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Curcumin is widely known for its antioxidant, anti-inflammatory, and antiproliferative activities in cell-culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvent oral bioavailability issues, and tested their potential against 17β-estradiol (E2)–mediated mammary tumorigenesis. Female Augustus Copenhagen Irish (ACI) rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2 cm; 200 mg each; 20% drug load) 4 days before grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Curcumin implants were changed after 4.5 months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months, and after the tumor incidence reached >80% (∼6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2 ± 1 vs. 5 ± 3; P = 0.001) and tumor volume (184 ± 198 mm3 vs. 280 ± 141 mm3; P = 0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity, whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in the presence of E2. Because CYP1A and CYP3A4 metabolize most of the E2 to its noncarcinogenic 2-OH metabolite, and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by high-performance liquid chromatography showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered. Cancer Prev Res; 7(4); 456–65. ©2014 AACR.

PDF file - 166K, Figure Legends for Supplementary data. Figure S-1. Structures of the three curcuminoids present in the commercially available curcumin powder: Curcumin or Curcumin I (diferuloylmethane; 75%); Curcumin II (demethoxycurcumin; 20%); and Curcumin III (Bisdemethoxycurcumin; 5%). Figure S-2. Tumor volume calculated from the tumor dimensions measured using digital vernier caliper. The delay in the appearance of first tumor and subsequent tumors with curcumin implants suggest that curcumin functions as a chemopreventive agent. The implants were replaced at about 135 days, which further retarded the tumor growth. The similarity in the tumor growth curves between sham and curcumin implants suggest that curcumin may not have any significant therapeutic role in this ACI rat mammary tumor model. Figure S-3. Mechanistic overview of cytochrome P450-mediated bioactivation of 17-beta-estradiol to initiate carcinogenesis and to promote proliferation.

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

Ischemic heart failure (HF) is associated with elevated inflammatory responses including pro-inflammatory cytokines, such as Tumor necrosis factor (TNF) α [and its cognate receptors, TNF receptor (TNFR) 1 and TNFR2] and pathological switching of CD4 + T-cells. TNF-TNFR2 signaling is known to promote CD4 + T-cell activation and proliferation. However, it is unknown if CD4 + T-cells also express TNFR1, and if elevated TNFα-TNFR1 signaling during HF mediates pathological effects of CD4 + T-cells. Using flow cytometry, we show that TNFR1 + CD4 + T-cells are amplified in the hearts at 3 days (d) and 8 weeks (wks) post-MI (HF) in mice. As compared to sham, while TNFα expression (and %) is blunted in CD4 + T-cells at 3 d post-MI, it is increased during HF with significantly higher expression in TNFR1 + T-cells as compared to TNFR1 - T-cells. Splenic CD4 + T-cells activated using anti-CD3/CD28 antibodies and treated with neutralizing anti-TNFR1 antibody showed increased T-cell proliferation, and Bcl-xL (pro-survival factor) and TNFα expression without affecting activation marker (CD69) suggesting a role of TNFR1 in mediating pro-apoptotic signaling without affecting activation. To confirm these results, we adoptively transferred (AT) HF-activated splenic WT and TNFR1 -/- T-cells (CD45.2) into CD45.1 naïve mice, and measured changes in cardiac function of recipient mice by echocardiography (as an indicator of pathological activity), and donor T-cells (as a measure of cell survival) in the blood, heart, spleen and mediastinal lymph nodes (mLNs) at 14 wks post-AT. Compared to WT, we observed amplified levels of donor TNFR1 -/- T-cells in the blood, spleen, and mLNs which were not different in the hearts. Consistently, recipient mice injected either with WT and TNFR1 -/- T-cells showed similar degrees of cardiac dysfunction. Interestingly, at 14 wks post-AT the expression of ki67, a proliferation marker, was magnified in HF-activated TNFR1 -/- T-cells (as compared to WT) infiltrated into the myocardium suggesting that TNF-TNFR1 mediates pro-survival and proliferative signaling in T-cells during HF, and not their pathogenicity. Our results also provide deeper insights into the paradoxical outcomes observed in the clinical trials antagonizing TNFα in human HF patients.

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Background: CD4 + T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. Methods: RNA-sequencing of cardiac CD4 + T cells and flow cytometric analysis of immune cells was conducted. Results: RNA-sequencing of CD4 + T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4 + T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERβ agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4 + T cells without affecting other myeloid and lymphoid cells in the HF mice. Conclusions: Our studies indicate that ERβ agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4 + T cells during chronic HF.

