Production Of Matrix Metalloproteinases Research Articles

Investigation of multinucleation and apoptosis of macrophages of BCG-infected mice and their production of cathepsins and matrix metalloproteinases

The relevance of the study of the role of macrophages and their multinucleated forms in the pathogenesis of tuberculous granulomatosis is determined by its wide prevalence, the presence of severe socio-economic consequences of its morbidity and necrotic complications, which are based on the high destructive potential of macrophages associated with the role of hydrolases in the degradation of extracellular matrix components. Aim of the study was to investigate the features of the multinucleation, apoptosis and expression of a number of hydrolases in macrophages of BCG-infected mice. Material and methods. The intensity of macrophage multinucleation and apoptosis, the peculiarities of their expression of matrix metalloproteinases (MMP-1, MMP-9), catepsins (CatB, CatD), caspase-3, and p53 protein were studied in peritoneal cells cultures of intact and BCG-infected BALB/c mice. Results. The number of multinucleated macrophages increased according to the terms of the experiment, having a maximum value for 3 months of observation, but after 2 months almost reaching this level. The realization of apoptosis, multinucleation of macrophages had a complex character, determining the composition of their subpopulations. The dynamics of the expression of the studied hydrolases by macrophages indicated their unequal role in tissue necrosis at various stages of granulomogenesis. The high functional ability of multinucleated macrophages to produce hydrolases of certain types is shown. Intense expression of MMP-1 in the early stages of granulomogenesis and its maximum value, as well as CatD expression for 3 months, and strong expression of MMP-9 for 6 months were noted. Conclusions. Stimulation of plastic processes in macrophages under conditions of BCG-granulomatosis determines the formation of multinucleated macrophages with high functional potential and intensive expression of hydrolases by macrophages for 2 and 3 months of granulomogenesis. These are periods of high risk of necrotic complications of tuberculous granulomatosis, which should be taken into account when developing methods for their prevention and therapeutic correction.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells

BackgroundExtracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs.MethodsCell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins.ResultsWe observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion.ConclusionsOur findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.Graphical

A Model for Membrane Degradation Using a Gelatin Invadopodia Assay.

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.

Ketosis prevents abdominal aortic aneurysm rupture through C–C chemokine receptor type 2 downregulation and enhanced extracellular matrix balance

Abdominal aortic aneurysms (AAAs) are prevalent with aging, and AAA rupture is associated with increased mortality. There is currently no effective medical therapy to prevent AAA rupture. The monocyte chemoattractant protein (MCP-1)/C–C chemokine receptor type 2 (CCR2) axis critically regulates AAA inflammation, matrix-metalloproteinase (MMP) production, and extracellular matrix (ECM) stability. We therefore hypothesized that a diet intervention that can modulate CCR2 axis may therapeutically impact AAA risk of rupture. Since ketone bodies (KBs) can trigger repair mechanisms in response to inflammation, we evaluated whether systemic ketosis in vivo could reduce CCR2 and AAA progression. Male Sprague–Dawley rats underwent surgical AAA formation using porcine pancreatic elastase and received daily β-aminopropionitrile to promote AAA rupture. Rats with AAAs received either a standard diet, ketogenic diet (KD), or exogenous KBs (EKB). Rats receiving KD and EKB reached a state of ketosis and had significant reduction in AAA expansion and incidence of rupture. Ketosis also led to significantly reduced aortic CCR2 content, improved MMP balance, and reduced ECM degradation. Consistent with these findings, we also observed that Ccr2−/− mice have significantly reduced AAA expansion and rupture. In summary, this study demonstrates that CCR2 is essential for AAA expansion, and that its modulation with ketosis can reduce AAA pathology. This provides an impetus for future clinical studies that will evaluate the impact of ketosis on human AAA disease.

Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

STAT3 ACTIVATION IN THE SYNOVIAL TISSUES FROM THE HIP JOINT IN THE EARLY STAGE OF RAPIDLY DESTRUCTIVE COXOPATHY

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC.Acknowledgements: This study was supported by Katakami Foundation for Clinical Research.

Urolithin B reduces cartilage degeneration and alleviates osteoarthritis by inhibiting inflammation.

