Novel Long Noncoding RNA, Macrophage Inflammation-Suppressing Transcript (MIST), Regulates Macrophage Activation During Obesity.

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.

The Role of the Mineralocorticoid Receptor in Inflammation: Focus on Kidney and Vasculature.

BACKGROUND: The remarkable success of clinical trials in mineralocorticoid receptor (MR) inhibition in heart failure has driven research on the physiological and pathological role(s) of nonepithelial MR expression. MR is widely expressed in the cardiovascular system and is a major determinant of endothelial function, smooth muscle tone, vascular remodeling, fibrosis, and blood pressure. An important new dimension is the appreciation of the role MR plays in immune cells and target organ damage in the heart, kidney and vasculature, and in the development of insulin resistance. SUMMARY: The mechanism for MR activation in tissue injury continues to evolve with the evidence to date suggesting that activation of MR results in a complex repertoire of effects involving both macrophages and T cells. MR is an important transcriptional regulator of macrophage phenotype and function. Another important feature of MR activation is that it can occur even with normal or low aldosterone levels in pathological conditions. Tissue-specific conditional models of MR expression in myeloid cells, endothelial cells, smooth muscle cells and cardiomyocytes have been very informative and have firmly demonstrated a critical role of MR as a key pathophysiologic variable in cardiac hypertrophy, transition to heart failure, adipose inflammation, and atherosclerosis. Finally, the central nervous system activation of MR in permeable regions of the blood-brain barrier may play a role in peripheral inflammation. Key Message: Ongoing clinical trials will help clarify the role of MR blockade in conditions, such as atherosclerosis and chronic kidney disease.

Midline-1 regulates effector T cell motility in experimental autoimmune encephalomyelitis via mTOR/microtubule pathway.

Background: Effector T cell activation, migration, and proinflammatory cytokine production are crucial steps in autoimmune disorders such as multiple sclerosis (MS). While several therapeutic approaches targeting T cell activation and proinflammatory cytokines have been developed for the treatment of autoimmune diseases, there are no therapeutic agents targeting the migration of effector T cells, largely due to our limited understanding of regulatory mechanisms of T cell migration in autoimmune disease. Here we reported that midline-1 (Mid1) is a key regulator of effector T cell migration in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS. Methods: Mid1-/- mice were generated by Crispr-Cas9 technology. T cell-specific Mid1 knockout chimeric mice were generated by adoptive transfer of Mid1-/- T cells into lymphocyte deficient Rag2-/- mice. Mice were either immunized with MOG35-55 (active EAE) or received adoptive transfer of pathogenic T cells (passive EAE) to induce EAE. In vitro Transwell® assay or in vivo footpad injection were used to assess the migration of T cells. Results: Mid1 was significantly increased in the spinal cord of wild-type (Wt) EAE mice and disruption of Mid1 in T cells markedly suppressed the development of both active and passive EAE. Transcriptomic and flow cytometric analyses revealed a marked reduction in effector T cell number in the central nervous system of Mid1-/- mice after EAE induction. Conversely, an increase in the number of T cells was observed in the draining lymph nodes of Mid1-/- mice. Mice that were adoptively transferred with pathogenic Mid1-/- T cells also exhibited milder symptoms of EAE, along with a lower T cell count in the spinal cord. Additionally, disruption of Mid1 significantly inhibited T-cell migration both in vivo and in vitro. RNA sequencing suggests a suppression in multiple inflammatory pathways in Mid1-/- mice, including mTOR signaling that plays a critical role in cell migration. Subsequent experiments confirmed the interaction between Mid1 and mTOR. Suppression of mTOR with rapamycin or microtubule spindle formation with colcemid blunted the regulatory effect of Mid1 on T cell migration. In addition, mTOR agonists MHY1485 and 3BDO restored the migratory deficit caused by Mid1 depletion. Conclusion: Our data suggests that Mid1 regulates effector T cell migration to the central nervous system via mTOR/microtubule pathway in EAE, and thus may serve as a potential therapeutic target for the treatment of MS.

