Objective-We investigated the comparative roles of mitogen-activated protein (MAP) kinases, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, in vascular smooth muscle cell (VSMC) proliferation, migration, and gene expression. Methods and Results-VSMCs were infected with recombinant adenovirus containing dominant-negative mutants of ERK, p38, and JNK (Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK, respectively) to specifically inhibit the respective MAP kinases and then stimulated with platelet-derived growth factor (PDGF)-BB. Ad-DN-ERK attenuated PDGF-BB-induced VSMC proliferation more potently than Ad-DN-p38 or Ad-DN-JNK, indicating the dominant role of ERK in VSMC proliferation. Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK similarly inhibited PDGF-induced VSMC migration. Ad-DN-ERK and Ad-DN-JNK suppressed PDGF-BB-induced downregulation of cyclin-dependent kinase inhibitor p27 Kip1 , whereas Ad-DN-p38 decreased PDGF-BB-induced upregulation of p21 Cip1 . Ad-DN-ERK inhibited PDGF-BB-induced plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1, and transforming growth factor-␤ 1 expressions, Ad-DN-p38 blocked monocyte chemoattractant protein-1 and transforming growth factor-␤ 1 expression but not PAI-1, whereas Ad-DN-JNK suppressed only PAI-1 expression. Moreover, in vivo gene transfer of Ad-DN-p38 to rat carotid artery caused the inhibition of intimal hyperplasia by balloon injury, indicating the involvement of p38 in vascular remodeling in vivo. Key Words: platelet-derived growth factor Ⅲ gene transfer Ⅲ vascular smooth muscle cell Ⅲ proliferation Ⅲ gene expression P latelet-derived growth factor-BB (PDGF-BB) is one of the most potent mitogens and chemoattractants for vascular smooth muscle cells (SMCs) and plays the central role in the onset and development of various vascular disorders. [1][2][3][4][5][6] As reviewed, 7,8 PDGF-BB, through interaction with PDGF, activates multiple signaling pathways in vascular SMCs, including SHP-2, Src, PLC-␥, Ras, protein kinase A, phosphatidylinositol 3-kinase (PI3-kinase), and mitogen-activated protein (MAP) kinases, which are supposed to play some role in PDGF-induced cellular responses. Ras, 9 Src, 10 and c-Jun 11 contribute to PDGFinduced vascular SMC proliferation. On the other hand, PI 3-kinase is known to participate in PDGF-induced vascular SMC migration. 12 However, the molecular mechanism of vascular SMC proliferation and migration by PDGF-BB remains to be fully understood. PDGF-BB not only stimulates proliferation and migration in vascular SMCs but also induces various genes. Interestingly, previous reports indicate that PDGF-BB induces plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-␤ 1 (TGF-␤ 1 ) in vascular SMCs. [13][14][15] Increased PAI-1 that leads to inhibition of plasminogen activation impairs fibrinolysis and thereby promotes thrombosis. 16 MCP-1 is the major chemotactic factor involved in the migration of monocytes into...

Abstract. Although liposome-encapsulated clodronate has been used as a means to deplete macrophages from certain tissues, target leukocyte subtypes within the kidney are not clearly known under normal and pathologic conditions. The present study was therefore conducted to examine the effects of liposome clodronate on renal infiltrating cell type following unilateral ureteral obstruction (UUO) and tried to correlate these changes to the mechanisms of early development of renal fibrosis. Renal infiltrating leukocyte subtypes and counts were determined by using multicolor flow cytometric analysis of cell suspensions from obstructed kidneys. UUO for 5 days elicited renal tubular apoptosis and renal fibrosis and showed 4-fold increase in renal leukocytes including monocytes/macrophages, dendritic cells, and T-cells. Repeated administration of liposome clodronate selectively depleted F4/80 + monocytes /macrophages and F4/80 + dendritic cells but not F4/80 − dendritic cells or other cell types in both obstructed and nonobstructed kidneys. Tubular apoptosis and renal fibrosis were also significantly attenuated by liposome clodronate. Increased gene expression of TNF-α and TGF-β observed in obstructed kidneys were markedly attenuated by depletion of renal mononuclear phagocytes. These findings suggest that F4/80 + monocytes /macrophages and/or F4/80 + dendritic cells play a pivotal role in the development of obstruction-induced tubular apoptosis and renal fibrosis, possibly through TNF-α and TGF-β dependent mechanisms.

