Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

[Figure: see text].

Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation.

The molecular mechanisms predisposing to atherosclerotic aneurysm formation remain undefined. Nevertheless, rupture of aortic aneurysms is a major cause of death in Western societies, with few available treatments and poor long-term prognosis. Indirect evidence suggests that matrix metalloproteinases (MMPs) and plasminogen activators (PAs) are involved in its pathogenesis. MMPs are secreted as inactive zymogens (pro-MMPs), requiring activation in the extracellular compartment. Plasmin, generated from the zymogen plasminogen by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA; refs 14,15), has been proposed as a possible activator in vitro, but evidence for such a role in vivo is lacking. Analysis of atherosclerotic aorta in mice with a deficiency of apoliprotein E (Apoe-/-; ref. 18), singly or combined with a deficiency of t-PA (Apoe-/-:Plat-/-) or of u-PA (Apoe-/-:Plau-/-; ref. 19), indicated that deficiency of u-PA protected against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. This genetic evidence suggests that plasmin is a pathophysiologically significant activator of pro-MMPs in vivo and may have implications for the design of therapeutic strategies to prevent aortic-wall destruction by controlling Plau gene function.

11beta-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice.

The enzyme 11beta-hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11beta-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11beta-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11beta-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11beta-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase.

A subset of patients with high plasma HDL concentrations have enhanced rather than reduced atherosclerosis. We have developed a new transgenic mouse model overexpressing human lecithin-cholesteryl acyltransferase (LCAT) that has elevated HDL and increased diet-induced atherosclerosis. LCAT transgenic mouse HDLs are abnormal in both composition and function. Liver uptake of [3H]cholesteryl ether incorporated in transgenic mouse HDL was reduced by 41% compared with control HDL, indicating ineffective transport of HDL-cholesterol to the liver and impaired reverse cholesterol transport. Analysis of this LCAT-transgenic mouse model provides in vivo evidence for dysfunctional HDL as a potential mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels.

Involvement of 15-lipoxygenase in early stages of atherogenesis.

The arachidonate 15-lipoxygenase which is expressed in atherosclerotic lesions is implicated in the oxidative modification of low density lipoproteins during atherogenesis. To obtain experimental in vivo evidence for this hypothesis, we analyzed the structure of oxygenated lipids isolated from the aorta of rabbits fed with a cholesterol-rich diet for different time periods and compared the pattern of oxygenation products with that isolated from low density lipoproteins treated in vitro with the pure rabbit 15-lipoxygenase and with oxygenated lipids isolated from advanced human atherosclerotic lesions. In early atherosclerotic lesions (12-wk cholesterol feeding), specific lipoxygenase products were detected whose structure was similar to those isolated from lipoxygenase-treated low density lipoproteins. The appearance of these products did coincide with the lipid deposition in the vessel wall. In later stages of atherogenesis (26-wk cholesterol feeding) the degree of oxidative modification of the tissue lipids did increase but the share of specific lipoxygenase products was significantly lower, suggesting an increasing overlay of the specific lipoxygenase products by nonenzymatic lipid peroxidation. In advanced human atherosclerotic lesions, large amounts of oxygenation products were detected whose structure suggests a nonenzymatic origin. These data suggest that the arachidonate 15-lipoxygenase is of pathophysiological importance during the early stages of atherogenesis. In later stages of plaque development nonenzymatic lipid peroxidation becomes more relevant.

The evolving role of C-reactive protein in atherothrombosis.

BACKGROUND: Inflammation is pivotal in all phases of atherosclerosis. Among the numerous inflammatory biomarkers, the largest amount of published data supports a role for C-reactive protein (CRP) as a robust and independent risk marker in the prediction of primary and secondary adverse cardiovascular events. In addition to being a risk marker, there is much evidence indicating that CRP may indeed participate in atherogenesis. CONTENT: In this review, we focus on the role of CRP in promoting atherothrombosis by discussing its effects on endothelial cells, endothelial progenitor cells, monocyte-macrophages, and smooth muscle cells. CONCLUSIONS: CRP is clearly a risk marker for cardiovascular disease and is recommended for use in primary prevention. In addition, CRP appears also to contribute to atherogenesis. However, much further research is needed, especially in appropriate animal models, to confirm the possible role of CRP in promoting atherothrombosis.

Clonal characteristics of experimentally induced "atherosclerotic" lesions in the hybrid hare.

The female hybrid hare (Lepus timidus x Lepus europaeus) is heterozygous for electrophoretically separable, X-linked isoenzymes of glucose-6-phosphate dehydrogenase. The isoenzymes of this animal have been used as cellular markers in the study of the clonal origins of experimentally induced atherosclerotic lesions. Aortic lesions produced in the hybrid hare by feeding cholesterol and injuring the aortic wall with a catheter have been shown to have polyclonal characteristics and in this way are fundamentally different from atherosclerotic fibrous plaques in man.

Elevation of aortic proline hydroxylase: a biochemical defect in experimental arteriosclerosis.

The relation of collagen synthesis to experimentally induced arteriosclerosis was studied by measuring proline hydroxylase activity. Gross aortic plaques were produced in rabbits by daily injection of epinephrine (intravenous) and thyroxine (intraperitoneal) for 4, 9, or 14 days. Activity of proline hydroxylase was significantly increased after 4 days of treatment and reached a peak, five- to sixfold increase, after 14 days of treatment. The increase in enzyme activity was correlated with the severity of observed arteriosclerosis. Increase in proline hydroxylase activity may be a possible biochemical defect in the aortas of rabbits with arteriosclerosis induced by injury.

Decreased obesity and atherosclerosis in human paraoxonase 3 transgenic mice.

Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Secretory phospholipase A2: a multifaceted family of proatherogenic enzymes.

Secretory phospholipase A(2) (sPLA(2)) represents a class of enzymes that hydrolyze phospholipids from cellular membranes and lipoproteins, resulting in multifarious proatherogenic actions in the vessel wall. Proatherogenic actions of sPLA(2) involve lipoprotein remodeling that facilitates proteoglycan binding and formation of lipid aggregates that are rapidly internalized by tissue macrophages. The hydrolysis of phospholipids on cell membranes generates bioactive lipids and lipolipoproteins with increased oxidative susceptibility. These particles and other bioactive lipids activate inflammatory pathways in various cells of the vessel wall. Transgenic mice overexpressing groups IIA, V, and X have increased atherosclerosis formation, whereas mice deficient in these sPLA(2) isoenzymes have less atherosclerosis formation. In apolipoprotein E knockout mice fed an atherosclerotic diet, sPLA(2) inhibition with varespladib reduced atherosclerosis formation. The potential for sPLA(2) inhibitors for preventing cardiovascular events is being investigated.

[aaRS--the etiological factor and the attractive target of many disorders]. / Znaczenie syntetaz aminoacylo-tRNA w patologiach i perspektywa ich wykorzystania w diagnostyce i terapii.

Aminoacyl-tRNA synthetases (aaRS) are essential proteins of all living organisms. It is known that they ensure the fidelity of transfer of genetic information from the DNA into the protein. Not far away it occurred that their role is not confined to catalyze the attachment of amino acids to transfer RNAs and thereby establish the rules of genetic code by virtue of matching the nucleotide triplet of anticodon with cognate amino acid. aaRSs are also engaged in the other crucial cellular processes. So the disturbance of function of any of them often causes serious disorders. Therefore this proteins could be an attractive target of drugs, not only against the mentioned illnesses but also against bacterial, fungal and parasitic infections. Constant progress on this field makes aaRSs still an interesting object of researches.

Association between a leukotriene C4 synthase gene promoter polymorphism and coronary artery calcium in young women: the Muscatine Study.

OBJECTIVE: A majority of the recognized risk factors for atherosclerosis and the development of cardiovascular disease have been derived from the study of older populations who have already manifested clinical symptoms. If risk factors can be identified earlier in life, such as genetic variation, preventive measures may be taken before overt symptoms of pathology have manifested, and when treatments may be most effective. METHODS AND RESULTS: In an effort to identify individuals at increased risk for cardiovascular disease, we genotyped 732 members of the Muscatine Study Longitudinal Adult Cohort for candidate genetic markers associated with several pathogenetic processes. We identified age-adjusted increased risks for coronary artery calcium (OR 4.29; 95% CI 1.78, 10.31) and increased mean carotid artery intimal-medial thickness associated with the (-444)A>C promoter polymorphism of Leukotriene C4 Synthase (LTC4S) in women. There were no similar associations in men. CONCLUSIONS: LTC4S plays a key role in the process of inflammation as the rate limiting enzyme in the conversion of arachidonic acid to cysteinyl-leukotrienes, important mediators of inflammatory responses. The (-444)C variant upregulates LTC4S mRNA expression, increasing the synthesis of proinflammatory leukotrienes. Our results support genetic variation modifying inflammatory pathways as an important mechanism in the development of atherosclerosis.

Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.

Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.

Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques.

Lipoprotein lipase (LPL), hydrolyzes the core triglycerides of lipoproteins, thereby playing a role in their maturation. LPL may be important in the metabolic pathways that lead to atherosclerosis, since it is secreted in vitro by both of the predominant cell types of the atherosclerotic plaque, i.e., macrophages and smooth muscle cells. Because of uncertainty concerning the primary cellular source of LPL in atherosclerotic lesions, in situ hybridization assays for LPL mRNA were performed on 12 coronary arteries obtained from six cardiac allograft recipients. Macrophages and smooth muscle cells were identified on adjacent sections with cell-specific antibodies and foam cells were identified morphologically. LPL protein was localized using a polyclonal antibody. LPL mRNA was produced by a proportion of plaque macrophages, particularly macrophage-derived foam cells, but was not detected in association with any intimal or medial smooth muscle cells. These findings were confirmed by combined immunocytochemistry and in situ hybridization on the same tissue sections. LPL protein was detected in association with macrophage-derived foam cells, endothelial cells, adventitial adipocytes, and medial smooth muscle cells, and, to a lesser extent, in intimal smooth muscle cells and media underlying well-developed plaque. These results indicate that macrophage-derived foam cells are the primary source of LPL in atherosclerotic plaques and are consistent with a role for LPL in the pathogenesis of atherosclerosis.

Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages.

The presence of oxysterols in macrophages isolated from atherosclerotic tissue and the effect of oxysterols on the regulation of lipoprotein lipase (LPL) mRNA were studied. Both rabbit and human macrophages, freshly isolated from atherosclerotic aorta, show about the same distribution of oxysterols, analyzed by isotope dilution mass spectrometry, except that all three preparations of human arterial-derived macrophages contained high levels of 27-hydroxycholesterol, which was not found in rabbit macrophages. To determine if oxysterols regulate LPL expression, human monocyte-derived macrophages were incubated with different oxysterols. Incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol resulted in a 70-75% reduction of LPL mRNA, analyzed by quantitative RT-PCR. Cholesterol and other tested oxysterols showed no effect on macrophage LPL mRNA expression compared with control. LPL activity in the medium was also reduced after exposure of the macrophages to 7 beta-hydroxycholesterol and 25-hydroxycholesterol. In conclusion, we have demonstrated accumulation of oxysterols in macrophage-derived foam cells isolated from atherosclerotic aorta. There was suppression of LPL mRNA in human monocyte-derived macrophages after incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol. It is tempting to suggest that an exposure to oxysterols may explain our earlier observation of a low level of LPL mRNA in arterial foam cells.

Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.

Aggregation and retention of LDL in the arterial wall are key events in atherogenesis, but the mechanisms in vivo are not known. Previous work from our laboratories has shown that exposure of LDL to bacterial sphingomyelinase (SMase) in vitro leads to the formation of LDL aggregates that can be retained by extracellular matrix and that are able to stimulate macrophage foam cell formation. We now provide evidence that retained LDL is hydrolyzed by an arterial-wall SMase activity. First, we demonstrated that SMase-induced aggregation is caused by an increase in particle ceramide content, even in the presence of excess sphingomyelin (SM). This finding is compatible with previous data showing that lesional LDL is enriched in SM, though its ceramide content has not previously been reported. To address this critical compositional issue, the ceramide content of lesional LDL was assayed and, remarkably, found to be 10-50-fold enriched compared with plasma LDL ceramide. Furthermore, the ceramide was found exclusively in lesional LDL that was aggregated; unaggregated lesional LDL, which accounted for 20-25% of the lesional material, remained ceramide poor. When [3H]SM-LDL was incubated with strips of rabbit aorta ex vivo, a portion of the LDL was retained, and the [3H]SM of this portion, but not that of unretained LDL, was hydrolyzed to [3H]ceramide by a nonlysosomal arterial hydrolase. In summary, LDL retained in atherosclerotic lesions is acted upon by an arterial-wall SMase, which may participate in LDL aggregation and possibly other SMase-mediated processes during atherogenesis.

Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms.

Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAA), a disorder characterized by chronic aortic wall inflammation and destruction of medial elastin. The purpose of this study was to determine if human macrophage elastase (HME; MMP-12) might participate in this disease. By reverse transcription-polymerase chain reaction, HME mRNA was consistently demonstrated in AAA and atherosclerotic occlusive disease (AOD) tissues (six of six), but in only one of six normal aortas. Immunoreactive proteins corresponding to proHME and two products of extracellular processing were present in seven of seven AAA tissue extracts. Total HME recovered from AAA tissue was sevenfold greater than normal aorta (P < 0.001), and the extracted enzyme exhibited activity in vitro. Production of HME was demonstrated in the media of AAA tissues by in situ hybridization and immunohistochemistry, but HME was not detected within the media of normal or AOD specimens. Importantly, immunoreactive HME was specifically localized to residual elastin fragments within the media of AAA tissue, particularly areas adjacent to nondilated normal aorta. In vitro, the fraction of MMP-12 sequestered by insoluble elastin was two- to fivefold greater than other elastases found in AAA tissue. Therefore, HME is prominently expressed by aneurysm-infiltrating macrophages within the degenerating aortic media of AAA, where it is also bound to residual elastic fiber fragments. Because elastin represents a critical component of aortic wall structure and a matrix substrate for metalloelastases, HME may have a direct and singular role in the pathogenesis of aortic aneurysms.

Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice.

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice.

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.