Arjan H. Schoneveld

Relation of arterial geometry to luminal narrowing and histologic markers for plaque vulnerability: the remodeling paradox ☆

OBJECTIVEnTo relate local arterial geometry with markers that are thought to be related to plaque rupture.nnnBACKGROUNDnPlaque rupture often occurs at sites with minor luminal stenosis and has retrospectively been characterized by colocalization of inflammatory cells. Recent studies have demonstrated that luminal narrowing is related with the mode of atherosclerotic arterial remodeling.nnnMETHODSnWe obtained 1,521 cross section slices at regular intervals from 50 atherosclerotic femoral arteries. Per artery, the slices with the largest and smallest lumen area, vessel area and plaque area were selected for staining on the presence of macrophages (CD68), T-lymphocytes (CD45RO), smooth muscle cells (alpha-actin) and collagen.nnnRESULTSnInflammation of the cap or shoulder of the plaque was observed in 33% of all cross sections. Significantly more CD68 and CD45RO positive cells, more atheroma, less collagen and less alpha-actin positive staining was observed in cross sections with the largest plaque area and largest vessel area vs. cross sections with the smallest plaque area and smallest vessel area, respectively. No difference in the number of inflammatory cells was observed between cross sections with the largest and smallest lumen area.nnnCONCLUSIONnIntraindividually, pathohistologic markers previously reported to be related to plaque vulnerability were associated with a larger plaque area and vessel area. In addition, inflammation of the cap and shoulder of the plaque was a common finding in the atherosclerotic femoral artery.

Atherosclerotic arterial remodeling and the localization of macrophages and matrix metalloproteases 1, 2 and 9 in the human coronary artery

Atherosclerotic luminal narrowing is determined by plaque mass and the mode of geometrical remodeling. Recently, we reported that the type of atherosclerotic remodeling is associated with the presence of histological markers for plaque vulnerability. Inflammation and matrix degrading proteases (MMPs) may play a role in both plaque vulnerability and in expansive arterial remodeling. The aim of the present study was to investigate the association between the remodeling mode and the localization of macrophages and MMPs in coronary atherosclerotic segments. From 36 atherosclerotic coronary arteries, 45 and 51 segments were selected with a vessel area that was >10% smaller and larger compared with the adjacent segments, respectively. No significant difference in staining for macrophages was observed between segments with expansive and constrictive remodeling. More MMP-2 and MMP-9 staining was observed in plaques of expansively remodeled segments compared with constrictively remodeled segments. In general, MMP-staining was less evident in the adventitial layer compared with the plaque. Zymography revealed more active MMP-2 in expansively remodeled segments compared with constrictively remodeled segments (340+/-319 vs. 199+/-181 (adjusted counts/mm(2)), respectively, P=0.019). Zymography did not show differences in inactive MMP-2 or MMP-9 among groups. It might be postulated that MMPs within the plaque play a causal role not only in plaque vulnerability but also in de novo atherosclerotic remodeling.

In Vivo Evidence for a Role of Toll-Like Receptor 4 in the Development of Intimal Lesions

Background—Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. Methods and Results—Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-&kgr;B and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area±SEM, 9134±1714 versus 2353±1076 &mgr;m2, P <0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859±904 &mgr;m2, P =0.02 versus wild type). Conclusions—A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Toll-Like Receptor 4 Is Involved in Outward Arterial Remodeling

Background—Toll-like receptor 4 (Tlr4) is the receptor for exogenous lipopolysaccharides (LPS). Expression of endogenous Tlr4 ligands, heat shock protein 60 (Hsp60) and extra domain A of fibronectin, has been observed in arthritic and oncological specimens in which matrix turnover is an important feature. In atherosclerosis, outward remodeling is characterized by matrix turnover and a structural change in arterial circumference and is associated with a vulnerable plaque phenotype. Since Tlr4 ligands are expressed during matrix turnover, we hypothesized that Tlr4 is involved in arterial remodeling. Methods and Results—In a femoral artery cuff model in the atherosclerotic ApoE3 (Leiden) transgenic mouse, Tlr4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling. With the use of the same model in wild-type mice, neointima formation and outward remodeling occurred. In Tlr4-deficient mice, however, no outward arterial remodeling was observed independent of neointima formation. Carotid artery ligation in wild-type mice resulted in outward remodeling without neointima formation in the contralateral artery. This was associated with an increase in Tlr4 expression and EDA and Hsp60 mRNA levels. In contrast, outward remodeling was not observed after carotid ligation in Tlr4-deficient mice. Conclusions—These findings provide genetic evidence that Tlr4 is involved in outward arterial remodeling, probably through upregulation of Tlr4 and Tlr4 ligands.

