PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

Inhibition of Urban Particulate Matter-Induced Airway Inflammation by RIPK3 through the Regulation of Tight Junction Protein Production.

Urban particulate matter (UPM) is a high-hazard cause of various diseases in humans, including in the respiratory tract, skin, heart, and even brain. Unfortunately, there is no established treatment for the damage caused by UPM in the respiratory epithelium. In addition, although RIPK3 is known to induce necroptosis, its intracellular role as a negative regulator in human lungs and bronchial epithelia remains unclear. Here, the endogenous expression of RIPK3 was significantly decreased 6 h after exposure to UPM. In RIPK3-ovexpressed cells, RIPK3 was not moved to the cytoplasm from the nucleus. Interestingly, the overexpression of RIPK3 dramatically decreased TEER and F-actin formation. Its overexpression also decreased the expression of genes for pro-inflammatory cytokines (IL-6 and IL-8) and tight junctions (ZO-1, -2, -3, E-cadherin, and claudin) during UPM-induced airway inflammation. Importantly, overexpression of RIPK3 inhibited the UPM-induced ROS production by inhibiting the activation of iNOS and eNOS and by regulating mitochondrial fission processing. In addition, UPM-induced activation of the iκB and NF-κB signaling pathways was dramatically decreased by RIPK3, and the expression of pro-inflammatory cytokines was decreased by inhibiting the iκB signaling pathway. Our data indicated that RIPK3 is essential for the UPM-induced inflammatory microenvironment to maintain homeostasis. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for UPM-induced pulmonary inflammation.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCß3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Resistin impairs mitochondrial homeostasis via cyclase-associated protein 1-mediated fission, leading to obesity-induced metabolic diseases.

OBJECTIVE: One of the suggested mechanisms of obesity-induced insulin resistance is mitochondrial dysfunction in target tissues such as skeletal muscle. In our study, we examined whether resistin, an adipokine associated with obesity-mediated insulin resistance, induced metabolic disorders by impairing mitochondrial homeostasis. METHODS: The morphology and function of mitochondria of skeletal muscle were examined in resistin-knockout and humanized resistin mice that were subjected to high-fat diet for 3 months. Morphology was examined by transmission electron microscopy. Mitochondria bioenergetics of skeletal muscle were evaluated using a Seahorse XF96 analyzer. Human skeletal myoblasts were used for in vitro studies on signaling mechanisms in responses to resistin. RESULTS: A high-fat diet in humanized resistin mice increased fragmented and shorter mitochondria in the skeletal muscle, whereas resistin-knockout mice had healthy normal mitochondria. In vitro studies showed that human resistin treatment impaired mitochondrial homeostasis by inducing mitochondrial fission, leading to a decrease in ATP production and mitochondrial dysfunction. Induction of mitochondrial fission by resistin was accompanied by increased formation of mitochondria-associated ER membranes (MAM). At the same time, resistin induced up-regulation of the protein kinase A (PKA) pathway. This activation of PKA induced phosphorylation of Drp1 at serine 616, leading to Drp1 activation and subsequent induction of mitochondrial fission. The key molecule that mediated human resistin-induced mitochondrial fission was adenylyl cyclase-associated protein 1 (CAP1), which was reported as a bona fide receptor for human resistin. Moreover, our newly developed biomimetic selective blocking peptide could repress human resistin-mediated mitochondrial dysfunction. High-fat diet-fed mice showed lower exercise capacity and higher insulin resistance, which was prevented by a novel peptide to block the binding of resistin to CAP1 or in the CAP1-knockdown mice. CONCLUSIONS: Our study demonstrated that human resistin induces mitochondrial dysfunction by inducing abnormal mitochondrial fission. This result suggests that the resistin-CAP1 complex could be a potential therapeutic target for the treatment of obesity-related metabolic diseases such as diabetes and cardiometabolic diseases.