Deciphering the therapeutic potential of Myeloid-Specific JAK2 inhibition in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by severe inflammation and pulmonary dysfunction. Despite advancements in critical care, effective pharmacological interventions for ARDS remain elusive. While Janus kinase 2 (JAK2) inhibitors have emerged as an innovative treatment for numerous autoinflammatory diseases, their therapeutic potential in ARDS remains unexplored. In this study, we investigated the contribution of JAK2 and its underlying mechanisms in ARDS utilizing myeloid-specific JAK2 knockout murine models alongside a pharmacological JAK2 inhibitor. Notably, myeloid-specific JAK2 knockout led to a notable attenuation of ARDS induced by intratracheal administration of LPS, accompanied by reduced levels of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue. Intriguingly, the ameliorative effects were abolished upon the depletion of monocyte-derived alveolar macrophages (Mo-AMs) rather than tissue-resident alveolar macrophages (TR-AMs). JAK2 deficiency markedly reversed LPS-induced activation of STAT5 in macrophages. Remarkably, pharmacological JAK2 inhibition using baricitinib failed to substantially alleviate neutrophils infiltration, implying that specific inhibition of JAK2 in Mo-AMs is imperative for ARDS amelioration. Collectively, our data suggest that JAK2 may mitigate ARDS progression through the JAK2 pathway in Mo-AMs, underscoring JAK2 in alveolar macrophages, particularly Mo-AMs, as a promising therapeutic target for ARDS treatment.

Chronic fine particulate matter exposure induces systemic vascular dysfunction via NADPH oxidase and TLR4 pathways.

RATIONALE: Chronic exposure to ambient air-borne particulate matter of < 2.5 µm (PM2.5) increases cardiovascular risk. The mechanisms by which inhaled ambient particles are sensed and how these effects are systemically transduced remain elusive. OBJECTIVE: To investigate the molecular mechanisms by which PM2.5 mediates inflammatory responses in a mouse model of chronic exposure. METHODS AND RESULTS: Here, we show that chronic exposure to ambient PM2.5 promotes Ly6C(high) inflammatory monocyte egress from bone-marrow and mediates their entry into tissue niches where they generate reactive oxygen species via NADPH oxidase. Toll-like receptor (TLR)4 and Nox2 (gp91(phox)) deficiency prevented monocyte NADPH oxidase activation in response to PM2.5 and was associated with restoration of systemic vascular dysfunction. TLR4 activation appeared to be a prerequisite for NAPDH oxidase activation as evidenced by reduced p47(phox) phosphorylation in TLR4 deficient animals. PM2.5 exposure markedly increased oxidized phospholipid derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in bronchioalveolar lavage fluid. Correspondingly, exposure of bone marrow-derived macrophages to oxPAPC but not PAPC recapitulated effects of chronic PM2.5 exposure, whereas TLR4 deficiency attenuated this response. CONCLUSIONS: Taken together, our findings suggest that PM2.5 triggers an increase in oxidized phospholipids in lungs that then mediates a systemic cellular inflammatory response through TLR4/NADPH oxidase-dependent mechanisms.

In vivo targeting of inflammation-associated myeloid-related protein 8/14 via gadolinium immunonanoparticles.

