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Background: Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity in atherosclerotic plaques, and among other approaches, has been modeled in vitro by cholesterol loading. Methods: Meta-analysis of scRNA-seq data from VSMC lineage traced cells across five experiments of murine atherosclerosis was performed. In vivo expression profiles were compared to three in vitro datasets of VSMCs loaded with cholesterol and three datasets of polarized macrophages. Results: We identified 24 cell clusters in the meta-analysis of single cells from mouse atherosclerotic lesions with notable heterogeneity across studies, especially for macrophage populations. Trajectory analysis of VSMC lineage positive cells revealed several possible paths of state transitions with one traversing from contractile VSMC to macrophages by way of a proliferative cell cluster. Transcriptome comparisons between in vivo and in vitro states underscored that data from three in vitro cholesterol-treated VSMC experiments did not mirror cell state transitions observed in vivo. However, all in vitro macrophage profiles analyzed (M1, M2, and oxLDL) were more similar to in vivo profiles of macrophages than in vitro VSMCs were to in vivo profiles of VSMCs. oxLDL loaded macrophages showed the most similarity to in vivo states. In contrast to the in vitro data, comparison between mouse and human in vivo data showed many similarities. Conclusions: Identification of the sources of variation across single cell datasets in atherosclerosis will be an important step towards understanding VSMC fate transitions in vivo. Also, we conclude that cholesterol-loading in vitro is insufficient to model the VSMC cell state transitions observed in vivo, which underscores the need to develop better cell models. Mouse models, however, appear to reproduce a number of the features of VSMCs in human plaques

Visual Abstract, Highlights • KUS121 was developed to selectively inhibit the adenosine triphosphatase activity of valosin-containing protein without affecting other cellular functions of valosin-containing protein. • KUS121 preserved adenosine triphosphate levels, reduced endoplasmic reticulum stress, and suppressed cell death in H9C2 rat cardiomyoblast cells, treated with tunicamycin or hydrogen peroxide, or cultured in glucose-free medium. • In murine ischemia and reperfusion injury models, KUS121 treatment after reperfusion attenuated the infarcted size and preserves cardiac function by maintaining adenosine triphosphate levels and reducing ER stress. • In porcine ischemia and reperfusion injury models, intracoronary administration of KUS121 also attenuated the infarcted area in a dose-dependent manner. • These results indicated that KUS121 is a promising novel therapeutic agent for myocardial infarction., Summary No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.

Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3′-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

Atherosclerosis is a disease of chronic inflammation. We investigated the roles of the cytokines IL-4 and IL-13, the classical activators of STAT6, in the resolution of atherosclerosis inflammation. Using Il4-/-Il13-/- mice, resolution was impaired, and in control mice, in both progressing and resolving plaques, levels of IL-4 were stably low and IL-13 was undetectable. This suggested that IL-4 is required for atherosclerosis resolution, but collaborates with other factors. We had observed increased Wnt signaling in macrophages in resolving plaques, and human genetic data from others showed that a loss-of-function Wnt mutation was associated with premature atherosclerosis. We now find an inverse association between activation of Wnt signaling and disease severity in mice and humans. Wnt enhanced the expression of inflammation resolving factors after treatment with plaque-relevant low concentrations of IL-4. Mechanistically, activation of the Wnt pathway following lipid lowering potentiates IL-4 responsiveness in macrophages via a PGE2/STAT3 axis.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33−/− mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress., 褐色脂肪細胞の燃焼を促す新たなメカニズムを解明 --体の熱産生にマイクロRNA-33が関与--. 京都大学プレスリリース. 2021-02-17.

Objective— Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results— Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E–deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions— Our data demonstrated critical roles of SREBF1 -miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.

Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown. In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn−/− mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls. Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.

Objective— Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. Approach and Results— MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II– and calcium chloride–induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride–induced AAA walls in miR-33 −/− mice. In vitro experiments revealed that peritoneal macrophages from miR-33 −/− mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33 −/− mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33 −/− mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33–deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. Conclusions— These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.

