Type I collagen gene expression in human atherosclerosis. Localization to specific plaque regions.

Because collagen is a major component of the human atherosclerotic plaque, factors controlling collagen synthesis may have a profound influence on the volume growth of these intimal lesions. In human arteries, we compared normal vs atherosclerotic media vs intimas for type I collagen gene expression using immunocytochemistry and in situ messenger RNA hybridization with subsequent correlations with plaque topographical features. We also determined the associations of such collagen gene expression with proximity to monocyte/macrophages and T lymphocytes. Type I collagen synthesis appears to be upregulated in atherosclerotic plaques compared with their underlying medias and normal internal mammary arteries and coronary diffuse intimal thickenings. At least in established and advanced coronary and carotid plaques, type I collagen gene expression is focal and especially prevalent in fibrous cap and vascularized regions. Although macrophages and type I procollagen messenger RNA and protein are both found in atherosclerotic plaques, no apparent spatial correlation between macrophage presence and type I procollagen presence was found within these atherosclerotic intimas. Type I procollagen presence appears to be negatively associated with the spatial presence of T cells. Thus, human atherosclerotic plaques exhibit nonuniform patterns of type I collagen gene expression. Although the biochemical determinants of this focal gene expression have yet to be determined, it is conceivable that stimulatory/inhibitory cytokines and other factors (eg hemodynamics) play important roles in determining the focal nature of collagen synthesis in atherosclerosis.     

人冠状动脉几种细胞外基质的动态变化与动脉粥样硬化形成的关系

观察Ⅰ、Ⅲ型前胶原ｍＲＮＡ和胶原蛋白及硫酸软骨素蛋白聚及其基因在正常和有动脉粥样硬化病变的人冠状动脉内的表达和分布,及Ⅰ、Ⅲ型胶原增生和硫酸软骨素蛋白聚糖增多在人冠状动脉粥样硬化病变形成过程中的作用。     

Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries.

Proteoglycans are important constituents of blood vessels and accumulate in various forms of vascular disease. Little is known concerning the proteoglycan composition of restenotic lesions formed after angioplasty and whether the proteoglycan composition of these lesions differs from that of primary atherosclerosis. Accordingly, we sought to characterize the distribution of two proteoglycans, biglycan and decorin, in primary atherosclerotic and restenotic lesions of human coronary arteries. Restenosis (n = 37) and primary (n = 11) lesions obtained from 48 patients by directional atherectomy of human coronary arteries were stained with antibodies against biglycan and decorin. To further characterize the extracellular matrix of restenotic tissues, we studied the co-distribution of these proteoglycans with collagen types I, III, and IV. The loose fibroproliferative tissue seen predominantly in restenosis lesions consistently stained positively for biglycan in patterns of deposition ranging from disseminated to homogeneous. The density and intensity of biglycan staining was correlated with the density of collagen type I and III fiber networks, both of which were observed to interweave among the loose fibroproliferative tissue. The compact connective tissue of primary atherosclerotic plaque was characterized by strong biglycan staining which co-localized with intense collagen type I and III staining. Only basement membrane-like structures rich in collagen type IV demonstrated negative biglycan staining. In contrast, loose fibroproliferative tissue exhibited no significant staining for decorin. Strong immunostaining for decorin, however, was found in primary atherosclerotic plaque. There are thus regional differences in the distribution of extracellular matrix proteoglycans of restenotic and primary human atherosclerotic lesions; these observations suggest that differences established for the biological roles of biglycan and decorin in other organ systems may extend as well to pathologically altered human coronary arteries.     

活性氧对平滑肌细胞合成基质金属蛋白酶-1, 3及其抑制物的影响

目的:探讨活性氧(ROS)对血管平滑肌细胞合成基质金属蛋白酶-1,3(MMP-1,3)和基质金属蛋白酶抑制物-1(TIMP-1)的影响,从而推测其是否促进动脉粥样硬化斑块的破裂。方法:体外培养胎儿主动脉平滑肌细胞,加入含100μmol/L黄嘌呤,5U/L黄嘌呤氧化酶的无血清培养液,孵育24h,收集细胞上清液。用Westernblotting方法检测浓缩后的细胞上清液中MMP-1,3和TIMP-1的含量。结果:黄嘌呤/黄嘌呤氧化酶组细胞上清液中的MMP-1含量明显少于正常对照组,并且转化成活性形式;MMP-3的含量明显多于正常对照组,并且转化成活性形式;TIMP-1的含量明显少于正常对照组。结论:ROS对MMPs-TIMPs平衡的影响很复杂,可能对动脉粥样硬化斑块的破裂起一定作用。     

