The expression of IGFs and IGF binding proteins in human carotid atherosclerosis, and the possible role of IGF binding protein-1 in the regulation of smooth muscle cell proliferation

ObjectiveProliferation of smooth muscle cells (SMCs) in the fibrous cap of atherosclerotic lesions has been proposed to be important for plaque stability. Since the insulin-like growth factor (IGF) system has been implicated to play a role in atherosclerosis and plaque stability, we investigated the expression of members of the IGF system in carotid plaques, in particular IGFBP-1 and its role in the regulation of SMC proliferation.Methods and resultsGene expression profiles of the IGF system in 164 human carotid plaques obtained from our Biobank of Karolinska Endarterectomies (BiKE) were analyzed. Expression of IGFBP-1 mRNA was significantly increased in carotid plaques compared with normal iliac arteries in contrast to IGF-1, IGF-2, and IGFBP-3 to IGFBP-6. The expression of IGFBP-1 mRNA correlated positively to that of CD163, CD68, IL-1β, IL-6, TNFα, IGFBP-4 and IGFBP-5. Immunohistochemistry demonstrated co-localization of IGFBP-1 with SMCs and macrophages. In vitro studies showed that IL-1β, IL-6 and TNFα stimulated IGFBP-1 mRNA expression in SMCs. IGFBP-1 stimulated SMC proliferation through ERK1/2 activation but independently of the IGF-1 receptor. In addition, IGFBP-1 modulated the effect of IGF-1 on SMC proliferation and ERK1/2 activation.ConclusionsOur results demonstrate that IGFBP-1 mRNA and protein is detected at increased levels in human carotid plaques, possibly as a consequence of plaque inflammation. IGFBP-1 affects SMC proliferation and may be involved in the regulation of plaque stability.     

Cathepsin L is significantly associated with apoptosis and plaque destabilization in human atherosclerosis

ObjectiveHuman atherosclerotic lesions overexpress elastolytic and collagenolytic cathepsins with unclear pathological implications. The aim of this study was to investigate the relationship among expression of cathepsin L, macrophage apoptosis in coronary artery disease (CAD) patients, clinical symptoms and plaque severity of human carotid atheroma.Methods and resultsQuantitative immunohistochemical analysis of human carotid atherosclerotic lesions (n = 49) showed that expression of lysosomal cathepsin L was significantly increased in atherosclerotic plaques with formation of the necrotic core and rupture of the cap. In those plaques, cathepsin L was associated mainly with CD68-positive macrophages, whereas significant lower levels of smooth muscle cell actin were detected. The expression of cathepsin L in these plaques was also correlated with apoptosis and the stress protein ferritin. Plaques from symptomatic patients showed greater increased levels of cathepsin L than those from asymptomatic patients. Human monocyte-derived macrophages from CAD patients (n = 7) showed significantly higher levels of cathepsin L, cellular lipids and apoptosis versus cells from matched healthy donors (n = 7). 7Beta-hydroxycholesterol significantly enhanced cathepsin L in cells from healthy donors but not in cells from CAD patients. Moreover, macrophage apoptosis was significantly correlated with expression of cathepsin L in cell nuclei and membranes.ConclusionThe results suggest that cathepsin L is involved in death of macrophages, necrotic core formation and development of atherosclerotic plaque instability. Macrophage lysosomal cathepsin L and related apoptosis may be potential targets for modulation or imaging of vulnerable plaques in human atherosclerosis.     

Activated platelets promote increased monocyte expression of CXCR5 through prostaglandin E2-related mechanisms and enhance the anti-inflammatory effects of CXCL13

Background

We have previously shown that the homeostatic chemokine CXCL13 is up-regulated in monocytes in atherosclerosis, mediating anti-apoptotic and anti-inflammatory effects.

Objective

To investigate the regulation of CXCL13s receptor, CXCR5.

Methods/patients

In vitro studies in THP-1 and primary monocytes and studies of CXCR5 expression in thrombus material obtained at the site of plaque rupture during myocardial infarction (MI).

