Release Of Matrix Metalloproteinases Research Articles

Secretion correlation between matrix metalloproteinases and extracellular vesicles revealed by synchronized dynamic monitoring of single cells on a microfluidic chip

Despite growing interest in matrix metalloproteinases (MMPs) as promising therapeutic targets for cancer treatments, cellular trafficking and release of MMPs remain underexplored. To evaluate to what content the MMP secretion is associated with extracellular vesicles (EVs), in this study, we report a droplet microfluidic assay for simultaneous dynamic monitoring of the MMP and EV secretion at the single-cell level. Microdroplets containing single cells were generated in a flow-focusing microchannel and then guided to a microcolumn array for on-chip patterning, incubation and time-lapse microscopy. Utilizing a green fluorescent substrate for MMP activity and a red fluorescent protein reporter for EVs, temporal secretion dynamics of MMPs and EVs from individual cells were recorded respectively. With this dual-reporter system, a strong correlation between MMP and EV secretion was revealed. We further demonstrated that promotion or inhibition of EV release by altering intracellular calcium levels would induce a rise or fall in single-cell MMP secretion, which was masked by population-based experiments. More importantly, the activated release of this EV subpopulation (exosomes) also resulted in a higher correlation coefficient between MMP and EV secretion, and vice versa. Therefore, compared to other EV subpopulations, exosomes should be more closely connected with MMP secretion, and thus may represent a potential target for combination therapy. When in contrast with bulk experiments, single-cell analysis of the secretion correlation between MMPs and EVs not only reveals extensive cellular heterogeneity, but also exhibits a higher resolution.

Long COVID elevated MMP-9 and release from microglia by SARS-CoV-2 Spike protein.

Long COVID is a major health concern because many patients develop chronic neuropsychiatric symptoms, but the precise pathogenesis is unknown. Matrix metalloproteinase-9 (MMP-9) can disrupt neuronal connectivity and be elevated in patients with long COVID. In this study, MMP-9 was measured in the serum of long COVID patients and healthy controls, as well as in the supernatant fluid of cultured human microglia cell line stimulated by recombinant severe acute respiratory syndrome coronavirus 2 Spike protein, as well as lipopolysaccharide (LPS) and neurotensin (NT) used as positive controls. MMP-9 was measured by commercial enzyme-linked immunosorbent assay. MMP-9 was significantly elevated in the serum of long COVID patients compared to healthy controls. Moreover, there was significant release of MMP-9 from a cultured human microglia cell line stimulated by LPS, NT, or Spike protein. We further show that pretreatment with the flavonoids luteolin and tetramethoxyluteolin (methlut) significantly inhibited the release of MMP-9 stimulated by the Spike protein. MMP-9 from Spike protein-stimulated microglia could contribute to the development of long COVID and may serve as a target for treatment including the use of luteolin.

Osteoarthritis induced by monosodium iodoacetate model

Objectives: this study aimed to highlight the evolution of the osteoarthritis (OA) model induced by monosodium iodoacetate (MIA) and its collaboration with the knowledge of the pathophysiology of inflammation, hypernociception and cartilage degeneration. Methods: PubMed and Science Direct were used with the descriptors: knee osteoarthritis or osteoarthritis of ankle) and (rat or osteoarthritis induced by monoiodoacetate) and (animal model of osteoarthritis or joint nociception). Studies containing rats, mice, and monoiodoacetate-induced osteoathritis were included. Exclusion criteria were: Animal models of osteoarthritis induced by destabilization; meniscectomy-induced osteoarthritis; non-invasive animal models of osteoarthritis induced by fracture, compression and tibial overload; animal models of osteoarthritis with large animals and models of osteoarthritis by papain or bacterial collagenase. We summarized studies that used MIA to induce knee or ankle OA in rats or mice and the evolution of the inflammatory markers. Results: a total of 38 original manuscripts met the inclusion criteria and were considered in the study. The model of OA induced by MIA is well-established and explored with several methodologies and has been widely used in different species. MIA induces cell death, progressive loss of chondrocytes and histological changes that mimics human OA. Studies with this model demonstrated inflammation, neurogenic hyperalgesia, release of cytokines and matrix metalloproteinases. More recently, molecular biology data confirm the activation of nuclear transcription factors, which modulates the expression of citokines in apoptotic chondrocytes. Conclusion: the MIA-induced OA has been useful in predicting several characteristics for the elucidation of osteoarthritis pathophysiology.

