眞鍋 一郎

Background: Although early detection is valuable for secondary lymphedema treatment, existing screening tests are not popular. This study aimed to propose a novel method of screening lymphedema patients based on the thickness of the subcutaneous fat measured with perioperative computed tomography (CT) and present the results from evaluation of patients who underwent those examinations was performed.Method: The medical records of 96 gynecological cancer patients and 189 breast cancer patients, whose lymphatic function was assessed with indocyanine green lymphography, were reviewed. In gynecological cancer patients, the perioperative temporal subcutaneous fat thickness index (T-SFTI) was calculated from presurgical and follow-up CT data, and in breast cancer patients, the postoperative crosswise subcutaneous fat thickness index (C-SFTI) was calculated. In lower extremity lymphedema patients, the effect of lymphaticovenular anastomosis (LVA) was also evaluated using T-SFTI.Results: Perioperative T-SFTI was assessed in 180 lower extremities, and it was significantly higher in 46 lymphatic dysfunction limbs (1.21 +/- 0.08) than in 134 normal lymphatic function limbs (1.03 +/- 0.08), (P < .01). Postoperative C-SFTI was assessed in 53 upper extremity, and it was significantly higher in 11 lymphatic dysfunction limbs (1.31 +/- 0.21) than in 42 normal lymphatic function limbs (1.01 +/- 0.06), (P < .01). In lower extremity lymphedema patients, T-SFTI improved significantly after planned conservative treatments and LVA (P = .04).Conclusion: Assessment of subcutaneous fat thickness using CT is useful for screening early stage lymphedema. If the efficacy of this method is validated, patients worldwide may be assessed using the same criterion.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells co-express lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4-regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4 While Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, it was downregulated in embryos overexpressing Klf5 Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)-extracellular signalregulated kinase (ERK) signalling is involved in segregation of the- EPI and PrE lineages; however, the mechanism involved in Fgf4 regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4. Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Klf5. Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Recurrent inactivating mutations have been identified in various hematological malignancies in the X-linked BCOR gene encoding BCL6 corepressor (BCOR); however, its tumor suppressor function remains largely uncharacterized. We generated mice missing Bcor exon 4, expressing a variant BCOR lacking the BCL6-binding domain. Although the deletion of exon 4 in male mice (Bcor(Delta E4/gamma)) compromised the repopulating capacity of hematopoietic stem cells, Bcor(Delta E4/gamma) thymocytes had augmented proliferative capacity in culture and showed a strong propensity to induce acute T-cell lymphoblastic leukemia (T-ALL), mostly in a Notch-dependent manner. Myc, one of the critical NOTCH1 targets in T-ALL, was highly up-regulated in Bcor(Delta E4/gamma) T-ALL cells. Chromatin immunoprecipitation/DNA sequencing analysis revealed that BCOR was recruited to the Myc promoter and restrained its activation in thymocytes. BCOR also targeted other NOTCH1 targets and potentially antagonized their transcriptional activation. Bcl6-deficient thymocytes behaved in a manner similar to Bcor(Delta E4/gamma) thymocytes. Our results provide the first evidence of a tumor suppressor role for BCOR in the pathogenesis of T lymphocyte malignancies.

Bmal1 (encoded by Arntl gene) is a core circadian clock gene that regulates various genes involved in circadian rhythm. Although Bmal1 is expressed rhythmically in macrophages, the role of Bmal1 in the regulation of their cellular function remains insufficiently understood. Here, we report that Bmal1 regulates time-dependent inflammatory responses following Toll-like receptor 4 (TLR4) activation by modulating enhancer activity. Global transcriptome analysis indicated that deletion of Arntl perturbed the time-dependent inflammatory responses elicited by TLR4 activation by Kdo2-lipid A (KLA). Although the recruitment of NF-kappa B p65 was unaffected, the acetylation status of lysine 27 of histone 3, which correlates positively with enhancer activity, was globally increased at PU. 1-containing enhancers in Arntl(-/-) macrophages as compared to wild-type cells. Expression of Nr1d1 and Nr1d2, encoding RevErb transcription factors, which repress enhancer RNA expression, was significantly decreased in Arntl(-/-) macrophages. Moreover, the level of H3K27 acetylation was increased by Arntl deletion at RevErb-dependent eRNA-expressing enhancers. These results suggest that Bmal1 controls KLA-responsive enhancers, in part by regulating RevErb-directed eRNA transcription. Taken together, the results of this study show that the clock transcription factor network containing Bmal1 controls the inflammatory responses of macrophages by regulating the epigenetic states of enhancers.

