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Background & Aims: Chronic liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH), pose a significant global health burden. Progressive liver fibrosis can lead to severe outcomes; however, there is a lack of effective therapies targeting advanced fibrosis. Hydrogen peroxide-inducible clone-5 (Hic-5), an adaptor protein in focal adhesion, is critical for promoting liver fibrosis in hepatic stellate cells. This study investigated its clinical applicability by examining hepatic Hic-5 expression in human fibrotic tissues, exploring its association with MASH, and assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting Hic-5 in a MASH mouse model., Methods: Hepatic Hic-5 expression in human fibrotic tissues underwent pathological image analysis and single-cell RNA sequencing. ASOs targeting Hic-5 were developed and tested using in vitro cell models. An in vivo MASH mouse model was used to evaluate the effects of anti- Hic-5 ASOs on advanced fibrosis and steatosis., Results: Hepatic Hic-5 expression increased with the progression of fibrosis, particularly in advanced stages. Single-cell RNA sequencing revealed Hic-5 expression primarily in hepatic stellate cells. In MASH-associated fibrosis, Hic-5 expression correlated with the expression of fibrotic genes. In the MASH mouse model, hepatic Hic-5 expression increased with disease progression. Anti- Hic-5 ASOs effectively suppressed Hic-5 expression in vitro and attenuated advanced fibrosis and steatosis in vivo , indicating their therapeutic potential., Conclusions: Hepatic Hic-5 expression is associated with advanced liver fibrosis and MASH. Anti- Hic-5 ASOs are promising therapeutic interventions for MASH accompanied by advanced fibrosis. These findings provide valuable insights into potential clinical treatments for advanced liver fibrosis., Impact and Implications: This study investigated the role of Hic-5 in liver fibrosis and steatohepatitis, highlighting its potential as a therapeutic target. We developed an antisense oligonucleotide (ASO) that was particularly transportable to the liver, and targeted Hic-5. Anti- Hic-5 ASO exhibited therapeutic efficacy for liver fibrosis and steatosis in vivo , indicating its therapeutic potential for liver fibrosis and steatosis. ASOs have already achieved dramatic therapeutic effects as approved nucleic acid drugs. Thus, anti- Hic-5 ASO is expected to lead the direct generation of seed compounds for the clinical development of drugs for liver fibrosis and steatosis., (© 2024 The Authors.)

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 ( P <0.001) and increased endothelial nitric oxide synthase phosphorylation in the aorta ( P <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, VCAM1 and IFNB ) 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 ( P <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.

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., (© 2023. The Author(s).)

Background: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells., Methods: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice., Results: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-β1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-β type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice., Conclusions: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

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.

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.

In the published article, the Fig. 4a was published incorrectly.

Excessive mechanical stress is a major cause of knee osteoarthritis. However, the mechanism by which the mechanical stress begets osteoarthritis development remains elusive. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFβ1i1), a TGF-β inducible focal adhesion adaptor, has previously been reported as a mediator of mechanotransduction. In this study, we analyzed the in vivo function of Hic-5 in development of osteoarthritis, and found that mice lacking Hic-5 showed a significant reduction in development of osteoarthritis in the knee. Furthermore, we found reduced expression of catabolic genes, such as metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 in osteoarthritic lesions in mice lacking Hic-5. During osteoarthritis development, Hic-5 is detected in chondrocytes of articular cartilage. To investigate the role of Hic-5 in chondrocytes, we isolated chondrocytes from articular cartilage of wild type and Hic-5-deficient mice. In these primary cultured chondrocytes, Hic-5 deficiency resulted in suppression of catabolic gene expression induced by osteoarthritis-related cytokines such as tumor necrosis factor α and interleukin 1β. Furthermore, Hic-5 deficiency in chondrocytes suppressed catabolic gene expression induced by mechanical stress. Revealing the regulation of chondrocyte catabolism by Hic-5 contributes to understanding the pathophysiology of osteoarthritis induced by mechanical stress.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks., Competing Interests: SH, MK, TT, KT, MS, MN, MO, IS, GH, VC, XL, TM, JK, TN, KM, HO, TT, TT, AM, KT No competing interests declared, (© 2019, Haraguchi et al.)