Immunocellular microenvironment of the vascular wall of cerebral aneurysms: What is the role of inflammatory cells in aneurysmal remodeling?

AbstractIntracranial aneurysms (IAs) may afflict up to 5% of the general population, or up to 15 million individuals in the US. The two forms of IAs that can be recognized by their shape are saccular and nonsaccular IAs, with uncommon aneurysm types, fusiform and dissecting aneurysms, comprising 13% of nonsaccular IAs. Conceivably, among the various risk factors for IA development, vessel wall inflammation represents a major cause. Accordingly, IAs may not necessarily be the result of passive widening of vessel wall structures, but may also be the result of inflammation and tissue degeneration. Thus, flow‐induced vascular remodeling during IA pathogenesis may reflect immune cell infiltration and consequent release of proinflammatory cytokine, chemokine, and matrix metalloproteinase that contribute to vessel wall degeneration and weakening. Thus, infiltrating neutrophils, macrophages, T‐lymphocytes and complement factors, and the resulting immune microenvironment may be pertinent in IA pathogenesis.

Impact of Hypoxia on Neutrophil Degranulation and Inflammatory Response in Alpha-1 Antitrypsin Deficiency Patients.

Alpha-1 antitrypsin deficiency (AATD) is an inflammatory disorder where neutrophils play a key role. Excessive neutrophil activation leads to local hypoxia and tissue damage. Most research on neutrophil function has been conducted under atmospheric conditions (21% O2), which may not represent physiological or pathological conditions. This study aimed to determine the effects of hypoxia on neutrophil degranulation and cytokine production in AATD patients. Neutrophils isolated from 54 AATD patients (31 MZ; 8 SZ; 15 ZZ) and 7 controls (MM) were exposed to hypoxia (1% O2) for 4 h. Neutrophil degranulation was assessed by measuring elastase (NE), myeloperoxidase (MPO), lactoferrin, and matrix metalloproteinase-9 (MMP-9) levels using immunoassay-based methods. Pro-inflammatory (IL-8, IL-1 beta, IL-6, and TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokine levels were assessed by a Luminex-based method. Our results indicate a significantly increased release of NE (p = 0.015), MPO (p = 0.042), lactoferrin (p = 0.015), and MMP-9 (p = 0.001) compared to controls. Pro-inflammatory cytokines show a significant rise in IL-8 (p = 0.019), a trend towards increased IL-1 beta (p = 0.3196), no change in IL-6 (p = 0.7329), and reduced TNF-alpha (p = 0.006). Anti-inflammatory cytokines show increased IL-4 (p = 0.057) and decreased IL-10 (p = 0.05703). Increased neutrophil degranulation and inflammatory phenotype are observed in AATD neutrophils under physiological hypoxia.

MicroRNAs in abdominal aortic aneurysms

The abdominal aortic aneurysm (AAA), representing 70% to 90% of all aortic aneurysms, is characterized by a widening of the diameter due to an irreversible weakening of the vascular walls. Risk factors include advanced age, male sex, smoking, hypertension, atherosclerosis, and genetic predisposition. From a pathophysiological point of view, AAA involves alterations in connective tissue proteins, chronic inflammation with the release of cytokines and matrix metalloproteinases, as well as changes in the extracellular matrix. MicroRNAs (miRNAs) have emerged as promising biomarkers and therapeutic targets in the regulation of gene expression and different specific signaling pathways in vascular pathologies. Among which some miRNAs have been highlighted such as: miR-29 and miR-27b-3p, involved in the degradation of the extracellular matrix and inflammatory processes during the progression of AAA. Some diagnostic methods such as computed tomography play a fundamental role, while some innovative therapeutic strategies, such as the inhibition of miRNAs, represent a leading role as possible diagnostic and therapeutic targets. A comprehensive approach includes surveillance and surgical treatment strategies to mitigate the progression and risk of AAA rupture, thus highlighting the relevance of miRNAs in the diagnosis and treatment of this pathology.

Investigating the Antifibrotic Effects of β-Citronellol on a TGF-β1-Stimulated LX-2 Hepatic Stellate Cell Model.

