MMP-1 In Fibroblasts Research Articles

Proteins extracted from pearl oyster (Pinctada martensii) with efficient accelerated wound healing in vitro through promoting cell proliferation, migration, and collagen formation

Ethnopharmacological relevancePearl oyster (Pinctada martensii) is used in Chinese traditional medicine use in photoprotective, anti-inflammatory, and wound treatment.Aim of the study: This study explored whether the mucus protein of Pearl oyster (protein of Pinctada martensii, PMP) affects human skin fibroblast (HSF) proliferation, migration, collagen-related gene expression related to collagen formation, and in vivo healing effects. Materials and methodsThe PMP component was analyzed by LC-MS/MS. The cell viability was evaluated using a CCK-8 kit. The expression genes were measured by reverse transcription polymerase chain reaction. A full-thickness excisional wounding model in Sprague-Dawley (SD) rats was used to test the repairing effect of PMP in vivo, and Hematoxylin-Eosin (H&E) and Masson's Trichrome staining were applied to evaluate skin structure. ResultsThe components of PMP were identified using LC-MS/MS proteomics, and a total of 3023 proteins were detected. The results of PMP-treated HSF showed that PMP effectively promoted cell proliferation by 1.6-fold and cell migration by 1.5-fold at a concentration of 1 mg/mL. Additionally, PMP treatment up-regulated the expression levels of collagen-related genes COL1A1, COL3A1, and MMP-1 in fibroblasts. Furthermore, PMP was applied in the therapy of full-thickness excisional wounds in rats. The results demonstrated that PMP significantly accelerated wound healing time, resulted in the recovery of dermal and epithelial thickness, and stimulated collagen regeneration. The regenerated skin closely resembled the structure of normal skin. ConclusionsThese findings provide solid evidence supporting the potential of PMP as a promising candidate for the treatment of skin wounds.

Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

Carboxyamidotriazole alleviates pannus formation and cartilage erosion in rats with adjuvant arthritis

Carboxyamidotriazole (CAI) was initially considered a non-cytotoxic anticancer agent. However, recently, pronounced anti-inflammatory properties of CAI have been reported. Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by aberrant activation of signaling pathways. Therefore, this study explored the therapeutic effects and potential mechanism of action of CAI on RA in the adjuvant arthritis (AA) model. The results showed that CAI reduced the severity of arthritis in AA rats as demonstrated by inhibited hind paw swelling, reduced body weight, and decreased infiltration of joint pathological inflammatory cells. Importantly, pathological scoring of new blood vessels and immunohistochemical assays revealed that CAI inhibited pannus formation. CAI decreased the expression of pro-angiogenic growth factors, such as vascular epidermal growth factor, basic fibroblast growth factor, and metalloproteinases (MMPs), namely, MMP-1 and MMP-3 in the synovium of AA rats. Furthermore, CAI significantly reduced the increased levels of phosphorylated p38, c-Jun N-terminal kinase (JNK)1/2, and extracellular signal-regulated kinase (ERK)1/2 proteins in AA rats. In addition, the proliferation of fibroblast-like synoviocytes (FLS) was downregulated by CAI both in vivo and in vitro. In conclusion, this investigation illustrates the therapeutic effect of CAI on synovitis and erosion of articular cartilage in RA. Furthermore, the mechanism might involve inhibition of aberrantly activated mitogen-activated protein kinase signaling, as well as a decrease in pro-angiogenic factors, MMP expression, and FLS proliferation.

Flavonoid components and anti-photoaging activity of flower extracts from six Paeonia cultivars

