Sara McCurdy

Chitinase Inhibition Promotes Atherosclerosis in Hyperlipidemic Mice

Chitinase 1 (CHIT1) is secreted by activated macrophages. Chitinase activity is raised in atherosclerotic patient sera and is present in atherosclerotic plaque. However, the role of CHIT1 in atherosclerosis is unknown. Preliminary studies of atherosclerosis in cynomolgous monkeys revealed CHIT1 to be closely correlated with areas of macrophage infiltration. Thus, we investigated the effects of a chitinase inhibitor, allosamidin, on macrophage function in vitro and on atherosclerotic development in vivo. In RAW264.7 cells, allosamidin elevated monocyte chemoattractant protein 1 and tumor necrosis factor alpha expression, and increased activator protein 1 and nuclear factor-κB transcriptional activity. Although inducible nitric oxide synthase, IL-6, and IL-1β expression were increased, Arg1 expression was decreased by chitinase inhibition, suggesting that suppression of CHIT1 activity polarizes macrophages into a M1 phenotype. Allosamidin decreased scavenger receptor AI, CD36, ABCA1, and ABCG1 expression which led to suppression of cholesterol uptake and apolipoprotein AI-mediated cholesterol efflux in macrophages. These effects were confirmed with CHIT1 siRNA transfection and CHIT1 plasmid transfection experiments in primary macrophages. Apolipoprotein E-deficient hyperlipidemic mice treated for 6 weeks with constant administration of allosamidin and fed an atherogenic diet showed aggravated atherosclerotic lesion formation. These data suggest that CHIT1 exerts protective effects against atherosclerosis by suppressing inflammatory responses and polarizing macrophages toward an M2 phenotype, and promoting lipid uptake and cholesterol efflux in macrophages.

Hyperlipidemia-induced cholesterol crystal production by endothelial cells promotes atherogenesis

Endothelial cells (EC) play a key role in atherosclerosis. Although EC are in constant contact with low density lipoproteins (LDL), how EC process LDL and whether this influences atherogenesis, is unclear. Here we show that EC take up and metabolize LDL, and when overburdened with intracellular cholesterol, generate cholesterol crystals (CC). The CC are deposited on the basolateral side, and compromise endothelial function. When hyperlipidemic mice are given a high fat diet, CC appear in aortic sinus within 1 week. Treatment with cAMP-enhancing agents, forskolin/rolipram (F/R), mitigates effects of CC on endothelial function by not only improving barrier function, but also inhibiting CC formation both in vitro and in vivo. A proof of principle study using F/R incorporated into liposomes, designed to target inflamed endothelium, shows reduced atherosclerosis and CC formation in ApoE−/− mice. Our findings highlight an important mechanism by which EC contribute to atherogenesis under hyperlipidemic conditions.Atherosclerosis is characterized by subendothelial lipid retention believed to be the result of endothelial trancytosis. Here, the authors show that endothelium can take up and process LDL, generating cholesterol crystals that are deposited on the basolateral side of the cells, causing their dysfunction that can be prevented by forskolin/rolipram treatment.

Time Series miRNA-mRNA integrated analysis reveals critical miRNAs and targets in macrophage polarization

Polarization of macrophages is regulated through complex signaling networks. Correlating miRNA and mRNA expression over time after macrophage polarization has not yet been investigated. We used paired RNA-Seq and miRNA-Seq experiments to measure the mRNA and miRNA expression in bone marrow-derived macrophages over a time-series of 8 hours. Bioinformatics analysis identified 31 differentially expressed miRNAs between M1 and M2 polarized macrophages. The top 4 M1 miRNAs (miR-155-3p, miR-155-5p, miR-147-3p and miR-9-5p) and top 4 M2 miRNAs (miR-27a-5p, let-7c-1-3p, miR-23a-5p and miR-23b-5p) were validated by qPCR. Interestingly, M1 specific miRNAs could be categorized to early- and late-response groups, in which three new miRNAs miR-1931, miR-3473e and miR-5128 were validated as early-response miRNAs. M1 polarization led to the enrichment of genes involved in immune responses and signal transduction, whereas M2 polarization enriched genes involved in cell cycle and metabolic processes. C2H2 zinc-finger family members are key targets of DE miRNAs. The integrative analysis between miRNAs and mRNAs demonstrates the regulations of miRNAs on nearly four thousand differentially expressed genes and most of the biological pathways enriched in macrophage polarization. In summary, this study elucidates the expression profiles of miRNAs and their potential targetomes during macrophage polarization.

