Intima Of Aortic Arch Research Articles

Abstract 462: Transcriptomic Analysis Reveals That Intimal Monocytes Drive Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor for atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. In Ldlr -/- mice HC induces intimal myeloid cells to develop into the foam cells of nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intimal myeloid cells during the earliest stages of atherogenesis, and correlate them to cellular responses. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, 14, and 56 days of CRD feeding using en face enzymatic digestion and cell microisolation. Bulk RNA-seq and RT-qPCR revealed that genes associated with lipid-loaded macrophages (e.g., Abcg1 , Lgals3 ) were progressively elevated over time. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid cells. With our intima specific approach, we have uncovered a diverse landscape of intimal myeloid cells; including monocytes, macrophages, foam cells, DCs, and neutrophils. Atherogenesis-associated inflammatory transcripts were scarcely detected in macrophage and foam cell clusters, but were prominent in non-macrophage cell types. Ccr2 + intimal monocytes exhibited a hypercholesterolemia induced increase in inflammatory pathways within 5 days of CRD feeding. Furthermore, analysis of monocyte and macrophage clusters suggests that this inflammatory signature dissipates as recently recruited monocytes transition into a macrophage/foam cell fate. We are using Ccr2 + fate mapping models, to interrogate the hypercholesterolemia-induced transition of inflammatory monocytes within a growing lesion. We suggest that the atherogenesis-associated inflammation may be initiated by recently recruited monocytes, and may not originate directly from lipid loaded macrophages.

Abstract 412: Transcriptomic Analysis Reveals That Lipid Loading Of Distinct Intimal Myeloid Cell Subpopulations Precedes Inflammation In Early Atherogenesis

Hypercholesterolemia (HC) is a key risk factor of atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. Dietary HC in Ldlr -/- mice induces intimal myeloid cells to develop into foam cells, constituting nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intima during the earliest stages of atherogenesis, and correlate them to cellular responses. Using en face enzymatic digestion and intimal cell microisolation, transcriptomic analysis was performed on the LC. Intimal cells were isolated from the LC of the ascending aortic arch of Ldlr -/- mice at 0, 5, and 14 days of CRD feeding. Bulk RNA-seq and RT-qPCR found that genes associated with lipid-loaded macrophages (e.g., Abcg1, Lgals3 ) were progressively elevated at 5 and 14 days of CRD. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not detected at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of Ldlr -/- mice revealed multiple sub-populations of myeloid, endothelial, and other cell types. A shift in the C1qb + , Mmp12 + myeloid subpopulations was observed as the duration of cholesterol feeding increased - with expansion of a macrophage population rich in lipid-responsive transcripts. This finding is consistent with an increase in macrophage number and the presence of Nile Red + CD68 + foam cells observed by immunoconfocal microscopy at 5 days of CRD. However, a robust inflammatory signature was not detected in this macrophage cluster. Interestingly, an inflammatory signature similar to that reported in early atherogenesis was identified in a distinct Ccr2 + myeloid population with no signature of lipid loading. This suggests that the inflammation associated with atherogenesis may be a result of intercellular crosstalk between distinct myeloid subpopulations within the intima and may not originate directly from lipid loaded macrophages.

Abstract 110: The Role Of Sphingosine-1-Phosphate In The Reverse Transendothelial Migration Of Aortic Intimal Myeloid Cells

CD11c + myeloid cells (MCs) reside in the normal aortic arch intima at regions predisposed to atherosclerosis. We showed that reverse transendothelial migration (RTM) of these intimal MCs into the arterial circulation is a protective immune response. RTM is triggered by systemic stimulation of cytokine and toll-like receptors (TLRs), e.g., TLR4 by lipopolysaccharide (LPS), and is inhibited by hypercholesterolemia and associated intimal MC lipid accumulation. We hypothesize that signaling by sphingosine-1-phosphate (S1P) and its receptors (S1PRs) is required for RTM of intimal MCs following systemic stimulation of TLRs, analogous to its role in immune cell trafficking in lymphoid tissues. In the lesser curvature of the aortic arch of wild type C57BL/6 mice, LPS induced the expression of S1P receptors 1 & 3, and sphingosine kinase 1 (SphK1). FTY720, a broad-spectrum S1P receptor modulator, blocked LPS-induced RTM, as determined by the enumeration of CD11c+ MCs by en face immunoconfocal microscopy. S1PR1 or S1PR3 inhibitors blocked RTM in a partial and additive manner, suggesting a role for both S1P receptors in intimal MC migration. Initial experiments show that Cre-mediated deletion of S1PR1 in myeloid cells partially blocks LPS-induced RTM, suggesting that myeloid-specific S1PR1 signaling is implicated in the migratory process. Furthermore, global loss or hematopoietic deficiency of SPHK1 reduced the abundance of CD11c+ MCs in the aortic intima, suggesting an additional role for myeloid S1P production in the homeostatic maintenance of resident aortic immune cells. Cre-mediated deletion of S1PR1 in endothelial cells also reduced the abundance of intimal MCs, providing further evidence that S1P signaling in the intima is important in maintaining the intimal MC population. Thus, functional and expression studies suggest that S1P signaling plays a role in both the maintenance of resident intimal MCs and LPS-induced RTM.

