Marked ventilation impairment due to progression of diffuse pleural thickening after cardiac surgery.

A 64-year-old Japanese man presented with dyspnea and shortness of breath during exertion. Chest computed tomography revealed bilateral pleural effusion. He was drowsy because of CO2 storage and died due to ventilatory impairment. His past medical history included a thymectomy and adjuvant radiotherapy with thymoma. He had undergone cardiac surgery and permanent pacemaker implantation. The autopsy examination revealed extensive bilateral pleural adhesions and diffuse visceral pleural thickening. An inspection of multiple lung sections failed to detect any asbestos body formation or mesothelioma. The patient's pleural effusion and diffuse pleural thickening may have exacerbated after cardiac surgery. In this case, the progression and pathophysiology of the pleural thickening could be traced by imaging and an autopsy, and we were able to estimate the factors that exacerbated the pleural thickening and ventilation impairment.

A case of pheochromocytoma presenting with cardiopulmonary arrest.

A 33-year-old woman complained of sudden chest pain and intense headache. She was unconscious and underwent defibrillation for ventricular fibrillation in the ambulance. In the emergency room, she was placed on an artificial respirator. Diffuse wall hypokinesis and decreased left ventricular ejection fraction (31%) were identified on transthoracic echocardiography, and an intra-aortic balloon pump was inserted to address the cardiogenic shock. A mass was identified in the right adrenal gland on abdominal ultrasonography. Since a pheochromocytoma was suspected, doxazosin and carvedilol were administered. Blood and urinary norepinephrine and dopamine levels were elevated, confirming the pheochromocytoma diagnosis, and right adrenalectomy was performed 23 days after the initial hospitalization. After surgery, the left ventricular wall motion and left ventricular ejection fraction had improved to 62% on echocardiography. Blood and urinary norepinephrine and dopamine levels also decreased to within the normal range. This case highlights that the patient returned to normalcy and recovered to a transient myocardial disorder or malignant arrhythmia after cardiopulmonary arrest due to early diagnosis of and accurate treatment for pheochromocytoma. .

A case of percutaneous coronary intervention for treatment of iatrogenic chronic total occlusion of the left circumflex artery after mitral valve repair.

A 55-year-old man had undergone mitral annuloplasty for mitral regurgitation with posterior mitral prolapse 3 years prior. He was examined at our hospital for dyspnea and fatigue. A coronary angiogram revealed iatrogenic chronic total occlusion (CTO) in the left circumflex coronary artery. We performed percutaneous coronary intervention (PCI) and successfully placed an everolimus-eluting stent. An intravascular ultrasound (IVUS) showed an impaired coronary artery at the occlusion site. To our knowledge, this is the first reported successful PCI for iatrogenic CTO after mitral valve repair. IVUS-guided PCI may help prevent complications in unusual CTO cases, such as coronary rupture.

A Case of Refractory Heart Failure in Becker Muscular Dystrophy Improved With Corticosteroid Therapy.

The patient was a 26 year-old man who was referred to our hospital in June 2011 because of severe heart failure. At age 24 years, he was found to have Becker muscular dystrophy. He received enalapril for cardiac dysfunction; however, he had worsening heart failure and was thus referred to our hospital. Echocardiography showed enlargement of the left ventricle, with a diastolic dimension of 77 mm and ejection fraction of 19%. His condition improved temporarily after an infusion of dobutamine and milrinone. He was then administered amiodarone for ventricular tachycardia; however, he subsequently developed hemoptysis. Amiodarone was discontinued and corticosteroid pulse therapy was administered followed by oral prednisolone (PSL). His creatinine phosphokinase (CPK) level and cardiomegaly improved after the corticosteroid therapy. The PSL dose was reduced gradually, bisoprolol was introduced, and the catecholamine infusion was tapered. A cardiac resynchronization device was implanted; however, the patient's condition gradually worsened, which necessitated dobutamine infusion for heart failure. We readministered 30 mg PSL, which decreased the CPK level and improved the cardiomegaly. The dobutamine infusion was discontinued, and the patient was discharged. He was given 7.5 mg PSL as an outpatient, and he returned to normal life without exacerbation of the heart failure. There are similar reports showing that corticosteroids are effective for skeletal muscle improvement in Duchenne muscular dystrophy; however, their effectiveness for heart failure has been rarely reported. We experienced a case of Becker muscular dystrophy in which corticosteroid therapy was effective for refractory heart failure.