Results

Our major findings were: (i) toll-like receptor agonists and particularly β-adrenergic receptor activation and releasate from thrombin-activated platelets increased CXCR5 mRNA levels in monocytes. (ii) The platelet-mediated induction of CXCR5 involved prostaglandin E₂/cAMP/protein kinase A-dependent as well as RANTES-dependent pathways with NFκB activation as a potential common down-stream mediator. (iii) Releasate from thrombin-activated platelets augmented the anti-inflammatory effects of CXCL13 in monocytes at least partly by enhancing the effects of CXCL13 on CXCR5 expression. (iv) We found strong immunostaining of CXCR5 in thrombus material obtained at the site of plaque rupture in patients with ST elevation MI (STEMI) and in unstable carotid lesions, co-localized with platelets.

Conclusion

Our findings suggest that platelet-mediated signaling through CXCR5 may be active in vivo during plaque destabilization, potentially representing a counteracting mechanism to inflammation.     

基于单细胞转录组鉴定动脉粥样硬化自身免疫表征

[目的]基于单细胞转录组生物信息学方法探讨动脉粥样硬化免疫微环境特征,挖掘免疫炎症与动脉粥样硬化之间潜在的联系。[方法]从GEO数据库中提取单细胞转录组数据集GSE159677,可视化分析颈动脉粥样硬化斑块区及其近心端毗邻非斑块区细胞组成成分,利用CellChat整合细胞间通讯网络,分析细胞间交互作用差异,识别动脉粥样硬化斑块免疫炎症信号通路差异,探索动脉粥样硬化免疫微环境中细胞间受体-配体特异性变化通路。[结果]本研究从单细胞测序的视角分析了动脉粥样硬化斑块内的细胞构成和细胞通讯。研究发现,在动脉粥样硬化斑块的细胞构成中内皮细胞和平滑肌细胞减少,而T细胞、单核细胞、巨噬细胞及软骨细胞明显增加。通过细胞通讯分析,发现树突状细胞、单核细胞、巨噬细胞、自然杀伤细胞与内皮细胞间的通讯作用及部分细胞与单核细胞间的通讯作用均有显著的改变,相关信号通路包括CXCL家族与ACRK1、CCL家族与ACRK1、MIF与CD74等配体-受体互作。MIF、ANXA1、YNF、RETN、LGASL9等对单核细胞以及NAMPT、CCL2、TNFSF12对内皮细胞的通讯改变在免疫炎症反应调控动脉粥样硬化机制中起着...     

Elevated levels of neopterin are associated with carotid plaques with complex morphology in patients with stable angina pectoris

ObjectiveNeopterin is an activation marker for monocytes/macrophages, and circulating levels of neopterin are elevated in patients with coronary complex lesions in unstable angina pectoris. We investigated the possible association between neopterin and complex carotid plaques which may be associated with the risk of ischemic stroke in patients with stable angina pectoris (SAP).MethodsWe measured plasma levels of neopterin in 102 patients with SAP and carotid ultrasound was performed for evaluation of the presence of carotid plaques and plaque surface characteristics categorized as complex or noncomplex. In addition, endarterectomy specimens of extracranial high-grade carotid stenosis with complex plaques from five patients with SAP were immunohistochemically examined with antibodies to smooth muscle cells, endothelial cells, platelets, macrophages, and T cells.ResultsPlasma neopterin levels were significantly higher in patients with complex carotid plaques than in those with noncomplex plaques (median [interquartile range]: 24.2 [19.2–39.3] nmol/L vs. 19.4 [11.9–25.1] nmol/L; P = 0.01) or without any plaques (18.8 [14.9–23.6] nmol/L; P = 0.001). On multivariate logistic analyses after adjustment for traditional atherosclerotic risk factors, multi-vessel coronary disease and high sensitivity C-reactive protein, neopterin levels were independently associated with the presence of complex carotid plaques (adjusted OR 2.21 per SD increase, 95%CI 1.13–4.33, P = 0.02). Immunohistochemical staining revealed abundant neopterin-positive macrophages in carotid complex lesions.ConclusionThese findings demonstrate that carotid plaques with complex morphology have increased circulating neopterin levels and immunohistochemical localization of neopterin in patients with SAP. Neopterin can be considered an important biomarker of plaque destabilization in carotid artery atherosclerotic lesions in this population.     