OBJECTIVE: Myeloid-related protein (Mrp) 8/14 complex (is a highly expressed extracellularly secreted protein, implicated in atherosclerosis. In this study, we evaluated the feasibility of targeting Mrp in vivo through synthetic immuno-nanoprobes. METHODS AND RESULTS: Anti-Mrp-14 and nonspecific IgG-conjugated gadolinium nanoprobes (aMrp-) were synthesized and characterized. Pharmacokinetics and vascular targeting via MRI of the formulations were assessed in vivo in high fat-fed apolipoprotein E deficient (ApoE(-/-)), ApoE(-/-)/Mrp14(-/-) (double knockout) and chow-fed wild-type (C57BL/6) mice. Bone marrow-derived myeloid progenitor cells were isolated from both ApoE(-/-) and double knockout mice, differentiated to macrophages, and were treated with LPS, with or without Mrp8, Mrp14, or Mrp8/14; conditioned media was used for in vitro studies. Mrp-activated cells secreted significant amounts of proinflammatory cytokines, which was abolished by pretreatment with aMrp-NP. We show in vitro that aMrp-NP binds endothelial cells previously treated with conditioned media containing Mrp8/14. MRI following intravenous delivery of aMrp-NP revealed prolonged and substantial delineation of plaque in ApoE(-/-) but not double knockout or wild-type animals. Nonspecific IgG-conjugated gadolinium nanoprobe-injected animals in all groups did not show vessel wall enhancement. Flow-cytometric analysis of aortic digesta revealed that aMrp-NP present in Ly-6G(+), CD11b(+), CD11c(+), and CD31(+) cells in ApoE(-/-) but not in double knockout animals. CONCLUSIONS: Targeted imaging with aMrp-NP demonstrates enhancement of plaque with binding to inflammatory cells and reduction in inflammation. This strategy has promise as a theranostic approach for atherosclerosis.

Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis.

BACKGROUND: Dipeptidyl-peptidase 4 (DPP-4) inhibitors are increasingly used to accomplish glycemic targets in patients with type II diabetes mellitus. Because DPP-4 is expressed in inflammatory cells, we hypothesized that its inhibition will exert favorable effects in atherosclerosis. METHODS AND RESULTS: Male LDLR(-/-) mice (6 weeks) were fed a high-fat diet or normal chow diet for 4 weeks and then randomized to vehicle or alogliptin, a high-affinity DPP-4 inhibitor (40 mg · kg(-1) · d(-1)), for 12 weeks. Metabolic parameters, blood pressure, vascular function, atherosclerosis burden, and indexes of inflammation were obtained in target tissues, including the vasculature, adipose, and bone marrow, with assessment of global and cell-specific inflammatory pathways. In vitro and in vivo assays of DPP-4 inhibition (DPP-4i) on monocyte activation/migration were conducted in both human and murine cells and in a short-term ApoE(-/-) mouse model. DPP-4i improved markers of insulin resistance and reduced blood pressure. DPP-4i reduced visceral adipose tissue macrophage content (adipose tissue macrophages; CD11b(+), CD11c(+), Ly6C(hi)) concomitant with upregulation of CD163. DPP-4 was highly expressed in bone marrow-derived CD11b(+) cells, with DPP-4i downregulating proinflammatory genes in these cells. DPP-4i decreased aortic plaque with a striking reduction in plaque macrophages. DPP-4i prevented monocyte migration and actin polymerization in in vitro assays via Rac-dependent mechanisms and prevented in vivo migration of labeled monocytes to the aorta in response to exogenous tumor necrosis factor-α and DPP-4. CONCLUSION: DPP-4i exerts antiatherosclerotic effects and reduces inflammation via inhibition of monocyte activation/chemotaxis. These findings have important implications for the use of this class of drugs in atherosclerosis.

Pulmonary T cell activation in response to chronic particulate air pollution.

The purpose of this study was to investigate the effects of chronically inhaled particulate matter <2.5 µm (PM(2.5)) on inflammatory cell populations in the lung and systemic circulation. A prominent component of air pollution exposure is a systemic inflammatory response that may exaggerate chronic diseases such as atherosclerosis and insulin resistance. T cell response was measured in wild-type C57B/L6, Foxp3-green fluorescent protein (GFP) "knockin," and chemokine receptor 3 knockout (CXCR3(-/-)) mice following 24-28 wk of PM(2.5) or filtered air. Chronic PM(2.5) exposure resulted in increased CXCR3-expressing CD4(+) and CD8(+) T cells in the lungs, spleen, and blood with elevation in CD11c(+) macrophages in the lung and oxidized derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine in wild-type mice. CXCR3 deficiency decreased T cells in the lung. GFP(+) regulatory T cells increased with PM(2.5) exposure in the spleen and blood of Foxp3-GFP mice but were present at very low levels in the lung irrespective of PM(2.5) exposure. Mixed lymphocyte cultures using primary, PM(2.5)-treated macrophages demonstrated enhanced T cell proliferation. Our experiments indicate that PM(2.5) potentiates a proinflammatory Th1 response involving increased homing of CXCR3(+) T effector cells to the lung and modulation of systemic T cell populations.

