Arteriosclerosis, Thrombosis, and Vascular Biology

DNA methylation is a form of epigenetic modification of the genome that can regulate gene expression. Hypermethylation of CpG islands in the promoter areas leads to decreased gene expression, whereas promoters of actively transcribed genes remain nonmethylated. Because of cellular proliferation and monoclonality of at least some of the lesion cells, atherosclerotic lesions have been compared with benign vascular tumors. ^1,2 However, although genetic and epigenetic background favors neoplastic transformation, atherosclerotic plaques never develop to malignant tumors. Among cancer cells, common features are genome-wide hypomethylation, which correlates with transformation and tumor progression. Recent studies have shown that DNA methylation changes occur also during atherogenesis and may contribute to the lesion development.

Objective— Activation of the endothelium by oxidized low-density lipoprotein (oxLDL) has been implicated in the development of atherosclerosis. Histone modifications impact on the transcriptional activity state of genes. We tested the hypothesis that oxLDL-induced inflammatory gene expression is regulated by histone modifications and experienced the effect of statins on these alterations.

Methods and Results— OxLDL-related interleukin-8 (IL-8) and monocyte-chemoattractant protein-1 (MCP-1) secretion in endothelial cells was reduced by statins but enhanced by histone deacetylase inhibitors. OxLDL induced lectin-like oxidized LDL receptor-1 (LOX-1) and extracellular regulated kinases (ERK1/2)-dependent acetylation of histone H3 and H4 as well as phosphorylation of histone H3, both globally and on the promoters of il8 and mcp1 . Pretreatment of oxLDL-exposed cells with statins reduced the above mentioned histone modification, as well as recruitment of CREB binding protein (CBP) 300, NF-κB, and of RNA polymerase II but prevented loss of binding of histone deacetylase (HDAC)-1 and -2 at the il8 and mcp1 gene promoters. OxLDL reduced HDAC1 and 2 expression, and statins partly restored global HDAC-activity. Statin-related effects were reverted with mevalonate. In situ experiments indicated decreased expression of HDAC2 in endothelial cells in atherosclerotic plaques of human coronary arteries.

Conclusions— Histone modifications seem to play an important role in atherosclerosis.

Objective— We tested the hypothesis that expression of transcripts adjacent to the chromosome 9p21 (Chr9p21) locus of coronary artery disease was affected by the genotype at this locus and associated with atherosclerosis risk.

Methods and Results— We replicated the locus for coronary artery disease ( P =0.007; OR=1.28) and other manifestations of atherosclerosis such as carotid plaque ( P =0.003; OR=1.31) in the Leipzig Heart Study, a cohort of 1134 patients with varying degree of angiographically assessed coronary artery disease. Expression analysis in peripheral blood mononuclear cells (n=1098) revealed that transcripts EU741058 and NR_003529 of antisense noncoding RNA in the INK4 locus ( ANRIL ) were significantly increased in carriers of the risk haplotype ( P =2.1×10 ⁻¹² and P =1.6×10 ⁻⁵ , respectively). In contrast, transcript DQ485454 remained unaffected, suggesting differential expression of ANRIL transcripts at Chr9p21. Results were replicated in whole blood (n=769) and atherosclerotic plaque tissue (n=41). Moreover, expression of ANRIL transcripts was directly correlated with severity of atherosclerosis ( EU741058 and NR_003529 ; P =0.02 and P =0.001, respectively). No consistent association of Chr9p21 or atherosclerosis was found with expression of other genes such as CDKN2A , CDKN2B , C9orf53 , and MTAP .

Conclusion— Our data provide robust evidence for an association of ANRIL but not CDKN2A, CDKN2B, C9orf53 , and MTAP , with atherosclerosis and Chr9p21 genotype in a large cohort.

Objective—With age, rat arterial walls thicken and vascular smooth muscle cells (VSMCs) exhibit enhanced migration and proliferation. Monocyte chemotactic protein-1 (MCP-1) affects these VSMC properties in vitro. Because arterial angiotensin II, which induces MCP-1 expression, increases with age, we hypothesized that aortic MCP-1 and its receptor CCR2 are also upregulated and affect VSMC properties.

Methods and Results—Both MCP-1 and CCR2 mRNAs and proteins increased in old (30-month) versus young (8-month) F344×BN rat aortas in vivo. Cellular MCP-1 and CCR2 staining colocalized with that of α-smooth muscle actin in the thickened aortas of old rats and were expressed by early-passage VSMCs isolated from old aortas, which, relative to young VSMCs, exhibited increased invasion, and the age difference was abolished by vCCI, an inhibitor of CCR2 signaling. MCP-1 treatment of young VSMCs induced migration and increased their ability to invade a synthetic basement membrane. The MCP-1–dependent VSMC invasiveness was blocked by vCCI. After MCP-1 treatment, migration and invasion capacities of VSMCs from young aortas no longer differed from those of VSMCs isolated from older rats.

Conclusions—Arterial wall and VSMC MCP-1/CCR2 increase with aging. MCP-1 enhances VSMC migration and invasion, and thus, MCP-1/CCR2 signaling may play a role in age-associated arterial remodeling.

Objective— Because late vein graft failure is caused by intimal hyperplasia (IH) and accelerated atherosclerosis, and these processes are thought to be inflammation driven, influx of monocytes is one of the first phenomena seen in IH, we would like to provide direct evidence for a role of the MCP-1 pathway in the development of vein graft disease.

