Framework nucleic acid-based hydrogel for sequential immune regulation and endogenous TGF-β1 capture in wound healing.

Ubiquitin C-terminal hydrolase L1 (UCHL1) plays vital roles in cell proliferation, angiogenesis, inflammation and oxidative stress. Nevertheless, it is unclear whether UCHL1 could regulate the biologic behaviour of cells and ultimately influences wound healing. We aim to illustrate the roles and the underlying mechanism of UCHL1 in cutaneous wound healing. Murine full-thickness excisional wound model was utilised to study the effects of UCHL1 on wound healing through topical administration of the UCHL1 inhibitor LDN57444, followed by assessment of wound areas and histological alterations. Subsequently, ethynyldeoxyuridine, scratch and transwell assays were performed to examine fibroblast migration and proliferation. The extracellular matrix (ECM)-related genes expression and transforming growth factor-β (TGF-β)/Smad signalling pathways activation were investigated by immuno-fluorescent staining, Western blots and quantitative reverse transcription polymerase chain reaction. We identified elevated UCHL1 expression in non-healing wound tissues. The UCHL1 expression displayed a dynamic change and reached a peak on Day-7 post-wounding during the healing process in mice. Cutaneous administration of LDN57444 promoted wound healing by facilitating collagen deposition, myofibroblast activation and angiogenesis. Invitro experiments demonstrated that UCHL1 concentration dependently inhibited migration, ECM synthesis and activation of human dermal fibroblasts, which was mechanistically related to downregulation of TGF-β/Smad signalling. Furthermore, these effects could be reversed by TGF-β inhibitor SB431542. Our findings reveal that UCHL1 is a negative regulator of cutaneous wound healing and considered as a novel prospective therapeutic target for effective wound healing.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome-lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

Collagenase-3 (MMP-13) Enhances Remodeling of Three-Dimensional Collagen and Promotes Survival of Human Skin Fibroblasts

Increased Expression of Integrin αvβ5 Induces the Myofibroblastic Differentiation of Dermal Fibroblasts

Matrix Metalloproteinase-3 Accelerates Wound Healing following Dental Pulp Injury

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

Rationale: Idiopathic Pulmonary Fibrosis (IPF) is a progressive pulmonary disorder usually associated with epithelial damage and fibroblast activation and involves numerous molecular signalling cascades governing activation, differentiation, proliferation and matrix remodelling. Canonical Wingless/integrase-1 (WNT) / s-catenin signalling pathway operates via phosphorylation of its co-receptor LRP5 upon Wnt ligand binding and positively regulates proliferation and cell survival in many types of cells. Moreover, WNT signalling is dysregulated in IPF and has been linked to pulmonary disease pathogenesis and progression. However, the direct role of LRP5 is not fully understood. Hypothesis: Decrease in Wnt/s-catenin pathway activity and stabilization of LRP5 plays a role in fibroblast activation and differentiation in pulmonary fibrosis. Methods and Results: Our study focused mainly on the contribution of Wnt ligand stimulation (Wnt1, 3A and 7A) on normal human lung fibroblast (NHLF) activity. Stimulation of NHLF (2 donors) with Wnt ligands for 24h induced fibroblast to myofibroblast transition as assessed by phase-contrast microscopy and by proliferation. We found a dramatic increase in aSMA expression and cell proliferation, with Wnt7A showing the most prominent effect. LRP5 knockdown followed by treatment with WNT ligands and/or TGF- s1 stimulation for 24h partially abrogated the WNT and TGF-s1 induced changes. Conclusions: Our findings suggest that elevation of aSMA expression and cell proliferation are modulated by LRP5 with a key role in fibroblast activation, differentiation and proliferation.

Tubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis

Introduction: Heart failure (HF) remains a major source of late morbidity and mortality after myocardial infarction (MI). Calcium handling and the signaling pathways that govern the functional remodeling associated with HF are unclear. Store-operated calcium entry (SOCE) is a key factor in modulating Ca2+ signals. SOCE is mediated by the STIM protein and Orai channels. Monitoring STIM and Orai distribution is of great interest for studying cardiac remodeling after MI. This study investigated the spatial distribution of STIM and Orai in the heart after MI injury. Methods: The myocardial injury was induced in adult mice via ligation of the left anterior descending artery. MI- and sham-operated animals were euthanized, and hearts were harvested for ex-vivo analyses at 3, 7, 14, and 28 days after MI. We used western blot coupled with immunostaining to detect temporal and spatial profiling of specific STIM and Orai isoforms in different physiological zones and timepoints of MI to generate an integrative high-resolution map of STIM-Orai expression during cardiac remodeling. Results: We found that STIM1 and Orai1 proteins were upregulated from 3-day post-MI throughout day 28 post-MI, (3-4 fold times higher than controls, p<0.001). STIM1 and Orai1 were the most abundant isoforms present in the injured heart. In contrast, Orai3 expression was found to transiently increase at 3- and 7-day post-MI (1.5 fold, p>0.01), STIM2 and Orai2 expression did not change. Spatial pattern analysis revealed a marked gradient of STIM1 and Orai1 expression (decreasing from the border zone, the infarcted area, and the distal area). Co-labeling with cell-specific markers revealed that cardiac fibroblasts exhibited a high level of STIM1 and Orai1 expression. In vitro activation of cardiac fibroblasts showed a marked increase of STIM1 and Orai1 protein expression. Conclusions: Taken together, these data demonstrate that STIM1 and Orai1 expression is associated with fibroblast activation during fibrotic remodeling in the MI. Ca2+ signaling pathways that drive the expansion of fibrosis may rely on sustained STIM1 and Orai1 expression in scar formation and fibroblast activation, of which could provide future targets to exploit for treatment to prevent HF.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and remodeling. In non-pathological conditions tissue homeostasis is maintained by the balance between extracellular matrix (ECM) synthesis and degradation. However, this equilibrium is altered in asthma and COPD leading to structural changes such as increased ECM deposition. Fibroblasts and airway smooth muscle cells (ASMC) are the main source of collagen and glycosaminoglycans (GAGs) and transforming growth factor-b1 (TGF-b1) is the most potent ECM stimulator in vitro. In turn, the ECM influences cell proliferation, migration, differentiation, and secretion of cytokines and growth factors. Thus, ECM deposition may perpetuate the inflammatory and remodeling processes, contributing to progression and severity of diseases. Inhaled corticosteroids and long acting b2-agonists (LABA) are the current therapy for asthma and COPD but their effect on airway remodeling is not clear. Therefore, the purpose of this thesis was to investigate the effect of corticosteroids and LABA on airway inflammation and tissue remodeling using a human cell based in vitro model. In the present thesis, primary human lung fibroblasts and ASMC were cultured under serumfree condition to reflect a non-inflammatory environment or with 5% fetal calf serum (FCS) and/or TGF-b1 to mimic inflammation. This experimental design allowed us to assess the effect of corticosteroids and LABA on total ECM, collagen and GAGs deposition, cell proliferation, cytokines release, collagen and ECM mediators mRNA expression, and gelatinolytic activity under both non-inflammatory and inflammatory conditions. Concerning the effect of corticosteroids and LABA on ECM deposition by fibroblasts, we showed that 5% FCS and TGF-b1 increased total ECM and collagen deposition. Under serumfree condition corticosteroids reduced ECM deposition and the effect was partly mediated by the glucocorticoid receptor and collagen de novo synthesis. Interestingly, in the presence of 5% FCS corticosteroids had the opposite effect. LABA reduced total ECM and collagen deposition under both conditions. Combined drugs further decreased ECM deposition under serum-free condition whereas they counteracted each other in 5% FCS, independently of TGF-b1. Furthermore, we showed that 5% FCS and TGF-b1 increased GAGs secretion and deposition. Under serum-free condition corticosteroids inhibited GAGs secretion and deposition whereas LABA alone had no effect but partly reversed the effect of corticosteroids. On the contrary, in 5% FCS corticosteroids and LABA increased GAGs deposition. Upon TGF-b1 stimulation, similar effects were obtained except in 5% FCS, where both drugs decreased the TGF-b1-induced GAGs secretion and deposition. Together, these findings indicate that the effect of corticosteroids but not of LABA, on ECM deposition by fibroblasts is altered by ongoing inflammation. The effects of corticosteroids and LABA on GAGs, matrix metalloproteinases (MMPs) and total ECM/collagen deposition by ASMC from healthy, asthma and COPD patients were also investigated. First, no difference in GAGs secretion and deposition was observed between ASMC from healthy, asthma and COPD patients. Under serum-free condition corticosteroids inhibited GAGs secretion and deposition while LABA had no clear effect nor modulated the effect of corticosteroids. In 5% FCS corticosteroids decreased GAGs secretion to a greater extent than LABA and their combination resulted in the same effect as corticosteroids alone. None of the drugs had any effect on GAGs deposition. Second, we assessed the effect of the drugs on ASMC-derived MMPs. In ASMC only proMMP-2 gelatinolytic activity was detected, which was decreased in asthma and COPD patients compared to healthy controls. However, corticosteroids and/or LABA had no effect on proMMP-2 activity. Lastly, we demonstrated that corticosteroids and LABA generated similar effects on total ECM and collagen deposition by ASMC from healthy subjects as observed with fibroblasts. In conclusion, our novel findings suggest that the action of corticosteroids and LABA on ECM deposition differs under non-inflammatory and inflammatory conditions in lung fibroblasts and ASMC. In the presence of acute inflammation with vessel leakage, increased ECM deposition may be regarded as an attempt of stromal cells to block further serum and cells infiltration into the tissue and this effect would be supported by corticosteroids. The addition of LABA would counteract the corticosteroid-induced ECM deposition and therefore their combination with corticosteroids may depend on the inflammatory status of the patient. Thus, when airway inflammation is resolved, combination therapy may beneficially reduce pathological tissue remodeling. Thus, our findings might have implications for the short- and long-term treatment strategies in regard to airway remodeling in asthma and COPD.