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

CD4 + T-cells turn pathological during chronic heart failure (HF). Phenotypic changes that mediate this transition are unknown. Thus, at 8 weeks post-infarction we conducted limited cell RNA-sequencing on 150 CD4 + T-cells isolated from rodent failing hearts and using ingenuity pathway analysis (z score>2) compared them either with CD4 + T-cells isolated from the mediastinal lymph nodes (mLNs) of the same mice or cardiac CD4 + T-cells from sham mice. T-cells isolated from the mLNs of HF mice showed enhanced TCR signaling (p-4 ) with 3-6 fold increase in several downstream mediators such as ITK, MAPK, LCK and Zap70. We also observed heightened leukocyte extravasation signaling (p-5 ) with significant increase in PKCθ, and Rho GTPase activating and Ras association domain proteins. These are consistent with enhanced antigen-presentation, and TCR activation during HF. Moreover, we observed significant upregulation of oxidative phosphorylation (p-15 ) in cardiac CD4 + T-cells as compared to mLN T-cells with 2-3 fold increase in the gene expression of ATP synthase subunits and cytochrome C indicative of their increased metabolic activity upon infiltration into the failing hearts. Compared to CD4 + T-cells from sham hearts, significant upregulation of inflammatory genes (p-5 ), chemotactic factors (p-4 ), and several pro-inflammatory cytokine-mediated pathways such as IL-6 (p-4 ), IL-1a (p-3 ) and TNFα (p-3 ) was observed in cardiac CD4 + T-cells from HF mice. Interestingly, we also observed a significant upregulation of homing (p-4 ) and connective tissue remodeling genes (p-3 ) with >8-fold increase in genes like LCN2, SLPI, IGF1, FGFR1, LTF, and TIMP2 required for transmigration and homing of CD4 + T-cells into the ischemic hearts. Our studies show enhanced antigenic CD4 + T-cell activation in the mLNs supporting enhanced pro-inflammatory signaling, metabolic activation, and extravasation into the ischemic hearts.

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-β) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.

Gender bias in cardiovascular disease has been extensively documented in epidemiological and clinical studies. Despite this, the precise molecular mechanisms underlying these disparities between men and women are poorly understood. It is clear that physiological concentrations of estradiol, such as those present in pre-menopausal women, exert cardioprotective effects that are absent in men or in post-menopausal women. These cardioprotective effects, in part, are due to the estrogen receptor-mediated modulation of the immune system including T-cells. Estrogen receptors (ERs) are widely expressed in different T-cell subsets which are known to play an indispensable role in the progression of cardiovascular disease. Because T-cells can be polarized into several distinct subsets depending on the activation milieu, they can have many different, potentially opposing functions, and it is unclear what roles estrogen receptor signaling may play in mediating these functions. This is further complicated by the discrete and often antagonistic actions of different ERs on T-cell biology which dictate the balance between numerous ER-dependent signaling pathways. While myriad effects of estrogen in T-cells are relevant for many cardiovascular diseases, their widespread effects on several other (patho)physiological systems introduce several obstacles to understanding ER signaling and its precise effects on the immune system. This review aims to provide a more comprehensive summary of the mechanisms of estrogen receptor-mediated modulation of T-cell function, polarization, and cytokine production in the context of cardiovascular disease.

Fidelity of wound healing after myocardial infarction (MI) is an important determinant of subsequent adverse cardiac remodeling and failure. Infiltrating Ly6C hi blood monocytes are a key component of this healing response; however, the importance of other monocyte/macrophage populations is unclear. Here, we demonstrate in mice an essential role for splenic CD169 + Tim4 + marginal zone metallophilic macrophages (MMMs) in post-MI wound healing. CD169 + Tim4 + MMMs circulate as Ly6C low monocytes, and dynamically populate the naïve heart. After acute MI, mobilization robustly increases to the heart, where these macrophages are obligatory for apoptotic neutrophil clearance and the induction of pro-resolving and reparative tissue macrophages. Splenic MMMs are both necessary and sufficient forpost-MI wound healing, and limit late pathological remodeling.Pharmacological expansion of the splenic marginal zone during acute MI alleviates inflammation and cardiac remodeling. Finally, humans with acute MI also exhibit expansion of blood CD169 + monocytes. Hence, splenic CD169 + Tim4 + MMMs are required for pro-resolving and reparative responses after MI, and can be manipulated for therapeutic benefit to limit long-term heart failure.

Inflammation is a double-edged sword for sterile tissue injury such as in myocardial infarction (MI). After ischemic injury, inflammatory immune responses activate repair processes, clear tissue-debris, form a stable scar and initiate angiogenesis in the myocardium for efficient wound-healing. However, incomplete immune resolution or sustained low-grade inflammation lead to ischemic cardiomyopathy (IC) characterized by maladaptive tissue remodeling and left-ventricular dilatation. It is clear that a delicate balance of cytokines, chemokines, prostaglandins, resolvins, and the innate and adaptive immune systems is critical for adequate healing as both insufficient- or overt-activation of inflammatory responses can either enhance rupture incidence or exacerbate cardiac dysfunction in the long-term. Among all the players, immune cells are the most critical as they are not only a source for all of the inflammatory protein mediators, but are also a target. However, phenotypic complexities associated with different immune subtypes, their interdependence, phasic-activations and varied functionalities often make it difficult to segregate the effects of one immune cell from another. In this review, we briefly summarize the role of several innate and adaptive immune cells to acquaint readers with complex immune-networks that dictate the extent of wound-healing post-MI and maladaptive remodeling during IC.

Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn(+/-);mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week–old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80(Hi), but lower percentages of F4/80(Lo) cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.

Ischemic heart failure (HF) results in the activation of self-reactive CD4+ T-lymphocytes against unknown cardiac antigens. The mechanisms that mediate the survival of self-reactive T-cells during ischemic HF are not known. TNF mediated NF-kB signaling regulates cellular apoptotic pathways and T cells express high levels of TNF receptor-1 (TNFR1) during chronic heart failure. Thus, we hypothesized that TNFR1 regulates pro-apoptotic signaling through NF-kB and is critical for the clearance of self-reactive T-cells during HF. To test this, male WT or TNFR1 -/- mice underwent coronary artery ligation to induce HF (10 wks post-ligation), followed by isolation and adoptive-transfer (AT) of splenic CD4+ T-cells (9X10 6 cells/mouse) into the tail-veins of naïve CD45.1 mice. Flow cytometric analysis showed ~7-fold higher levels of HF-activated TNFR1 -/- T cells in recipient mice when compared with WT T-cells at 2d post-AT, and were not different between the 2-groups at 1 wk. Interestingly, TNFR1 -/- T cells were significantly higher again at 2 wks (as compared to WT T-cells) and the levels were sustained for up to 14 wks post-AT. In contrast, donor WT T-cells were slowly cleared from the recipient mice. In addition, as compared to WT T-cells, ~11-fold higher levels of adoptively transferred TNFR1 -/- T cells were observed in the spleens of recipient mice at 14 wks suggesting that TNFR1 -/- T cells had higher propensity to seed into the lymphoid tissues. This data also suggests that TNFR1 -/- T cells either have enhanced pro-survival pathways or were less prone to activation-induced cell death. In-vitro T-cell proliferation assays using anti-CD3/CD28 antibodies showed enhanced proliferation and expression of anti-apoptotic Bcl-xL upon treatment with a TNFR1 neutralization antibody corroborating in-vivo results. Intriguingly, as opposed to WT T-cells, adoptive transfer of HF-activated TNFR1 -/- T-cells induced significant cardiac dysfunction and the ejection fraction (EF) of naïve recipient mice was significantly decreased from 63±6% to 45±6% at 14 weeks post-AT. Our data show that TNFR1 signaling regulates pro-vs anti-apoptotic pathways in a context-dependent manner and potentiate pathological phenotype of HF activated T-cells by enhancing their life-span.

CD4+ T-cells mediate wound-healing post-myocardial infarction (MI) but exacerbate left-ventricular (LV) remodeling during chronic heart failure (HF). Mechanisms that lead to this transition are unknown. Therefore, we hypothesized that as opposed to MI, T-cells activate specific pathological signals to promote LV-remodeling during chronic HF. To identify such signals, we conducted limited cell RNA-sequencing of CD4+ T-cells sorted from the failing hearts (8 weeks post-MI) of male mice and, surprisingly, found activation of estrogen receptor (ER)-α signaling. As ERα effects are antagonized by ERβ, we tested a novel ERβ agonist (OSU-ERB-012) to modulate T-cell activity and LV remodeling. In-vitro assays showed that OSU-ERB-012 dose dependently inhibit activation and proliferation of T-cells isolated from male mice (IC 50 3.4 μM). In-vivo assays (60 mg/kg/day; oral) showed no overt toxicity and a significant reduction in circulating T cells without affecting neutrophils, monocytes, or B-cells indicating specificity against T-cells. Furthermore, this effect was specific to TCR-mediated T-cell activation and the drug did not affect T-cells stimulated with PMA/Ionomycin suggesting preferential inhibition of antigenically-activated T-cells. To test therapeutic efficacy, male 10-12 week old mice underwent coronary ligation or sham operation and at 4 weeks post-MI randomized according to their cardiac function to receive either the vehicle or OSU-ERB-012 (60 mg/kg/day, oral) for the next 4 weeks. Consistently, at 8 weeks we observed a significant reduction in T-cells in the circulation and the spleens of drug-treated mice when compared with the vehicle treatment. As expected, vehicle treated HF mice showed progressive LV dilatation with significantly increased end-diastolic and end-systolic volumes (EDV and ESV, respectively) from 4-8 weeks post-MI. Importantly, treatment with OSU-ERB-012 significantly inhibited these changes and blunted LV remodeling from 4-8 weeks post-MI. Significant reduction in tibia-normalized heart weights supported these results. Our studies indicate that OSU-ERB-012 is a novel orally-active drug molecule that selectively inhibits T-cell activation and blunts pathological LV-remodeling during chronic HF.

Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties, however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.

Aims Heart failure (HF) is characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines and HF. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The objective of this study was to investigate the role of TWEAK/Fn14 pathway in promoting cardiac inflammation under non ischemic stress conditions. Main methods Wild type (WT) and Fn14 knock out (Fn14−/−) mice were subjected to pressure overload [transaortic constriction (TAC)] for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 antagonist L524-0366. Cardiac function was measured by echocardiography. Cardiac fibrosis and macrophage infiltration were quantified using immunohistochemistry and flow cytometry, respectively. Cardiac fibroblasts were isolated for quantifying TWEAK-induced chemokine release. Key findings Fn14−/− mice displayed improved cardiac function, reduced fibrosis and lower macrophage infiltration in heart compared to WT following TAC. L524-0366 mitigated maladaptive remodeling with TAC. TWEAK induced secretion of the pro-inflammatory chemokine, monocyte chemoattractant protein 1 from WT but not Fn14−/− fibroblasts in vitro, in part through activation of non-canonical NF-κB signaling. Finally, Fn14 expression was increased in mouse following TAC and in human failing hearts. Significance Our findings support an important role for the TWEAK/Fn14 promoting macrophage infiltration and fibrosis in heart under non-ischemic stress, with potential for therapeutic intervention to improve cardiac function in the setting of HF.

Inflammation has recently gained tremendous attention as a key contributor in several chronic diseases. While physiological inflammation is essential to counter a wide variety of damaging stimuli and to improve wound healing, dysregulated inflammation such as in the myocardium and vasculature can promote cardiovascular diseases. Given the high severity, prevalence, and economic burden of these diseases, understanding the factors involved in the regulation of physiological inflammation is essential. Like other complex biological phenomena, RNA-based processes are emerging as major regulators of inflammatory responses. Among such processes are cis-regulatory elements in the mRNA of inflammatory genes, noncoding RNAs directing the production or localization of inflammatory cytokines/chemokines, or pathogenic RNA driving inflammatory responses. In this review, we describe several specific RNA-based molecular mechanisms by which physiological inflammation pertaining to cardiovascular diseases is regulated. These include the role of AU-rich element-containing mRNAs, long non-coding RNAs, microRNAs, and viral RNAs.

Background: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling. Methods: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1) − /− mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays. Results: Compared with wild-type sham mice, CD4 + Foxp3 + Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4 + T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34 + Flk1 + circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization. Conclusions: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.

Primary or secondary hypogonadism results in a range of signs and symptoms that compromise quality of life and requires life-long testosterone replacement therapy. In this study, an implantable nanochannel system is investigated as an alternative delivery strategy for the long-term sustained and constant release of testosterone. In vitro release tests are performed using a dissolution set up, with testosterone and testosterone:2-hydroxypropyl-β-cyclodextrin (TES:HPCD) 1:1 and 1:2 molar ratio complexes release from the implantable nanochannel system and quantify by HPLC. 1:2 TES:HPCD complex stably achieve 10-15 times higher testosterone solubility with 25-30 times higher in vitro release. Bioactivity of delivered testosterone is verified by LNCaP/LUC cell luminescence. In vivo evaluation of testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels by liquid chromatography mass spectrometry (LC/MS) and multiplex assay is performed in castrated Sprague-Dawley rats over 30 d. Animals are treated with the nanochannel implants or degradable testosterone pellets. The 1:2 TES:HPCD nanochannel implant exhibits sustained and clinically relevant in vivo release kinetics and attains physiologically stable plasma levels of testosterone, LH, and FSH. In conclusion, it is demonstrated that by providing long-term steady release 1:2 TES:HPCD nanochannel implants may represent a major breakthrough for the treatment of male hypogonadism.

Curcumin is widely known for its antioxidant, anti-inflammatory, and antiproliferative activities in cell-culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvent oral bioavailability issues, and tested their potential against 17β-estradiol (E2)–mediated mammary tumorigenesis. Female Augustus Copenhagen Irish (ACI) rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2 cm; 200 mg each; 20% drug load) 4 days before grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Curcumin implants were changed after 4.5 months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months, and after the tumor incidence reached >80% (∼6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2 ± 1 vs. 5 ± 3; P = 0.001) and tumor volume (184 ± 198 mm3 vs. 280 ± 141 mm3; P = 0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity, whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in the presence of E2. Because CYP1A and CYP3A4 metabolize most of the E2 to its noncarcinogenic 2-OH metabolite, and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by high-performance liquid chromatography showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered. Cancer Prev Res; 7(4); 456–65. ©2014 AACR.