Abstract. We investigated the involvement of reactive oxygen species (ROS) and intracellular calcium in nephrotoxicity related to an antitumor agent, cisplatin. In this study, we employed cultured renal epithelial cells (LLC-PK 1 ). Cisplatin at 500 µM significantly increased the production of ROS 5 h and caused cell injury. This agent significantly increased the intracellular calcium level ([Ca 2+ ] i ) in a dose-dependent manner 1 h or more after exposure. DPPD (N,N'-diphenyl-p-phenylenediamine), an antioxidant, inhibited a cisplatin-related increase in active oxygen production and cell injury but did not inhibit an early increase in the [Ca 2+ ] i level. An intracellular calcium-chelating compound BAPTA-AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester) inhibited an increase in ROS production and cell injury induced by cisplatin. Furthermore, BAPTA-AM suppressed the rise of [Ca 2+ ] i level in 1 h after exposure; however, an extracellular calcium chelator EGTA and a calcium antagonist nicardipine did not inhibit the rise in [Ca 2+ ] i level in the early phase. An NADPH oxidase inhibitor inhibited a cisplatin-related increase in ROS production and cell disorder. These results suggest that cisplatin-related calcium release from the site of intracellular calcium storage in the early phase causes oxidative stress in renal tubular epithelial cells. Cisplatin may increase the intracellular production of ROS via NADPH oxidase.

Cancer-specific cell-surface antigens are ideal targets for monoclonal antibody (mAb)-based immunotherapy but are likely to have previously been identified in transcriptome or proteome analyses. Here, we show that the active conformer of an integrin can serve as a specific therapeutic target for multiple myeloma (MM). We screened >10,000 anti-MM mAb clones and identified MMG49 as an MM-specific mAb specifically recognizing a subset of integrin β molecules. The MMG49 epitope, in the N-terminal region of the β chain, is predicted to be inaccessible in the resting integrin conformer but exposed in the active conformation. Elevated expression and constitutive activation of integrin β conferred high MMG49 reactivity on MM cells, whereas MMG49 binding was scarcely detectable in other cell types including normal integrin β lymphocytes. T cells transduced with MMG49-derived chimeric antigen receptor (CAR) exerted anti-MM effects without damaging normal hematopoietic cells. Thus, MMG49 CAR T cell therapy is promising for MM, and a receptor protein with a rare but physiologically relevant conformation can serve as a cancer immunotherapy target.

