IL-37-a putative therapeutic agent in cardiovascular diseases.

Although it is a member of the Interleukin (IL)-1 family, IL-37 is unique in that it has wide-ranging anti-inflammatory characteristics. It was originally thought to prevent IL-18-mediated inflammation by binding to the IL-18-binding protein. However, upon discovery that it binds to the orphan receptor, IL-1R8, further studies have revealed an expanded role of IL-37 to include several intracellular and extracellular pathways that affect various aspects of inflammation. Its potential role specifically in cardiovascular diseases (CVD) stemmed initially from the discovery of elevated plasma IL-37 levels in human patients with acute coronary syndrome and atrial fibrillation. Other studies using mouse models of ischemia/reperfusion injury, vascular calcification and myocardial infarction have revealed that IL-37 can have a beneficial role in these conditions. This review will explore recent research on the effects of IL-37 on the pathogenesis of CVD.

Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells that Contribute to the Fibrous Cap.

TCF21 is a basic helix-loop-helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461).

Leukocyte transglutaminase 2 expression limits atherosclerotic lesion size.

OBJECTIVE: Transglutaminase 2 (TG2), a broadly expressed regulator of protein cross-linking, wound healing, and tissue fibrosis, mediates apoptotic cell ingestion and transforming growth factor-beta release by macrophages and thereby can limit leukocyte-mediated inflammation. In atherosclerosis, oxidative stress and accumulation of unesterified cholesterol stimulate atherosclerotic lesion cell apoptosis. Cell death in advanced atherosclerotic lesions promotes lesion expansion and vulnerable plaques prone to rupture. Hence, we tested the hypothesis that leukocyte TG2 expression limits atherosclerosis. METHODS AND RESULTS: We transplanted TG2-/- or TG2+/+ bone marrow into lethally irradiated low-density lipoprotein receptor (LDLR)-/- mice and evaluated diet-induced atherosclerosis after 16 weeks. We subsequently studied cultured TG2-/- and congenic TG2+/+ mouse macrophages for selected atherogenesis regulatory functions. Atherosclerotic aortic valve lesions in LDLR-/- recipients of TG2-/- bone marrow were larger and more subintimal lesional macrophage penetration than in TG2+/+ marrow recipients. Lesion intimal TG2 expression appeared robust in TG2+/+ but not TG2-/- marrow recipients. Cultured TG2-/- macrophages demonstrated diminished phagocytosis of apoptotic leukocytes, unaltered endocytosis, and degradation of oxidized LDL but decreased retinoic acid induction of the reverse cholesterol transport and apoptotic cell uptake mediator ABCA1. CONCLUSIONS: We conclude that macrophage TG2 expression promotes both apoptotic cell clearance and ABCA1 expression in vitro and limits atherosclerotic lesion size in vivo.

Effect of gamma-irradiation and bone marrow transplantation on atherosclerosis in LDL receptor-deficient mice.

Bone marrow transplantation (BMT) is commonly used to study the participation of bone marrow-derived cells in atherosclerosis. To determine the effect of this methodology on lesions, 16 male low density lipoprotein (LDL) receptor knockout (LDLr-/-) mice were reconstituted with bone marrow from syngeneic LDLr-/- mice after 10 Gy gamma-irradiation and compared with 12 male LDLr-/- littermates that did not undergo BMT (no-BMT group). Mice were fed a high fat diet (HFD) for 16 weeks to induce atherosclerosis. Sixteen additional LDLr-/- mice underwent BMT, and 12 male LDLr-/- mice that did not undergo BMT were fed a chow diet for 56 weeks. Thoracic aorta lesion areas were smaller in BMT mice than in no-BMT mice fed the HFD (P<0.0001). In contrast, aortic root lesion areas were greater in the BMT mice fed the HFD (P<0.0001) as well as in those fed the chow diet (P=0.0001). Abdominal aorta free cholesterol and cholesteryl ester mass were minimal in all groups studied. Aortic root lesions from all no-BMT mice were densely collagenous and encapsulated by a cellular cap, whereas lesions in the BMT mice contained lipid cores and minimal collagen staining. Although the reason for these differences in lesion size and composition remains unresolved, this study suggests that multiple parameters of lesion formation should be examined to assess atherosclerosis.