Heart failure is a complex clinical syndrome characterized by insufficient cardiac function. In addition to abnormalities intrinsic to the heart, dysfunction of other organs and dysregulation of systemic factors greatly affect the development and consequences of heart failure. Here we show that the heart and kidneys function cooperatively in generating an adaptive response to cardiac pressure overload. In mice subjected to pressure overload in the heart, sympathetic nerve activation led to activation of renal collecting-duct (CD) epithelial cells. Cell-cell interactions among activated CD cells, tissue macrophages and endothelial cells within the kidney led to secretion of the cytokine CSF2, which in turn stimulated cardiac-resident Ly6C(lo) macrophages, which are essential for the myocardial adaptive response to pressure overload. The renal response to cardiac pressure overload was disrupted by renal sympathetic denervation, adrenergic beta 2-receptor blockade or CD-cell-specific deficiency of the transcription factor KLF5. Moreover, we identified amphiregulin as an essential cardioprotective mediator produced by cardiac Ly6C(lo) macrophages. Our results demonstrate a dynamic interplay between the heart, brain and kidneys that is necessary for adaptation to cardiac stress, and they highlight the homeostatic functions of tissue macrophages and the sympathetic nervous system.

Macrophages play pivotal roles in both the induction and resolution phases of inflammatory processes. Macrophages have been shown to synthesize anti-inflammatory fatty acids in an LXR-dependent manner, but whether the production of these species contributes to the resolution phase of inflammatory responses has not been established. Here, we identify a biphasic program of gene expression that drives production of anti-inflammatory fatty acids 12-24 hr following TLR4 activation and contributes to downregulation of mRNAs encoding pro-inflammatory mediators. Unexpectedly, rather than requiring LXRs, this late program of anti-inflammatory fatty acid biosynthesis is dependent on SREBP1 and results in the uncoupling of NF kappa B binding from gene activation. In contrast to previously identified roles of SREBP1 in promoting production of IL1 beta during the induction phase of inflammation, these studies provide evidence that SREBP1 also contributes to the resolution phase of TLR4-induced gene activation by reprogramming macrophage lipid metabolism.

Chronic inflammation in visceral adipose tissue (VAT) precipitates the development of cardiometabolic disorders. Although changes in T cell function associated with visceral obesity are thought to affect chronic VAT inflammation, the specific features of these changes remain elusive. Here, we have determined that a high-fat diet (HFD) caused a preferential increase and accumulation of CD44(hi)CD62L(lo)CD4(+) T cells that constitutively express PD-1 and CD153 in a B cell-dependent manner in VAT. These cells possessed characteristics of cellular senescence and showed a strong activation of Spp1 (encoding osteopontin [OPN]) in VAT. Upon T cell receptor stimulation, these T cells also produced large amounts of OPN in a PD-1resistant manner in vitro. The features of CD153(+)PD-1(+)CD44(hi)CD4(+) T cells were highly reminiscent of senescence-associated CD4(+) T cells that normally increase with age. Adoptive transfer of CD153(+)PD-1(+)CD44(hi)CD4(+) T cells from HFD-fed WT, but not Spp1-deficient, mice into the VAT of lean mice fed a normal diet recapitulated the essential features of VAT inflammation and insulin resistance. Our results demonstrate that a distinct CD153(+)PD1(+)CD44(hi)CD4(+) T cell population that accumulates in the VAT of HFD-fed obese mice causes VAT inflammation by producing large amounts of OPN. This finding suggests a link between visceral adiposity and immune aging.

Chronic hypertension causes vascular remodeling that is associated with an increase in periostin- (postn) positive cells, including fibroblasts and smooth muscle cells. Kruppel-like factor (KLF) 5, a transcription factor, is also observed in vascular remodeling; however, it is unknown what role KLF5 plays in postn-positive cells during vascular remodeling induced by deoxycorticosterone-acetate (DOCA) salt. We used postn-positive cell-specific Klf5-deficient mice (Klf5(Postn)KO: Klf5(flox/flox); Postn(Cre/-)) and wild-type mice (WT: Klf5(flox/flox); Postn(-/-)). We implanted a DOCA pellet and provided drinking water containing 0.9% NaCl for 8 weeks. The DOCA-salt treatment induced hypertension in both genotypes, as observed by increases in systolic blood pressure. In WT animals, DOCA-salt treatment increased the aortic medial area compared with the non-treated controls. Similarly, Tgfb1 was overexpressed in the aortas of the DOCA-salt treated WT mice compared with the controls. Immunofluorescence staining revealed that fibroblast-specific protein 1 (FSP1)(+)-alpha smooth muscle actin (alpha SMA)(+) myofibroblasts exist in the medial area of the WT aortas after DOCA-salt intervention. Importantly, these changes were not observed in the Klf5(Postn)KO animals. In conclusion, the results of this study suggest that the presence of KLF5 in postn-positive cells contributes to the pathogenesis of aortic thickening induced by DOCA-salt hypertension.

