Gerhard Bauriedel

Role of smooth muscle cell death in advanced coronary primary lesions: implications for plaque instability.

OBJECTIVE Instability of coronary atheroma leads to the onset of acute coronary syndromes including myocardial infarction and death, as well as to the progression of the arteriosclerotic disease. As yet, the underlying factors and mechanisms causing plaque rupture are not completely understood. Since a low content of smooth muscle cells (SMCs) apparently plays a key role, the question points to the events leading to the loss of intimal SMCs. METHODS We compared coronary atherectomy specimens from 25 patients with unstable angina to those from 25 patients with stable angina. Transmission electron microscopy was used to identify intimal cell population, to detect stage and cell type of apoptosis, and to differentiate between apoptosis and necrosis. RESULTS Plaques associated with unstable angina contained more macrophages/lymphocytes and significantly less SMCs (P = 0.01), compared with stable angina plaques. Specific cell death forms, apoptosis and necrosis, were present in all coronary atheroma. As key findings, both the proportion of SMCs undergoing apoptosis and the frequency of cytoplasmic remnants of apoptotic SMCs (matrix vesicles) were significantly increased in unstable versus stable angina lesions (P = 0.002 and P = 0.002). In addition, cellular necrosis was more frequent in the first coronary atheroma group (P = 0.02). Positive correlations were found between the frequency of apoptotic cells and necrosis (r = 0.41, P = 0.04), and that of matrix vesicles and necrosis (r = 0.63, P = 0.001) only in plaques with unstable angina, but not in those with stable angina. CONCLUSIONS Our data demonstrate that high cell death due to apoptosis and necrosis is a basic in situ feature found in advanced coronary primary lesions associated with unstable angina, possibly explaining their low density of (viable) SMCs. Thus, antagonization of intimal cell death should be considered in order to stabilize the intimal plaque texture of coronary atheroma with the ultimate goal to prevent plaque rupture.

Caspase-3 and Tissue Factor Expression in Lipid-Rich Plaque Macrophages Evidence for Apoptosis as Link Between Inflammation and Atherothrombosis

Background—Macrophages associated with arterial wall lipid deposition contribute to inflammatory processes. Tissue factor (TF) has been implicated in the thrombogenicity of atherosclerotic plaques. Intimal cells undergoing apoptosis have been postulated as a source for TF. However, there is only limited knowledge of cell type, plaque component, and conditions associated with TF expression and apoptosis. We examined the hypothesis that macrophages exposed to conditions of lipid-rich plaque undergo apoptosis and express TF. Methods and Results—In human carotid (n=15) and coronary (n=6) atherosclerotic plaques, TF and caspase-3 mRNA and protein expression (evaluated by in situ hybridization and immunohistochemistry) were increased significantly in lipid-rich compared with fibrous plaque components (P <0.01) and correlated with high macrophage content (P <0.05). Double-labeling studies demonstrated colocalization of TF and active caspase-3. In hyperlipidemic mice, expression of TF and active caspase-3 was observed simultaneously and colocalized in neointimal macrophages after arterial injury. In neointima of normolipidemic animals, TF and active caspase-3 were absent after arterial injury. In monocytes cultured in the presence of oxidized LDL, strong induction and colocalization of TF and active caspase-3 were found compared with baseline (P <0.05). Both antigens were significantly decreased after cotreatment with a caspase inhibitor (P <0.05) and were absent in untreated control cells. Conclusions—The expression of TF as the primary cell-associated activator of the coagulation pathway proves to be closely related to macrophages undergoing apoptosis in conditions of lipid-rich plaque, pointing to a key role of lipid content and inflammatory cell viability in determining plaque thrombogenicity.

Apoptosis in Restenosis Versus Stable-Angina Atherosclerosis Implications for the Pathogenesis of Restenosis

Decreases in programmed cell death (apoptosis) may contribute to restenotic hyperplasia by prolonging the life span of intimal cells. Apoptotic events were compared in restenotic versus primary lesions, by using atherectomy samples from 16 restenotic and 30 primary human peripheral and coronary lesions from patients presenting with stable angina. We used transmission electron microscopy to identify apoptosis, quantify its frequency, distinguish apoptosis from necrosis, and relate these events to cellular composition. Smooth muscle cell (SMC) density was higher in restenotic versus primary lesions (P<0.0001), whereas the number of macrophages was significantly reduced (P<0.01) and the number of lymphocytes was lower, but not significantly (P=0.06). As the main finding, restenotic lesions contained fewer apoptotic cells compared with primary lesions (3% versus 13%, P=0.002), whereas no differences were found for cellular necrosis. With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to both SMCs and macrophages. The key finding of less apoptosis in restenotic versus primary lesions was in agreement with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis (2% versus 9%, P<0.001). For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). No relationship was seen between the frequency of apoptosis and the density of macrophages. In conclusion, the data of the present study indicate that a low level of apoptosis may be an important mechanism leading to restenotic intimal lesion development after interventional procedures.