A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis.

Previous work has implicated PPAR gamma in the regulation of CD36 expression and macrophage uptake of oxidized LDL (oxLDL). We provide evidence here that in addition to lipid uptake, PPAR gamma regulates a pathway of cholesterol efflux. PPAR gamma induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor LXR alpha. Ligand activation of PPAR gamma leads to primary induction of LXR alpha and to coupled induction of ABCA1. Transplantation of PPAR gamma null bone marrow into LDLR -/- mice results in a significant increase in atherosclerosis, consistent with the hypothesis that regulation of LXR alpha and ABCA1 expression is protective in vivo. Thus, we propose that PPAR gamma coordinates a complex physiologic response to oxLDL that involves particle uptake, processing, and cholesterol removal through ABCA1.

PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification.

Inogranic pyrophosphate (PPi) inhibits hydroxyapatite deposition, and mice deficient in the PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) Plasma cell membrane glycoprotein-1 (PC-1) develop peri-articular and arterial calcification in early life. In idiopathic infantile arterial calcification (IIAC), hydroxyapatite deposition and smooth muscle cell (SMC) proliferation occur, sometimes associated with peri-articular calcification. Thus, we assessed PC-1 expression and PPi metabolism in a 25-month-old boy with IIAC and peri-articular calcifications. Plasma PC-1 was <1 ng/ml by enzyme-linked immunosorbent assay in the proband, but 10 to 30 ng/ml in unaffected family members and controls. PC-1 functioned to raise extracellular PPi in cultured aortic SMCs. However, PC-1 was sparse in temporal artery lesion SMCs in the proband, unlike the case for SMCs in atherosclerotic carotid artery lesions of unrelated adults. Proband plasma and explant-cultured dermal fibroblast NTPPPH and PPi were markedly decreased. The proband was heterozygous at the PC-1 locus, and sizes of PC-1 mRNA and polypeptide, and the PC-1 mRNA-coding region sequence were normal in proband fibroblasts. However, immunoreactive PC-1 protein was relatively sparse in proband fibroblasts. In conclusion, deficient extracellular PPi and a deficiency of PC-1 NTPPPH activity can be associated with human infantile arterial and peri-articular calcification, and may help explain the sharing of certain phenotypic features between some IIAC patients and PC-1-deficient mice.

Apolipoprotein E and atherosclerosis.

Apolipoprotein E plays a key protective role in atherosclerosis. Its capacity to safeguard against this disease can be attributed to at least three distinct functions. First, plasma apolipoprotein E maintains overall plasma cholesterol homeostasis by facilitating efficient hepatic uptake of lipoprotein remnants. Second, lesion apolipoprotein E in concert with apolipoprotein A-I facilitates cellular cholesterol efflux from macrophage foam cells within the intima of the lesion. Third, lesion apolipoprotein E directly modifies both macrophage- and T lymphocyte-mediated immune responses that contribute to this chronic inflammatory disease.

Leukocyte CD11b expression is not essential for the development of atherosclerosis in mice.

