眞鍋 一郎

Vascular endothelial growth factor-A (VEGF-A) is one of the major angiogenic factors, and its actions are primarily mediated through its two membrane receptors, VEGFR-1 and VEGFR-2. A soluble form of VEGFR-1 (sVEGFR-1) sequesters the free form of VEGF-A, and acts as a potent anti-angiogenic factor. While sVEGFR-1 is synthesized as a splice variant of VEGF-R1 gene, the interactions between VEGF-A and sVEGFR-1 remain largely unknown. Here, we show that VEGF-A upregulates sVEGF-R1 expression in human vascular endothelial cells but leaves full-length VEGF-R1 expression unchanged, and that this induction was dependent on the VEGFR-2-protein kinase C-MEK signaling pathway. The VEGF-A-induced sVEGFR-1 upregulation can operate as a negative feedback system, which if modulated can become a novel therapeutic target for regulating pathological angiogenesis. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Atherosclerosis is a chronic inflammatory disease driven by lipids and other atherogenic factors. It is characterized by a dynamic and complex pathological process of bone marrow-derived cells playing divergent roles. Recent studies have begun unraveling the contribution of growing varieties of subsets of immune cells and other bone marrow-derived cells to atherogenesis. For example, bone marrow-derived vascular progenitor cells have been shown to play an important role in the pathogenesis of atherosclerosis. This review provides an overview of the current understanding of contributions of bone marrow-derived cells to atherosclerosis. Particular focus is placed on myeloid cells and vascular progenitor cells. We also summarize the uncertainty surrounding cellular lineage identity and functions. Expansion of our understanding of pathological roles of various subsets of bone marrow-derived cells in atherosclerosis may lead to identification of novel cellular and molecular targets for development of therapeutic strategies.

Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1-specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis.

Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS(eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.

心臓が悪くなり腎血流量が低下し、腎臓が悪くなる。または、逆に腎臓が悪くなると体液貯留傾向となり心不全が悪化するという現象は、臨床的によく経験される。これらは、心腎症候群(cardio-renal syndrome)と呼ばれ、心疾患と腎疾患がどのように共存しているかを示す症候群である。これに対して「心腎連関」とは、心臓と腎臓はさまざまな因子などを介してお互いにクロストークし、生体の恒常性維持を行っており、その破綻が心腎疾患となって現れると考え、メカニズムを理解しようとするものである。本稿では、心腎連関をつかさどる分子について、現在どこまで解明されているか広く解説する。(著者抄録)

SUS316L stainless steel and cobalt-chromium and platinum-chromium alloys are widely used platforms for coronary stents. These alloys also contain nickel (Ni), which reportedly induces allergic reactions in some subjects and is known to have various cellular effects. The effects of Ni on neointima formation after stent implantation remain unknown, however. We developed coronary stents made of Ni-free high-nitrogen austenitic stainless steel prepared using a N-2-gas pressurized electroslag remelting (P-ESR) process. Neointima formation and inflammatory responses following stent implantation in porcine coronary arteries were then compared between the Ni-free and SUS316L stainless steel stents. We found significantly less neointima formation and inflammation in arteries implanted with Ni-free stents, as compared to SUS316L stents. Notably, Ni2+ was eluted into the medium from SUS316L but not from Ni-free stainless steel. Mechanistically, Ni2+ increased levels of hypoxia inducible factor protein-1 alpha (HIF-1 alpha) and its target genes in cultured smooth muscle cells. HIF-1 alpha and their target gene levels were also increased in the vascular wall at SUS316L stent sites but not at Ni-free stent sites. The Ni-free stainless steel coronary stent reduces neointima formation, in part by avoiding activation of inflammatory processes via the Ni-HIF pathway. The Ni-free-stainless steel stent is a promising new coronary stent platform.