Rationale: Heart failure and atherosclerosis share the underlying mechanisms of chronic inflammation followed by fibrosis. A highly conserved microRNA (miR), miR-33, is considered as a potential therapeutic target for atherosclerosis because it regulates lipid metabolism and inflammation. However, the role of miR-33 in heart failure remains to be elucidated. Objective: To clarify the role of miR-33 involved in heart failure. Methods and Results: We first investigated the expression levels of miR-33a/b in human cardiac tissue samples with dilated cardiomyopathy. Increased expression of miR-33a was associated with improving hemodynamic parameters. To clarify the role of miR-33 in remodeling hearts, we investigated the responses to pressure overload by transverse aortic constriction in miR-33–deficient (knockout [KO]) mice. When mice were subjected to transverse aortic constriction, miR-33 expression levels were significantly upregulated in wild-type left ventricles. There was no difference in hypertrophic responses between wild-type and miR-33KO hearts, whereas cardiac fibrosis was ameliorated in miR-33KO hearts compared with wild-type hearts. Despite the ameliorated cardiac fibrosis, miR-33KO mice showed impaired systolic function after transverse aortic constriction. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart. Deficiency of miR-33 impaired cardiac fibroblast proliferation, which was considered to be caused by altered lipid raft cholesterol content. Moreover, cardiac fibroblast–specific miR-33–deficient mice also showed decreased cardiac fibrosis induced by transverse aortic constriction as systemic miR-33KO mice. Conclusion: Our results demonstrate that miR-33 is involved in cardiac remodeling, and it preserves lipid raft cholesterol content in fibroblasts and maintains adaptive fibrotic responses in the remodeling heart.

Background Micro RNA (miR)‐33 targets cholesterol transporter ATP ‐binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR‐33 family, miR‐33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR‐33a and miR‐33b using genetically modified mice. We generated 4 strains with or without miR‐33a and miR‐33b. Comparison between mice with only miR‐33a (wild‐type mice) and mice with only miR‐33b (miR‐33a −/− /miR‐33b +/+ ) revealed the dominant expression of miR‐33b in the liver. To evaluate the whole body atherogenic potency of miR‐33a and miR‐33b, we developed apolipoprotein E–deficient/miR‐33a +/+ /miR‐33b −/− mice and apolipoprotein E–deficient/miR‐33a −/− /miR‐33b +/+ mice. With a high‐fat and high‐cholesterol diet, the apolipoprotein E–deficient/miR‐33a −/− /miR‐33b +/+ mice developed increased atherosclerotic plaque versus apolipoprotein E–deficient/miR‐33a +/+ /miR‐33b −/− mice, in line with the predominant expression of miR‐33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR‐33a and miR‐33b in bone marrow cells. Conclusions The miR‐33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR‐33b would be more potent than miR‐33a.

Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.

Atherosclerosis is a leading cause of death worldwide in industrialized countries. Disease progression and regression are associated with different activation states of macrophages derived from inflammatory monocytes entering the plaques. The features of monocyte-to-macrophage transition and the full spectrum of macrophage activation states during either plaque progression or regression, however, are incompletely established. Here, we use a combination of single-cell RNA sequencing and genetic fate mapping to profile, for the first time to our knowledge, plaque cells derived from CX3CR1(+) precursors in mice during both progression and regression of atherosclerosis. The analyses revealed a spectrum of macrophage activation states with greater complexity than the traditional M1 and M2 polarization states, with progression associated with differentiation of CXC3R1(+) monocytes into more distinct states than during regression. We also identified an unexpected cluster of proliferating monocytes with a stem cell–like signature, suggesting that monocytes may persist in a proliferating self-renewal state in inflamed tissue, rather than differentiating immediately into macrophages after entering the tissue.