The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP-1 and MMP-9 in human macrophages in vitro

The complement component C5a is formed during activation of the complement cascade and exerts chemotactic and proinflammatory effects. Macrophages, which are localized in the rupture‐prone shoulder regions of coronary plaques, are thought to play a major role in plaque destabilization and rupture through the production of matrix metalloproteinases (MMPs). When human monocyte‐derived macrophages were stimulated in vitro with C5a, MMP‐1 and MMP‐9 mRNA levels were significantly increased. Furthermore, C5a up‐regulated MMP‐1 and MMP‐9 antigens and activity, as determined by ELISA and specific activity assays. These effects were blocked by antibodies against the receptor C5aR/CD88. In addition, blocking experiments revealed that MMP‐1 expression was mediated by activation of the transcription factor AP‐1, and MMP‐9 expression was induced by activation of NF‐κB and AP‐1. Immunohistochemical analysis of human coronary plaques demonstrated the colocalization of C5a, MMP‐1, and MMP‐9 in vivo. Together, these observations indicate that activation of the complement cascade and formation of C5a may play a role in the onset of acute coronary events by induction of MMPs in atherosclerotic lesions.—Speidl, W. S., Kastl, S. P., Hutter, R., Katsaros, K. M., Kaun, C., Bauriedel, G., Maurer, G., Huber, K., Badimon, J. J., Wojta, J. The complement component C5a is present in human coronary lesions in vivo and induces the expression of MMP‐1 and MMP‐9 in human macrophages in vitro. FASEB J. 25, 35–44 (2011). www.fasebj.org

Migratory activity of human smooth muscle cells cultivated from coronary and peripheral primary and restenotic lesions removed by percutaneous atherectomy.

BackgroundThe successful cultivation of human smooth muscle cells (SMC) from coronary and peripheral atherosclerotic lesions removed by percutaneous directional atherectomy is described. Methods and ResultsSixty-seven patients in whom plaque material was obtained compose the study population. A total of 73 lesions from both coronary (n=38) and peripheral (n=35) arteries of primary (n=50) and restenotic origin (n=23) were studied. Successful cultivation was significantly (p<0.001) dependent on the quantity of plaque material submitted. Fifty-five percent of patients in whom atherectomy specimens were removed from coronary lesions yielded an adequate SMC population in comparison to 89% of those from peripheral arteries (p<0.01). Cultivation was not dependent on the age and sex of patients, lesion origin, risk factors, medications, or incidence of unstable angina. In an attempt to quantify SMC activity, migratory velocity was measured with a computer-assisted motion analysis system. SMC migratory velocity was found to be significantly (p<0.001) greater in restenotic than in primary plaque material. This finding was confirmed for both coronary and peripheral lesions. ConclusionsOur data suggest that elevated SMC migratory activity may be an important mechanism in the development of restenotic lesions.

Cell constitution and characteristics of human atherosclerotic plaques selectively removed by percutaneous atherectomy

The Simpson atherectomy device used for the recanalization of severely stenosed peripheral arteries is able to collect plaque material which can be further characterized. This study reports histological, immunohistochemical and transmission electron microscopic findings on advanced human primary atherosclerotic plaques of peripheral arteries percutaneously removed by a Simpson atherectomy catheter. Material from stenosing plaques consisted of dense connective tissue with abundant amounts of concentrically arranged elastic fibers and lamellae. This meshwork contained numerous cells, often arranged in clusters and oriented with their longer axis parallel to the direction of blood flow. The vast majority of these cells could be easily identified as vimentin-positive and desmin-negative smooth muscle cells containing lipid deposits in the perinuclear region and numerous glycogen particles. Monocytes/macrophages were observed only very infrequently. Plaque tissue contained a range of smooth muscle cell phenotypes. Most of the cells were of an intermediate phenotype, i.e. sparsely filled with myofilament bundles at the cell periphery and a high amount of organelles such as mitochondria, rough endoplasmic reticulum and Golgi cisterns. An intact lining of pieces of intimal tissue with endothelial cells was not observed. Two-dimensional gel electrophoresis of plaque tissue showed the presence of alpha-, beta- and gamma-actin isoforms with a clear predominance of the beta-isoform.