Tamibarotene-loaded biodegradable matrices with antithrombogenic and drug-releasing properties were prepared in a crosslinking reaction between amino groups of alkali-treated collagen (AlCol) and active ester groups of trisuccinimidyl citrate. The resulting matrices were characterized by their residual amino group concentrations, swelling ratios and thermal, antithrombogenic and drug-releasing properties. It was clarified that the addition of tamibarotene does not inhibit matrix formation. After immersion in water, the swelling ratio of a matrix became lower than that prior to immersion. Thermal analysis indicated that AlCol interacted with tamibarotene. The addition of tamibarotene to the matrix did not influence the antithrombogenic property of the resulting matrix. A matrix with a high crosslinking density had a prolonged tamibarotene elution time. These results demonstrate that tamibarotene-loaded matrices have great potential as a coating material for drug-eluting stents.

Modulator recognition factor-2 (Mrf2/AT-rich interaction domain (Arid)5b) has been revealed to be involved in pathogenesis of atherosclerosis and adipogenesis. Single-nucleotide polymorphisms (SNPs) in the MRF2/ARID5B gene are associated with coronary artery disease (CAD) and has been proposed as a candidate gene for type 2 diabetes (T2D). The study was aimed to determine whether any of the four MRF2/ARID5B SNPs (rs2893880, rs10740055, rs7087507 and rs10761600) associated with susceptibility to CAD are also associated with T2D, and to determine whether SNP genotype influences the levels of adiponectin and other clinical factors. Association of MRF2/ARID5B SNPs was investigated in 500 diabetic patients from the Department of Metabolic Diseases at the University of Tokyo and 243 hospital-based nondiabetic individuals from the Institute for Adult Disease Asahi Life Foundation Hospital and 500 community-based nondiabetic individuals from the Hiroshima Atomic Bomb Casualty Council Health Management Center. Associations of haplotypes of these SNP with levels of adiponectin and other clinical factors were evaluated when the data was available. We found rs2893880C, rs10740055A, rs7087507A and rs10761600T were increasingly associated with T2D in terms of allele/genotype frequencies of each SNP and their haplotype combinations. Individuals with haplotype CAAT indicated an 1.86 times higher prevalence of diabetes compared with individuals with GCGA (OR 1.86 (95% confidence interval (CI) 1.43-2.41)). Furthermore, CAAT significantly associated with adiponectin levels and other clinical factors. In conclusion, polymorphisms on the MRF2/ARID5B gene were associated with susceptibility to T2D as well as adiponectin and other clinical factors, which was in a completely concordant way with their associations with CAD. Journal of Human Genetics (2012) 57, 727-733; doi:10.1038/jhg.2012.101; published online 13 September 2012

Consumption of foods high in saturated fatty acids (FAs) as well as elevated levels of circulating free FAs are known to be associated with T2D. Though previous studies showed inflammation is crucially involved in the development of insulin resistance, how inflammation contributes to beta cell dysfunction has remained unclear. We report here the saturated FA palmitate induces beta cell dysfunction in vivo by activating inflammatory processes within islets. Through a combination of in vivo and in vitro studies, we show beta cells respond to palmitate via the TLR4/MyD88 pathway and produce chemokines that recruit CD11b(+)Ly-6C(+) M1-type proinflammatory monocytes/macrophages to the islets. Depletion of M1-type cells protected mice from palmitate-induced beta cell dysfunction. Islet inflammation also plays an essential role in beta cell dysfunction in T2D mouse models. Collectively, these results demonstrate a clear mechanistic link between beta cell dysfunction and inflammation mediated at least in part via the FFA-TLR4/MyD88 pathway.

Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM alpha-actin and SM22 alpha were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photo-chemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-alpha and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIb alpha to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases. (Blood. 2012; 119(8): e45-e56)

The 'evidence' in evidence-based medicine (EBM) is often limited to knowledge obtained from randomized controlled clinical trials (RCT). Most RCTs, however, have strict enrollment criteria which make patient background characteristics and clinical histories significantly different from those encountered in actual practice. Thus it is important to accumulate and analyze data obtained in daily practice to gain insight into a larger clinical picture. Recent developments in information technology and its lowered cost have enabled us to record clinical activity in much greater detail at a lower cost. These factors prompted us to design and develop a coronary angiography and intervention reporting system (CAIRS) to collect data and analyze outcomes of coronary intervention. The resulting advanced CAIRS can record detailed data on coronary angiographic and interventional procedures.To date, data on 10,025 cases of coronary angiography, of which 3,574 were interventional, have been collected over a 5.5 year period. There were 4,343 unique patients, 3,115 (71.7%) of which were male. The overall mean age was 67.0 +/- 11.5. The mean age of males was 66.3 +/- 11.4 and that of females was 69.0 +/- 11.4. About one-third of the patients never underwent a PCI procedure at our institution. For patients that underwent at least one PCI procedure at our institution, the prescription rate of stain increased from 50.8% in 2005 to 80.3% in 2011, while those of nitrate and ticlopidine decreased from 36.7% and 90.8% in 2005 to 21.3% and 0.8% in 2011, respectively. We have also implemented the same system at another institution and compared the data on stem usage between the two institutions, which revealed vastly different stent usage profiles.In conclusion, we have successfully developed and implemented an advanced coronary angiography and intervention reporting system which we call CAIRS. Implementing the same system at multiple institutions and analyzing data collected from several institutions will provide detailed and timely insight into the 'real world' of coronary angiography and interventional procedures and their outcome. (Int Heart J 2012; 53: 35-42)

Chronic inflammation appears to underlie most, if not all, the chronic diseases of today, including cardiovascular disease, type 2 diabetes, chronic kidney disease, Alzheimer's disease and cancer. We have demonstrated that obesity induces chronic local inflammation in adipose tissue. We also found that chronic inflammation is crucially involved in the development of heart failure and chronic kidney disease. In this article, I review recent findings reported by my group and others regarding the mechanisms underlying the chronic inflammatory processes commonly observed in adipose tissue, heart and kidney. I then discuss the key features of the chronic inflammation seen in chronic diseases. (Circ J 2011; 75: 2739-2748)

BACKGROUND & AIMS: Kruppel-like factor 5 (KLF5) is transcription factor that is expressed by dividing epithelial cells of the intestinal epithelium. KLF5 promotes proliferation in vitro and in vivo and is induced by mitogens and various stress stimuli. To study the role of KLF5 in intestinal epithelial homeostasis, we examined the phenotype of mice with conditional deletion of Klf5 in the gut. METHODS: Mice were generated with intestinal-specific deletion of Klf5 (Vil-Cre; Klf5fl/fl). Morphologic changes in the small intestine and colon were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction. RESULTS: Klf5 mutant mice were born at a normal Mendelian ratio but had high mortality compared with controls. Complete deletion of Klf5 from the intestinal mucosa resulted in neonatal lethality that corresponded with an absence of epithelial proliferation. Variegated intestinal-specific deletion of Klf5 in adult mice resulted in morphologic changes that included a regenerative phenotype, impaired barrier function, and inflammation. Adult mutant mice exhibited defects in epithelial differentiation and migration. These changes were associated with reduced expression of Caudal type homeobox (Cdx) 1, Cdx2, and Eph and ephrin signaling proteins. Concomitantly, Wnt signaling to beta-catenin was reduced. Proliferation in regenerative crypts was associated with increased expression of the progenitor cell marker Sox9. CONCLUSIONS: Deletion of Klf5 in the gut epithelium of mice demonstrated that KLF5 maintains epithelial proliferation, differentiation, and cell positioning along the crypt radial axis. Morphologic changes that occur with deletion of Klf5 are associated with disruption of canonical Wnt signaling and increased expression of Sox9.

