Sex-specific phenotypes in the aging mouse heart and consequences for chronic fibrosis.

The incidence of diastolic dysfunction increases with age in both humans and mice. This is characterized by increased passive stiffness and slower relaxation of the left ventricle. The stiffness arises at least partially from progressively increased interstitial collagen deposition because of highly secretory fibroblasts. In the past, we demonstrated that AMPK activation via the drug 5-aminoimidazole-4-carboxamide riboside (AICAR) in middle-aged mice reduced adverse remodeling after myocardial infarction. Therefore, as an attempt to normalize the fibroblast phenotype, we used 21-mo-old male and female mice and treated them with AICAR (0.166 mg/g body wt) where each mouse was followed in a functional study over a 3-mo period. We found sex-related differences in extracellular matrix (ECM) composition as well as heart function indices at baseline, which were further accentuated by AICAR treatment. AICAR attenuated the age-related increase in left atrial volume (LAV, an indicator of diastolic dysfunction) in female but not in male hearts, which was associated with reduced collagen deposition in the old female heart, and reduced the transcription factor Gli1 expression in cardiac fibroblasts. We further demonstrated that collagen synthesis was dependent on Gli1, which is a target of AMPK-mediated degradation. By contrast, AICAR had a minor impact on cardiac fibroblasts in the old male heart because of blunted AMPK phosphorylation. Hence, it did not significantly improve old male heart function indices. In conclusion, we demonstrated that male and female hearts are phenotypically different, and sex-specific differences need to be considered when analyzing the response to pharmacological intervention.NEW & NOTEWORTHY The aging heart develops diastolic dysfunction because of increased collagen deposition. We attempted to reduce collagen expression in the old heart by activating AMPK using AICAR. An improvement of diastolic function and reduction of cardiac fibrosis was found only in the female heart and correlated with decreased procollagen expression and increased degradation of the transcription factor Gli1. Male hearts display blunted AICAR-dependent AMPK activation and therefore this treatment had no benefits for the male mice.

Cleavage stimulating factor 64 depletion mitigates cardiac fibrosis through alternative polyadenylation.

Alternative polyadenylation (APA) regulates gene expression by cleavage and addition of poly(A) sequence at different polyadenylation sites (PAS) in 3'UTR, thus, generating transcript isoforms with different lengths. Cleavage stimulating factor 64 (CstF64) is an APA regulator which plays a role in PAS selection and determines the length of 3'UTR. CstF64 favors the use of proximal PAS, resulting in 3'UTR shortening, which enhances the protein expression by increasing the stability of the target genes. The aim of this study is to investigate the role of CstF64 in cardiac fibrosis, a key event leading to heart failure (HF). We determined the expression of CstF64, key profibrotic genes, and their 3'UTR changes by calculating distal PAS (dPAS) usage in left ventricular (LV) tissues and cardiac fibroblasts from HF patients. CstF64 was upregulated in HF LV tissues and cardiac fibroblasts along with increased deposition of fibrosis genes such as COL1A and FN1 and significant shortening in their 3'UTR. In addition, HF cardiac fibroblasts showed increased transforming growth factor receptor ß1 (TGFßR1) expression consistent with significant shortening in 3'UTR of TGFßR1. Upon knockdown of CstF64 from HF fibroblasts, downregulation in pro-fibrotic genes corresponding to lengthening in their 3'UTR was observed. Our finding suggests an important role of CstF64 in myofibroblast activation and promotion of cardiac fibrosis during HF through APA. Therefore, targeting CstF64 mediated RNA processing approach in human HF could provide a new therapeutic treatment strategy for limiting fibrotic remodeling.

Changes in cardiac resident fibroblast physiology and phenotype in aging.