Matrix Metalloproteinase 2 Is Associated With Stable and Matrix Metalloproteinases 8 and 9 With Vulnerable Carotid Atherosclerotic Lesions: A Study in Human Endarterectomy Specimen Pointing to a Role for Different Extracellular Matrix Metalloproteinase Inducer Glycosylation Forms

Background and Purpose— We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. Methods— Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. Results— Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. Conclusions— MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Athero-express: differential atherosclerotic plaque expression of mRNA and protein in relation to cardiovascular events and patient characteristics. Rationale and design.

In clinical practice, biological markers are not available to routinely assess the progression of atherosclerotic disease or the development of restenosis following endarterectomy or catheter based interventions. Endarterectomy procedures provide an opportunity to study mechanisms of restenosis and progression of atherosclerotic disease since atherosclerotic tissue is obtained. Athero-Express is an ongoing prospective study, initiated in 2002, with the objective to investigate the etiological value of plaque characteristics for long term outcome. Patients are included who undergo an endarterectomy of the carotid artery. At baseline blood is withdrawn, patients fill in an extensive questionnaire and diagnostic examinations are performed. Atherosclerotic plaques are freshly harvested, immunohistochemically stained and examined for the presence of macrophages, smooth muscle cells, collagen and fat. Parts of the atherosclerotic plaques are freshly frozen to study protease activity and protein and RNA expressions. Patients undergo a duplex follow up to assess procedural restenosis (primary endpoint) at 3xa0months, 1xa0year and 2xa0years. Secondary endpoints encompass major adverse cardiovascular events. In the future, the creation of this biobank with atherosclerotic specimen will allow the design of cross-sectional and follow up studies with the objective to investigate the expression of newly discovered genes and proteins and their interaction with patients and plaque characteristics in the progression of atherosclerotic disease. Objective is to include 1000–1200 patients in 5xa0years. In January 2004, 289 patients had been included. It is expected that 250 patients will be included yearly.

The Impact of Atherosclerotic Arterial Remodeling on Percentage of Luminal Stenosis Varies Widely Within the Arterial System A Postmortem Study

Luminal stenosis can be based on large atherosclerotic plaques in compensatory enlarged segments or on relatively little plaques in shrunken segments. In the present study, the contribution of plaque formation and remodeling to luminal narrowing was compared among six types of arteries prone to symptomatic atherosclerosis. Cross-sections (n = 5195) were obtained at regular intervals from 329 arteries. For each artery, the cross-section that contained the least amount of plaque was considered to be the reference. For each cross-section, the percentage of lumen area decrease was expressed as a percentage of the lumen area at the reference site (luminal stenosis). Similarly, the area encompassed by the internal elastic lamina (IEL area) was expressed as a percentage of the IEL area at the reference site (relative IEL area). All cross-sections were categorized in three groups: relative IEL area > 105% (enlargement), 95% to 105% (no remodeling), and < 95% (shrinkage). The prevalence of enlargement (50% to 75%) was significantly higher compared with shrinkage (8% to 25%). Shrinkage was observed most frequently in the femoral arteries (25%) and infrequently in the renal arteries (8%). For all types of arteries, the relative IEL area correlated negatively with luminal stenosis (P < .001). Regression analysis of relative IEL area on luminal stenosis, however, showed significant differences in the first-order regression coefficients among artery types. On average, plaque increase was more compensated for by enlargement in the coronary, common carotid, and renal arteries compared with the arteries obtained from the lower extremities. Anatomic regional differences were observed in the impact of arterial wall remodeling on percent luminal stenosis in de novo atherosclerotic lesions.