Liver fibrosis, a consequence of chronic liver damage or inflammation, is characterized by the excessive buildup of extracellular matrix components. This progressive condition significantly raises the risk of severe liver diseases like cirrhosis and hepatocellular carcinoma. The lack of approved therapeutics underscores the urgent need for novel anti-fibrotic drugs. Hepatic stellate cells (HSCs), key players in fibrogenesis, are promising targets for drug discovery. This study investigated the anti-fibrotic potential of Citrus hystrix DC. (KL) and its bioactive compound, β-citronellol (β-CIT), in a human HSC cell line (LX-2). Cells exposed to TGF-β1 to induce fibrogenesis were co-treated with crude KL extract and β-CIT. Gene expression was analyzed by real-time qRT-PCR to assess fibrosis-associated genes (ACTA2, COL1A1, TIMP1, SMAD2). The release of matrix metalloproteinase 9 (MMP-9) was measured by ELISA. Proteomic analysis and molecular docking identified potential signaling proteins and modeled protein-ligand interactions. The results showed that both crude KL extract and β-CIT suppressed HSC activation genes and MMP-9 levels. The MAPK signaling pathway emerged as a potential target of β-CIT. This study demonstrates the ability of KL extract and β-CIT to inhibit HSC activation during TGF-β1-induced fibrogenesis, suggesting a promising role of β-CIT in anti-hepatic fibrosis therapies.

Progress in the treatment of Osteoarthritis with avocado-soybean unsaponifiable.

Osteoarthritis (OA) is one of the leading causes of joint dysfunction and disability in the elderly, posing serious social problems and a huge socio-economic burden. Existing pharmacological treatments have significant drawbacks, and searching for an effective pharmacological intervention is an urgent priority. Recent studies have demonstrated the chondroprotective, anabolic, and anti-catabolic properties of avocado-soybean unsaponifiable (ASU), a natural plant extract made from avocado and soybean oils, consisting of the remainder of the saponified portion of the product that cannot be made into soap. The main components of ASU are phytosterols, beta-sitosterol, canola stanols, and soya stanols, which are rapidly incorporated into cells. Studies have confirmed the anti-inflammatory, antioxidant, and analgesic properties of phytosterols. ASU slows down the progression of OA primarily by inhibiting pathways involved in the development of OA disease. ASU prevents cartilage degradation by inhibiting the release and activity of matrix metalloproteinases and by increasing the tissue inhibition of these catabolic enzymes; ASU is also involved in the inhibition of the activation of nuclear factor κB (NF-κB) which is a transcriptional inhibitor that regulates the inflammatory response of chondrocytes. NF-κB is a transcription factor that regulates the inflammatory response of chondrocytes, and inhibition of the transfer of the transcription factor NF-κB from the cytoplasm to the nucleus regulates the transcription of many pro-inflammatory factors. By appealing to the mechanism of action and thus achieving anti-inflammatory, anti-catabolic, and pro-synthetic effects on cartilage tissues, AUS is clinically responsive to the reduction of acute pain and OA symptom progression. This paper aims to summarize the studies on the use of avocado-soybean unsaponifiable in the pharmacological treatment of osteoarticular.

Activity of hydrogel-vitamin D3 /bacterioruberin nanoparticles on imiquimod-induced fibroblasts-keratinocytes spheroids

Topical administration of vitamin D3 (VD3) analogs and corticosteroids is the mainstay treatment for mild localized psoriasis. VD3 however is an unstable class II drug (insoluble and permeable), classically formulated as unattractive viscous and oil-based ointments or creams. In this work, nanostructured archaeolipid carriers (NAC) with a core loaded with VD3 and the C50 dipolar carotenoid from halophilic archaea bacterioruberin (BR): NAC-VD3, were formulated into a carbopol hydrogel: gNAC-VD3. After its rheological characterization, its anti-inflammatory and antioxidant activity was assessed on bi-cellular spheroids, consisting of a core of fibroblasts surrounded by a ring of keratinocytes activated with imiquimod (IMQ) as a psoriasis model. gNAC-VD3 (0.8 mg VD3/135 μg BR/gram gel) conserved the antioxidant activity, showed pseudoplastic non-Newtonian behaviour, stability over time, good occlusion capacity and spreadability, all suitable properties for topical application in patients with psoriasis. gNAC-VD3 did not decrease the viability of spheroids but significantly reduced the release of proinflammatory cytokines (IL-8, IL-6, TNF-α), oxidative stress (ROS), and matrix metalloproteinases from IMQ-induced spheroids to levels similar to the uninduced spheroids. Results suggest that gNAC-VD3 may constitute a potential anti-inflammatory agent for the topical treatment of mild psoriasis and deserves further in vivo exploration.