Kruppel-like factor 5 (Klf5) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. We show that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation. During muscle regeneration after injury (cardiotoxin injection), Klf5 was induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impaired muscle regeneration, and myotube formation was suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppressed induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD and Mef2, and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment was greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

Angiopoietin-like protein 2 (ANGPTL2) is a chronic inflammatory mediator that, when deregulated, is associated with various pathologies. However, little is known about its activity in lung. To assess a possible lung function, we generated a rabbit monoclonal antibody that specifically recognizes mouse ANGPTL2 and then evaluated protein expression in mouse lung tissue. We observed abundant ANGPTL2 expression in both alveolar epithelial type I and type II cells and in resident alveolar macrophages under normal conditions. To assess ANGPTL2 function, we compared lung phenotypes in Angptl2 knockout (KO) and wild-type mice but observed no overt changes. We then generated a bleomycin-induced interstitial pneumonia model using wild-type and Angptl2 KO mice. Bleomycin-treated wild-type mice showed specifically upregulated ANGPTL2 expression in areas of severe fibrosing interstitial pneumonia, while Angptl2 KO mice developed more severe lung fibrosis than did comparably treated wild-type mice. Lung fibrosis seen following bone marrow transplant was comparable in wild-type or Angptl2 KO mice treated with bleomycin, suggesting that Angptl2 loss in myeloid cells does not underlie fibrotic phenotypes. We conclude that Angptl2 deficiency in lung epithelial cells and resident alveolar macrophages causes severe lung fibrosis seen following bleomycin treatment, suggesting that ANGPTL2 derived from these cell types plays a protective role against fibrosis in lung.

Psoriasis is a chronic inflammatory skin disease marked by aberrant tissue repair. Mutant mice modeling psoriasis skin characteristics have provided useful information relevant to molecular mechanisms and could serve to evaluate therapeutic strategies. Here, we found that epidermal ANGPTL6 expression was markedly induced during tissue repair in mice. Analysis of mice overexpressing ANGPTL6 in keratinocytes (K14-Angptl6 Tg mice) revealed that epidermal ANGPTL6 activity promotes aberrant epidermal barrier function due to hyperproliferation of prematurely differentiated keratinocytes. Moreover, skin tissues of K14-Angptl6 Tg mice showed aberrantly activated skin tissue inflammation seen in psoriasis. Levels of the proteins S100A9, recently proposed as therapeutic targets for psoriasis, also increased in skin tissue of K14-Angptl6 Tg mice, but psoriasis-like inflammatory phenotypes in those mice were not rescued by S100A9 deletion. This finding suggests that decreasing S100A9 levels may not ameliorate all cases of psoriasis and that diverse mechanisms underlie the condition. Finally, we observed enhanced levels of epidermal ANGPTL6 in tissue specimens from some psoriasis patients. We conclude that the K14-Angptl6 Tg mouse is useful to investigate psoriasis pathogenesis and for preclinical testing of new therapeutics. Our study also suggests that ANGPTL6 activation in keratinocytes enhances psoriasis susceptibility.

A cardioprotective response that alters ventricular contractility or promotes cardiomyocyte enlargement occurs with increased workload in conditions such as hypertension. When that response is excessive, pathological cardiac remodelling occurs, which can progress to heart failure, a leading cause of death worldwide. Mechanisms underlying this response are not fully understood. Here, we report that expression of angiopoietin-like protein 2 (ANGPTL2) increases in pathologically-remodeled hearts of mice and humans, while decreased cardiac ANGPTL2 expression occurs in physiological cardiac remodelling induced by endurance training in mice. Mice overexpressing ANGPTL2 in heart show cardiac dysfunction caused by both inactivation of AKT and sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) 2a signalling and decreased myocardial energy metabolism. Conversely, Angptl2 knockout mice exhibit increased left ventricular contractility and upregulated AKT-SERCA2a signalling and energy metabolism. Finally, ANGPTL2-knockdown in mice subjected to pressure overload ameliorates cardiac dysfunction. Overall, these studies suggest that therapeutic ANGPTL2 suppression could antagonize development of heart failure.

PurposeTo determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model.MethodsWe investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6C(hi), Ly6C(int), and Ly6C(lo)) after laser injury in mice using flow cytometry,and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the beta 3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6C(hi) cells were transferred into Ly5.2mice, the proportions of intraocular Ly5.1(+)Ly6C(hi) cells were compared.ResultsIn WT mice, the proportion of CD4(+) T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8(+) T cells did not change significantly. Proportions of B220(+) cells, granulocytes, and two subtypes of intraocular macrophages (Ly6C(hi) and Ly6C(lo)) peaked at day 3 following laser injury. In contrast, Ly6C(int/lo)CD64(+) subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between CD4(-/-) or Rag2(-/)-mice and controls, whereas lesion size was significantly reduced in CCR2(-/-) mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with beta 3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6C(hi) macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6C(hi) cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6C(hi) cells recruited into the eye were reduced in splenic-denervated and splenectomized mice.ConclusionsAlthough lymphocytes had little effect on CNV formation, Ly6C(hi) macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.

