Cardiovascular hemodynamics in mice with tumor necrosis factor receptor-associated factor 2 mediated cytoprotection in the heart.

Introduction: Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, end-diastolic diameter, and others are used to assess cardiac function, their dependency on loading conditions somewhat limits their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include the interaction between the ventricle and the aorta (ventricular-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity. Methods: We measured cardiac Doppler velocities, blood pressures, along with VVC, aortic impedance, and pulse wave velocity to evaluate global cardiac function in a mouse model of cardiac-restricted low levels of TRAF2 overexpression that conferred cytoprotection in the heart. Results: While previous studies reported that response to myocardial infarction and reperfusion was improved in the TRAF2 overexpressed mice, we found that TRAF2 mice had significantly lower cardiac systolic velocities and accelerations, diastolic atrial velocity, aortic pressures, rate-pressure product, LV contractility and relaxation, and stroke work when compared to littermate control mice. Also, we found significantly longer aortic ejection time, isovolumic contraction and relaxation times, and significantly higher mitral early/atrial ratio, myocardial performance index, and ventricular vascular coupling in the TRAF2 overexpression mice compared to their littermate controls. We found no significant differences in the aortic impedance and pulse wave velocity. Discussion: While the reported tolerance to ischemic insults in TRAF2 overexpression mice may suggest enhanced cardiac reserve, our results indicate diminished cardiac function in these mice.

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.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmias.

AIMS: Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the 'NACHT, LRR, and PYD Domains-containing Protein 3' (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF. METHODS AND RESULTS: Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3-/-) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3-/- mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K+-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca2+ release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3-/-) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca2+ release in DIO mice. CONCLUSION: These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation.

Aortic acceleration as a noninvasive index of left ventricular contractility in the mouse.

The maximum value of the first derivative of the invasively measured left ventricular (LV) pressure (+ dP/dtmax or P') is often used to quantify LV contractility, which in mice is limited to a single terminal study. Thus, determination of P' in mouse longitudinal/serial studies requires a group of mice at each desired time point resulting in "pseudo" serial measurements. Alternatively, a noninvasive surrogate for P' will allow for repeated measurements on the same group of mice, thereby minimizing physiological variability and requiring fewer animals. In this study we evaluated aortic acceleration and other parameters of aortic flow velocity as noninvasive indices of LV contractility in mice. We simultaneously measured LV pressure invasively with an intravascular pressure catheter and aortic flow velocity noninvasively with a pulsed Doppler probe in mice, at baseline and after the administration of the positive inotrope, dobutamine. Regression analysis of P' versus peak aortic velocity (vp), peak velocity squared/rise time (vp2/T), peak (+ dvp/dt or v'p) and mean (+ dvm/dt or v'm) aortic acceleration showed a high degree of association (P' versus: vp, r2 = 0.77; vp2/T, r2 = 0.86; v'p, r2 = 0.80; and v'm, r2 = 0.89). The results suggest that mean or peak aortic acceleration or the other parameters may be used as a noninvasive index of LV contractility.

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.

Spironolactone in pulmonary arterial hypertension: results of a cross-over study.