Endothelial cell-specific deletion of a microRNA accelerates atherosclerosis

Background and aimsChronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis. Methods and ResultsUsing a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6Clow or the pro-inflammatory Ly6Cinterm and Ly6Chigh fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis. ConclusionsOur study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis.

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation

Rationale: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT. Objective: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms. Methods and Results: Ldlr-/- mice underwent total body γ-irradiation, bone marrow reconstitution and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared to mice without BMT. In BMT mice neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet (CRD) for 3, 6 and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70kD dextran. LDL accumulation was dramatically higher in the intima of Ldlr-/- relative to Ldlr+/+ mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single cell resolution, was performed to give insights into pathways modulated by BMT. Conclusions: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.

Pharmaceutical stabilization of mast cells attenuates experimental atherogenesis in low-density lipoprotein receptor-deficient mice

Mast cells (MCs) contribute to atherogenesis by releasing pro-inflammatory mediators to activate vascular cells and other inflammatory cells. This study examined whether MC activation or stabilization affects diet-induced atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr–/–) mice. When Ldlr–/– mice consumed an atherogenic diet for 3 or 6 months, MC activation with compound 48/80 (C48/80) increased aortic arch intima and total lesion areas, and plasma total cholesterol, LDL, and triglyceride levels, whereas MC stabilization with cromolyn reduced these parameters. There were significant differences in arch intima and total lesion areas, and plasma total cholesterol, LDL, and triglyceride levels between C48/80-treated and cromolyn-treated mice. To examine a therapeutic application of cromolyn in atherosclerosis, we fed Ldlr–/– mice an atherogenic diet for 3 months followed by giving mice cromolyn for additional 3 months. Cromolyn did not affect aortic arch intima area, but significantly reduced lipid deposition in the thoracic-abdominal aortas. In aortic arches, however, cromolyn treatment significantly reduced lesion contents of Mac-3+ macrophages, CD4+ T cells, activated MCs, and lesion cell proliferation. While plasma total cholesterol and LDL levels increased and high-density lipoprotein (HDL) levels decreased from 3 months to 6 months of an atherogenic diet, cromolyn treatment decreased significantly plasma total cholesterol, LDL, and triglyceride levels and increased HDL levels above those of 3-month time point. These observations demonstrate that MC stabilization reduces lesion inflammation, ameliorates plasma lipid profiles, and may serve as a potential therapy for this cardiovascular disease.

Overexpression of TGF-ß1 in Macrophages Reduces and Stabilizes Atherosclerotic Plaques in ApoE-Deficient Mice

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Foam cells generated by a combination of hyperglycemia and hyperlipemia in rats

Diabetes is a major risk factor for atherosclerosis, as well as hyperlipemia. Investigators have suggested that denatured lipoprotein in hyperglycemia transforms macrophages into foam cells, which correlates with the development or progression of atherosclerosis. In the present study, we examined the generation of foam cells in rats caused by a combination of hyperglycemia and hyperlipemia. Streptozotocin-induced diabetic male Wister rats were fed a high cholesterol diet (HCD) containing 1% cholesterol and 0.5% cholic acid to maintain a hyperglycemic and hyperlipemic state. Animals fed the HCD for 8 weeks or longer showed a high incidence of foam cell accumulation in the renal glomerulus, intima of aortic arch, splenic red pulp and marginal zone, liver sinusoid and intestinal lamina propria. The foam cells exhibited positive staining for antimonocyte/macrophage antibody and lipids in all these tissues. Anti-rat apolipoprotein B (apo B) antibody revealed that positive staining existed only in the cytoplasm of glomerular foam cells. These results suggest that the origin of these foam cells can be attributed to lipid-laden macrophages. The generation of foam cells in the hyperglycemia-hyperlipidemia supervening rat model presented in the present study might be a useful tool for investigations of the pathogenesis of foam cells.