Methods and Results— MCP-1 expression is demonstrated in various stages of vein graft disease in a murine model in which venous interpositions are placed in the carotid arteries of hypercholesterolemic ApoE3Leiden mice and in cultured human saphenous vein (HSV) segments in which IH occurs. The functional involvement of MCP-1 in vein graft remodeling is demonstrated by blocking the MCP-1 receptor CCR-2 using 7ND-MCP-1. 7ND-MCP1 gene transfer resulted in 51% reduction in IH in the mouse model, when compared with controls. In HSV cultures neointima formation was inhibited by 53%. In addition, we demonstrate a direct inhibitory effect of 7ND-MCP-1 on the proliferation of smooth muscle cell (SMC) in HSV cultures and in SMC cell cultures.

Conclusion— These data, for the first time, prove that MCP-1 has a pivotal role in vein graft thickening due to intimal hyperplasia and accelerated atherosclerosis.

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.

Objective— We hypothesized that reactive oxygen species (ROS) contribute to progression of aortic valve (AV) calcification/stenosis.

Methods and Results— We investigated ROS production and effects of antioxidants tempol and lipoic acid (LA) in calcification progression in rabbits given 0.5% cholesterol diet +10 ⁴ IU/d Vit.D ₂ for 12 weeks. Superoxide and H ₂ O ₂ microfluorotopography and 3-nitrotyrosine immunoreactivity showed increased signals not only in macrophages but preferentially around calcifying foci, in cells expressing osteoblast/osteoclast, but not macrophage markers. Such cells also showed increased expression of NAD(P)H oxidase subunits Nox2, p22phox, and protein disulfide isomerase. Nox4, but not Nox1 mRNA, was increased. Tempol augmented whereas LA decreased H ₂ O ₂ signals. Importantly, AV calcification, assessed by echocardiography and histomorphometry, decreased 43% to 70% with LA, but increased with tempol ( P ≤0.05). Tempol further enhanced apoptosis and Nox4 expression. In human sclerotic or stenotic AV, we found analogous increases in ROS production and NAD(P)H oxidase expression around calcifying foci. An in vitro vascular smooth muscle cell (VSMC) calcification model also exhibited increased, catalase-inhibitable, calcium deposit with tempol, but not with LA.

Conclusions— Our data provide evidence that ROS, particularly hydrogen peroxide, potentiate AV calcification progression. However, tempol exhibited a paradoxical effect, exacerbating AV/vascular calcification, likely because of its induced increase in peroxide generation.

Diabetes mellitus increasingly afflicts our aging and dysmetabolic population. Type 2 diabetes mellitus and the antecedent metabolic syndrome represent the vast majority of the disease burden—increasingly prevalent in children and older adults. However, type 1 diabetes mellitus is also advancing in preadolescent children. As such, a crushing wave of cardiometabolic disease burden now faces our society. Arteriosclerotic calcification is increased in metabolic syndrome, type 2 diabetes mellitus, and type 1 diabetes mellitus—impairing conduit vessel compliance and function, thereby increasing the risk for dementia, stroke, heart attack, limb ischemia, renal insufficiency, and lower extremity amputation. Preclinical models of these dysmetabolic settings have provided insights into the pathobiology of arterial calcification. Osteochondrogenic morphogens in the BMP-Wnt signaling relay and transcriptional regulatory programs driven by Msx and Runx gene families are entrained to innate immune responses—responses activated by the dysmetabolic state—to direct arterial matrix deposition and mineralization. Recent studies implicate the endothelial–mesenchymal transition in contributing to the phenotypic drift of mineralizing vascular progenitors. In this brief overview, we discuss preclinical disease models that provide mechanistic insights—and point to challenges and opportunities to translate these insights into new therapeutic strategies for our patients afflicted with diabetes mellitus and its arteriosclerotic complications.

Các nghiên cứu quan sát về sự phát triển nhân hoại tử xác định xuất huyết trong mảng là yếu tố quan trọng trong sự tăng trưởng và mất ổn định của mảng xơ vữa động mạch. Sự tích tụ nhanh chóng của màng hồng cầu gây ra sự thay đổi đột ngột trong nền của mảng với đặc trưng là sự gia tăng cholesterol tự do trong lõi lipid và xâm nhập quá mức của đại thực bào. Neoangiogenesis liên quan chặt chẽ với sự phát triển của mảng xơ vữa, và sự thiếu năng lực vi mạch có khả năng là nguồn gốc của xuất huyết trong mảng. Tân mạch nội mạc chủ yếu được cho là xuất phát từ ngoại mạc, nơi có nhiều mạch máu vasa vasorum tồn tại trước đó. Trong các tổn thương có nhân hoại tử sớm, đa số các mạch xâm nhập từ ngoại mạc xảy ra tại những vị trí cụ thể của sự phá hủy thành giữa. Một sự rạn nứt ở thành giữa có khả năng tạo điều kiện cho sự phát triển nhanh chóng của các vi mạch từ ngoại mạc, và việc tiếp xúc với môi trường xơ vữa động mạch kích thích sự phát triển mạch máu bất thường, được đặc trưng bởi sự phân nhánh không tổ chức và các ống nội mạc chưa trưởng thành với lớp lót “rò rỉ” không hoàn thiện. Mạng lưới các mạch máu chưa trưởng thành này là nguồn cung cấp khả thi cho xuất huyết trong mảng, cung cấp phospholipid từ hồng cầu và cholesterol tự do. Sự thay đổi nhanh chóng trong nền của mảng gây ra bởi sự tích tụ quá mức của hồng cầu có thể thúc đẩy sự chuyển đổi từ tổn thương ổn định sang tổn thương không ổn định. Bài tổng quan này thảo luận về vai trò tiềm năng của vasa vasorum trong mảng trong sự không ổn định của tổn thương liên quan đến sự vỡ của mảng.