Social disruption stress increases IL-6 levels and accelerates atherosclerosis in ApoE-/- mice

IntroductionWe have previously shown that different forms of stress have distinctive effects on atherogenesis in mice. We showed that social stress increase atherosclerosis in ApoE^?/? mice, while more physical forms of stress do not. Here we evaluated the effect of social disruption (SDR) stress on atherogenesis and evaluated cytokine release after SDR-stress and five more physical stressors.MethodsMale ApoE^?/? mice were exposed to SDR-stress during 12 weeks, and atherosclerotic plaque area was assessed in aorta, aortic root and innominate artery. Further, male C57BL/6 mice were exposed to SDR-stress or five physical stressors, and cytokine and corticosterone levels were analyzed in plasma/serum samples immediately after stress.ResultsWe found a correlation between the level of SDR-stress and atherosclerotic plaque area in aorta and a numerical increased plaque area in aortic root. SDR stress did not affect histological features of plaque composition. However, SDR-stress increased levels of corticosterone, IL-6 and CXCL1. Plasma corticosterone increased for all five physical stressors, but IL-6 and CXCL1 only increased in the group exposed to restraint combined with rat odor.ConclusionsThese findings suggest that SDR-stress is indeed atherogenic, in contrast to our previous results using the physical stressors. A possible explanation to this difference is that SDR-stress, but not physical stressors, leads to release of the pro-inflammatory cytokines IL-6 and CXCL1.     

In vivo 18F-fluorodeoxyglucose positron emission tomography imaging provides a noninvasive measure of carotid plaque inflammation in patients.

OBJECTIVES: Given the importance of inflammation in atherosclerosis, we sought to determine if atherosclerotic plaque inflammation could be measured noninvasively in humans using positron emission tomography (PET). BACKGROUND: Earlier PET studies using fluorodeoxyglucose (FDG) demonstrated increased FDG uptake in atherosclerotic plaques. Here we tested the ability of FDG-PET to measure carotid plaque inflammation in patients who subsequently underwent carotid endarterectomy (CEA). METHODS: Seventeen patients with severe carotid stenoses underwent FDG-PET imaging 3 h after FDG administration (13 to 25 mCi), after which carotid plaque FDG uptake was determined as the ratio of plaque to blood activity (target to background ratio, TBR). Less than 1 month after imaging, subjects underwent CEA, after which carotid specimens were processed to identify macrophages (staining with anti-CD68 antibodies). RESULTS: There was a significant correlation between the PET signal from the carotid plaques and the macrophage staining from the corresponding histologic sections (r = 0.70; p < 0.0001). When mean FDG uptake (mean TBR) was compared with mean inflammation (mean percentage CD68 staining) for each of the 17 patients, the correlation was even stronger (r = 0.85; p < 0.0001). Fluorodeoxyglucose uptake did not correlate with plaque area, plaque thickness, or area of smooth muscle cell staining. CONCLUSIONS: We established that FDG-PET imaging can be used to assess the severity of inflammation in carotid plaques in patients. If subsequent natural history studies link increased FDG-PET activity in carotid arteries with clinical events, this noninvasive measure could be used to identify a subset of patients with carotid atherosclerosis in need of intensified medical therapy or carotid artery intervention to prevent stroke.     

A novel chemokine, Leukotactin-1, induces chemotaxis, pro-atherogenic cytokines, and tissue factor expression in atherosclerosis

Chemokines such as monocyte chemoattractant protein (MCP) -1 and interleukin (IL)-8 are known to be involved in various processes in atherosclerosis such as plaque formation, plaque rupture, and thrombus formation. We investigated whether a new chemokine, Leukotactin (LKN)-1, is involved in atherosclerosis. We tested the expression of LKN-1 by immunohistochemical methods in carotid atherosclerotic plaque specimen. Induction of pro-inflammatory cytokines, transmigration, and tissue factor (TF) expression were tested in THP-1 cells and human peripheral blood monocytes treated with recombinant human LKN-1. Immunohistochemical analyses revealed that expression of LKN-1 occurs in regions of plaques rich in foam cells. In a Boyden chamber assay, THP-1 cells treated with 0.01--10 nM of LKN-1 transmigrated through gelatin coated filters in a dose dependent manner. LKN-1 also induced the transient expression of TNF-alpha, IL-8, and MCP-1 within 15 min of the treatment of the THP-1 cells. When peripheral blood monocytes were treated with LKN-1, expression levels of TF and TF-mediated procoagulating activity were induced in a time- and dose-dependent manner. These results raise the possibility that LKN-1 is another chemokine that is involved in the atherogenesis. LKN-1 may chemoattract immune cells into the plaque, induce pro-inflammatory cytokines, and produce thrombi by inducing TF expression.     

