Release Of Matrix Metalloproteinases Research Articles

Protective Effect of Persimmon Calyx Extract against UVB‐induced Skin Photoaging in Human Skin Fibroblasts

The persimmon is the edible fruit, which has been widely cultivated in the countries of East Asia. The persimmon calyx is a by‐product generated during persimmon processing. It has been reported that persimmon calyx possesses anti‐oxidative, anti‐inflammatory, and anti‐coagulant properties. However, few reports have been published on the protective activities of persimmon calyx on photoaging in skin fibroblast cells. Therefore, the purpose of this study was to evaluate the protective activities of persimmon calyx methanol extract (PCME) on UVB‐induced photoaging in human skin fibroblasts (Hs68 cells). The cells were treated with PCME at concentrations ranging from 5 to 20 □g/mL for 24 h and then the cells were exposed to UVB (30 mJ/cm2) irradiation. The results revealed that PCME showed cytoprotective effects in a dose‐dependent manner. PCME also significantly inhibited the release of matrix metalloproteinase 1 and 3. Furthermore, PCME reduced UVB‐induced production of reactive oxygen species and collagen degradation in Hs68 cells. Taken together, our data suggest that PCME has strong protective activity against UVB‐induced skin photoaging and thus can be a useful agent for anti‐photoaging cosmetic ingredient.

Symurban Nanocrystals for Advanced Anti-Pollution Skincare

Several of most common dermatoses worldwide, e.g., psoriasis and atopic dermatitis, are worsened in their clinical picture when the skin is regularly exposed to an increased air pollution level, e.g., particulate matter. This is explainable by the activation of the aryl hydrocarbon receptor (AhR) in the skin, which results in an increased release of proinflammatory cytokines and matrix metalloproteinases. Symurban is a competitive AhR antagonist and thus allows the effective protection of skin. In order to improve its dermal bioavailability as a poorly soluble active agent (0.25 µg/mL), nanocrystals were prepared and evaluated. Nanocrystals are pure active crystals reduced in particle size to the submicron range of 100 to 1000 nm. They feature the properties of nanocrystals, such as increased saturation solubility and dissolution velocity, without having to be declared as nanomaterial. Production methods and parameters were systematically investigated. Wet bead milling at 2000 rpm for 30 min yielded the best results. A z-average of 280 nm was achieved for a 10% Symurban suspension with a polydispersity index of 0.20, indicating a narrow size distribution. For the long-term stabilization of the nanocrystal suspension, the performance of 15 surfactants of different categories and HLB values were investigated and evaluated. It was found that non-ionic surfactants in general were better able to stabilize the system than anionic or amphoteric surfactants. Highest stability of over 12 months at 25 °C was achieved with 2% Plantacare 810 UP, an ECOCERT surfactant with high skin tolerance. The suspension was also chemically long-term stable with >97% of remaining Symurban over 12 months. The saturation solubility of Symurban as nanocrystals was significantly increased from 0.25 to 2.9 μg/mL, which corresponds to a factor of >11. In a case study of one male volunteer with healthy skin conditions, penetration profiles of Symurban nanocrystal hydrogel and commercial anti-pollution serum containing an identical amount of Symurban were determined and compared. After 20 min of exposure, the relative Symurban concentration in the deeper skin layers (tape 19-30) was more than two times higher for nanocrystal hydrogel (16%) than the commercial serum (7%). These results suggest that nanocrystals are a promising delivery system for the poorly soluble anti-pollution agent Symurban.

Assessment of recombinant tissue plasminogen activator (rtPA) toxicity in cultured neural cells and subsequent treatment with poly-arginine peptide R18D.

