Takashi Sonobe

Intracellular calcium accumulation following eccentric contractions in rat skeletal muscle in vivo: role of stretch-activated channels

Although the accumulation of intracellular calcium ions ([Ca2+]i) is associated with muscle damage, little is known regarding the temporal profile of muscle [Ca2+]i under in vivo conditions, and, specifically, the effects of different contraction types [e.g., isometric (ISO); eccentric (ECC)] on [Ca2+]i remain to be determined. The following hypotheses were tested. 1) For 90 min at rest, an in vivo vs. in vitro preparation would better maintain initial [Ca2+]i. 2) Compared with ISO, ECC contractions (50 contractions, 10 sets, 5-min interval) would lead to a greater increase of [Ca2+]i. 3) Elevated [Ca2+]i during ECC would be reduced or prevented by the stretch-activated ion channel blockers streptomycin and gadolinium (Gd3+). Spinotrapezius muscles of Wistar rats were exteriorized (in vivo) or excised (in vitro). [Ca2+]i was evaluated by loading the muscle with fura 2-AM using fluorescence imaging. [Ca2+]i rose progressively beyond 40 min at rest under in vitro but not in vivo conditions during the 90-min protocol. In vivo [Ca2+]i increased more rapidly during ECC (first set) than ISO (fifth set) (P < 0.05 vs. precontraction values). The peak level of [Ca2+]i was increased by 21.5% (ISO) and 42.8% (ECC) after 10 sets (both P < 0.01). Streptomycin and Gd3+ abolished the majority of [Ca2+]i increase during ECC (69 and 86% reduction, respectively; P < 0.01 from peak [Ca2+]i of ECC). In conclusion, in vivo quantitative analyses demonstrated that ECC contractions elevate [Ca2+]i significantly more than ISO contractions and that stretch-activated channels may play a permissive role in this response.

Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension

Significance Pulmonary arterial hypertension (PAH) is a serious disease characterized by vascular remodeling in pulmonary arteries. Although an elevated IL-6 serum level correlates with poor prognosis of PAH patients, it is unclear how IL-6 promotes PAH. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. In mice with hypoxia-induced pulmonary hypertension (HPH), Th17 cells and M2 macrophages accumulate in the lungs after hypoxia exposure. IL-21 primarily derived from Th17 cells promotes M2 macrophage polarization. Consistently, IL-21 receptor-deficient mice show resistance to HPH with no accumulation of M2 macrophages in the lungs. IL-21 and M2 macrophage markers were upregulated in the lungs of patients with end-stage idiopathic PAH. These findings suggest promising therapeutic strategies for PAH targeting IL-6/IL-21–signaling axis. IL-6 is a multifunctional proinflammatory cytokine that is elevated in the serum of patients with pulmonary arterial hypertension (PAH) and can predict the survival of patients with idiopathic PAH (IPAH). Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6–mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly up-regulated after hypoxia exposure in the lungs of mice treated with control antibody but not in the lungs of mice treated with MR16-1. Although IL-17 blockade with an anti–IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. In accordance with these findings, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Of note, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH in association with M2 macrophage polarization, downstream of IL-6-signaling. The IL-6/IL-21–signaling axis may be a potential target for treating PAH.

Docking Protein Gab1 Is an Essential Component of Postnatal Angiogenesis After Ischemia via HGF/c-Met Signaling

Rationale: Grb2-associated binder (Gab) docking proteins, consisting of Gab1, Gab2, and Gab3, have crucial roles in growth factor–dependent signaling. Various proangiogenic growth factors regulate angiogenesis and endothelial function. However, the roles of Gab proteins in angiogenesis remain elusive. Objective: To elucidate the role of Gab proteins in postnatal angiogenesis. Methods and Results: Endothelium-specific Gab1 knockout (Gab1ECKO) mice were viable and showed no obvious defects in vascular development. Therefore, we analyzed a hindlimb ischemia (HLI) model of control, Gab1ECKO, or conventional Gab2 knockout (Gab2KO) mice. Intriguingly, impaired blood flow recovery and necrosis in the operated limb was observed in all of Gab1ECKO, but not in control or Gab2KO mice. Among several proangiogenic growth factors, hepatocyte growth factor (HGF) induced the most prominent tyrosine phosphorylation of Gab1 and subsequent complex formation of Gab1 with SHP2 (Src homology-2–containing protein tyrosine phosphatase 2) and phosphatidylinositol 3-kinase subunit p85 in human endothelial cells (ECs). Gab1-SHP2 complex was required for HGF-induced migration and proliferation of ECs via extracellular signal-regulated kinase (ERK)1/2 pathway and for HGF-induced stabilization of ECs via ERK5. In contrast, Gab1-p85 complex regulated activation of AKT and contributed partially to migration of ECs after HGF stimulation. Microarray analysis demonstrated that HGF upregulated angiogenesis-related genes such as KLF2 (Krüppel-like factor 2) and Egr1 (early growth response 1) via Gab1-SHP2 complex in human ECs. In Gab1ECKO mice, gene transfer of vascular endothelial growth factor, but not HGF, improved blood flow recovery and ameliorated limb necrosis after HLI. Conclusion: Gab1 is essential for postnatal angiogenesis after ischemia via HGF/c-Met signaling.