CD11b is an alpha chain of the leukocyte beta(2)-integrin, Mac-1, which mediates binding and extravasation of leukocytes. Because this event is critical in atherosclerosis, we examined the role of CD11b in lesion formation. Atherosclerosis-susceptible, low density lipoprotein receptor-deficient (LDL-R(-/)-) mice were irradiated and repopulated with bone marrow cells from CD11b-deficient (CD11b(-/)-) mice. After 4 weeks, <2% of the peripheral blood leukocytes of the CD11b(-/)- bone marrow-transplanted LDL-R(-/)- mice expressed CD11b, whereas approximately 25% of the CD11b(+/)+ bone marrow-transplanted LDL-R(-/)- mice expressed CD11b. After consuming a high-fat diet for 16 weeks the mean lesion aortic valve area, cholesterol accumulation in the aorta, and the degree of intimal macrophage infiltration were similar in mice reconstituted with either CD11b(+)(/+) or CD11b(-/)- bone marrow cells. The studies confirm that CD11b expression of bone marrow-derived cells does not influence the development of atherosclerosis in hypercholesterolemic LDL-R(-/)- mice.

Interleukin-8 and its receptor CXCR2 in atherosclerosis.

The participation of inflammatory cells in atherosclerosis is a well-known process that involves numerous molecules including chemotactic cytokines (chemokines) for their entry into the vessel wall. Although the C-C chemokine monocyte chemoattractant protein-1 and its receptor, CCR2, have been implicated in atherosclerosis, the role of the classic C-X-C chemokine, interleukin-8 (KC/growth-related oncogene alpha in mice) and its receptor CXCR2 has not been studied in the pathogenesis of atherosclerosis. Our research has shown that CXCR2 is strongly expressed on macrophages (Mphi) in atherosclerotic lesion. This CXCR2 expression is proatherogenic in that CXCR2 deficiency significantly reduces the progression of advanced atherosclerosis in mice. Although the mechanism still needs to be worked out, it appears that CXCR2 expression on lesion Mphi is essential for these cells to be retained in the lesion.

Participation of innate and acquired immunity in atherosclerosis.

Coronary artery disease, the major manifestation of atherosclerosis, is the leading cause of death in the Western world. However, the pathogenesis of atherosclerosis is still poorly understood. Controversy exists regarding the participation of innate immunity involving macrophages and natural killer (NK) cells vs antigen-specific acquired immunity involving lymphocytes. Macrophages predominate in atherosclerotic lesions. NK cells, although smaller in number, are present as well. Furthermore, T lymphocytes that participate in acquired immunity are frequently observed in lesions and can modulate lesion progression. By using mouse models of hyperlipidemia, our laboratory is addressing in vivo the participation of both innate inflammatory responses and acquired immune responses in atherosclerosis.

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice.

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.

ApoA1 reduces free cholesterol accumulation in atherosclerotic lesions of ApoE-deficient mice transplanted with ApoE-expressing macrophages.

Along with apolipoprotein (apo) E, which promotes cholesterol efflux from foam cells, apoA1-containing high density lipoprotein (HDL) is thought to facilitate the transport of cholesterol from lesions. This role for apoA1 was tested in vivo by lethally irradiating apoE-deficient and apoE- plus apoA1-deficient mice and reconstituting them with bone marrow cells isolated from wild-type (WT) mice. ApoE, but not apoA1, was synthesized by the transplanted bone marrow-derived cells. Therefore, this transplantation procedure generated apoE-deficient animals with atherosclerotic lesions that contained both apoE and apoA1 (E/A1 mice) and apoE-deficient animals with lesions that contained apoE but no apoA1 (E/A1o mice). As shown previously, the transplanted WT macrophage-derived apoE dramatically lowered the plasma hypercholesterolemia in both groups. On feeding with an atherogenic diet after transplantation, plasma cholesterol levels were raised in both groups of mice, but the levels in the E/A1 mice at 20 weeks were 2- to 3-fold higher than in E/A1o mice. Immunohistochemical staining verified that apoE was abundant in lesions of both groups, whereas apoA1 was detected in the lesions of E/A1 mice only. Despite a 2- to 3-fold lower total plasma cholesterol in the E/A1o mice, the free cholesterol recovered from isolated aortas was approximately 60% higher and the mean lesion area in serial sections of the aortic valves 45% larger. Therefore, apoA1 reduces free cholesterol accumulation in vivo in atherosclerotic lesions.