Genus Paeonia is an important ornamental and medicinal crop. The flowers of Paeonia are rich in flavonoids, and flavonoids have excellent antioxidant and anti-inflammatory activities, which have the potential to slow down skin aging. In this study, flavonoids from whole flowers of six popular Paeonia cultivars were extracted using three different solvents. The composition and content of peony flowers flavonoids extracted with three solvents were determined by high performance liquid chromatography quadrupole time-of-flight mass spectrometry / mass spectrometry (HPLC-Q-TOF-MS/MS) and HPLC-diode array detection (DAD). A total of 26 flavonoids were isolated and identified, and 60 % ethanol showed the highest extraction efficiency. Not only flavonoids composition but also the content was significantly different in different peony flowers, among which ‘Hei Tian E’ had the most abundant composition (17 flavonoids) and the highest total content of flavonoids (57.75 mg/g DW by using 60 % ethanol). After that, the anti-skin photoaging effects of the 60 % ethanol extracts of the six Paeonia cultivars flowers were evaluated by UVR-Fulkutis and UVA-Fibroblasts photoaging models. Under exposure to UV radiation, some Paeonia flowers’ extracts can prevent the decrease of skin surface thickness and reduce the loss of Collagen IV and VII. In addition, the production and accumulation of ROS, MMP-1 and MMP-3 in fibroblasts were inhibited, indicating the attenuation of skin photoaging by the extracts. Among the six samples tested, ‘Hei Tian E’ whole flower extract (BH-HT) had the best comprehensive effect. Finally, the correlations between components and anti-photoaging efficacy were analyzed. We speculate that the anti-photoaging effect was significantly related to its richness in quercetin 3-O-di-glucoside, quercetin 3-O-glucoside, isorhamnetin 3-O-galloyl arabinoside, cyanidin 3,5-di-O-glucoside, cyanidin 3-O-glucoside, and peonidin 3-O-glucoside. In general, the results show that Paeonia flowers have potential for development of anti-photoaging herbal essence products.

Staphylococcus aureus enhances gelatinase activities in monocytic U937 cells and in human gingival fibroblasts

Background/purposeStaphylococcus aureus (S. aureus) has been suggested to be an initiative pathogen in peri-implantitis because of the solid affinity to titanium. However, the detail pathogenesis for the peri-implantitis initiation by S. aureus is still lacking. This study aimed to in vitro examine the gelatinases’ activities of monocytic U937 cell and human gingival fibroblast after challenges with S. aureus lipoteichoic acid (LTA) and peptidoglycan (PGN). Materials and methodsReleases of gelatinases, including matrix metalloproteinase (MMP)-2 and −9, from cells were measured by zymography. The releases were further examined after being given the S. aureus LTA/PGN. Roles of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways on the enzyme releases were examined by administrating inhibitors. ResultsS. aureus LTA and PGN increased the activities of pro-MMP-9 from U937 cells and pro-MMP-2 and MMP-2 from gingival fibroblasts. By giving the NF-κB inhibitor, the enhanced gelatinase activities in both cells were attenuated. In U937 cells, the enhanced pro-MMP-9 could further be attenuated by MAPK inhibitors, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), P38 MAPK, and c-Jun N-terminal kinase (JNK) inhibitors; however, the attenuation by MAPK inhibitors could not be observed for MMP-2 in gingival fibroblasts. Nevertheless, in gingival fibroblasts, the pro-MMP-2 could be attenuated by JNK inhibitor. ConclusionS. aureus could enhance gelatinase activities of gingival fibroblasts and U937 cells, via NF-κB. The MAPK pathway was also involved in MMP-9 activity of U937 cells; however, the involvement of MAPK in MMP-2 activity of gingival fibroblasts was questioned.

Transglutaminase 2 mediates UVB-induced matrix metalloproteinase-1 expression by inhibiting nuclear p65 degradation in dermal fibroblasts.

Matrix metalloproteinases (MMPs) play a key role in tissue remodelling by cleaving extracellular matrix (ECM) components. In the skin, UV irradiation increases expression of MMPs that causes dysregulation of ECM homeostasis in dermis, leading to acceleration of skin aging. However, the mediator(s) that links UV irradiation to the upregulation of MMPs have not been fully defined. Previously, we showed that UVB irradiation activated transglutaminase 2 (TG2) in keratinocytes, eliciting an inflammatory response by activating NF-κB signalling. In this study, we reported the role of TG2 in mediating the UVB-induced expression of MMP-1. In human dermal fibroblasts, UVB irradiation enhanced the expression and activity of TG2, which in turn promotes the expression of MMP-1. Analyses of MMP-1 promoter showed that activation of the NF-κB signalling pathway, rather than AP-1, was responsible for the TG2-mediated upregulation of MMP-1. Moreover, Western blot analysis revealed that TG2 increased the activity of NF-κB by inhibiting degradation of p65 in the nucleus. Furthermore, ex vivo skin from TG2-knockout mice exhibited significantly reduced levels of MMP-1 compared to that from wild-type mice. These results indicate that TG2 functions as a mediator for the UVB-induced expression of MMP-1 in dermal fibroblasts, providing a new target for preventing skin photodamage.