Monocyte/macrophage suppression in CD11b diphtheria toxin receptor transgenic mice differentially affects atherogenesis and established plaques

Although monocytes/macrophages are considered important in atherogenesis, their role in established plaques is unclear. For example, macrophage content is associated with plaque instability, but their loss through cell death is observed at sites of plaque rupture. To examine the role of monocytes/macrophages in atherosclerosis, we developed CD11b-diphtheria toxin (DT) receptor (DTR) transgenic mice, whereby administration of DT selectively kills monocytes/macrophages. DT treatment reduced peripheral blood monocytes and tissue macrophages and inhibited macrophage function in CD11b-DTR mice and apolipoprotein E-null (apoE(-/-)) mice transplanted with CD11b-DTR bone marrow. In atherogenesis experiments, DT markedly reduced plaque development and altered plaque composition, reducing collagen content and necrotic core formation. In mice with established plaques, acute DT treatment induced macrophage apoptosis and reduced macrophage content but did not induce plaque inflammation, thrombosis, or rupture. Furthermore, despite a 50% reduction in monocytes, chronic DT treatment of these mice did not alter plaque extent or composition, most likely because of ongoing recruitment/proliferation of monocytes with recovery of macrophage content. We conclude that monocytes/macrophages are critical to atherogenesis, but established plaques are more resistant to reductions in monocytes.     

Comparative immunohistochemical staining of atherosclerotic plaques using F16, F8 and L19: Three clinical-grade fully human antibodies

ObjectiveF16, F8 and L19 are three fully human monoclonal antibodies, specific to splice isoforms of tenascin-C and fibronectin, which stain sites of active tissue remodeling and which are currently in Phase I and II clinical trials as radio-immunoconjugates and immunocytokines in patients with cancer and arthritis. The characterization of atherosclerosis using these antibodies may open novel pharmacodelivery options for the imaging and treatment of cardiovascular conditions. It may also allow a better assessment of the corresponding immunoconjugates in polymorbid patients with atherosclerotic plaques.MethodsWe performed a comparative immunohistochemical analysis with the F16, F8 and L19 antibodies in 28 freshly frozen human carotid plaques and in 11 normal arteries. Furthermore, we assessed the localization of the antibodies in relation to the infiltrating macrophages, vasa vasorum and Ki67-positive proliferating cells of the plaque.ResultsThe F16 antibody, specific to the extra-domain A1 of tenascin-C, stained plaques with a selective and intense pattern, while F8 and L19, specific to the EDA and EDB domains of fibronectin, respectively, exhibited a less selective and intense staining. In immunofluorescence, F16 was found to bind regions rich in macrophages, vasa vasorum and proliferating cells, while showing no detectable vs. weak staining of normal arteries and of quiescent plaque structures.ConclusionThe human monoclonal antibody F16 stains areas of active tissue remodeling in atherosclerotic plaques and may thus deserve to be investigated as a suitable building block for the development of radiopharmaceuticals for plaque imaging or for the antibody-based targeted delivery of therapeutic agents to atherosclerotic lesions.     

TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages.

The expression of TWEAK (TNFSF12) and TweakR/Fn14 was detected in regions rich in macrophage/foam cells in atherosclerotic plaques. The role of TWEAK in monocytes in relation to atherogenesis was investigated by analyzing the cellular events induced by TWEAK in a human macrophage-like cell line, THP-1. TWEAK induced various molecular mediators of atherogenesis, such as IL-6, MCP-1, IL-8 and MMP-9, and the induction was augmented by interferon-gamma. TWEAK-induced activation of MMP-9 was mediated by activation of NF-kappaB. These results suggest that TWEAK is involved in atherosclerosis by inducing pro-inflammatory cytokines and extracellular matrix degrading enzymes, which reduce plaque stability.     