Tobacco smoke condensate-induced senescence in endothelial cells was ameliorated by colchicine treatment via suppression of NF-κB and MAPKs P38 and ERK pathways activation

Smoking is the major cause of cardiovascular diseases and cancer. It induces oxidative stress, leading to DNA damage and cellular senescence. Senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase, which are known to play a vital role in the initiation and progression of cardiovascular diseases and metastasis in cancer. The current study investigated the smoking induced cellular senescence and employed colchicine that blocked senescence in endothelial cells exposed to tobacco smoke condensate. Colchicine prevented oxidative stress and DNA damage in tobacco smoke-condensate-treated endothelial cells. Colchicin reduced β-gal activity, improved Lamin B1, and attenuated cell growth arrest markers P21 and P53. Colchicine also ameliorated the expression of SASP factors and inhibited the activation of NF-kB and MAPKs P38 and ERK. In summary, colchicine inhibited tobacco smoke condensate-induced senescence in endothelial cells by blocking the activation of NF-kB and MAPKs P38 and ERK.Graphical

The pathological function of neutrophils in pemphigoid diseases

Abstract Pemphigoid diseases (PDs) are a group of autoimmune blistering diseases, including bullous pemphigoid, epidermolysis bullosa acquisita, mucous membrane pemphigoid, linear immunoglobulin A disease, and other rare variants. These diseases are characterized by the presence of autoantibodies that target proteins at the dermal-epidermal junction, resulting in the formation of tense blisters and erosions on the skin and/or mucosa. The current therapeutic approaches, such as systemic corticosteroid, are associated with significant adverse effects, highlighting that safer and more effective treatment options are an urgent clinical need. To address this unmet need, a comprehensive understanding of the detailed mechanisms underlying PDs is essential. Based on their histopathological infiltration in pemphigoid lesions, neutrophils have long been implicated as major contributors to the initiation and progression of the diseases. Numerous in vivo and in vitro studies have investigated the role of neutrophils in the pemphigoid pathology, revealing various pathological mechanisms induced by these cells, including the release of neutrophil elastase and matrix metalloproteinase-9, as well as the formation of neutrophil extracellular traps. The present review provides a comprehensive summary and critical evaluation of the current understanding regarding the role of neutrophils in PDs. In addition, it discusses the potential of targeting neutrophil-associated pathways as a novel therapeutic approach for the diseases.

Differential biological activity of two extracts of ripe fruits of Carica candamarcensis Hook on human neutrophils

We evaluated the effect of the total macerate (TM) and seed oil (SO) of mature Carica candamarcensisfruits, on the release of Matrix metalloproteinase 9 (MMP9) and the phosphorylation of MAPK in neutrophils. The antioxidant capacity of these extracts was evaluated by ABTS assay. Neutrophils stimulated with different dilutions of TM or SO were analyzed for cytotoxicity, MMP9 release, and MAPK phosphorylation, using trypan blue exclusion assays, zymography, and immunoblotting, respectively. Both extracts show antioxidant activity, being higher in TM; none presented cytotoxic effect. The 5% and 2.5% dilutions of TM significantly reduced MMP9 release, and all decreased MAPK phosphorylation. SO significantly increased the release of MMP9 and MAPK phosphorylation, the effect being greater when they were prestimulated with lipopolysaccharide.TM may have anti-inflammatory potential, while SO could have a priming effect that needs to be confirmed.

Glyoxal-derived advanced glycation end products (GO-AGEs) with UVB critically induce skin inflammaging: in vitro and in silico approaches

Advanced glycation end products (AGEs) have potential implications on several diseases including skin inflammation and aging. AGEs formation can be triggered by several factors such as UVB, glyoxal and methylglyoxal etc. However, little attention has been paid to glyoxal-derived AGEs (GO-AGEs) and UVB-induced skin inflammaging, with none have investigated together. This study aimed to investigate the possible role of GO-AGEs and UVB in skin inflammaging focusing on revealing its molecular mechanisms. The effects of GO-AGEs in the presence or absence of UVB were studied by using enzyme linked immunosorbent assay, western blotting, qPCR, flow cytometry and in silico approaches. In HaCaT cells, GO-AGEs in the presence of UVB irradiation (125 mJ/cm2) dramatically enhanced the release of different pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) with further activation of RAGE signaling pathways (NF-κB, COX 2, and IL- 1β) and increased oxidative stress also noticed in NHEK cells. In NHDF cells, extracellular matrix disruption noted via increasing matrix metalloproteinase release and decreasing collagen type 1 and SIRT1 expression. Besides that, the docking scores obtained from the molecular docking study support the above-mentioned results. This study strongly suggests the pivotal role of GO-AGEs in skin inflammaging and illuminates novel molecular pathways for searching most effective and updated anti-aging therapy.

The Impact of 45S5-Bioactive Glass on Synovial Cells in Knee Osteoarthritis-An In Vitro Study.

Synovial inflammation in osteoarthritis (OA) is characterized by the release of cartilage-degrading enzymes and inflammatory cytokines. 45S5-bioactive glass (45S5-BG) can modulate inflammation processes; however, its influence on OA-associated inflammation has hardly been investigated. In this study, the effects of 45S5-BG on the release of cartilage-degrading metalloproteinases and cytokines from synovial membrane cells (SM) isolated from patients with knee OA was assessed in vitro. SM were cultivated as SM monocultures in the presence or absence of 45S5-BG. On day 1 (d1) and d7 (d7), the concentrations of Matrix Metalloproteinases (MMPs) and cytokines were assessed. In 45S5-BG-treated SM cultures, MMP9 concentration was significantly reduced at d1 and d7, whilst MMP13 was significantly increased at d7. Concentrations of interleukin (IL)-1B and C-C motif chemokine ligand 2 (CCL2) in 45S5-BG-treated SM cultures were significantly increased at both time points, as were interferon gamma (IFNG) and IL-6 at d7. Our data show an effect of 45S5-BG on SM activity, which was not clearly protective, anti-inflammatory, or pro-inflammatory. The influence of 45S5-BG on MMP release was more suggestive of a cartilage protective effect, but 45S5-BG also increased the release of pro-inflammatory cytokines. Further studies are needed to analyze the effect of BGs on OA inflammation, including the anti-inflammatory modification of BG compositions.

Use of reconstructed skin model to assess the photoprotection afforded by three sunscreen products having different SPF values against DNA lesions and cellular alterations

IntroductionBoth UVB and UVA rays induce biological damages in the epidermis and the dermis that contribute to photo-carcinogenesis and photoaging. In the present study, the photoprotective effect of 2 ISO standard sunscreens, P3 (Sun Protection Factor [SPF]15) and P6 (SPF40) and of an SPF50+ labeled commercial sunscreen product was tested in reconstructed skin tissues exposed to increasing doses of UV Solar Simulated Radiation (UV-SSR). UV-induced damages were evaluated using several biological markers, including DNA lesions in the presence or absence of sunscreen protection. MethodT-Skin™ model samples (EPISKIN), composed of a fibroblast-populated dermal equivalent and a fully differentiated epidermis, were protected with the test sunscreens (1.3 mg/cm² topically applied on molded polymethyl methacrylate plate) before being exposed to increasing UV doses (0 – 2.5 – 5 - 25 – 40 J/cm²). Twenty-four hours after exposure, tissues with and without sunscreen protection, were analyzed for skin viability and morphology, DNA lesions (cyclobutane pyrimidine dimer) and inflammatory mediator quantification. Results were compared to untreated exposed tissues using a Wilcoxon non-parametric test. ResultsFor untreated tissues, UV-SSR exposure induced a dose-dependent decrease in epidermal and dermal viabilities, an increase in release of proinflammatory cytokines and matrix metalloproteinases and were associated with morphological damages at doses as low as 5 J/cm2. DNA lesions were even detected at the lowest dose of 2.5 J/cm2, and their number increased with the UV-SSR dose. In the samples protected with sunscreens, these abnormalities were partially or totally prevented with P6 providing a better protection compared to P3, and the SPF50+ sunscreen showing a trend for better protection than P6, for example against DNA damage. ConclusionsThis study demonstrates that photoprotective effects of different sunscreens can be discriminated and ranked on reconstructed skin tissues (T-Skin™ model) exposed to UV-SSR. Showing significant differences between the reference products P3 and P6 in line with their respective SPF values, such study allows the evaluation of epidermal and dermal damages at the tissue, cellular and molecular levels. It thus opens the way to a new model of integrated assessment of sunscreens. In line with its labeled 50+ SPF, the commercial test product confirmed its improved protection especially on DNA damage prevention.

MTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

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.

To gain insights into how proteases signal to connective tissues cells in the periodontium. 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. 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. 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. 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.