Sodium/glucose Co-Transporter 2 Inhibitor, Empagliflozin, Alleviated Transient Expression of SGLT2 after Myocardial Infarction.

BACKGROUND AND OBJECTIVES: Large clinical studies of sodium/glucose cotransporter 2 (SGLT2) inhibitors have shown a significant beneficial effect on heart failure-associated hospitalization and cardiovascular events. As SGLT2 is known to be absent in heart cells, improved cardiovascular outcomes are thought to be accounted for by the indirect effects of the drug. We sought to confirm whether such benefits were mediated through SGLT2 expressed in the heart using myocardial infarction (MI) model. METHODS: Mice pre-treated with empagliflozin (EMPA), an SGLT2 inhibitor, showed a significantly reduced infarct size compared with the vehicle group three days post-MI. Interestingly, we confirmed SGLT2 localized in the infarct zone. The sequential changes of SGLT2 expression after MI were also evaluated. RESULTS: One day after MI, SGLT2 transiently appeared in the ischemic areas in the vehicle group and increased until 72 hours. The appearance of SGLT2 was delayed and less in amount compared with the vehicle group. Additionally, there was a significant difference in metabolites, including glucose and amino acids in the ¹H nuclear magnetic resonance analysis between groups. CONCLUSIONS: Our work demonstrates that SGLT2 is transiently expressed in heart tissue early after MI and EMPA may directly operate on SGLT2 to facilitate metabolic substrates shifts.

Cyclase-associated protein 1 is a binding partner of proprotein convertase subtilisin/kexin type-9 and is required for the degradation of low-density lipoprotein receptors by proprotein convertase subtilisin/kexin type-9.

AIMS: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. METHODS AND RESULTS: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. CONCLUSION: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.

COMP-Angiopoietin-1 accelerates muscle regeneration through N-cadherin activation.

Angiopoietin-1 modulates vascular stability via Tie2 on endothelial cells. In our previous study, we also showed it acts as an inhibitor of cardiomyocyte death. However, it remains poorly understood how Ang1 regulates myogenesis during muscle regeneration. Here we found that COMP-Ang1 (cAng1) enhances muscle regeneration through N-cadherin activation. Muscle fiber regeneration after limb muscle damage by ischemic injury was enhanced with cAng1 treatment. Mechanistically cAng1 directly bound to N-cadherin on the myoblast surface in a Ca2+ dependent manner. The interaction enhanced N-cadherin activation via N-cadherin/p120-catenin complex formation, which in turn activated p38MAPK (but not AKT or ERK) and myogenin expression (but not myoD) as well as increasing myogenin+ cells in/ex vivo. After transplantation of GFP-expressing myoblasts (GFP-MB), we showed an increased generation of GFP+ myotubes with adenovirus cAng1 (Adv-cAng1) injection. Adv-cAng1, however, could not stimulate myotube formation in N-cadherin-depleted GFP-MB. Taken together, this study uncovers the mechanism of how cAng1 promotes myoblast differentiation and muscle regeneration through the N-cadherin/p120-catenin/p38MAPK/myogenin axis.

Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.

Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

Magnetic bionanoparticle enhances homing of endothelial progenitor cells in mouse hindlimb ischemia.

BACKGROUND AND OBJECTIVES: Poor homing efficiency is one of the major limitations of current stem cell therapy. Magnetic bionanoparticles (MPs) obtained from Magnetospirillum sp. AMB-1 have a lipid bilayer membrane and ferromagnetic properties. We evaluated a novel priming strategy using MPs to enhance the homing of transplanted progenitor cells to target tissue. MATERIALS AND METHODS: Effects of MP on proliferation, viability, and migration of late human endothelial progenitor cells (EPCs) were examined in vitro. Additionally, effects of MP on gene and protein expression related to survival and adhesion were evaluated. Homing and angiogenic efficiency of MP transferred late EPCs was evaluated in nude mouse hindlimb ischemia model. RESULTS: Below threshold concentration, MP transfer did not influence proliferation or survival of late EPCs, but enhanced migration and trans-endothelial migration of late EPCs toward magnet. Below threshold concentration, MP transfer did not influence gene and protein expression related to survival. In the mouse hindlimb ischemia model, late EPCs treated with high dose MP (5 ug/mL) showed enhanced homing of injected late EPCs in the ischemic limb by magnet, compared to low dose MP (1 ug/mL) treated late EPCs. In addition, high dose MP transferred EPC showed significantly better improvement of perfusion in ischemic limb compared to untreated EPC. CONCLUSION: MP transfer with magnet application can be a promising novel strategy to enhance homing efficacy and outcomes of current stem cell therapy.