Role of Rho-kinase signaling and endothelial dysfunction in modulating blood flow distribution in pulmonary hypertension

Rho-kinase-mediated vasoconstriction and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). However, their contribution to the adverse changes in pulmonary blood flow distribution associated with PAH has not been addressed. This study utilizes synchrotron radiation microangiography to assess the specific role, and contribution of, Rho-kinase-mediated vasoconstriction and endothelial dysfunction in PAH. Male adult Sprague-Dawley rats were injected with saline (Cont-rats) or monocrotaline (MCT-rats) 3 wk before microangiography was performed on the left lung. We assessed dynamic changes in vessel internal diameter (ID) in response to 1) the Rho-kinase inhibitor fasudil (10 mg/kg iv); or 2) ACh (3 μg · kg⁻¹ · min⁻¹), sodium nitroprusside (SNP, 5 μg · kg⁻¹ · min⁻¹), and N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg iv). We observed that MCT-rats had fewer vessels of the microcirculation compared with Cont-rats. The fundamental result of this study is that fasudil improved pulmonary blood flow distribution and reduced pulmonary pressure in PAH rats, not only by dilating already-perfused vessels (ID > 100 μm), but also by restoring blood flow to vessels that had previously been constricted closed (ID < 100 μm). Endothelium-dependent vasodilation was impaired in MCT-rats primarily in vessels with an ID < 200 μm. Moreover the vasoconstrictor response to l-NAME was accentuated in MCT-rats, but only in the 200- to 300-μm vessels. These results highlight the importance of Rho-kinase-mediated control and endothelial control of pulmonary vascular tone in PAH. Indeed, an effective therapeutic strategy for treating PAH should target both the smooth muscle Rho-kinase and endothelial pathways.

Dynamic Synchrotron Imaging of Diabetic Rat Coronary Microcirculation In Vivo

Objective—In diabetes, long-term micro- and macrovascular damage often underlies the functional decline in the cardiovascular system. However, it remains unclear whether early-stage diabetes is associated with in vivo functional impairment in the coronary microvasculature. Synchrotron imaging allows us to detect and quantify regional differences in resistance microvessel caliber in vivo, even under conditions of high heart rate. Methods and Results—Synchrotron cine-angiograms of the coronary vasculature were recorded using anesthetized Sprague-Dawley rats 3 weeks after treatment with vehicle or streptozotocin (diabetic). In the early diabetic state, in the presence of nitric oxide and prostacyclin, vessel diameters were smaller (P<0.01) and endothelium-dependent vessel recruitment was already depressed (P<0.05). Endothelium-dependent and -independent vasodilatory responses in individual coronary vessels were not different in vivo. Inhibition of NO and PGI2 production in diabetes uncovered early localized impairment in dilation. Diabetic animals displayed focal stenoses and segmental constrictions during nitric oxide synthase/cyclooxygenase blockade, which persisted during acetylcholine infusion (P<0.05), and a strong trend toward loss of visible microvessels. Conclusion—Synchrotron imaging provides a novel method to investigate coronary microvascular function in vivo at all levels of the arterial tree. Furthermore, we have shown that early-stage diabetes is associated with localized coronary microvascular endothelial dysfunction.

APJ Regulates Parallel Alignment of Arteries and Veins in the Skin

Molecular pathways regulating the development of arterial and venous endothelial cells (ECs) are now well established, but control of parallel arterial-venous alignment is unclear. Here we report that arterial-venous alignment in the skin is determined by apelin receptor (APJ) expression in venous ECs. One of the activators of APJ is apelin. We found that apelin is produced by arterial ECs during embryogenesis, induces chemotaxis of venous ECs, and promotes the production of secreted Frizzled-related protein 1 (sFRP1) by APJ(+) ECs. sFRP1 stimulates matrix metalloproteinase production by Ly6B.2(+) neutrophil-like cells located between the arteries and veins, resulting in remodeling of extracellular matrices to support venous displacement. Moreover, using apelin- or APJ-deficient mice, which exhibit arterial-venous disorganization, we found that arterial-venous alignment is involved in thermoregulation, possibly by regulating countercurrent heat exchange. We hypothesize that the evolution of parallel juxtapositional arterial-venous alignment was an adaptation to reduce body heat loss.