The cardiac fibroblast plays a central role in tissue homeostasis and in repair after injury. With aging, dysregulated cardiac fibroblasts have a reduced capacity to activate a canonical transforming growth factor-ß-Smad pathway and differentiate poorly into contractile myofibroblasts. That results in the formation of an insufficient scar after myocardial infarction. In contrast, in the uninjured aged heart, fibroblasts are activated and acquire a profibrotic phenotype that leads to interstitial fibrosis, ventricular stiffness, and diastolic dysfunction, all conditions that may lead to heart failure. There is an apparent paradox in aging, wherein reparative fibrosis is impaired but interstitial, adverse fibrosis is augmented. This could be explained by analyzing the effectiveness of signaling pathways in resident fibroblasts from young versus aged hearts. Whereas defective signaling by transforming growth factor-ß leads to insufficient scar formation by myofibroblasts, enhanced activation of the ERK1/2 pathway may be responsible for interstitial fibrosis mediated by activated fibroblasts. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/fibroblast-phenotypic-changes-in-the-aging-heart/ .

Aicar treatment reduces interstitial fibrosis in aging mice: Suppression of the inflammatory fibroblast.

In 2030, elderly people will represent 20% of the United States population. Even now, chronic cardiac diseases, especially heart failure with preserved systolic function (HFpEF), are the most expensive DRGs for Medicare. Progressive interstitial fibrosis in the aging heart is well recognized as an important component of HFpEF. Our recent studies suggested an important pathophysiologic role for reduced TGF-ß receptor 1 (TGFßR1) signaling in mesenchymal stem cells (MSCs) and their mesenchymal fibroblast progeny in the development of interstitial fibrosis. This report arises from our previous studies, which suggest that an inflammatory phenotype exists in these mesenchymal fibroblasts as a result of a reduced TGF-ß-Smad-dependent pathway but upregulated farnesyltransferase (FTase)-Ras-Erk signaling. In this report we provide evidence for a therapeutic approach that downregulates Erk activation through an adenosine monophosphate-activated kinase (AMPK) pathway. Aging C57BL/6J mice were treated with AICAR (an AMPK activator) for a 30-day period. This treatment suppressed excessive monocyte chemoattractant protein-1 (MCP-1) generation, which diminished leukocyte infiltration and in consequence suppressed the formation of macrophage-derived myeloid fibroblasts. Interestingly, the number of mesenchymal fibroblasts was also reduced. In addition, we observed changes in extracellular matrix (ECM) deposition, specifically that collagen type I and the alternatively spliced variant of fibronectin (EDA) expressions were reduced. These data suggest that the upregulation of AMPK activity is a potential therapeutic approach to fibrosis in the aging heart.

Dissecting the role of myeloid and mesenchymal fibroblasts in age-dependent cardiac fibrosis.

Aging is associated with increased cardiac interstitial fibrosis and diastolic dysfunction. Our previous study has shown that mesenchymal fibroblasts in the C57BL/6J (B6J) aging mouse heart acquire an inflammatory phenotype and produce higher levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) secreted by these aged fibroblasts promotes leukocyte uptake into the heart. Some of the monocytes that migrate into the heart polarize into M2a macrophages/myeloid fibroblasts. The number of activated mesenchymal fibroblasts also increases with age, and consequently, both sources of fibroblasts contribute to fibrosis. Here, we further investigate mechanisms by which inflammation influences activation of myeloid and mesenchymal fibroblasts and their collagen synthesis. We examined cardiac fibrosis and heart function in three aged mouse strains; we compared C57BL/6J (B6J) with two other strains that have reduced inflammation via different mechanisms. Aged C57BL/6N (B6N) hearts are protected from oxidative stress and fibroblasts derived from them do not develop an inflammatory phenotype. Likewise, these mice have preserved diastolic function. Aged MCP-1 null mice on the B6J background (MCP-1KO) are protected from elevated leukocyte infiltration; they develop moderate but reduced fibrosis and diastolic dysfunction. Based on these studies, we further delineated the role of resident versus monocyte-derived M2a macrophages in myeloid-dependent fibrosis and found that the number of monocyte-derived M2a (but not resident) macrophages correlates with age-related fibrosis and diastolic dysfunction. In conclusion, we have found that ROS and inflammatory mediators are necessary for activation of fibroblasts of both developmental origins, and prevention of either led to better functional outcomes.

Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.

Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

Mesenchymal stem cell-derived inflammatory fibroblasts promote monocyte transition into myeloid fibroblasts via an IL-6-dependent mechanism in the aging mouse heart.

