Exposure of articular cartilage to excessive mechanical loading is deeply involved in the pathogenesis of osteoarthritis. Here, we identify gremlin-1 as a mechanical loading-inducible factor in ...chondrocytes, detected at high levels in middle and deep layers of cartilage after cyclic strain or hydrostatic pressure loading. Gremlin-1 activates nuclear factor-κB signalling, leading to subsequent induction of catabolic enzymes. In mice intra-articular administration of gremlin-1 antibody or chondrocyte-specific deletion of Gremlin-1 decelerates osteoarthritis development, while intra-articular administration of recombinant gremlin-1 exacerbates this process. Furthermore, ras-related C3 botulinum toxin substrate 1 activation induced by mechanical loading enhances reactive oxygen species (ROS) production. Amongst ROS-activating transcription factors, RelA/p65 induces Gremlin-1 transcription, which antagonizes induction of anabolic genes such as Sox9, Col2a1, and Acan by bone morphogenetic proteins. Thus, gremlin-1 plays essential roles in cartilage degeneration by excessive mechanical loading.
Abstract
Aims
Lifestyle-related diseases promote atherosclerosis, a chronic inflammatory disease; however, the molecular mechanism remains largely unknown. Endogenous DNA fragments released under ...over-nutrient condition provoke sterile inflammation through the recognition by DNA sensors. Here, we investigated the role of stimulator of interferon genes (STING), a cytosolic DNA sensor, in atherogenesis.
Methods and results
Apolipoprotein E-deficient (Apoe−/−) mice fed a western-type diet (WTD), a hypercholesterolaemic mouse model, showed higher STING expression and markers for DNA damage such as γH2AX, p53, and single-stranded DNA (ssDNA) accumulation in macrophages in the aorta compared with wild-type (WT) mice. The level of cGAMP, a STING agonist, in the aorta was higher in Apoe−/− mice. Genetic deletion of Sting in Apoe−/− mice reduced atherosclerotic lesions in the aortic arch, lipid, and macrophage accumulation in plaques, and inflammatory molecule expression in the aorta compared with the control. Pharmacological blockade of STING using a specific inhibitor, C-176, ameliorated atherogenesis in Apoe−/− mice. In contrast, bone marrow-specific STING expression in Apoe−/− mice stimulated atherogenesis. Expression or deletion of STING did not affect metabolic parameters and blood pressure. In vitro studies revealed that STING activation by cGAMP or mitochondrial DNA accelerated inflammatory molecule expression (e.g. TNF-α or IFN-β) in mouse and human macrophages. Activation of nuclear factor-κB and TANK binding kinase 1 was involved in STING-associated vascular inflammation and macrophage activation. Furthermore, human atherosclerotic lesions in the carotid arteries expressed STING and cGAMP.
Conclusion
Stimulator of interferon genes stimulates pro-inflammatory activation of macrophages, leading to the development of atherosclerosis. Stimulator of interferon genes signalling may serve as a potential therapeutic target for atherosclerosis.
Graphical Abstract
STING signalling, originally associated with innate immune system, may provide a novel mechanism of atherogenesis by linking lifestyle-related diseases to chronic inflammatory disease and serve as a potential therapeutic target for atherosclerosis.
Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL-derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid ...deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex-driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor-deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.