Chemokines and atherosclerosis.

The recruitment of mononuclear leukocytes, and the migration, growth and activation of macrophages, lymphocytes and smooth muscle cells within lesions, are critical features of the chronic inflammatory response that typifies atherogenesis. Chemokines are members of a superfamily of small polypeptides that mediate not only migration, but also growth and activation of leukocytes and a variety of other cells. Monocyte chemoattractant and activating protein-1 was the first chemokine to be implicated in leukocyte-mediated inflammation in atherosclerosis. This review emphasizes new information on the potential atherogenic roles of monocyte chemoattractant and activating protein-1 and several other closely related chemokines of the C-C subfamily. We focus particular attention on the newly recognized atherogenic role of a subgroup of closely related chemokines of the C-X-C subfamily that includes interleukin-8 and growth regulated oncogene alpha. We also discuss new studies that reveal how CD40 ligand and certain other stimuli can promote chemokine expression in atherosclerosis.

A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.

Chronic macrophage-mediated inflammation is central to atherosclerosis. A role of the monocyte chemotactic and activating C-C chemokine JE/monocyte chemotactic protein-1 has been proposed. However, the human C-X-C chemokines growth-regulated oncogene (GROalpha) and IL-8, and their shared receptor, CXCR-2, also can be expressed at sites of chronic inflammation. Because we detected CXCR-2 in the intima of human atherosclerotic lesions, we examined the role of leukocyte CXCR-2 expression in affecting lesion cellularity. Atherosclerosis-susceptible LDL receptor-deficient mice were irradiated, successfully repopulated with bone marrow cells that either lacked or expressed mIL-8RH (the homologue of CXCR-2), and fed an atherogenic diet for 16 wk. In recipients of mIL-8RH+/+ marrow, mIL-8RH colocalized with densely accumulated intimal MOMA-2 positive macrophages. In contrast, lesions in recipients of mIL-8RH-/- marrow lacked mIL-8RH, had little intimal MOMA-2 staining, and were less extensive. The mIL-8RH ligand KC/GROalpha was detected in the intima of all aortic atherosclerotic lesions. Thus, the capacity of leukocytes to express mIL-8RH, and associated intralesional expression of its ligands such as KC/GROalpha, mediated the intimal accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.

Role of leukocyte-specific LDL receptors on plasma lipoprotein cholesterol and atherosclerosis in mice.

Bone marrow-derived macrophages and lymphocytes express LDL receptors (LDL-R), which allow these cells to take up cholesterol-rich lipoproteins. Although these cells are ubiquitously distributed in the body, it is not known whether they influence plasma cholesterol. Macrophages and T lymphocytes also are found in atherosclerotic lesions, but it is not known whether their LDL-R expression plays a role in atherosclerosis. To address these questions, we subjected LDL-R -/-mice to total body irradiation to eliminate their endogenous bone marrow-derived cells and repopulated them with either LDL-R-expressing wild-type bone marrow (treated mice) or LDL-R -/- bone marrow (control mice). Thus, the only difference between the two groups of mice was the ability of the bone marrow-derived cells to express the LDL-R in the treated mice. Plasma cholesterol levels were similar in the two groups of mice at 8 and 16 weeks after transplantation. Chromatographic separation of the lipoproteins revealed similar lipoprotein cholesterol distributions. Although the extent of lesion area in the aortic valves of the high-fat-diet-fed mice was more severe than that in the chow-fed mice, lesions appeared similar between control and treated mice given either chow or high-fat diet. Abundant LDL-R expression was detected in the lesions of treated mice, whereas the lesions of control mice showed no LDL-R expression, indicating that donor-derived leukocytes had migrated into the lesions of the recipient mice. Thus, bone marrow transplantation can be used as a tool to replace the endogenous bone marrow-derived cells in the artery wall with those of the donor origin.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation.

