Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Improved gene transfer efficiency in liver with vesicular stomatitis virus G-protein pseudotyped retrovirus after partial liver resection and thymidine kinase-ganciclovir pre-treatment.

Liver-directed gene therapy is a promising alternative for the treatment of various liver diseases. Pseudotyped (VSV-G) retroviruses can be produced in high titres which is essential to overcome the problem of low gene transfer efficiency detected previously with first generation Moloney murine (MMLV) retroviruses and plasmid vectors. We compared the lacZ gene transfer efficiency of MMLV retroviruses and VSV-G retroviruses in Watanabe heritable hyperlipidaemic rabbit liver using an intraportal administration route. Hepatocyte proliferation was stimulated by a partial (10%) liver resection and a thymidine kinase-ganciclovir treatment. We also studied the safety of the gene transfer by clinical chemistry, tissue pathology and PCR analysis of lung, kidney, spleen and gonads. Gene transfer efficiency with the VSV-G retrovirus was significantly higher than with the traditional MMLV-based retrovirus (9.5+/-5.26 vs 0.21+/-0.10 positive hepatocytes mm(-2), P<0.05). After a 12-month follow-up period no lacZ expression was detected in liver samples. No transgene was detected in plasma or in lung, kidney, spleen and gonads by PCR analysis 7 days after gene transfer. Transient increases were found in plasma c-reactive protein, aspartyl aminotransferase and alanine aminotransferase levels shortly after the operation with both types of retroviruses. VSV-G retrovirus was well tolerated and may become an efficient new tool in liver gene therapy. The absence of transgene in systemic circulation or in extrahepatic tissues including gonads is an important safety feature required for in vivo gene therapy.

Autoantibodies against oxidized LDL and endothelium-dependent vasodilation in insulin-dependent diabetes mellitus.

We determined whether autoantibodies against oxidized LDL are increased in patients with IDDM, and if so, whether they are associated with endothelial dysfunction in vivo. Autoantibodies against oxidized LDL (ratio of antibodies against oxidized vs. native LDL, oxLDLab) were determined in 38 patients with IDDM (HbA(1c) 8.4+/-0.2%), who were clinically free of macrovascular disease, and 33 healthy normolipidemic subjects (HbA(1c) 5.1+/-0.1%, P<0.001 vs. IDDM). The groups had comparable serum total-, LDL- (2. 9+/-0.1 vs. 2.8+/-0.1 mmol/l, IDDM vs. controls), and HDL-cholesterol concentrations. OxLDLab were 1.5-fold higher in the IDDM patients (1.8+/-0.1) than in the normal subjects (1.2+/-0.1, P<0.001). OxLDLab were correlated with age in normal subjects, but not with age, duration of disease, LDL-cholesterol, HbA(1c) or degree of microvascular complications in patients with IDDM. To determine whether oxLDLab are associated with endothelial dysfunction in vivo, blood flow responses to intrabrachial infusions of acetylcholine, sodium nitroprusside and L-NMMA were determined in 23 of the patients with IDDM (age 33+/-1 years, body mass index 24. 3+/-0.6 kg/m(2), HbA(1c) 8.5+/-0.3%) and in the 33 matched normal males. OxLDLab were 41% increased in IDDM (1.7+/-0.2 vs. 1.2+/-0.1, P<0.01). Within the group of IDDM patients, HbA(1c) but not oxLDLab or LDL-cholesterol, was inversely correlated with the forearm blood flow response to acetylcholine (r=-0.51, P<0.02), an endothelium-dependent vasodilator, but not to sodium nitroprusside (r=0.06, NS). These data demonstrate that oxLDLab concentrations are increased in patients with IDDM, but show that chronic hyperglycemia rather than oxLDLab, is associated with impaired endothelium-dependent vasodilation in these patients.

Expression of inducible nitric oxide synthase in macrophages and smooth muscle cells in various types of human atherosclerotic lesions.