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) in ischaemic stroke has been associated with neurotoxicity, blood brain barrier (BBB) disruption and intra-cerebral hemorrhage. To examine rtPA cellular toxicity we investigated the effects of rtPA on cell viability in neuronal, astrocyte and brain endothelial cell (bEnd.3) cultures with and without prior exposure to oxygen-glucose deprivation (OGD). In addition, the neuroprotective peptide poly-arginine-18 (R18D; 18-mer of D-arginine) was examined for its ability to reduce rtPA toxicity. Studies demonstrated that a 4- or 24-h exposure of rtPA was toxic, affecting neuronal cell viability at ≥ 2µM, and astrocyte and bEnd.3 cells viability at ≥ 5μM. In addition, a 4-h exposure to rtPA after a period of OGD (OGD/rtPA) exacerbated toxicity, affecting neuronal, astrocyte and bEnd.3 cell viability at rtPA concentrations as low as 0.1µM. Treatment of cells with low concentrations of R18D (0.5 and 1µM) reduced the toxic effects of rtPA and OGD/rtPA, while on some occasions a higher 2µM R18D concentrations exacerbated neuronal and bEnd.3 cell toxicity in OGD/rtPA exposed cultures. In exploratory studies we also demonstrated that OGD activates matrix metalloproteinase-9 (MMP-9) release into the supernatant of astrocyte and bEnd.3 cell cultures, but not neuronal cultures, and that OGD/rtPA increases MMP-9 activation. Furthermore, R18D decreased MMP-9 activation in OGD/rtPA treated astrocyte and bEnd.3 cell cultures. In summary, the findings show that rtPA can be toxic to neural cells and that OGD exacerbates toxicity, while R18D has the capacity to reduce rtPA neural cellular toxicity and reduce MMP-9 activation in astrocytes and bEnd.3. Poly-arginine-18 peptides, which are being developed as neuroprotective therapeutics for ischaemic stroke, therefore have the additional potential of reducing cytotoxic effects associated with rtPA thrombolysis in the treatment of ischaemic stroke.

Analysis of the association of MPO and MMP-9 with stroke severity and outcome: Cohort study.

In acute cerebral ischemia, circulating neutrophil count and neutrophil-to-lymphocyte ratio (NLR) are positively associated with stroke severity and worse outcomes. Mediators of this effect are unknown. We aimed to investigate (1) the relationship between plasma matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) concentrations with stroke severity and outcome and (2) MMP-9 and MPO release after ex vivo stimulation of neutrophils by recombinant tissue plasminogen activator (rtPA). We analyzed data collected in 255 patients with supratentorial cerebral infarcts recruited within 48 hours of symptoms onset irrespective of rtPA treatment. The endpoints were excellent outcome (modified Rankin Scale score 0-1), symptomatic intracerebral hemorrhage (European Cooperative Acute Stroke Study-II definition), and death at 3 months. The role of rtPA treatment on peripheral neutrophil degranulation was investigated in 18 patients within 4.5 hours and after 72 hours. Neutrophil counts, NLR, and MPO plasma concentrations, but not MMP-9, were positively correlated with stroke severity. Higher neutrophil counts and NLR were independently associated with worse outcomes and higher mortality rates at month 3. Higher MPO plasma concentrations, but not MMP-9, were associated with worse outcome. Neutrophil-derived MMP-9, after ex vivo rtPA stimulation, but not MPO, were higher after 72 hours in patients treated by IV rtPA but not associated with hemorrhagic transformation. Neutrophil counts, NLR, and MPO plasma concentrations are associated with worse outcome in patients with acute cerebral ischemia, in contrast to MMP-9. Further investigations are needed to deepen our knowledge on MPO's role in the deleterious effect of neutrophils because it could represent a potential therapeutic target.