Renal tubulointerstitial damage is the final common pathway leading from chronic kidney disease to end-stage renal disease. Inflammation is clearly involved in tubulointerstitial injury, but it remains unclear how the inflammatory processes are initiated and regulated. Here, we have shown that in the mouse kidney, the transcription factor Kruppel-like factor-5 (KLF5) is mainly expressed in collecting duct epithelial cells and that Klf5 haploinsufficient mice (Klf5(+/-) mice) exhibit ameliorated renal injury in the unilateral ureteral obstruction (UUO) model of tubulointerstitial disease. Additionally, Klf5 haploinsufficiency reduced accumulation of CD11b(+)F4/80(lo) cells, which expressed proinflammatory cytokines and induced apoptosis among renal epithelial cells, phenotypes indicative of M1-type macrophages. By contrast, it increased accumulation of CD11b(+)F4/80(hi) macrophages, which expressed CD206 and CD301 and contributed to fibrosis, in part via TGF-beta production - phenotypes indicative of M2-type macrophages. Interestingly, KLF5, in concert with C/EBP alpha, was found to induce expression of the chemotactic proteins S100A8 and S100A9, which recruited inflammatory monocytes to the kidneys and promoted their activation into M1-type macrophages. Finally, assessing the effects of bone marrow-specific Klf5 haploinsufficiency or collecting duct- or myeloid cell-specific Klf5 deletion confirmed that collecting duct expression of Klf5 is essential for inflammatory responses to UUO. Taken together, our results demonstrate that the renal collecting duct plays a pivotal role in the initiation and progression of tubulointerstitial inflammation.

Hypertension is a typical modern lifestyle-related disease that is closely associated with the development of cardiovascular disorders. Elevation of angiotensin II (ANG II) is one of several critical factors for hypertension and heart failure; however, the mechanisms underlying the ANG II-mediated pathogenesis are still poorly understood. Here, we show that ANG II-mediated cardiac fibrosis, but not hypertrophy, is regulated by interferon regulatory factor 3 (IRF3), which until now has been exclusively studied in the innate immune system. In a ANG II-infusion mouse model (3.0 mg/kg/d), we compared IRF3-deficient mice (Irf3(-/-)/Bcl2l12(-/-)) with matched wild-type (WT) controls. The development of cardiac fibrosis [3.95 +/- 0.62% (WT) vs. 1.41 +/- 0.46% (Irf3(-/-)/Bcl2l12(-/-)); P < 0.01] and accompanied reduction in left ventricle end-diastolic dimension [2.89 +/- 0.10 mm (WT) vs. 3.51 +/- 0.15 mm (Irf3(-/-)/Bcl2l12(-/-)); P=0.012] are strongly suppressed in Irf3(-/-)/Bcl2l12(-/-) mice, whereas hypertrophy still develops. Further, we provide evidence for the activation of IRF3 by ANG II signaling in mouse cardiac fibroblasts. Unlike the activation of IRF3 by innate immune receptors, IRF3 activation by ANG II is unique in that it is activated through the canonical ERK signaling pathway. Thus, our present study reveals a hitherto unrecognized function of IRF3 in cardiac remodeling, providing new insight into the progression of hypertension-induced cardiac pathogenesis.-Tsushima, K., Osawa, T., Yanai, H., Nakajima, A., Takaoka, A., Manabe, I., Ohba, Y., Imai, Y., Taniguchi, T., Nagai, R. IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II-induced hypertension. FASEB J. 25, 1531-1543 (2011). www.fasebj.org

Background: The association between vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase 1 (sFlt-1), and coronary artery disease (CAD) was investigated. Methods: We enrolled 112 hypertensive patients with proven CAD and 112 hypertensive controls matched for age and gender. The severity of CAD was assessed by the most severe clinical presentation of CAD in patients' history and by the number of diseased vessels. Results: Vascular endothelial growth factor level was lower, whereas sFlt-1 level was higher in the CAD group compared to the controls. Diabetes mellitus (P = .001), smoking (P = .004), and higher sFlt-1 level (P = .01) were independently associated with CAD. Younger age (P = .02), smoking (P = .049), and higher VEGF levels (P = .02) were independently associated with a history of myocardial infarction (MI), whereas higher sFlt-1 level (P = .01) was independently associated with multivessel disease. Conclusion: Plasma sFlt-1 levels are positively associated with the presence of CAD and are associated with angiographical severity of CAD.