LXRα activation in macrophages enhances regression of atherosclerotic plaques in mice by regulating genes crucial for cholesterol efflux, cell motility and inflammation. Diabetes, however, impairs plaque regression in mice. LXRα is phosphorylated at serine 198 (pS198), which affects the expression of genes controlling inflammation, lipid metabolism and cell movement. We hypothesize that LXRα function is affected by hyperglycemia through changes in LXRα pS198. Indeed, macrophages cultured in diabetes relevant high glucose versus normal glucose display alterations in LXR-dependent gene expression and increased LXRα pS198. We therefore examined the consequence of disrupting LXRα phosphorylation (S196A in mouse LXRα) during regression of atherosclerosis in normal and diabetic mice. We find that phosphorylation deficient LXRα S196A reduces macrophage retention in plaques in diabetes, which is predicted to be anti-atherogenic and enhance plaque regression. However, this favorable effect on regression is masked by increased monocyte infiltration in the plaque attributed to leukocytosis in LXRα S196A mice. RNA-seq of plaque macrophages from diabetic S196A mice shows increased expression of chemotaxis and decreased expression of cell adhesion genes, consistent with reduced macrophage retention by LXRα S196A. Thus, the non-phosphorylated form of LXRα precludes macrophage retention in the plaque. Our study provides the first evidence for a physiological role of LXRα phosphorylation in modulating atherosclerosis regression. Compounds that prevent LXRα phosphorylation or ligands that induce the conformation of non-phosphorylated LXRα may selectively enhance macrophage emigration from atherosclerotic plaques.

MicroRNA 33 (miR-33) targets ATP-binding cassette transporter A1 (ABCA1), and its deficiency increases serum high-density lipoprotein (HDL)-cholesterol (HDL-C) and ameliorates atherosclerosis. Although we previously reported that miR-33 deficiency increased peripheral Ly6Chigh monocytes on an ApoE-deficient background, the effect of miR-33 on the monocyte population has not been fully elucidated, especially in a wild-type (WT) background. We found that Ly6Chigh monocytes in miR-33−/− mice were decreased in peripheral blood and increased in bone marrow (BM). Expansion of myeloid progenitors and decreased apoptosis in Lin− Sca1+ c-Kit+ (LSK) cells were observed in miR-33−/− mice. A BM transplantation study and competitive repopulation assay revealed that hematopoietic miR-33 deficiency caused myeloid expansion and increased peripheral Ly6Chigh monocytes and that nonhematopoietic miR-33 deficiency caused reduced peripheral Ly6Chigh monocytes. Expression of high-mobility group AT-hook 2 (HMGA2) targeted by miR-33 increased in miR-33-deficient LSK cells, and its knockdown abolished the reduction of apoptosis. Transduction of human apolipoprotein A1 and ABCA1 in WT mouse liver increased HDL-C and reduced peripheral Ly6Chigh monocytes. These data indicate that miR-33 deficiency affects distribution of inflammatory monocytes through dual pathways. One pathway involves the enhancement of Hmga2 expression in hematopoietic stem cells to increase Ly6Chigh monocytes, and the other involves the elevation of HDL-C to decrease peripheral Ly6Chigh monocytes.

Atherosclerosis can be induced by the injection of a gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 (PCSK9)–encoding adeno-associated viral vector (AAVmPCSK9), avoiding the need for knockout mice models, such as low-density lipoprotein receptor deficient mice. As regression of atherosclerosis is a crucial therapeutic goal, we aimed to establish a regression model based on AAVmPCSK9, which will eliminate the need for germ-line genetic modifications. C57BL6/J mice were injected with AAVmPCSK9 and were fed with Western diet for 16 weeks, followed by reversal of hyperlipidemia by a diet switch to chow and treatment with a microsomal triglyceride transfer protein inhibitor (MTPi). Sixteen weeks following AAVmPCSK9 injection, mice had advanced atherosclerotic lesions in the aortic root. Surprisingly, diet switch to chow alone reversed hyperlipidemia to near normal levels, and the addition of MTPi completely normalized hyperlipidemia. A six week reversal of hyperlipidemia, either by diet switch alone or by diet switch and MTPi treatment, was accompanied by regression of atherosclerosis as defined by a significant decrease of macrophages in the atherosclerotic plaques, compared to baseline. Thus, we have established an atherosclerosis regression model that is independent of the genetic background.

