Abstract-Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder. Key Words: pulmonary hypertension Ⅲ Rho-kinase Ⅲ vascular smooth muscle cells Ⅲ endothelial nitric oxide synthase Ⅲ macrophages P rimary pulmonary hypertension (PPH) is a lifethreatening disease characterized by a marked and sustained elevation of pulmonary artery pressure. The disease has no obvious causes and ultimately results in right ventricular (RV) failure and death. The pathological changes of hypertensive pulmonary arteries include endothelial injury, proliferation and hypercontraction of vascular smooth muscle cells (VSMCs), and migration of macrophages. 1-3 PPH continues to be a serious clinical problem with high morbidity and mortality. 4 In 1990s, Rho-kinase/ROK/ROCK was identified as an effector of the small GTPase Rho, 5-7 which plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expression. 8 -10 In a series of experimental and clinical studies, we have demonstrated that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of arteriosclerosis. 11-17 These Rho-kinase-mediated alterations in blood vessels also may be involved in the pathogenesis of pulmonary hypertension (PH). In this study, we examined whether Rho-kinasemediated pathway is involved in the pathogenesis of rat model of fatal PH in vivo. Materials and MethodsThe present study was...

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types.

Background-Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo. Methods and Results-Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg · kg Ϫ1 · d Ϫ1 in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (PϽ0.05, nϭ15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both PϽ0.01, nϭ6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-␤ 2 , TGF-␤ 3 , and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both PϽ0.05, nϭ10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (PϽ0.05, nϭ5). Conclusions-These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.

Abstract-Intracellular signaling pathway mediated by small GTPase Rho and its effector Rho-kinase plays an important role in regulation of vascular smooth muscle contraction and other cellular functions. We have recently demonstrated that Rho-kinase is substantially involved in angiotensin II-induced gene expressions and various cellular responses in vitro. However, it remains to be examined whether Rho-kinase is involved in the angiotensin II-induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. Long-term infusion of angiotensin II for 4 weeks caused hypertrophic changes of vascular smooth muscle and cardiomyocytes in rats. Both changes were significantly suppressed by concomitant oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor, hydroxyfasudil, after oral administration. Angiotensin II caused a perivascular accumulation of macrophages and Rho-kinase activation, both of which were also significantly suppressed by fasudil. Vascular NAD(P)H oxidase expression (nox1, nox4, gp91phox, and p22phox) and endothelial production of superoxide anions were markedly increased by angiotensin II, both of which were also significantly suppressed by fasudil. Thus, fasudil ameliorated the impaired endothelium-dependent relaxations caused by angiotensin II without affecting vasodilator function of vascular smooth muscle. These results provide evidence that Rho-kinase is substantially involved in the angiotensin II-induced cardiovascular hypertrophy in rats in vivo. The suppression of endothelial NAD(P)H oxidase upregulation and resultant superoxide production and the amelioration of endothelial vasodilator function may be involved in this process. 4 It has been demonstrated that Rho-kinase/ROK␣/ROCK II (an isoform to p160ROCK/ROK␤/ROCK I), 5 which is an effector of the small GTPase Rho, inhibits MLC phosphatase activity by phosphorylating its myosin-binding subunit 6,7 and thus plays a central role in agonist-induced Ca 2ϩ sensitization and hypercontraction of VSMCs. 4,5,7 We have demonstrated that Rho-kinase plays an important role in angiotensin II-induced mRNA expression of monocyte chemoattractant protein-1 8 and of plasminogen activator inhibitor-1 9 in cultured rat aortic VSMCs. We have recently demonstrated that in cultured human coronary VSMCs, the expression of Rho-kinase is enhanced by inflammatory stimuli, such as angiotensin II and interleukin-1␤ (IL-1␤). 10 However, it remains to be determined whether Rho-kinase is involved in angiotensin II-induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. The present study was thus designed to examine these points in rats in vivo. Materials and Methods AnimalsThis experiment was reviewed and approved by the Committee on Ethics of Animal Experiments of the Kyushu University. A total of 111 adult male WKY rats (14 to 17 weeks old, weighing 300 to 350 g) obtained from the colony at the Kyushu University were used. Animals were anesthetized with intraperitoneal pentobarbital

These findings show that milnacipran acts as a SNRI in vitro and in vivo and may be useful for the treatment of anxiety as well as depression.

