. Consequently, these bioactive compounds emerge as promising preventive agents for UVB-induced skin damage, promoting skin health.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site.

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

Effect of collagen sponge combined with epidermal growth factor in repairing maxillofacial head and neck wounds in rats.

Collagen sponge and epidermal growth factor (EGF) promote wound healing. However, the effect of collagen sponge combined with EGF in repairing maxillofacial head and neck wounds remains unclear. The rats were divided into 3 groups, including experimental group 1 (Vaseline gauze+EGF), experimental group 2 (collagen sponge+EGF) with control group (Vaseline+normal saline), and maxillofacial head and neck wounds were simulated. Wound pathological morphology was detected by HE staining; wound EGF, IL-1ß, IL-6 along with TNF-α contents by ELISA and MMP1 level by western blot. At 7 and 14 days after treatment, wound healing rate of two experimental groups was higher than that of control group, and that of experimental group 2 presented higher than that of experimental group 1. Compared with control group, experimental group 1 had significantly fewer inflammatory cells in the wound tissue, local erythrocyte spillage outside the vascular walls, more collagen deposition and more granulation tissue. Compared with experimental group 1, inflammatory cells in wound tissues of experimental group 2 were significantly reduced, the collagen tissues were visible and arranged, and the growth of the wound granulation tissue was obvious. IL-1ß, IL-6 along with TNF-α levels in two experimental groups presented lower than control group, and EGF level was higher. More importantly, in contrast to experimental group 1, IL-1ß, IL-6 along with TNF-α in experimental group 2 presented lower, and EGF level presented higher. At 14 days after treatment, MMP1 level in two experimental groups was lower than control group. In contrast to experimental group 1, MMP1 level in experimental group 2 was lower. In summary, collagen sponge combined with EGF for the first time significantly improved the healing speed of maxillofacial head and neck wounds and reduced the scar left after wound healing.

SPTLC2 ameliorates chondrocyte dysfunction and extracellular matrix metabolism disturbance in vitro and in vivo in osteoarthritis.

Disturbances in chondrocyte extracellular matrix (ECM) metabolism in osteoarthritis (OA) are a major cause of OA and potentially lead to personal disability, placing a huge burden on society. Chondrocyte apoptosis and ECM catabolism have a major role in the OA process. Firstly, bioinformatics analysis was performed to screen differentially expressed genes (DEGs) in OA, and serine palmitoyltransferase subunit 2 (SPTLC2) was chosen, which had high-level expression in the OA cartilage tissues and OA chondrocytes. Overexpression and knockdown of SPTLC2 were achieved in OA chondrocytes and normal chondrocytes respectively to study the effect of SPTLC2 upon ECM metabolism of chondrocytes. Cell viability and apoptosis were measured using MTT and flow cytometry analyses; SPTLC2 overexpression enhanced the OA chondrocyte viability and decreased apoptotic rate. In addition, Western blot detection of ECM-related factors (Collagen I, Collage II, MMP-1, MMP-3, and MMP-13) revealed that SPTLC2 overexpression promoted the expression of collagens (Collagen I and Collage II) and suppressed matrix metalloproteinase (MMP-1, MMP-3, and MMP-13) level. In contrast, SPTLC2 knockdown in normal chondrocytes showed opposite effects on cell viability, apoptosis, and ECM degeneration. The articular cartilage of OA rats was transfected with lentivirus overexpressing SPTLC2; HE and Safranin-O fast green demonstrated that SPTLC2 overexpression could alleviate chondrocyte injuries and slow down the development of OA. In conclusion, SPTLC2 plays a role in OA and may be a potential target gene for the treatment of OA.

Seabuckthorn (Hippophae rhamnoides, L.) pulp oil prevents ultraviolet-induced damage in human fibroblasts.

Seabuckthorn pulp oil (SBO) is used in beauty products because of its rich lipophilic substances with high nutraceutical and cosmeceutical potential. However, the mechanism through which SBO enhances skin elasticity remains unclear. Therefore, in this study, we examined the anti-photoaging activity of SBO in normal human dermal fibroblasts (NHDF) under ultraviolet (UV) irradiation. Pretreatment with SBO significantly suppressed UV-B-induced cell toxicity and collagen degradation, suggesting that SBO contains anti-photoaging substances. Further, palmitoleic acid, the main component of SBO, maintained cell viability and collagen levels in UV-B-irradiated NHDF by suppressing the expression of matrix metalloproteinase 1 and acted on the inhibition of p38 and JNK phosphorylation and nuclear translocation of nuclear factor-kappa B. These findings suggest the utility of SBO as an anti-photoaging agent.

Evaluation test and analysis of a microneedle and iontophoresis based medical device "CELLADEEP Patch" in skin improvement on ex vivo human-derived skin tissue models.