Potential role of IL-37 in atherosclerosis

IL-37 is a member of the IL-1 family, but unlike most other members of this family of cytokines, it has wide-ranging anti-inflammatory properties. Initially shown to bind IL-18 binding protein and prevent IL-18-mediated inflammation, its known role has been expanded to include distinct pathways, both intracellular involving the transcription factor Smad3, and extracellular via binding to the orphan receptor IL-1R8. A number of recent publications investigating the role of IL-37 in atherosclerosis and ischemic heart disease have revealed promising therapeutic value of the cytokine. Although research concerning the role of IL-37 and its mechanism in atherosclerosis is relatively scant, there are a number of well-known atherosclerotic processes that this cytokine can mediate with the potential of modulating the disease progression itself. This review will probe in detail the effects of IL-37 on important pathological processes such as inflammation, dysregulated lipid metabolism, and apoptosis, by analyzing existing data as well as exploring the potential of this cytokine to influence these properties.

Defective Fas Expression on Bone Marrow Derived Cells Alters Atherosclerotic Plaque Morphology in Hyperlipidemic Mice.

Fas (CD95) is a member of the TNF-receptor family expressed on a wide range of cells. Interaction of Fas with its receptor, Fas ligand (Fas-L), stimulates an intracellular cascade of events that leads to apoptosis. Because apoptosis of inflammatory cells plays a key role in atherosclerosis we sought to determine the role of Fas in the development of atherosclerosis by repopulating the bone marrow cells of atherosclerosis-prone low density lipoprotein receptor null (LDL-R-/-) mice with either cells from lpr mice (lpr-BMT) that have defective Fas expression or from control mice (WT-BMT). The lpr-BMT mice exhibited no peripheral blood Fas expression 4 weeks after BMT. After consuming an atherogenic diet for 16 weeks, lpr-BMT mice developed atherosclerotic lesions characterized by smaller fibrous area with thinner fibrous cap and less TUNEL-positive staining compared to WT-BMT mice, although overall lesion size in lpr-BMT mice was similar to that of WT-BMT mice. Examination of a series of human atherosclerotic lesions revealed that many Fas-positive cells were colocalized with CD68-positive macrophages. Although apoptotic cells were rarely observed in the foam cell-rich fatty streak lesions, apoptotic CD68-positive macrophages in advanced lesions were detected in areas rich with inflammatory cells near the necrotic core. These observations suggest that Fas expression by the macrophages in atherosclerotic lesions can influence the plaque morphology towards a more fibrous type.

Cluster of Differentiation 43 Deficiency in Leukocytes Leads to Reduced Atherosclerosis—Brief Report

Objective—The aim of this study was to investigate the role of cluster of differentiation 43 (CD43), an integral membrane glycoprotein with both proadhesive and antiadhesive activities, in atherosclerosis. Approach and Results—Low-density lipoprotein receptor–deficient mice were lethally irradiated and reconstituted with either bone marrow from CD43−/− mice or from wild-type controls. We found that mice lacking the CD43 on their leukocytes had significantly less severe atherosclerosis and that, contrary to our expectation, macrophage infiltration into the vessel wall was not affected by the lack of CD43 in the leukocytes. However, we found that CD43 mediates cholesterol homeostasis in macrophages by facilitating cholesterol efflux. This resulted in a significant reduction in storage of cholesterol in the aorta of mice lacking CD43 in the leukocytes. Conclusions—CD43 may be an important mediator of macrophage lipid homeostasis, thereby affecting macrophage foam cell formation and ultimately atherosclerotic plaque development.