Cholesterol homeostasis is a tightly regulated process. Previous work has been dominated by sterol-response element—related mechanisms, but the influence of micro-RNAs (miRs) is increasingly recognized, with miR-223 becoming a recent member of this group. Several micro-RNAs (miRNAs or miR-) have been reported to be involved in cholesterol homeostasis.1–4 In a recent study, Vickers et al5 presented miR-223 as an important new player in this arena, one that they propose to be a coordinator of cholesterol homeostasis.5 This miRNA had been previously found to be rich in myeloid cells, where it regulates differentiation and inflammation,6,7 but the authors found that its abundance in hepatic cells is at least comparable with that of many functionally validated miRNAs. A relationship between miR-223 and cholesterol metabolism was suggested by finding its expression in human hepatoma (Huh7) cells was positively associated with intracellular cholesterol levels. In a series of studies in Huh7 cells, it was then shown that miR-223 (1) inhibited high-density lipoprotein cholesterol (HDL-C) uptake, (2) promoted cholesterol efflux, and (3) suppressed cholesterol biosynthesis. The authors also showed increased cholesterol levels in liver and plasma of miR-223 knockout mice, extending its involvement in cholesterol homeostasis in vitro to the in vivo setting. The experimental approaches taken are well established in the miRNA field and were coupled to a productive use of bioinformatics. This included investigating whether the effects of miR-223 on a particular RNA were direct, by cloning into a luciferase reporter plasmid the putative target sequence in the 3′-UTR of that mRNA (or this sequence deleted in nucleotides required for miR-223 binding). The effect of exogenously supplied miR-223 on luciferase activity in transfected HEK293 cells was then measured. Thus, they determined that the molecular mechanisms for the functional reductions in HDL-C uptake and cholesterol biosynthesis …

Sterol regulatory element-binding protein 2 (SREBP-2) transcription factor has been identified as a key protein in cholesterol metabolism through the transactivation of the LDL receptor and cholesterol biosynthesis genes. Here, we generated mice lacking microRNA (miR)-33, encoded by an intron of the Srebp2, and showed that miR-33 repressed the expression of ATP-binding cassette transporter A1 (ABCA1) protein, a key regulator of HDL synthesis by mediating cholesterol efflux from cells to apolipoprotein A (apoA)-I. In fact, peritoneal macrophages derived from miR-33-deficient mice showed a marked increase in ABCA1 levels and higher apoA-I-dependent cholesterol efflux than those from WT mice. ABCA1 protein levels in liver were also higher in miR-33-deficient mice than in WT mice. Moreover, miR-33-deficient mice had significantly higher serum HDL cholesterol levels than WT mice. These data establish a critical role for miR-33 in the regulation of ABCA1 expression and HDL biogenesis in vivo., コレステロールの調節メカニズムを解明（蛋白をコードしない短いRNAであるmicroRNA-33を欠損させると血中HDLが上昇することを見いだした）. 京都大学プレスリリース. 2010-09-21.

Atherosclerosis is a leading cause of death worldwide in industrialized countries. We have previously shown that disease progression and regression are strongly associated with the M1/M2 activation status of macrophages derived from inflammatory monocytes in the plaques with large differences in transcriptional profiles. However, the features of monocyte to macrophage transitions during either plaque progression or regression are poorly understood. Here, we use a combination of single-cell RNA sequencing and tamoxifen-inducible fate mapping approaches to profile 3157 and 2198 arterial cells derived from CX3CR1+ precursor in atherosclerotic mice during plaque progression and regression. There was surprising heterogeneity among the cell populations, despite derivation from a common precursor. We compared the distinct cell clusters with the IMMGEN database to define each population and then used pseudo-time analysis to model the differentiation process from monocyte precursors to activated macrophages under progression and regression conditions. Notably, distinct cell clusters with a type 1 interferon (IFN) signature was observed during progression, which was significantly rare in regression. Whereas a unique cluster in regression was marked by increased expression of various heat shock proteins (HSP). Overall, this study highlights previously unidentified genes and programs in macrophages associated with atherosclerosis progression and regression, uncovering the unexpected plasticity of CX3CR1+ monocyte derived cells.