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.

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.

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.

Carcinoma-associated fibroblasts (CAFs) influence tumor initiation, progression, and metastasis within the tumor-associated stroma. This suggests that CAFs would be a potential target for tumor therapy. Here we found that Hydrogen peroxide-inducible clone-5 (Hic-5), also named transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was strongly induced in CAFs found in human colorectal cancer. To investigate the role of Hic-5 in CAFs, we isolated CAFs and the control counterpart normal fibroblasts (NFs) from human colorectal cancer and non-cancerous regions, respectively. Hic-5 was highly expressed in isolated human CAFs and strongly induced in NFs in culture by the supernatant from cultured colorectal cancer cells as well as cytokines such as TGF-β, IL-1β and stromal cell-derived factor 1 (SDF-1/CXCL12). Furthermore, tumor growth was inhibited in a co-culture assay with Hic-5 knockdown fibroblasts compared with control fibroblasts. To clarify the function and significance of Hic-5 in colorectal cancer in vivo, we utilized a mouse model of azoxymethane (AOM)-induced colorectal cancer using Hic-5-deficient mice. Lack of Hic-5 in CAFs completely prevented AOM-induced colorectal cancer development in the colon tissues of mice. Mechanistic investigation revealed that Hic-5 promoted the expression of lysyl oxidase and collagen I in human control counterpart fibroblasts. Taken together, these results demonstrate that Hic-5 in CAFs is responsible for orchestrating or generating a tumor-promoting stroma.

The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7 cells are used for transfection experiments to analyze the actions of functional molecules including steroids. However, a preliminary study suggested that COS-7 cells metabolize [ 3 H]testosterone to [ 3 H]androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the present study investigated the expression of steroidogenic enzymes and the metabolism of steroids in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase, 17β-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome P450 11β-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β, and 17β-HSD actively metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17β-HSD activity toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.

The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of Paired-homeobox 6 (PAX6) is regulated by multiple cis-DNA elements including the intronic α-enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the α-enhancer in the post-natal mouse retina. Tgfb1i1 -/- mice show elevated α-enhancer activity leading to overproduction of Pax6ΔPD isoform that supports the GABAergic amacrine cell fate maintenance. Consequently, the Tgfb1i1 -/- mouse retinas show a sustained light response, which becomes more transient in mice with the auto-stimulation-defective Pax6 ΔPBS/ΔPBS mutation. Together, we show the antagonistic regulation of the α-enhancer activity by Pax6 and the LIM protein complex is necessary for the establishment of an inner retinal circuitry, which controls visual adaptation., Competing Interests: The authors declare that no competing interests exist.

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 p47 ph ° x were manifested, and it was noted that the hydrogen peroxide-generating enzyme NOX4 possesses atheroprotective effects. Loss of NOX1 or p47 ph ° 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., Competing Interests: None.

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.

Oxidized cholesterols (oxycholesterols) in food have been recognized as strong atherogenic components, but their tissue distributions and roles in cardiovascular diseases remain unclear. To investigate whether accumulation of oxycholesterols is linked to cardiac morphology and function, and whether reduction of oxycholesterols can improve cardiac performance, domestic male swine were randomized to a control diet (C), high caloric diet (HCD) or HCD+Ezetimibe, an inhibitor of intestinal cholesterol absorption, group (HCD+E) and evaluated for: (1) distribution of oxycholesterol components in serum and tissues, (2) levels of oxycholesterol-related enzymes, (3) paracardial and epicardial coronary fat thickness, and (4) cardiac performance. Ezetimibe treatment for 8weeks attenuated increases in oxycholesterols in the HCD group almost completely in liver, but reduced only levels of 4β-hydroxycholesterol in left ventricular (LV) myocardium. Ezetimibe treatment altered the expression of genes for cholesterol and fatty acid metabolism and decreased the expression of CYP3A46, which catabolizes cholesterol to 4β-hydroxycholesterol, strongly in liver. An increase in epicardial fat thickness and impaired cardiac performance in the HCD group were improved by ezetimibe treatment, and the improvement was closely related to the reduction in levels of 4β-hydroxycholesterol in LV myocardium. In conclusion, an increase in oxycholesterols in the HCD group was closely related to cardiac hypertrophy and dysfunction, as well as an increase in epicardial fat thickness. Ezetimibe may directly reduce oxycholesterol in liver and LV myocardium, and improve cardiac morphology and function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes., (© 2016 Petropoulos et al.)