Combined use of gemcitabine (Gem) and LY-364947 (LY), a TGF-β1 receptor inhibitor, has shown promise for the treatment of fibrotic pancreatic cancer, by reducing collagen production and improving tumor drug penetration. The preparation and optimization of novel Gem and LY formulations, including co-encapsulation in liposomes, require a validated method for the simultaneous quantification of both drugs, a method that had yet to be developed. Here we demonstrate an RP-HPLC protocol for the simultaneous detection of Gem and LY at 266 and 228 nm with retention times of 3.37 and 11.34 mins, respectively. The method, which uses a C18 column and a KH2PO4 (10 mM)-methanol mobile phase, was validated for linearity, precision, accuracy, limits of detection, and robustness. Co-loaded liposomes with both Gem and LY (Gem/LY liposomes) were developed to investigate the protocol applicability to pharmacokinetic analysis and formulation characterization. The method specificity was evaluated in presence of liposomal components in fetal bovine serum (FBS). Finally, the method was demonstrated by quantifying Gem/LY liposomal encapsulation efficiency and concentration liposomes-spiked FBS.

Background— Inappropriately sustained inflammation is a hallmark of chronic ischemic heart failure (HF); however, the pathophysiological role of T lymphocytes is unclear. Methods and Results— Permanent coronary ligation was performed in adult C57BL/6 mice. When compared with sham-operated mice, mice with HF (8 weeks after ligation) exhibited the following features: (1) significant ( P + CD8 + cytotoxic and CD3 + CD4 + helper (Th) T lymphocytes, together with increased Th1, Th2, Th17, and regulatory T-cell (Treg) CD4 + subsets; (2) significant expansion of CD8 + and CD4 + T cells in failing myocardium, with increased Th1, Th2, Th17, and Treg CD4 + subsets, marked reduction of the Th1/Th2 ratio, augmentation of the Th17/Treg ratio, and upregulation of Th2 cytokines; and (3) significantly increased Th1, Th2, Th17 cells, and Tregs, in the spleen and mediastinal lymph nodes, with expansion of splenic antigen-experienced effector and memory CD4 + T cells. Antibody-mediated CD4 + T-cell depletion in HF mice (starting 4 weeks after ligation) reduced cardiac infiltration of CD4 + T cells and prevented progressive left ventricular dilatation and hypertrophy, whereas adoptive transfer of splenic CD4 + T cells (and, to a lesser extent, cardiac CD3 + T cells) from donor mice with HF induced long-term left ventricular dysfunction, fibrosis, and hypertrophy in naive recipient mice. Conclusions— CD4 + T lymphocytes are globally expanded and activated in chronic ischemic HF, with Th2 (versus Th1) and Th17 (versus Treg) predominance in failing hearts, and with expansion of memory T cells in the spleen. Cardiac and splenic T cells in HF are primed to induce cardiac injury and remodeling, and retain this memory on adoptive transfer.

Background Although cardiac and splenic mononuclear phagocytes (MPs), i.e., monocytes, macrophages and dendritic cells (DCs), are key contributors to cardiac remodeling after myocardial infarction, their role in pressure-overload remodeling is unclear. We tested the hypothesis that these immune cells are required for the progression of remodeling in pressure-overload heart failure (HF), and that MP depletion would ameliorate remodeling. Methods and Results C57BL/6 mice were subjected to transverse aortic constriction (TAC) or sham operation, and assessed for alterations in MPs. As compared with sham, TAC mice exhibited expansion of circulating LyC6hi monocytes and pro-inflammatory CD206− cardiac macrophages early (1 w) after pressure-overload, prior to significant hypertrophy and systolic dysfunction, with subsequent resolution during chronic HF. In contrast, classical DCs were expanded in the heart in a biphasic manner, with peaks both early, analogous to macrophages, and late (8 w), during established HF. There was no significant expansion of circulating DCs, or Ly6C+ monocytes and DCs in the spleen. Periodic systemic MP depletion from 2 to 16 w after TAC in macrophage Fas-induced apoptosis (MaFIA) transgenic mice did not alter cardiac remodeling progression, nor did splenectomy in mice with established HF after TAC. Lastly, adoptive transfer of splenocytes from TAC HF mice into naïve recipients did not induce immediate or long-term cardiac dysfunction in recipient mice. Conclusions Mononuclear phagocytes populations expand in a phasic manner in the heart during pressure-overload. However, they are dispensable for the progression of remodeling and failure once significant hypertrophy is evident and blood monocytosis has normalized.

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS−/− mice (iNOS−/−c) or WT mice (WTc). Chi-meric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ∼twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ∼sevenfold higher cardiac M1 macrophages, which were primarily CCR2– cells. In contrast, as compared with WTc HF mice, iNOS−/− c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS−/−c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomy-ocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS−/− HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.

Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant formulation (“coated” implants) using curcumin, individual curcuminoids, withaferin A and oltipraz. This method involves the coating of blank polycaprolactone implants with 20–30 layers of 10–20% polycaprolactone solution in dichloromethane containing 0.5–2% of the test agent. The in vitro release showed that while oltipraz was released with almost zero-order kinetics over 8 weeks, curcumin, individual curcuminoids and withaferin A were released with some initial burst. The in vivo release was determined by grafting implants subcutaneously in A/J mice. When delivered by coated implants, oltipraz significantly diminished lung DNA adducts in mice treated with dibenzo[ a,l ]pyrene compared with sham treatment (28 ± 7 versus 54 ± 17 adducts/10 9 nucleotides). Withaferin A also diminished DNA adducts, but it was insignificant. Curcumin and individual curcuminoids were ineffective. Analysis of lung, liver and brain by UPLC-fluorescence showed the presence of the three test curcuminoids indicating effectiveness of the implant delivery system. Further, based on its known antitumor activity in vivo , withaferin A given via the implants significantly inhibited human lung cancer A549 xenograft in athymic nude mice, while it was ineffective when the same total dose was administered i.p. and required over 2-fold higher dose to elicit effectiveness. Together, our data suggest that coated polymeric implants can accommodate heat-labile compounds, can furnish sustained release for long duration, and elicit DNA damage-inhibiting and anti-tumor activities.

Since ancient times, chemopreventive agents have been used to treat/prevent several diseases including cancer. They are found to elicit a spectrum of potent responses including anti-inflammatory, antioxidant, antiproliferative, anticarcinogenic, and antiangiogenic activity in various cell cultures and some animal studies. Research over the past 4 decades has shown that chemopreventives affect a number of proteins involved in various molecular pathways that regulate inflammatory and carcinogenic responses in a cell. Various enzymes, transcription factors, receptors, and adhesion proteins are also affected by chemopreventives. Although, these natural compounds have shown significant efficacy in cell culture studies, they elicited limited efficacy in various clinical studies. Their introduction into the clinical setting is hindered largely by their poor solubility, rapid metabolism, or a combination of both, ultimately resulting in poor bioavailability upon oral administration. Therefore, to circumvent these limitations and to ease their transition to clinics, alternate strategies should be explored. Drug delivery systems such as nanoparticles, liposomes, microemulsions, and polymeric implantable devices are emerging as one of the viable alternatives that have been shown to deliver therapeutic concentrations of various potent chemopreventives such as curcumin, ellagic acid, green tea polyphenols, and resveratrol into the systemic circulation. In this review article, we have attempted to provide a comprehensive outlook for these delivery approaches, using curcumin as a model agent, and discussed future strategies to enable the introduction of these highly potent chemopreventives into a physician's armamentarium. Cancer Prev Res; 4(8); 1158–71. ©2011 AACR.

Curcumin, an anti-oxidant, anti-inflammatory, and anti-neoplastic agent, exhibited limited oral efficacy due to its poor bioavailability. To overcome this limitation, polymeric implants for continuous systemic delivery of curcumin were developed and tested for their safety and biocompatibility. Two 2-cm polycaprolactone implants containing polyethylene glycol and 20% (w/w) curcumin were grafted subcutaneously at the back of the Augustus Copenhagen Irish rats. Rats were euthanized and blood was analyzed for various hematological parameters; biochemical markers of liver/kidney function and local tissues were analyzed for local inflammatory reactions. Curcumin implants exhibited biphasic release kinetics with ∼3.6 + 0.8, 5.8 ± 1.1, 13.1 ± 2.1, 21.8 ± 0.3, 38.1 ± 0.6, and 47.2 ± 1.6 mg cumulative curcumin being released from both the implants after 1, 4, 12, 25, and 90 days. No significant differences in various hematological parameters (like white blood cells, red blood cells, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin), liver enzymes (like aspartate transaminase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, amylase, or lipase), or biochemical parameters of kidney function (like blood urea nitrogen, creatinine, Ca(2+), Na(+), and Cl(-) levels) were observed at any of these time points. However, a significant increase in serum phosphorus levels was observed at all the time points in sham implants as well as in curcumin diet and implant groups. Local implantation site showed foreign body granulomatous reaction with influx of histiocytes and occasional multi-nucleated giant cells with sham implants and was minimal around the curcumin implants. These polymeric implants were found to have little or no systemic toxicity with an acute reaction at local site which was reduced significantly by curcumin implants.