Peroxisome proliferator-activated receptor-gamma agonists suppress tissue factor overexpression in rat balloon injury model with paclitaxel infusion.

The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF), a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK), which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1), was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI) in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group) with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001) in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

Adipokine resistin is a key player to modulate monocytes, endothelial cells, and smooth muscle cells, leading to progression of atherosclerosis in rabbit carotid artery.

OBJECTIVES: We investigated the effects of human resistin on atherosclerotic progression and clarified its underlying mechanisms. BACKGROUND: Resistin is an adipokine first identified as a mediator of insulin resistance in murine obesity models. But, its role in human pathology is under debate. Although a few recent studies suggested the relationship between resistin and atherosclerosis in humans, the causal relationship and underlying mechanism have not been clarified. METHODS: We cloned rabbit resistin, which showed 78% identity to human resistin at the complementary deoxyribonucleic acid level, and its expression was examined in 3 different atherosclerotic rabbit models. To evaluate direct role of resistin on atherosclerosis, collared rabbit carotid arteries were used. Histological and cell biologic analyses were performed. RESULTS: Rabbit resistin was expressed by macrophages of the plaque in the 3 different atherosclerotic models. Peri-adventitial resistin gene transfer induced macrophage infiltration and expression of various inflammatory cytokines, resulting in the acceleration of plaque growth and destabilization. In vitro experiments elucidated that resistin increased monocyte-endothelial cell adhesion by upregulating very late antigen-4 on monocytes and their counterpart vascular cell adhesion molecule-1 on endothelial cells. Resistin augmented monocyte infiltration in collagen by direct chemoattractive effect as well as by enhancing migration toward monocyte chemotactic protein-1. Administration of connecting segment-1 peptide, which blocks very late antigen-4 × vascular cell adhesion molecule-1 interaction, ameliorated neointimal growth induced by resistin in vivo. CONCLUSIONS: Our results indicate that resistin aggravates atherosclerosis by stimulating monocytes, endothelial cells, and vascular smooth muscle cells to induce vascular inflammation. These findings provide the first insight on the causal relationship between resistin and atherosclerosis.

Oscillating pressure treatment upregulates connexin43 expression in skeletal myoblasts and enhances therapeutic efficacy for myocardial infarction.

Transplantation of autologous skeletal myoblasts (SMBs) is a potential therapeutic approach for myocardial infarction. However, their clinical efficacy and safety is still controversial. Electrical coupling through gap junction between SMBs and host myocardium is essential for synchronized contraction and electrical stability. Here, we investigated the effect of heart beat-simulating environment, oscillating pressure, on the expression of connexin43 in two types of SMBs from rat and mouse. We found that connexin43 is markedly decreased under ischemia-mimicking conditions such as serum starvation and hypoxia (1% O(2)) in rat primary cultured SMBs and mouse C2C12 SMB cell line. Interestingly, the decrease of connexin43 expression under serum starvation was attenuated by oscillating pressure. Oscillating pressure treatment increased the expression of connexin43 twofold through AP-1 stimulation, which was blocked by PD98059, ERK inhibitor. In coculture of cardiomyocytes and C2C12, pressure-treated C2C12 and cardiomyocytes were able to form functional gap junction, which was demonstrated by both calcein-AM dye transfer assay and measurement of simultaneous contraction. In rat myocardial infarction model, transplantation of SMBs pretreated with oscillating pressure resulted in lesser ventricular dilatation and better systolic function than transplantation of untreated SMBs and control group. These results suggested that application of oscillating pressure on SMBs before transplantation may be useful to promote therapeutic efficacy for myocardial infarction by enhancing gap junction formation between transplanted and host cells.