Chronic inflammation is the common pathological basis for such age-associated diseases as cardiovascular disease, diabetes, cancer and Alzheimer's disease. A multitude of bodily changes occur with aging that contribute to the initiation and development of inflammation. In particular, the immune system of elderly individuals often exhibits diminished efficiency and fidelity, termed immunosenescence. But, although immune responses to new pathogens and vaccines are impaired, immunosenescence is also characterized by a basal systemic inflammatory state. This alteration in immune system function likely promotes chronic inflammation. Changes in the tissue microenvironment, such as the accumulation of cell debris, and systemic changes in metabolic and hormonal signals, also likely contribute to the development of chronic inflammation. Monocyte/macrophage lineage cells are crucial to these age-associated changes, which culminate in the development of chronic inflammatory diseases. In this review, we will summarize the diverse physiological and pathological roles of macrophages in the chronic inflammation underlying age-associated diseases.

Proinflammatory cytokine production in macrophages involves multiple regulatory mechanisms, which are affected by environmental and intrinsic stress. In particular, accumulating evidence has suggested epigenetic control of macrophage differentiation and function mainly in vitro. SET domain, bifurcated 1 (Setdb1, also known as Eset) is a histone 3 lysine 9 (H3K9)-specific methyltransferase and is essential for early development of embryos. Here we demonstrate that Setdb1 in macrophages potently suppresses Toll-like receptor 4 (TLR4)-mediated expression of proinflammatory cytokines including interleukin-6 through its methyltransferase activity. As a molecular mechanism, Setdb1-deficiency decreases the basal H3K9 methylation levels and augments TLR4-mediated NF-kappa B recruitment on the proximal promoter region of interleukin-6, thereby accelerating interleukin-6 promoter activity. Moreover, macrophage-specific Setdb1-knockout mice exhibit higher serum interleukin-6 concentrations in response to lipopolysaccharide challenge and are more susceptible to endotoxin shock than wildtype mice. This study provides evidence that the H3K9 methyltransferase Setdb1 is a novel epigenetic regulator of proinflammatory cytokine expression in macrophages in vitro and in vivo. Our data will shed insight into the better understanding of how the immune system reacts to a variety of conditions.

In severely hypoxic condition, HIF-1 alpha-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1 alpha-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1 alpha-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Doxorubicin (Dox) is a potent anticancer agent that is widely used in the treatment of a variety of cancers, but its usage is limited by cumulative dose-dependent cardiotoxicity mainly due to oxidative damage. Ataxia telangiectasia mutated (ATM) kinase is thought to play a role in mediating the actions of oxidative stress. Here, we show that ATM in cardiac fibroblasts is essential for Dox-induced cardiotoxicity.ATM knockout mice showed attenuated Dox-induced cardiotoxic effects (e.g. cardiac dysfunction, apoptosis, and mortality). As ATM was expressed and activated predominantly in cardiac fibroblasts, fibroblast-specific Atm-deleted mice (Atm(fl/fl);Postn-Cre) were generated to address cell type-specific effects, which showed that the fibroblast is the key lineage mediating Dox-induced cardiotoxicity through ATM. Mechanistically, ATM activated the Fas ligand, which subsequently regulated apoptosis in cardiomyocytes at later stages. Therapeutically, a potent and selective inhibitor of ATM, KU55933, when administered systemically was able to prevent Dox-induced cardiotoxicity.ATM-regulated effects within cardiac fibroblasts are pivotal in Dox-induced cardiotoxicity, and antagonism of ATM and its functions may have potential therapeutic implications.

Lymphedema is a debilitating progressive condition that severely restricts quality of life and is frequently observed after cancer surgery. The mechanism underlying lymphedema development remains poorly understood, and no effective pharmacological means to prevent or alleviate the ailment is currently available. Using a mouse model of lymphedema, we show here that excessive generation of immature lymphatic vessels is essential for initial edema development and that this early process is also important for later development of lymphedema pathology. We found that CD4(+) T cells interact with macrophages to promote lymphangiogenesis, and that both lymphangiogenesis and edema were greatly reduced in macrophage-depleted mice, lymphocyte-deficient Rag2(-/-) mice or CD4(+) T-cell-deficient mice. Mechanistically, T helper type 1 and T helper type 17 cells activate lesional macrophages to produce vascular endothelial growth factor-C, which promotes lymphangiogenesis, and inhibition of this mechanism suppressed not only early lymphangiogenesis, but also later development of lymphedema. Finally, we show that atorvastatin suppresses excessive lymphangiogenesis and lymphedema by inhibiting T helper type 1 and T helper type 17 cell activation. These results demonstrate that the interaction between CD4(+) T cells and macrophages is a potential therapeutic target for prevention of lymphedema after surgery.