The introduction of curcumin into clinics is hindered by its low water solubility and poor bioavailability. To overcome these limitations, we developed curcumin implants using poly (e-caprolactone) as the polymeric matrix. Implants were prepared by melt-extrusion method; in vitro drug release was optimized for effects of polymer composition, drug load, surface area and water-soluble additives. Implants were also tested under in vivo conditions for cumulative curcumin release, and liver concentration was correlated with its efficacy to modulate selected xenobiotic-metabolizing enzymes (CYP1A1 and GSTM). Drug release from implants followed biphasic release pattern with Higuchi kinetics and was proportional to the surface area of implants. Drug release increased proportionately from 2 to 10% (w/w) drug load, and incorporation of 10% (w/w) of water-soluble additives (F-68, PEG 8000 and cyclodextrin) did not significantly alter the drug release. In vivo drug release was found to be ∼1.8 times higher than in vitro release. Curcumin was detected at 60 ± 20 ng/g in the liver after four days of implantation and was almost constant (8–15 ng/g) for up to 35 days. This time-dependent drop in curcumin level was found to be due to induction of CYP1A1 and GSTM (μ) enzymes which led to increased metabolism of curcumin. Our data showed that these implants were able to release curcumin for long duration and to modulate liver phase I and phase II enzymes, demonstrating curcumin’s biological efficacy delivered via this delivery system.

Heart failure (HF) is characterized by exaggerated systemic and myocardial pro-inflammatory responses, which impede myocardial healing and promote pathological remodeling and dysfunction. Currently, there are no strategies available to restrain and/or modulate immune cell responses triggered by myocardial injury. 4F, an apolipoprotein A-I mimetic, exhibits anti-inflammatory properties and influences macrophage polarity. We hypothesized that 4F treatment following reperfused myocardial infarction (MI) in mice would attenuate post-MI cardiac remodeling by inducing an anti-inflammatory and tissue healing monocyte polarity. C57BL/6 mice (6-8/group) were intravenously injected with either PBS or L-4F (100 μg/day) for 5 d starting at day 3 after reperfused MI (60 min ischemia). Flow cytometry revealed that compared to PBS injected MI mice, 4F significantly (p < 0.05) decreased levels of pro-inflammatory monocytes (CD45+Cd11b+Ly6Chigh) in blood (20.4 ± 4.6 vs 9.4 ± 1.7%), spleen (25.5 ± 7.4 vs 8.0 ± 6.0%), and heart (0.9 ± 0.4 vs 0.2 ± 0.1%); and decreased myocardial macrophage (CD45+CD11b+F4/80+) infiltration (1.6 ± 0.8 vs 0.28 ± 0.2%). The levels of anti-inflammatory monocytes, however, were unchanged. Echocardiography revealed that these changes in immune cell profiles in 4F-injected mice were also accompanied by improvements in cardiac function. Compared to PBS injected MI mice, 4F treatment attenuated post-MI LV chamber dilatation (84.1 ± 16 vs 65.0 ± 8.5 uL and 59.15 ± 16.6 vs 38.4 ± 11.8 uL; end-diastolic and systolic volumes, respectively), and improved LV ejection fraction (28.4 ± 10.8 vs 41.7 ± 12.8%). In addition, in vitro studies using mouse peritoneal macrophages indicated that 4F promotes an anti-inflammatory macrophage phenotype potentially by inhibiting the glycolytic metabolic profile typical of pro-inflammatory macrophages. These effects on macrophage polarity by 4F were associated with parallel alterations in the expression of hypoxia inducible factor isoforms 1 and 2α, known markers of macrophage polarity. We conclude that 4F could be used therapeutically after MI to augment reparative immune responses and thereby limit the long-term maladaptive effects of exaggerated inflammation on adverse post-MI cardiac remodeling.

The functional role of regulatory T-cells (Tregs) in chronic heart failure (HF) is unknown. Using flow cytometry, we assessed Tregs during the progression of ischemic HF in male C57Bl/6 mice after coronary ligation and myocardial infarction (MI). At 7 d post-MI, circulating Tregs (% CD4+ T-cells) were comparable to sham-operated mice. Thereafter, blood Tregs significantly (p < 0.05 vs sham) increased ~1.5 fold at 2, 4, 6, and 8 w, and exhibited increased levels of pro-inflammatory intracellular IFNγ (40.5 ± 5.9 vs 28.4 ± 5.1%) and TNFα (34.6 ± 8.8 vs 21.7 ± 6.9%). Moreover, at 8 w post-MI, Tregs were increased in the spleen (24.1 ± 3.9 vs 18.3 ± 1.4%) and failing heart (0.95 ± 0.42 vs 0.33 ± 0.23%) as compared with sham mice. In contrast to splenic Tregs from sham mice, Tregs isolated from HF mice (8 w post-MI) failed to suppress effector T-cell proliferation, and were robustly antiangiogenic as indexed by mouse coronary endothelial cell tube formation assays. Transgenic mice expressing diphtheria toxin (DT) receptor (DTR) under control of the Foxp3 promoter underwent coronary ligation and DT-induced selective Treg ablation starting at 4 w post-MI (PBS control). Echocardiography before and after (8 w post-MI) PBS or DT injection revealed that in contrast to progressive LV dysfunction and remodeling after PBS, DT-mediated Treg ablation and subsequent reconstitution reversed pathological LV remodeling (decreased EDV and ESV, and increased EF). As compared with PBS injection, DT-induced Treg ablation also significantly (p < 0.05) reduced LV hypertrophy; enhanced circulating CD34+Flk1+ cells, cardiac CCR5 gene expression, and myocardial capillary density; reduced border zone fibrosis; and decreased pro-inflammatory (IL-17, IL-2 and IL-6) and pro-fibrotic (IL-4 and IL-10) serum cytokines. Similar results were obtained upon Treg depletion with anti-CD25 antibody in HF. We conclude that Tregs in chronic HF acquire pathological pro-inflammatory, profibrotic, and antiangiogenic properties, and lose their immunosuppressive capacity. Dysfunctional Tregs promote inflammation and adverse LV remodeling in HF; depletion of dysfunctional Tregs and subsequent restoration to a normal Treg phenotype may represent a novel approach to therapeutic immunomodulation in HF.