Background-It has been proposed that bone marrow-derived cells infiltrate the neointima, where they differentiate into smooth muscle (SM) cells; however, technical limitations have hindered clear identification of the lineages of bone marrow-derived "SM cell-like" cells.Methods and Results-Using a specific antibody against the definitive SM cell lineage marker SM myosin heavy chain (SM-MHC) and mouse lines in which reporter genes were driven by regulatory programs for either SM-MHC or SM alpha-actin, we demonstrated that although some bone marrow-derived cells express SM alpha-actin in the wire injury-induced neointima, those cells did not express SM-MHC, even 30 weeks after injury. Likewise, no SM-MHC+ bone marrow-derived cells were found in vascular lesions in apolipoprotein E-/- mice or in a heart transplantation vasculopathy model. Instead, the majority of bone marrow-derived SM alpha-actin(+) cells were also CD115(+)CD11b(+)F4/80(+)Ly-6C(+), which is the surface phenotype of inflammatory monocytes. Moreover, adoptively transferred CD11b(+)Ly-6C(+) bone marrow cells expressed SM alpha-actin in the injured artery. Expression of inflammation-related genes was significantly higher in neointimal subregions rich in bone marrow-derived SM alpha-actin(+) cells than in other regions.Conclusions-It appears that bone marrow-derived SM alpha-actin(+) cells are of monocyte/macrophage lineage and are involved in vascular remodeling. It is very unlikely that these cells acquire the definitive SM cell lineage. (Circulation. 2010; 122: 2048-2057.)

Background: The relationship between fms-like tyrosine kinase-1 (sFlt-1), a soluble receptor for vascular endothelial growth factor (VEGF), and vascular disease has not been established, so this study aimed to elucidate the association between sFlt-1 and the progression of carotid intima media thickness (IMT) in hypertensive patients.Methods and Results: The 120 hypertensive patients under medical control were enrolled and 112 completed the study (age 59 9 years, 57 females). Plasma VEGF and sFlt-1 levels were measured at enrollment. At baseline and 24-month visit, carotid IMT was measured and the association between sFlt-1 and IMT progression was assessed by linear regression. At baseline, age (r=0.186) and low level of high-density lipoprotein-cholesterol (HDL-C <40mg/dl, r=0.214) were significantly related to carotid IMT. Over the 24 months, carotid IMT increased from 0.670 +/- 0.089mm to 0.696 +/- 0.095mm. There was a positive correlation between sFlt-1 tertiles and IMT change (P=0.05 by ANOVA). Upon multivariate analysis, log-transformed sFlt-1 level (beta=0.137, P=0.003) and low HDL-C (beta=0.048, P=0.04) were identified as predictors of IMT progression, independent of other confounding variables.Conclusions: High sFlt-1 level is predictive of carotid IMT progression in hypertensive patients. Low HDL-C level was also associated with IMT change. These observations support a high sFlt-1 level being indicative of progression of atherosclerosis. (Circ J 2010; 74: 2211-2215)