Fibrosis in the old mouse heart arises partly as a result of aberrant mesenchymal fibroblast activation. We have previously shown that endogenous mesenchymal stem cells (MSCs) in the aged heart are markedly resistant to TGF-ß signaling. Fibroblasts originating from these MSCs retain their TGF-ß unresponsiveness and become inflammatory. In current studies, we found that these inflammatory fibroblasts secreted higher levels of IL-6 (3-fold increase, P < 0.05) when compared with fibroblasts derived from the young hearts. Elevated IL-6 levels in fibroblasts derived from old hearts arose from up-regulated expression of Ras protein-specific guanine nucleotide releasing factor 1 (RasGrf1), a Ras activator (5-fold, P < 0.01). Knockdown of RasGrf1 by gene silencing or pharmacologic inhibition of farnesyltransferase (FTase) or ERK caused reduction of IL-6 mRNA (more than 65%, P < 0.01) and decreased levels of secreted IL-6 (by 44%, P < 0.01). In vitro, IL-6 markedly increased monocyte chemoattractant protein-1-driven monocyte-to-myeloid fibroblast formation after transendothelial migration (TEM; 3-fold, P < 0.01). In conclusion, abnormal expression of RasGrf1 promoted production of IL-6 by mesenchymal fibroblasts in the old heart. Secreted IL-6 supported conversion of monocyte into myeloid fibroblasts. This process promotes fibrosis and contributes to the diastolic dysfunction in the aging heart.

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-ß), due to reduced expression of TGF-ß receptor I (TßRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-ß promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-ß signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TßRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ".

Aberrant differentiation of fibroblast progenitors contributes to fibrosis in the aged murine heart: role of elevated circulating insulin levels.

With age, the collagen content of the heart increases, leading to interstitial fibrosis. We have shown that CD44(pos) fibroblasts derived from aged murine hearts display reduced responsiveness to TGF-ß but, paradoxically, have increased collagen expression in vivo and in vitro. We postulated that this phenomenon was due to the defect in mesenchymal stem cell (MSC) differentiation in a setting of elevated circulating insulin levels and production that we observed in aging mice. We discovered that cultured fibroblasts derived from aged but not young cardiac MSCs of nonhematopoietic lineage displayed increased basal and insulin-induced (1 nM) collagen expression (2-fold), accompanied by increased farnesyltransferase (FTase) and Erk activities. In a quest for a possible mechanism, we found that a chronic pathophysiologic insulin concentration (1 nM) caused abnormal fibroblast differentiation of MSCs isolated from young hearts. Fibroblasts derived from these MSCs responded to insulin by elevating collagen expression as seen in untreated aged fibroblast cultures, suggesting a causal link between increased insulin levels and defective MSC responses. Here we report an insulin-dependent pathway that specifically targets collagen type I transcriptional activation leading to a unique mechanism of fibrosis that is TGF-ß and inflammation-independent in the aged heart.

AICAR-dependent AMPK activation improves scar formation in the aged heart in a murine model of reperfused myocardial infarction.

We have demonstrated that scar formation after myocardial infarction (MI) is associated with an endogenous pool of CD44(pos)CD45(neg) multipotential mesenchymal stem cells (MSC). MSC differentiate into fibroblasts secreting collagen that forms a scar and mature into myofibroblasts that express alpha smooth muscle actin (α-SMA) that stabilizes the scar. In the aging mouse, cardiac repair after MI is associated with impaired differentiation of MSC; MSC derived from the aged hearts form dysfunctional fibroblasts that deposit less collagen in response to transforming growth factor beta-1 (TGF-ß1) and poorly mature into myofibroblasts. We found in vitro that the defect in myofibroblast maturation can be remedied by AICAR, which activates non-canonical TGF-ß signaling through AMP-activated protein kinase (AMPK). In the present study, we injected aged mice with AICAR and subjected them to 1h occlusion of the left anterior descending artery (LAD) and then reperfusion for up to 30days. AICAR-dependent AMPK signaling led to mobilization of an endogenous CD44(pos)CD45(neg) MSC and its differentiation towards fibroblasts and myofibroblasts in the infarct. This was accompanied by enhanced collagen deposition and collagen fiber maturation in the scar. The AICAR-treated group has demonstrated reduced adverse remodeling as indicated by improved apical end diastolic dimension but no changes in ejection fraction and cardiac output were observed. We concluded that these data indicate the novel, previously not described role of AMPK in the post-MI scar formation. These findings can potentially lead to a new therapeutic strategy for prevention of adverse remodeling in the aging heart.

