Xiao-Feng Lei

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.

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A(-/-)) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β were significantly increased in SR-A(-/-) mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A(-/-) macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A(-/-) macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.

Delayed growth of EL4 lymphoma in SR-A-deficient mice is due to upregulation of nitric oxide and interferon-γ production by tumor-associated macrophages

Class A scavenger receptors (SR‐A, CD204) are highly expressed in tumor‐associated macrophages (TAM). To investigate the function of SR‐A in TAM, wild‐type and SR‐A‐deficient (SR‐A−/−) mice were injected with EL4 cells. Although these groups of mice did not differ in the numbers of infiltrating macrophages and lymphocytes and in neovascularization, SR‐A−/− mice had delayed growth of EL4 tumors. Expression of inducible nitric oxide (NO) synthase and interferon (IFN)‐γ mRNA increased significantly in tumor tissues from SR‐A−/− mice. Engulfment of necrotic EL4 cells induced upregulation of NO and IFN‐γ production by cultured macrophages, and production of NO and IFN‐γ increased in SR‐A−/− macrophages in vitro. IFN‐β production by cultured macrophages was also elevated in SR‐A−/− macrophages in vitro. These results suggested that the antitumor activity of macrophages increased in SR‐A−/− mice because of upregulation of NO and IFN‐γ production. These data indicate an important role of SR‐A in regulating TAM function by inhibiting toll‐like receptor (TLR)4–IFN‐β signaling. (Cancer Sci 2009); 00: 000–000)

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.

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.

Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein.

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.

Identification of Hic-5 as a novel regulatory factor for integrin αIIbβ3 activation and platelet aggregation in mice

Summary.  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.