Tumor necrosis factor receptor superfamily 12 may destabilize atherosclerotic plaques by inducing matrix metalloproteinases

Immunohistochemical staining of human atherosclerotic plaques revealed expression of the tumor necrosis factor receptor superfamily (TNFRSF) 12 in regions rich in macrophage/foam cells. The role of TNFRSF12 in the functioning of monocytes in relation to atherogenesis was investigated by analysis of cellular events after stimulation of TNFRSF12 in a human macrophage-like cell line, THP-1. Activation of the THP-1 cells on plates coated with monoclonal antibody against TNFRSF12 induced the expression of matrix metalloproteinases (MMPs) -1, -9, and -13. Furthermore, the expression patterns of TNFRSF12 and the MMPs overlapped in atherosclerotic plaques. Signaling of TNFRSF12 may thus contribute to the induction of extracellular matrix degrading enzymes in macrophages.     

Vascular neuropeptide Y contributes to atherosclerotic plaque progression and perivascular mast cell activation

Aim

Neuropeptide Y is an abundantly expressed neurotransmitter capable of modulating both immune and metabolic responses related to the development of atherosclerosis. NPY receptors are expressed by a number of vascular wall cell types, among which mast cells. However, the direct effects of NPY on atherosclerotic plaque development and progression remain to be investigated. In this study we thus aimed to determine whether NPY is expressed in atherosclerotic plaques and to establish its role in atherosclerotic plaque development.

Methods and results

NPY expression was seen to be increased up to 2-fold in unstable human endarterectomy plaques, as compared to stable plaques, and to be significantly upregulated during lesion progression in apoE^−/− mice. In apoE^−/− mice focal overexpression of NPY in the carotid artery significantly increased atherosclerotic plaque size compared to controls, while plaque composition was unaffected. Interestingly, perivascular mast cell activation was significantly higher in the NPY-overexpressing mice, suggesting that NPY may impact plaque progression in part via mast cell activation. Furthermore, in vitro NPY-induced murine mast cell activation resulted in the release of pro-atherogenic mediators including IL-6 and tryptase.

Conclusions

Our data show that NPY expression is increased during atherogenesis and in particular in unstable plaques. Furthermore, perivascular overexpression of NPY promoted plaque development and perivascular mast cell activation, suggestive of a role for NPY-induced mast cell activation in lesion progression.     

EGF mediates monocyte chemotaxis and macrophage proliferation and EGF receptor is expressed in atherosclerotic plaques

The recruitment of peripheral monocytes to the sub-endothelial space, their development into macrophages and subsequent proliferation are critical events during atherosclerosis. Receptors for epidermal growth factor (EGF) have been identified on cells of the myeloid lineage, but a role for them in atherogenesis has yet to be described. We have identified functional EGF receptors (EGFR, ErbB1/HER-1) on peripheral blood monocytes and monocyte-derived macrophages. Uniquely, these receptors were found to mediate both chemotaxis in monocytes and macrophages and proliferation in macrophages. EGFR mRNA was detected in atherosclerotic plaques, but not in morphologically normal aortae and EGFR receptor staining co-localised with macrophage staining in these plaques. The identification of receptors for EGF on peripheral blood monocytes, macrophages and atherosclerotic lesions, together with their transduction of two functionally important cellular events, heightens the potential importance of members of the EGF super-family in atherogenesis and other chronic inflammatory processes.     

Factor Seven Activating Protease (FSAP) expression in human monocytes and accumulation in unstable coronary atherosclerotic plaques