Objective: Prevalence of ankle osteoarthritis (OA) is lower than that of knee OA, however, the molecular mechanisms underlying the difference remain unrevealed. In the present study, we developed mouse ankle OA models for use as tools to investigate pathophysiology of ankle OA and molecular characteristics of ankle cartilage., Design: We anatomically and histologically examined ankle and knee joints of C57BL/6 mice, and compared them with human samples. We examined joints of 8-week-old and 25-month-old mice. For experimental models, we developed three different ankle OA models: a medial model, a lateral model, and a bilateral model, by resection of respective structures. OA severity was evaluated 8 weeks after the surgery by safranin O staining, and cartilage degradation in the medial model was sequentially examined., Results: Anatomical and histological features of human and mouse ankle joints were comparable. Additionally, the mouse ankle joint was more resistant to cartilage degeneration with aging than the mouse knee joint. In the medial model, the tibiotalar joint was markedly affected while the subtalar joint was less degenerated. In the lateral model, the subtalar joint was mainly affected while the tibiotalar joint was less altered. In the bilateral model, both joints were markedly degenerated. In the time course of the medial model, TdT-mediated dUTP nick end labeling (TUNEL) staining and Adamts5 expression were enhanced at early and middle stages, while Mmp13 expression was gradually increased during the OA development., Conclusion: Since human and mouse ankles are comparable, the present models will contribute to ankle OA pathophysiology and general cartilage research in future., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

Background & Aim: Hydrogen peroxide-inducible clone-5 (Hic-5), also named as transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was found to be induced by TGF-β. Previous studies have shown that TGF-β is a principal mediator of hepatic stellate cell (HSC) activation in liver fibrosis. However, this process remains elusive. In this study, we aimed to define the role of Hic-5 in HSC activation and liver fibrosis., Methods: We examined the expression levels of Hic-5 during HSCs activation and in fibrotic liver tissues by quantitative real-time reverse transcriptase polymerase chain reaction, Western blot and immunohistochemistry. Hic-5 knockout (KO) and wild-type (WT) mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) injection to induce liver fibrosis., Results: Hic-5 expression was strongly upregulated in activated HSCs of the human fibrotic liver tissue and BDL or CCl4-induced mouse liver fibrosis. Hic-5 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Furthermore, Hic-5 knockdown by siRNA in vivo repressed CCl4-induced liver fibrosis in mice. Mechanistically, the absence of Hic-5 significantly inhibited the TGF-β/Smad2 signaling pathway, proved by increasing Smad7 expression, resulting in reduced collagen production and α-smooth muscle actin expression in the activated HSCs., Conclusion: Hic-5 deficiency attenuates the activation of HSCs and liver fibrosis though reducing the TGF-β/Smad2 signaling by upregulation of Smad7. Thus, Hic-5 can be regarded as a potential therapeutic target for liver fibrosis., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor (TGF)-β1-inducible focal adhesion protein. We previously demonstrated that Hic-5 was localized in mesangial cells and its expression was associated with glomerular cell proliferation and matrix expansion in human and rat glomerulonephritis (GN). In the present study, we first assessed the role of Hic-5 in mesangioproliferative GN by injecting Habu venom into heminephrectomized wild type (Hic-5+/+) and Hic-5-deficient (Hic-5-/-) mice. Hic-5+/+ GN mice exhibited glomerular cell proliferation on day 7. Surprisingly, glomerular cell number and Ki-67-positive cells in Hic-5-/- GN mice were significantly greater than those in Hic-5+/+ GN mice on day 7, although the number of glomerular apoptotic cells and the expression of growth factors (platelet-derived growth factor-BB and TGF-β1) and their receptors were similarly increased in both Hic-5+/+ and Hic-5-/- GN mice. In culture experiments, proliferation assays showed that platelet-derived growth factor-BB and TGF-β1 enhanced the proliferation of Hic-5-/- mesangial cells compared with Hic-5+/+ mesangial cells. In addition, mitogenic regulation by Hic-5 was associated with altered and coordinated expression of cell cycle-related proteins including cyclin D1 and p21. The present results suggest that Hic-5 might regulate mesangial cell proliferation in proliferative GN in mice. In conclusion, modulation of Hic-5 expression might have a potential to prevent mesangial cell proliferation in the acute mitogenic phase of glomerulonephritis.