Skin flap-induced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression

Previous studies have shown that covering granulation tissue of a full-thickness skin wound by a vascularized skin flap induces tissue remodeling, with a rapid loss of granulation tissue cells by apoptosis. In the present study, in situ hybridization has been used to examine mRNA expression for several factors that may be implicated in the apoptosis seen in this tissue. Skin wounds were made on the dorsal skin of 8-week-old rats. Ten days after wounding, skin flaps were created surgically and sutured over the granulation tissue. Tissue sections of granulation tissue from various times after addition of the skin flap were hybridized with 33P-labelled cRNA probes for transforming growth factor-beta1 (TGF-beta1), beta-inducible gene H3 (beta-ig-h3), alpha1 (1) procollagen, alpha-smooth muscle actin, matrix metalloproteinase-13 (MMP-13) and -2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), and inducible nitric oxide synthase (iNOS). Control granulation tissue prior to addition of the skin flap showed high levels of TGF-beta1, beta-ig-h3, alpha1 (1) procollagen, alpha-smooth muscle actin, and TIMP-1 expression. MMP-13, MMP-2, and iNOS mRNA were low in 10-day granulation tissue. Addition of a skin flap resulted in a decrease in the expression of TGF-beta1, beta-ig-h3, alpha1 (I) procollagen, alpha-smooth muscle actin, and TIMP-1, but increased expression of MMP-13 and MMP-2. Similarly, an increase in iNOS mRNA expression was observed in the granulation tissue after addition of the skin flap. Addition of a vascularized skin flap may result in rapid remodelling of granulation tissue due to a decrease in expression of the trophic growth factor TGF-beta1 and increased degradation of extracellular matrix due to an alteration in the balance between MMPs and their inhibitor, TIMP-1. Additionally, increased iNOS expression may also favour apoptosis through the generation of free radicals. The additive effect of reduced growth factor expression, increased extracellular matrix turnover, and nitric oxide generation may result in the fibroblast and vascular cell apoptosis seen during the rapid remodelling of this tissue.     

病毒性心脏病小鼠心脏胶原代谢的动态变化

目的：探讨急、慢性病毒性心肌炎和扩张型心肌病小鼠心肌组织中胶原代谢的动态变化和特征。方法：以柯萨奇病毒B3感染BALB/c小鼠分别建立病毒性心脏病动物模型，同期均设正常对照。组织病理学方法和心脏超声确认动物模型后，以酶联免疫吸附法（ELISA）检测各组小鼠胶原前肽（PINP、PICP和PIIINP）的血清浓度；以免疫印迹法（Western blotting）检测间质胶原酶（MMP-1）及其组织抑制物（TIMP-1）在心肌组织中的表达；同时检测MMP-1活性变化结果：各期感染小鼠心脏均出现明显心肌纤维化，急性期为修复性心肌纤维化，胶原合成和降解均增强；慢性期反应性纤维化和修复性纤维化并存，胶原合成增多降解减少；心肌病理主要为反应性纤维化，胶原合成增多；MMP-1表达量和活性随病程进行性减少，TIMP-1的表达无变化，MMP-1/TIMP-1进行性降低。结论：病毒性心脏病不同时期胶原代谢有其各自的特点，对病程和预后的影响不同。     

Differences in connective tissue gene expression between normally functioning, polycystic and post-menopausal ovaries

Arrested follicular maturation is a characteristic feature of polycystic ovary syndrome (PCOS). Follicles mature in ovarian stroma composed of extracellular matrix (ECM). However, little is known of the expression of ECM genes in polycystic ovaries. The present study compares the expression levels of genes coding for collagens, matrix metalloproteinases (MMP), their inhibitors (TIMP) and cathepsins in polycystic ovaries using fertile and post-menopausal ovaries as controls. In northern analyses, the gene expression profiles of type I and III collagen of PCOS samples resembled those observed in normal follicular phase ovaries, while mRNA levels of proalpha1(IV) collagen and TIMP-3 mRNA were significantly lower in polycystic than control ovaries. During the normal menstrual cycle, an increase was observed in MMP-9 gene expression during the luteal phase. In post-menopausal ovaries, mRNA levels for type I, III and IV collagens and osteonectin were reduced, while the MMP, TIMP (excluding TIMP-3) and cathepsins did not reflect this metabolic down-regulation. Immunohistochemical staining for MMP-9 and TIMP-4 suggested differences between polycystic and normally functioning ovaries. These data demonstrate that normal ovarian functions are associated with changes in production and degradation of ECM. The alterations observed in the production and/or distribution of type IV collagen, TIMP-3 and TIMP-4 suggest involvement of basement membranes in the pathogenesis of PCOS.     

