Multimerization of TREM2 is impaired by Alzheimer's disease-associated variants.

INTRODUCTION: The immune receptor triggering receptor expressed on myeloid cells 2 (TREM2) is among the strongest genetic risk factors for Alzheimer's disease (AD) and is a therapeutic target. TREM2 multimers have been identified in crystallography and implicated in the efficacy of antibody therapeutics; however, the molecular basis for TREM2 multimerization remains poorly understood. METHODS: We used molecular dynamics simulations and binding energy analysis to determine the effects of AD-associated variants on TREM2 multimerization and validated with experimental results. RESULTS: TREM2 trimers remained stably bound, driven primarily by salt bridge between residues D87 and R76 at the interface of TREM2 units. This salt bridge was disrupted by the AD-associated variants R47H and R98W and nearly ablated by the D87N variant. This decreased binding among TREM2 multimers was validated with co-immunoprecipitation assays. DISCUSSION: This study uncovers a molecular basis for TREM2 forming stable trimers and unveils a novel mechanism by which TREM2 variants may increase AD risk by disrupting TREM2 oligomerization to impair TREM2 normal function. HIGHLIGHTS: Triggering receptor expressed on myeloid cells 2 (TREM2) multimerization could regulate TREM2 activation and function. D87-R76 salt bridges at the interface of TREM2 units drive the formation of stable TREM2 dimers and trimers. Alzheimer's disease (AD)-associated R47H and R98W variants disrupt the D87-R76 salt bridge. The AD-associated D87N variant leads to complete loss of the D87-R76 salt bridge.

Genetic responses to adding nitrates to improve hydrophilic yellow pigment in Monascus fermentation.

Nitrates can stimulate the biosynthesis of hydrophilic yellow pigments (HYPs) in Monascus ruber CGMCC 10910. To explore the molecular mechanisms whereby nitrates (NaNO3 and NH4NO3) regulate HYP production, an integrated transcriptomic and proteomic analysis was conducted in this study. Nitrate addition led to an approximately 75% higher HYP production compared with the untreated group, especially compounds Y3 and Y4. Comparative transcriptomic analysis found that mpigsA, H, K, L, and P genes involved in yellow pigment biosynthesis were significantly upregulated. In addition, pigment biosynthesis-related (carbon catabolism, amino acid metabolism, polyketide synthesis, and fatty acid metabolism) genes were upregulated to provide precursors and energy for HYP biosynthesis and cell growth. Secretion-related (cytomembrane ergosterol biosynthetic, and transport) pathways were also noticeably regulated to accelerate transmembrane transport of HYPs. Meanwhile, proteomic analysis showed that nitrates improved the protein expression of hybrid polyketide synthase-nonribosomal peptide synthetase, oxidoreductase, glucoamylase, endo-1,4-beta-xylanase, O-acetylhomoserine, and isocitrate lyase to enhance HYP production. These findings demonstrated the regulatory mechanism of nitrates for enhancing HYP production in Monascus. KEY POINTS: • Nitrates stimulated the biosynthesis of Monascus hydrophilic yellow pigments (HYPs) • Nitrates affected transcriptional level of pigment biosynthesis- and transport genes • Increased expression of hybrid PKS-NRPS and transporters promoted production of HYPs.

Sodium starch octenyl succinate facilitated the production of water-soluble yellow pigments in Monascus ruber fermentation.

Natural water-soluble Monascus pigments (WSMPs) have been in increasing demand but have not been able to achieve industrial production due to the low production rate. This study aimed to improve the biosynthesis and secretion of extracellular yellow pigments (EYPs) through submerged fermentation with Monascus ruber CGMCC 10,910 supplemented with sodium starch octenyl succinate (OSA-SNa). The results demonstrated that the yield was 69.68% and 48.89% higher than that without OSA-SNa in conventional fermentation (CF) and extractive fermentation (EF), respectively. The mainly increased EYP components were Y3 and Y4 in CF, but they were mainly Y1 and Y2 as well as secreted intracellular pigments, including Y5, Y6, O1, and O2, in EF. Scanning electron microscopy analysis revealed that the mycelium presented an uneven surface profile with obvious wrinkles and small fragments with OSA-SNa. It was found that a higher unsaturated/saturated fatty acids ratio in the cell membrane resulted in increased permeability and facilitated the export of intracellular yellow pigments into the broth with OSA-SNa treatment. In addition, a higher NAD+/NADH ratio and glucose-6-phosphate dehydrogenase activity provided a reducing condition for yellow pigment biosynthesis. Gene expression analysis showed that the expression levels of the key genes for yellow pigment biosynthesis were significantly upregulated by OSA-SNa. This study provides an effective strategy to promote the production of WSMPs by microparticle-enhanced cultivation using OSA-SNa. KEY POINTS: • OSA-SNa addition facilitated the production of Monascus yellow pigments. • Mycelial morphology and membrane permeability were affected by OSA-SNa. • The key gene expression of yellow pigments was upregulated.