Rationale: Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations., Objective: To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis., Methods and Results: The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated μ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects., Conclusions: Loss of CAST in angiogenic ECs facilitates μ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis., (© 2015 American Heart Association, Inc.)

Aims: The adhesion of circulating monocytes to endothelial cells (ECs) is an early and critical event in the formation of atherosclerotic plaques. Hydrogen peroxide-inducible clone 5 (Hic-5) serves as an adaptor molecule in cell adhesion complexes. However, the role of endothelial Hic-5 in monocyte-EC interaction and atherogenesis remains unclear. We examined the roles of endothelial Hic-5 in monocyte-EC interaction and atherogenesis using mouse models of atherosclerosis and cultured human umbilical vein endothelial cells (HUVECs)., Methods and Results: Hic-5 was expressed in ECs, but not in monocytes/macrophages. An ex vivo monocyte adhesion assay revealed that adhesion of THP-1 monocytes to aortas isolated from Apoe(-/-) and LDLR(-/-) mice stimulated by TNF-α or oxidized LDL was suppressed by Hic-5 deficiency. Scanning electron microscopic observations of aortas harvested from Apoe(-/-) mice revealed that TNF-α- or oxidized LDL-induced microvilli-like structures were markedly suppressed by Hic-5 deficiency. Relative Hic-5 deficiency suppressed 60% of the atherosclerotic lesions in aortas from Apoe(-/-) and LDLR(-/-) mice. In contrast, overexpression of Hic-5 in HUVECs promoted induction of microvilli-like structures and adherence of THP-1 cells in an adhesion receptor such as intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-dependent manner., Conclusion: Hic-5 in ECs plays an important role in the formation of microvilli-like structures and in the interaction between ECs and monocytes, leading to monocyte recruitment and subsequent development of atherosclerosis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Objective: Although nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) is reportedly essential for phagocyte host defenses, it has been found to aggravate atherosclerosis in apolipoprotein E (Apoe)-null mice through excess production of superoxide. We therefore assessed the role of NOX2 in an experimental model of abdominal aortic aneurysm (AAA) and assessed the mechanism of NOX2 action in AAA., Approach and Results: AAA was induced in low-density lipoprotein receptor-null (Ldlr(-/-)) mice by infusing angiotensin II. Nox2 expression was elevated in the abdominal aortae of these mice during infusion of angiotensin II, with enhanced Nox2 expression mainly because of the recruitment of NOX2-enriched macrophages into AAA lesions. Unexpectedly, systemic Nox2 deficiency promoted AAA development but reduced the level of reactive oxygen species in AAA lesions. Nox2 deficiency stimulated macrophage conversion toward the M1 subset, enhancing expression of interleukin (IL)-1β and matrix metalloproteinase-9/12 mRNA. Administration of neutralizing antibody against IL-1β abolished AAA development in Nox2-deficient mice. Bone marrow transplantation experiments revealed that AAA aggravation by Nox2 deficiency is because of bone marrow-derived cells. Isolated bone marrow-derived macrophages from Nox2-null mice could not generate reactive oxygen species. In contrast, IL-1β expression in peritoneal and bone marrow-derived macrophages, but not in peritoneal neutrophils, was substantially enhanced by Nox2 deficiency. Pharmacological inhibition of Janus kinase/signal transducers and activators of transcription signaling inhibited excess IL-1β expression in Nox2-deficient macrophages, whereas matrix metalloproteinase-9 secretion was constitutively stimulated via nuclear factor-κB signals., Conclusions: Nox2 deficiency enhances macrophage secretion of IL-1β and matrix metalloproteinase-9, disrupting tissue-remodeling functions in AAA lesions. These actions are unfavorable if NOX2 is to serve as a molecular target for AAA., (© 2014 American Heart Association, Inc.)