Time courses of HMGB1 and other inflammatory markers after generalized convulsive seizures

PurposeNeuroinflammation and disruption of blood brain barrier (BBB) are important players in epileptogenesis, ictogenesis and pharmacoresistance. In this context, we investigated blood levels of HMGB1 and other inflammatory and BBB markers after generalized and focal to bilateral tonic-clonic seizures in serum, summarized under the term generalized convulsive seizures (GCS). MethodsWe included consenting adults who were admitted to the epilepsy monitoring unit. Blood samples were drawn at baseline and immediately after a GCS as well as after 2, 6 and 24 h. We measured leukocytes, c-reactive protein (CRP), the danger-associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1) and S100, receptor of advanced glycation end products (RAGE) alongside the BBB markers intercellular adhesion molecule-1 (ICAM1) and matrix metalloproteinase 9 (MMP9). Noradrenaline and lactate measurements were available from a previous study. P-levels <0.05 were regarded as significant. ResultsTwenty-eight patients with 28 GCS were included. Leukocytosis occurred immediately after GCS and normalized within two hours (p < 0.001). S100 and HMGB1 both increased by ∼80 % (p < 0.001). MMP9 peaked after six hours with levels at 48.6 % above baseline. RAGE decreased by 17.6 % with a nadir at 24 h. CRP increased by 118 % with a peak at 24 h. ICAM1 remained stable (p = 0.068). Postictal HMGB1 correlated with postictal leukocytosis (r = 0.42; p = 0.025) and with MMP9 levels six hours later (r = 0.374; p = 0.05). Postictal lactate levels correlated with MMP9 at 6 h (r = 0.48; p = 0.01) and CRP at 24 h (r = 0.39; p = 0.04). Postictal noradrenaline correlated with lactate (r = 0.57; p = 0.02) and leukocytes (r = 0.39; p = 0.047). DiscussionThe serum level of the DAMPs HMGB1 and S100 increase immediately after GCS. The hypothetical mechanism includes central nervous processes, such as glutamate toxicity and ROS release from seizing neurons but also muscular tissues. BBB breakdown is marked by the release of MMP9. Further research is needed to understand the complex interactions between electrical and metabolic stress, neuroinflammation and BBB mechanics in seizures and epilepsy. ConclusionOur study reveals signs of inflammation, neuronal damage and transitory disruption of BBB following single GCS, underscoring the widespread and possibily detrimental effects of recurrent seizures on brain properties. The long term impact on the disease course, however, is unclear.

Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation

BackgroundGlioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release.MethodsAstrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes.ResultsImmunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highly-glycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels.ConclusionsThe results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness.1D3uFmTqHFEH8Tq79aQmnJVideo abstractGraphical abstract

Effect of Different-Volume Fluid Resuscitation on Organ Functions in Severe Acute Pancreatitis and Therapeutic Effect of Poria cocos.

Objective To explore the effect of different-volume fluid resuscitation (FR) on organ functions in severe acute pancreatitis (SAP) and to elucidate the therapeutic effect and mechanism of Poria cocos on organ injuries caused by high-volume FR. Methods 1. Clinical study: retrospective analysis of thirty-one patients about the effect of titrated fluid resuscitation protocol (TFR) on the occurrence of acute kidney injury (AKI) secondary to SAP. 2. Experimental study: rats (N = 30) were randomly divided into five groups: sham, model, low-volume FR (1.5 ml/kg/h), high-volume FR (10 ml/kg/h), and Poria cocos combined with high-volume FR (10 ml/kg/h + intraintestinal administration Poria cocos 5 g/kg); serum or plasma indicators and histopathologic scores were compared to explore the effect and mechanism of different fluid volumes and Poria cocos on organ function in SAP. Results The occurrence of AKI, fluid volume, and fluid velocity in TFR group was lower than that in the control group. Logistic regression analysis showed that increased Marshall scores and fluid velocity were risk factors for predicting occurrence of AKI in SAP. Low-volume FR decreased the levels of blood urea nitrogen (BUN), serum creatinine (Cr), matrix metalloproteinase (MMP), and pathologic scores of the pancreas and kidney. High-volume FR increased ascites, MMPs, and kidney pathologic scores. Poria cocos decreased the levels of BUN, Cr, MMPs, and pathologic scores of the pancreas and kidney and increased the arterial oxygen saturation. Conclusion TFR-associated lower fluid volume and velocity reduced the occurrence of AKI secondary to SAP. High volume might aggravate AKI via increased MMP release leading to endothelial glycocalyx damage and vascular endothelial dysfunction. Poria cocos reduced MMP release, relieved glycocalyx damage, and alleviated the pancreas and kidney injury aggravated by high fluid volume in SAP. Therefore, endothelial glycocalyx protection might be a new strategy in the treatment of SAP.