The renin-angiotensin-aldosterone system is implicated in the pathophysiology of pulmonary arterial hypertension. We undertook this study to determine the effects of spironolactone, a mineralocorticoid receptor blocker, on collagen metabolism in pulmonary arterial hypertension patients. After obtaining institutional review board approval and informed consent, 42 pulmonary arterial hypertension patients were prospectively enrolled and 35 patients completed the 16-week randomized double-blinded crossover clinical trial. Subjects received 50 mg spironolactone or placebo and at the end of week 8, treatment arm was switched. Circulating levels of collagen biomarkers, brain natriuretic peptide, and aldosterone levels were measured, and six-minute walk distance, liver function tests, and echocardiogram data were collected at weeks 0, 8, and 16. Mean age was 45 ± 15 years and 87% were females. At baseline, brain natriuretic peptide and aldosterone levels were 74 ± 95 pg/ml and 7 ± 8 pg/ml, respectively. There was no change in the levels of amino-terminal propeptide of procollagen type III (PIIINP), MMP-9, TIMP-1, and MMP-9/TIMP-1 ratio at weeks 8 and 16 compared to baseline values in placebo arm and treatment arm. The baseline six-min walk distance was 436 ± 115 meters at baseline and no change in walk distance was noted at weeks 8 and 16 (P = 0.372). None of the patients developed hyperkalemia or liver function test abnormalities at weeks 8 and 16 requiring discontinuation of study drug. Our study showed no change in collagen metabolite levels in pulmonary arterial hypertension patients treated with spironolactone. Spironolactone was safe and well tolerated by pulmonary arterial hypertension patients with no increased hyperkalemia or liver function test abnormalities.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

Coordinated changes in signaling pathways and gene expression in hearts subjected to prolonged stress maintain cardiac function. Loss of steroid receptor coactivator-2 (SRC-2) results in a reversal to the fetal gene program and disrupts the response to pressure overload, accompanied by prominent effects on metabolism and growth signaling, including increased AMPK activation. We proposed that early metabolic stress driven by AMPK activation induces contractile dysfunction in mice lacking SRC-2. We used 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK transiently before transverse aortic constriction (TAC) in wild-type and cardiomyocyte-specific SRC-2 knockout (CKO) animals. In contrast to AMPK activities during stress, in unstressed hearts, AICAR induced a mild activation of Akt signaling, and, in SRC-2-CKO mice, partially relieved an NAD+ deficiency and increased antioxidant signaling. These molecular changes translated to a mild hypertrophic response to TAC with decreased maladaptive remodeling, including markedly decreased fibrosis. Additionally, preactivation of AMPK in SRC-2-CKO mice was accompanied by a dramatic improvement in cardiac function compared with saline-treated SRC-2-CKO mice. Our results show that altered molecular signaling before stress onset has extended effects on sustained cardiac stress responses, and prestress modulation of transient growth and metabolism pathways may control those effects.-Nam, D. H., Kim, E., Benham, A., Park, H.-K., Soibam, B., Taffet, G. E., Kaelber, J. T., Suh, J. H., Taegtmeyer, H., Entman, M. L., Reineke, E. L. Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

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.

AMP-activated protein kinase/myocardin-related transcription factor-A signaling regulates fibroblast activation and renal fibrosis.

Chronic kidney disease is a major cause of death, and renal fibrosis is a common pathway leading to the progression of this disease. Although activated fibroblasts are responsible for the production of the extracellular matrix and the development of renal fibrosis, the molecular mechanisms underlying fibroblast activation are not fully defined. Here we examined the functional role of AMP-activated protein kinase (AMPK) in the activation of fibroblasts and the development of renal fibrosis. AMPKα1 was induced in the kidney during the development of renal fibrosis. Mice with global or fibroblast-specific knockout of AMPKα1 exhibited fewer myofibroblasts, developed less fibrosis, and produced less extracellular matrix protein in the kidneys following unilateral ureteral obstruction or ischemia-reperfusion injury. Mechanistically, AMPKα1 directly phosphorylated cofilin leading to cytoskeleton remodeling and myocardin-related transcription factor-A nuclear translocation resulting in fibroblast activation and extracellular matrix protein production. Thus, AMPK may be a critical regulator of fibroblast activation through regulation of cytoskeleton dynamics and myocardin-related transcription factor-A nuclear translocation. Hence, AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

Steroid receptor coactivator-2 (SRC-2) coordinates cardiomyocyte paracrine signaling to promote pressure overload-induced angiogenesis.

Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1α and -2α in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.

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.

The role of C-reactive protein in innate and acquired inflammation: new perspectives.