A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart.

The cardiac fibroblast interacts with an extracellular matrix (ECM), enabling myofibroblast maturation via a process called mechanosensing. Although in the aging male heart, ECM is stiffer than in the young mouse, myofibroblast development is impaired, as demonstrated in 2-D and 3-D experiments. In old male cardiac fibroblasts, we found a decrease in actin polymerization, α-smooth muscle actin (α-SMA), and Kindlin-2 expressions, the latter an effector of the mechanosensing. When Kindlin-2 levels were manipulated via siRNA interference, young fibroblasts developed an old-like fibroblast phenotype, whereas Kindlin-2 overexpression in old fibroblasts reversed the defective phenotype. Finally, inhibition of overactivated extracellular regulated kinases 1 and 2 (ERK1/2) in the old male fibroblasts rescued actin polymerization and α-SMA expression. Pathological ERK1/2 overactivation was also attenuated by Kindlin-2 overexpression. In contrast, old female cardiac fibroblasts retained an operant mechanosensing pathway. In conclusion, we identified defective components of the Kindlin/ERK/actin/α-SMA mechanosensing axis in aged male fibroblasts.

Defective myofibroblast formation from mesenchymal stem cells in the aging murine heart rescue by activation of the AMPK pathway.

Aged mice in a murine model of myocardial infarction exhibit less effective myocardial repair. We hypothesized that the deficiency arises from altered lineage choice of endogenous mesenchymal stem cells (MSCs) and faulty maturation of myofibroblasts. Examination of cardiac MSCs revealed a substantial reduction in the pluripotency marker Nanog in cells from aged mice. In addition, the aged MSCs demonstrated a redirected lineage choice that favored adipocytic commitment over fibroblast or myofibroblast differentiation. Fibroblasts derived from aged MSCs demonstrated reduced expression of transforming growth factor-ß (TGF-ß) receptors I and II and diminished SMAD3 phosphorylation, associated with attenuated contractility and migration. Overexpression of constitutively active TGF-ß receptor I in aged cardiac fibroblasts ameliorated their defective motility but did not improve their contractility. Culturing of MSCs and fibroblasts with AICAR (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside) to activate adenosine monophosphate-activated kinase resulted in TGF-ß-dependent development of myofibroblasts with markedly enhanced contractility despite no reduction in adipocytic commitment or increased expression of TGF-ß receptors and SMAD3 phosphorylation. The data suggest an adenosine monophosphate-activated kinase-dependent gain of function as mediated by phosphorylation of TGF-ß-activated kinase 1 and p38 mitogen-activated protein kinase, which amplifies the response to TGF-ß1 via a non-canonical pathway, thus compensating for the reduced expression of TGF-ß receptors.

Immune-inflammatory dysregulation modulates the incidence of progressive fibrosis and diastolic stiffness in the aging heart.

Diastolic dysfunction in the aging heart is a grave condition that challenges the life and lifestyle of a growing segment of our population. This report seeks to examine the role and interrelationship of inflammatory dysregulation in interstitial myocardial fibrosis and progressive diastolic dysfunction in aging mice. We studied a population of C57BL/6 mice that developed progressive diastolic dysfunction over 30 months of life. This progressive dysfunction was associated with increasing infiltration of CD45(+) fibroblasts of myeloid origin. In addition, increased rates of collagen expression as measured by cellular procollagen were apparent in the heart as a function of age. These cellular and functional changes were associated with progressive increases in mRNA for MCP-1 and IL-13, which correlated both temporally and quantitatively with changes in fibrosis and cellular procollagen levels. MCP-1 protein was also increased and found to be primarily in the venular endothelium. Protein assays also demonstrated elevation of IL-4 and IL-13 suggesting a shift to a Th2 phenotype in the aging heart. In vitro studies demonstrated that IL-13 markedly enhanced monocyte-fibroblast transformation. Our results indicate that immunoinflammatory dysregulation in the aging heart induces progressive MCP-1 production and an increased shift to a Th2 phenotype paralleled by an associated increase in myocardial interstitial fibrosis, cellular collagen synthesis, and increased numbers of CD45(+) myeloid-derived fibroblasts that contain procollagen. The temporal association and functional correlations suggest a causative relationship between age-dependent immunoinflammatory dysfunction, fibrosis and diastolic dysfunction.

