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 ...

Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. In this study, we examined the role of pravastatin in ischemia-induced angiogenesis. Unilateral hindlimb ischemia was surgically induced in C57BL/6J mice. Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and endothelial NO synthase (eNOS) was increased in ischemic tissues. Furthermore, mice treated with pravastatin showed higher increases in phosphorylation than did untreated mice. Laser Doppler analysis has shown that pravastatin treatment accelerates the development of collateral vessels and angiogenesis in response to hindlimb ischemia. Capillary density in the ischemic hindlimb was also increased by pravastatin treatment. An in vitro study on human umbilical vein endothelial cells (HUVECs) revealed that pravastatin increased the phosphorylation of AMPK. Pravastatin-induced phosphorylation of eNOS, one of the downstreams of AMPK, was inhibited by compound C, an AMPK antagonist. The increased migration and tube formation of HUVECs by pravastatin were significantly blocked by compound C treatment. The accelerated angiogenesis by pravastatin after hindlimb ischemia was significantly reduced after treatment with compound C. Thus, ischemia induced AMPK phosphorylation in vivo. Furthermore, pravastatin could also activate AMPK in vivo and in vitro. Such phosphorylation results in eNOS activation and angiogenesis, which provide a novel explanation for one of the pleiotropic effects of statins that is beneficial for angiogenesis. Keywords: angiogenesis; endothelium; ischemia; nitric oxide synthase; statins INTRODUCTIONThe AMP-activated protein kinase (AMPK) is a trimeric enzyme comprising a catalytic a-subunit and regulatory-b, g-subunits. AMPK was identified as an upstream kinase that phosphorylates and hence inactivates 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acetyl-coenzyme A carboxylase (ACC), key enzymes that control choresterol/isoprenoid and fatty acid biosynthesis, respectively. AMPK is considered to be a cellular energy sensor that stimulates ATP-producing catabolic pathways and inhibits ATP-consuming anabolic pathways. 1 Thus, the AMPK pathway is thought to be a regulator of stress responses and cellular energy homeostasis. However, recent studies have shown that AMPK also plays an important role in maintaining endothelial functions. 2 AMPK activation has beneficial effects on endothelial functions and in antiatherogenesis. These effects include the induction of the endothelial nitric oxide synthase (eNOS) pathway to increase NO bioavailability; the suppression of endothelial reactive oxygen species production when stimulated by hyperglycemia or high free fatty acids to improve endothelial free fatty acid oxidation and limit lipid accumulation; the inhibition of apoptosis and inflammation; and the modulation of the vascular tone. 3,4

Abstract. The ebb and flow of cellular life depends largely on signaling pathways and networks, which are regulated by specific protein-protein interactions. These interactions often involve assembly of large signaling complexes containing many different protein kinases, protein phosphatases, their substrates, and scaffold proteins. Identification of protein complexes is the key to understanding cellular functions. One of the techniques used for the isolation of protein complexes is the affinity purification system. Inhibitors of 3-hydroxyl-3-methyglutaryl coenzyme A (HMG-CoA) reductase (i.e., statins) exert cholesterol-independent vasoprotective effects that are mediated, in part, through the activation of Akt. However, the molecular mechanism remains unknown. To elucidate the molecular mechanisms of the pleioptropic effects of statins, we searched for the binding molecule of Akt1 by using a combined mass spectrometry and affinity purification strategy. By this technique, we identified the protein-protein interactions of 23 proteins from statin-treated rat aortic endothelial cells (rAECs). Our results suggest that this approach is very effective and statin activates many Akt down-stream targets, not only endothelial nitric oxide synthase (eNOS). The methodology presented here would provide a new tool for chemical proteomics in medicinal science.

The HMG-CoA reductase inhibitors (statins) have been shown to exert several protective effects on the vasculature that are unrelated to changes in the cholesterol profile, and to induce angiogenesis. The proangiogenic effect exerted by statins has been attributed to the activation of the PI3K/Akt pathway in endothelial cells; however, it is unclear how statins activate this pathway. Pravastatin-mediated activation of Akt and MAPK occurs rapidly (within 10 min.) and at low doses (10 nM). Here, we hypothesized that FGF-2 contributes to the proangiogenic effect of statins. We found that pravastatin, a hydrophilic statin, induced phosphorylation of the FGF receptor (FGFR) in human umbilical vein endothelial cells. SU5402, an inhibitor of FGFR, abolished pravastatin-induced PI3K/Akt and MAPK activity. Likewise, anti-FGF-2 function-blocking antibodies inhibited Akt and MAPK activity. Moreover, depletion of extracellular FGF-2 by heparin prevented pravastatin-induced phosphorylation of Akt and MAPK. Treatment with FGF-2 antibody inhibited pravastatin-enhanced endothelial cell proliferation, migration and tube formation. These observations indicate that pravastatin exerts proangiogenic effects in endothelial cells depending upon the extracellular FGF-2.

It is well recognized that sodium/glucose cotransporter 2 inhibitors (SGLT2i) are associated with weight loss in people with type 2 diabetes. However, the time-dependent associations of weight loss with the anthropometric and body fluid measurements and serum parameters during SGLT2i intervention remain unclear. Thus, we aimed to clarify weight loss-related factors, carefully focusing on the clinical process during the long-term intervention with a SGLT2i, tofogliflozin in people with type 2 diabetes. We analyzed data from 775 people with type 2 diabetes that participated in tofogliflozin phase 3 trials. Participants received tofogliflozin (20, or 40 mg; n = 235, and 540, respectively) orally once daily for 52 weeks. The correlation analysis was performed at week 4, 24 and 52. Baseline characteristics showed men (66 %), age (mean: 58 years), HbA1c (8.0 %), BMI (26 kg/m2) and eGFR (84 mL/min/1.73m2). Tofogliflozin significantly reduced body weight at week 4, 24 and 52 (mean: -1.4, -3.0 and -3.0 kg, respectively). At week 4, weight loss was negatively correlated with both the change in body fluid measures (hematocrit, RBC count, serum creatinine level, and uric acid level) and beta-hydroxybutyrate (BHB). At week 24, weight loss was positively correlated with anthropometric measurements (waist and hip circumferences) and hepatic enzymes levels, and negatively correlated with adiponectin, HDL-C, and BHB levels, but not with body fluid measures. Although the values at week 52 were in concert with the results at week 24, there was a modest but significant correlation between weight loss and BHB level. Our results clarified that tofogliflozin reduces weight, especially that of visceral fat in people with type 2 diabetes, at least due to fluid loss initially and to lipolysis in late. Weight loss might contribute to decrease in the hepatic enzymes, whereas it also accompanies an increase in beta-hydroxybutyrate, the safety issue of which to be investigated in future. Disclosure A. Yoshida: Employee; Self; Kowa Pharmaceutical Co. Ltd. H. Kusakabe: Employee; Self; Kowa Pharmaceutical Co.Ltd. T. Takamura: None. K. Kaku: Speaker's Bureau; Self; AstraZeneca. Advisory Panel; Self; Boehringer Ingelheim GmbH. Speaker's Bureau; Self; Mitsubishi Tanabe Pharma Corporation, MSD K.K.. Advisory Panel; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Ono Pharmaceutical Co., Ltd.. Advisory Panel; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. Speaker's Bureau; Self; Taisho Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceuticals, Japan Inc., Kowa Pharmaceuticals, Japan Inc.. Advisory Panel; Self; Astellas, Japan Inc. H. Suganami: Employee; Self; Kowa Company, Ltd..