Effect of co-exposure to nickel and particulate matter on insulin resistance and mitochondrial dysfunction in a mouse model.

BACKGROUND: It has been well recognized that toxicity of fine ambient air particulate matter (PM(2.5)) may depend on its chemical constituents, including components such as soluble metals that may theoretically exert distinctive effects. We have recently demonstrated an important effect of PM(2.5) on metabolic function. Since transition metals, such as nickel (Ni), represent an important component of exposure in certain environments, and may significantly influence the toxicity of inhalational exposure, we investigated the effects of Ni as a variable component of ambient PM(2.5) exposure. METHODS: Male ApoE knockout mice were exposed to filtered air (FA), fine-sized nickel sulfate particles alone (Ni) at 0.44 µg/m(3), concentrated ambient air PM(2.5) (CAPs) at a mean of 70 µg/m(3), or CAPs+Ni in Tuxedo, NY, 6 hours/day, 5 days/week, for 3 months. RESULTS: Exposure to Ni, irrespective of co-exposure to CAPs, resulted in body weight gain, while exposure to CAPs+Ni significantly enhanced fasting glucose and worsened insulin resistance measures (HOMA-IR), when compared with exposure to CAPs alone. CAPs+Ni exposure induced a significant decrease in phosphorylation of AMP-activated protein kinase (AMPK) α. Exposure to Ni or CAPs+Ni significantly induced microcirculatory dysfunction and increased monocytic cell infiltration into lung and adipose, and decreased uncoupling protein 1 expression at gene and protein levels and several brown adipocyte-specific genes in adipose tissue. CONCLUSIONS: Ni exposure has effects on metabolic and inflammatory parameters that are comparable to that of CAPs. Additionally, Ni synergistically exacerbates CAPs-induced adverse effects on some of, but not all of, these parameters, that may be mediated via the AMPK signaling pathway. These findings have important implications for inhaled transition metal toxicity that may exert synergistic effects with other PM(2.5) components.

Ambient air pollution exaggerates adipose inflammation and insulin resistance in a mouse model of diet-induced obesity.

BACKGROUND: There is a strong link between urbanization and type 2 diabetes mellitus. Although a multitude of mechanisms have been proposed, there are no studies evaluating the impact of ambient air pollutants and the propensity to develop type 2 diabetes mellitus. We hypothesized that exposure to ambient fine particulate matter (<2.5 mum; PM(2.5)) exaggerates diet-induced insulin resistance, adipose inflammation, and visceral adiposity. METHODS AND RESULTS: Male C57BL/6 mice were fed high-fat chow for 10 weeks and randomly assigned to concentrated ambient PM(2.5) or filtered air (n=14 per group) for 24 weeks. PM(2.5)-exposed C57BL/6 mice exhibited marked whole-body insulin resistance, systemic inflammation, and an increase in visceral adiposity. PM(2.5) exposure induced signaling abnormalities characteristic of insulin resistance, including decreased Akt and endothelial nitric oxide synthase phosphorylation in the endothelium and increased protein kinase C expression. These abnormalilties were associated with abnormalities in vascular relaxation to insulin and acetylcholine. PM(2.5) increased adipose tissue macrophages (F4/80(+) cells) in visceral fat expressing higher levels of tumor necrosis factor-alpha/interleukin-6 and lower interleukin-10/N-acetyl-galactosamine specific lectin 1. To test the impact of PM(2.5) in eliciting direct monocyte infiltration into fat, we rendered FVBN mice expressing yellow fluorescent protein (YFP) under control of a monocyte-specific promoter (c-fms, c-fms(YFP)) diabetic over 10 weeks and then exposed these mice to PM(2.5) or saline intratracheally. PM(2.5) induced YFP cell accumulation in visceral fat and potentiated YFP cell adhesion in the microcirculation. CONCLUSIONS: PM(2.5) exposure exaggerates insulin resistance and visceral inflammation/adiposity. These findings provide a new link between air pollution and type 2 diabetes mellitus.

