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Radiation-induced pulmonary fibrosis is a severe complication of patients treated with thoracic irradiation. We have previously shown that syndecan-2 reduces fibrosis by exerting alveolar epithelial cytoprotective effects. Here, we investigate whether syndecan-2 attenuates radiation-induced pulmonary fibrosis by inhibiting fibroblast activation. C57BL/6 wild-type mice and transgenic mice that overexpress human syndecan-2 in alveolar macrophages were exposed to 14 Gy whole-thoracic radiation. At 24 weeks after irradiation, lungs were collected for histological, protein, and mRNA evaluation of pulmonary fibrosis, profibrotic gene expression, and α-smooth muscle actin (α-SMA) expression. Mouse lung fibroblasts were activated with transforming growth factor (TGF)-β1 in the presence or absence of syndecan-2. Cell proliferation, migration, and gel contraction were assessed at different time points. Irradiation resulted in significantly increased mortality and pulmonary fibrosis in wild-type mice that was associated with elevated lung expression of TGF-β1 downstream target genes and cell death compared with irradiated syndecan-2 transgenic mice. In mouse lung fibroblasts, syndecan-2 inhibited α-SMA expression, cell contraction, proliferation, and migration induced by TGF-β1. Syndecan-2 attenuated phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and serum response factor binding to the α-SMA promoter. Syndecan-2 attenuates pulmonary fibrosis in mice exposed to radiation and inhibits TGF-β1–induced fibroblast–myofibroblast differentiation, migration, and proliferation by down-regulating phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and blocking serum response factor binding to the α-SMA promoter via CD148. These findings suggest that syndecan-2 has potential as an antifibrotic therapy in radiation-induced lung fibrosis.

Lung transplantation is the only viable option for patients suffering from otherwise incurable end-stage pulmonary diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Despite aggressive immunosuppression, acute rejection of the lung allograft occurs in over half of transplant recipients, and the factors that promote lung acceptance are poorly understood. The contribution of lymphatic vessels to transplant pathophysiology remains controversial, and data that directly address the exact roles of lymphatic vessels in lung allograft function and survival are limited. Here, we have shown that there is a marked decline in the density of lymphatic vessels, accompanied by accumulation of low-MW hyaluronan (HA) in mouse orthotopic allografts undergoing rejection. We found that stimulation of lymphangiogenesis with VEGF-C156S, a mutant form of VEGF-C with selective VEGFR-3 binding, alleviates an established rejection response and improves clearance of HA from the lung allograft. Longitudinal analysis of transbronchial biopsies from human lung transplant recipients demonstrated an association between resolution of acute lung rejection and decreased HA in the graft tissue. Taken together, these results indicate that lymphatic vessel formation after lung transplantation mediates HA drainage and suggest that treatments to stimulate lymphangiogenesis have promise for improving graft outcomes.

Background Sepsis is a complex and life-threatening disease process related to a systemic response to severe infection. Due to the challenges of treating patients with sepsis, new therapies are being investigated, including cell-based approaches. Trophoblast stem cells (TSCs) are immune privileged cells with immunomodulatory properties. Thus, we proposed that TSCs may be beneficial in experimental models of sepsis to regulate the immune response and curtail organ injury. Methods Sepsis was induced by experimental models in mice; cecal ligation and puncture (CLP) and lung infection with Streptococcus (S.) pneumoniae. TSCs were isolated from the chorionic villi of human (h) term placentas, and from mouse (m) placentas using anti-CD117 MicroBeads, and were administered intravenously 6 h after CLP or S. pneumoniae infection. We assessed mortality, bacterial clearance, organ injury, inflammatory response, and production of cytokines and chemokines. Results CD117 + hTSCs did not express human leukocyte antigen (HLA) I or II, and were clonogenic and self-renewing. CLP led to severe mortality by 7 days, and administration of either hTSCs or mTSCs resulted in markedly improved survival compared with control cells or vehicle. hTSCs promoted bacterial clearance and decreased organ injury in the liver, kidney, spleen, and bowel. The elevated innate immune response in the peritoneum, predominantly neutrophils, was attenuated by hTSCs. In addition, neutrophil infiltration into the spleen was less in mice receiving hTSCs, which corresponded with reduced plasma pro-inflammatory cytokines and chemokines. When assessing the lung response to S. pneumoniae infection, administration of hTSCs resulted in fewer bacteria in bronchoalveolar lavage fluid (BALF) and lung tissue, and less lung edema and injury. Neutrophils, which were markedly increased in BALF, were diminished and infiltration of neutrophils and macrophages into the lungs was decreased by hTSCs. BALF pro-inflammatory cytokines and chemokines were mitigated by hTSCs to levels of Sham mice, and systemic injury to the liver and spleen was attenuated. Conclusions CD117 + hTSCs are immune privileged cells that when given after the onset of experimental models of infection/sepsis resulted in improved outcomes due to enhanced bacterial clearance, resolving inflammation, and less organ injury. These data support hTSCs as a potential cell-based therapy for sepsis.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Oxidative stress resulting from inflammatory responses that occur during acute lung injury and sepsis can initiate changes in mitochondrial function. Autophagy regulates cellular processes in the setting of acute lung injury, sepsis, and oxidative stress by modulating the immune response and facilitating turnover of damaged cellular components. We have shown that mesenchymal stromal cells (MSCs) improve survival in murine models of sepsis by also regulating the immune response. However, the effect of autophagy on MSCs and MSC mitochondrial function during oxidative stress is unknown. This study investigated the effect of depletion of autophagic protein microtubule–associated protein 1 light chain 3B (LC3B) and beclin 1 (BECN1) on the response of MSCs to oxidative stress. MSCs were isolated from wild-type (WT) and LC3B−/− or Becn1+/− mice. MSCs from the LC3B−/− and Becn1+/− animals had increased susceptibility to oxidative stress–induced cell death as compared with WT MSCs. The MSCs depleted of autophagic proteins also had impaired mitochondrial function (decreased intracellular ATP, reduced mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production) under oxidative stress as compared with WT MSCs. In WT MSCs, carbon monoxide (CO) preconditioning enhanced autophagy and mitophagy, and rescued the cells from oxidative stress–induced death. CO preconditioning was not able to rescue the decreased survival of MSCs from the LC3B−/− and Becn1+/− animals, further supporting the tenet that CO exerts its cytoprotective effects via the autophagy pathway.