Macrophage-Specific Expression of IL-37 in Hyperlipidemic Mice Attenuates Atherosclerosis

Atherosclerosis, the progressive buildup of plaque within arterial blood vessels, can lead to fatal downstream events, such as heart attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of monocytes and its associated inflammation, as well as the formation of lipid-laden macrophage foam cells within the vessel wall. IL-37 is recognized as an important anti-inflammatory cytokine expressed especially by immune cells. This study was undertaken to elucidate the role of macrophage-expressed IL-37 in reducing the production and effects of proinflammatory cytokines, preventing foam cell formation, and reducing the development of atherosclerosis. Expression of human IL-37 was achieved with a macrophage-specific overexpression system, using the CD68 promoter in mouse primary bone marrow–derived macrophages via retroviral transduction. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted protein production (e.g., IL-6, M-CSF, and ICAM-1) of key inflammatory mediators. IL-37 expression also inhibited macrophage proliferation, apoptosis, and transmigration, as well as reduced lipid uptake, compared with controls in vitro. The in vivo effects of macrophage-expressed IL-37 were investigated through bone marrow transplantation of transduced hematopoietic stem cells into irradiated atherosclerosis-prone Ldlr−/− mice. After 10 wk on a high-fat/high-cholesterol diet, mice with IL-37–expressing macrophages showed reduced disease pathogenesis, which was demonstrated by significantly less arterial plaque development and systemic inflammation compared with control mice. The athero-protective effect of macrophage-expressed IL-37 has implications for development of future therapies to treat atherosclerosis, as well as other chronic inflammatory diseases.

CD98 Regulates Vascular Smooth Muscle Cell Proliferation in Atherosclerosis

Conclusions: After adjustment for baseline characteristics, patients with symptomatic peripheral artery disease (PAD) based on prior revascularization, rather than an abnormal ABI, had a higher rate of myocardial infarction (MI) and acute limb ischemia but with a similar composite rate of cardiovascular death, MI and stroke. No significant difference was found between ticagrelor and clopidogrel for reduction of cardiovascular or acute limb ischemic events. Summary: The EUCLID trial (Examining Use of Ticagrelor vs Clopidogrel In PAD) randomized 13, 885 patients with peripheral artery disease to treatment with ticagrelor 90 mg twice daily or clopidogrel 75 mg daily. Patients were enrolled based on an abnormal ankle-brachial index #0.80 or a previous lower extremity revascularization. This analysis focuses on the 7875 (57%) patients enrolled based on the previous lower extremity revascularization criterion. Important exclusion criteria were that patients could not be enrolled within 30 days of most recent revascularization nor if on dual antiplatelet therapy. The primary efficacy end point was a composite rate of cardiovascular death, MI, or ischemic stroke. The primary safety end point was major bleeding. Patients with a previous revascularization had a mean age of 66 years, 73% were male, and the median baseline ankle-brachial index was 0.78. When compared to those entered into the study based on ABI criteria, there was a lower % of women (27% vs 29.3%; P 1⁄4 .004) but higher % of carotid stenosis (19% vs 15.3%; P 1⁄4 .001), prior coronary intervention (26.3% vs 19.1%; P 1⁄4 .001), and multilevel vascular disease (46.2% vs 40.7%; P 1⁄4 .001). They were more commonly smokers, hyperlipidemia and more were being treated with cardioprotective medications (antiplatelets, statins) prior to enrollment. There were fewer diabetics in this group. 31.3% were asymptomatic while 4.5% were experiencing critical limb ischemia. 63.5% had endovascular revascularization while 36.3% required an open operation. The timing of most recent revascularization was nearly a third of patients in each of three time intervals: <6 months, 6-24 months, and >2 years. 7.5% of patients had prior amputation; 1.7% above knee, 1.1% below knee and the remainder less extensive. Compared with patients enrolled based on the ABI criterion, patients enrolled based previous revascularization had higher rates of the primary composite end point (11.4% vs 9.9%; P 1⁄4 .02), myocardial infarction (5.9% vs 3.6%; P < .001), acute limb ischemia (2.5% vs 0.6%; P < .001), and major bleeding (1.8% vs 1.3%; P 1⁄4 .01). After adjustment for baseline characteristics, patients enrolled based on previous revascularization vs those with abnormal ABI criteria had similar rates of the primary composite end point (P 1⁄4 .12) but significantly higher rates of myocardial infarction (P 1⁄4 .005) and acute limb ischemia (P < .001). Ticagrelor was not found to statistically improve the rates of primary or secondary efficacy or primary safety end points vs clopidogrel treatment: primary efficacy end point (11.4% vs 11.3%; P 1⁄4 .90), all-cause mortality (9.2% vs 9.2%; P 1⁄4 .93), acute limb ischemia (2.5% vs 2.5%; P 1⁄4 .84), or major bleeding (1.9% vs 1.8%; P 1⁄4 .41). The bleeding rate was no different for intracranial bleeds (both 0.6%), fatal bleeds (0.1% ticagrelor vs 0.3% clopidogrel) or minor bleeds (1.6% ticagrelor vs 1.3% clopidogrel). The median duration of follow-up was w30 months with 19 patients either lost to follow-up or of unknown status. Comments: This subgroup analysis of the EUCLID study provides some insight into the care of patient with a prior revascularization. We must be more aware of the increased risk of myocardial infarction in these patients since it affects our attention and intensity of medical treatment. The increased incidence of acute limb ischemia is more difficult to interpret since we do not know the specific type of revascularization (angioplasty, bypass (synthetic or autogenous), location, etc.), which would affect the type of medical adjuvants we might chose to improve patency. The bottom-line for this study, however, is that either of these antiplatelets have a similar effect on cardiovascular risk reduction. It adds understanding to our use of monotherapy in these patients but much work needs to be done to determine optimal therapy. CD98 Regulates Vascular Smooth Muscle Cell Proliferation in Atherosclerosis Baumer Y, McCurdy S, Alcala M, Mehta N, Lee BH, Ginsberg MH, et al. Atherosclerosis 2017;256:105-14.

Natural sea salt consumption confers protection against hypertension and kidney damage in Dahl salt-sensitive rats

ABSTRACT Although sea salts are widely available to consumers nowadays, whether its consumption over refined salt has any real health benefits is largely unknown. This study was conducted to compare hypertension-inducing propensity of natural sea salt (SS) to refined salt (RS) in a well-established animal model of hypertension. Five groups of male Dahl salt-sensitive rats were fed rat chow diet supplemented with various amounts of salt for 15 weeks. The groups were: control (CON, n = 10), 4% RS (RS4), 4% SS (SS4), 8% RS (RS8), 8% SS (SS8) (n = 12 for each group). After 15 weeks, both SS4 and SS8 groups had significantly lower systolic (SBP) and diastolic blood pressure (DBP) compared to RS4 and RS8 rats, respectively. RS8 rats had markedly higher SBP and DBP compared to all other groups. Echocardiography just prior to sacrifice showed abnormalities in RS4, SS8 and RS8 hearts, while CON and SS4 hearts displayed normal measurements. Plasma renin and aldosterone levels of high salt groups were lower than those of CON, and serum electrolytes were similar amongst all groups. Abnormal kidney pathology and high glomerulosclerosis index scores were seen in RS4 and RS8 rats, but SS4 and SS8 kidneys showed relatively normal morphology similar to CON kidneys. Our findings show that consumption of natural sea salt induces less hypertension compared to refined salt in the Dahl salt-sensitive rat.

CD43 Deficiency in Leukocytes Leads to Reduced Atherosclerosis – Brief Report

Objective—The aim of this study was to investigate the role of cluster of differentiation 43 (CD43), an integral membrane glycoprotein with both proadhesive and antiadhesive activities, in atherosclerosis. Approach and Results—Low-density lipoprotein receptor–deficient mice were lethally irradiated and reconstituted with either bone marrow from CD43−/− mice or from wild-type controls. We found that mice lacking the CD43 on their leukocytes had significantly less severe atherosclerosis and that, contrary to our expectation, macrophage infiltration into the vessel wall was not affected by the lack of CD43 in the leukocytes. However, we found that CD43 mediates cholesterol homeostasis in macrophages by facilitating cholesterol efflux. This resulted in a significant reduction in storage of cholesterol in the aorta of mice lacking CD43 in the leukocytes. Conclusions—CD43 may be an important mediator of macrophage lipid homeostasis, thereby affecting macrophage foam cell formation and ultimately atherosclerotic plaque development.