Tissue resident C reactive protein in degenerative aortic valves: correlation with serum C reactive protein concentrations and modification by statins

Objective: To assess aortic valve probes for valvar C reactive protein (CRP) presence, the relation between valvar and serum CRP, and a possible modification of CRP by statin medication. Setting: Tertiary referral centre. Patients and design: End stage, degenerative valve tissue was taken from 81 patients, 57 with non-rheumatic aortic valve stenosis (AS) and 24 with degenerative aortic valve bioprosthesis (BP). Five non-stenosed valves served as controls. Tissue from four non-implanted bioprostheses was also examined. The presence and location of CRP was analysed by use of immunostaining and morphometry. Serum CRP concentrations were measured preoperatively. Results: The majority of AS and BP valves exhibited CRP labelled cells, predominantly localised to the valvar fibrosa. The expression of CRP was much higher in BP than in AS (by a factor of 3.7, p  =  0.03). Notably, non-stenosed aortic valves and non-implanted bioprostheses did not have CRP signalling. Serum CRP was also increased with BP (by a factor of 2.5, p  =  0.02) and was significantly correlated with valvar CRP expression (r  =  0.54, p < 0.001). The main finding in patients with (n  =  26) and without statin treatment (n  =  55) was that both valvar CRP expression (p  =  0.02) and serum CRP concentrations (p  =  0.04) were lower in the statin treated group. Conclusions: CRP was found in a large series of degenerative aortic valves, more often in bioprostheses than in native cusps. Serum CRP concentrations may reflect inflammatory processes within the aortic valve. The association of statin treatment with decreases in both valvar and serum CRP concentrations may explain known pleiotropic effects of statins in patients with aortic stenosis.

Effects of vanadate in cultured rat heart muscle cells. Vanadate transport, intracellular binding and vanadate-induced changes in beating and in active cation flux.

Cultured rat heart muscle cells have been used to study uptake and intracellular binding of Na483VO4 (vanadate), as well as the influence of vanadate on beating and 86Rb+ uptake of these cells. 1. Vanadate is taken up into cultured rat heart muscle cells in an energy-independent manner by a saturable transport system (Km approximately 60 microM, V approximately 200 pmol per mg protein per min at 37 degrees C). Analysis of intracellular binding of vanadate reveals a curved Scatchard plot indicating more than one binding site. Maximal binding amounts to 3 . 10(9) molecules of vanadate per cell. 2. Vanadate exerts a positive chronotropic and inotropic effect and increases automaticity. First effects can be seen at 1 . 10(-7) M Na3VO4. Concentrations higher than 1. 10(-3) M induce toxic effects (arrhythmias, fibrillation and stand-still of the cell). 3. Vanadate-induced alterations of beating is paralleled by a vanadate-induced stimulation of (86Rb+ + K+) uptake into the cells of up to 75%. Maximal stimulation is obtained at concentrations of 1 . 10(-4)--1 . 10(-3) M vanadate. The stimulation is thought to be due to an increased activity of (Na+ + K+)-ATPase, since it can be inhibited by ouabain. This result is in contrast to in vitro experiments with purified membrane preparations of (Na+ + K+)-ATPase of different organs, where an inhibition of (Na+ + K+)-ATPase by vanadate has been found. 4. The results indicate a possible role of vanadate as an endogenous regulator of active cation flux in heart tissue.

Role of L-type calcium channels on stimulated calcium influx and on proliferative activity of human coronary smooth muscle cells.

Dihydropyridine (DHP) calcium channel blockers are widely used in treatment of coronary artery disease. To evaluate the specific role of L-type calcium channels in the antianginal and possibly antiatherosclerotic properties of DHP inhibitors, we examined the effects of a 1,4-DHP agonist and antagonist on angiotensin II (ANG II)- and serum-stimulated calcium influx and proliferation of human coronary smooth muscle cells (cSMC). Fluorometry of fura-2 was used to measure changes in free cytosolic Ca2+ concentration ([Ca2+]i) in cSMC after short- and long-term pretreatment with the calcium agonist Bay K 8644 or the antagonist nitrendipine, respectively. Proliferative activity was quantified during exponential growth in serum-supplemented medium with or without both DHPs. Short- and long-term pretreatment with Bay K 8644 increased basal [Ca2+]i significantly in resting cells and augmented ANG II- and serum-induced sustained [Ca2+]i responses. Concordantly, proliferation rate was increased. In contrast, nitrendipine had no significant effect on basal or stimulated [Ca2+]i after short-term treatment, but decreased [Ca2+]i after 24-h incubation, attenuated the plateau phase of ANG II- and serum-evoked [Ca2+]i transients, and reduced proliferative activity of these cells. The results indicate that 1,4-DHPs modulate ANG II- and serum-induced Ca2+ influx in cSMC. Thus, L-type calcium channels may contribute to [Ca2+]i transients evoked by ANG II and serum. Moreover, the modulating effects of both DHPs on proliferative activity suggest involvement of DHP-sensitive calcium channels. Calcium influx through L-type channels may be one of the mechanisms that determine responsiveness to vasoconstrictors and proliferative activity of human cSMC.

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na3VO4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10(-4)-10(-3) M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na+ influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10(-4)--10(-3) M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation--concerning beating properties, potassium uptake and cellular potassium--can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1-10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy-D-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state of vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.