Iron–sulfur (Fe‐S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR‐210‐ISCU1/2 axis cause Fe‐S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR‐210 and repression of the miR‐210 targets ISCU1/2 down‐regulated Fe‐S levels. In mouse and human vascular and endothelial tissue affected by PH, miR‐210 was elevated accompanied by decreased ISCU1/2 and Fe‐S integrity. In mice, miR‐210 repressed ISCU1/2 and promoted PH. Mice deficient in miR‐210, via genetic/pharmacologic means or via an endothelial‐specific manner, displayed increased ISCU1/2 and were resistant to Fe‐S‐dependent pathophenotypes and PH. Similar to hypoxia or miR‐210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise‐induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR‐210‐ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe‐S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings. Synopsis Hypoxia‐inducible miR‐210 down‐regulates its targets ISCU1/2 to regulate pulmonary vascular and endothelial levels of Fe‐S clusters upon acquired injury or mutations. A patient with ISCU loss‐of‐function mutation presents with pulmonary vasculopathy. In mouse and human pulmonary vascular and endothelial tissue affected by PH, hypoxic induction of miR‐210 and repression of its targets ISCU1/2, down‐regulates iron‐sulfur levels. Using genetic and pharmacologic methods to perform gain‐of‐function and loss‐of‐function analyses of miR‐210 and ISCU1/2 in the pulmonary vasculature of mice, the miR‐210‐ISCU1/2 axis is shown to be necessary and sufficient for induction of hypoxic PH. Cardiopulmonary exercise testing of a woman with ISCU mutations revealed exercise‐induced pulmonary vascular dysfunction. Graphical Abstract Hypoxia‐inducible miR‐210 down‐regulates its targets ISCU1/2 to regulate pulmonary vascular and endothelial levels of Fe‐S clusters upon acquired injury or mutations. A patient with ISCU loss‐of‐function mutation presents with pulmonary vasculopathy.

Background Autosomal‐recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation–contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi‐omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions. Methods Skeletal muscles from 2‐month‐old SPEG‐deficient (Speg‐CKO) and wild‐type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg‐CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg‐CKO mice. Based on the multi‐omics results, we performed quantitative real‐time PCR, co‐immunoprecipitation and immunoblot to verify the findings. Results We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg‐CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24‐fold], S162 [1.61 ± 0.37‐fold] and S165 [1.66 ± 0.13‐fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix–receptor interaction (P < 1e−15) and peroxisome proliferator‐activated receptor signalling (P < 9e−14). Conclusions We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG‐interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi‐omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-β inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-β resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-β-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-β resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-β in β-catenin stability through protein phosphorylation, we found that GSK3-β inhibition resulted in an increase in β-catenin levels. Simultaneous silencing of β-catenin and inhibition of GSK3-β demonstrated that β-catenin is required for GSK3-β-induced lymphangiogenesis.

Background Therapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction. Methods Lung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and β-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-α-induced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lung-slices ex vivo and on hyaluronan uptake in vitro . Results In vitro , FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo . We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis. Conclusion FK506 alters lymphatic endothelial cell molecular characteristics and causes lymphatic endothelial cell dysfunction in vitro and ex vivo. These effects of FK506 on lymphatic endothelial cell may impair the ability of the transplanted lung to drain hyaluronan macromolecules in vivo . The implications of our findings on the long-term health of lung allografts merit more investigation.