Nitric oxide (NO) is an important regulatory agent in blood vessels. We studied the expression of inducible nitric oxide synthase (iNOS) in different types of human atherosclerotic lesions using simultaneous in situ hybridization and immunocytochemistry. Since nitric oxide and its derivates or reaction products can have both oxidative and antioxidative effects, we also studied the presence of oxidized low-density lipoproteins (ox-LDL) and peroxynitrite-modified proteins in the same lesions as indicators of oxidative damage. Twenty-seven aortic samples were studied from seven autopsies. Samples were classified microscopically as normal areas, initial lesions (type I), fatty streaks (type II), intermediate lesions (type III), atheroma (type IV), fibroatheroma lesions (type Va) and fibrotic lesions (type Vc). In normal arterial wall iNOS mRNA was expressed at a low level in smooth muscle cells (SMCs). Absence of, or a low level of, epitopes characteristic of ox-LDL was found in the normal arterial wall. The expression of iNOS mRNA and protein was induced in macrophages and SMCs in the majority of early lesions and in all advanced atherosclerotic lesions. Epitopes characteristic of ox-LDL and peroxynitrite-modified proteins tended to be colocalized in iNOS-positive lesions. We consider that iNOS and oxidative injuries may play an important part in atherogenesis.

Enhanced plasma cholesterol lowering effect of retrovirus-mediated LDL receptor gene transfer to WHHL rabbit liver after improved surgical technique and stimulation of hepatocyte proliferation by combined partial liver resection and thymidine kinase--ganciclovir treatment.

In this study we report an improved method for in vivo gene transfer to liver. Repeated injections of Moloney murine leukemia virus-derived retroviruses containing LDL receptor cDNA were given to the portal vein in combination with a 10% partial liver resection and stimulation of hepatocyte proliferation by plasmid/liposome-mediated thymidine kinase gene transfer and ganciclovir treatment. The method was used for the treatment of LDL receptor deficiency in Watanabe heritable hyperlipidemic rabbits. We demonstrate an increase in hepatocyte proliferation index by thymidine kinase and ganciclovir treatment from 0.9 to 1.35% and a maximum of 35% decrease in total plasma cholesterol level 2-3 months after the gene transfer. A 20% decline was still present after a 52-week follow-up period. A 50% decrease was also observed in plasma triglycerides. Liver function tests indicated a transient increase in plasma alkaline phosphatase level up to 12 weeks after the gene transfer. In situ PCR and RT-PCR analyses indicated that the transgene was present in periportal areas and was transcribed to mRNA 1 week after the gene transfer. Because of the relatively simple and controllable technique we suggest that repeated retrovirus injections via a portal vein catheter together with the limited partial liver resection and plasmid/liposome-mediated thymidine kinase gene transfer-ganciclovir treatment may be used to improve the results of retrovirus-mediated liver gene therapy.

Autoantibodies against oxidized low density lipoprotein in patients with angiographically verified coronary artery disease.

Oxidation of low density lipoproteins (LDL) obviously plays an important role in the pathogenesis of atherosclerosis. The purpose of the study was to determine whether antibodies against oxidized LDL are associated with coronary artery disease (CAD). We determined the serum levels of antibodies against copper-oxidized LDL by enzyme-linked immunosorbent assay in 58 patients with angiographically verified CAD and 34 controls without CAD. The mean antibody level, expressed in optical density units, was significantly higher in patients than in controls (0.150+/-0.088 versus 0.094+/-0.054, respectively; P=0.00089). In logistic regression analysis, high antibody level against oxidized LDL was associated significantly with CAD (P=0.0114), independent of age (P=0.00137), gender (P=0.0021), body mass index (P=0.5947), triglyceride concentration (P=0.9813), and total cholesterol-high density lipoprotein (HDL) cholesterol (P=0.0080) group. Similar analysis in nondiabetic subjects (n=79) and in men only (n=75) showed analogous results, with only minor changes in P values. The antibody level against oxidized LDL differed significantly between nonsmokers and smokers in CAD patients (P<0.00197) but not in controls (P=NS). In addition, the antibody level against oxidized LDL differed significantly between nonsmokers and smokers in subjects with low HDL cholesterol (0.9 mmol/L). In conclusion, elevated levels of antibodies against oxidized LDL were associated with CAD. The data suggest that oxidized LDL plays a role in the pathogenesis of atherosclerosis and suggest a protective function for HDL against LDL oxidation.