Anti-MMP-2 and MMP-9 activity of Salsola komarovii Iljin extract and its solvent fractions

Objective: To investigate matrix metalloproteinases (MMP)-2 and MMP-9 inhibitory effect of Salsola komarovii Iljin, an edible halophyte with health beneficial effects. Methods: Salsola komarovii crude extracts (SKI), and solvent (n-hexane, 85% aq. MeOH, n-BuOH, and H2O) fractionated extracts of SKI were prepared. Gelatin zymography was carried out to observe MMP enzymatic activity. The release of the MMP enzymes was measured by enzyme-linked immunosorbent assay. Expression of MMPs in mRNA and protein level were investigated by polymerase chain reaction analysis and immunoblotting, respectively. Results: SKI and SKI fractions inhibited active MMP-2 and MMP-9 amount in the treated cell culture medium. Also, SKI suppressed the release of MMP-2 and MMP-9 from stimulated HT1080 human fibrosarcoma cells. Furthermore, SKI suppressed the mRNA and protein expression of MMP-2 and MMP-9. SKI fractions showed parallel effects except for H2O fraction which did not yield any significant MMP inhibitory effect. Among fractions, 85% aq. MeOH was the most active fraction to inhibit both the enzymatic effect and expression of MMP-2 and MMP-9. Conclusions: SKI may contain potential MMP release inhibitory compounds. Salsola komarovii is a promising source of compounds against MMP and could be utilized in the development of antitumor agents.

Statin treatment increases the clinical risk of tendinopathy through matrix metalloproteinase release \u2013 a cohort study design combined with an experimental study

Recent experimental evidence indicates potential adverse effects of statin treatment on tendons but previous clinical studies are few and inconclusive. The aims of our study were, first, to determine whether statin use in a cohort design is associated with tendinopathy disorders, and second, to experimentally understand the pathogenesis of statin induced tendinopathy. We studied association between statin use and different tendon injuries in two population-based Swedish cohorts by time-dependent Cox regression analysis. Additionally, we tested simvastatin in a 3D cell culture model with human tenocytes. Compared with never-users, current users of statins had a higher incidence of trigger finger with adjusted hazard ratios (aHRs) of 1.50 for men (95% confidence interval [CI] 1.21–1.85) and 1.21 (1.02–1.43) for women. We also found a higher incidence of shoulder tendinopathy in both men (aHR 1.43; 1.24–1.65) and women (aHR 1.41; 0.97–2.05). Former users did not confer a higher risk of tendinopathies. In vitro experiments revealed an increased release of matrix metalloproteinase (MMP)-1 and MMP-13 and a weaker, disrupted matrix after simvastatin exposure. Current statin use seems to increase the risk of trigger finger and shoulder tendinopathy, possibly through increased MMP release, and subsequently, a weakened tendon matrix which will be more prone to injuries.