ObjectiveThe Factor Seven Activating Protease (FSAP) is known to influence fibrinolysis and to play a critical role in the inhibition of vascular smooth muscle cell (VSMC) proliferation and migration as well as neointima formation. In order to define the role of FSAP in vascular pathophysiology we have investigated the expression of FSAP protein and mRNA in human vascular cells and coronary atherosclerotic plaques with defined clinical features.Methods and resultsDirectional coronary atherectomy (DCA) specimens from 40 lesions were analyzed for FSAP antigen and mRNA expression. Higher level of FSAP mRNA (p < 0.001) as well as FSAP immunostaining (p < 0.005) was observed in patients with acute coronary syndromes compared to patients with stable angina pectoris. FSAP antigen was found to be focally accumulated in hypocellular and lipid-rich areas within the necrotic core of atherosclerotic plaques. FSAP was also co-localized with CD11b/CD68 expressing cells in macrophage-rich shoulder regions of the plaques. Monocyte-derived macrophages expressed FSAP in vitro and this was further induced by pro-inflammatory mediators.ConclusionsFSAP accumulation in coronary atherosclerotic lesions is due to either local synthesis by monocytes/macrophages, or uptake from the plasma due to plaque hemorrhage. The higher expression of FSAP in unstable plaques suggests that it may destabilise plaque through reducing VSMC proliferation/migration and altering the hemostatic balance.     

Enhanced cyclooxygenase 2-mediated vasorelaxation in coronary arteries from insulin-resistant obese Zucker rats

Objective

The expression of p53 has been associated with DNA damage, cell senescence, proliferation and apoptosis in human atherosclerotic plaques. However, it is largely unknown whether p53 expression is related to the stability and clinical manifestations of atherosclerotic plaques in humans. In the present study, we examined whether p53 expression is related to clinical symptoms and plaque integrity in patients with carotid atherosclerosis (n = 62). We also investigated p53 expression and its relation to apoptosis and apoptosis-related cathepsin L and ferritin in the carotid lesions.

Methods and results

We found that smooth muscle cells often had nuclear p53 in the shoulder region of carotid lesions while CD68-positive macrophages, which had both nuclear and cytoplasmic p53, frequently appeared in the surrounding areas of necrotic cores or plaque cap regions. Quantitative image analysis of immunohistochemistry showed that p53 expression was significantly increased in plaques with necrotic core formation or cap rupture and lesions from patients with transient ischemic attacks (TIAs). The levels of p53 expression was significantly increased in more severe stenosed lesions but decreased with prolonged time between symptom onset and carotid endarterectomy. Furthermore, p53 expression was significantly correlated with the expression of ferritin, lysosomal cathepsin L, and apoptosis.

Conclusion

The increased p53 expression, particularly macrophage p53 levels, is associated with the enlargement of necrotic cores, plaque rupture and clinical manifestations of carotid plaques. Concomitant increases of lysosomal cathepsin, ferritin, and p53 levels may promote the apoptosis and atheroma progression in patients with carotid atherosclerosis.     

老年患者股动脉和颈动脉及冠状动脉粥样硬化斑块稳定性的比较

目的　探讨股动脉、颈动脉、冠状动脉粥样硬化斑块的稳定性。方法　收集我院老年尸体解剖病例 15例 ,将所有病例的两侧股动脉、两侧颈动脉、左冠状动脉前降支进行连续取材 ,常规病理检查 ,部分节段行α 平滑肌肌动蛋白、CD6 8、bax染色。结果　股动脉粥样硬化斑块中的平滑肌细胞、巨噬细胞数量与颈动脉相近。与冠状动脉比较 ,股动脉粥样硬化斑块中的平滑肌细胞相对多 ,巨噬细胞相对少 ;bax在巨噬细胞的表达多 ,在平滑肌细胞的表达少。结论　平滑肌细胞、巨噬细胞数量的不同导致了 3种动脉粥样硬化斑块不同的稳定性。股动脉中的粥样硬化斑块较冠状动脉更稳定。     

Endothelial insulin receptor expression in human atherosclerotic plaques: Linking micro- and macrovascular disease in diabetes?

