Nature Medicine 11,1173 - 1179 (2005)

Regulation of lung injury and repair by Toll-like receptors and hyaluronan

僕の実験でも当たり前のことしかデータに出ていないが
Nature Medicineにでるということはすごい。
Alert cell starategyを早くまとめたいものです。

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88-/- and Tlr4-/-Tlr2-/- mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell–specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell–surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-B. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.

J. Clin. Invest. 115:3527-3535 (2005).

The mitochondrial origin of postischemic arrhythmias
Fadi G. Akar, Miguel A. Aon, Gordon F. Tomaselli and Brian O’Rourke
Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Brian O’Rourke, Division of Cardiology, Johns Hopkins University, 720 Rutland Avenue, Ross 844, Baltimore, Maryland 21205, USA. Phone: (410) 614-0034; Fax: (410) 955-7953; E-mail: bor@jhmi.edu.

Recovery of the mitochondrial inner membrane potential (m) is a key determinant of postischemic functional recovery of the heart. Mitochondrial ROS-induced ROS release causes the collapse of m and the destabilization of the action potential (AP) through a mechanism involving a mitochondrial inner membrane anion channel (IMAC) modulated by the mitochondrial benzodiazepine receptor (mBzR). Here, we test the hypothesis that this mechanism contributes to spatiotemporal heterogeneity of m during ischemia-reperfusion (IR), thereby promoting abnormal electrical activation and arrhythmias in the whole heart. High-resolution optical AP mapping was performed in perfused guinea pig hearts subjected to 30 minutes of global ischemia followed by reperfusion. Typical electrophysiological responses, including progressive AP shortening followed by membrane inexcitablity in ischemia and ventricular fibrillation upon reperfusion, were observed in control hearts. These responses were reduced or eliminated by treatment with the mBzR antagonist 4'-chlorodiazepam (4'-Cl-DZP), which blocks depolarization of m. When applied throughout the IR protocol, 4'-Cl-DZP blunted AP shortening and prevented reperfusion arrhythmias. Inhibition of ventricular fibrillation was also achieved by bolus infusion of 4'-Cl-DZP just before reperfusion. Conversely, treatment with an agonist of the mBzR that promotes m depolarization exacerbated IR-induced electrophysiological changes and failed to prevent arrhythmias. The effects of these compounds were consistent with their actions on IMAC and m. These findings directly link instability of m to the heterogeneous electrophysiological substrate of the postischemic heart and highlight the mitochondrial membrane as a new therapeutic target for arrhythmia prevention in ischemic heart disease.

J. Clin. Invest. 115:3378-3384, 2005

Platelets in inflammation and atherogenesis
Meinrad Gawaz, Harald Langer and Andreas E. May

Meinrad Gawaz, Medizinische Klinik III, Eberhard Karls Universität Tübingen, Otfried-Müller-Straße 10, D-72076 Tübingen, Germany. Phone: 49-7071-29-83688; Fax: 49-7071-29-5749; E-mail: meinrad.gawaz@med.uni-tuebingen.de.


Platelet-derived mediators stimulate inflammation
During the adhesion process, platelets become activated and release an arsenal of potent inflammatory and mitogenic substances into the local microenvironment, thereby altering chemotactic, adhesive, and proteolytic properties of ECs (24). These platelet-induced alterations of the endothelial phenotype support chemotaxis, adhesion, and transmigration of monocytes to the site of inflammation (Figure 2).
Released from dense granules, -granules, lysosomes, the canalicular system, or the cytosol, platelets secrete or expose adhesion proteins (e.g., fibrinogen, fibronectin, vWF, thrombospondin, vitronectin, P-selectin, GPIIb/IIIa), growth factors (e.g., PDGF, TGF-ß, EGF, bFGF), chemokines (e.g., RANTES, platelet factor 4 [CXC chemokine ligand 4], epithelial neutrophil-activating protein 78 [CXC chemokine ligand 5]), cytokine-like factors (e.g., IL-1ß, CD40 ligand, ß-thromboglobulin), and coagulation factors (e.g., factor V, factor XI, PAI-1, plasminogen, protein S). These proteins act in a concerted and finely regulated manner to influence widely differing biological functions such as cell adhesion, cell aggregation, chemotaxis, cell survival and proliferation, coagulation, and proteolysis, all of which accelerate inflammatory processes and cell recruitment. For example, IL-1ß has been identified as a major mediator of platelet-induced activation of ECs (25, 26). The IL-1ß activity expressed by platelets appears to be associated with the platelet surface, and coincubation of ECs with thrombin-activated platelets induces IL-1ß–dependent secretion of IL-6 and IL-8 from ECs (26). Furthermore, incubation of cultured ECs with thrombin-stimulated platelets significantly enhances the secretion of endothelial monocyte chemoattractant protein-1 (MCP-1) in an IL-1ß–dependent manner (12). MCP-1 belongs to the CC family of chemokines and is thought to play a key role in the regulation of monocyte recruitment to inflamed tissue and in atherosclerosis (27, 28).