Metabolic effects of air pollution exposure and reversibility.

Air pollution involving particulate matter smaller than 2.5 µm in size (PM2.5) is the world's leading environmental risk factor contributing to mortality through cardiometabolic pathways. In this study, we modeled early life exposure using chow-fed C57BL/6J male mice that were exposed to real-world inhaled, concentrated PM2.5 (~10 times ambient levels/~60-120 µg/m3) or filtered air over a 14-week period. We investigated the effects of PM2.5 on phenotype, the transcriptome, and chromatin accessibility and compared these with the effects of a prototypical high-fat diet (HFD) as well as cessation of exposure on phenotype reversibility. Exposure to PM2.5 impaired glucose and insulin tolerance and reduced energy expenditure and 18FDG-PET uptake in brown adipose tissue. Multiple differentially expressed gene clusters in pathways involving metabolism and circadian rhythm were noted in insulin-responsive tissues. Although the magnitude of transcriptional change detected with PM2.5 exposure was lower than that observed with a HFD, the degree of alteration in chromatin accessibility after PM2.5 exposure was significant. The novel chromatin remodeler SMARCA5 (SWI/SNF complex) was regulated in response to PM2.5 exposure, the cessation of which was associated with a reversal of insulin resistance and restoration of chromatin accessibility and nucleosome positioning near transcription start sites, as well as a reversal of exposure-induced changes in the transcriptome, including SMARCA5. These changes indicate pliable epigenetic control mechanisms following cessation of exposure.

Exposure to Air Pollution Disrupts Circadian Rhythm through Alterations in Chromatin Dynamics.

Particulate matter ≤2.5µm (PM2.5) air pollution is a leading environmental risk factor contributing disproportionately to the global burden of non-communicable disease. We compared impact of chronic exposure to PM2.5 alone, or with light at night exposure (LL) on metabolism. PM2.5 induced peripheral insulin resistance, circadian rhythm (CR) dysfunction, and metabolic and brown adipose tissue (BAT) dysfunction, akin to LL (with no additive interaction between PM2.5 and LL). Transcriptomic analysis of liver and BAT revealed widespread but unique alterations in CR genes, with evidence for differentially accessible promoters and enhancers of CR genes in response to PM2.5 by ATAC-seq. The histone deacetylases 2, 3, and 4 were downregulated with PM2.5 exposure, with increased promoter occupancy by the histone acetyltransferase p300 as evidenced by ChIP-seq. These findings suggest a previously unrecognized role of PM2.5 in promoting CR disruption and metabolic dysfunction through epigenetic regulation of circadian targets.

Differential contribution of bone marrow-derived infiltrating monocytes and resident macrophages to persistent lung inflammation in chronic air pollution exposure.

Chronic exposure to particulate matter < 2.5µ (PM2.5) has been linked to cardiopulmonary disease. Tissue-resident (TR) alveolar macrophages (AΦ) are long-lived, self-renew and critical to the health impact of inhalational insults. There is an inadequate understanding of the impact of PM2.5 exposure on the nature/time course of transcriptional responses, self-renewal of AΦ, and the contribution from bone marrow (BM) to this population. Accordingly, we exposed chimeric (CD45.2/CD45.1) mice to concentrated PM2.5 or filtered air (FA) to evaluate the impact on these end-points. PM2.5 exposure for 4-weeks induced an influx of BM-derived monocytes into the lungs with no contribution to the overall TR-AΦ pool. Chronic (32-weeks) PM2.5 exposure on the other hand while associated with increased recruitment of BM-derived monocytes and their incorporation into the AΦ population, resulted in enhanced apoptosis and decreased proliferation of TR-AΦ. RNA-seq analysis of isolated TR-AΦ and BM-AΦ from 4- and 32-weeks exposed mice revealed a unique time-dependent pattern of differentially expressed genes. PM2.5 exposure resulted in altered histological changes in the lungs, a reduced alveolar fraction which corresponded to protracted lung inflammation. Our findings suggest a time-dependent entrainment of BM-derived monocytes into the AΦ population of PM2.5 exposed mice, that together with enhanced apoptosis of TR-AΦ and reorganization of transcriptional responses, could collectively contribute to the perpetuation of chronic inflammation.