Effects of 17beta-estradiol on matrix metalloproteinase-1 synthesis by human dermal fibroblasts

BACKGROUND: Hormone replacement therapy (HRT) has been used in treatment of various menopausal disorders. It has been well documented that HRT increases the amount of dermal collagen and skin thickness in vivo. However little is known about the effects of female sex hormones on dermal fibroblasts in vitro. OBJECTIVE: The aim of this study is to determine whether or not 17beta-estradiol affects mRNA expression and production of type I collagen, matrix metalloproteinases-1 (MMP-1), tissue inhibitor metalloproteinases-1 (TIMP-1) or transforming growth factor-beta1 (TGF-beta1) by human dermal fibroblasts. METHODS: Fibroblasts were cultured with and without 17beta-estradiol for 6h. We evaluated the changes of mRNA expressions and protein production of type I collagen, MMP-1, TIMP-1 and TGF-beta1. RESULTS: The mRNA expressions of collagen alpha1(I), MMP-1, TIMP-1, TGF-beta1 were not changed by 17beta-estradiol stimulations at a concentration of 10(-12) to 10(-8) M. However, 17beta-estradiol at concentrations of 10(-12) and 10(-10) M exhibited inhibitory effects on proMMP-1, but not type I collagen or TIMP-1 synthesis. The synthesis of TGF-beta1 by fibroblasts stimulated with 10(-8) M of estradiol was significantly increased as compared with the control. However, the level of TGF-beta type II receptor phosphorylation was not elevated under the same conditions. CONCLUSION: Suppressed synthesis of MMP-1 at a low concentration of 17beta-estradiol may be partly involved in the dermal tissue remodeling to inhibit the degradative change.     

Aspirin Inhibits MMP-2 and MMP-9 Expression and Activity Through PPARα/γ and TIMP-1-Mediated Mechanisms in Cultured Mouse Celiac Macrophages

Aspirin is an anti-inflammatory drug, and has been widely used for the prevention of cardio-cerebrovascular events. Matrix metalloproteinase (MMP)-2 and MMP-9 can degrade the extracellular matrix and may be critical for the development and disruption of atherosclerotic plaques, while tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit the degradation of extracellular matrix. The purpose of present study was to investigate the inhibitory effects of aspirin on MMP-2 and MMP-9 expression and activity in cultured mouse celiac macrophages, and to determine the possible mechanisms. The results showed that MMP-2/9 mRNA expression and release were significantly decreased after cultured mouse celiac macrophages were treated with aspirin 12.5–50 μg/ml for 24 h, while the TIMP-1 mRNA expression and release, and peroxisome proliferator-activated receptor (PPAR) α/γ mRNA expression were increased after the same treatment. Moreover the aspirin-induced down-regulation of MMP-2/9 mRNA expression and reduction of MMP-9 release were notably alleviated after pretreatment with specific inhibitors of PPARα/γ. These results suggested that aspirin could inhibit the expression and release of MMP-2/9 by up-regulation of PPARα/γ gene expression, and also inhibit the activity of MMP-2/9 by induction of TIMP-1 expression, which might be good for the stabilization of atherosclerotic plaques and the prevention of cardio-cerebrovascular events.     

Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions

Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease.     

Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin

Human skin is daily exposed to infrared (IR) radiation from natural sunlight. However, the effects of IR irradiation on collagen metabolism have not been investigated in human skin in vivo. Here, we examined whether single or repeated (three times a week for 4 weeks) exposure to IR irradiation changes the expressions of type I procollagen and interstitial collagenase (MMP-1). By using immunostaining, Western blotting, and semi-quantitative RT-PCR, we analyzed the protein and mRNA levels of type I procollagen and MMP-1 in young buttock skin. A single dose of IR to human skin increased the expression of type I procollagen within 24h, but did not change the expression of MMP-1. On the other hand, multiple IR doses reduced the expression of type I procollagen and increased the expression of MMP-1. We also found that TGF-betas may mediate type I procollagen synthesis in IR-irradiated human skin. Our results demonstrate that the regulations of the expressions of type I procollagen and MMP-1 differ in acute and chronically IR-irradiated skin. In particular, decreased collagen levels and increased MMP-1 levels in chronic IR-irradiated skin may be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo.     

