Katsuhito Fujiu

Renal collecting duct epithelial cells regulate inflammation in tubulointerstitial damage in mice

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 Krüppel-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-β production--phenotypes indicative of M2-type macrophages. Interestingly, KLF5, in concert with C/EBPα, 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.

SUMOylation of Kruppel-like transcription factor 5 acts as a molecular switch in transcriptional programs of lipid metabolism involving PPAR-delta

Obesity and metabolic syndrome are increasingly recognized as major risk factors for cardiovascular disease. Herein we show that Krüppel-like transcription factor 5 (KLF5) is a crucial regulator of energy metabolism. Klf5+/− mice were resistant to high fat–induced obesity, hypercholesterolemia and glucose intolerance, despite consuming more food than wild-type mice. This may in part reflect their enhanced energy expenditure. Expression of the genes involved in lipid oxidation and energy uncoupling, including those encoding carnitine-palmitoyl transferase-1b (Cpt1b) and uncoupling proteins 2 and 3 (Ucp2 and Ucp3), was upregulated in the soleus muscles of Klf5+/− mice. Under basal conditions, KLF5 modified with small ubiquitin-related modifier (SUMO) proteins was associated with transcriptionally repressive regulatory complexes containing unliganded peroxisome proliferator–activated receptor-δ (PPAR-δ) and co-repressors and thus inhibited Cpt1b, Ucp2 and Ucp3 expression. Upon agonist stimulation of PPAR-δ, KLF5 was deSUMOylated, and became associated with transcriptional activation complexes containing both the liganded PPAR-δ and CREB binding protein (CBP). This activation complex increased the expression of Cpt1b, Ucp2 and Ucp3. Thus, SUMOylation seems to be a molecular switch affecting function of KLF5 and the transcriptional regulatory programs governing lipid metabolism.

Bone Marrow–Derived Cells Contribute to Vascular Inflammation but Do Not Differentiate Into Smooth Muscle Cell Lineages

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 &agr;-actin, we demonstrated that although some bone marrow–derived cells express SM &agr;-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 &agr;-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 &agr;-actin in the injured artery. Expression of inflammation-related genes was significantly higher in neointimal subregions rich in bone marrow–derived SM &agr;-actin+ cells than in other regions. Conclusions— It appears that bone marrow–derived SM &agr;-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.

Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis

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 leukemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to Fli1 silencing-dependent collagen induction, 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.

Thoracic Spinal Cord Stimulation for Heart Failure as a Restorative Treatment (SCS HEART study): first-in-man experience

BACKGROUND Preclinical studies suggest that neuromodulation with thoracic spinal cord stimulation (SCS) improves left ventricular (LV) function and remodeling in systolic heart failure (HF). OBJECTIVE The purpose of this study was to evaluate the safety and efficacy of a SCS system for the treatment of systolic HF. METHODS We performed a prospective, multicenter pilot trial in patients with New York Heart Association (NYHA) class III HF, left ventricular ejection fraction (LVEF) 20%-35%, and implanted defibrillator device who were prescribed stable optimal medical therapy. Dual thoracic SCS leads were used at the T1-T3 level. The device was programmed to provide SCS for 24 hours per day (50 Hz at pulse width 200 μs). RESULTS We enrolled 22 patients from 5 centers:17 patients underwent implantation of a SCS device and 4 patients who did not fulfill the study criteria served as nontreated controls. No deaths or device-device interactions were noted during the 6-month period in the 17 SCS-treated patients. Fifteen of 17 completed the efficacy endpoint assessments: composite score improved by 4.2 ± 1.3, and 11 patients (73%) showed improvement in ≥4 of 6 efficacy parameters. There was significant improvement in NYHA class (3.0 vs 2.1, P = .002; 13/17 improved); Minnesota Living with Heart Failure Questionnaire (42 ± 26 vs 27 ± 22, P = .026; 12/17 improved); peak maximum oxygen consumption (14.6 ± 3.3 vs 16.5 ± 3.9 mL/kg/min, P = .013; 10/15 improved); LVEF (25% ± 6% vs 37% ± 8%, P<.001; 14/16 improved); and LV end-systolic volume (174 ± 57 vs 137 ± 37 mL, P = .002; 11/16 improved) but not in N-terminal prohormone brain natriuretic peptide. No such improvements were observed in the 4 nontreated patients. CONCLUSION The results of this first-in-human trial suggest that high thoracic SCS is safe and feasible and potentially can improve symptoms, functional status, and LV function and remodeling in patients with severe, symptomatic systolic HF.

Cardioprotective function of cardiac macrophages

The heart is composed of several cell types including cardiomyocytes, cardiac fibroblasts, endothelial, and smooth muscle cells. In addition to these major cell types, cardiac macrophages are also present in small numbers under physiological conditions. Recently, the resident macrophage is considered to have vital functions in the maintenance of tissues and homeostasis in many organs, including brain, liver, adipose tissue, lymphatic tissue, and intestinal tract. However, detailed functions of the cardiac resident macrophage are not fully understood. Although the removal of debris arising from damaged cardiomyocytes and pro-inflammatory effects after heart injuries are conventional tasks of cardiac macrophages (classically activated macrophage or M1 macrophage), novel functions like anti-inflammatory roles, adaptive response, and tissue maintenance have also been reported in recent years. Macrophages that possess these novel functions are generally so-called M2 macrophages, which are alternatively activated and show anti-inflammatory phenotype under pathological conditions. In this review, we focus on the cardioprotective function of the cardiac macrophage and discuss in light of unveiled fundamental functions of macrophages that have been also found in other organs.

Contributions of cardiomyocyte–cardiac fibroblast–immune cell interactions in heart failure development

The heart contains various types of cells, including cardiomyocytes, cardiac fibroblasts, many kinds of immune cells and vascular cells. Initial studies mainly focused on cardiomyocytes, which directly reflect the contractile function of the heart. Recently, pivotal functions of cardiac fibroblasts have been revealed in the maintenance of cardiac function, physiological cardiac remodeling after heart stress and pathological remodeling using genetically engineered mouse models, like the fibroblast-specific gene knockout mouse, bone marrow transplantation and immune cell-specific gene knockout. Moreover, chronic inflammation is considered to be a basic pathological mechanism that underlies various diseases, including heart failure. In the development of heart failure, the contributions of immune cells like T lymphocytes and monocyte/macrophage lineage cells have been also reported. Immune cells have diverse and multiple functions in regulating both pro-inflammatory effects and the resolution of heart failure. On the one hand, immune cells have protective effects to compensate for and overcome heart stresses. On the other hand, they also contribute to sustained inflammation and result in the development of heart failure. These observations prompted a shift in the heart-related studies to include the complex communications between cardiomyocytes and other kinds of cardiac cells, including inflammatory cells residing in or recruited to the heart. This review will summarize the current knowledge regarding cell–cell interactions during cardiac remodeling and the development of heart failure. We will especially focus on the interactions among cardiomyocytes, cardiac fibroblasts and immune cells.

Regulatory polymorphism in transcription factor KLF5 at the MEF2 element alters the response to angiotensin II and is associated with human hypertension

Kru¨ppel‐like factor 5 (KLF5) is a zincfinger‐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 ω-3 polyunsaturated fatty acid, eicosapentaenoic acid, attenuates abdominal aortic aneurysm development via suppression of tissue remodeling.

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, ω-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-α 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.

Saturated Fatty Acid Palmitate Aggravates Neointima Formation by Promoting Smooth Muscle Phenotypic Modulation

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 &agr;-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-&kgr;B/NADPH oxidase 1/reactive oxygen species signaling pathway: nuclear factor-&kgr;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.