Osteoarthritis is the most prevalent degenerative joint disease reported worldwide. Conventional treatment strategies mainly focus on medication and involve surgical joint replacement. The use of these therapies is limited by gastrointestinal complications and the lifespan of joint prostheses. Hence, safe and efficacious drugs are urgently needed to impede the osteoarthritis progression. Urolithin B, a metabolite of ellagic acid in the gut, exhibits anti-inflammatory and antioxidant properties; however, its role in osteoarthritis remains unclear. In this study, we demonstrated that urolithin B efficiently inhibits the inflammatory factor-induced production of matrix metalloproteinases (MMP3 and MMP13) in vitro and upregulates the expression of type II collagen and aggrecan. Urolithin B alleviates cartilage erosion and osteophyte formation induced by anterior cruciate ligament transections. Moreover, urolithin B inhibits the activation of the NF-κB pathway by reducing the phosphorylation of Iκb-α and the nuclear translocation of P65. In summary, urolithin B significantly inhibits inflammation and alleviates osteoarthritis. Hence, urolithin B can be considered a potential agent suitable for the effective treatment of osteoarthritis in the future.

The protective effect of Ergolide in osteoarthritis: In vitro and in vivo studies

Osteoarthritis (OA), a prevalent degenerative condition, occurs due to the deterioration of joint tissues and cells. Consequently, safeguarding chondrocytes against damage caused by inflammation is an area of future research emphasis. There is growing evidence that Ergolide (ERG) has multiple biological functions. Nevertheless, it is still uncertain whether it can hinder the advancement of OA. In this study, we investigate the ERG's potential to reduce inflammation and protect cartilage. ERG treatment in vitro effectively inhibited the excessive production of pro-inflammatory substances, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and tumor necrosis factor-α (TNF-α), leading to their complete suppression. Furthermore, ERG suppressed the production of matrix-degrading enzymes (ADAMTS-5) and matrix metalloproteinase 13 (MMP13), consequently impeding the breakdown of extracellular matrix (ECM) and restraining the synthesis of collagenase II and Aggrecan. Through the P38/MAPK pathway, we discovered that ERG hinders the activation of NF-κB in chondrocytes induced by IL-1β. The protective effect of ERG was enhanced by the p38 MAPK inhibitor SB203580. In vivo, ERG further demonstrated protective effects on cartilage in animal models of DMM. In conclusion, the study has discovered that ERG exhibits innovative therapeutic potential in the context of OA.

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

CD146 deficiency aggravates chronic obstructive pulmonary disease via the increased production of S100A9 and MMP-9 in macrophages

Chronic obstructive pulmonary disease (COPD) is a leading cause of global death. As a molecule beyond adhesion, CD146 is involved in COPD pathogenesis. However, the mechanisms of CD146 in COPD remain largely elusive. We hypothesized that CD146 regulates the production of matrix metalloproteinase-9 (MMP-9) in macrophages and thereby contributes to COPD. Here, we constructed a murine model of COPD using lipopolysaccharide (LPS) and porcine pancreatic elastase (PPE). In COPD-like mice, LPS and PPE decreased the pulmonary expression of CD146. MMP-9 expression and bioactivity were increased in CD146 knockout COPD-like mice. In vitro, LPS decreased CD146 expression in macrophages. With or without LPS challenge, CD146-defective macrophages produced more MMP-9. Transcriptome analysis based on next-generation sequencing (NGS) revealed that S100A9 regulated MMP-9 production in CD146-defective macrophages. Targeting S100A9 with paquinimod decreased lung inflammation and alleviated alveolar destruction in COPD-like mice. Collectively, our study suggests that CD146 negatively regulates MMP-9 production in macrophages via the S100A9 pathway in COPD.

Neuropeptide Y, a potential marker for lupus, promotes lupus development

ObjectiveRelationship between neuropeptide Y (NPY) serum levels, NPY genetic mutation with systemic lupus erythematosus (SLE) pathogenesis is yet to be clarified, and role of NPY in development of SLE needs elucidation. MethodThis study included 460 SLE patients, 472 non-SLE cases, 500 healthy volunteers. Serum NPY, matrix metalloproteinase-1 (MMP-1) and MMP-8 levels were tested by ELISA. Genotyping 7 NPY single nucleotides polymorphisms (SNPs) (rs5573, rs5574, rs16129, rs16138, rs16140, rs16147, rs16478) was obtained by Kompetitive Allele-Specific PCR (KASP) method. Pristane-induced lupus mice were treated with NPY-Y1 receptor antagonist, and histological analysis, serological changes of the mice were evaluated. ResultsNPY serum concentrations were significantly increased in SLE patients when compared to that in healthy volunteers, non-SLE cases. Rs5573 G allele, rs16129 T allele, rs16147 G allele frequencies were significantly different between SLE cases and healthy controls. Rs5574 TT + TC genotypes were related to levels of IgG, C3, C4 and erythrocyte sedimentation rate, and rs16138 GG + GC genotypes correlated with SLE cases with anti-double-stranded deoxyribonucleic acid antibody (anti-dsDNA) (+). Serum MMP-1, MMP-8 concentrations were higher in SLE patients, and NPY levels were significantly related to MMP-1, MMP-8 levels. After treatment of lupus mice with NPY-Y1 receptor antagonist, damage of liver, spleen and kidney was alleviated, production of autoantibodies (anti-nuclear antibody (ANA), total IgG, anti-dsDNA) and MMP-1, MMP-8 was down-regulated, and differentiation of CD3+, CD8+ T cells, B cells, monocytes, macrophages, T helper 1 (Th1), Th2, Th17 cells was reversed. ConclusionNPY may be a biomarker for lupus, which may promote occurrence and development of lupus.