In early age-related macular degeneration (AMD), complement component C3 can be observed in drusen, which is the accumulation of material beneath the retinal pigment epithelium. The complement pathways, via the activation of C3, can upregulate the expression of cytokines and their receptors and the recruitment of inflammatory leukocytes, both of which play an important role in the development of choroidal neovascularization (CNV) in exudative AMD. Laser-induced CNV lesions were found to be significantly smaller in C3(-/-) mice than in wild-type mice. By using flow cytometry, we demonstrated that the proportions of intraocular granulocytes, CD11b(+) F4/80(+) Ly6C(hi) and CD11b(+) F4/80(+) Ly6C(lo) cells, were lower in C3(-/-) mice than in wild-type mice as early as day 1 after laser injury, and the proportions of granulocytes and three macrophage/monocyte subsets were significantly lower on day 3. In contrast, C3(-/-) mice had more granulocytes and CD11b(+) F4/80(+) Ly6C(hi) cells in peripheral blood than wild-type mice after injury. Further, the expression levels of Vegfa164 were upregulated in intraocular Ly6C(hi) macrophages/monocytes of C3(-/-) mice, but not as much as in wild-type mice. Collectively, our data demonstrate that despite a more pronounced induction of systemic inflammation, inhibition of complement factor C3 suppresses CNV by decreasing the recruitment of inflammatory cells to the lesion.

Aims Kruppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study.Methods and results Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis.Conclusion Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts.

The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.

Background: Recent research indicates that the adipose tissue of nonvascularized grafts is completely remodeled within 3 months, although origins of next-generation cells are unclear.Methods: Inguinal fat pads of green fluorescent protein mice and wild-type mice were cross-transplanted beneath the scalp. At 1, 2, 4, and 12 weeks after transplantation, grafted fat was harvested, weighed, and analyzed through immunohistochemistry, whole-mount staining, and flow cytometry of cell isolates. Bone marrow of green fluorescent protein mice was transplanted to wild-type mice (after irradiation). Eight weeks later, these mice also received fat grafts, which were analyzed as well.Results: The majority of host-derived cells detected during remodeling of grafted fat were macrophages (> 90 percent at the early stage; 60 percent at 12 weeks). Cell origins were analyzed at 12 weeks (i.e., when completely regenerated). At this point, mature adipocytes were largely derived from adipose-derived stem/stromal cells of grafts. Although vascular wall constituents were chiefly graft derived, vascular endothelial cells originated equally from graft and host bone marrow. Adipose-derived stem/stromal cells of regenerated fat were an admixture of grafted, host nonbone marrow, and host bone marrow cells.Conclusions: The above findings underscore the importance of adipose stem/stromal cells in the grafted fat for adipocyte regeneration. Host bone marrow and local tissues contributed substantially to capillary networks and provided new adipose-derived stem/stromal cells. An appreciation of mechanisms that are operant in this setting stands to improve clinical outcomes of fat grafting and cell-based therapies.

Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1 alpha were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1 alpha administration rapidly induced MK rupture-dependent thrombopoiesis and increased platelet counts. IL-1 alpha-IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1 alpha determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs.

Aortic dissection and intramural haematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear. Here we show that granulocyte macrophage colony-stimulating factor (GM-CSF) is a triggering molecule for this condition. Transcription factor Kruppel-like factor 6 (KLF6)-myeloid-specific conditional deficient mice exhibit this aortic phenotype when subjected to aortic inflammation. Mechanistically, KLF6 downregulates expression and secretion of GM-CSF. Administration of neutralizing antibody against GM-CSF prevents the condition in these mice. Conversely, administration of GM-CSF in combination with aortic inflammation to wild-type mice is sufficient to induce the phenotype, suggesting the general nature of effects. Moreover, patients with this condition show highly increased circulating levels of GM-CSF, which is also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory molecule causative of this aortopathy, and modulation of this cytokine might be an exploitable treatment strategy for the condition.