Artificial Neural Network Modeling of Nanostructured Lipid Carriers Containing 5-O-Caffeoylquinic Acid-Rich Cratoxylum formosum Leaf Extract for Skin Application.

Purpose: To investigate the in vitro anti-skin-aging properties of Cratoxylum formosum extract and encapsulate this plant extract in nanostructured lipid carriers (CFE-NLCs) for dermal application. Methods: The biological properties of the plant extract, including enhanced procollagen type I synthesis and anti-matrix metalloproteinase activity, were evaluated to assess its cosmetic benefits. An artificial neural network (ANN) coupled with K-fold cross-validation was applied to investigate the effects of the formulants and optimize the CFE-NLCs. The physicochemical properties, percutaneous absorption, and irritation potential of the CFE-NLCs were analyzed. Results: Liquid chromatography-mass spectrometry analysis revealed that CFE contained 5-O-caffeoylquinic acid as the vital constituent. Appropriate skin-care properties were also demonstrated with respect to enhanced type I procollagen synthesis and the inhibition of MMP-1, MMP-3, and MMP-9 in primary human dermal fibroblasts. The optimal CFE-NLCs exhibited better skin absorption and biocompatibility and lower irritation potential than the free botanical extract solution. Conclusion: The findings obtained highlight CFE-NLCs as promising skin-care ingredients.

Irinotecan and its metabolite SN38 inhibits procollagen I production of dermal fibroblasts from Systemic Sclerosis patients

Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by a microangiopathy and fibrosis of the skin and internal organs. No treatment has been proved to be efficient in case of early or advanced SSc to prevent or reduce fibrosis. There are strong arguments for a key role of topo-I in the pathogenesis of diffuse SSc. Irinotecan, a semisynthetic derivative of Camptothecin, specifically target topo-I. This study was undertaken to evaluate the effects of noncytotoxic doses of irinotecan or its active metabolite SN38 on collagen production in SSc fibroblasts. Dermal fibroblasts from 4 patients with SSc and 2 healthy donors were cultured in the presence or absence of irinotecan or SN38. Procollagen I release was determined by ELISA and expression of a panel of genes involved in fibrosis was evaluated by qRT-PCR. Subcytotoxic doses of irinotecan and SN38 caused a significant and dose-dependent decrease of the procollagen I production in dermal fibroblasts from SSc patients, respectively − 48 ± 3%, p < 0.0001 and − 37 ± 6.2%, p = 0.0097. Both irinotecan and SN38 led to a global downregulation of genes involved in fibrosis such as COL1A1, COL1A2, MMP1 and ACTA2 in dermal fibroblasts from SSc patients (respectively − 27; − 20.5; − 30.2 and − 30% for irinotecan and − 61; − 55; − 50 and − 54% for SN38). SN38 increased significantly CCL2 mRNA level (+ 163%). The inhibitory effect of irinotecan and its active metabolite SN38 on collagen production by SSc fibroblasts, which occurs through regulating the levels of expression of genes mRNA, suggests that topoisomerase I inhibitors may be effective in limiting fibrosis in such patients.

Effect of the natural flavonoids myricetin and dihydromyricetin on the wound healing process in vitro.

Myricetin (MYR) and dihydromyricetin (DHM) are classified as natural flavonoids. Both substances are known for their anti-inflammatory and antioxidant properties. In this study, an in vitro model of inflammation was demonstrated on monolayers of scratched fibroblasts or keratinocytes exposed to LPS from Pseudomonas aeruginosa for six hours. MYR and DHM were subsequently applied to the cells for 24 hours at sub toxic concentrations (5-15 µM). Inflammatory parameters were analysed in collected cell medium and lysate after the incubation period using the Enzyme-Linked ImmuneSorbent Assay (ELISA) and Western blot. Both flavonoids inhibit the production of pro-inflammatory cytokines (IL-6, IL-8) in LPS-stimulated skin cells as well as the decreased level of MMP-1 in fibroblasts. However, the application of MYR and DHM dose dependently increased the level of MMP-1 in keratinocytes. In our experiments, we focused on the anti-glycation activity of MYR and DHM, where the higher concentration of MYR seems to be more effective.