Abstract 13229: Coiled-Coil Domain-Containing Protein 80 Protects Against Aortic Dissection by Maintaining the Contractile Smooth Muscle Cell Phenotype

Background: Aortic dissection (AD) is a life-threatening medical emergency characterized by adverse vascular remodeling. Effective prevention and treatment options are lacking for AD. Coiled-coil domain-containing protein 80 (CCDC80) plays an essential role in regulating vascular remodeling. In the present study, we aimed to define the role of CCDC80 in the formation and development of AD. Methods: The expression and subcellular localization of CCDC80 were determined in human and mouse AD. CCDC80 knockout mice (CCDC80 –/– ) and vascular smooth muscle cell (VSMC)-specific CCDC80 knockout mice (CCDC80 fl/fl SM22α Cre + ) were treated with angiotensin II (Ang II) or Ang II combined with β-aminopropionitrile monofumarate (BAPN) to induce AD models. RNA-Seq analysis combined with immunoprecipitation and blockage of the JAK2/STAT3 signaling pathway in CCDC80 –/– mice were used to evaluate the effect of CCDC80 deficiency on this pathway. Results: CCDC80 was downregulated in VSMCs in human and mouse AD. CCDC80 – / – mice challenged with Ang II and BAPN developed AD with higher frequency and severity compared with their littermate controls (66.7% vs. 100%, p < 0.05). Remarkably, compared with littermate controls treated with Ang II, CCDC80 –/– mice (8.3% vs. 87.5%, p < 0.05) and CCDC80 fl/fl SM22α Cre + mice (12.5% vs. 80%, p < 0.05) frequently developed AD with severe elastin fragmentation and collagen deposition. CCDC80 interacted with JAK2; CCDC80 deficiency could promote VSMC phenotype switching, proliferation, and migration as well as matrix metalloproteinase production by activating the JAK2/STAT3 signaling pathway. The JAK2/STAT3 pathway-specific inhibitor ameliorated adverse vascular remodeling and reduced AD formation (90% vs. 55%, p < 0.05) in CCDC80 knockout mice. Conclusion: CCDC80 deficiency exacerbates the progression of AD by activating the JAK2/STAT3 pathway involved in regulating the phenotype switching and function of VSMCs. Our findings suggest that CCDC80 is a potential target for the prevention and treatment of AD.

Cyanidin‐3‐galactoside from Aronia melanocarpa ameliorates PM10‐induced pulmonary inflammation by promoting PINK1/Parkin signaling pathway‐mediated alveolar macrophage mitophagy

AbstractAronia melanocarpa is rich in anthocyanin, among which cyanidin‐3‐galactoside (C3G) is the most abundant. Here, we established an experimental model of mice and alveolar macrophage cell line MH‐S exposed to PM10, and C3G was administered to explore the underlying mechanism of C3G exerting protective effects on PM10‐induced lung inflammation. The results showed C3G alleviated PM10‐induced lung inflammation by reducing matrix metalloproteinases production at both gene and protein levels as well as pro‐inflammatory cytokine levels. Autophagy was activated in PM10‐injured alveolar macrophages (AMs), and C3G promoted autophagy indicated by LC3, BECN1, Atg5, Bcl‐2, and P62 expression, and C3G further resisted PM10‐induced AMs apoptosis. Besides, C3G could promote Pink1/Parkin‐mediated mitochondrial autophagy of AMs to alleviate the overexpression of mitochondrial reactive oxygen species (ROS) so that maintain mitochondrial membrane potential and ATP productions to restrict the distribution of Cyt‐c and further reduce the increasing of caspase 3/7 activity. Therefore, C3G enhanced the clearance of PM10‐damaged mitochondria by promoting AMs mitophagy, thereby reducing the activation of mitochondria‐dependent apoptosis pathway and the production of excess ROS, which reduced AMs apoptosis and secretion of pro‐inflammatory factors to ameliorate PM10‐induced pulmonary inflammation, suggesting that C3G may be as a functional food ingredient with the potential to improve lung health.