However, platelet IL-1ß does not only modify endothelial release of chemotactic proteins. IL-1ß additionally can increase endothelial expression of adhesion molecules. Surface expression of ICAM-1 and vß3 on ECs is significantly enhanced by activated platelets via IL-1ß (12). Both enhanced chemokine release and upregulation of endothelial adhesion molecules through platelet-derived IL-1ß act in concert and promote neutrophil and monocyte adhesion to the endothelium. IL-1ß–dependent expression of early inflammatory genes, such as MCP-1 or ICAM-1, involves the activation of the transcription factor NF-B. Transient adhesion of platelets to the endothelium initiates degradation of IB and supports activation of NF-B in ECs, thereby inducing NF-B–dependent chemokine gene transcription (29, 30). Likewise, platelet-induced NF-B activation was largely reduced by IL-1ß antagonists, which supports the notion that platelet IL-1ß is the molecular determinant of platelet-dependent activation of the transcription factor. Activation of NF-B involves a cascade of phosphorylation processes. One family of kinases that is involved in NF-B–dependent gene expression is the MAPKs, such as p38 MAPK. In a manner similar to that of recombinant human IL-1ß, activated platelets have the potential to induce phosphorylation of p38 MAPK. Correspondingly, transfection of a dominant-negative p38 mutant significantly reduced platelet-induced MCP-1 secretion in ECs (31).

Once recruited to the vascular wall, platelets may promote inflammation by chemoattraction of leukocytes through mediators such as platelet-activating factor and macrophage inflammatory protein-1, may stimulate smooth muscle cell proliferation (TGF-ß, PDGF, serotonin) (32), and may contribute to matrix degradation by secretion of MMP-2 (33).

A finely regulated functional interaction of platelets with chemokines has also been implicated in atherogenesis (34). Activated platelets can release chemokines and can induce the secretion of chemokines in various cells of the vascular wall; in turn, certain chemokines can enhance platelet aggregation and adhesion in combination with primary agonists and can trigger monocyte recruitment (35). One such candidate for monocyte recruitment is RANTES, which has been shown to trigger monocyte arrest on inflamed and atherosclerotic endothelium (35). Deposition of platelet-derived RANTES induces monocyte recruitment mediated by P-selectin (36, 37). Another platelet-derived chemokine is platelet factor 4 (PF4), the most abundant protein secreted by activated platelets. First, PF4 acts as a chemoattractant for monocytes promoting their differentiation into macrophages (38). Second, PF4 may directly aggravate the atherogenic actions of hypercholesterolemia by promoting the retention of lipoproteins. Sachais and colleagues have recently shown that PF4 can facilitate the retention of LDL on cell surfaces by inhibition of its degradation by the LDL receptor (39). In addition, PF4 markedly enhances the esterification and uptake of oxidized LDL by macrophages (40). The fact that PF4 has been found in human atherosclerotic lesions and was found associated with macrophages in early lesions and with foam cells in more advanced lesions (41) supports the concept that PF4 released from locally activated platelets enters the vessel wall and promotes vascular inflammation and atherogenesis.

Furthermore, release of platelet-derived CD40 ligand (CD40L, CD154) induces inflammatory responses in endothelium. Henn et al. (42) showed that platelets store CD40L in high amounts and release it within seconds after activation in vitro. Ligation of CD40 on ECs by CD40L expressed on the surface of activated platelets increased the release of IL-8 and MCP-1, the principal chemoattractants for neutrophils and monocytes (42). In addition, platelet CD40L enhanced the expression of endothelial adhesion receptors including E-selectin, VCAM-1, and ICAM-1, all molecules that mediate the attachment of neutrophils, monocytes, and lymphocytes to the inflamed vessel wall (42). Moreover, CD40L induces endothelial tissue factor expression (43). Hence, like IL-1ß, CD40L expressed on platelets induces ECs to release chemokines and to express adhesion molecules, thereby generating signals for the recruitment of leukocytes in the process of inflammation. CD40 ligation on ECs, smooth muscle cells, and macrophages initiates the expression and release of matrix-degrading enzymes, the MMPs. These enzymes, which degrade ECM proteins, significantly contribute to destruction and remodeling of inflamed tissue. Activated platelets release MMP-2 during aggregation (33, 44). Furthermore, adhesion of activated platelets to ECs results in generation and secretion of MMP-9 and of the protease receptor urokinase-type plasminogen activator receptor (uPAR) on cultured endothelium (45). The endothelial release of MMP-9 is dependent on both the fibrinogen receptor GPIIb/IIIa and CD40L, since inhibition of either mechanism resulted in reduction of platelet-induced matrix degradation activity of ECs. Moreover, GPIIb/IIIa ligation results in substantial release of CD40L in the absence of any further platelet agonist (45, 46) (Figure 2). These results suggest that the release of platelet-derived proinflammatory mediators like CD40L is dependent on GPIIb/IIIa–mediated adhesion. This mechanism may be pathophysiologically important to localize platelet-induced inflammation of the endothelium at a site of firm platelet-endothelium adhesion.