Treatment with a DC-SIGN ligand reduces macrophage polarization and diastolic dysfunction in the aging female but not male mouse hearts.

Cardiac diastolic dysfunction in aging arises from increased ventricular stiffness caused by inflammation and interstitial fibrosis. The diastolic dysfunction contributes to heart failure with preserved ejection fraction (HFpEF), which in the aging population is more common in women. This report examines its progression over 12 weeks in aging C57BL/6J mice and correlates its development with changes in macrophage polarization and collagen deposition.Aged C57BL/6J mice were injected with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) ligand 1 (DCSL1, an anti-inflammatory agent) or saline for 12 weeks. Echo and Doppler measurements were performed before and after 4 and 12 weeks of treatment. DCSL1 prevented the worsening of diastolic dysfunction over time in females but not in males. Cardiac single cell suspensions analyzed by flow cytometry revealed changes in the inflammatory infiltrate: (1) in males, there was an increased total number of leukocytes with an increased pro-inflammatory profile compared with females and they did not respond to DCSL1; (2) by contrast, DCSL1 treatment resulted in a shift in macrophage polarization to an anti-inflammatory phenotype in females. Notably, DCSL1 preferentially targeted tumor necrosis factor-α (TNFα+) pro-inflammatory macrophages. The reduction in pro-inflammatory macrophage polarization was accompanied by a decrease in collagen content in the heart.Age-associated diastolic dysfunction in mice is more severe in females and is associated with unique changes in macrophage polarization in cardiac tissue. Treatment with DCSL1 mitigates the changes in inflammation, cardiac function, and fibrosis. The characteristics of diastolic dysfunction in aging female mice mimic similar changes in aging women.

Mechanosensing dysregulation in the fibroblast: A hallmark of the aging heart.

The myofibroblast is a specialized fibroblast that expresses α-smooth muscle actin (α-SMA) and participates in wound contraction and fibrosis. The fibroblast to myofibroblast transition depends on chemical and mechanical signals. A fibroblast senses the changes in the environment (extracellular matrix (ECM)) and transduces these changes to the cytoskeleton and the nucleus, resulting in activation or inhibition of α-SMA transcription in a process called mechanosensing. A stiff matrix greatly facilitates the transition from fibroblast to myofibroblast, and although the aging heart is much stiffer than the young one, the aging fibroblast has difficulties in transitioning into the contractile phenotype. This suggests that the events occurring downstream of the matrix, such as activation or changes in expression levels of various proteins participating in mechanotransduction can negatively alter the ability of the aging fibroblast to become a myofibroblast. In this review, we will discuss in detail the changes in ECM, receptors (integrin or non-integrin), focal adhesions, cytoskeleton, and transcription factors involved in mechanosensing that occur with aging.

Peroxisome proliferator-activated receptor-delta upregulates 14-3-3 epsilon in human endothelial cells via CCAAT/enhancer binding protein-beta.

