Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different ...roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1
−/−) and ROCK2-deficient (cROCK2
−/−) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1
−/− mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2
−/− mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1
−/− hearts and down-regulated in cROCK2
−/− hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1
−/− mice, whereas their expressions were significantly lower in cROCK2
−/− mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.
Endothelial AMP-activated protein kinase (AMPK) plays an important role for vascular homeostasis, and its role is impaired by vascular inflammation. However, the role of endothelial AMPK in the ...pathogenesis of pulmonary arterial hypertension (PAH) remains to be elucidated.
To determine the role of endothelial AMPK in the development of PAH.
Immunostaining showed that endothelial AMPK is downregulated in the pulmonary arteries of patients with PAH and hypoxia mouse model of pulmonary hypertension (PH). To elucidate the role of endothelial AMPK in PH, we used endothelial-specific AMPK-knockout mice (eAMPK(-/-)), which were exposed to hypoxia. Under normoxic condition, eAMPK(-/-) mice showed the normal morphology of pulmonary arteries compared with littermate controls (eAMPK(flox/flox)). In contrast, development of hypoxia-induced PH was accelerated in eAMPK(-/-) mice compared with controls. Furthermore, the exacerbation of PH in eAMPK(-/-) mice was accompanied by reduced endothelial function, upregulation of growth factors, and increased proliferation of pulmonary artery smooth muscle cells. Importantly, conditioned medium from endothelial cells promoted pulmonary artery smooth muscle cell proliferation, which was further enhanced by the treatment with AMPK inhibitor. Serum levels of inflammatory cytokines, including tumor necrosis factor-α and interferon-γ were significantly increased in patients with PAH compared with healthy controls. Consistently, endothelial AMPK and cell proliferation were significantly reduced by the treatment with serum from patients with PAH compared with controls. Importantly, long-term treatment with metformin, an AMPK activator, significantly attenuated hypoxia-induced PH in mice.
These results indicate that endothelial AMPK is a novel therapeutic target for the treatment of PAH.
Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinases and promotes inflammation. However, the role of Bsg in the pathogenesis of cardiac hypertrophy and failure ...remains to be elucidated. We examined the role of Bsg in cardiac hypertrophy and failure in mice and humans.
We performed transverse aortic constriction in Bsg(+/-) and in wild-type mice. Bsg(+/-) mice showed significantly less heart and lung weight and cardiac interstitial fibrosis compared with littermate controls after transverse aortic constriction. Both matrix metalloproteinase activities and oxidative stress in loaded left ventricle were significantly less in Bsg(+/-) mice compared with controls. Echocardiography showed that Bsg(+/-) mice showed less hypertrophy, less left ventricular dilatation, and preserved left ventricular fractional shortening compared with littermate controls after transverse aortic constriction. Consistently, Bsg(+/-) mice showed a significantly improved long-term survival after transverse aortic constriction compared with Bsg(+/+) mice, regardless of the source of bone marrow (Bsg(+/+) or Bsg(+/-)). Conversely, cardiac-specific Bsg-overexpressing mice showed significantly poor survival compared with littermate controls. Next, we isolated cardiac fibroblasts and examined their responses to angiotensin II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion, and extracellular signal-regulated kinase/Akt/JNK activities in Bsg(+/+) cardiac fibroblasts, all of which were significantly less in Bsg(+/-) cardiac fibroblasts. Consistently, extracellular and intracellular Bsg significantly promoted cardiac fibroblast proliferation. Finally, serum levels of Bsg were significantly elevated in patients with heart failure and predicted poor prognosis.
These results indicate the crucial roles of intracellular and extracellular Bsg in the pathogenesis of cardiac hypertrophy, fibrosis, and failure in mice and humans.
Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and ...adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH.
We aimed to discover a novel drug for PAH that inhibits PASMC proliferation.
We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs.
These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.
Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits ...fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients.
To determine the role of activated TAFI (TAFIa) in the development of CTEPH.
Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats.
These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.
A total of 850 birds belonging to 40 species within 13 orders were examined for mycoplasmas during 1968 through 1977. In addition, 2, 848 dead-in-shell chicks, 275 pathological specimens and 285 ...normal semen samples of chickens were subjected to the study. It was revealed that birds belonging to either Galliformes or Columbiformes harbored mycoplasmas in high incidence. Serological identification of 440 isolates proved that both Mycoplasma gallisepticum and M. synoviae parasitized among chickens, with an exception of sparrows. Host range of M. gallinarum was the widest of all species or serogroups of avian mycoplasmas. M. iners and serogroups C, D, and I-J-K-N-Q-R were detected from various tissues and sperm samples of domesticated and wild birds belonging to Galliformes. Both M. columbinum and M. columborale were detected only from pigeons, which were considered as the common hosts for these two species of mycoplasmas.
In cartilage regenerative medicine, autologous chondrocyte implantation (ACI) has been applied clinically for partial defects of joint cartilage or nasal augmentation. To make treatment with ACI more ...effective and prevalent, modalities to evaluate the quality of transplanted constructs noninvasively are necessary. In this study, we compared the efficacy of several noninvasive modalities for evaluating the maturation of tissue-engineered auricular cartilage containing a biodegradable polymer scaffold. We first transplanted tissue-engineered cartilage consisting of human auricular chondrocytes, atelocollagen gel, and a poly-l-lactic acid (PLLA) porous scaffold subcutaneously into the back of athymic nude rats. Eight weeks after transplantation, the rats were examined by magnetic resonance imaging (MRI), X-ray, and ultrasound as noninvasive modalities. Then, the excised constructs were examined by histological and biochemical analysis including toluidine blue (TB) staining, glycosaminoglycans content, and enzyme-linked immunosorbent assay of type II collagen. Among the modalities examined, transverse relaxation time (T2) and apparent diffusion coefficient of MRI showed quite a high correlation with histological and biochemical results, suggesting that these can effectively detect the maturation of tissue-engineered auricular cartilage. Since these noninvasive modalities would realize time-course analysis of the maturation of tissue-engineered auricular cartilage, this study provides a substantial insight for improving the quality of tissue-engineered cartilage, leading to improvement of the quality and technique in cartilage regenerative medicine.
Vascular endothelial cell death contributes to the progression of atherosclerotic lesion, and several transcriptional regulators are involved in the process. Activating transcription factor 3/liver ...regenerating factor-1 (ATF3/LRF-1), a stress-inducible transcriptional repressor, was shown to be highly expressed in vascular endothelial cells and macrophages of human atherosclerotic lesions by immunohistological assay. The expression was colocalized in these cells which were positive for TdT-mediated dUTP nick-end labeling (TUNEL) and annexin V. Treatment of human umbilical vein endothelial cells (HUVECs) by tumor necrosis factor (TNF)-α, oxidized low density lipoprotein (oxLDL), and lysophosphatidylcholine (LPC) rapidly induced ATF3/LRF-1, which showed an increased DNA binding to the consensus ATF/CRE sequence by supershift of gel shift assay. Flow cytometry analysis and immunostaining analysis with TUNEL assay showed that ATF3/LRF-1 was highly expressed in cell death induced by these agents. Moreover, antisense ATF3/LRF-1 cDNA partly suppressed the cell death induced by TNF-α, oxLDL, and LPC. From these results, it is indicated that ATF3/LRF-1 is one of the immediate early response genes in vascular endothelial cells in response to atherogenic stimuli, and may play a role in the endothelial cell death associated with atherogenesis.
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