Ventilation/perfusion mismatch during lung aeration at birth.

At birth, the transition to newborn life is triggered by lung aeration, which stimulates a large increase in pulmonary blood flow (PBF). Current theories predict that the increase in PBF is spatially related to ventilated lung regions as they aerate after birth. Using simultaneous phase-contrast X-ray imaging and angiography we investigated the spatial relationships between lung aeration and the increase in PBF after birth. Six near-term (30-day gestation) rabbits were delivered by caesarean section, intubated and an intravenous catheter inserted, before they were positioned for X-ray imaging. During imaging, iodine was injected before ventilation onset, after ventilation of the right lung only, and after ventilation of both lungs. Unilateral ventilation increased iodine levels entering both left and right pulmonary arteries (PAs) and significantly increased heart rate, iodine ejection per beat, diameters of both left and right PAs, and number of visible vessels in both lungs. Within the 6th intercostal space, the mean gray level (relative measure of iodine level) increased from 68.3 ± 11.6 and 70.3 ± 7.5%·s to 136.3 ± 22.6 and 136.3 ± 23.7%·s in the left and right PAs, respectively. No differences were observed between vessels in the left and right lungs, despite the left lung not initially being ventilated. The increase in PBF at birth is not spatially related to lung aeration allowing a large ventilation/perfusion mismatch, or pulmonary shunting, to occur in the partially aerated lung at birth.

Centrally administered ghrelin activates cardiac vagal nerve in anesthetized rabbits.

Although central ghrelin has cardioprotective effect through inhibiting sympathetic nerve activity, the effects of central ghrelin on cardiac vagal nerve remain unknown. We investigated the effects of centrally administered ghrelin on cardiac autonomic nerve activities using microdialysis technique. A microdialysis probe was implanted in the right atrial wall adjacent to the sinoatrial node of an anesthetized rabbit and was perfused with Ringers solution containing a cholinesterase inhibitor, eserine. After injection of ghrelin (1 nmol) into the right lateral cerebral ventricle, norepinephrine (NE) and acetylcholine (ACh) concentrations in the dialysate samples were measured as indices of NE and ACh release from nerve endings to the sinoatrial node using high-performance liquid chromatography. Heart rate was 270±4 bpm at baseline and decreased gradually after ghrelin injection to 234±9 bpm (P<0.01) at 60-80 min, followed by gradual recovery. Dialysate ACh concentration was 5.5±0.8 nM at baseline and increased gradually after ghrelin injection to 8.8±1.2 nM (P<0.01) at 60-80 min; the concentration started to decrease gradually from 100 to 120 min after injection reaching 5.6±0.8 nM at 160-180 min. Central ghrelin did not change mean arterial pressure or dialysate NE concentration. The elevated dialysate ACh concentration declined rapidly after transection of cervical vagal nerves. These results indicate that centrally administered ghrelin activates cardiac vagal nerve.

Oxidative stress augments pulmonary hypertension in chronically hypoxic mice overexpressing the oxidized LDL receptor

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

Imaging of the closed-chest mouse pulmonary circulation using synchrotron radiation microangiography

Structural and functional changes of pulmonary circulation related to pathophysiology of pulmonary arterial hypertension (PAH) remain to be fully elucidated. Angiographic visualization in in vivo animals provided a powerful tool for assessing the major indexes associated with the pathogenesis of PAH. In this study, we have exploited the full potential of synchrotron radiation (SR) microangiography to show the ability to visualize pulmonary hemodynamics in a closed-chest mouse. Male adult mice were anesthetized and cannulated with a customized 24-gauge catheter into the right ventricle via the jugular vein for administering iodine contrast agent. The microangiography was performed on the left lung. We measured dynamic changes in vessel diameter in response to acetylcholine (ACh) and acute exposure to hypoxic gas (10% O(2)). Moreover, the pulmonary transit time was estimated by the time of contrast agent circulating. We were able to visualize the pulmonary arteries from the left pulmonary artery (LPA) to the third generation of branching (inner diameter <100 μm). ACh and acute hypoxia induced vascular responses chiefly in the second and third branching vessels rather than the LPA and the first branching vessels. The transit time was only 0.83 s. These results demonstrate the effectiveness of SR for visualizing the pulmonary circulation in a closed-chest mouse. Future studies using SR microangiography on specific gene-targeted knockout and transgenic mice will provide new insights into the pathophysiology of pulmonary dysfunction and functional adaptation to survive in hypoxic condition.