Aims A significant increase in congestive heart failure (CHF) was reported when the anti-ErbB2 antibody trastuzumab was used in combination with the chemotherapy drug doxorubicin (Dox). The aim of the present study was to investigate the role(s) of miRNAs in acute Dox-induced cardiotoxicity. Methods and results Neuregulin-1-ErbB signalling is essential for maintaining adult cardiac function. We found a significant reduction in ErbB4 expression in the hearts of mice after Dox treatment. Because the proteasome pathway was only partially involved in the reduction of ErbB4 expression, we examined the involvement of microRNAs (miRs) in the reduction of ErbB4 expression. miR-146a was shown to be up-regulated by Dox in neonatal rat cardiac myocytes. Using a luciferase reporter assay and overexpression of miR-146a, we confirmed that miR-146a targets the ErbB4 3′UTR. After Dox treatment, overexpression of miR-146a, as well as that of siRNA against ErbB4, induced cell death in cardiomyocytes. Re-expression of ErbB4 in miR-146a-overexpressing cardiomyocytes ameliorated Dox-induced cell death. To examine the loss of miR-146a function, we constructed ‘decoy’ genes that had tandem complementary sequences for miR-146a in the 3′UTR of a luciferase gene. When miR-146a ‘decoy’ genes were introduced into cardiomyocytes, ErbB4 expression was up-regulated and Dox-induced cell death was reduced. Conclusion These findings suggested that the up-regulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from CHF.

MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADP-ribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3'-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart.

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3'UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.

The accumulation of visceral adipose tissue is closely associated with insulin resistance and metabolic syndrome. Therefore, it is important to identify genes that are required for adipocyte differentiation. To identify genes that are required for the differentiation of 3T3-L1 preadipocytes into mature adipocytes, we used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 cell lines that lose their ability to differentiate into mature adipocytes. One of the genes identified was TG-interacting factor (TGIF), a DNA binding homeodomain protein that has been demonstrated to suppress Smad-mediated activation of transforming growth factor beta (TGF-beta)-regulated transcription. In the TGIF-disrupted clone of 3T3-L1 preadipocytes, the rate of differentiation into mature adipocytes was clearly reduced compared with that in the wild-type clone. Suppression of TGIF by lentivirus-mediated RNAi also inhibited the differentiation of 3T3-L1 cells. Insulin specifically increased the abundance of TGIF protein, primarily by enhancing its stability. In addition, insulin caused the rapid accumulation of TGIF in the nuclei. Forced expression of exogenous TGIF repressed both endogenous and overexpressed Smad2/3-mediated promoter activity in 3T3-L1. These findings suggest that insulin specifically antagonizes TGF-beta signaling in preadipocytes by stabilizing the putative Smad transcriptional corepressor TGIF and regulates adipocyte differentiation.

Objective— We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques, it has become appreciated that ≈40% of cells classified as macrophages by histological markers may be of VSMC origin. Therefore, we sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results— VSMC of mouse (or human) origin were incubated with cyclodextrin–cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions— Cholesterol loading of VSMC converts them to a macrophage-appearing state by downregulating the miR-143/145–myocardin axis. Although these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.

[Background]The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. [Methods and Results]We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβ[kd] TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβ[kd] TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβ[kd] TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. [Conclusions]These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.

As drug therapy is of limited efficacy in the treatment of heart diseases related to loss of cardiomyocytes, which have very poor division potential, regenerative medicine is expected to be a new strategy to address regenerative treatment in cardiac diseases. To achieve myocardial regeneration, elucidation of the mechanism of myocardial differentiation from stem cells is essential. Myocardial differentiation from embryonic pluripotent stem cells has been investigated worldwide, and remarkable developments such as establishment of induced pluripotent stem cells and transformation of somatic cells to cardiomyocytes have recently been made, markedly changing the strategy of regenerative medicine. At the same time, the close involvement of microRNA in the maintenance, proliferation, differentiation, and reprogramming of these stem cells has been revealed. In this report, microRNA is outlined, focusing on its role in myocardial differentiation.