A mouse model of yellow fluorescent protein (YFP) expression in hematopoietic cells to assess leukocyte-endothelial interactions in the microcirculation.

In this study, we describe the use of intravital microscopy in a transgenic mouse model expressing yellow fluorescent protein (YFP) under the control of a monocyte specific promoter c-fms (CD115) to track and quantify specific leukocyte subsets. Flow cytometry on peripheral and bone marrow leukocytes revealed that YFP was predominantly expressed by CD11a(+), CD11b(+), and CD14(+) monocytes. In the bone marrow, 67+/-4% of Ly6C(high) F4/80(+) cells were YFP(high) while 55+/-1% of Ly6C(low) F4/80(+) cells were YFP(low) supporting the use of c-fms(YFP) expression as a marker of monocyte lineage. 70+/-7% of CD11b(+) F4/80(+) Ly6C(+) ("triple positive") cells expressed YFP. To assess leukocyte-endothelial interactions in YFP(+) cells in c-fms(YFP+) mice, we evaluated leukocyte adhesion, rolling and local shear stress responses in the cremasteric endothelium 4 h following administration of TNFalpha. TNFalpha resulted in a five-fold increase in adhesion of YFP(+) cells to the endothelium and provided superior discriminative ability in assessing rolling and adhesion events when compared with bright field microscopy. Additionally, when compared with Rhodamine-6G labeled leukocytes or GFP(+) cells in mice transplanted with green fluorescent protein (GFP) positive bone marrow, the level of detail observed in the c-fms(YFP+) was greater, with both GFP(+) and YFP(+) cells demonstrating superior signal to noise compared to bright field microscopy. A weak positive linear correlation between wall shear stress and YFP(+) cell adhesion (r(2)=0.20, p<0.05) was seen in the cremasteric microcirculation. Taken together, these data demonstrate the use of c-fms(YFP+) mice in identifying distinct monocyte subsets and highlight the potential of this model for real-time monocyte-endothelial interactions using intravital microscopy.

Developmental, hormonal, and nutritional regulation of porcine adipose triglyceride lipase (ATGL).

Adipose triglyceride lipase (ATGL) is a newly identified lipase. We report for the first time the porcine ATGL sequence and characterize ATGL gene and protein expression in vitro and in vivo. Adult pig tissue expresses ATGL at high levels in the white adipose and muscle tissue relative to other tested tissues. We show that within the white adipose tissue ATGL is expressed at higher levels in the adipocyte than in the stromal-vascular fraction. Additionally, ATGL expression increases dramatically in the subcutaneous adipose during adipose development and maturation, as well as during in vitro adipogenesis. Peroxisome proliferator-activated receptor gamma transcript levels increased concomitant with ATGL gene expression, suggesting a possible role in the regulation of ATGL by adipogenic regulators. In vitro treatment of differentiated primary pig preadipocytes with insulin and forskolin decreased ATGL gene expression in a dose-dependent manner, suggesting ATGL transcript levels are hormone sensitive. In vivo experimentation showed that calorie-restriction in gilts resulted in increased ATGL mRNA and protein levels in subcutaneous and peri-renal fat tissues. Our data demonstrate that ATGL expression reacts to hormonal stimuli and plays a role in catecholamine-induced lipolysis in porcine adipose tissue.

Noncoding RNAs in Cardiovascular Disease: Pathological Relevance and Emerging Role as Biomarkers and Therapeutics.