Intravascular ultrasound and magnetic resonance imaging in the assessment of atherosclerotic lesions in rabbit aorta. Correlation to histopathologic findings.

RATIONALE AND OBJECTIVES: The authors compare the usefulness of intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI) for quantitation of atherosclerosis in hyperlipidemic rabbits, correlated with histopathology. METHODS: Magnetic resonance imaging with T1- and T2-weighted spin echo sequences and three-dimensional time-of-flight MR angiography of the abdominal aorta was performed on seven rabbits using a 1.5 T MR imager and a standard head coil. X-ray angiography and IVUS examination (3.5 F/30 MHz imaging catheter) was performed via carotid artery access. RESULTS: Time-of-flight MR angiography source images provided the best resolution and plaque-lumen contrast in visual comparison between the different MRI sequences. Intra- and interobserver reproducibilities of the lesion thickness and area measurements were similar in IVUS and MRI (Pearson correlations 0.52-0.97; P < 0.01). The measurements from IVUS and MRI correlated closely with each other as well as with those made from histopathologic specimens (Pearson correlations 0.50-0.79; P < 0.001). The measurements from IVUS were somewhat more accurate than those made from MRI. CONCLUSIONS: Both MRI and IVUS with clinically available imaging equipments are feasible and accurate for the quantitation of experimental atherosclerosis of rabbit aorta.

Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia.

Arterial gene transfer offers a promising new approach for the treatment of vascular disorders. However, no data are available about the gene transfer efficiency in human arteries in vivo. The aim of this study was to evaluate the safety and feasibility of catheter-mediated adenoviral gene transfer in human peripheral arteries. Ten patients (8 females, 2 males, mean age 80 +/- 8 years) suffering from chronic critical leg ischemia with a prior decision for amputation were recruited in the study. Gene transfer was performed in eight patients in conjunction with a conventional percutaneous transluminal angioplasty, using a perfusion coil balloon catheter. Two patients served as controls. Increasing concentrations of replication-deficient adenoviruses (titers from 1 x 10(8) to 4 x 10(10) PFU) containing a nuclear-targeted beta-galactosidase marker gene were administered into the arteries over 10 min via the catheter. Amputations were performed 20 to 51 hr after the procedures and gene transfer efficiency was evaluated in the transduced arteries using X-Gal staining for beta-galactosidase activity. Beta-galactosidase gene transfer was well tolerated and no adverse tissue responses or systemic complications were observed in any of the patients. Gene transfer was successful in six of the eight patients. Gene transfer efficiency varied between 0.04 and 5.0% of all arterial cells. Transgene expression was detected in smooth muscle cells, endothelial cells, and macrophages and in tunica adventitia. However, transgene activity was not evenly distributed in the arterial wall and no transgene activity was found beneath advanced atherosclerotic lesions. The safety and feasibility of in vivo gene transfer by adenoviral vectors to human peripheral arteries were established. Although improvements are still required in gene transfer efficiency, these findings suggest that adenoviruses can be used to deliver therapeutically active genes into human arteries.

Characterization of atherosclerotic lesions in apo E3-leiden transgenic mice.

Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam cell formation and determine whether advanced atherosclerotic lesions develop in these animals. Special attention was given to the presence of oxidized lipoproteins and expression of lipoprotein receptors. Plasma cholesterol levels in apo E3-leiden mice on an atherogenic diet increased from 2 to 36 mmol/l in 4 months. At this time apo E3-leiden mice had developed lesions, which ranged from early fatty streaks in thoracic and abdominal aorta to advanced lesions in aortic arch. Early fatty streaks were entirely composed of macrophages which also expressed scavenger receptors. Epitopes characteristic of oxidized LDL were present in macrophage-rich foam cells. Advanced atherosclerotic lesions also developed in apo E3-leiden mice including smooth muscle cell cap formation and erosion of the media. Macrophages and epitopes characteristic of oxidized LDL were present in core and shoulder regions. Scavenger receptors were expressed in macrophages in advanced lesions, whereas LDL-receptor-related protein (LRP) was mainly expressed in smooth muscle cells. It is concluded that: (1) macrophages are the major cell type in both early and advanced atherosclerotic lesions; (2) scavenger receptors and oxidized lipoproteins are present in lesion macrophages; and (3) LRP is mostly expressed in smooth muscle cells. Thus, lesions in apo E3-leiden transgenic mice have features in common with human atherosclerosis. Since lesion macrophages also retain their ability to synthesize endogenous apo E, apo E3-leiden transgenic mouse may be a useful model for studies on the development and genetics of atherosclerosis.