L型カルシウムチャネル J. Clin. Invest. 115:3306-3317, 2005

The L-type calcium channel in the heart: the beat goes on
Ilona Bodi, Gabor Mikala, Sheryl E. Koch, Shahab A. Akhter, and Arnold Schwartz

Sydney Ringer would be overwhelmed today by the implications of his simple experiment performed over 120 years ago showing that the heart would not beat in the absence of Ca2+. Fascination with the role of Ca2+ has proliferated into all aspects of our understanding of normal cardiac function and the progression of heart disease, including induction of cardiac hypertrophy, heart failure, and sudden death. This review examines the role of Ca2+ and the L-type voltage-dependent Ca2+ channels in cardiac disease.

When Sydney Ringer (1) discovered the vital role of Ca2+ in the heart, investigations took a leap forward and have continued unabated (2). Austrian scientist Otto Loewi, best known for his work on autonomic transmitters and discovery of "chemical vagusstoff," recognized the connection between digitalis and Ca2+ in 1917–1918. Although he always believed that Ca2+ was the key to understanding life’s processes, the Nobel Prize in Physiology and Medicine was awarded to Loewi and Sir Henry Hallett Dale in 1936 for their studies on neurotransmitters.

Ca2+ is the link in excitation-contraction (EC) coupling (Figure 1), which starts during the upstroke of the action potential (AP) and causes the opening of the L-type voltage-dependent Ca2+ channel (L-VDCC). Interest in high-voltage–activated L-VDCCs began with biochemical and continued with molecular characterizations, culminating in the cloning of the pore-forming 1 subunit and the auxiliary channel subunit 2/ from rabbit skeletal muscle (3-5). Although the L-VDCC subunits are most abundant in fast skeletal transverse tubules, Ca2+ influx is not required for contraction in skeletal muscle, unlike cardiac muscle, which requires Ca2+ entry with each beat and triggers Ca2+ release from the sarcoplasmic reticulum (SR) via Ca2+-release channels, e.g., ryanodine receptor 2 (RyR2). This amplifying process, termed Ca2+-induced Ca2+ release (CICR) by A. Fabiato, causes a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) (from 100 nM to 1 µM) to a level required for optimal binding of Ca2+ to troponin C and induction of contraction (2). There is a close correlation between activation of the L-type Ca2+ current (ICa,L) and cardiac contraction. Contraction is followed by Ca2+ release from troponin C and its reuptake by the SR via activation of the SR Ca2+-ATPase 2a (SERCA2a) Ca2+ pump in addition to extrusion across the sarcolemma via the Na+/Ca2+ exchanger (NCX). In the human heart under resting conditions, the time required for cardiac myocyte depolarization, Ca2+-induced Ca2+ release, contraction, relaxation, and recovery is 600 ms. This process occurs approximately 70 times a minute or over 2 billion times in the average lifespan. Ca2+ is also required for maintenance of cell integrity and gene expression (6) relevant to the growth and development of the embryonic heart (7). L-VDCCs are regulated by the adrenergic nervous system and may interact with G protein–coupled receptors (8).



Model for CDI and VDI. (A) Ca2+ channel at rest when no Ca2+ influx occurs. At rest, in the absence of Ca2+, the CaM binds to peptide A, located between the EF hand and the IQ motif of the C terminus of the L-VDCC 1C subunit. In response to a depolarizing stimulus, Ca2+ enters through the L-VDCC and binds to CaM. In the open Ca2+ channel state, the EF hand prevents structural conformation of the I–II loop required to block Ca2+ entry through the channel pore (B). In addition, the hydrophobic I1654 in the IQ motif is a stabilizing factor preventing the occlusion of the pore. Upon elevation of [Ca2+]i (depolarization), the Ca2+/CaM complex undergoes the Ca2+-dependent conformational change that relieves the inhibition of EF hand, permitting the I–II loop to interact with the pore and accelerate the fast inactivation process (C). The graph shows representative ICa traces evoked by depolarization from –50 mV to +40 mV, as labeled, using –60 mV as holding potential. (D) Involvement of CaM and CaMKII in the facilitation process. CaMKII enhances the ICa through phosphorylation of L-VDCC. We show murine whole-cell ICa generated from paired depolarizing pulses (–60 mV ± 10 mV at 0.5 Hz) representing Ca2+-dependent facilitation (graph).