Rescue balloon pulmonary angioplasty under veno-arterial extracorporeal membrane oxygenation in a patient with acute exacerbation of chronic thromboembolic pulmonary hypertension.

We describe a case of a 41-year-old woman with acute exacerbation of chronic thromboembolic pulmonary hypertension (CTEPH) complicated by rapidly progressive respiratory failure and right heart failure with cardiogenic shock. A computed tomography (CT) showed thrombi in the right main pulmonary artery and bilateral peripheral pulmonary arteries, and echocardiography showed right ventricular dilatation and tricuspid regurgitation, with an estimated pressure gradient of 80 mmHg. The patient was initially diagnosed with acute pulmonary thromboembolism, and thrombolytic therapy was administered. Her condition subsequently deteriorated, however, necessitating mechanical ventilation and veno-arterial extracorporeal membrane oxygenation (VA-ECMO). We performed emergency catheter-directed thrombectomy and thrombus aspiration. Pulmonary hypertension (PH) temporarily improved, but subsequently worsened, and the patient was diagnosed with CTEPH. Pulmonary endarterectomy (PEA) was performed. After PEA, we were unable to wean the patient off VA-ECMO, and rescue balloon pulmonary angioplasty (BPA) to the middle and inferior lobe branches of the right lung was performed. Five days after BPA, the patient was removed from VA-ECMO and on the 57th day of hospitalization, she was weaned off the ventilator. The patient was discharged after 139 days of hospitalization. Rescue BPA represents a useful intervention for improving PH and weaning off VA-ECMO in a patient with acute exacerbation of CTEPH.

GIT1 mediates VEGF-induced podosome formation in endothelial cells: critical role for PLCgamma.

OBJECTIVE: We and others showed that tyrosine kinase receptors (TKRs) such as the epidermal growth factor receptor stimulate G protein-coupled receptor (GPCR) kinase-interacting protein 1 (GIT1) phosphorylation via c-Src, which is required for phospholipase C-gamma (PLCgamma) activation, indicating that GIT1 participates in TKR signaling. VEGF is the most important TKR in endothelial cells (ECs); essential for cell survival, migration, and angiogenesis. Podosomes, actin-rich structures, were found to contribute to EC migration, tissue invasion, and matrix remodeling, suggesting a role for podosomes in angiogenesis. Because GIT1 is a substrate of c-Src, and podosome formation is c-Src dependent, we hypothesized that GIT1 plays an important role in VEGF-induced EC podosome formation and cell migration. METHODS AND RESULTS: Exposure of ECs to VEGF for 30 minutes stimulated GIT1 colocalization with podosomes. Depletion of GIT1 by siRNA significantly decreased VEGF-induced podosome formation. A key role for PLCgamma was suggested by several experiments. Double staining PLCgamma and actin showed colocalization of PLCgamma with podosomes. Podosome formation was dramatically reduced by PLCgamma inhibitor U73122, Src inhibitor PP2, or expression of dominant negative small GTPases. Therefore, VEGF-induced EC podosome formation is dependent on Src, GIT1, PLCgamma, and small GTPases. In addition, matrix metalloprotease 2 (MMP2) and MT-MMP1 were detected at sites of VEGF-induced podosomes. Depletion of GIT1 by siRNA also significantly inhibited VEGF-induced MMP2 activation and extracellular matrix (ECM) degradation. Therefore, GIT1 mediates VEGF-induced matrix metalloproteinase (MMP) activation and ECM degradation by regulating podosome formation. Finally, depletion of GIT1 by siRNA significantly decreased VEGF-induced cell migration. CONCLUSIONS: These data indicate that GIT1 is an essential mediator for VEGF-induced EC podosome formation and cell migration via PLCgamma.

Fluid shear stress inhibits TNF-mediated JNK activation via MEK5-BMK1 in endothelial cells.

Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-alpha (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress=12dyn/cm(2)) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2. These results support a key role for the MEK5-BMK1 signaling pathway in the atheroprotective effects of blood flow.

Celecoxib inhibits the expression of survivin via the suppression of promoter activity in human colon cancer cells.