The aim of this study was to compare the effects of 3 different statin regimens that have equivalent low-density lipoprotein cholesterol (LDL-C) lowering efficacy on the apolipoprotein B/A1 ratio and glucose metabolism. After a 4-week dietary lead-in, 90 hypercholeserolemic patients were randomly assigned to 1 of 3 treatment groups for 8 weeks: atorvastatin 20 mg, rosuvastatin 10 mg, or atorvastatin/ezetimibe 5 mg/5 mg. At drug treatment week 8, we compared the percentage changes in lipid parameters, apolipoprotein B/A1 ratio, hemoglobin A1c, and homeostasis model assessment-insulin resistance (HOMA-IR) from baseline. Seventy-six patients completed the study and the percentage changes in LDL-C were comparable among the groups. However, the percentage reduction in the apolipoprotein B/A1 ratio was significantly greater in the rosuvastatin group (-47% +/- 14%, P = .04) and the combination group (-46% +/- 8%, P = .05) than in the atorvastatin group (-39% +/- 11%). The percentage increase in hemoglobin A1c was small but significantly greater in the atorvastatin group compared to the combination group (3.0% +/- 5.2% and -0.4% +/- 4.0%, P = .03). The effect of rosuvastatin on hemoglobin A1c was not different from those of the other 2 regimens. The effects of 3 statin regimens were similar on HOMA-IR. In conclusion, 3 statin regimens have differential effect on apolipoprotein B/A1 and glycemic control after comparable LDL-C reduction.

Kruppel-like factor 5 (KLF5) is a zinc-finger-type transcription factor that mediates the tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Our previous studies have shown that KLF5 is induced by angiotensin II (AII), although the precise molecular mechanism is not yet known. Here we analyzed regulatory single nucleotide polymorphisms (SNPs) within the KLF5 locus to identify clinically relevant signaling pathways linking AII and KLF5. One SNP was located at -1282 bp and was associated with an increased risk of hypertension: subjects with the A/A and A/G genotypes at -1282 were at significantly higher risk for hypertension than those with the G/G genotype. Interestingly, a reporter construct corresponding to the -1282G genotype showed much weaker responses to AII than a construct corresponding to -1282A. Electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays collectively showed that the -1282 SNP is located within a functional myocyte enhancer factor 2 (MEF2) binding site, and that the -1282G genotype disrupts the site and reduces the AII responsiveness of the promoter. Moreover, MEF2 activation via reactive oxygen species and p38 mitogen-activated protein kinase induced KLF5 expression in response to AII, and KLF5 and MEF2 were coexpressed in coronary atherosclerotic plaques. These results suggest that a novel signaling and transcription network involving MEF2A and KLF5 plays an important role in the pathogenesis of cardiovascular diseases such as hypertension.-Oishi, Y., Manabe, I., Imai, Y., Hara, K., Horikoshi, M., Fujiu, K., Tanaka, T., Aizawa, T., Kadowaki, T., Nagai, R. Regulatory polymorphism in transcription factor KLF5 at the MEF2 element alters the response to angiotensin II and is associated with human hypertension. FASEB J. 24, 1780-1788 (2010). www.fasebj.org

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provi

Fibroblasts, which are the most numerous cell type in the heart, interact with cardiomyocytes in vitro and affect their function; however, they are considered to play a secondary role in cardiac hypertrophy and failure. Here we have shown that cardiac fibroblasts are essential for the protective and hypertrophic myocardial responses to pressure overload in vivo in mice. Haploinsufficiency of the transcription factor-encoding gene Kruppel-like factor 5 (Klf5) suppressed cardiac fibrosis and hypertrophy elicited by moderate-intensity pressure overload, whereas cardiomyocyte-specific Klf5 deletion did not alter the hypertrophic responses. By contrast, cardiac fibroblast-specific Klf5 deletion ameliorated cardiac hypertrophy and fibrosis, indicating that KLF5 in fibroblasts is important for the response to pressure overload and that cardiac fibroblasts are required for cardiomyocyte hypertrophy. High-intensity pressure overload caused severe heart failure and early death in mice with Klf5-null fibroblasts. KLF5 transactivated Igf1 in cardiac fibroblasts, and IGF-1 subsequently acted in a paracrine fashion to induce hypertrophic responses in cardiomyocytes. Igf1 induction was essential for cardioprotective responses, as administration of a peptide inhibitor of IGF-1 severely exacerbated heart failure induced by high-intensity pressure overload. Thus, cardiac fibroblasts play a pivotal role in the myocardial adaptive response to pressure overload, and this role is partly controlled by KLF5. Modulation of cardiac fibroblast function may provide a novel strategy for treating heart failure, with KLF5 serving as an attractive target.

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk(-/-) mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alpha IIb beta 3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk(-/-) mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk(-/-) platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta 3 integrin subunit, and reduced binding of Fyn to integrin alpha IIb beta 3. These results provide new insight into the mechanism of alpha IIb beta 3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

Background: Although omega-3 polyunsaturated fatty acids are known to have beneficial effects on cardiovascular diseases. their prognostic value has not been studied prospectively in patients with acute myocardial infarction (AMI).Methods and Results: The plasma levels of phospholipids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (% of total fatty acids), were measured in 508 patients (365 males;, mean age, 63 years) with AMI. Clinical and biomarker predictors of all-cause and cardiovascular mortality were identified by stepwise Cox regression model. During a mean follow-up of 16.1 months, 36 (7.1%) patients died. After controlling for confounding variables, age (hazard ratio (HR): 1.09, P<0.001), renal insufficiency (HR: 2.84, P=0.01) and EPA level (HR: 0.29 P=0.004) were identified as independent predictors of all cause-mortality. When stratified by gender, age (HR: 1.08, P=0.001) and renal insufficiency (HR: 4.49, P=0.003) were predictors of all-cause-mortality in males, whereas EPA level (HR: 0.18, P=0.009) and angiotensin-converting enzyme inhibitor use (HR: 0.24, P=0.03) were identified as predictive of all-cause-mortality in females.Conclusions: Lower plasma level of EPA, but not DHA, was an independent predictor for all-cause-mortality in patients with AMI, but this relationship was significant only in female patients. (Circ J 2009; 73: 2250-2257)

Metabolic syndrome is a major risk factor for cardiovascular and metabolic diseases. Playing a central role in the development of metabolic syndrome and in its clinical consequences is visceral obesity. Adipose tissue is now considered to be an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that is seen in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Recent studies have shown that obesity induces chronic local inflammation in adipose tissue, and that cells of the innate immune system, particularly macrophages, are crucially involved in adipose inflammation and systemic metabolic abnormalities. Moreover, we and others recently revealed that T cells are key regulators of adipose inflammation, and that the adaptive immune system is also crucially important. In mouse models modulation of T cell function ameliorated not only adipose inflammation but also systemic insulin resistance induced by obesity. Thus clarification of the inflammatory processes ongoing in obese adipose tissue would seem essential for the understanding of metabolic syndrome and for developing novel therapeutic strategies to treat it.

Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals. In particular, obese adipose tissue shows features characteristic of active local inflammation. At present, however, little is known about the sequence of events that comprises the inflammatory cascade or the mechanism by which inflammation develops. We found that large numbers of CD8(+) effector T cells infiltrated obese epididymal adipose tissue in mice fed a high-fat diet, whereas the numbers of CD4(+) helper and regulatory T cells were diminished. The infiltration by CD8(+) T cells preceded the accumulation of macrophages, and immunological and genetic depletion of CD8(+) T cells lowered macrophage infiltration and adipose tissue inflammation and ameliorated systemic insulin resistance. Conversely, adoptive transfer of CD8(+) T cells to CD8-deficient mice aggravated adipose inflammation. Coculture and other in vitro experiments revealed a vicious cycle of interactions between CD8(+) T cells, macrophages and adipose tissue. Our findings suggest that obese adipose tissue activates CD8(+) T cells, which, in turn, promote the recruitment and activation of macrophages in this tissue. These results support the notion that CD8(+) T cells have an essential role in the initiation and propagation of adipose inflammation.

Use of short interfering RNA (siRNA) is a promising new approach thought to have a strong potential to lead to rapid development of gene-oriented therapies. Here, we describe a newly developed, systemically injectable siRNA vehicle, the "wrapsome" (WS), which contains siRNA and a cationic lipofection complex in a core that is fully enveloped by a neutral lipid bilayer and hydrophilic polymers. WS protected siRNA from enzymatic digestion, providing a long half-life in the systemic circulation. Moreover, siRNA/WS leaked from blood vessels within tumors into the tumor tissue, where it accumulated and was subsequently transfected into the tumor cells. Because the transcription factor KLF5 is known to play a role in tumor angiogenesis, we designed KLF5-siRNA to test the antitumor activity of siRNA/WS. KLF5-siRNA/WS exhibited significant antitumor activity, although neither WS containing control scrambled-siRNA nor saline containing KLF5-siRNA affected tumor growth. KLF5-siRNA/WS inhibited Klf5 expression within tumors at both mRNA and protein levels, significantly reducing angiogenesis, and we detected no significant acute or long-term toxicity. Our findings support the idea that siRNA/WS can be used to knock down specific genes within tumors and thereby exert therapeutic effects against cancers. [Cancer Res 2009;69(16):6531-8]

Metabolic syndrome is a major risk factor of cardiovascular events, and obese visceral adipose tissue remodeling and malfunctioning based on chronic inflammation play a central role. To assess dynamic multi-cellular interplay, a novel ex vivo and in vivo adipose tissue imaging method was developed. We found close spatial and temporal interrelationships between angiogenesis and adipogenesis, and both were augmented in obese adipose. In addition, we found increased leukocyte-platelet-endothelial cell interactions in the microcirculation of obese visceral adipose that were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Both macrophages and endothelial cells showed increased adhesion molecules, and platelets were also activated locally in obese adipose. Up-regulated expression of adhesion molecules on multiple cell types suggests that their increased interactions contribute to local activation of inflammatory processes within visceral obese adipose tissue. Interestingly, the heightened leukocyte-platelet-endothelial interactions were not observed in obese subcutaneous fat pads. Our results demonstrated the power of our imaging technique to analyze complex cellular interplays in vivo and to evaluate new therapeutic interventions against them. Results also indicate that visceral obese adipose tissue is an inflammatory site itself.

Recent advances in our understanding of the disease biology of KLFs have spurred considerable interest in their potential to serve as therapeutic targets. Results obtained with small molecules and nucleic acids (e.g., siRNA) targeting KLFs in vitro and in vivo strongly support the feasibility of therapeutic modulation of KLFs for the treatment of cancer as well as cardiovascular and metabolic diseases. Nonetheless, a better understanding of the precise mode of action of KLF in its transcription network, particularly its interaction with other transcription factors and cofactors and its posttranslational modification, would further facilitate development of KLF therapeutics. Moreover, development of improved drug delivery systems would increase the number of diseases that could be targeted by siRNA against KLFs. KLFs have also been used to induce pluripotency in induced pluripotent stern (iPS) cells, suggesting that pharmacological modulation of KLFs may be a useful approach to tailoring iPS cells and their derivatives.

Kruppel-like transcription factors (KLFs) participate in diverse physiological and pathological processes, such as cell growth, cell differentiation, tumorigenicity, metabolism, inflammation, and tissue remodeling in response to diverse external stress. The importance of KLFs has recently been appreciated as detailed mechanisms of their molecular functions have been rapidly unraveled. However, many questions remain to be addressed: for instance, (1) how is gene expression of KLFs regulated-in a developmental stage-specific manner or in terms of cell-cell interaction; (2) how do KLFs interplay with other cofactors amid the transcriptional network; and (3) need to explore the tertiary structure of KLFs. Given the importance of KLFs in disease biology, extensive investigations on KLFs are expected to lead to identification of therapeutic targets for many diseases.

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs. STEM CELLS 2009; 27: 238-249