Background Toll-like receptor ( TLR ) 9 recognizes bacterial DNA , activating innate immunity, whereas it also provokes inflammation in response to fragmented DNA released from mammalian cells. We ...investigated whether TLR 9 contributes to the development of vascular inflammation and atherogenesis using apolipoprotein E-deficient ( Apoe
) mice. Methods and Results Tlr9-deficient Apoe
( Tlr9
Apoe
) mice and Apoe
mice on a Western-type diet received subcutaneous angiotensin II infusion (1000 ng/kg per minute) for 28 days. Angiotensin II increased the plasma level of double-stranded DNA, an endogenous ligand of TLR 9, in these mice. Genetic deletion or pharmacologic blockade of TLR 9 in angiotensin II-infused Apoe
mice attenuated atherogenesis in the aortic arch ( P<0.05), reduced the accumulation of lipid and macrophages in atherosclerotic plaques, and decreased RNA expression of inflammatory molecules in the aorta with no alteration of metabolic parameters. On the other hand, restoration of TLR 9 in bone marrow in Tlr9
Apoe
mice promoted atherogenesis in the aortic arch ( P<0.05). A TLR 9 agonist markedly promoted proinflammatory activation of Apoe
macrophages, partially through p38 mitogen-activated protein kinase signaling. In addition, genomic DNA extracted from macrophages promoted inflammatory molecule expression more effectively in Apoe
macrophages than in Tlr9
Apoe
macrophages. Furthermore, in humans, circulating double-stranded DNA in the coronary artery positively correlated with inflammatory features of coronary plaques determined by optical coherence tomography in patients with acute myocardial infarction ( P<0.05). Conclusions TLR 9 plays a pivotal role in the development of vascular inflammation and atherogenesis through proinflammatory activation of macrophages. TLR 9 may serve as a potential therapeutic target for atherosclerosis.
Accumulated evidence suggests that activated pancreatic stellate cells (PSCs) serve as the main source of the extracellular matrix proteins accumulated under the pathological conditions leading to ...pancreatic fibrosis in chronic pancreatitis (CP). However, little is known about the mechanisms of PSC activation. PSCs have morphologic and functional similarities to hepatic stellate cells, which are activated by hydrogen peroxide-inducible clone-5 (Hic-5), a TGF-β1-induced protein. In this study, we investigated whether Hic-5 activates PSCs, which promote pancreatic fibrosis development in CP. Hic-5-knockout and wild type mice were subjected to caerulein injection to induce CP. Hic-5 expression was strongly upregulated in activated PSCs from human CP tissue and from mouse pancreatic fibrosis in caerulein-induced CP. Hic-5 deficiency significantly attenuated mouse pancreatic fibrosis and PSC activation in the experimental murine CP model. Mechanistically, Hic-5 knock down significantly inhibited the TGF-β/Smad2 signaling pathway, resulting in reduced collagen production and α-smooth muscle actin expression in the activated PSCs. Taken together, we propose Hic-5 as a potential marker of activated PSCs and a novel therapeutic target in CP treatment.
NADPH oxidases (NOX) are enzymes that catalyze the production of reactive oxygen species (ROS). Four species of NOX catalytic homologs (NOX1, NOX2, NOX4, and NOX5) are reportedly expressed in ...vascular tissues. The pro-atherogenic roles of NOX1, NOX2, and their organizer protein p47ph°x were manifested, and it was noted that the hydrogen peroxide-generating enzyme NOX4 possesses atheroprotective effects. Loss of NOX1 or p47ph°x appears to ameliorate murine aortic dissection and subsequent aneurysmal diseases; in contrast, the ablation of NOX2 exacerbates the aneurysmal diseases. It is possible that the loss of NOX2 activates inflammatory cascades in macrophages in the lesions. Roles of NOX5 in vascular functions are currently undetermined, owing to the absence of this enzyme in rodents and the limitation of the experimental procedure. Thus, it is possible that the NOX family of enzymes exhibits heterogeneity in the atherosclerotic diseases. In this aspect, subtype-selective NOX inhibitor may be promising when NOX systems serve as a molecular target for atherosclerotic and aneurysmal diseases.