Noncoding RNAs (ncRNA) include a diverse range of functional RNA species-microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) being most studied in pathophysiology. Cardiovascular morbidity is associated with differential expression of myriad miRNAs; miR-21, miR-155, miR-126, miR-146a/b, miR-143/145, miR-223, and miR-221 are the top 9 most reported miRNAs in hypertension and atherosclerotic disease. A single miRNA may have hundreds of messenger RNA targets, which makes a full appreciation of the physiologic ramifications of such broad-ranging effects a challenge. miR-21 is the most prominent ncRNA associated with hypertension and atherosclerotic disease due to its role as a "mechano-miR", responding to arterial shear stresses. "Immuno-miRs", such as miR-155 and miR-223, affect cardiovascular disease (CVD) via regulation of hematopoietic cell differentiation, chemotaxis, and activation in response to many pro-atherogenic stimuli. "Myo-miRs", such as miR-1 and miR-133, affect cardiac muscle plasticity and remodeling in response to mechanical overload. This in-depth review analyzes observational and experimental reports of ncRNAs in CVD, including future applications of ncRNA-based strategies in diagnosis, prediction (e.g., survival and response to small molecule therapy), and biologic therapy.

CITED2 Restrains Proinflammatory Macrophage Activation and Response.

Macrophages are strategically distributed in mammalian tissues and play an essential role in priming the immune response. However, macrophages need to constantly strike a balance between activation and inhibition states to avoid a futile inflammatory reaction. Here, we identify the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) as a potent repressor of macrophage proinflammatory activation. Gain- and loss-of-function studies revealed that CITED2 is required for optimal peroxisome proliferator-activated receptor gamma (PPARγ) activation and attendant select anti-inflammatory gene expression in macrophages. More importantly, deficiency of CITED2 resulted in significant attenuation of rosiglitazone-induced PPARγ activity, PPARγ recruitment to target gene promoters, and anti-inflammatory target gene expression in macrophages. Interestingly, deficiency of Cited2 strikingly heightened proinflammatory gene expression through stabilization of hypoxia-inducible factor 1 alpha (HIF1α) protein in macrophages. Further, overexpression of Egln3 or inhibition of HIF1α in Cited2-deficient macrophages completely reversed elevated proinflammatory cytokine/chemokine gene expression. Importantly, mice bearing a myeloid cell-specific deletion of Cited2 were highly susceptible to endotoxin-induced sepsis symptomatology and mortality. Collectively, our observations identify CITED2 as a novel negative regulator of macrophage proinflammatory activation that protects the host from inflammatory insults.

Alternative splicing of delta-like 1 homolog (DLK1) in the pig and human.

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be present in mouse and cattle. Surprisingly, DLK1-A was undetected in various tissues from fetal and postnatal pigs. Instead, DLK1-C2 was the most abundant isoform while DLK1-B was expressed to a lesser extent. In fractionated adipose tissue, pDLK1 was most highly expressed in the stromal-vascular cell fraction. In addition, total pDLK1 was highly expressed in fetal adipose tissue but dramatically decreased postnatally. Our data suggests that expression of DLK1-B and -C2 isoforms is sufficient for normal pig development. Furthermore, human and pig samples showed no alterations in species-specific splicing, but expression levels decreased with age, suggesting that regulation of expression, not splicing, is the most likely mechanism controlling the biological function of DLK1.

"Eat me" imaging and therapy.

Clearance of apoptotic debris is a vital role of the innate immune system. Drawing upon principles of apoptotic clearance, convenient delivery vehicles including intrinsic anti-inflammatory characteristics and specificity to immune cells can be engineered to aid in drug delivery. In this article, we examine the use of phosphatidylserine (PtdSer), the well-known "eat-me" signal, in nanoparticle-based therapeutics making them highly desirable "meals" for phagocytic immune cells. Use of PtdSer facilitates engulfment of nanoparticles allowing for imaging and therapy in various pathologies and may result in immunomodulation. Furthermore, we discuss the targeting of the macrophages and other cells at sites of inflammation in disease. A thorough understanding of the immunobiology of "eat-me" signals is requisite for the successful application of "eat-me"-bearing materials in biomedical applications.

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages.

Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.