P6394Neutrophil activation patterns in acute coronary syndrome with intact fibrous cap - results from the OPTICO-ACS study

Abstract Background In up to one third of all cases, acute coronary syndrome occurs without signs of plaque rupture. Instead endothelial cell erosion is considered to be the hallmark of acute coronary syndrome with intact fibrous cap (IFC-ACS), with matrix metalloproteinase 9 (MMP9) directly linked to this pathology. The main source of MMP9 immediately after ACS are the neutrophil granulocytes. Therefore, their molecular activation patterns and subsequent MMP9 production are the objectives of the ongoing, translational OPTICO-ACS-Study, aiming to compare the mechanisms and prognosis of IFC-ACS and ACS with ruptured fibrous cap (RFC-ACS). Methods Local and systemic blood samples were simultaneously obtained from the site of the ACS-causing culprit lesion (LOC) using an aspiration catheter and from the systemic circulation (SYS). Using optical coherence tomography (OCT) the ACS-causing culprit lesion was characterized and two patient groups, patients with ACS caused by intact (IFC-ACS) and by ruptured fibrous cap (RFC-ACS) were compared. Each group consists of twenty patients (n=20) matched by age, gender and diabetes. Neutrophil counts and expression of activation markers were immediately quantified by whole-blood flow cytometry. Release of active MMP9 into the plasma was assessed by fluorescence-based zymography. Activation profiles of freshly isolated neutrophils, including MMP9 activity and effect on endothelial cell death, in response to toll-like receptor 2 (TLR2) stimulation was studied. Results Local neutrophils of patients with IFC-ACS show significantly higher expression of TLR2 in comparison to RFC-ACS neutrophils (LOC: 1866±382.1 vs. 1498±426.9; IFC-ACS vs. RFC-ACS, p=0.03). MMP9 activity is significant higher (p=0.01) in plasma obtained from the culprit site of IFC-ACS (74.1 U/ml±4.1) compared to those of RFC-ACS patients (70.0 U/ml±5.1) indicating secretion and activation of the enzyme during IFC-ACS. Importantly, in patients with IFC-ACS, TLR2-stimulation using Pam3CSK4 triggers higher activity rates of MMP9 only in neutrophils isolated directly from the culprit site (LOC), but not systemically (+27%±17.2% IFC-LOC vs. IFC-SYS; p=0.003). This effect was not observed in RFC-derived neutrophils. Inhibiting TLR2 by a monoclonal antibody, strongly reduced secretion of activated MMP9 only in the local neutrophils from IFC-ACS-patients (−54.6%±6.5% in IFC-LOC-anti-TLR2 vs. IFC-LOC-vehicle; p=0.008), but not from systemic IFC-neutrophils, nor from RFC-neutrophils. Furthermore, LOC IFC-ACS neutrophils aggravate endothelial cell death upon TLR2-activation in comparison to LOC RFC-ACS neutrophils (58.4±4.96% vs. 20±1.89%, IFC-ACS vs. RFC-ACS; p=0.0023). Conclusion We newly describe differential kinetics of MMP9 release by neutrophils in ACS patients with IFC versus RFC. Our data support a role of a TLR-2 activated and neutrophil-MMP9-mediated mechanism leading to endothelial cell erosion in patients with IFC-ACS. Acknowledgement/Funding DZHK (German Centre for Cardiovascular Research); BIH (Berlin Institute of Health)

Endothelial dysfunction in cerebral aneurysms

Endothelial cell (EC) dysfunction is known to contribute to cerebral aneurysm (CA) pathogenesis. Evidence shows that damage or injury to the EC layer is the first event in CA formation. The mechanisms behind EC dysfunction in CA disease are interrelated and include hemodynamic stress, hazardous nitric oxide synthase (NOS) activity, oxidative stress, estrogen imbalance, and endothelial cell-to-cell junction compromise. Abnormal variations in hemodynamic stress incite pathological EC transformation and inflammatory zone formation, ultimately leading to destruction of the vascular wall and aneurysm dilation. Hemodynamic stress activates key molecular pathways that result in the upregulation of chemotactic cytokines and adhesion molecules, leading to inflammatory cell recruitment and infiltration. Concurrently, oxidative stress damages EC-to-EC junction proteins, resulting in interendothelial gap formation. This further promotes leukocyte traffic into the vessel wall and the release of matrix metalloproteinases, which propagates vascular remodeling and breakdown. Abnormal hemodynamic stress and inflammation also trigger adverse changes in NOS activity, altering proper EC mediation of vascular tone and the local inflammatory environment. Additionally, the vasoprotective hormone estrogen modulates gene expression that often suppresses these harmful processes. Crosstalk between these sophisticated pathways contributes to CA initiation, progression, and rupture. This review aims to outline the complex mechanisms of EC dysfunction in CA pathogenesis.