Mechanism of colorectal cancer cell exosome-derived circ_0044366 in regulating release of MMP2 in fibroblasts and promoting metastasis of colorectal cancer

Mechanism of colorectal cancer cell exosome-derived circ_0044366 in regulating release of MMP2 in fibroblasts and promoting metastasis of colorectal cancer

MicroRNA-mediated extracellular matrix remodeling in squamous cell carcinoma of the oral cavity.

We evaluated microRNAs and extracellular matrix component profiles in squamous cell carcinoma of the oral cavity (OSCC) in comparison to healthy mucosa. Retrospective study investigating 64 microRNAs related to oncogenic process and to constituents of the extracellular matrix. We also performed immunohistochemical assays for molecules involved in the same biological processes. High expression of miR-21-5p (p < 0.001) and miR-106-5p (p < 0.001) and low expression of miR-320a (p = 0.001) and miR-222-3p (p = 0.001) were predictors of malignancy. Individually, miR-21-5p exhibited the best statistical performance (area under the curve = 0.972; 95% confidence interval: 0.911-1.000) in the differentiation between tumor tissue and healthy mucosa. Moreover, tumor sample showed increased expression of MMP-2, MMP-9, α-laminin, and β-laminin in tumor-related fibroblasts and lower continuity of type IV collagen in the basement membrane. The present study demonstrates the biological effects of microRNAs on the carcinogenesis of OSCC as well as the intense modification of the tumor microenvironment.

Adiponectin, but Not TGF-β1, CTGF, IL-6 or TNF-α, May Be a Potential Anti-Inflammation and Anti-Fibrosis Factor in Keloid.

IntroductionNumerous studies have elucidated adiponectin as a negative impact on inflammation and tissue fibrosis. However, little is known about the relevance between adiponectin and inflammatory factors in keloid.MethodsTo clarify whether adiponectin plays a role in the inflammation and fibrosis of keloid, 50 patients with keloid and 50 healthy subjects were enrolled, We examined the serum and mRNA expression levels of adiponectin, TGF-β1, CTGF, IL-6 and TNF-α in normal skin tissues and keloid tissues by ELISA and qPCR, respectively. Correlation analysis between serum concentration of adiponectin with Vancouver Scar Scale (VSS) scores and the age of patients with keloid was evaluated, and the adiponectin concentrations in patients with keloid between different genders were measured. We further examined the effects of adiponectin on TGF-β1 mediated expression of collagen I, FN and MMP-1 in normal fibroblasts (NFs) and keloid fibroblasts (KFs).ResultsWe discovered that lower serum concentration and mRNA expression of adiponectin, but higher TGF-β1, CTGF, IL-6 and TNF-α levels were measured in patients with keloid compared with those in normal controls. Furthermore, there was a strong inverse correlation between the serum adiponectin levels and VSS scores in patients with keloid, but not in ages, and there was no statistically difference between different genders. Moreover, adiponectin attenuated TGF-β1 mediated expression of collagen I and FN, and upregulated the expression level of MMP-1 in KFs, but not in NFs. In addition, the inhibitory effect of adiponectin on TGF-β1 was attenuated by AMPK inhibitor Compound C, but not PI3K/Akt inhibitor LY294002.DiscussionAdiponectin may exert an anti-inflammation and anti-fibrosis role in the development of keloid. One of the underlying mechanisms may be the activation of the AMPK signaling pathway.

Long-term exposure to butyric acid induces excessive production of matrix metalloproteases in human gingival fibroblasts

ObjectiveThe purpose of this study was to clarify the relationship between bacteria-induced butyric acid and periodontal disease progression. DesignNormal human gingival fibroblasts were exposed to butyric acid (0, 1, 5, 10, and 15 mM) adjusted to a pH of 7.2–7.4 using sodium hydroxide for 0–96 h and cell viability was evaluated. In addition, the effects of butyric acid on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in gingival fibroblasts were analyzed by real-time RT-PCR, ELISA, western blotting, and stromelysin zymography. ResultsButyric acid reduced the viability of gingival fibroblasts in a concentration- and time-dependent manner. Furthermore, butyric acid promoted production of MMP-1, MMP-3, and MMP-10 in gingival fibroblasts and suppressed TIMP-2 protein production. ConclusionsButyric acid promoted overproduction of MMPs, resulting in a disruption of the balance between MMPs and TIMPs expression in gingival fibroblasts. Our study suggests that the butyric acid produced by causative bacteria stimulates excessive MMP expression in periodontal tissue, leading to destruction of the tissue.