Thickened intima of the aortic arch is a risk factor for stroke with coronary artery bypass grafting

Background. Perioperative stroke is one of the most serious complications of cardiac surgery. Methods. Using transesophageal echocardiography, we estimated the intimal thickness of the thoracic aorta as an index of the severity of aortic atherosclerosis to determine the risk of stroke in coronary artery bypass grafting (CABG) patients. The study population comprised 315 consecutive patients who underwent isolated CABG with cardiopulmonary bypass. Results. Five patients (1.6%) had perioperative cerebral stroke or systemic emboli. We compared the mean intimal thicknesses of the ascending aorta, aortic arch, and descending aorta. Mean thicknesses in patients without stroke were 2.07 ± 0.76, 2.78 ± 1.15, and 2.32 ± 1.21 mm, respectively, and mean thicknesses in the stroke patients were 1.94 ± 0.55, 6.94 ± 3.79, and 3.39 ± 1.85 mm, respectively. The patients with an intima of more than 5 mm at the aortic arch had a significantly greater incidence of perioperative stroke ( p = 0.007). Conclusions. These results suggest that patients who have an aortic arch intima thickened to more than 5 mm are at a significantly high risk for perioperative stroke, and thus, the CABG procedure should be carefully evaluated to prevent such complications.

Ultrastructural changes in the aortic arch intima caused by water-immersion stress in rats

Ultrastructural changes in the aortic arch intima caused by water-immersion stress in rats

動脈硬化症の発生進展過程における血液線溶活性の意義

A total of 29 young female arbino rabbits weighing around 2kg each were used in 2 experimental studies.Ex. 1: In 9 rabbits, the determination of dose and method of UK administration, which enoughly provoked of blood fibrinolysis, initially examined.Ex. 2: In order to evaluate the significance of the accerelated blood fibrinolysis on the pathogenesis and the development of atherosclerosis, 20 rabbits were divided into 2 subgroups (UK group, Control group). The UK group was administered UK via marginal ear vein 2 times every day during only initial 2 weeks and simultaneously fed a lanolin diet contained 1.4% cholesterol throughout experimental period. The control group was fed a lanoline diet alone in the same period.We concluded followings.Ex. 1: When 30, 000 IU/kg/day of UK was initially administered during first one week and then grdually decreased bolus of UK next week, it could be enoughly accerelated blood fibrinolysis.Ex. 2: (1) In both groups, TC remarkably increased at 1 and 4 weeks. No significant difference between both groups was observed throughout the experiment.(2) Although no remarkable changes of fibrinogen contents were recognized in UK group throughout experimental period, in control group it remarkably increased at 4 weeks and after that gradually increased up to 10 weeks. However, there was significant difference between both groups at 2 weeks.(3) In control group, ELT grdually prolonged up to 2 weeks.In contrast to it significantly shortened at 1 and 2 weeks in UK group.(4) In UK group, a lysis area was inclined to increase up to 2 weeks, and after 6 weeks slightly weakened lysis area were recognized.(5) FDP significantly incerased at 2 weeks in control group, but significantly increased at 1 and 8 weeks in UK group.(6) A proportion of atheromatous lesion of thoracic aorta was less recognized in UK group than that in control group.(7) The stratification of foam cells in the intima of aortic arch were prominently observed in control group than in UK group. And the proliferation of intimal smooth muscle cells were remarkably recognized in UK group than in control group.Above the results lead us to believe that the accerelated blood fibrinolysis due to administration of UK during the initial 2 weeks of experimental period would be one of the important retarding factors of the generation and the development of experimental atherosclerosis in rabbits.

Effect of hypertension on the entry of 125I-labelled low density lipoprotein into the aortic intima in normal-fed rabbits

The entry of [ 125I]-labelled low density lipoprotein (LDL) into different regions of the aortic intima has been studied over a 6 hour period in both normotensive and renal hypertensive rabbits fed a normal diet. Studies have also been carried out in previously hypertensive rabbits in which the blood pressure was normalized with parenteral hydralazine during the six hour period, in which entry was studied. In the normotensive rabbits entry into the aortic intima was less than 1 μg of LDL protein/100 mg dry defatted weight over the 6 hour period with greatest entry into aortic arch intima and significantly less into both the thoracic and abdominal aortic intimae. Hypertension increased the entry into the arch and into the thoracic and abdominal segments but this was only statistically significant for the aortic arch. The entry of [ 125I] LDL into the intima in those rabbits in which the hypertension had been normalized was similar to that for the hypertensive rabbits. The results suggest that hypertension in the normal fed rabbit increases lipoprotein entry into the arterial wall by an effect on vessel wall permeability rather than by a direct effect of filtration pressure.