Assessing the respiratory toxicity of dihydroxyacetone using an in vitro human airway epithelial tissue model

Dihydroxyacetone (DHA) is an approved color additive used in sunless tanning lotions. Recently, there has been an increased use of DHA in sunless tanning booths in a manner that could result in its inhalation during application. In the present study, we have evaluated the potential for DHA causing toxicity via inhalation using a human air-liquid-interface (ALI) in vitro airway epithelial tissue model. ALI airway models have a close structural and functional resemblance to the in vivo airway epithelium, and thus data generated in these models may have relevance for predicting human responses. To simulate in vivo exposure conditions, we employed a method for liquid aerosol generation that mimics the physical form of inhaled chemicals and used doses of DHA and an exposure frequency reflecting human respiratory exposures during tanning sessions. Compared to the vehicle control, cilia beating frequency (CBF) and MUC5AC secretion were significantly decreased after each exposure. However, time-course studies indicated that both CBF and MUC5AC secretion returned to normal levels within 3 days after the treatment. Matrix metalloproteinase (MMP) release, on the other hand, was decreased 24 h after the first exposure and its level returned to baseline after 5 exposures. No significant morphological changes occurred in the DHA-treated cultures after 5 weekly exposures. Our findings indicate that DHA, at concentrations likely to be experienced by humans, has transient toxic effects on human airway ALI cultures.

Matrix Metalloproteinase‐9 Release from Porphyromonas gingivalis Lipopolysaccharide (LPS)‐treated Rat Brain Microglia In Vitro

BackgroundThe putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.MethodsP. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&amp;D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.ResultsMMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.ConclusionsBoth ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.Support or Funding InformationSupport by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes.

PurposeWhile VZV DNA and antigen have been detected in acute and chronic VZV keratitis, it is unclear whether productive infection of corneal cells is ongoing or whether residual, noninfectious VZV antigens elicit inflammation. Herein, we examined VZV-infected primary human corneal epithelial cells (HCECs) and keratocytes (HKs) to elucidate the pathogenesis of VZV keratitis.MethodsHCECs and HKs were mock- or VZV infected. Seven days later, cells were examined for morphology, proinflammatory cytokine and matrix metalloproteinase (MMP) release, ability to recruit peripheral blood mononuclear cells (PBMCs) and neutrophils, and MMP substrate cleavage.ResultsBoth cell types synthesized infectious virus. VZV-infected HCECs proliferated, whereas VZV-infected HKs died. Compared to mock-infected cells, VZV-infected HCECs secreted significantly more IL-6, IL-8, IL-10, and IL-12p70 that were confirmed at the transcript level, and MMP-1 and MMP-9; conditioned supernatant attracted PBMCs and neutrophils and cleaved MMP substrates. In contrast, VZV-infected HKs suppressed cytokine secretion except for IL-8, which attracted neutrophils, and suppressed MMP release and substrate cleavage.ConclusionsOverall, VZV-infected HCECs recapitulate findings of VZV keratitis with respect to epithelial cell proliferation, pseudodendrite formation and creation of a proinflammatory environment, providing an in vitro model for VZV infection of corneal epithelial cells. Furthermore, the proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the finding that VZV-infected HKs die and suppress most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.