The participation of C-reactive protein (CRP) in host defense against microorganisms has been well described. More controversial has been its role in chronic conditions such as cardiovascular disease. Our recent publications explain the reasons for some of the confusion concerning CRP as a risk factor for disease and whether it is pro-inflammatory or anti-inflammatory. We found that two isoforms of CRP, pentameric (pCRP) and monomeric (mCRP), on microparticles (MPs), were not measureable by standard clinical assays. When we investigated MPs by imaging cytometry in plasma from controls versus patients with peripheral artery disease, we found that MPs from endothelial cells bearing mCRP were elevated. This elevation did not correlate with the soluble pCRP measured by high-sensitivity CRP assays. The data suggest that detection of mCRP on MPs may be a more specific marker in diagnosis, measurement of progression, and risk sensitivity in chronic disease. In an in vitro model of vascular inflammation, pCRP was anti-inflammatory and mCRP was pro-inflammatory for macrophage and T cell polarization. When we further investigated pCRP under defined conditions, we found that pCRP in the absence of a phosphocholine ligand had no inflammatory consequences. When combined with phosphocholine ligands, pCRP signaled through two Fcγ receptors (FcγRI and FcγRII) via phosphorylation of spleen tyrosine kinase (pSYK) to activate monocytes. Phosphocholine itself, when bound to pCRP, induced a congruent M2 macrophage and Th2 response. Phosphocholine is also the head group on the lipid phosphatidylcholine, which can become oxidized. Liposomes bearing oxidized phosphatidylcholine without pCRP promoted a uniform M1 macrophage and Th1 pro-inflammatory response. When oxidized liposomes were bound to pCRP, there was a disjunction in the macrophage and T cell response: monocytes matured into M2 macrophages, but the T cells polarized into a Th1 phenotype. The CRP-bound liposomes signaled monocytes via FcγRII to promote an anti-inflammatory M2 macrophage state, whereas the lack of FcγR on T cells allowed their liposome-induced polarization to a pro-inflammatory Th1 phenotype unopposed by the contribution of the pCRP/FcγR interaction. Different isoforms of CRP and its binding to complex ligands may determine its biological activities and their contribution to inflammatory states.

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.

TNF/Ang-II synergy is obligate for fibroinflammatory pathology, but not for changes in cardiorenal function.

Angiotensin-II (Ang-II) infusion is associated with the development of interstitial fibrosis in both heart and kidney as a result of chemokine-dependent uptake of monocytes and subsequent development of myeloid fibroblasts. This study emphasizes on the synergistic role of tumor necrosis factor (TNF) on the time course of Ang-II-induced fibrosis and inflammation in heart and kidney. In wild-type (WT) hearts, Ang-II-induced fibrosis peaked within 1 week of infusion and remained stable over a 6-week period, while the myeloid fibroblasts disappeared; TNF receptor-1-knockout (TNFR1-KO) hearts did not develop a myeloid response or cardiac fibrosis during this time. WT hearts developed more accelerated cardiac hypertrophy and remodeling than TNFR1-KO In the kidney, 1-week Ang-II infusion did not evoke a fibrotic response; however, after 6 weeks, WT kidneys displayed modest but significant tubulointerstitial collagen deposition associated with the appearance of myeloid cells and profibrotic gene activation. Renal fibrosis was not seen in Ang-II-infused TNFR1-KO By contrast, while hypertension increased and cardiac function decreased more slowly in TNFR1-KO than WT, they were equivalently abnormal at 6 weeks. Similarly, serum markers for renal dysfunction were not different after 6 weeks. In conclusion, Ang-II infusion initiated fibroinflammatory responses with different time courses in heart and kidney, both requiring TNFR1 signaling, and both associated with monocyte-derived myeloid fibroblasts. TNFR1 deletion obviated the fibroinflammatory effects of Ang-II, but did not alter changes in blood pressure and cardiorenal function after 6 weeks. Thus, the synergy of TNF with Ang-II targets the fibroinflammatory component of Ang-II signaling.