BACKGROUND: Microneedles are tiny needles, typically ranging from tens to hundreds of micrometers in length, used in various medical procedures and treatments. The tested medical device named "CELLADEEP Patch" a dissolvable microneedle therapy system (MTS), made of hyaluronic acid and collagen. And the iontophoresis technique is also applied in the system. The study aimed to evaluate the effectiveness of the "CELLADEEP Patch" in skin improvement. METHODS: Ex vivo human-derived skin tissue models were used in this study and they were divided into three different groups, namely, the Untreated Group, the Negative Control Group, and the Test Group respectively. The Untreated Group received no treatment measures, the Negative Control Group was exposed to ultraviolet B radiation (UVB) irradiation, and the Test Group was exposed to UVB irradiation and treated with "CELLADEEP Patch". Skin moisture content, transdermal water loss, and skin elasticity were evaluated by three clinical devices. Additionally, histological staining and related mRNA expression levels were also analyzed. RESULTS: The results of skin moisture content, transdermal water loss, and skin elasticity evaluation consistently illustrated that the application of "CELLADEEP Patch" led to remarkable skin improvement. And the analysis of histological staining images also confirmed the effectiveness of the "CELLADEEP Patch", especially for increasing collagen density. Moreover, the upregulation of Collagen type 1 a (COL1A1) and hyaluronan synthase 3 mRNA expression and the decrease of Matrix metalloproteinase 1 (MMP-1) and Interleukin-1 beta (IL-1ß) mRNA expression reflected its wrinkle improvement, moisturizing and anti-inflammation function. CONCLUSION: "CELLADEPP Patch", the MTS combined with the iontophoresis technique, exhibits its effectiveness in moisturizing, skin elasticity improvement, and anti-inflammatory function when applied to ex vivo human-derived skin tissue models in experiments. The study has contributed to the understanding of the "CELLADEPP Patch" and laid the foundation for subsequent animal experiments and clinical trials.

Ultraviolet Protection From a Patented Amino Acid Complex Technology.

OBJECTIVE:   This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment.  Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used.   J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.

Interleukin-24: A molecular mediator of particulate matter's impact on skin aging.

Air pollution, a global health concern, has been associated with adverse effects on human health. In particular, particulate matter (PM), which is a major contributor to air pollution, impacts various organ systems including the skins. In fact, PM has been suggested as a culprit for accelerating skin aging and pigmentation. In this study, using single-cell RNA sequencing, IL-24 was found to be highly upregulated among the differentially expressed genes commonly altered in keratinocytes and fibroblasts of ex vivo skins exposed to PM. It was verified that PM exposure triggered the expression of IL-24 in keratinocytes, which subsequently led to a decrease in type I procollagen expression and an increase in MMP1 expression in fibroblasts. Furthermore, long-term treatment of IL-24 induced cellular senescence in fibroblasts. Through high-throughput screening, we identified chemical compounds that inhibit the IL-24-STAT3 signaling pathway, with lovastatin being the chosen candidate. Lovastatin not only effectively reduced the expression of IL24 induced by PM in keratinocytes but also exhibited a capacity to restore the decrease in type I procollagen and the increase in MMP1 caused by IL-24 in fibroblasts. This study provides insights into the significance of IL-24, illuminating mechanisms behind PM-induced skin aging, and proposes IL-24 as a promising target to mitigate PM-associated skin aging.

Asiatic acid inhibits osteosarcoma cell migration and invasion via the AKT/Sp1/MMP1 axis.

Osteosarcoma is a malignant bone tumor affecting adolescents and children. No effective treatment is currently available. Asiatic acid (AA), a triterpenoid compound found in Centella asiatica, possesses anti-tumor, anti-inflammatory, and anti-oxidant properties in various types of tumor cells. This study aims to determine whether AA exerts antitumor effects in human osteosarcoma cells. Our results indicate that AA does not influence the viability, proliferative rate, or cell cycle phase of human osteosarcoma cells under non-toxic conditions. AA suppressed osteosarcoma cell migration and invasion by down-regulating matrix metalloproteinase 1 (MMP1) expression. Data in the TNMplot database suggested MMP1 expression was higher in osteosarcoma than in normal tissues, with associated clinical significance observed in osteosarcoma patients. Overexpression of MMP1 in osteosarcoma cells reversed the AA-induced suppression of cell migration and invasion. AA treatment decreased the expression of specificity protein 1 (Sp1), while Sp1 overexpression abolished the effect of AA on MMP1 expression and cell migration and invasion. AA inhibited AKT phosphorylation, and treatment with a PI3K inhibitor (wortmannin) increased the anti-invasive effect of AA on osteosarcoma cells via the p-AKT/Sp1/MMP1 axis. Thus, AA exhibits the potential for use as an anticancer drug against human osteosarcoma.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.