Background: Although increased amounts of reactive oxygen species in the pathogenesis of abdominal aortic aneurysm (AAA) are well documented, the precise molecular mechanisms by which reactive oxygen species induce AAAs have not been fully elucidated. This study focused on the role of hydrogen peroxide-inducible clone 5 (Hic-5), which is induced by hydrogen peroxide and transforming growth factor-β, in the cellular signaling of AAA pathogenesis., Methods and Results: Using the angiotensin II-induced AAA model in Apoe(-/-) mice, we showed that Apoe(-/-)Hic-5(-/-) mice were completely protected from AAA formation and aortic rupture, whereas Apoe(-/-) mice were not. These features were similarly observed in smooth muscle cell-specific Hic-5-deficient mice. Furthermore, angiotensin II treatment induced Hic-5 expression in a reactive oxygen species-dependent manner in aortic smooth muscle cells in the early stage of AAA development. Mechanistic studies revealed that Hic-5 interacted specifically with c-Jun N-terminal kinase p54 and its upstream regulatory molecule mitogen-activated protein kinase kinase 4 as a novel scaffold protein, resulting in the expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase 2 activation in aortic smooth muscle cells., Conclusion: Hic-5 serves as a novel scaffold protein that specifically activates the mitogen-activated protein kinase kinase 4/p54 c-Jun N-terminal kinase pathway, thereby leading to the induction and activation of matrix metalloproteinases in smooth muscle cells and subsequent AAA formation. Our study provided a novel therapeutic option aimed at inhibiting the mitogen-activated protein kinase kinase 4-Hic-5-p54 c-Jun N-terminal kinase pathway in the vessel wall, particularly through Hic-5 inhibition, which may be used to produce more precise and effective therapies.

This review highlights the pro-atherogenic roles of Ca(2+)-sensitive intracellular protease calpains. Among more than ten species of calpain isozymes, µ- and m-calpains have been characterized most extensively. These two isozymes are ubiquitously expressed in mammalian tissues, including blood vessels, and tightly regulate functional molecules in the vascular component cells through limited proteolytic cleavage. Indeed, previous cell-based experiments showed that calpains play significant roles in nitric oxide production in vascular endothelial cells (ECs), maintenance of EC barrier function and angiogenesis for maintaining vascular homeostasis. Recently, we demonstrated that modified-low density lipoprotein (LDL)-induced m-calpain causes hyperpermeability in ECs, leading to the infiltration of monocytes/macrophages and plasma lipids into the intimal spaces (Miyazaki T. et al., Circulation. 2011; 124: 2522-2532). Calpains also mediate oxidized LDL-induced apoptotic death in ECs. In monocytes/macrophages, calpains induce proteolytic degradation of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which results in impaired cholesterol efflux and subsequent macrophage foam cell formation. In vascular smooth muscle cells, calpains may be involved in the conversion from contractile phenotype to proliferative phenotype. In hepatocytes, calpains disrupt the biogenesis of high-density lipoprotein via proteolytic degradation of ABCA1. Thus, calpains may serve as novel candidate molecular targets for control of atherosclerosis.

Background: Integrin αIIbβ3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic strand., Objectives: Hic-5 function in αIIbβ3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic-5(-/-) mice were analyzed., Methods and Results: Hic-5(-/-) mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin-induced aggregation in Hic-5(-/-) platelets. Mechanistically, Hic-5(-/-) platelets showed limited activation of αIIbβ3 upon thrombin treatment. Morphological alteration in Hic-5(-/-) platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co-immunoprecipitating with the activated αIIbβ3 was smaller in Hic-5(-/-) platelets than in wild-type platelets., Conclusion: We identified Hic-5 as a novel and specific regulatory factor for thrombin-induced αIIbβ3 activation and subsequent platelet aggregation in mice., (© 2012 International Society on Thrombosis and Haemostasis.)

