Asako Katsume

Pressure-Mediated Hypertrophy and Mechanical Stretch Induces IL-1 Release and Subsequent IGF-1 Generation to Maintain Compensative Hypertrophy by Affecting Akt and JNK Pathways

Rationale: It has been reported that interleukin (IL)-1 is associated with pathological cardiac remodeling and LV dilatation, whereas IL-1&bgr; has also been shown to induce cardiomyocyte hypertrophy. Thus, the role of IL-1 in the heart remains to be determined. Objective: We studied the role of hypertrophy signal-mediated IL-1&bgr;/insulin-like growth factor (IGF)-1 production in regulating the progression from compensative pressure-mediated hypertrophy to heart failure. Methods and Results: Pressure overload was performed by aortic banding in IL-1&bgr;–deficient mice. Primarily cultured cardiac fibroblasts (CFs) and cardiac myocytes (CMs) were exposed to cyclic stretch. Heart weight, myocyte size, and left ventricular ejection fraction were significantly lower in IL-1&bgr;–deficient mice (20%, 23% and 27%, respectively) than in the wild type 30 days after aortic banding, whereas interstitial fibrosis was markedly augmented. DNA microarray analysis revealed that IGF-1 mRNA level was markedly (≈50%) decreased in the IL-1&bgr;–deficient hypertrophied heart. Stretch of CFs, rather than CMs, abundantly induced the generation of IL-1&bgr; and IGF-1, whereas such IGF-1 induction was markedly decreased in IL-1&bgr;–deficient CFs. IL-1&bgr; released by stretch is at a low level unable to induce IL-6 but sufficient to stimulate IGF-1 production. Promoter analysis showed that stretch-mediated IL-1&bgr; activates JAK/STAT to transcriptionally regulate the IGF-1 gene. IL-1&bgr; deficiency markedly increased c-Jun N-terminal kinase (JNK) and caspase-3 activities and enhanced myocyte apoptosis and fibrosis, whereas replacement of IGF-1 or JNK inhibitor restored them. Conclusions: We demonstrate for the first time that pressure-mediated hypertrophy and mechanical stretch generates a subinflammatory low level of IL-1&bgr;, which constitutively causes IGF-1 production to maintain adaptable compensation hypertrophy and inhibit interstitial fibrosis.

Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function

Background— The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. Methods and Results— Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2−/− mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)–mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor–dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor–induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor–mediated Src association with PLC&ggr;1 and phosphorylation of 783Tyr-PLC&ggr;1 also were abolished by PYK2 deficiency. Conclusion— These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLC&ggr;1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase–mediated vasoactive function and angiogenic response.

Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit α-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.

Early Inflammatory Reactions in Atherosclerosis Are Induced by Proline-Rich Tyrosine Kinase/Reactive Oxygen Species–Mediated Release of Tumor Necrosis Factor-α and Subsequent Activation of the p21Cip1/Ets-1/p300 System

Objective—Reactive oxygen species (ROS) are involved in the initial process of atherosclerosis, whereas it remains to be determined how atherogenic stimulus causes ROS-mediated proinflammatory reactions. Here, we focused on proline-rich tyrosine kinase (PYK2)–mediated ROS generation and examined how atherogenic stimulus causes early proinflammatory reactions. Methods and Results—PYK2-deficient (knockout [KO]) (PYK2-KO) mice were crossbred with apolipoprotein E (ApoE)–deficient (PYK2-KO/ApoE-KO) mice. PYK2-KO/ApoE-KO mice and endothelial cells (EC) were used for the study. Aortic atherogenic lesions in PYK2-KO/ApoE-KO mice were markedly decreased (55% versus ApoE-KO) after 8 weeks of a Western diet. Aortic PYK2 was activated as early as 7 days after the Western diet, when inflammatory cells were not yet activated. Addition of the proatherogenic oxidized phospholipid lysophosphatidylcholine caused activation of endothelial PYK2. Lysophosphatidylcholine-activated PYK2 induced NADPH oxidase–mediated ROS generation and ROS-mediated synthesis of tumor necrosis factor-&agr; (TNF&agr;), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and p21Cip1/Ets-1. Neutralizing anti-TNF&agr; antibody or knockdown of p21Cip1/Ets-1 system blocked the induction of VCAM-1 and MCP-1. PYK2 deficiency abolished these ROS-mediated proinflammatory reactions. Further analysis revealed that PYK2/ROS-mediated p21Cip1/Ets-1 activation upregulated the transcription of the MCP-1 gene in collaboration with p300 transcription coactivator. Conclusion—PYK2 is a key tyrosine kinase activated by high cholesterol exposure, which causes ROS-mediated TNF&agr; release and induces TNF&agr;-dependent expression of proinflammatory molecules via the p21Cip1/Ets-1/p300 transcription system.