Tissue-specific patterns of gene expression play an important role in the distinctive features of each organ. Small CTD phosphatases (SCPs) 1-3 are recruited by repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. SCPs are highly expressed in the heart and contain microRNAs (miR)-26b, 26a-2, and 26a-1 with the same seed sequence in their introns. Therefore, we tried to investigate the roles of miR-26b and its host gene in neonatal rat cardiomyocytes. Overexpression of miR-26b suppressed the mRNA expression levels of ANF, βMHC, and ACTA1 and reduced the cell surface area in cardiomyocytes. We confirmed that miR-26b targets the 3' untranslated region (3'UTR) of GATA4 and canonical transient receptor potential channel (TRPC) 3. Conversely, silencing of the endogenous miR-26b family enhanced the expression levels of TRPC3 and GATA4. On the other hand, overexpression of SCP1 induced the mRNA expression of ANF and βMHC and increased the cell surface area in cardiomyocytes. Next, we compared the effect of overexpression of SCP1 with its introns and SCP1 cDNA to observe the net function of SCP1 expression on cardiac hypertrophy. When the expression levels of SCP1 were the same, the overexpression of SCP1 cDNA had a greater effect at inducing cardiac hypertrophy than SCP1 cDNA with its intron. In conclusion, SCP1 itself has the potential to induce cardiac hypertrophy; however, the effect is suppressed by intronic miR-26b in cardiomyocytes. miR-26b has an antagonistic effect on its host gene SCP1.

As drug therapy is of limited efficacy in the treatment of heart diseases related to loss of cardiomyocytes, which have very poor division potential, regenerative medicine is expected to be a new strategy to address regenerative treatment in cardiac diseases. To achieve myocardial regeneration, elucidation of the mechanism of myocardial differentiation from stem cells is essential. Myocardial differentiation from embryonic pluripotent stem cells has been investigated worldwide, and remarkable developments such as establishment of induced pluripotent stem cells and transformation of somatic cells to cardiomyocytes have recently been made, markedly changing the strategy of regenerative medicine. At the same time, the close involvement of microRNA in the maintenance, proliferation, differentiation, and reprogramming of these stem cells has been revealed. In this report, microRNA is outlined, focusing on its role in myocardial differentiation.

Background— Recently, elevation of circulating muscle-specific microRNA (miRNA) levels has been reported in patients with acute myocardial infarction. However, it is still unclear from which part of the myocardium or under what conditions miRNAs are released into circulating blood. The purpose of this study was to identify the source of elevated levels of circulating miRNAs and their function in cardiovascular diseases. Methods and Results— Serum levels of miRNA (miR)-1 and miR-133a were increased significantly in patients not only with acute myocardial infarction but also with unstable angina pectoris and Takotsubo cardiomyopathy without elevation of serum creatine phosphokinase or cardiac troponin. MicroRNA microarray analysis of the heart from a mouse model of myocardial infarction indicated that the levels of miR-1, miR-133a, miR-208a, and miR-499 were significantly reduced in the infarcted myocardium. In situ hybridization of miR-133a also showed that miR-133a levels were very low in the infarcted and peri-infarcted myocardium. It has been shown that circulating miRNAs are localized inside exosomes, which are released after Ca 2+ stimulation. We stimulated H9c2 cardiomyoblasts with A23187 and measured miR-133a levels in the exosome fraction of the culture medium. A23187 induced a dose-dependent release of miR-133a, and significant elevation was observed only at concentrations where dead cells were detected. We also found that miR-133a–containing exosomes reduced the luciferase activity of 293FT cells transfected with an miR-133a sensor vector. Conclusions— These results suggest that elevated levels of circulating miR-133a in patients with cardiovascular diseases originate mainly from the injured myocardium. Circulating miR-133a can be used as a marker for cardiomyocyte death, and it may have functions in cardiovascular diseases.

MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene expression and play important roles in gene regulation in the heart. To examine the role of miRNAs in the expression of the two isoforms of the cardiac myosin heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility, endogenous miRNAs were downregulated in neonatal rat ventricular myocytes (NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer, an essential enzyme for miRNA biosynthesis, and MHC expression levels were examined. As a result, Dicer siRNA could downregulate endogenous miRNAs simultaneously and the β-MHC gene but not α-MHC, which implied that specific miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC protein was downregulated by silencing of endogenous miR-27a. Through a bioinformatics screening using TargetScan, we identified thyroid hormone receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation via thyroid hormone signaling and to be upregulated during the differentiation of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC gene expression by targeting TRβ1 in cardiomyocytes.