Mannan-binding lectin ameliorates renal fibrosis by suppressing macrophage-to-myofibroblast transition

Mannan-binding lectin (MBL) is a pattern-recognition molecule that plays a crucial role in innate immunity. MBL deficiency correlates with an increased risk of chronic kidney disease (CKD). However, the molecular mechanisms are not fully defined. Here, we established a CKD model in wild-type (WT) and MBL-deficient (MBL−/−) mice via unilateral ureteral obstruction (UUO). The result showed that MBL deficiency aggravated the pathogenesis of renal fibrosis in CKD mice. Strikingly, the in vivo macrophage depletion investigation revealed that macrophages play an essential role in the MBL-mediated suppression of renal fibrosis. We found that MBL limited the progression of macrophage-to-myofibroblast transition (MMT) in kidney tissues of UUO mice. Further in vitro study showed that MBL−/− macrophages exhibited significantly increased levels of fibrotic-related molecules compared with WT cells upon transforming growth factor beta (TGF-β) stimulation. We demonstrated that MBL inhibited the MMT process by suppressing the production of matrix metalloproteinase 9 (MMP-9) and activation of Akt signaling. In summary, our study revealed an expected role of MBL on macrophage transition during renal fibrosis, thus offering new insight into the potential of MBL as a therapeutic target for CKD.

Protective effect of the brown seaweed Padina boryana against UVB-induced photoaging in vitro in skin cells and in vivo in zebrafish

The ethanol extract of Padina boryana (PB-EtOH) containing phlorotannin effectively inhibits oxidative stress and melanin synthesis, suggesting the potential use of PB-EtOH in the cosmetics industry. To further investigate the cosmetic effects of PB-EtOH, its photoprotective effects against ultraviolet (UV) irradiation was investigated. The results displayed that PB-EtOH remarkably improved cell viability by suppressing the apoptosis via cleaning the intracellular reactive oxygen species (ROS) in UVB-exposed human keratinocytes. In addition, PB-EtOH effectively suppressed oxidative stress, increased collagen synthesis, and reduced the matrix metalloproteinase production in UVB-exposed human dermal fibroblasts in a concentration-dependent manner. Furthermore, the in vivo anti-photoaging effect was determined using the zebrafish model. The results displayed that PB-EtOH remarkably attenuated UVB-stimulated photodamage. The photoprotective effect was displays in reducing the levels of ROS, nitric oxide, lipid peroxidation, and cell death. These results demonstrate that PB-EtOH possesses anti-photoaging effects and suggest that PB-EtOH may be an ideal ingredient to develop a skincare product in the cosmetic industry.

Immunomodulatory effect of Red Moringa oleifera Leaves Fermentation Extract on Il-21 And Il-22 Expressions in Balb/C Mice Exposed to Salmonella typhi

Interleukin-21(IL-21) and interleukin-22(IL-22) expressions in the substantial intestine increase when inflammation occurs. They can also induce the production of pro-inflammatory cytokines and Matrix Metallo Proteinases (MMP) in subepithelial fibroblasts. To evaluate at IL-21 and IL-22 expressions in BALB/c mice after administering red M. oleifera leaf fermented extracts exposed to Salmonella typhi. The expression of IL-21 and IL-22 were evaluated for immunomodulatory effect was analyzed by flowcytometer. Data were analyzed by one-way ANOVA and continued with the Tukey test (p<0.05). The results showed that the fermented extract of red M. oleifera leaves could act as an immunosuppressor characterized by decreased levels of IL-21 and IL-22 both on CD4 T and CD8 T cells in mice injected with Salmonella typhi ranging from 14 mg/Kg BW to 42 mg/Kg BW and Pyrex analysis explains that the cathecin compound has the same active side as 8MR in forming bonds with MMP-9. The results of this study provide is the fermented extract of red M. oleifera leaves decreasing the expression of CD4+IL21+, CD8+IL21+ and CD4+IL22+, CD8+IL22+ through inhibition of MMP-9

Integrin-dependent recruitment of Src to ROS-producing endosomes in osteoarthritic cartilage.

Fibronectin (FN) fragments stimulate catabolic signaling, and, by binding to integrins, they induce chondrocytes to increase the production of matrix metalloproteinases, including MMP-13. In this issue of Science Signaling, Miao etal. reveal that internalization of a FN fragment, but not intact FN, by α5β1 integrin results in the formation of ROS-producing endosomes (redoxosomes) through which chondrocytes detect and respond to damaged matrix by producing more MMP-13.