The bioactive peptide salusin-β is highly expressed in human atheromas; additionally, infusion of antiserum against salusin-β suppresses the development of atherosclerosis in atherogenic mice. This study examined the roles of salusin-β in vascular inflammation during atherogenesis. Infusion of antiserum against salusin-β attenuated the induction of VCAM-1, monocyte chemoattractant protein (MCP)-1, and IL-1β and as well as nuclear translocation of NF-κB in aortic endothelial cells (ECs) of LDL receptor-deficient mice, which led to the prevention of monocyte adhesion to aortic ECs. In vitro experiments indicated that salusin-β directly enhances the expression levels of proinflammatory molecules, including VCAM-1, MCP-1, IL-1β, and NADPH oxidase 2, as well as THP-1 monocyte adhesion to cultured human umbilical vein ECs (HUVECs). Both salusin-β-induced VCAM-1 induction and monocyte/HUVEC adhesion were suppressed by pharmacological inhibitors of NF-κB, e.g., Bay 11-7682 and curcumin. Furthermore, the VCAM-1 induction was significantly prevented by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002, whereas it was accelerated by the ERK inhibitor, U-0126. Treatment of HUVECs with salusin-β, but not with salusin-α, accelerated oxidative stress and nuclear translocation of NF-κB as well as phosphorylation and degradation of IκB-α, an endogenous inhibitor of NF-κB. Thus, salusin-β enhanced monocyte adhesion to vascular ECs through NF-κB-mediated inflammatory responses in ECs, which can be modified by PI3K or ERK signals. These findings are suggestive of a novel role of salusin-β in atherogenesis.

Hydrogen peroxide-inducible clone-5 (Hic-5) is a focal adhesion scaffold protein primarily expressed in vascular and visceral smooth muscle cells. We recently generated mice lacking Hic-5, which grew with no apparent abnormality (Kim-Kaneyama J, et al. J Mol Cell Cardiol. 2011;50(1):77-86). However, we discovered that recovery of arterial media following vascular injury is delayed significantly in Hic-5 knockout mice consequent to enhanced apoptosis of cultured vascular smooth muscle cells after mechanical stress; thus, Hic-5 is regarded as a novel factor in vascular remodeling. The Hic-5 gene is also induced by transforming growth factor-β, a well-known accelerator in fibrosis. Hic-5 involvement in various fibrotic disorders, e.g., scar formation, keloid formation and glomerulosclerosis, has been proposed. siRNA silencing of Hic-5 in a breast cancer cell line reduces its invasiveness; moreover, Hic-5 serves as a steroid hormone co-activator and likely participates in endometriosis and prostate cancer. Thus, functional characterization of Hic-5 in various pathophysiological conditions may afford novel mechanistic insights into a wide variety of diseases.

Forces associated with blood flow are crucial not only for blood vessel development but also for regulation of vascular pathology. Although there have been many studies characterizing the responses to mechanical stimuli, molecular mechanisms linking biological responses to mechanical forces remain unclear. Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion adaptor protein proposed as a candidate for a mediator of mechanotransduction. In the present study, we generated Hic-5-deficient mice by targeted mutation. Mice lacking Hic-5 are viable and fertile, and show no obvious histological abnormalities including vasculature. However, after wire injury of the femoral artery in Hic-5 deficient mice, histological recovery of arterial media was delayed due to enhanced apoptosis of vascular wall cells, whereas neointima formation was enhanced. Stretch-induced apoptosis was enhanced in cultured vascular smooth muscle cells (vascular SMCs) from Hic-5 deficient mice. Mechanical stress also induced the alteration of intracellular distribution of vinculin from focal adhesions to the whole cytoplasm in SMCs. Immunoelectron microscopic study of vascular SMCs from a wire-injured artery demonstrated that vinculin was dispersed in the nucleus and the cytoplasm in Hic-5-deficient mice whereas vinculin was localized mainly in the sub-plasma membrane region in wild type mice. Our findings indicate that Hic-5 may serve as a key regulator in mechanosensitive vascular remodeling., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Focal adhesion components are targets for biochemical and mechanical stimuli that evoke crucial injury. Hic-5 (hydrogen peroxide-inducible clone 5) is a multidomain adaptor protein which is implicated in the regulation of integrin signaling in focal adhesion. The aim of this research was to test the hypothesis that Hic-5, a focal adhesion LIM protein expressed in smooth muscle cells, is involved in dynamic processes by pathological stimuli in the vessel wall. Here, we describe the analysis of the function of Hic-5 using a mouse model of vascular injury that may mimic balloon angioplasty. At 4 days after vascular injury, marked down-regulation of the Hic-5 expression was observed in the smooth muscle layer, and local delivery of the Hic-5 using adenovirus vectors repressed injury-induced neointimal expansion. In addition, Hic-5 reduced cells migration into three-dimensional collagen gels, and the forced expression of Hic-5 in cells embedded in the collagen gel matrix repressed the expression of uPA that participates in smooth muscle cell migration. These results suggest that Hic-5 modulates cellular responses to pathological stimuli in the vessel wall.