心房纤颤模型犬心房组织基质金属蛋白酶9及组织抑制因子1与纤维化的关系***

背景：基质金属蛋白酶及其组织抑制因子在心房组织中的相互作用及动态平衡与心房纤颤的发生及维持密切相关。 目的：构建持续性心房纤颤犬模型,观察其心房肌组织基质金属蛋白酶9及其组织抑制因子1的基因表达与心房纤颤及心肌纤维化的关系。 方法：采用慢性快速心房起搏诱发持续性心房纤颤犬模型,并设置假手术组。通过Masson三色法染色计算胶原容积分数来评估纤维化程度,左心房心肌基质金属蛋白酶9及组织抑制因子1的mRNA水平表达使用反转录聚合酶联反应检测,其蛋白水平表达通过蛋白质印迹法测定。 结果与结论：与假手术组相比,持续性心房纤颤模型组心房肌纤维化程度明显增高,胶原容积分数明显增加(P < 0.01),且基质金属蛋白酶9 mRNA及蛋白表达水平明显增加(P < 0.01),组织抑制因子1的mRNA及蛋白表达水平明显下降        (P < 0.01)。结果证实,心房纤颤心房组织中基质金属蛋白酶9/组织抑制因子1基因表达的调控失衡以及基质金属蛋白酶9活性的增高与组织抑制因子1活性降低可能是影响胶原代谢、促进或抑制心肌纤维化,造成心房纤颤时心房结构重构的分子机制之一。     

内源性一氧化碳对低氧大鼠肺动脉转化生长因子-β3和Ⅰ型胶原表达的影响

目的:探讨内源性一氧化碳(CO)对低氧大鼠肺动脉胶原代谢的作用及其机制。方法:采用常压低氧大鼠肺动脉高压模型,观察血红素氧合酶(HO)抑制剂锌原卟啉-Ⅸ(ZnPP-Ⅸ)对肺动脉平均压(PAMP)和肺组织匀浆碳氧血红蛋白(HbCO)含量的影响,并用免疫组织化学和核酸原位杂交法分别观察ZnPP-Ⅸ对肺动脉转化生长因子-β₃(TGF-β₃)、Ⅰ型胶原蛋白的表达和肺动脉TGF-β₃mRNA、Ⅰ型前胶原mRNA和金属蛋白酶组织抑制因子(TIMP-1)mRNA表达的影响。结果:ZnPP-Ⅸ使低氧大鼠PAMP明显升高,肺组织匀浆CO含量明显降低;ZnPP-Ⅸ能促进TGF-β₃蛋白表达和TGF-β₃mRNA表达,显著促进低氧大鼠肺动脉Ⅰ型胶原蛋白表达和Ⅰ型前胶原mRNA表达,上调TIMP-1mRNA的表达。结论:内源性CO可能通过抑制TGF-β₃mRNA和TGF-β₃蛋白表达而抑制胶原蛋白的合成,促进胶原的降解,从而对低氧大鼠肺动脉胶原代谢发挥重要的调节作用。     

Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro

Human periodontal ligament fibroblasts were subjected to 10% cyclic equibiaxial tensional and compressive forces in vitro. Media supernatants were analyzed for changes in total protein, extracellular matrix proteins type I collagen and fibronectin, as well as MMP expression by gelatin zymography and Western blot. RNA analyses for changes in collagen, MMP-2, and TIMP-2 were carried out by either Real-time PCR and/or Northern blot. Application of compressional forces resulted in decreases in type I collagen and fibronectin protein, Col1A1 RNA, and increases in total protein, MMP-2 protein (latent and active), and MMP-2 RNA. TIMP-2 RNA was unchanged by compressive forces. In contrast, tensional forces increased total protein, type I collagen, Col1A1 RNA, as well as MMP-2 and TIMP-2 RNA. These studies show that cells can perceive two different forms of mechanical stimuli and respond in a differential manner relative to extracellular matrix synthesis and degradation.     

Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.     

Expression and Localization of Matrix Metalloproteinase-12 in the Aorta of Cholesterol-Fed Rabbits : Relationship to Lesion Development

Degradation of extracellular matrix (ECM) proteins in the aorta is a critical step for the development of atherosclerosis. Expression of matrix metalloproteinase (MMP)-12 (macrophage elastase), an elastin-degrading proteinase in the MMP family, was investigated in the thoracic aorta of rabbits fed a 1% cholesterol-containing diet for 16 weeks. In the atherosclerotic lesions, MMP-12 was produced abundantly at both the mRNA and protein levels, whereas no expression was observed in the normal rabbit aortas. The principal source of MMP-12 was macrophage foam cells (MFCs) that had infiltrated the atherosclerotic intima; this was demonstrated in both in vitro culture studies of MFCs purified from atherosclerotic lesions and immunohistochemical studies of aortic lesions. Additional biochemical studies using recombinant rabbit MMP-12 revealed that MMP-12 digested elastin, type IV collagen, and fibronectin and also activated MMP-2 and MMP-3. Expression of MMP-12 by human macrophage cell lines was increased by stimulation with acetylated low-density lipoprotein, implying augmentation of MMP-12 production during foam cell formation. Increased expression of MMP-12 in atherosclerotic lesions, concomitant with foam cell generation, which triggers the acceleration of ECM breakdown, is likely to be a critical step in the initiation and progression of the atherosclerotic cascade.     

生长抑素及奥曲肽对肝星状细胞基质代谢的负调节作用

目的： 探讨生长抑素（SST）及其类似物奥曲肽（OCT）对大鼠肝星状细胞（HSCs）细胞外基质（ECM）代谢的影响。方法: 采用原位杂交、［3H］-脯氨酸掺入、酶联免疫吸附（ELISA）、免疫细胞化学染色法，分别检测SST、OCT处理后HSC中I型及III型胶原mRNA、基质金属蛋白酶（MMP）、基质金属蛋白酶抑制物（TIMP）的水平和胶原合成率，以及培养上清液中层黏连蛋白（LM）、透明质酸（HA）、III型前胶原（PCIII）的含量。结果: 10^-7 mol/L-10^-6 mol/L SST、10^-7 mol/L-10^-5mol/L OCT均可显著抑制HSC中I、III型胶原mRNA及胶原的表达，明显减少HA、LM、PCIII的分泌，且呈现剂量依赖关系。10^-6 mol/L SST及10^-6 mol/L-10^-5 mol/L OCT共孵育还可使HSC的TIMP-1合成明显下降，从而降低MMP-1/TIMP-1比例。结论: SST及其类似物可通过转录、翻译水平的调节，减少ECM合成，加速其降解，从而发挥抗肝纤维化作用。     

Augmented Expression of Cyclooxygenase-2 in Human Atherosclerotic Lesions

Cyclooxygenase-1 (Cox-1) and Cox-2 convert arachidonic acid to prostaglandin H(2), the precursor of other prostaglandins and thromboxanes, eicosanoids important in vascular pathophysiology. However, knowledge of the expression of cyclooxygenases within atherosclerotic lesions is scant. This study tested the hypothesis that human atheroma and nonatherosclerotic arteries express the two Cox isoforms differentially. Cox-1 mRNA and protein localized on endothelial and medial smooth muscle cells of normal arteries (n = 5), whereas Cox-2 expression was not detectable. In contrast, atheromatous (n = 7) lesions contained both Cox-1 and Cox-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery, whereas smooth muscle cells showed lower levels, as demonstrated by immunohistochemical and in situ hybridization analysis. Furthermore, microvascular endothelium in plaques showed notable staining for both isoforms. In accord with immunohistochemical studies, Western blot analysis of protein extracts from normal arteries revealed constitutive Cox-1, but not Cox-2, expression. Extracts of atheromatous lesions, however, contained both Cox-1 and Cox-2 protein, detected as two immunoreactive proteins of approximately 70 and 50 kd. Macrophages expressed the short form of Cox-1/-2 constitutively after several days of in vitro culture, rather than the 70-kd protein. These results shed new light on the inflammatory pathways that operate in human atheroma. In particular, the expression of Cox-2 in atheromatous, but not in unaffected, arteries has therapeutic implications, given the advent of selective Cox-2 inhibitors.