Peroxisome proliferator-activated receptor delta (PPARdelta) agonists are promising new agents for treatment of the metabolic syndrome. Although they possess antiatherosclerotic properties in vivo and promote endothelial cell survival, their mechanism of action is incompletely understood. 14-3-3epsilon is a critical component of the endothelial cell antiapoptotic machinery, which is essential to maintain homeostasis of the vascular wall. To test the hypothesis that PPARdelta targets 14-3-3epsilon in endothelial cells, we studied the response of the gene that encodes 14-3-3epsilon in humans, YWHAE, to PPARdelta ligands (L-165,041 and GW501516). We found that PPARdelta activates YWHAE promoter in a concentration and time-dependent manner. Consistent with these findings, L-165,041 increased 14-3-3epsilon mRNA and protein level, whereas PPARdelta small interfering RNA suppressed both basal and L-165,041-dependent YWHAE transcription and 14-3-3epsilon protein expression. Surprisingly, PPAR response elements in YWHAE promoter were not required for upregulation by PPARdelta, whereas a CCAAT/enhancer binding protein (C/EBP) site located at -160/-151 bp regulated both basal and PPARdelta-dependent promoter activity. Intriguingly, activation or knock down of endogenous PPARdelta regulated C/EBPbeta protein expression. Chromatin immunoprecipitation assays demonstrated that L-165,041 determines the localization of C/EBPbeta to the region spanning this C/EBP response element, whereas sequential chromatin immunoprecipitation analysis revealed that C/EBPbeta and PPARdelta form a transcriptional activating complex on this C/EBP site. Our work uncovers a novel role for C/EBPbeta as a mediator of PPARdelta-dependent 14-3-3epsilon gene regulation in human endothelial cells and provides insight into the mechanism by which PPARdelta agonists may be beneficial in atherosclerosis.

Improved Cardiovascular Function in Old Mice After N-Acetyl Cysteine and Glycine Supplemented Diet: Inflammation and Mitochondrial Factors.

Metabolic, inflammatory, and functional changes occur in cardiovascular aging which may stem from oxidative stress and be remediable with antioxidants. Glutathione, an intracellular antioxidant, declines with aging, and supplementation with glutathione precursors, N-acetyl cysteine (NAC) and glycine (Gly), increases tissue glutathione. Thirty-month old mice were fed diets supplemented with NAC or NAC+Gly and, after 7 weeks, cardiac function and molecular studies were performed. The NAC+Gly supplementation improved diastolic function, increasing peak early filling velocity, and reducing relaxation time, left atrial volume, and left ventricle end diastolic pressure. By contrast, cardiac function did not improve with NAC alone. Both diet supplementations decreased cardiac levels of inflammatory mediators; only NAC+Gly reduced leukocyte infiltration. Several mitochondrial genes reduced with aging were upregulated in hearts by NAC+Gly diet supplementation. These Krebs cycle and oxidative phosphorylation enzymes, suggesting improved mitochondrial function, and permeabilized cardiac fibers from NAC+Gly-fed mice produced ATP from carbohydrate and fatty acid sources, whereas fibers from control old mice were less able to utilize fatty acids. Our data indicate that NAC+Gly supplementation can improve diastolic function in the old mouse and may have potential to prevent important morbidities for older people.

Phosphocholine-containing ligands direct CRP induction of M2 macrophage polarization independent of T cell polarization: Implication for chronic inflammatory states.

INTRODUCTION: We studied monocyte transendothelial migration and subsequent polarization into M1/M2 macrophages in response to C-reactive protein (CRP) with two disease-related ligands: (1) phosphocholine (PC) and (2) multilamellar liposomes containing both unoxidized and oxidized forms of the lipid, phosphatidylcholine. These ligands differ in biological origin: PC is present on bacterial cell walls while oxidized lipids are present in atherogenic lipids. METHODS: We used an in vitro model of human monocyte transendothelial migration and assessed the polarization of monocytes and T cells and signaling through Fcγ receptors in monocytes. RESULTS: CRP without ligands did not promote M2 macrophage differentiation over background levels. However, when paired with either ligand, it increased M2 numbers. M2 differentiation was dependent on IL-13, and in the case of CRP with PC, was associated with a Th2 response. Paradoxically, while CRP with PC initiated a Th2 response, the combination of liposomes with CRP resulted in a Th1 response without any change in Th2 numbers despite association with M2 macrophage polarization. To resolve the conundrum of an anti-inflammatory macrophage response coexisting with a proinflammatory T cell response, we investigated signaling of CRP and its ligands through Fcγ receptors, which leads to macrophage activation independent of T cell signaling. We found that CRP plus PC acted via FcγRI, whereas CRP with liposomes bound to FcγRII. Both were activating signals as evidenced by SYK phosphorylation. CONCLUSION: We conclude that CRP with ligands can promote M2 macrophage differentiation to fibroblasts through FcγR activation, and this may result in an anti-inflammatory influence despite a proinflammatory T cell environment caused by oxidized lipids. The potential relationship of this mechanism to chronic inflammatory disease is discussed.