We investigated the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on human colon cancer cell lines to clarify the mechanisms underlying the chemopreventive effect of NSAIDs. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, induced apoptosis and strongly reduced the expression of an anti-apoptotic protein, survivin, in both protein and mRNA levels in HCT-116 cells. Subsequently, we conducted luciferase reporter assay using a reporter gene driven by the human survivin promoter. A series of analyses using luciferase reporter constructs containing fragments of the survivin promoter and electrophoretic mobility shift assay indicated that the -75/-66 bp region relative to the initiating codon was involved in celecoxib action to suppress survivin promoter activity. Celecoxib also suppressed the activity of TOPflash, T-cell factor reporter plasmid, and the reporter gene driven by the human cyclin D1 promoter, suggesting that this compound inhibited the expression of Wnt/beta-catenin signaling target genes. Further, we found that other NSAIDs including indomethacin, resveratrol, and SC-560 induced apoptosis and suppressed the expression of survivin and the Wnt/beta-catenin signaling pathway in HCT-116 cells, indicating that these effects were likely to be common among NSAIDs. Moreover, NSAIDs (celecoxib, SC-560 and indomethacin) also suppressed the expression of cyclin D1 and survivin on other colon cancer cell lines (DLD-1 and SW-620). Our results suggested that NSAIDs could inhibit proliferation and induce apoptosis in colon cancer cells by inhibition of survivin expression and the Wnt/beta-catenin signaling pathway.

Physiology and pharmacology of the prostaglandin J2 family.

The prostaglandin (PG) J(2) family including PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are metabolites of PGD(2). They had been known as powerful inhibitors of cell proliferation and viral replication until 15d-PGJ(2) was found to be a natural ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). Since then, several new pharmacological actions of the PGJ(2) family have been found, such as pro- and anti-apoptotic effects, cell differentiation-inducing effects, and inhibitory effects on inflammatory processes, whether they depend on PPAR gamma or not. We reported that the PGJ(2) family, particularly 15d-PGJ(2), inhibits cell proliferation by reducing the expression of G(1) cyclins and inducing the expression of cyclin-dependent kinase inhibitor p21 and moreover, induces cell differentiation in vascular smooth muscle cells. In vascular endothelial cells, we found that 15d-PGJ(2) inhibits apoptotic cell death at least in part by the induction of the inhibitor of apoptosis protein c-IAP1. More importantly, physiological levels of laminar fluid shear stress loaded on endothelial cells upregulate the expression of lipocalin-type PGD(2) synthase, which converts PGH(2) to PGD(2), the precursor of the PGJ(2) family. Based on these results, we have hypothesized that the PGJ(2) family synthesized in vascular wall plays an important physiological role to protect vascular cells from atherogenic stimuli.

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells.

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.

Dictyostelium differentiation-inducing factor-3 activates glycogen synthase kinase-3beta and degrades cyclin D1 in mammalian cells.

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

Transcriptional and posttranscriptional regulation of cyclooxygenase-2 expression by fluid shear stress in vascular endothelial cells.

OBJECTIVE: Fluid shear stress induces cyclooxygenase (COX)-2 gene expression in vascular endothelial cells. We investigated the underlying mechanism of this induction. METHODS AND RESULTS: Exposure of human umbilical vein endothelial cells to laminar shear stress in the physiological range (1 to 30 dyne/cm2) upregulated the expression of COX-2 but not COX-1, a constitutive isozyme of COX. The expression of COX-2 mRNA began to increase within 0.5 hour after the loading of shear stress and reached a maximal level at 4 hours. Roles of the promoter region and the 3'-untranslated region in the human COX-2 gene were evaluated by the transient transfection of luciferase reporter vectors into bovine arterial endothelial cells. Shear stress elevated luciferase activity via the region between -327 and 59 bp. Mutation analysis indicated that cAMP-responsive element (-59/-53 bp) was mainly involved in this response. On the other hand, shear stress selectively stabilized COX-2 mRNA. Moreover, shear stress elevated luciferase activity when a 3'-untranslated region of COX-2 gene containing 17 copies of the AUUUA mRNA instability motif was inserted into the vector. CONCLUSIONS: Transcriptional activation and posttranscriptional mRNA stabilization contribute to the rapid and sustained expression of COX-2 in response to shear stress.

Shear stress induces expression of vascular endothelial growth factor receptor Flk-1/KDR through the CT-rich Sp1 binding site.

Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by approximately 3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5'-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress-responsive element resided in the sequence between -94 and -31 bp, which contained putative nuclear factor-kappaB, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.

Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library.

To investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells (HUVEC). We used a microcarrier culture system and a combination of subtraction and reverse-subtraction methods to isolate a number of genes upregulated by shear stress. The resultant subtracted library includes several known genes (e.g. MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (e.g. CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel genes. Some responsive to centrifugal force and shear stress (RECS) genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis.