Inhibition of accelerated atherosclerosis in vein grafts by placement of external stent in apoE*3-Leiden transgenic mice.

OBJECTIVE: Vein grafts fail because of the development of intimal hyperplasia and accelerated atherosclerosis. Placement of an external stent around vein grafts resulted in an inhibition of intimal hyperplasia in several animal studies. Here, we assess the effects of external stenting on accelerated atherosclerosis in early vein grafts in carotid arteries in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice. METHODS AND RESULTS: Venous interposition grafting was performed in apolipoprotein E*3-Leiden mice fed standard chow or a highly cholesterol-rich diet for 4 weeks. After engraftment, external stents with different inner diameters (0.4 or 0.8 mm) were placed. In unstented vein grafts in hypercholesterolemic mice, thickening up to 50 times the original thickness, with foam cell-rich lesions, calcification, and necrosis, was observed within 28 days. The atherosclerotic lesions observed show high morphological resemblance to atherosclerotic lesions observed in human vein grafts. In stented vein grafts in hypercholesterolemic mice, no foam cell accumulation or accelerated atherosclerosis was observed. Compared with unstented vein grafts, stenting of vein grafts in a hypercholesterolemic environment resulted in a 94% reduction of vessel wall thickening. These effects were independent of stent size. CONCLUSIONS: Extravascular stent placement results in strong inhibition of accelerated vein graft atherosclerosis in hypercholesterolemic transgenic mice and thereby provides a perspective for therapeutic intervention in vein graft diseases.

Apoptotic versus autophagic cell death in heart failure.

OBJECTIVE: Progressive loss of cardiomyocytes is one of the most important pathogenic characteristics of heart failure. Apoptosis may be an important mode of cell death in heart failure but it must be demonstrated by multiple criteria and not just TUNEL staining alone. Previously, we and others have demonstrated that besides apoptosis other phenomena like active gene transcription can result in TUNEL positivity. Moreover, other types of cell death that are caspase-independent could be important in heart failure. This study examined the hypothesis whether TUNEL labeling parallels caspase activation. METHODS: Cardiac tissue of patients in the terminal stage of heart failure as a consequence of ischaemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) were studied. Embryonic mice hearts were used for positive control for detection of the classical apoptosis. RESULTS: In mice embryonic hearts we could clearly find apoptotic cell death detected by TUNEL labeling and immunohistochemistry for activated caspase-3. In heart failure, TUNEL-positive cardiomyocytes were negative for active caspase-3 but showed signs of active gene transcription (SC-35). However, autophagic cell death could be found in 0.3% of the cardiomyocytes. Autophagic cell death was demonstrated by granular cytoplasmic ubiquitin inclusions, an established marker of autophagocytosis in neurons. Interestingly, these autophagic cardiomyocytes were TUNEL and activated caspase-3 negative but were also negative for C9, a marker for necrosis. Western blot analysis confirmed that in cardiomyopathies no cleavage of caspase-3 and caspase-7 occurred. CONCLUSION: The present study demonstrates two fundamentally different situations of cell death in cardiac tissue. In embryonic mice, cardiomyocytes undergo caspase-dependent cell death. However, cardiomyocytes in heart failure show caspase-independent autophagic cell death rather than apoptotic cell death.

Oxidative DNA damage and repair in experimental atherosclerosis are reversed by dietary lipid lowering.

Increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis. The oxidative environment is mainly created by the production of reactive oxygen species, which are assumed to mediate vascular tissue injury. Oxidative DNA damage resulting from free radical attack remains, however, a poorly examined field in atherosclerosis. Male New Zealand White rabbits were fed a cholesterol-rich diet (0.3%) for 24 weeks. The induced atherosclerotic plaques showed elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine (8-oxoG) as demonstrated by immunohistochemistry. 8-oxoG immunoreactivity was found predominantly in the superficial layer of the plaque containing numerous macrophage-derived foam cells but not in the media or in arteries of age-matched control animals. Alkaline single-cell gel electrophoresis revealed that the number of DNA strand breaks was significantly higher in the plaque as compared with control samples of normolipemic animals. These changes were associated with the upregulation of DNA repair enzymes (poly[ADP-ribose] polymerase-1, p53, phospho-p53 [phosphorylated at Ser392], and XRCC1 [x-ray repair cross-complementing 1]). DNA strand breaks normalized after 4 weeks of dietary lipid lowering. However, a significant reduction of 8-oxoG immunoreactivity was only observed after a prolonged period of lipid lowering (12 to 24 weeks). Repair pathways started to decline progressively when cholesterol-fed animals were placed on a normal diet. In conclusion, oxidative DNA damage and increased levels of DNA repair, both associated with diet-induced hypercholesterolemia, are strongly reduced during dietary lipid lowering. These findings may provide a better insight into the benefits of lipid-lowering therapy on plaque stabilization.

The role of apoptosis in vascular disease.

Normal arteries are characterized by a low turnover of endothelial (EC) and smooth muscle cells (SMC). Different mechanisms protect the EC and SMC against apoptosis in the normal artery. In hypertension, SMC replication is increased but this is not counterbalanced by increased apoptosis, resulting in thickening of the media of arteries and arterioles. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and SMC undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. SMC apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in the early stages of atherosclerosis SMC become susceptible to apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that SMC may be killed by activated macrophages. The loss of the SMC can be detrimental for plaque stability since most of the interstitial collagen fibres, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies were removed. Apoptotic cells that are not scavenged in the plaque activate thrombin, which could further induce intraplaque thrombosis. It can be concluded that apoptosis in primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreased after lipid lowering which could be important in the understanding of the cell biology of plaque stabilization.

Vascular remodeling in varicose veins.

The present study describes the histopathologic aspects of varicose (n=29; mean age, 52 +/- 12 years) and normal saphenous veins (n=17; mean age, 51 +/- 12 years) of patients from a similar age group. We focused on the changes that occur in the circular layer of the venous wall. We examined the venous walls by light microscopy and transmission electronmicroscopy. A semiquantitative grading system was used to assess the smooth muscle cell (SMC) hypertrophy and the change that occurs in the elastin pattern. The volume densities (Vv) of SMC and collagen were measured as well as the diameter of the SMC, and the nuclei of SMC per fixed area were counted. The varicose vein wall differed from the normal saphenous vein by the presence of hypertrophic SMC as well as disorganized elastin patterns. A correlation between the hypertrophic SMC and an abnormal elastin pattern was observed (r=0.658, p<0.001). Ultrastructurally, the SMC show prominent microherniations and vesicles that bud from the cell. These vesicles contain microfilaments and microtubuli, although no other organelles could be detected. The elastin fibers are disrupted from the hypertrophic SMC. No significant difference could be detected in both the Vv of SMC and the Vv of collagen. The diameter of the SMC in the varicose vein (d=9.45 +/- 1.22 microm) differs significantly from that in the normal saphenous vein (d=6.22 +/- 1.47 microm) (p<0.001). Also, the nuclei of SMC per fixed area differs significantly between the varicose (87 +/- 18) and nonvaricose (117 +/- 24) veins (p<0.001). We conclude that the cellular hypertrophy of the SMC and the microherniations could be the basis for disruption of the elastin fibers connected to the SMC in varicose veins. Disrupted connections between SMC and elastin fibers could in turn induce the weakness of the venous wall observed in varicose vein disease.

Cell composition, replication, and apoptosis in atherosclerotic plaques after 6 months of cholesterol withdrawal.

Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8+/-0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.

RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis.

The detection of DNA fragmentation by the use of the TUNEL technique has become a standard technique for the detection of apoptosis in tissue sections. DNA cleavage, detected by the TUNEL technique, is the last irreversible stage of the apoptosis cascade. When the nuclear DNA is cleaved in oligonucleosomal-sized fragments, processes such as transcription are completely abolished. The values of apoptotic cell death that were obtained for atherosclerotic plaques by the TUNEL technique ranged from less than 2% up to 60%. The latter value would indicate that plaques are in an imminent state of collapse, which is certainly not the case. Other factors that could result in TUNEL labeling have to be considered. Therefore, we developed a co-localization system and studied TUNEL labeling together with markers of RNA transcription and splicing. The present study demonstrates that, besides apoptotic nuclei, non-apoptotic nuclei that show signs of active gene transcription are labeled by the TUNEL technique. The fact that the TUNEL technique is not specific for the executive phase of apoptosis is not surprising, as the technique is only selective (rather than specific) for apoptotic nuclei as these contain a far greater degree of DNA fragmentation than non-apoptotic nuclei. Therefore, we think that the TUNEL technique should be combined with additional techniques, such as markers of transcription and morphological criteria.

Ultrastructural changes of the myocardium in the embryonic rat heart.

BACKGROUND: Ultrastructural changes of the embryonic heart have been described, and quantitative studies have reported the changes of cellular organelles in late fetal and postnatal development. However, no specific data are available on the quantitative morphology of the individual segments and intersegmental junctions of the early embryonic heart, although these components must have different functions. METHODS: We measured the absolute volumes of glycogen, Golgi complex, myofibrils, mitochondria, and the surface areas of the rough endoplasmic reticulum and mitochondrial cristae in the different regions of the embryonic rat heart by using stereological tools. RESULTS: During embryonic development, the cardiac segments and intersegmental junctions increase their glycogen volume. The sinoatrial junction and primary fold show a more rapid increase than all the other cardiac regions, whereas the atrioventricular canal shows a high level of glycogen content throughout the period studied. The Golgi complex and rough endoplasmic reticulum show a conspicuous decrease from day 15 onward. The cellular content of myofibrils and mitochondria and the surface area of the mitochondrial cristae show a gradual increase from day 11 to day 17 of development, but full maturation apparently takes place in late fetal and early postnatal stages. At day 15 of development, the cellular volumes of myofibrils and mitochondria show a temporary decrease. CONCLUSIONS: The glycogen content cannot be explained on the basis of metabolism alone. The storage of glycogen is hypothesized to serve mechanical cell stability and may also be related to a target mechanism for ingrowing nerves. Myofibrillar and mitochondrial contents of the myocytes indicate a relatively late differentiation of the venous pole of the heart. Uninterrupted maturation is only started at the time of septation.