ObjectiveExogenous insulin use in patients with type 2 diabetes (DM2) has been associated with an increased risk of cardiovascular events. Through which mechanisms insulin may increase atherosclerotic plaque vulnerability is currently unclear. Because insulin has been suggested to promote angiogenesis in diabetic retinopathy and tumors, we hypothesized that insulin enhances intra-plaque angiogenesis.MethodsAn in vitro model of pathological angiogenesis was used to assess the potential of insulin to enhance capillary-like tube formation of human microvascular endothelial cells (hMVEC) into a three dimensional fibrin matrix. In addition, insulin receptor expression within atherosclerotic plaques was visualized in carotid endarterectomy specimens of 20 patients with carotid artery stenosis, using immunohistochemical techniques. Furthermore, microvessel density within atherosclerotic plaques was compared between 68 DM2 patients who received insulin therapy and 97 DM2 patients who had been treated with oral glucose lowering agents only.ResultsInsulin, at a concentration of 10^?8 M, increased capillary-like tube formation of hMVEC 1.7-fold (p < 0.01). Within human atherosclerotic plaques, we observed a specific distribution pattern for the insulin receptor: insulin receptor expression was consistently higher on the endothelial lining of small nascent microvessels compared to more mature microvessels. There was a trend towards an increased microvessel density by 20% in atherosclerotic plaques derived from patients using insulin compared to plaques derived from patients using oral glucose lowering agents only (p = 0.05).ConclusionExogenous insulin use in DM2 patients may contribute to increased plaque vulnerability by stimulating local angiogenesis within atherosclerotic plaques.     

Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression

Background and aimsAtherosclerosis is a chronic inflammatory process involving the activity of several cytokines and growth factors. Platelet-derived growth factor-A (PDGF-A) and PDGF-B are important mitogens and chemoattractants for monocytes as well as smooth muscle cells. We sought to identify the role of PDGF-C and PDGF-D, two new members of the PDGF family, in monocyte migration and differentiation. We also assessed their effects in regulating matrix metalloproteinase-2 (MMP-2) and MMP-9, which are important for cell migration.Methods and resultsPDGF-C and PDGF-D were expressed in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic plaques, as shown by immunohistochemical analysis. PDGF-C and PDGF-D mRNA and protein expression was induced after differentiation of THP-1 monocytes to macrophages, and both PDGF-C and PDGF-D induced MMP-9 mRNA expression in a concentration-dependent manner. Treatment of cells with PDGF-C or PDGF-D enhanced the secretion of MMP-2 and MMP-9 in a cell-dependent manner. In a migration assay using a Boyden chamber with 8 μm pore size, PDGF-C and PDGF-D attracted THP-1 monocytes in a concentration-dependent manner.ConclusionsOur data suggest that PDGF-C and PDGF-D, like PDGF-A and PDGF-B, play important roles in atherosclerosis by stimulating MMP activity and influencing monocyte migration.     

Rapid Screening for Subclinical Atherosclerosis by Carotid Ultrasound Examination: The HAPPY (Heart Attack Prevention Program for You) Substudy

BackgroundCardiovascular disease (CVD)-related death rates have been escalating in emerging economies such as India. A strategy to initiate prophylactic medical intervention by direct identification of subclinical atherosclerotic burden may be appropriate in rural populations where assessment based on traditional risk factors is not available.ObjectivesThis study sought to investigate the feasibility of performing rapid automated carotid ultrasound studies in a rural setting and to measure the prevalence of carotid plaques and age-specific distribution of carotid intima-media thickness (IMT) as an index of subclinical atherosclerosis.MethodsScreening of the extracranial carotid system with automated B-mode ultrasound was performed along with health questionnaire assessments in 771 asymptomatic volunteers (ages 40 ± 14 years; 626 men and 145 women) with no known CVD. Measurements of IMT were recorded as the mean of 24 spatial measurements performed over a 1-cm region in the far wall of the common carotid artery at end diastole; the prevalence of the plaque (focal IMT >1.5 mm) was determined.ResultsA total of 69 (8.9%) subjects had atherosclerotic plaques. Of these, 16 (2.1%) exhibited bilateral plaques, 28 (3.6%) left carotid plaque only, and 25 (3.2%) had right carotid plaques. Patients even under 50 years showed a high prevalence of carotid plaques (7%), which increased with age (25% and 35% for 51 to 70 and >70 years, respectively). Only 3 (4.3%) participants with plaques were former smokers. Global mean IMT was 0.55 ± 0.13 mm and correlated with age for both left and right carotid arteries (r = 0.61 and 0.60, p < 0.001 for both) in male as well as female subjects (r = 0.70 and 0.67, p < 0.001 for both), respectively.ConclusionsRapid community screening for subclinical atherosclerosis is feasible with automated carotid ultrasound examination and may be beneficial in rural communities of industrializing nations where traditional CVD risk factor data are not yet readily available.