As opposed to chronic ischemic heart failure (HF), spleen-derived macrophages (Mf) are not prominent disease mediators during the chronic stages of pressure-overload (PO) HF. Increases in Mf during the early stages of PO, however, may serve as important drivers of both remodeling and activation of T-cell responses during later stages. Additionally, recent evidence indicates distinct groups of resident and circulating monocyte-derived infiltrating Mf that have different reparative vis-à-vis pro-inflammatory capacities. We hypothesized that cardiac Mf are expanded during the early stages of PO, primarily due to an increase in infiltrating Mf, and contribute to long-term T-cell activation in the PO heart. Cardiac Mf were characterized by flow cytometry as MerTK + CD64 + F480 + MHCII + cells, with infiltrating Mf populations identified as expressing chemokine receptor 2 (CCR2). One week after transverse aortic constriction (TAC), C57Bl/6 mice exhibited significant (p < 0.05) hypertrophy (heart weight/body weight 7.7 ± 0.5 vs 5.7 ± 0.2 mg/g; LV wall thickness 0.84 ± 0.03 vs 0.74 ± 0.02), without change in LVEF as compared with sham. There were robust increases in cardiac macrophages (3.15 ± 0.64 vs 1.09 ± 0.24%), specifically in the infiltrating CCR2 + MHCII hi population (1.6 ± 0.5 vs 0.5 ± 0.1%). TAC mice also exhibited higher circulating Gr1 – CD11b + Ly6C hi monocytes (2.6 ± 0.5 vs 1.3 ± 0.2%) that are recruited to tissue via CCR2. To determine activation of the adaptive immune response, we analyzed the mediastinal heart draining lymph nodes (MLN) for T-cells and antigen presenting cells. As compared with sham mice, the MLN of TAC mice were larger with greater cellularity (10.8 x 10 5 vs 4.2 x 10 5 ), especially related to MerTK + CD169 + Mf and CD3 + CD8 + T cells. We conclude that there is both an organ-specific and systemic immune response during compensatory PO hypertrophy, despite the absence of ischemic tissue injury. As infiltrating Mf are known to be less reparative, potently pro-inflammatory, and robust activators of adaptive immune cells, specifically targeting Ly6C hi monocyte recruitment and CCR2 + infiltrating Mf during the early compensated stages of pressure-overload may prevent subsequent inflammatory activation, adverse remodeling, and the transition to HF.

Obstructive biological barriers limit the transport and efficacy of cancer nanotherapeutics. Creative manipulation of tumor microenvironment provides promising avenues towards improving chemotherapeutic response. Such strategies include the use of mechanical stimuli to overcome barriers, and increase drug delivery and therapeutic efficacy. The rational use of gold nanorod-mediated mild hyperthermia treatment (MHT) alters tumor transport properties, increases liposomal gemcitabine (Gem Lip) delivery, and antitumor efficacy in pancreatic cancer CAPAN-1 tumor model. MHT treatment leads to a threefold increase in accumulation of 80-nm liposomes and enhances spatial interstitial distribution. I.v. injection of Gem Lip and MHT treatment lead to a threefold increase in intratumor gemcitabine concentration compared to chemotherapeutic infusion alone. Furthermore, combination of MHT treatment with infusion of 12 mg kg(-1) Gem Lip leads to a twofold increase in therapeutic efficacy and inhibition of CAPAN-1 tumor growth when compared to equimolar chemotherapeutic treatment alone. Enhanced therapeutic effect is confirmed by reduction in tumor size and increase in apoptotic index where MHT treatment combined with 12 mg kg(-1) Gem Lip achieves similar therapeutic efficacy as the use of 60 mg kg(-1) free gemcitabine. In conclusion, improvements in vivo efficacy are demonstrated resulting from MHT treatment that overcome transport barriers, promote delivery, improve efficacy of nanomedicines.