Disease-related responses induced by cadmium in an in vitro human airway tissue model

Cadmium (Cd) is found at high concentrations in tobacco smoke due to its volatility when tobacco is burned. Inhaled Cd is linked to smoking-related respiratory diseases, such as chronic obstructive pulmonary disease and lung cancer. Alterations in mucociliary clearance, squamous metaplasia, and carcinoma are commonly observed in the respiratory tract of animals exposed to Cd. In vitro cell models widely used to study mechanisms underlying Cd toxicity are not suitable for studying its effects on mucociliary clearance and airway tissue remodeling. Herein we assess Cd-induced functional and structural changes in a well-differentiated human air-liquid-interface (ALI) airway tissue model. Acute treatments with Cd induced aberrant expression and secretion of mucins, impaired cilia functions, and squamous differentiation, and produced persistent oxidative stress and enhanced release of pro-inflammatory cytokines and matrix metalloproteinases. Accumulation of intracellular Cd was associated with sustained oxidative stress and inflammation, which, in turn, may have initiated squamous differentiation in ALI cultures. These observations demonstrate that ALI airway tissue models can recapitulate the functional and structural alterations in Cd-exposed animals, suggesting their potential application for studying tissue responses related to respiratory toxicants like those present in tobacco smoke.

Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease-Activated Receptor-2, in Human Corneal Epithelial Cells.

Cathepsin S (CTSS) activity is increased in tears of Sjögren’s syndrome (SS) patients. This elevated CTSS may contribute to ocular surface inflammation. Human corneal epithelial cells (HCE-T cells) were treated with recombinant human CTSS at activity comparable to that in SS patient tears for 2, 4, 8, and 24 h. Acute CTSS significantly increased HCE-T cell gene and protein expression of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) from 2 to 4 h, while matrix metalloproteinase 9 (MMP-9), CTSS, and protease-activated receptor-2 (PAR-2) were increased by chronic CTSS (24 h). To investigate whether the increased pro-inflammatory cytokines and proteases were induced by CTSS activation of PAR-2, HCE-T cells were transfected with PAR-2 siRNA, reducing cellular PAR-2 by 45%. Cells with reduced PAR-2 expression showed significantly reduced release of IL-6, TNF-α, IL-1β, and MMP-9 into culture medium in response to acute CTSS, while IL-6, TNF-α, and MMP-9 were reduced in culture medium, and IL-6 and MMP-9 in cell lysates, after chronic CTSS. Moreover, cells with reduced PAR-2 expression showed reduced ability of chronic CTSS to induce gene expression of pro-inflammatory cytokines and proteases. CTSS activation of PAR-2 may represent a potential therapeutic target for amelioration of ocular surface inflammation in SS patients.

Telmisartan inhibits Ang II-induced MMP-9 expression in macrophages in stabilizing atheromatous plaque.

To investigate the effects of telmisartan on matrix metalloproteinase-9 (MMP-9) expression in macrophages induced by angiotensin II (Ang II) and its mechanism. THP-1 cells were adopted for research, and phorbol-12-myristate-13-acetate (PMA) was utilized to induce THP-1 cells to be transformed into macrophages, with Ang II as a stimulating factor and telmisartan as a therapeutic drug. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) were applied to detect cell viability and toxicity. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the MMP-9 release level. Polymerase Chain Reaction (PCR) and Western blotting were conducted to detect the expressions of MMP-9 messenger ribonucleic acid (mRNA) and protein, respectively. The mechanism of action was further studied, and the activity of cyclooxygenase-2 (COX2)/macrophage-expressed gene 1 (mPEG1) pathway was determined via PCR and Western blotting. The 1 mM Ang II could remarkably activate the synthesis and release of MMP-9 as well as the COX2/mPEG1 pathway in macrophages. However, telmisartan could effectively repress the Ang II-induced MMP-9 synthesis and release in the macrophages, and suppress the COX2/mPEG1 pathway in the macrophages activated by Ang II. Telmisartan can inhibit the activation of MMP-9 in the macrophages by suppressing the COX2/mPEG1 pathway.

Protective activity of caffeic acid and sinapic acid against UVB-induced photoaging in human fibroblasts.