ABSTRACT
The transformation of fibroblasts to myofibroblasts plays a major role in fibrogenic responses during dermal wound healing. We show a contribution of calpain systems (intracellular ...regulatory protease systems) in vascular endothelial cells (ECs) to myofibroblast differentiation in wound sites. Dermal wound healing experiments in mice found that calpastatin (an endogenous inhibitor of calpains) is enriched in preexisting vessels but not in newly formed capillaries. Transgenic overexpression of calpastatin in ECs delayed wound healing in mice as well as reducing the keratinocyte layer, extracellular matrix deposition, and myofibroblast accumulation in wound sites. EC and leukocyte markers, however, remain unchanged. Calpastatin overexpression reduced the expression of genes encoding platelet‐derived growth factor‐B and PDGF receptor‐β (PDGFR‐β). Topical application of platelet‐derived growth factor‐BB–containing ointment to wounds accelerated healing in control mice, but calpastatin overexpression prevented this acceleration. In cultured human dermal fibroblasts, α‐smooth muscle actin and PDGFR‐β were up‐regulated by coculturing with ECs, but this action was inhibited by suppression of EC calpain activity. EC‐driven transformation of mouse dermal fibroblasts was also suppressed by calpastatin overexpression in ECs. These results suggest that endothelial calpain systems influence PDGFR‐β signaling in fibroblasts, EC‐driven myofibroblast differentiation, and subsequent fibrogenic responses in wounds.—Miyazaki, T., Haraguchi, S., Kim‐Kaneyama, J.‐R., Miyazaki, A. Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing. FASEB J. 33, 2037–2046 (2019). www.fasebj.org
Osteoarthritis (OA) is the most common joint disease associated with articular cartilage destruction. Matrix metalloproteinase-13 (MMP-13) has an essential role in OA pathogenesis by degradation of ...collagen II, a major component of articular cartilage. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFB1I1), a transforming growth factor-β-inducible mechanosensor, has previously been reported to promote OA pathogenesis by upregulating MMP-13 expression in mouse osteoarthritic lesions. In our current study, immunohistochemical analysis showed that Hic-5 protein expression was increased in human OA cartilage compared with normal cartilage. Functional experiments demonstrated that Hic-5 and MMP-13 expression was increased by mechanical stress, and mechanical stress-induced MMP-13 expression was suppressed by Hic-5 siRNA in human chondrocytes. Moreover, intracellular localization of Hic-5 shifted to the nucleus from focal adhesions in human chondrocytes subjected to mechanical stress, and nuclear Hic-5 increased MMP-13 gene expression. In vivo, intra-articular injection of Hic-5 siRNA decreased the Osteoarthritis Research Society International score and MMP-13 protein expression in articular cartilage of OA rats. Our findings suggest that Hic-5 regulates transcription of MMP-13 in human chondrocytes, and Hic-5 may be a novel therapeutic target for OA because OA progression was suppressed by intra-articular injection of Hic-5 siRNA in rats.
Background Sterile inflammation caused by metabolic disorders impairs endothelial function; however, the underlying mechanism by which hyperglycemia induces inflammation remains obscure. Recent ...studies have suggested that stimulator of interferon genes (STING), a key cytosolic DNA sensor in the innate immune system, contributes to the pathogenesis of inflammatory diseases. This study examines the role of the STING in endothelial dysfunction in streptozotocin-induced diabetic mice. Methods and Results Injection of streptozotocin promoted the expression of STING and DNA damage markers in the aorta of wild-type mice. Streptozotocin elevated blood glucose and lipid levels in both wild-type and STING-deficient mice, which showed no statistical differences. Genetic deletion of STING ameliorated endothelial dysfunction as determined by the vascular relaxation in response to acetylcholine (
<0.001) and increased endothelial nitric oxide synthase phosphorylation in the aorta (
<0.05) in STZ-injected mice. Endothelium-independent vascular response to sodium nitroprusside did not differ. Treatment with a direct STING agonist, cyclic GMP-AMP, or mitochondrial DNA increased inflammatory molecule expression (eg,
and
) and decreased endothelial nitric oxide synthase phosphorylation in human umbilical vein endothelial cells, partially through the STING pathway. Cyclic GMP-AMP significantly impaired endothelial function of aortic segments obtained from wild-type mice, which was ameliorated in the presence of C-176, a STING inhibitor, or a neutralizing interferon-β antibody. Furthermore, the administration of C-176 ameliorated endothelial dysfunction in STZ-induced diabetic mice (
<0.01). Conclusions The DNA damage response regulated by STING impairs endothelial function. STING signaling may be a potential therapeutic target of endothelial dysfunction caused by hyperglycemia.
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