Expression of LDL receptor, VLDL receptor, LDL receptor-related protein, and scavenger receptor in rabbit atherosclerotic lesions: marked induction of scavenger receptor and VLDL receptor expression during lesion development.

BACKGROUND: Atherosclerotic lesions contain foam cells that arise from monocyte-macrophages and smooth muscle cells (SMCs) by excessive uptake of lipoproteins. There are many candidate receptors for the lipid accumulation, such as LDL receptor (LDLR), VLDL receptor (VLDLR), LDL receptor-related protein (LRP), and scavenger receptors (SRs). However, little quantitative information exists on the expression of these receptors in normal and atherosclerotic arteries. METHODS AND RESULTS: Competitive reverse transcription-polymerase chain reaction and in situ hybridization were used for the studies in New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbit aortic intima-medias. NZW rabbits were fed a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. LDLR mRNA expression was low in aortic intima-medias of all groups. Of the analyzed receptors, LRP had the highest expression in the control group, and its mRNA was induced threefold in the 14-week group, the aortas of which had extensive lesions. SR expression was low and VLDLR expression moderate in the control group. Both receptors were highly induced during cholesterol feeding (SRs, 3-fold and 270-fold induction; VLDLR, 15-fold and 100-fold induction in the 3-week and 14-week groups, respectively). Comparable results were obtained from WHHL rabbits: high basal LRP mRNA in normal intima-medias; moderate induction of LRP and marked induction of SRs and VLDLR in fatty streaks and fatty plaques. In situ hybridization indicated that LRP and VLDLR were expressed in SMCs and macrophages. VLDLR expression was also observed in endothelial cells. SR expression was detected only in macrophages. CONCLUSIONS: SR and VLDLR mRNAs were highly induced in atherosclerotic lesions. VLDLR and LRP may be involved in the formation of both SMC-and macrophage-derived foam cells, whereas SRs play an important role in lipid uptake in macrophages.

Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins.

Oxidative processes play an important role in atherogenesis. Because superoxide anion and nitric oxide (NO) are important mediators in vascular pathology, we studied the expression of extracellular superoxide dismutase (EC-SOD) and inducible nitric oxide synthase (iNOS) in human and rabbit atherosclerotic lesions by using simultaneous in situ hybridization and immunocytochemistry and EC-SOD enzyme activity measurements. We also analyzed the presence in the arterial wall of oxidized lipoproteins and peroxynitrite-modified proteins as indicators of oxidative damage and possible mediators in vascular pathology. EC-SOD and iNOS mRNA and protein were expressed in smooth muscle cells and macrophages in early and advanced lesions. The expression of both enzymes was especially prominent in macrophages. As measured by enzyme activity, EC-SOD was the major SOD isoenzyme in the arterial wall. EC-SOD activity was higher in highly cellular rabbit lesions but lower in advanced, connective tissue-rich human lesions. Despite the abundant expression of EC-SOD, malondialdehyde-lysine and hydroxynonenal-lysine epitopes characteristic of oxidized lipoproteins and nitrotyrosine residues characteristic of peroxynitrite-modified proteins were detected in iNOS-positive, macrophage-rich lesions, thus implying that malondialdehyde, hydroxynonenal, and peroxynitrite are important mediators of oxidative damage. We conclude that EC-SOD, iNOS, and the balance between NO and superoxide anion play important roles in atherogenesis. EC-SOD and iNOS are highly expressed in lesion macrophages. High EC-SOD expression in the arterial wall may be required not only to prevent deleterious effects of superoxide anion but also to preserve NO activity and prevent peroxynitrite formation. Modulation of arterial EC-SOD and iNOS activities could provide means to protect arteries against atherosclerotic vascular disease.