Bone metastasis of breast cancer cells is a major concern, as it causes increased morbidity and mortality in patients. Bone tissue-derived CXCL12 preferentially recruits breast cancer cells expressing CXCR4 to bone metastatic sites. Thus, understanding how CXCR4 expression is regulated in breast cancer cells could suggest approaches to decrease bone metastasis of breast tumor cells. Here, we show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) increases responsiveness of breast cancer cells to CXCL12 by promoting up-regulation of CXCR4 in those cells. In addition, we used a xenograft mouse model established by intracardiac injection of tumor cells to show that ANGPTL2 knockdown in breast cancer cells attenuates tumor cell responsiveness to CXCL12 by decreasing CXCR4 expression in those cells, thereby decreasing bone metastasis. Finally, we found that ANGPTL2 and CXCR4 expression levels within primary tumor tissues from breast cancer patients are positively correlated. We conclude that tumor cell-derived ANGPTL2 may increase bone metastasis by enhancing breast tumor cell responsiveness to CXCL12 signaling through up-regulation of tumor cell CXCR4 expression. These findings may suggest novel therapeutic approaches to treat metastatic breast cancer.

Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates beta-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of beta-catenin signalling in hypertensive arterial remodelling. Activation of b-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of beta-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension- induced beta-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-induced activation of beta-catenin signalling becomes a novel therapeutic target to prevent arteriosclerosis in patients with hypertension.

Chronic inflammation underlies an array of chronic, non-communicable diseases, including various cardiovascular and metabolic diseases and cancer. Recent studies have also shown that the mechanisms involved in regulating immunity and metabolism are intricately linked. The term immunometabolism refers to that linkage. In this review, we will discuss immune cell-mediated regulation of metabolic homeostasis and pathology in major metabolic tissues. A particular focus is on macrophages, which play diverse roles in the inflammatory processes induced in non-communicable diseases.

Congenital contractural arachnodactyly (CCA) is a rare connective tissue disorder characterized by marfanoid habitus with camptodactyly. However, cardiac features have rarely been documented in adults. We herein report a sporadic case of CCA in a 20-year-old woman who developed decompensated dilated cardiomyopathy. The patient did not have any mutations in the FBN1 or FBN2 genes, which are most commonly associated with Marfan syndrome and CCA, respectively. Although whether these two diseases are caused by a mutation(s) in the same gene or two different genes remains unknown, this case provides new clinical insight into the cardiovascular management of CCA.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis by activating tumor angiogenesis and tumor cell chemotaxis and invasiveness. However, it is unclear whether ANGPTL2 expression has an effect on tumor cell survival. Here, we explored that possibility by determining whether ANGPTL2 expression altered survival of human colorectal cancer cell lines treated with antineoplastic drugs. To do so, we generated SW480 cells expressing ANGPTL2 (SW480/ANGPTL2) and control (SW480/Ctrl) cells. Apoptosis induced by antineoplastic drug treatment was significantly decreased in SW480/ANGPTL2 compared to control cells. Expression of anti-apoptotic BCL-2 family genes was upregulated in SW480/ANGPTL2 compared to SW480/Ctrl cells. To assess signaling downstream of ANGPTL2 underlying this effect, we carried out RNA sequencing analysis of SW480/ANGPTL2 and SW480/Ctrl cells. That analysis, combined with in vitro experiments, indicated that Syk-PI3K signaling induced expression of BCL-2 family genes in SW480/ANGPTL2 cells. Furthermore, ANGPTL2 increased its own expression in a feedback loop by activating the spleen tyrosine kinase-nuclear factor of activated T cells (Syk-NFAT) pathway. Finally, we observed a correlation between higher ANGPTL2 expression in primary unresectable tumors from colorectal cancer patients who underwent chemotherapy with a lower objective response rate. These findings suggest that attenuating ANGPTL2 signaling in tumor cells may block tumor cell resistance to antineoplastic therapies.

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukaemia integration 1 (Fli1) and Kruppel-like factor 5 (KLF5). In addition to the Fli1 silencing-dependent collagen induction, the simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B-cell activation and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.

Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in I kappa B-zeta, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of I kappa B-zeta in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, I kappa B-zeta-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in I kappa B-zeta-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in I kappa B-zeta-deficient B cells relative to I kappa B-zeta-expressing B cells. These results indicate that I kappa B-zeta regulates TLR-mediated CSR by inducing AID. Moreover, I kappa B-zeta defines differences in the transcriptional regulation of different antibody responses.

The nuclear I kappa B family protein I kappa B-NS is expressed in T cells and plays an important role in Interferon (IFN)-gamma and Interleukin (IL)-2 production. I kappa B-zeta, the most similar homolog of I kappa B-NS, plays an important role in the generation of T helper (Th)17 cells in cooperation with ROR gamma t, a master regulator of Th17 cells. Thus, I kappa B-zeta deficient mice are resistant to Th17-dependent experimental autoimmune encephalomyelitis (EAE). However, I kappa B-zeta deficient mice develop the autoimmune-like Sjogren syndrome with aging. Here we found that I kappa B-NS-deficient (Nfkbid(-/-)) mice show resistance against developing Th17-dependent EAE. We found that Nfkbid(-/-) T cells have decreased expression of IL-17-related genes and ROR gamma t in response to Transforming Growth Factor (TGF)-beta 1 and IL-6 stimulation. Thus, I kappa B-NS plays a pivotal role in the generation of Th17 cells and in the control of Th17-dependent EAE.