Objective-Heat shock protein 70s (Hsp70s) are molecular chaperones that protect cells from damage in response to various stress stimuli. However, the functions and mechanisms in endothelial cells (ECs) have not been examined. Herein, we investigate the role of Hsp70s, including heat shock cognate protein 70 (Hsc70), which is constitutively expressed in nonstressed cells (ie, ECs). Hsp72, a member of the Hsp70 family, protects cells, tissues, and organs from various harmful conditions in blood vessels. Previous work 4,5 has shown that an increase in levels of a particular Hsp72, induced by heat stress, is associated with the protection of ventricular and endothelial function after ischemia-reperfusion injury. Several studies 6 -9 have been reported that upregulation of Hsp72 in cells by gene transfer greatly increases the resistance of myocardial cells in vitro and in transgenic mice. On the other hand, deletion of Hsp72 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp72-knockout hearts against ischemia/ reperfusion. 10 Thus, Hsp72 may be expected to play a protective role by reducing the risk of myocardial cell injury and exerting its beneficial effects on endothelial function. However, little is known about the functional role of Hsp70 family members in response to ischemic injury. Methods and Results-TheThe human Hsp70 family, which is the largest and most conserved Hsp family, contains at least eight homologous chaperone proteins. This family includes the Hsp72-inducible protein and constitutively expresses the heat shock cognate protein 70 (Hsc70) isoform, both of which are localized to the cytoplasm. Two members of the Hsp70 family, Hsc70 and Hsp72, have a high degree of sequence homology (86% sequence identity), and both proteins copurify with one another. Hsc70 is abundantly and ubiquitously expressed in all cells, whereas Hsp72 is expressed only at low levels in most unstressed healthy cells and tissues. However, its expression is rapidly induced by a variety of physical and chemical stresses; therefore, it is often called the major stress-inducible Hsp70. It is suggested that Hsp72 and Hsc70 can substitute for each other in healthy cells, whereas Hsp72 expression is essential for certain cells to respond to some cytotoxic factors. There are several chaperone mechanisms based on inducible Hsp72 and constitutive Hsc70. Although both bind to misfolded proteins, newly synthesized polypeptides (ie, Hsp72 and Hsc70) function in the cytosol, suggesting that they display specificity for their client proteins or, alternatively, serve particular chaperone-independent functions. 11 Previous studies mainly analyzed Hsp72 under vari- ous stress conditions; however, there is little research on Hsc70 in the cardiovascular system. In the present study, we investigated whether Hsp70s modulate the angiogenic process. Recently, it has been reported that RNAi-mediated knockdown of human Hsc 70 A12B, a member of the Hsp70 family, disrupted normal zebra fish blood vessel development and inhibited in ...

Takotsubo cardiomyopathy, alternatively known as stress cardiomyopathy, is an increasingly recognized clinical syndrome characterized by acute reversible apical ventricular dysfunction. To elucidate the mechanism, we tried to make a new model of takotsubo-like cardiomyopathy in non-human primates. Echocardiography revealed that repeated intravenous infusion of epinephrine overdose in cynomolgus monkeys induced takotsubo-like cardiomyopathy, which is characterized by progressive left ventricle and depressed systolic function with severe hypokinesis in apical regions and hyperkinesis in the basal region. Although this cardiac dysfunction almost normalized after a month even without any treatment, metoprolol, a b-blocker, improved the decreased ejection fraction earlier than in the control. Luxol fast blue staining, which is useful for estimating myocytolysis, showed that increased myocytolysis was observed in the apical ventricle of the epinephrine-infused heart. Metoprolol diminished epinephrine-induced cardiomyocytolysis. To explain the mechanism of takotsubo myopathy and the effect of metoprolol, gene expressions in apical or basal ventricle were compared. Heart failure-related genes, such as brain natriuretic peptide, connective tissue growth factor and osteopontin; calcium signaling-related genes, such as ryanodine receptor 2, sarcoendoplasmic reticulum Ca 2+ -ATPase 2A2 and adenylate cyclase 7; renin-angiotensin system-related genes, such as angiotensinogen, angiotensin II receptor, type 1 and type 2; and mitochondria-related genes, such as peroxisome proliferatoractivated receptor-c co-activator-1a, cytochrome c and transcription factor A mitochondrial, were significantly changed at the apical ventricle rather than at the basal ventricle. The changes of some genes improved with metoprolol treatment. These results indicate that this model is valuable in understanding the pathogenesis of takotsubo cardiomyopathy and the effectivity of b-blockers.

Renal cortical AP-1 and NF-kappaB DNA binding were activated in chronic CsA nephrotoxicity. These activations were induced largely by means of RAS-independent mechanisms. It is suggested that prevention of CsA-induced DNA-binding activation of these transcription factors is at least in part responsible for the beneficial effects of Mg supplementation on CsA nephrotoxicity.

CGA is an effective tool for predicting outcomes in older patients with DLBCL. The patients treated with curative intent had significantly better outcomes compared with those receiving palliation, irrespective of CGA stratification. Curative treatment should be considered even for 'unfit' patients.