C9orf72-ALS human iPSC microglia are pro-inflammatory and toxic to co-cultured motor neurons via MMP9

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, with additional pathophysiological involvement of non-neuronal cells such as microglia. The commonest ALS-associated genetic variant is a hexanucleotide repeat expansion (HRE) mutation in C9orf72. Here, we study its consequences for microglial function using human iPSC-derived microglia. By RNA-sequencing, we identify enrichment of pathways associated with immune cell activation and cyto-/chemokines in C9orf72 HRE mutant microglia versus healthy controls, most prominently after LPS priming. Specifically, LPS-primed C9orf72 HRE mutant microglia show consistently increased expression and release of matrix metalloproteinase-9 (MMP9). LPS-primed C9orf72 HRE mutant microglia are toxic to co-cultured healthy motor neurons, which is ameliorated by concomitant application of an MMP9 inhibitor. Finally, we identify release of dipeptidyl peptidase-4 (DPP4) as a marker for MMP9-dependent microglial dysregulation in co-culture. These results demonstrate cellular dysfunction of C9orf72 HRE mutant microglia, and a non-cell-autonomous role in driving C9orf72-ALS pathophysiology in motor neurons through MMP9 signaling.

The Eotaxin-1/CCR3 Axis and Matrix Metalloproteinase-9 Are Critical in Anti-NC16A IgE-Induced Bullous Pemphigoid.

Bullous pemphigoid (BP) is the most common autoimmune bullous skin disease of humans and is characterized by eosinophilic inflammation and circulating and tissue-bound IgG and IgE autoantibodies directed against two hemidesmosomal proteins: BP180 and BP230. The noncollagenous 16A domain (NC16A) of BP180 has been found to contain major epitopes recognized by autoantibodies in BP. We recently established the pathogenicity of anti-NC16A IgE through passive transfer of patient-derived autoantibodies to double-humanized mice that express the human high-affinity IgE receptor, FcεRI, and human NC16A domain (FcεRI/NC16A). In this model, anti-NC16A IgEs recruit eosinophils to mediate tissue injury and clinical disease in FcεRI/NC16A mice. The objective of this study was to characterize the molecular and cellular events that underlie eosinophil recruitment and eosinophil-dependent tissue injury in anti-NC16A IgE-induced BP. We show that anti-NC16A IgEs significantly increase levels of key eosinophil chemoattractants, eotaxin-1 and eotaxin-2, as well as the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) in the lesional skin of FcεRI/NC16A mice. Importantly, neutralization of eotaxin-1, but not eotaxin-2, and blockade of the main eotaxin receptor, CCR3, drastically reduce anti-NC16A IgE-induced disease activity. We further show that anti-NC16A IgE/NC16A immune complexes induce the release of MMP-9 from eosinophils, and that MMP-9-deficient mice are resistant to anti-NC16A IgE-induced BP. Lastly, we find significantly increased levels of eotaxin-1, eotaxin-2, and MMP-9 in blister fluids of BP patients. Taken together, this study establishes the eotaxin-1/CCR3 axis and MMP-9 as key players in anti-NC16A IgE-induced BP and candidate therapeutic targets for future drug development and testing.

Asiaticoside Attenuates Blood-Spinal Cord Barrier Disruption by Inhibiting Endoplasmic Reticulum Stress in Pericytes After Spinal Cord Injury.

The blood-spinal cord barrier (BSCB) plays a vital role in the recovery of spinal cord function after spinal cord injury (SCI). Pericytes, pluripotent members of the neurovascular unit (NVU), receive signals from neighboring cells and are critical for maintaining CNS function. Therapeutic targets for the BSCB include endothelial cells (ECs) and glial cells, but few drugs target pericytes. This study was designed to explore whether asiaticoside has a positively effect on pericytes and the integrity of the BSCB. In this study, we found that asiaticoside could inhibit the loss of junction proteins just 1 day after SCI in vivo, but our in vitro study showed no significant differences in the expression of endothelial junction proteins between the control and asiaticoside treatment groups. We also found that asiaticoside could inhibit endoplasmic reticulum (ER) stress and pericyte apoptosis, which might be associated with the inhibition of junction protein reduction in ECs. Thus, we investigated the interactions between pericytes and ECs. Our results showed that asiaticoside could decrease the release of matrix metalloproteinase (MMP)-9 in pericytes and therefore upregulate the expression of junction proteins in ECs. Furthermore, the protective effect of asiaticoside on pericytes is related to the inhibition of ER stress via the MAPK signaling pathway. Taken together, our results demonstrate that asiaticoside treatment inhibits BSCB disruption and enhances functional recovery after SCI.