In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.

The intestinal epithelium maintains homeostasis by a self-renewal process involving resident stem cells, including Lgr5(+) crypt-base columnar cells, but core mechanisms and their contributions to intestinal cancer are not fully defined. In this study, we examined a hypothesized role for KLF5, a zinc-finger transcription factor that is critical to maintain the integrity of embryonic and induced pluripotent stem cells, in intestinal stem-cell integrity and cancer in the mouse. Klf5 was indispensable for the integrity and oncogenic transformation of intestinal stem cells. In mice, inducible deletion of Klf5 in Lgr5(+) stem cells suppressed their proliferation and survival in a manner associated with nuclear localization of beta-catenin (Catnb), generating abnormal apoptotic cells in intestinal crypts. Moreover, production of lethal adenomas and carcinomas by specific expression of an oncogenic mutant of beta-catenin in Lgr5(+) stem cells was suppressed completely by Klf5 deletion in the same cells. Given that activation of the Wnt/beta-catenin pathway is the most frequently altered pathway in human colorectal cancer, our results argue that KLF5 acts as a fundamental core regulator of intestinal oncogenesis at the stem-cell level, and they suggest KLF5 targeting as a rational strategy to eradicate stem-like cells in colorectal cancer. (C) 2014 AACR.

Abdominal aortic aneurysm (AAA) is a prevalent vascular disease that can progressively enlarge and rupture with a high rate of mortality. Inflammation and active remodeling of the aortic wall have been suggested to be critical in its pathogenesis. Meanwhile, omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) are known to reduce cardiovascular events, but its role in AAA management remains unclear. Here, we show that EPA can attenuate murine CaCl2-induced AAA formation. Aortas from BALB/c mice fed an EPA-diet appeared less inflamed, were significantly smaller in diameter compared to those from control-diet-fed mice, and had relative preservation of aortic elastic lamina. Interestingly, CT imaging also revealed markedly reduced calcification of the aortas after EPA treatment. Mechanistically, MMP2, MMP9, and TNFSF11 levels in the aortas were reduced after EPA treatment. Consistent with this finding, RAW264.7 macrophages treated with EPA showed attenuated Mmp9 levels after TNF-alpha simulation. These results demonstrate a novel role of EPA in attenuating AAA formation via the suppression of critical remodeling pathways in the pathogenesis of AAAs, and raise the possibility of using EPA for AAA prevention in the clinical setting.

The epidemic growth in the prevalence of obesity has made the impact of metabolic syndrome on cardiovascular events increasingly significant. Elevated visceral adiposity, the indispensable component of metabolic syndrome, is thought to play a primary role in the increasing incidence of cardiometabolic disorders. Importantly, obesity is not merely the simple expansion of adipose tissue mass; it also involves the activation of inflammatory processes within visceral adipose tissue. Adipose tissue inflammation on the one hand enhances the production of proinflammatory adipokines and on the other hand increases the release of free fatty acids via the activation of lipolysis. The adipokines and free fatty acids secreted from visceral fat then contribute to a cardiometabolic pathology. We herein summarize recent advances in our understanding of the mechanisms by which visceral obesity leads to the activation of inflammation in cardiovascular and metabolic tissues and promotes cardiometabolic disease. Our focus is on Toll-like receptor 4 signaling and free fatty acids as mediators of chronic inflammation in patients with metabolic syndrome and atherosclerosis.

The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation-related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin a5b1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

Distinct B cell populations, designated regulatory B (B-reg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified B-reg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific Il10 deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue B-reg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support B-reg cell function, and B-reg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue B-reg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that B-reg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.

Objective Obesity is a major risk factor of atherosclerotic cardiovascular disease. Circulating free fatty acid levels are known to be elevated in obese individuals and, along with dietary saturated fatty acids, are known to associate with cardiovascular events. However, little is known about the molecular mechanisms by which free fatty acids are linked to cardiovascular disease.Approach and Results We found that administration of palmitate, a major saturated free fatty acid, to mice markedly aggravated neointima formation induced by carotid artery ligation and that the neointima primarily consisted of phenotypically modulated smooth muscle cells (SMCs). In cultured SMCs, palmitate-induced phenotypic modulation was characterized by downregulation of SMC differentiation markers, such as SM -actin and SM-myosin heavy chain, and upregulation of mediators involved in inflammation and remodeling of the vessel wall, such as platelet-derived growth factor B and matrix metalloproteinases. We also found that palmitate induced the expression of proinflammatory genes via a novel toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor-B/NADPH oxidase 1/reactive oxygen species signaling pathway: nuclear factor-B was activated by palmitate via toll-like receptor 4 and its adapter, MyD88, and once active, it transactivated Nox1, encoding NADPH oxidase 1, a major reactive oxygen species generator in SMCs. Pharmacological inhibition and small interfering RNA-mediated knockdown of the components of this signaling pathway mitigated the palmitate-induced upregulation of proinflammatory genes. More importantly, Myd88 knockout mice were resistant to palmitate-induced exacerbation of neointima formation.Conclusions Palmitate seems to promote neointima formation by inducing inflammatory phenotypes in SMCs.