Development of myocardial fiber organization in the rat heart.

Confocal laser-scanning microscopy of phalloidine-stained actin fibers is a relatively new tool for studying the development of myocardial fiber organization. It seems to show orientation of myocytes in rather early embryonic stages. To further evaluate the differentiation of the myocardium, this technique was compared with transmission electron microscopy in rat embryos aged between 11 and 18 days. Although the confocal images of actin filament patterns pointed to early myocyte orientation, the electron micrographs revealed that even at 17 days the ventricular myocardium was far from mature. Myofibrils never completely filled the myocytes, and lack of organization was the rule rather than the exception. The organized structure as revealed by confocal microscopy was based on cell-to-cell continuity, whereas electron microscopy showed crossing and disarray within individual myocytes. Exceptions were in the ventricular trabeculations, which showed precocious myofiber differentiation. The trabeculations probably support ventricular systole in those stages in which the free walls do not yet provide efficient contractions. The other exception was the wall of the outflow tract, which showed well-oriented myofibrils from early stages onwards. Apparently, the outflow tract has a different function in these stages. The differences found between confocal microscopy and electron microscopy suggest that some caution is indicated in the interpretation of fluorescent images of relatively low magnification.

Nuclear and cellular size of myocytes in different segments of the developing rat heart.

BACKGROUND: In the embryonic heart, the individual cardiac segments show different growth rates. For the analysis of changing form in relation with changing function, data on number and shape of cardiomyocytes are necessary. Such data will give insight into the process of hypertrophy and/or hyperplasia as they may take place in the myocardium in the embryonic period. METHODS: We have measured the volumes of the nuclei and myocytes as well as the surface areas of the nuclear envelope and cellular membrane using stereological tools in rat embryos from 11 days postcoitum to 17 days postcoitum. From the data of the cellular volume of the myocytes and the myocardial volume of the individual segments, we have calculated the total number of myocytes during the developmental period. RESULTS: It is shown that the sinus venosus, sinu-atrial junction, and atrium increase their cellular volume during development, whereas the other cardiac segments show no difference in cellular volume. Similarly, the surface area of the cell membrane of the sinus venosus and sinu-atrial junction had increased during development. The nuclear volume and the surface area of the nuclear envelope did not differ during the period studied. The total number of myocytes showed a conspicuously smaller increase in the atrio-ventricular canal and distal outlet segment than in the other segments. CONCLUSIONS: The increase of the cellular volume in the segments sinus venosus and sinu-atrial junction seems to be due to a late differentiation process. In general, however, the increase of the myocardial volume in the individual cardiac segments is caused by hyperplasia of the cardiomyocytes in these segments and not by hypertrophy. The surface area of cells has a fixed relationship with cell volume, indicating that no important changes take place in the developmental period studied.

Growth of the myocardial volumes of the individual cardiac segments in the rat embryo.

BACKGROUND: Although the growth of the developing heart in relation to an increase of ventricular systolic pressure and the growth of the entire embryo during development has been described, no data are available on the growth of the individual segments and intersegmental junctions. Because these different portions are known to function differently, the need for data on their individual development is obvious. METHODS: We have measured the volumes of these different compartments by Cavalieri's point counting method in rat embryos from 11 to 17 days. RESULTS: It is shown that sinus venosus and sinu-atrial junction as well as the main compartments atrium, inlet, and proximal outlet segment grow roughly proportional to the total myocardial volume. Atrio-ventricular canal and distal outlet segment show a restricted growth and their proportional volumes decrease in time. The inlet segment is the most important part of the ventricular mass at 11 days of gestation, when it is still larger than the proximal outlet segment and, thus, takes the greater part in systolic action of the ventricular mass. The growth of the primary fold increases from day 13 onwards and can be considered as part of the wall of the inlet segment which gives rise to the main part of the ventricular septum. CONCLUSIONS: The timing of the septal volume increase fits with qualitative descriptions of ventricular septation. The atrio-ventricular canal and distal outlet segment have an important constrictive function in early stages, when valves are not yet present. Slow conduction and contraction patterns have been reported to be a characteristic feature of these portions of the embryonic heart. With development of valves these segments are loosing their mechanical function and, thus, their proportional volume declines.