Retrovirus insertion-mediated random mutagenesis was applied in 3T3-L1 preadipocyte cells to better understand the molecular basis of obesity (the expansion of individual adipocytes). We found that tryptophan hydroxylase-1, a rate-limiting enzyme for the synthesis of serotonin (5-HT), is expressed in adipocytes and is required for their differentiation. A 5-HT type 2A receptor (5-HT(2A)R) antagonist, ketanserin, and a 5-HT(2c)R antagonist, SB-242084, inhibited adipocyte differentiation. Because 5-HT(2c)R mRNA levels are up-regulated during adipocyte differentiation and micro-RNA (miR)-448 is located in the fourth intron of Htr2c, we also studied the role of miR-448 in 3T3-L1 cells. Through a bioinformatics approach, Kruppel-like factor 5 (KLF5) was identified as a potential target of miR-448. Using a luciferase reporter assay, we confirmed that miR-448 targets the Klf5 3'-intranslated region. Overexpression of miR-448 reduced the expression of Klf5 and adipocyte differentiation, which was confirmed by the reduced expression of adipogenic genes and triglyceride accumulation. To examine the loss of miR-448 function, we constructed a decoy gene that had tandem complementary sequences for miR-448 in the 3'-untranslated region of a luciferase gene under the control of a cytomegalovirus promoter. When the miR-448 decoy gene was introduced into 3T3-L1 preadipocytes, KLF5 was up-regulated and triglyceride concentration was increased. In this study, we identified the regulation of adipocyte differentiation by 5-HT, 5-HT(2A)R, and 5-HT(2C)R. miR-448-mediated repression of KLF5 was identified as a negative regulator for adipocyte differentiation.

We report a case of 79-year-old man, who was diagnosed to have transient ACTH deficiency associated with polymyalgia rheumatica (PMR). The patient presented with sudden onset bilateral shoulder pain, which was gradually aggravated. Plasma ACTH was undetectable, and both serum cortisol and urinary 17-OHCS were very low. Other pituitary hormones were normal, suggesting that hypothalamo-pituitary-adrenal (HPA) axis is selectively damaged. However, within several weeks, plasma ACTH returned to normal, and showed a normal increase response to corticotropin-releasing hormone stimulation test. These results indicated that ACTH deficiency was only transient. After hydrocortisone (10 mg/day) was administered, his symptoms became suddenly improved. Based on those results and clinical course, ie, elevated erythrocyte sedimentation rate, negative rheumatoid factor and the typical symptoms, which showed improvement to glulcocorticoid therapy, the final diagnosis was PMR, which was associated with transient ACTH deficiency. This is the first report of a case of PMR, in which the HPA axis was examined in its very acute phase. It was demonstrated that the case was associated with the transient adrenocortical hypofunction, which was recovered during a short time. It is therefore possible that PMR may show a different responsiveness of HPA axis depending on its phases.

Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F0F1 ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.

In response to metabolic stress, GLUT4, the most abundant glucose transporter, translocates from intracellular vesicles to the plasma membrane. This appears to play an important role in protecting cardiac myocytes from ischemic injury. To investigate the precise mechanisms of GLUT4 translocation in cardiomyocytes, we have established a method for quantifying the relative proportion of sarcolemmal GLUT4 to total GLUT4 in these cells. Stimulation with H2O2 resulted in a concentration-dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. The dominant-negative form (DN) of AMP-activated protein kinase (AMPK) alpha2 inhibited the H2O2-induced translocation of GLUT4. We further examined the role of two known AMPK kinases (AMPKKs), calmodulin-dependent protein kinase kinase (CaMKK)beta and LKB1. The DN of CaMKKbeta or LKB1 alone inhibited H2O2-induced GLUT4 translocation only partially compared to the inhibition produced by the DN of AMPKalpha2. However, the combination of DN-LKB1 and DN-CaMKKbeta inhibited translocation to an extent similar to with DN-AMPKalpha2. Stimulation with H2O2 also activated Akt and the inhibition of PI3-K/Akt prevented GLUT4 translocation to the same extent as with AMPK inhibition. When the DN of AMPKalpha2 was applied with DN-PI3-K, there was a complete reduction in the GLUT4 membrane level similar to that seen at the 0 time-point. These results demonstrate that AMPK and PI3-K/Akt have an additive effect on oxidative stress-mediated GLUT4 translocation.