Melatonin is well known to have a beneficial effect on the cardiovascular system, but it remains to be elucidated whether melatonin has a therapeutic effect on the vascular damage induced by the potential vasoactive substance angiotensin II (Ang II). In this study, the effects of melatonin on Ang II-induced vascular endothelial damage were investigated. In cultured vascular endothelial cells, Ang II stimulation increased ROS generation and inhibited eNOS phosphorylation (Ser1177), both of which were clearly restored by pretreatment with melatonin. The translocation of p47(phox) subunit of NADPH oxidase from the cytosol to plasma membrane was promoted in Ang II-treated vascular endothelial cells, which was canceled by melatonin pretreatment. In Ang II-infused rats, increased ROS generation in the aortic wall and impaired endothelial function of the aortic ring were observed, which were rescued by coadministration of melatonin. In vasculature, melatonin receptor agonist ramelteon had the antioxidative effect in the same manner as melatonin by itself. These findings suggest that melatonin directly ameliorates Ang II-induced vascular endothelial damage partly via its antioxidative properties, providing with us the potential rationale for clinical application of melatonin to the prevention from cardiovascular diseases.

Background: Restenosis after percutaneous coronary intervention (PCI) is still a great concern even in the recent drug-eluting stent (DES) era. As less invasive and sensitive parameter to detect restenosis is needed, this study was aimed to assess whether the clinical implication of temporal change in plasma BNP levels might be a useful indicator of restenosis after DES implantation.Methods and results: 847 consecutive patients who underwent elective PCI using silorimus-eluting sent (SES) between 2005 and 2009 were analyzed. Primary endpoint was subsequent target-lesion revascularization (TLR) after PCI. There was no significant difference in either baseline (TLR + vs. TLR-: 107.2 +/- 172.2 vs. 96.2 +/- 175.5 pg/mL, P=0.53) or follow-up plasma B-type natriuretic peptide (BNP) levels (TLR + vs. TLR-: 88.6 +/- 111.6 vs. 68.5 +/- 226.0 pg/mL, P=0.35) between patients with and without subsequent TLR. Conversely, ratio of follow-up to baseline BNP was significantly higher in patients with TLR (TLR + vs. TLR-: 1.55 +/- 1.58 vs. 1.07 +/- 1.04, P<0.001). Multivariate analysis using logistic regression showed log transformed BNP-ratio was an independent predictor of TLR (adjusted odds ratio (aOR): 1.94, 95%CI: 1.42-2.66, P<0.001). A closer relationship between BNP elevation greater than 2-fold and subsequent TLR was found (aOR: 2.69, 95%CI: 1.27-5.69, P<0.009). Furthermore, propensity score matching analysis showed that the incidence of subsequent TLR was significantly higher in patients with BNP elevation (P<0.001).Conclusion: Serial measurement of plasma BNP levels and its change might be a useful approach to predict restenosis in patients without typical chest symptoms receiving SES. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Biodegradable composite matrices comprising poly-(L-lactic acid) (PLLA) and citric acid-crosslinked alkali-treated gelatin (AlGelatin) with endothelialization, antithrombogenic, and drug release properties were prepared. The characterization of composite matrices with various mixing ratios was performed by evaluating their swelling ratio, endothelial cell culture, antithrombogenic tests, and drug release behavior. Tamibarotene (Am80), which specifically inhibits smooth muscle cell proliferation, was employed as the drug. The swelling ratio of composite matrices decreased as the PLLA content decreased. The number of endothelial cells cultured on the surfaces of composite matrices was maximal at the PLLA/AlGelatin-TSC ratio of 80/20. Antithrombogenic tests revealed that the levels of platelets and fibrin network formation decreased as the AlGelatin-TSC content increased. The Am80 release test indicated that the release rate decreased as PLLA content increased. Using the resulting composite matrix, Am80-eluting stents possessing a smooth surface and a coating thickness of approximate to 15 m were successfully obtained. Am80 was continuously released from the resulting stent at approximate to 40%, up to 28 days without burst release. Therefore, Am80-eluting stent with its antithrombogenic and endothelialization properties has great potential for clinical use. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.