Recent evidence suggests that bone marrow-derived cells may contribute to repair and lesion formation following vascular injury. In most studies, bone marrow-derived cells were tracked by transplanting exogenous cells into bone marrow that had been compromised by irradiation. It remains to be determined whether endogenous circulating progenitors actually contribute to arterial remodeling under physiological conditions. Here, we established a parabiotic model in which two mice were conjoined subcutaneously without any vascular anastomosis. When wild-type mice were joined with transgenic mice that expressed green fluorescent protein (GFP) in all tissues, GFP-positive cells were detected not only in the peripheral blood but also in the bone marrow of the wild-type mice. The femoral arteries of the wild-type mice were mechanically injured by insertion of a large wire. At 4 wk, there was neointima hyperplasia that mainly consisted of alpha-smooth muscle actin-positive cells. GFP-positive cells were readily detected in the neointima (14.8+/-4.5%) and media (31.1+/-8.8%) of the injured artery. Some GFP-positive cells expressed alpha-smooth muscle actin or an endothelial cell marker. These results indicate that circulating progenitors contribute to re-endothelialization and neointimal formation after mechanical vascular injury even in nonirradiated mice.

Jumping translocation breakpoint (JTB) is a gene located on human chromosome 1 at q21 that suffers an unbalanced translocation in various types of cancers, and potentially encodes a transmembrane protein of unknown function. The results of cancer profiling indicated that its expression was suppressed in many cancers from different organs, implying a role in the neoplastic transformation of cells. Recently, we isolated JTB as a TGF-beta1-inducible clone by differential screening. In this study, we characterized its product and biological functions. We found that it was processed at the N-terminus and located mostly in mitochondria. When expressed in cells, JTB-induced clustering of mitochondria around the nuclear periphery and swelling of each mitochondrion. In those mitochondria, membrane potential, as monitored with a JC-1 probe, was significantly reduced. Coinciding with these changes in mitochondria, JTB retarded the growth of the cells and conferred resistance to TGF-beta1-induced apoptosis. These activities were dependent on the N-terminal processing and induced by wild-type JTB but not by a mutant resistant to cleavage. These findings raised the possibility that aberration of JTB in structure or expression induced neoplastic changes in cells through dysfunction of mitochondria leading to deregulated cell growth and/or death.

Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacted with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affected the subcellular distribution of PINCH but directed its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments.

Hic-5 is a focal adhesion protein belonging to the paxillin LIM family that shuttles in and out of the nucleus. In the present study, we examined the expression of Hic-5 among mouse tissues by immunohistochemistry and found its expression only in smooth-muscle cells in several tissues. This result is consistent with a previous report on adult human tissues and contradicts the relatively ubiquitous expression of paxillin, the protein most homologous to Hic-5. One factor characterizing smooth-muscle cells in vivo is a continuous exposure to mechanical stretching in the organs. To study the involvement of Hic-5 in cellular responses to mechanical stress, we exposed mouse embryo fibroblasts to a uni-axial cyclic stretching and found that Hic-5 was relocalized from focal adhesions to stress fibers through its C-terminal LIM domains during the stress. In sharp contrast to this, paxillin did not change its focal-adhesion-based localization. Of the factors tested, which included interacting partners of Hic-5, only CRP2 (an only-LIM protein expressed in vascular smooth-muscle cells) and GIT1 were, like Hic-5, localized to stress fibers during the cyclic stretching. Interestingly, Hic-5 showed a suppressive effect on the contractile capability of cells embedded in three-dimensional collagen gels, and the effect was further augmented when CRP2 co-localized with Hic-5 to fiber structures of those cells. These results suggested that Hic-5 was a mediator of tensional force, translocating directly from focal adhesions to actin stress fibers upon mechanical stress and regulating the contractile capability of cells in the stress fibers.