Redox-active endosomes mediate α5β1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5β1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5β1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Impact of Metabolic Stress on BeWo Syncytiotrophoblast Function.

During placental formation, cytotrophoblasts (CTBs) fuse into multinucleate, microvilli-coated syncytiotrophoblasts (STBs), which contact maternal blood, mediating nutrient, metabolite, and gas exchange between mother and fetus, and providing a barrier against fetal infection. Trophoblasts remodel the surrounding extracellular matrix through the secretion of matrix metalloproteinases (MMPs). Maternal obesity and diabetes mellitus can negatively impact fetal development and may impair trophoblast function. We sought to model the impact of metabolic stress on STB function by examining MMP and hormone secretion. The BeWo CTB cell line was syncytialized to STB-like cells with forskolin. Cell morphology was examined by electron microscopy and immunofluorescence; phenotype was further assessed by ELISA and RT-qPCR. STBs were exposed to a metabolic stress cocktail (MetaC: 30 mM glucose, 10 nM insulin, and 0.1 mM palmitic acid). BeWo syncytialization was demonstrated by increased secretion of HCGβ and progesterone, elevated syncytin gene expression (ERVW-1 and ERVFRD-1), loss of tight junctions, and increased surface microvilli. MetaC strongly suppressed syncytin gene expression (ERVW-1 and ERVFRD-1), suppressed HCGβ and progesterone secretion, and altered both MMP-9 and MMP-2 production. Metabolic stress modeling diabetes and obesity altered BeWo STB hormone and MMP production in vitro.

Flavonoid extract from propolis alleviates periodontitis by boosting periodontium regeneration and inflammation resolution via regulating TLR4/MyD88/NF-κB and RANK/NF-κB pathway

Ethnopharmacological relevanceIn traditional Chinese medicine, propolis has been used for treating oral diseases for centuries, widely. Flavonoid extract is the main active ingredient in propolis, which has attracted extensive attention in recent years. Aim of the studyThe objective and novelty of the current study aims to identify the mechanism of total flavonoid extract of propolis (TFP) for the treatment of periodontitis, and evaluate the therapeutic effect of TFP-loaded liquid crystal hydrogel (TFP-LLC) in rats with periodontitis. MethodsIn this study, we used lipopolysaccharide-stimulated periodontal ligament stem cells (PDLSCs) to construct in vitro inflammation model, and investigated the anti-inflammatory effect of TFP by expression levels of inflammatory factors. Osteogenic differentiation was assessed using alkaline phosphatase activity and alizarin red staining. Meanwhile, the expression of toll like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor-kappa B (NF-κB), receptor activator of NF-κB (RANK) etc, were quantitated to investigate the therapeutic mechanism of TFP. Finally, we constructed TFP-LLC using a self-emulsification method and administered it to rats with periodontitis via periodontal pocket injection to evaluate the therapeutic effects. The therapeutic index, microcomputed tomography (Micro-CT), H&E staining, TRAP staining, and Masson staining were used for this evaluation. ResultsTFP reduced the expression of TLR4, MyD88, NF-κB and inflammatory factor in lipopolysaccharide-stimulated PDLSCs. Meanwhile, TFP simultaneously regulating alkaline phosphatase, RANK, runt-associated transcription factor-2 and matrix metalloproteinase production to accelerate osteogenic differentiation and collagen secretion. In addition, TFP-LLC can stably anchor to the periodontal lesion site and sustainably release TFP. After four weeks of treatment with TFP-LLC, we observed a decrease in the levels of NF-κB and interleukin-1β (IL-1β) in the periodontal tissues of rats, as well as a significant reduction in inflammation in HE staining. Similarly, Micro CT results showed that TFP-LLC could significantly inhibit alveolar bone resorption, increase bone mineral density (BMD) and reduce trabecular bone space (Tb.Sp) in rats with periodontitis. ConclusionCollectively, we have firstly verified the therapeutic effects and mechanisms of TFP in PDLSCs for periodontitis treatment. Our results indicate that TFP perform anti-inflammatory and tissue repair activities through TLR4/MyD88/NF-κB and RANK/NF-κB pathways in PDLSCs. Meanwhile, for the first time, we employed LLC delivery system to load TFP for periodontitis treatment. The results showed that TFP-LLC could be effectively retained in the periodontal pocket and exerted a crucial role in inflammation resolution and periodontal tissue regeneration.