This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-beta(1) (TGF-beta(1))-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-beta(1), with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-beta(1)-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-beta(1) signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-beta(1), which is associated with differences in HA generation. TGF-beta(1) regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response.

Diabetic periodontitis presents a unique therapeutic challenge, primarily due to its chronic inflammatory profile and the associated bone loss driven by immune dysregulation. Metformin (Met) is recognized for its potent anti-inflammatory properties, yet its limited cellular uptake has hindered its clinical effectiveness in diabetic periodontitis. A tetrahedral framework nucleic acid (tFNA)-based delivery system is developed to enhance Met cellular uptake and investigate its effects on diabetic periodontitis in this study. The tFNA-loaded Met complex (TMC) demonstrates efficient Met loading efficiency and rapid cellular internalization. These results show that in vitro, TMC significantly inhibited pro-inflammatory cytokines and matrix metalloproteinaseat both mRNA and protein levels under high glucose conditions. Mechanistically, TMC suppresses NLRP3-mediated pyroptosis by downregulating NLRP3, Caspase-1, Caspase-11, and GSDMD, ultimately reducing Mature-IL-1β and Mature-IL-18 production. Furthermore, TMC activates AMPK while inhibiting NF-κB signaling, thus mitigating the inflammatory response in diabetic periodontitis. In vivo, intraperitoneal administration of TMC in a diabetic periodontitis mouse model significantly reduces inflammatory cell infiltration, collagen degradation, and osteoclast formation, thus alleviating alveolar bone loss. These findings highlight the therapeutic potential of tFNA as an efficient Met delivery vehicle to tackle diabetic periodontitis-associated inflammation and bone resorption, providing a promising strategy for managing diabetic complications.

The effect of sesamin on airway fibrosis in vitro and in vivo.