Anticatabolic and Anti-Inflammatory Effects of Myricetin 3-O-β-d-Galactopyranoside in UVA-Irradiated Dermal Cells via Repression of MAPK/AP-1 and Activation of TGFβ/Smad.

UV irradiation is one of the main causes of extrinsic skin aging. UV-mediated skin aging, also known as photoaging, causes excessive breakdown of extracellular matrix which leads skin to lose its elasticity and strength. Several phytochemicals are known to exert anti-photoaging effects via different mechanisms, partly due to their antioxidant properties. The current study has been carried out to determine the potential anti-photoaging properties of myricetin 3-O-β-d-galacto-pyranoside (M3G), a flavonol glycoside isolated from L. tetragonum, in UVA-irradiated in vitro models; HaCaT keratinocytes and human dermal fibroblasts (HDFs). UVA-induced changes in MMP-1 and collagen production have been observed in HaCaT keratinocytes and HDFs. Further, UVA-induced activation of MAPK signaling, and pro-inflammatory cytokine production have been investigated. TGFβ/Smad pathway has also been analyzed in UVA-irradiated HDFs. Treatment with M3G reversed the UVA-induced changes in MMP-1 and collagen production both in HaCaT keratinocytes and HDFs. UVA-mediated activation of p38, ERK and JNK MAPK activation was also inhibited by M3G treatment in HaCaT keratinocytes. In HDFs, M3G was able to upregulate the TGFβ/Smad pathway activation. In addition, M3G downregulated the UVA-induced pro-inflammatory cytokines in keratinocytes and HDFs. It has been suggested that the M3G has exerted potential antiphotoaging properties in vitro, by attenuating UVA-induced changes in MMP-1 and collagen production in keratinocytes and dermal fibroblasts.

Opsin 3 is a key regulator of ultraviolet A-induced photoageing in human dermal fibroblast cells.

SummaryBackgroundChronic exposure to ultraviolet (UV) radiation (mainly UVA) induces a sustained increase of matrix metalloproteinases (MMPs), especially MMP1, MMP2, MMP3 and MMP9 in human skin fibroblasts. MMPs can lead to the degradation of fibrous connective tissue, which is the main cause of skin photoageing. The molecular mechanisms through which fibroblasts sense UVA and trigger the cell signalling pathways involved in the upregulation of MMPs have not been well elucidated.ObjectivesHere, we investigated the function and potential mechanisms of photoageing of opsin (OPN)3 in normal human dermal fibroblasts (NHDFs).MethodsReal‐time polymerase chain reaction and Western blot analysis were used to analyse the expression levels of OPN3 in NHDFs and photoageing models. Subsequently, NHDFs transfected with OPN3 inhibitors and indicators related to photoageing before and after UVA irradiation included expression levels of MMP1, MMP2, MMP3 and MMP9, and signalling pathway protein molecules were examined.ResultsWe provide evidence that OPN3 initiates UVA phototransduction in NHDFs. OPN3 triggers phosphorylation of activator protein 1 and ultimately upregulates MMP1, MMP2, MMP3 and MMP9 in NHDFs through activating Ca2+/calmodulin‐dependent protein kinase II, cyclic adenosine monophosphate response element‐binding protein, extracellular signal‐regulated kinase, c‐JUN N‐terminal kinase and p38. Here, we demonstrate for the first time that OPN3 is the key sensor responsible for upregulating MMP1, MMP2, MMP3 and MMP9 in NHDFs following UVA exposure via the calcium‐dependent G protein‐coupled signalling pathway.ConclusionsOur studies provide insights into the understanding of the molecular mechanisms through which human skin cells respond to UVA radiation and may reveal molecular targets for skin photoageing prevention or clinical management. What's already known about this topic? Photoaged fibroblasts accumulate with long‐term ultraviolet (UV) exposure.Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of photoageing.MMP1, MMP2, MMP3 and MMP9 are responsible for the destruction of fibroblast collagen in severely photodamaged skin.Opsins (OPNs) are light‐sensitive members of the superfamily of heptahelical G protein‐coupled receptors, a family of cell surface receptors that are activated by a variety of stimuli and mediate signalling across membranes. What does this study add? OPN3 is highly expressed in fibroblasts and responds to UVA irradiation.OPN3 regulates the expression of MMP1, MMP2, MMP3 and MMP9 via the calcium‐dependent G protein‐coupled signalling pathway.OPN3 is a light‐sensitive sensor that plays an important role in photoageing of the skin.

Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice

Induced pluripotent stem cells (iPSCs) provide new approaches for the management of severe skin wound healing due to their infinite proliferative capacity, pluripotency into multiple lineages, and important ethical acceptability. In this study, we aimed to differentiate iPSCs into keratinocytes and to observe the therapeutic effects of transplanted iPSCs-derived keratinocytes on wound healing in mice. Here, mouse iPSCs had been successfully differentiated into keratinocytes. Next, iPSCs-derived keratinocytes labeled by CSFE were injected directly into the full-thickness skin wound. Hematoxylin & Eosin, Masson's trichrome, EdU staining and immunohistochemical staining were performed to assess the effects of iPSCs-derived keratinocytes on wound healing. Our results showed that transplantation of iPSCs-derived keratinocytes into full-thickness skin wound site accelerated re-epithelialization and reduced scar formation. In addition, we found that conditioned medium of iPSCs-derived keratinocytes reduced the expression of α-SMA and COL1 and increased the expression of MMP1 in fibroblasts in vitro. Further mechanism studies show the TNF-α-induced activation of NF-κB is involved in the effect of conditioned medium of iPSCs-derived keratinocytes on fibroblasts. In conclusion, this study has shown that iPSCs-derived keratinocytes decrease the healing time by increasing the epithelization rate and reduce scarring, suggesting a possible new treatment for skin wound healing.

Abstract 257: Cortical Bone Stem Cell-Derived Exosomes Alter Wound Healing Response in Cardiac Fibroblasts and Cardiac Endothelial Cells

Rationale: Post-myocardial infarction (MI) results in remodeling that leads to the development of heart failure. We have previously seen improvements in post-MI wound healing and scar formation as a result of administration of cortical bone stem cell derived exosomes (CBSC exosomes). Objectives: We sought to further elucidate the mechanism through which CBSC exosomes improved scar formation and altered wound healing through in vitro experimentation. We continued to explore the unique characteristics of CBSC exosomes in order to understand why they have anti-fibrotic effects. Methods and Results: We performed migration assays on cardiac endothelial cells and cardiac fibroblasts, and saw alterations in cell migration in both cell types upon treatment with mCBSC CM and mCBSC exosomes. We discovered that migration of CECs was reduced by 20% under the influence of mCBSC CM and mCBSC exosomes, but rescued when exosomes were removed from mCBSC CM. We discovered that the removal of exosomes from mCBSC CM slowed cardiac fibroblast migration by 40%. We saw a decrease in the quantity of pro-fibrotic mRNA in cardiac endothelial cells and cardiac fibroblasts after treatment with mCBSC exosomes. Adult cardiac endothelial cell mRNA levels of VEGFA, Col4A1, and VCAM were reduced nearly two fold from control untreated cell levels following mCBSC exosome treatment. Adult cardiac fibroblast Col1A1, Col3A1, Col4A1, and MMP2 mRNA levels were reduced one-fold from control untreated cell levels following mCBSC exosome treatment. Conclusions: Our findings show that the wound healing induced by CBSC treatment post-MI involves a mechanism altering the migration of endothelial cells, sustaining the migratory capabilities of fibroblasts, and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.

