Takuro Miyazaki

m-Calpain Induction in Vascular Endothelial Cells on Human and Mouse Atheromas and Its Roles in VE-Cadherin Disorganization and Atherosclerosis

Background— Although dysfunction of VE-cadherin–mediated adherence junctions in vascular endothelial cells (ECs) is thought to be one of the initial steps of atherosclerosis, little is known regarding how VE-cadherin is disrupted during atherogenic development. This study focused on the role of calpain, an intracellular cysteine protease, in the proteolytic disorganization of VE-cadherin and subsequent progression of atherosclerosis. Methods and Results— Increased expression of m-calpain was observed in aortic ECs in atherosclerotic lesions in humans and low-density lipoprotein receptor–deficient (ldlr−/−) mice. Furthermore, proteolytic disorganization of VE-cadherin was shown in aortic ECs in ldlr−/− and apolipoprotein E–deficient (apoE−/−) mice. Long-term administration of calpain inhibitors into these mice attenuated atherosclerotic lesion development and proinflammatory responses, as well as VE-cadherin disorganization, without normalization of plasma lipid profiles. Furthermore, in vivo transfection of m-calpain siRNA to ldlr−/− mice prevented disorganization of VE-cadherin and proatherogenic hyperpermeability in aortic ECs. Treatment of cultured ECs with oxidized LDL, lysophosphatidylcholine, or LDL pretreated with secreted phospholipase A2 led to the induction of m-calpain but not of &mgr;-calpain, thereby eliciting selective m-calpain overactivation. These data suggest that lysophosphatidylcholine-induced m-calpain directly cleaves a juxtamembrane region of VE-cadherin, resulting in dissociation of &bgr;-catenin from the VE-cadherin complex, disorganization of adherence junctions, and hyperpermeability in ECs. Conclusions— Subtype-selective induction of m-calpain in aortic ECs during atherosclerotic progression is associated with proteolytic disorganization of VE-cadherin and proatherogenic hyperpermeability in cells. Thus, a strategy to selectively inhibit m-calpain may be useful for the therapeutic treatment of patients with atherosclerosis.

Distinct Effects of Tissue-Type Plasminogen Activator and SMTP-7 on Cerebrovascular Inflammation Following Thrombolytic Reperfusion

Background and Purpose— Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. Methods— The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. Results— Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1&bgr;, TNF-&agr;, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 &mgr;g/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. Conclusions— Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

Salusin-β accelerates inflammatory responses in vascular endothelial cells via NF-κB signaling in LDL receptor-deficient mice in vivo and HUVECs in vitro.

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.

Dysregulation of Calpain Proteolytic Systems Underlies Degenerative Vascular Disorders

Chronic vascular diseases such as atherosclerosis, aneurysms, diabetic angiopathy/retinopathy as well as fibrotic and proliferative vascular diseases are generally complicated by the progression of degenerative insults, which are characterized by endothelial dysfunction, apoptotic/necrotic cell death in vascular/immune cells, remodeling of extracellular matrix or breakdown of elastic lamella. Increasing evidence suggests that dysfunctional calpain proteolytic systems and defective calpain protein metabolism in blood vessels contribute to degenerative disorders. In vascular endothelial cells, the overactivation of conventional calpains consisting of calpain-1 and -2 isozymes can lead to the disorganization of cell-cell junctions, dysfunction of nitric oxide synthase, sensitization of Janus kinase/signal transducer and activator of transcription cascades and depletion of prostaglandin I2, which contributes to degenerative disorders. In addition to endothelial cell dysfunctions, calpain overactivation results in inflammatory insults in macrophages and excessive fibrogenic/proliferative signaling in vascular smooth muscle cells. Moreover, calpain-6, a non-proteolytic unconventional calpain, is involved in the conversion of macrophages to a pro-atherogenic phenotype, leading to the pinocytotic deposition of low-density lipoprotein cholesterol in the cells. Here, we discuss the recent progress that has been made in our understanding of how calpain contributes to degenerative vascular disorders.

Calpastatin Counteracts Pathological Angiogenesis by Inhibiting Suppressor of Cytokine Signaling 3 Degradation in Vascular Endothelial Cells

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.

NADPH Oxidase Deficiency Exacerbates Angiotensin II–Induced Abdominal Aortic Aneurysms in Mice

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)-1&bgr; and matrix metalloproteinase-9/12 mRNA. Administration of neutralizing antibody against IL-1&bgr; 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-1&bgr; 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-1&bgr; expression in Nox2-deficient macrophages, whereas matrix metalloproteinase-9 secretion was constitutively stimulated via nuclear factor-&kgr;B signals. Conclusions—Nox2 deficiency enhances macrophage secretion of IL-1&bgr; 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.

m-Calpain antagonizes RhoA overactivation and endothelial barrier dysfunction under disturbed shear conditions

AIMS It has been reported that laminar shear flow (LF) improves barrier functions in vascular endothelial cells (ECs), whereas disturbed flow (DF) impairs the barrier. Our previous study showed that LF stimulus led to the activation of the cysteine protease, m-calpain, in ECs, which can influence RhoA activity. We hypothesized that m-calpain participates in the shear pattern-dependent EC barrier maintenance through RhoA signalling. METHODS AND RESULTS m-Calpain expression levels in the intima in the inferior aspect of mouse aortic arch where DF dominates were higher than those in adjacent regions. Elevation in transendothelial albumin permeability, which was induced by administration of a calpain inhibitor (ALLM), was prominent in the inferior arch; moreover, this elevation was abolished by Rho kinase (ROCK) inhibitor (Y-27632). Similarly, short interfering RNA (siRNA)-induced silencing of m-calpain resulted in increased RhoA activity and hyperpermeability in the aortic arch, which was accompanied by ROCK inhibitor-sensitive phosphorylation of downstream effecter LIM kinase 2 (LIMK2), stress fibre accumulation in endothelium and enhanced interendothelial gaps. Exposure of human umbilical vein endothelial cells to LF diminished RhoA activity; in contrast, DF facilitated the activity. siRNA-induced m-calpain silencing further accelerated the DF-induced RhoA overactivation, phosphorylation of LIMK2, and cytoskeletal rearrangement, resulting in barrier dysfunction in the cells. CONCLUSION Our findings revealed relatively high m-calpain expression levels in the inferior arch. The m-calpain activity antagonizes DF-induced overactivation of RhoA/ROCK/LIMK2 signalling and subsequent cytoskeletal rearrangement in ECs, which leads to barrier improvement.

Hic-5 deficiency attenuates the activation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice

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.

Identification of Hic-5 as a novel scaffold for the MKK4/p54 JNK pathway in the development of abdominal aortic aneurysms.

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.

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

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.