The LIM protein Hic-5 is a focal adhesion protein shuttling in and out of the nucleus through the redox-sensitive nuclear export signal, and unlike other focal adhesion proteins including paxillin, the protein most homologous to Hic-5, it accumulates in the nucleus under oxidative conditions and participates in the transcription of c-fos and p21(Cip1) genes. Here, we examined the roles of the interacting partners of Hic-5, focal adhesion kinase (FAK) and protein tyrosine phosphatase PEST (PTP-PEST), in the nuclear translocation of Hic-5 and found that they were inhibitory. Interestingly, the interaction of Hic-5 with FAK was regulated by specific cysteines near the binding site and decreased in cells under oxidative conditions. Its interaction with PTP-PEST was also sensitive to the oxidant. These results suggest that the nuclear-cytoplasmic shuttling of Hic-5 is regulated by its interacting partners at focal adhesions or in the cytoplasm in a redox-sensitive manner, coordinating its role at focal adhesions with that in the nucleus, depending on the redox state of cells. Cytochalasin D or a phorbol ester also induced nuclear accumulation of Hic-5, which was inhibited by scavengers of reactive oxygen species (ROS), suggesting that besides oxidants, endogenously produced ROS induced the nuclear accumulation of Hic-5.

Hic-5 is a LIM protein with striking similarity to paxillin, and shuttles between focal adhesions and the nucleus. Our previous study suggested that Hic-5 participates in the transcriptional control of several genes such as the c-fos and p21 genes. In the present study, we examined the function of Hic-5 in the nucleus using the transcriptional promoter region of the p21 gene. When localized to the nucleus, Hic-5 was found to transactivate the p21 promoter through two of five Sp1 sites in the region proximal to the TATA box. The Hic-5 effect was mediated by a transactivation domain of Sp1 and functional interaction with p300 through the LIM4 domain. Hic-5 was also shown to interact functionally and physically with Smad3 through the LIM domains and to potentiate p21 promoter activity together with Smad3 and Sp1. These properties were confirmed in an artificial system using GAL4-fusion protein. Thus, Hic-5 was suggested to have a potential function as a cofactor in the transcriptional complex that contains Sp1, playing a role in gene transcription in the nucleus as well as in integrin signaling at focal adhesion sites., (Copyright 2004 Wiley-Liss, Inc.)

hic-5 was originally isolated as an H(2)O(2)-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H(2)O(2). Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H(2)O(2) treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

Hic-5 is a member of LIM family proteins with a striking similarity to paxillin and localizes primarily in the focal adhesion. We recently reported that Hic-5 translocated to the nucleus under oxidative stress and was involved in transcriptional regulation. In the present study, we extended these findings to show that transcription of c-fos gene was up-regulated by overexpression of Hic-5. In clonal stable transformants established from human immortalized fibroblasts by transfection of an expression vector of Hic-5, the constitutive level of c-fos mRNA was well correlated with that of Hic-5. In reporter assays using the luciferase gene under control of the human c-fos 5(')-upstream region from -2.2kb to +1, expression of Hic-5, that was engineered to accumulate in the nucleus, stimulated the transcriptional activity of the c-fos enhancer. From experiments using various deletions and point mutations, it was revealed that multiple sequences including GC/Sp1, Ets, and ERE/AP-1 elements found around the -1.3kb region were responsible for the activation by Hic-5. Hic-5 itself did not bind to these elements in a sequence specific manner, but p300 appeared to be involved in the induction of c-fos. These results suggest that Hic-5 participates in the transcriptional regulation of c-fos as a scaffold in transcriptional complexes.