In this study, the protective effects of caffeic acid (CA) and sinapic acid (SA) on photoaging in human skin fibroblasts induced by UVB irradiation (30mJ/cm2 ) were examined. The results revealed that exposure to UVB irradiation reduced cell viability by approximately 33% compared with the non-UVB irradiated control. However, CA and SA blocked the UVB irradiation-induced cytotoxicity greater than the other phenolic acids. CA and SA also significantly inhibited the release of matrix metalloproteinase 1 (MMP-1) and reduced the expression of MMP-1 mRNA in UVB-irradiated Hs68 cells. Furthermore, CA and SA reduced UVB-induced production of reactive oxygen species (ROS) and collagen degradation in Hs68 cells. Finally, CA and SA effectively downregulated activation of the UVB-induced mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) signaling pathways. These results imply that hydroxylated cinnamate derivatives can be therapeutic agents against UVB-induced skin photoaging. PRACTICAL APPLICATIONS: Hydroxylated cinnamate derivatives such as CA and SA have a high cytoprotective effect against UV irradiation-induced photoaging in human fibroblasts via the inactivation of the MAPKs/NFκB signaling pathway. The present study suggests that CA and SA may be useful in therapeutic and cosmetic applications for the treatment of skin photoaging.

Effect of microfragmented adipose tissue on osteoarthritic synovial macrophage factors.

Cell-based therapies using adipose-derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti-inflammatory activities. Recently, an innovative one-step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF-ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL-6), CC-chemokine ligand 5/receptor-activated normal T-cell expressed and secreted (CCL5/RANTES), CC-chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and CC-chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α), and a higher level only of CXC-chemokine ligand 8/interleukin 8 compared with MF-ADMSCs. Matrix metalloproteinase 9 (MMP-9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP-1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL-6. In contrast, MF, not MF-ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage-specific chemokines (CCL2/MCP-1 and CCL3/MIP-1α) and degradative marker MMP-9. Interestingly, MF increased TIMP-1 (the MMP-9 inhibitor) and down-modulated toll-like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF-ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling.

Peptide binding to cleaved CD31 dampens ischemia/reperfusion-induced intestinal injury

BackgroundCD31 is a key transmembrane neutrophil immunoregulatory receptor. Mesenteric ischemia/reperfusion-induced neutrophil activation leads to a massive cleavage and shedding of the most extracellular domains of CD31 into plasma, enhancing the deleterious effect of neutrophil activation. We have evaluated the preventive therapeutic potential of an engineered synthetic octapeptide (P8RI), which restores the inhibitory intracellular signaling of cleaved CD31, in an experimental model of acute mesenteric ischemia/reperfusion.MethodsIn a randomized, controlled, and experimenter-blinded preclinical study, mesenteric ischemia/reperfusion (I/R) was induced in Wistar rats by superior mesenteric artery occlusion for 30 min followed by 4 h of reperfusion. Three groups of rats were compared: I/R + saline perfusion (I/R controls group, n = 7), I/R + preventive P8RI perfusion (P8RI group, n = 7), and sham-operated rats + saline perfusion (sham group, n = 7).ResultsCompared with I/R controls, P8RI perfusion significantly decreased intestinal ischemia/reperfusion injury (Chiu’s score, P = 0.01; epithelial area, P = 0.001) and bacterial translocation (plasma Escherichia coli DNA, P = 0.04) and could limit intestinal bleeding (P = 0.09). P8RI decreased neutrophil activation assessed by matrix metalloproteinase-9 release in plasma (P < 0.001) and in the intestinal wall, albeit without statistical significance (P = 0.06 and P = 0.058 for myeloperoxydase). Inhibition of CD31 cleavage from neutrophils could play a major role in the protective effects of P8RI (P < 0.0001).ConclusionsPreventive administration of P8RI, a CD31-agonist peptide, could decrease I/R-induced intestinal injury by potentially limiting neutrophil activation.