G-CSF exerts dual effects on endothelial cells--opposing actions of direct eNOS induction versus indirect CRP elevation.

Granulocyte-colony stimulating factor (G-CSF) has been shown to have protective effects in the heart and brain. However, it may also be involved in the acute inflammatory response which may be harmful. The effects of G-CSF on endothelial cells (ECs) and the vasculature are mostly unknown. To study the possible dual effects of G-CSF on ECs, we investigated whether G-CSF induces release of C-reactive protein (CRP) by hepatocytes and whether the direct beneficial effects of G-CSF could protect ECs from the detrimental effects of CRP. G-CSF treatment significantly induced monocytes to produce IL-6, and culture supernatants of G-CSF-stimulated monocytes induced CRP production in hepatocytes. On the other hand, G-CSF directly promoted EC proliferation and migration and reversed the deleterious effects of CRP. In mechanistic analyses, G-CSF increased not only the protein expression of endothelial nitric oxide synthase (eNOS), but also its transcription. Furthermore, it enhanced eNOS phosphorylation and activation, leading to increased production of NO. Thus, G-CSF reversed the attenuated production of NO by CRP. These effects of G-CSF on eNOS transcription, translation, and activation were blunted by the PI3K inhibitor, suggesting that EC protective effects of G-CSF were associated with the activation of the Akt/eNOS pathway. In conclusion, although G-CSF induces an inflammatory reaction leading to CRP production, it has direct beneficial effects protecting ECs from the deleterious effects of CRP through activation of Akt/eNOS pathway, leading to an increase in NO production. Our data suggests that G-CSF may exert dual opposing effects on endothelial cells.

Identification and characterization of arginase II as a chondrocyte phenotype-specific gene.

We have previously shown that activation of extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) causes chondrocyte dedifferentiation, which contributes to the destruction of arthritic cartilage. In the present study, we identified genes involved in the ERK1/2 regulation of chondrocyte dedifferentiation. Several genes were identified by subtractive hybridization, and, of these, arginase II was selected for further functional characterization. Similar to the pattern of type II collagen expression, which is a hallmark of chondrocyte differentiation, arginase II expression was increased during chondrogenesis of mesenchymal cells. The high expression level of arginase II was decreased during dedifferentiation of chondrocytes, whereas its expression was restored during redifferentiation of the dedifferentiated chondrocytes. Inhibition of ERK1/2 signaling in chondrocytes enhanced type II collagen expression with a concomitant increase in expression and activity of arginase II. However, ectopic expression of arginase II or inhibition of its activity did not affect chondrocyte differentiation. The results collectively indicate that expression of arginase II is specific to the chondrocyte phenotype, although the expression of arginase II alone is not sufficient for articular chondrocytes to maintain a differentiated phenotype.

Exacerbation of Avellino corneal dystrophy after laser in situ keratomileusis.

PURPOSE: To report a case of Avellino corneal dystrophy that increased in severity 1 year after uncomplicated laser in situ keratomileusis (LASIK) for myopia. METHODS: Avellino dystrophy was confirmed by polymerase chain reaction sequencing of DNA from the patient and her parents. RESULTS: Best spectacle-corrected visual acuity decreased from 20/20 to 20/30 12 to 20 months after LASIK owing to opacities that appeared centrally in the corneal stroma and the LASIK flap and remaining posterior stroma interface. CONCLUSIONS: LASIK is contraindicated in patients with Avellino corneal dystrophy because vision may be reduced by corneal opacities that appear in the interface of the flap and remaining posterior stroma postoperatively.