ERK1/2 mediates the lipopolysaccharide-induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts

Myocardial fibrosis is a critical event during septic shock. Upregulation in the fibrosis signaling cascade proteins such as fibroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and activation of matrix metalloproteinases (MMPs) are widely associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac fibrosis and heart failure. However, evidences suggest that the common upstream mediators of fibrosis cascade play little role in cardiac fibrosis induced by LPS; further, it is unknown if LPS directly triggers the expressions and/or activity of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac fibroblasts. In the present study, we treated primary cultures of cardiac fibroblasts with LPS to explore whether LPS upregulates FGF-2, uPA, tPA, MMP-2, MMP-9 and enhance cellular migration. Further the precise molecular and cellular mechanisms behind these LPS induced responses were identified. Inhibition assays on MAPKs using U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) and QNZ (NFκB inhibitor) show that LPS-induced upregulation of FGF-2, uPA, MMP-2 and MMP-9 in cardiac fibroblasts was mediated through ERK1/2 signaling. Collectively, our results provide a link between LPS-induced cardiac dysfunction and ERK1/2 signaling pathway and thereby implies ERK1/2 as a possible target to regulate LPS induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts.

Attenuation of aquaporin-3 and epidermal growth factor receptor expression and activation in systemic sclerosis dermal fibroblasts.

Impaired wound healing and skin dehydration are the mainstay of systemic sclerosis (SSc) cutaneous manifestations. Aquaporin-3 (AQP3) has a pivotal role in skin hydration and wound healing. Epidermal growth factor receptor (EGFR) activation is impaired in SSc fibroblasts. It is unclear whether AQP3 downregulation or epidermal growth factor (EGF) signaling are the primary points of dysregulation in SSc patients. Skin punch biopsies were obtained from 10 SSc patients and 10 healthy subjects. The mRNA and/or protein expression levels of AQP3, EGFR/p-EGFR, matrix metalloproteinase-1/2/9 (MMP-1/2/9), and tissue inhibitors of metalloproteinase-1 (TIMP1) at baseline and after EGF and transforming growth factor-β1 (TGF-β1) treatment was evaluated in extracted fibroblasts using real-time polymerase chain reaction and western blot analysis. SSc fibroblasts expressed lower AQP3 and EGFR, compared with normal fibroblasts. Normal fibroblasts increased AQP3 expression in response to EGF whereas AQP3 expression had no change in EGF-treated-SSc fibroblasts. Likewise, EGFR was activated in response to EGF in the normal group but not SSc group. Baseline expression of MMP-1/2/9 and TIMP1 was not different between SSc and controls. EGF treatment did not result in alteration of any MMPs expression in either of the groups. Combination treatment resulted in a significant upregulation of MMP-1 in normal fibroblasts compared with SSc fibroblasts, while in SSc fibroblasts MMP-9 expression was upregulated in response to treatment with TGF-β1 only. Downregulation of AQP3 expression in SSc fibroblasts may be related to reduced EGFR expression and activation. TGF-β1 (alone or in combination with EGF) only can upregulate AQP3 expression in SSc fibroblasts so, TGF-β1 affect MMP-1 and MMP-9 just in SSc fibroblasts.

Stromal Fibroblasts Mediate Anti-PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma.

Although anti-PD-1 therapy has improved clinical outcomes for select patients with advanced cancer, many patients exhibit either primary or adaptive resistance to checkpoint inhibitor immunotherapy. The role of the tumor stroma in the development of these mechanisms of resistance to checkpoint inhibitors remains unclear. We demonstrated that pharmacologic inhibition of the TGFβ signaling pathway synergistically enhanced the efficacy of anti-CTLA-4 immunotherapy but failed to augment anti-PD-1/PD-L1 responses in an autochthonous model of BRAFV600E melanoma. Additional mechanistic studies revealed that TGFβ pathway inhibition promoted the proliferative expansion of stromal fibroblasts, thereby facilitating MMP-9-dependent cleavage of PD-L1 surface expression, leading to anti-PD-1 resistance in this model. Further work demonstrated that melanomas escaping anti-PD-1 therapy exhibited a mesenchymal phenotype associated with enhanced TGFβ signaling activity. Delayed TGFβ inhibitor therapy, following anti-PD-1 escape, better served to control further disease progression and was superior to a continuous combination of anti-PD-1 and TGFβ inhibition. This work illustrates that formulating immunotherapy combination regimens to enhance the efficacy of checkpoint blockade requires an in-depth understanding of the impact of these agents on the tumor microenvironment. These data indicated that stromal fibroblast MMP-9 may desensitize tumors to anti-PD-1 and suggests that TGFβ inhibition may generate greater immunologic efficacy when administered following the development of acquired anti-PD-1 resistance.See related Spotlight on p. 1444.