Sahohime Matsumoto

Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload

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 Krüppel-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 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.

Excess Lymphangiogenesis Cooperatively Induced by Macrophages and CD4+ T Cells Drives the Pathogenesis of Lymphedema

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.

A heart-brain-kidney network controls adaptation to cardiac stress through tissue macrophage activation.

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 Ly6Clo 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 β2-receptor blockade or CD-cell-specific deficiency of the transcription factor KLF5. Moreover, we identified amphiregulin as an essential cardioprotective mediator produced by cardiac Ly6Clo 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.

Screening and determination of gene function using randomized ribozyme and siRNA libraries.

Rapid progress in the sequencing of the genomes of model organisms, such as the mouse, rat, nematode, fly, and Arabidopsis, as well as the human genome, has provided abundant sequence information, but functions of long stretches of these genomes remain to be determined. RNA-based technologies hold promise as tools that allow us to identify the specific functions of portions of these genomes. In particular, catalytic RNAs, known also as ribozymes, can be engineered for optimization of their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, with subsequent screening for phenotypic changes, can be used for the rapid identification ofa gene function. Ribozyme technology complements another RNA-based tool for the determination of gene function, which is based on libraries of small interfering RNAs (siRNAs).

Genome-Wide Screening by Using Small-Interfering RNA-Expression Libraries

RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is silenced by double-stranded RNA (dsRNA) in a sequence-specific manner. This technology has the potential to affect all aspects of target discovery and validation. With the completion of the human genome, it is now possible to design small-interfering RNA (siRNA) libraries targeting every human gene. Specific siRNAs, libraries containing a pathway, gene family, or gene set of interest, are expected to unsecure new targets in pathways of therapeutic interest. Here, we highlight the potential of siRNA screens for target identification by using cell-based assays.

Stromal Vascular Cells

In addition to being an energy reservoir, adipose tissue is now regarded as an active endocrine organ that is crucial to the maintenance of systemic energy homeostasis. However, obese adipose tissue, particularly visceral adipose tissue, is thought to be a key contributor to the development of insulin resistance and the clinical consequences of metabolic syndrome, including cardiovascular disease. In addition to the adipocytes themselves, adipose tissue also contains vascular cells and a variety of other cells within the stroma, as well as extracellular matrix (ECM), which provides mechanical support for the tissue. These stromal cells and ECM play important roles in the dynamic processes involved in adipose tissue development, expansion, and inflammation.