Endothelial FGF receptor signaling accelerates atherosclerosis

Members of the fibroblast growth factor (FGF) family have been clinically applied to the treatment of ischemic diseases because of their strong angiogenic actions. Although tissue ischemia is predominantly caused by atherosclerosis, the roles of endothelial FGF receptors (FGF-Rs) in atherosclerosis remain obscure. We generated endothelial cell (EC)-targeted constitutively active FGF-R2-overexpressing mice, using the Tie2 promoter (Tie2-FGF-R2-Tg), and crossed them with apolipoprotein E (ApoE)-deficient mice (ApoE-KO) to generate Tie2-FGF-R2-Tg/ApoE-deficient mice (Tie2-FGF-R2-Tg/ApoE-KO). After being fed a Western diet for 8 wk, the Tie2-FGF-R2-Tg/ApoE-KO demonstrated 2.0-fold greater atherosclerotic lesion area on the luminal surfaces of the aortas than the ApoE-KO (P < 0.01). The level of p21(Cip1) protein, a cell cycle inhibitor, in the FGF-R2-overexpressing EC was 2.5-fold greater than that in the wild-type (WT) EC at the baseline (P < 0.01). FGF-R2 overexpression in the EC resulted in increased expression of VCAM-1 and ICAM-1, acceleration of apoptosis, and decreased proliferative activity, all of which were normalized by small interfering RNA (siRNA)-mediated knockdown of p21(Cip1) (75% reduction in protein level, P < 0.01). Furthermore, the expression of PDGF-B and Egr-1, a PDGF/p21(Cip1)-inducible transcription factor, in the aortic endothelium of Tie2-FGF-R2-Tg/ApoE-KO was significantly greater than that in ApoE-KO. The proliferation of vascular smooth muscle cells in the aortic media of Tie2-FGF-R2-Tg/ApoE-KO was 2.0-fold higher than that in ApoE-KO (P < 0.01). Thus our study reveals that endothelial FGF-R2 signaling aggravates atherosclerosis by promoting p21(Cip1)-mediated EC dysfunction and cautions against the use of FGF for therapeutic angiogenesis in the setting of atherosclerosis.

Placement of pacemaker leads via the extrathoracic subclavian vein guided by fluoroscopy and venography in the oblique projection

Blind needle puncture of the subclavian vein, which is the standard method used for insertion of pancemaker electrodes, causes an increased risk of lead fractures due to entrapment of the lead by the costoclavicular ligament and/or subclavius muscle. The extrathoracic lead insertion technique was developed to prevent such lead fractures. The present study was performed to evaluate the usefulness of extrathoracic subclavian vein puncture under the guidance of both fluoroscopy and venography in the oblique beam projection. Pacemaker leads were implanted in ten patients under the guidance of both fluoroscopy and venography in the ipsilateral anterior oblique projection. The angle of projection was set as large as possible between 35° and 45°. The needle was held parallel to the X-ray angle of incidence and inserted toward the first rib, then withdrawn until the tip entered the subclavian vein. This modified method of pacemaker implantation was successful in all patients, with no complications during the follow-up period ranging from 4 to 19 months. It also prevented pneumothorax and lead entrapment in soft tissue associated with the clavicle that might be caused by the conventional technique.