Apo E, a key regulator of cholesterol-rich lipoprotein metabolism, is synthesized by numerous extrahepatic tissues. Although its synthesis in macrophages is documented, the contribution of macrophage-derived apo E to hepatic clearance of serum cholesterol is unknown. To address this issue bone marrow transplantation was performed on hypercholesterolemic apo E-deficient mice with either syngeneic apo E-deficient mouse bone marrow cells (E0-control) or wild-type mouse bone marrow cells expressing apo E (E0-treated). E0-control and E0-treated mice were fed either a regular chow diet or an atherogenic diet (designated E0-control-HF and E0-treated-HF). Serum cholesterol levels dropped dramatically in the E0-treated mice largely due to a reduction in their VLDL cholesterol. No changes were seen in the E0-control mice. After 4 wk serum cholesterol in E0-treated-HF mice was about four-fold lower compared to E0-control-HF animals. Moreover, the extent of atherosclerosis in the E0-treated-HF mice after 14-16 wk was greatly reduced. Wild-type apo E mRNA was detected in the liver, spleen, and brain of the E0-treated mice indicating that apo E gene transfer was successfully achieved through bone marrow transplantation. More importantly, the level of apo E expression was sufficient to reduce the severe hypercholesterolemia of the apo E-deficient mice fed either chow or atherogenic diets.

Prevalence of riboflavin deficiency among Guatemalan elderly people and its relationship to milk intake.

Six groups of elderly subjects from central Guatemala were assessed for riboflavin status by using the erythrocyte glutathione reductase activity coefficient (EGRAC). The prevalence of riboflavin deficiency ranged from 50% to 76% among the free-living rural elderly subjects. Milk intake data that were collected from some of the subjects showed a significant correlation (P < 0.0001) between frequency of milk intake and riboflavin status. In a short-term riboflavin supplementation experiment in which nine riboflavin-deficient subjects were given 10 mg riboflavin/d for 3 d, all the subjects' EGRACs were normalized by the supplementation. However, they returned to a state of deficiency within 2 wk while consuming their usual diets without supplementation. It appears that the high prevalence of riboflavin deficiency in elderly Guatemalan people is caused by inadequate intake of riboflavin-rich foods such as dairy products, and that sufficient amounts of riboflavin need to be ingested regularly to maintain satisfactory riboflavin status.

Riboflavin requirement of healthy elderly humans and its relationship to macronutrient composition of the diet.

The riboflavin requirements of two groups of riboflavin-deficient, but otherwise healthy, Guatemalan elderly persons over the age of 60 y were studied by varying the fat:carbohydrate ratio in two diets. The first group consumed a diet similar in macronutrient content to a Western-type diet with low carbohydrate and high fat; the second group consumed a typical Guatemalan diet with high carbohydrate and low fat. Energy and protein intakes of both groups were similar. Riboflavin status was monitored by weekly measurements of erythrocyte glutathione reductase activity coefficient (EGRAC) and urinary riboflavin excretion. Increasing increments of riboflavin were added to the subjects' diets until their status was normalized, as indicated by EGRAC of < 1.34 and a sharp increase in urinary riboflavin excretion. Using the EGRAC method, the mean value of riboflavin intake at which the subjects' EGRAC reached the limit of normality was 1.37 +/- 0.03 mg/d in the first phase and 1.29 +/- 0.03 mg/d in the second phase. The sharp increase in urinary excretion occurred at riboflavin intakes of 1.13 and 1.03 mg/d for Groups 1 and 2, respectively. Thus, the differences between the two groups suggest that diets with a lower fat:carbohydrate ratio can decrease the dietary need for riboflavin. The dietary requirement of riboflavin, as estimated by the more reliable urinary excretion method, was 1.1-1.3 mg/d for those consuming the Western-type diet, which is similar to values found over 40 y ago in young adults. We conclude that the dietary requirements of riboflavin in the elderly do not differ from those of young adults.