MicroRNA (miRNA) is a small noncoding RNA that modulates posttranscriptional translation. Growing evidence indicates that miRNA is involved in basic cell functions and oncogenesis. In order to find a possible functional role of miRNA in the heart, we first suppressed the expression of Dicer and Ago2, which are required for miRNA biogenesis and function, by lenti-virus-mediated siRNA in neonatal rat cardiomyocytes. Knockdown of Dicer and Ago2 expression significantly increased their beating rate; therefore, we hypothesized that miRNA may regulate the expression of cardiomyocyte contractile protein since cardiac contractility depends on the expression of two myosin heavy chain (MHC) genes, α and β, which are regulated in an antithetical manner by developmental, physiological, and pathological signals. As siRNA against Dicer, which results in the loss of all miRNA function, upregulated α-MHC and downregulated β-MHC mRNA expression, we tried to identify functional miRNAs which have an opposite effect against Dicer siRNA on α/β-MHC expression in the heart. We first performed miRNA microarray analysis in the heart of Dahl salt-sensitive rats in which systemic hypertension caused compensated concentric left ventricular hypertrophy (LVH) at the age of 11 weeks, followed by congestive heart failure (CHF) at the age of 17 weeks. In this model, decreased α/β-MHC ratio is observed at LVH and CHF stages. Next we overexpressed miRNAs which are upregulated in LVH and CHF stages in neonatal rat cardiomyocytes by lenti-virus. After screening these miRNAs, miR-206 was found to decrease the α/β-MHC mRNA expression ratio. The computational miRNA target prediction algorithm showed one of the targets of miR-206 is RXR-α, which regulates α/β-MHC transcription. Forced expression of miR-206 reduced the translation of RXR-α. Moreover, when the expression of RXR-α was downregulated by siRNA, the α/β-MHC mRNA expression ratio decreased in the same manner as miR-206. In conclusion, miR-206 controls the gene expression of MHC isoforms by targeting RXR-α, and these results suggest that miR-206 regulates cardiac contractility.

The differentiation of preadiopocytes (preAdps) into mature adipocytes (Adps) is closely associated with the expression of adipocyte (Adp)-specific gene products that are deeply involved in insulin resistance; therefore, it is critical to identify genes required for Adp differentiation. We used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 preAdp cells which lose their ability to differentiate into mature Adps. Using this approach, we discovered that tryptophan hydroxylase-1 (TPH1), a rate-limiting enzyme for the biosynthesis of serotonin (Ser), was required for Adp differentiation. While differentiation was clearly reduced in a mutant 3T3-L1 cell line that lacked expression of the normal TPH1 gene, reconstitution of the TPH1 gene in this cell line restored its differentiation potential. The administration of Ser enhanced differentiation in 3T3-L1 cells and in cells of the stromal vascular fraction from adult mice. On the other hand, differentiation was inhibited by 5-hydroxy tryptophan type 2A (5HT2A) receptor antagonists as well as the Ser synthesis inhibitor. Differentiation into mature Adp was significantly impaired in preAdps prepared from TPH1−/− mice compared to those from wild-type mice. We observed no differences in the histology of adipose tissue between TPH1−/−and wild-type mice at the basal state; however, after obesity was induced by a high-fat diet, increases in body weight and mesenteric fat weight were more prominent in TPH1−/− mice than in wild-type mice. In obese groups, we observed extremely large (140μm∼) Adps without any small Adps in TPH1−/− mice, in contrast to usually hypertrophied (μ80∼m) Adps and small Adps in wild-type mice. Interestingly, Ser treatment of mature cultured Adps enhanced lipolysis, which was blocked by the 5HT7 receptor antagonist but not by the 5HT2A receptor antagonist. Oral administration of 5HT2A receptor antagonist in high fat diet-induced obese mice reduced mesenteric fat weight and the number of small adipocytes; however, it did not increase the number of extremely large adipocytes as seen in TPH1−/−mice. Thus, Ser in adipose tissue acts as an autocrine factor required for Adp differentiation and lipolysis. Distinct receptor subtypes appear to mediate these two processes.