Inflammation of the Atherosclerotic Cap and Shoulder of the Plaque Is a Common and Locally Observed Feature in Unruptured Plaques of Femoral and Coronary Arteries

-Retrospectively, plaque rupture is often colocalized with inflammation of the cap and shoulder of the atherosclerotic plaque. Local inflammation is therefore considered a potential marker for plaque vulnerability. However, high specificity of inflammation for plaque rupture is a requisite for application of inflammation markers to detect rupture-prone lesions. The objective of the present study was to investigate the prevalence and distribution (local versus general) of inflammatory cells in nonruptured atherosclerotic plaques. The cap and shoulder of the plaque were stained for the presence of macrophages and T lymphocytes in 282 and 262 cross sections obtained from 74 coronary and 50 femoral arteries, respectively. From most cases, 2 atherosclerotic arteries were studied to gain insight into the local and systemic distribution of the inflammatory process. In 45% and 41% of all cross sections, staining for macrophages was observed in the femoral and coronary arteries, respectively. Rupture of the fibrous cap was observed in 2 femoral and 3 coronary artery segments and was always colocalized with inflammatory cells. At least 1 cross section stained positively for CD68 or acid phosphatase in 84% and 71% of all femoral and coronary arteries, respectively. Only 1 femoral and 6 coronary arteries revealed a positive stain for CD68 in all investigated segments. Inflammation of the cap and shoulder of the plaque is a common feature, locally observed, in atherosclerotic femoral and coronary arteries. The high prevalence of local inflammatory responses should be considered if they are used as a diagnostic target to detect vulnerable, rupture-prone lesions.

Local Atherosclerotic Plaques Are a Source of Prognostic Biomarkers for Adverse Cardiovascular Events

Objective—Atherosclerotic cardiovascular disease is a major burden to health care. Because atherosclerosis is considered a systemic disease, we hypothesized that one single atherosclerotic plaque contains ample molecular information that predicts future cardiovascular events in all vascular territories. Methods and Results—AtheroExpress is a biobank collecting atherosclerotic lesions during surgery, with a 3-year follow-up. The composite primary outcome encompasses all cardiovascular events and interventions, eg, cardiovascular death, myocardial infarction, stroke, and endovascular interventions. A proteomics search identified osteopontin as a potential plaque biomarker. Patients undergoing carotid surgery (n=574) served as the cohort in which plaque osteopontin levels were examined in relation to their outcome during follow-up and was validated in a cohort of patients undergoing femoral endarterectomy (n=151). Comparing the highest quartile of carotid plaque osteopontin levels with quartile 1 showed a hazard ratio for the primary outcome of 3.8 (95% confidence interval, 2.6–5.9). The outcome did not change after adjustment for plaque characteristics and traditional risk factors (hazard ratio, 3.5; 95% confidence interval, 2.0–5.9). The femoral validation cohort showed a hazard ratio of 3.8 (95% confidence interval 2.0 to 7.4) comparing osteopontin levels in quartile 4 with quartile 1. Conclusion—Plaque osteopontin levels in single lesions are predictive for cardiovascular events in other vascular territories. Local atherosclerotic plaques are a source of prognostic biomarkers with a high predictive value for secondary manifestations of atherosclerotic disease.

Significance of Peptidoglycan, a Proinflammatory Bacterial Antigen in Atherosclerotic Arteries and Its Association With Vulnerable Plaques

Peptidoglycan (PG) is a major component of the cell wall of gram-positive bacteria that is abundantly present in all human mucosa. PG is a functional lipopolysaccharide analog that binds to CD14 on macrophages and induces proinflammatory cytokine production and metalloproteinases. We investigated the hypothesis that bacterial PG is present in atherosclerotic tissue. In addition, plaque phenotypes were characterized in relation to presence of PG. Immunohistology of carotid (n = 15) and femoral (n = 6) endarterectomy specimens revealed the presence of PG in the cytoplasm of cells located in plaques. PG was detected in 14 of 15 carotid arteries and 5 of 6 femoral arteries. From the 14 coronary arteries, 31 atherosclerotic segments were selected. PG was detected within 19 of 31 of these coronary segments. Western blot demonstrated the presence of the toll-like receptor (TLR-2), the co-receptor for PG, in coronary artery tissue. The number of PG-containing cells in coronary arteries was significantly higher when the histologic features of plaque vulnerability were evident. Inflammation of the cap or shoulder was observed in 11 of 19 PG-positive versus 2 of 12 PG-negative segments (p = 0.023). More than 50% of the plaque area consisted of atheroma in 7 of 19 PG-positive segments and 0 of 12 PG-negative segments (p = 0.025). Heavy smooth muscle cell staining occurred in the plaque cap and shoulder in 3 of 19 PG-positive segments versus 9 of 12 PG-negative segments. Proinflammatory bacterial PG and its co-receptor have been observed in atherosclerotic arteries, in association with the vulnerable plaque phenotype.