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a (1;11) (q42.1;q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. To investigate how DISC1 confers susceptibility to psychiatric disorders, we previously identified fasciculation and elongation protein zeta-1 and Kendrin as DISC1-interacting molecules in a yeast twohybrid screen of a human brain complementary DNA library. Here, we have further identified a novel DISC1-interacting protein, termed DISC1-Binding Zinc-finger protein (DBZ), which has a predicted C 2 H 2 -type zinc-finger motif and coiled-coil domains. DBZ was co-immunoprecipitated with DISC1 in lysates of PC12 cells and rat brain tissue. The domain of DISC1 interacting with DBZ was close to the translocation breakpoint in the DISC1 gene. DBZ messenger RNA (mRNA) was expressed in human brains, but not in peripheral tissues. In situ hybridization revealed high expression of DBZ mRNA in the hippocampus, olfactory tubercle, cerebral cortex and striatum in rats. Because this pattern of localization was similar to that of the pituitary adenylate cyclase (PAC 1 ) receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which has recently been implicated in neuropsychological functions, we examined whether DISC1/DBZ interaction was involved in the PACAP signaling pathway. PACAP upregulated DISC1 expression and markedly reduced the association between DISC1 and DBZ in PC12 cells. A DISC1-binding domain of DBZ reduced the neurite length in PC12 cells after PACAP stimulation and in primary cultured hippocampal neurons. The present results provide some new molecular insights into the mechanisms of neuronal development and neuropsychiatric disorders.

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a translocation, t(1;11) (q42.1;q14.3), that segregates with major psychiatric disorders, including schizophrenia, recurrent major depression and bipolar affective disorder, in a Scottish family. Here we report that mammalian DISC1 endogenously expressed in oligodendroglial lineage cells negatively regulates differentiation of oligodendrocyte precursor cells into oligodendrocytes. DISC1 expression was detected in oligodendrocytes of the mouse corpus callosum at P14 and P70. DISC1 mRNA was expressed in primary cultured rat cortical oligodendrocyte precursor cells and decreased when oligodendrocyte precursor cells were induced to differentiate by PDGF deprivation. Immunocytochemical analysis showed that overexpressed DISC1 was localized in the cell bodies and processes of oligodendrocyte precursor cells and oligodendrocytes. We show that expression of the myelin related markers, CNPase and MBP, as well as the number of cells with a matured oligodendrocyte morphology, were decreased following full length DISC1 overexpression. Conversely, both expression of CNPase and the number of oligodendrocytes with a mature morphology were increased following knockdown of endogenous DISC1 by RNA interference. Overexpression of a truncated form of DISC1 also resulted in an increase in expression of myelin related proteins and the number of mature oligodendrocytes, potentially acting via a dominant negative mechanism. We also identified involvement of Sox10 and Nkx2.2 in the DISC1 regulatory pathway of oligodendrocyte differentiation, both well-known transcription factors involved in the regulation of myelin genes.

Abstract-Cardiac allograft vasculopathy (CAV) continues to be a major cause of late graft failure after cardiac transplantation. We have demonstrated that Rho-kinase, an effector of the small GTPase Rho, plays an important role in the pathogenesis of arteriosclerosis. In this study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of CAV using a specific Rho-kinase inhibitor and a dominant-negative Rho-kinase. Hearts from AKR mice were heterotopically transplanted to C3H/He (allograft) or AKR mice (isograft), and the effects of long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil, on CAV were examined at 2 and 4 weeks after the transplantation. Coronary remodeling in the allografts characterized by intimal thickening and perivascular fibrosis was dose-dependently suppressed in the fasudil group compared with the control group (PϽ0.01, nϭ9 to 10). The inhibitory effects of hydroxyfasudil were mimicked by in vivo gene transfer of dominant-negative Rho-kinase (PϽ0.05, nϭ4). Among the proinflammatory cytokines examined, those of macrophage migration inhibitory factor, interferon-␥, and transforming growth factor-␤1 were upregulated in the control group and were dose-dependently inhibited in the fasudil group (PϽ0.01, nϭ5