Inducing red pigment and inhibiting citrinin production by adding lanthanum(III) ion in Monascus purpureus fermentation.

Monascus pigments (MPs) are widely used natural colorants in Asian countries. The problems of low extracellular red pigment (ERP) and high citrinin remain to be solved in Monascus pigment production. The effect of lanthanum(III) ion (LaCl3) on Monascus purpureus fermentation was investigated in this study. The yields of ERP and biomass respectively reached maxima of 124.10 U/mL and 33.10 g/L by adding 0.4 g/L La3+ on the second day in the total 8-day fermentation; simultaneously, citrinin was decreased by 59.93% and 38.14% in the extracellular and intracellular fractions, respectively. Reactive oxygen species (ROS) levels were obviously improved by La3+ treatment, while the activities of catalase (CAT) and superoxide dismutase (SOD) were increased compared with the control. The ratio of unsaturated/saturated fatty acids in mycelia was increased from 2.94 to 3.49, indicating that the permeability and fluidity of the cell membrane were enhanced under La3+ treatment. Gene expression analysis showed that the relative expression levels of Monascus pigment synthesis genes (pksPT, mppB, mppD, MpFasB2, and MpPKS5) were significantly upregulated by La3+ treatment, and in contrast, the relative expression levels of citrinin synthesis genes (ctnA, pksCT and mppC) were markedly downregulated. This work confirmed that LaCl3 possesses the potential to induce red pigment biosynthesis and inhibit citrinin production in M. purpureus fermentation. KEY POINTS: • La3+ induced red pigment and inhibited citrinin production in Monascus fermentation. • La3+ regulated genes expression up for Monascus pigment and down for citrinin. • La3+ increased the UFAs in cell membrane to enhance the permeability and fluidity.

Regulation of pancreatic cancer TRAIL resistance by protein O-GlcNAcylation.

TRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling.

Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation.

Extreme environments, for example high-salt-stress condition, that can induce secondary metabolite biosynthesis in fungi are a promising and effective strategy for producing natural Monascus pigments used as food colourants and nutraceutical supplements. In this study, the relationship between the mycelial morphology and expression of pigment biosynthetic genes in high-salt-stress fermentation (HSF) with Monascus ruber CGMCC 10910 was investigated. The Monascus fungus grew well under HSF conditions with 35 g/l NaCl, and the intracellular yellow pigment yield in HSF was 40% higher than that in conventional batch fermentation (CBF). Moreover, the mycelial morphology was maintained in a better state, with a hyphal diameter of 5-6 µm in HSF, indicating good biocatalytic activity for pigment synthesis. The rate of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p < 0.05) changed, and the extracellular yellow pigments were transformed into each other, indicating that the pigment biosynthesis pathway was changed to promote yellow pigment accumulation in HSF. The pigment biosynthesis genes MpPKS5, MpFasB2, mppE, mppD and mppB were significantly (p < 0.05) up-regulated by approximately 58.4-106.1%, whereas the regulatory genes mppR1 and mppR2 were significantly (p < 0.05) down-regulated by approximately 23.2% and 59.0% in HSF. Notably, the mppE gene was highly correlated with (r > 0.95, p < 0.05) hyphal diameter. These findings indicated that the cultivation of the Monascus fungus under high-salt-stress conditions was beneficial for pigment biosynthesis by controlling the mycelial morphology to regulate gene expression. This study first described the relationship between the mycelial morphology and expression of pigment biosynthetic genes in Monascus during fermentation. KEY POINTS: • High-salt-stress fermentation (HSF) was first performed to improve Monascus pigment yield. • Pigment biosynthesis was enhanced by maintaining the mycelial morphology in an improved state in HSF. • Gene expression was up-/downregulated to promote yellow pigment accumulation in HSF. • The mycelial morphology was highly related to the expression of pigment biosynthetic genes in HSF.

Improving mycelial morphology and adherent growth as well as metabolism of Monascus yellow pigments using nitrate resources.

Mycelial adhesion affects cell growth and the production of water-soluble extracellular yellow pigment (EYP) in submerged fermentation with Monascus ruber CGMCC 10910. Two nitrates, NaNO3 and KNO3, were used as nitrogen sources for mitigating mycelial adhesion and improving the production of EYP in this study. The results showed that the adhesion of mycelia in the fermentation broth significantly decreased by adding 5 g/L NaNO3, which prevented mycelia from attaching to the inner wall of the Erlenmeyer flask. It was suggested that NaNO3 reduced the total amount of extracellular polysaccharides, increased extracellular proteins, and decreased the viscosity of the fermentation broth. Scanning electron microscopy (SEM) analysis revealed that the mycelial morphology was shorter and more dispersed and vigorous under NaNO3 conditions than under the control conditions. The biomass increased by 49.2% and 45.4% with 5 g/L NaNO3 and 6 g/L KNO3 treatment, respectively, compared with that of the control, and the maximum production of EYP was 267.1 and 241.8 AU350, which increased by 70.0% and 53.9% compared with that of the control, respectively. Simultaneously, the ratios of intracellular yellow pigment to orange pigment increased significantly with 5 g/L of NaNO3 addition (p < 0.05). Genetic analysis found that the expression levels of the key genes for Monascus pigment biosynthesis were significantly upregulated by NaNO3 addition (p < 0.05 or p < 0.01). This study provides an effective strategy for the production of water-soluble Monascus yellow pigments.Key Points• Nitrate addition decreased mycelial adhesion and improved cell growth in Monascus pigment fermentation.• The biosynthesis genes of water-soluble extracellular yellow pigment (EYP) were upregulated by nitrate addition.• The mycelial morphology was significantly influenced to enhance EYP biosynthesis with nitrate addition.

Cytoplasmic PARP-1 promotes pancreatic cancer tumorigenesis and resistance.

The poly(ADP-ribose) polymerases (PARP) play important roles in repairing damaged DNA during intrinsic cell death. We recently linked PARP-1 to death receptor (DR)-activated extrinsic apoptosis, the present studies sought to elucidate the function of cytoplasmic PARP-1 in pancreatic cancer tumorigenesis and therapy. Using human normal and pancreatic cancer tissues, we analyzed the prevalence of cytoplasmic PARP-1 expression. In normal human pancreatic tissues, PARP-1 expression was present in the nucleus; however, cytoplasmic PARP-1 expression was identified in pancreatic cancers. Therefore, cytoplasmic PARP-1 mutants were generated by site-direct mutagenesis, to determine a causative effect of cytoplasmic PARP-1 on pancreatic cancer tumorigenesis and sensitivity to therapy with TRA-8, a humanized DR5 antibody. PARP-1 cytoplasmic mutants rendered TRA-8 sensitive pancreatic cancer cells, BxPc-3 and MiaPaCa-2, more resistant to TRA-8-induced apoptosis; whereas wild-type PARP-1, localizing mainly in the nucleus, had no effects. Additionally, cytoplasmic PARP-1, but not wild-type PARP-1, increased resistance of BxPc-3 cells to TRA-8 therapy in a mouse xenograft model in vivo. Inhibition of PARP enzymatic activity attenuated cytoplasmic PARP-1-mediated TRA-8 resistance. Furthermore, increased cytoplasmic PARP-1, but not wild-type PARP-1, was recruited into the TRA-8-activated death-inducing signaling complex and associated with increased and sustained activation of Src-mediated survival signals. In contrast, PARP-1 knockdown inhibited Src activation. Taken together, we have identified a novel function and mechanism underlying cytoplasmic PARP-1, distinct from nuclear PARP-1, in regulating DR5-activated apoptosis. Our studies support an innovative application of available PARP inhibitors or new cytoplasmic PARP-1 antagonists to enhance TRAIL therapy for TRAIL-resistant pancreatic cancers.

The long non-coding RNA HOTAIR enhances pancreatic cancer resistance to TNF-related apoptosis-inducing ligand.

Pancreatic cancer is a malignant neoplasm with a high mortality rate. Therapeutic agents that activate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis have shown promising efficacy, but many pancreatic cancers are resistant to TRAIL therapy. Epigenetic regulation plays important roles in tumor pathogenesis and resistance, and a recent study indicated that the long non-coding RNA HOX transcript antisense RNA (HOTAIR) is overexpressed in pancreatic cancer. However, the role of HOTAIR in pancreatic cancer resistance to anticancer agents is unknown. The present study determined the role of HOTAIR in pancreatic cancer TRAIL resistance and investigated the underlying molecular mechanisms. We observed that TRAIL-resistant pancreatic cancer cells had higher levels of HOTAIR expression, whereas TRAIL-sensitive pancreatic cancer cells had lower HOTAIR levels. Overexpressing HOTAIR in TRAIL-sensitive cells attenuated TRAIL-induced apoptosis, and shRNA-mediated HOTAIR knockdown in TRAIL-resistant PANC-1 cells sensitized them to TRAIL-induced apoptosis. These results support a causative effect of HOTAIR on TRAIL sensitivity. Mechanistically, we found that increased HOTAIR expression inhibited the expression of the TRAIL receptor death receptor 5 (DR5), whereas HOTAIR knockdown increased DR5 expression. We further demonstrated that HOTAIR regulates DR5 expression via the epigenetic regulator enhancer of zeste homolog 2 (EZH2) and that EZH2 controls histone H3 lysine 27 trimethylation on the DR5 gene. Taken together, these results demonstrate that high HOTAIR levels increase the resistance of pancreatic cancer cells to TRAIL-induced apoptosis via epigenetic regulation of DR5 expression. Our study therefore supports the notion that targeting HOTAIR function may represent a strategy to overcome TRAIL resistance in pancreatic cancer.

MicroRNA let-7c regulates macrophage polarization.

Macrophages demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. The role of microRNAs (miRNAs) in regulating macrophage polarization has been largely undefined. In this study, we found that miRNA let-7c is expressed at a higher level in M-BMM (M2 macrophages) than in GM-BMM (M1 macrophages). let-7c levels are also greater in alveolar macrophages from fibrotic lungs as compared with those from normal lungs. let-7c expression was decreased when M-BMM converted to GM-BMM, whereas it increased when GM-BMM converted to M-BMM. LPS stimulation reduced let-7c expression in M-BMM. We found that overexpression of let-7c in GM-BMM diminished M1 phenotype expression while promoting polarization to the M2 phenotype. In contrast, knockdown of let-7c in M-BMM promoted M1 polarization and diminished M2 phenotype expression. We found that let-7c targets C/EBP-δ, a transcriptional factor that plays an important role in inflammatory response. Furthermore, we found that let-7c regulates bactericidal and phagocytic activities of macrophages, two functional phenotypes implicated in macrophage polarization. Our data suggest that the miRNA let-7c plays an important role in regulating macrophage polarization.

miR-125a-5p regulates differential activation of macrophages and inflammation.

Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.

miR-145 regulates myofibroblast differentiation and lung fibrosis.

The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung myofibroblast differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung myofibroblast differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-ß1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-ß1 induced SMA-α expression. miR-145 did not affect the activity of TGF-ß1, but promoted the activation of latent TGF-ß1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the differentiation of lung myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.

Beyond Small Molecular Cations: Elucidating the Alkaline Stability of Cationic Moieties at the Membrane Scale.

A major hindrance in the commercialization of alkaline polyelectrolyte-based electrochemical energy conversion devices is the development of durable anion exchange membranes (AEMs). Despite many alkali-stable cations that have been explored, the stability of these cationic moieties at the membrane scale is in the blind. Herein, we present a molecularly designed polyaromatic AEM with cationic moieties in an alternating manner to unbiasedly compare the alkaline stability of piperidinium and ammonium groups at the membrane state. Using nuclear magnetic resonance spectroscopy, we demonstrate that the pentyltrimethyl group is about 2-fold more stable than piperidinium within a polyaromatic scaffold, either in ex-situ alkaline soaking or in-situ cell operation. This finding challenges the judgment extrapolated from the stability trend of cations, that is, the piperidinium-functionalized AEM is more alkali-stable than the counterparts based on quaternary ammoniums. Moreover, the deterioration mechanism of piperidinium moiety after being embedded in polyaromatic backbone is rationalized by density functional theory.

Participation of miR-200 in pulmonary fibrosis.

Excessive extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). Epithelial-mesenchymal transition, involving transition of alveolar epithelial cells (AECs) to pulmonary fibroblasts, appears to be an important contributory process to lung fibrosis. Although aberrant expression of microRNAs (miRs) is involved in a variety of pathophysiologic processes, the role of miRs in fibrotic lung diseases is less well understood. In the present study, we found that miR-200a, miR-200b, and miR-200c are significantly down-regulated in the lungs of mice with experimental lung fibrosis. Levels of miR-200a and miR-200c were reduced in the lungs of patients with IPF. miR-200 had greater expression in AECs than in lung fibroblasts, and AECs from mice with experimental pulmonary fibrosis had diminished expression of miR-200. We found that the miR-200 family members inhibit transforming growth factor-ß1-induced epithelial-mesenchymal transition of AECs. miR-200 family members can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Indeed, the introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Thus, the miR-200 family members participate importantly in fibrotic lung diseases and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach in treating pulmonary fibrotic diseases.

miR-31 is a negative regulator of fibrogenesis and pulmonary fibrosis.

Aberrant expression of miRNAs is closely associated with initiation and progression of pathological processes, including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in lung fibrosis is not well characterized. We sought to determine the role of miR-31 in regulating the fibrogenic, contractile, and migratory activities of lung fibroblasts and modulating of pulmonary fibrosis in vivo. In vivo lung fibrosis models and ex vivo cell culture systems were employed. Real-time PCR and Western blot analysis were used to determine gene expression levels. miR-31 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic, contractile, and migratory activities of lung fibroblasts were determined. We found that miR-31 expression is reduced in the lungs of mice with experimental pulmonary fibrosis and in IPF fibroblasts. miR-31 inhibits the profibrotic activity of TGF-ß1 in normal lung fibroblasts and diminishes the fibrogenic, contractile, and migratory activities of IPF fibroblasts. In these experiments, miR-31 was shown to directly target integrin α(5) and RhoA, two proteins that have been shown to regulate activation of fibroblasts. We found that levels of integrin α(5) and RhoA are up-regulated in fibrotic mouse lungs. Knockdown of integrin α(5) and RhoA attenuated fibrogenic, contractile, and migratory activities of IPF fibroblasts, in a manner similar to that observed with miR-31. We also found that introduction of miR-31 ameliorated experimental lung fibrosis in mice. Our data suggest that miR-31 is an important regulator of the pathological activities of lung fibroblasts and may be a potential target in the development of novel therapies to treat pathological fibrotic disorders, including pulmonary fibrosis.

Mitochondrial fission and bioenergetics mediate human lung fibroblast durotaxis.

Pulmonary fibrosis is characterized by stiffening of the extracellular matrix. Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we found primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness-regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a dynamin-related protein 1/mitochondrial fission factor-dependent (DRP1/MFF-dependent) pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. These findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a potentially new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of human idiopathic pulmonary fibrosis.

miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling.

Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases.

Identification of a microRNA signature in renal fibrosis: role of miR-21.

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-ß1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-ß1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.

Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

Aging is a strong risk factor and an independent prognostic factor for progressive human idiopathic pulmonary fibrosis (IPF). Aged mice develop nonresolving pulmonary fibrosis following lung injury. In this study, we found that mouse double minute 4 homolog (MDM4) is highly expressed in the fibrotic lesions of human IPF and experimental pulmonary fibrosis in aged mice. We identified MDM4 as a matrix stiffness-regulated endogenous inhibitor of p53. Reducing matrix stiffness down-regulates MDM4 expression, resulting in p53 activation in primary lung myofibroblasts isolated from IPF patients. Gain of p53 function activates a gene program that sensitizes lung myofibroblasts to apoptosis and promotes the clearance of apoptotic myofibroblasts by macrophages. Destiffening of the fibrotic lung matrix by targeting nonenzymatic cross-linking or genetic ablation of Mdm4 in lung (myo)fibroblasts activates the Mdm4-p53 pathway and promotes lung fibrosis resolution in aged mice. These findings suggest that mechanosensitive MDM4 is a molecular target with promising therapeutic potential against persistent lung fibrosis associated with aging.

Synergy between dynamic covalent boronic ester and boron-nitrogen coordination: strategy for self-healing polyurethane elastomers at room temperature with unprecedented mechanical properties.

Achieving mechanical robustness and highly efficient self-healing simultaneously at room temperature is always a formidable challenge for polymeric materials. Herein, a series of novel supramolecular polyurethane elastomers (SPUEs) are developed by incorporating dynamic covalent boronic ester and boron-nitrogen (B-N) coordination. The SPUEs demonstrate the highest tensile toughness (∼182.2 MJ m-3) to date for room-temperature self-healable polymers, as well as an excellent ultimate tensile strength (∼10.5 MPa) and ultra-high fracture energy (∼72 100 J m-2), respectively, owing to a synergetic quadruple dynamic mechanism. It is revealed that the B-N coordination not only facilitates the formation and dissociation of boronic ester at room temperature but also dramatically enhances the mechanical properties by the intermolecular coordinated chain crosslinking and intramolecular coordinated chain folding. Meanwhile, the B-N coordination and urethane hydrogen interaction also serve as sacrificial bonds, which rupture during stretching to dissipate energy and recover after release, leading to superior notch insensitiveness and recoverability. The SPUEs restore their mechanical robustness after self-healing at room temperature and the self-healing efficiency can be dramatically accelerated by surface wetting.