Background. Assessment of heart rate, pre- and afterload and load-independent cardiac contractility is important for a meaningful interpretation of pathophysiological changes in animal models of cardiovascular disease. Arterial elastance (Ea) is a frequently used parameter for afterload, though it is unclear whether it correlates with important clinical features, such as atherosclerosis. Mice with combined leptin and low-density receptor deficiency (DKO) show spontaneous age-dependent atherosclerotic plaque-formation (from only very small fatty streaks at 12 weeks of age, to massive lesions at 36 weeks of age).

OBJECTIVEWe investigated the effect of a dual peroxisome proliferator-activated receptor (PPAR)alpha,gamma-agonist on atherosclerosis and cardiac function in mice with combined leptin and low-density lipoprotein receptor deficiency (DKO). In these mice, obesity, diabetes, and hyperlipidemia are associated with accelerated atherosclerosis and loss of cardiac function.METHODS AND RESULTSWe treated 12-week-old DKO mice with the PPARalpha,gamma-agonist (S)-3-(4-(2-carbazol-9-yl-ethoxy) phenyl-2-ethoxy-propionic-acid) for 12 weeks. The agonist lowered free fatty acids with 42% and increased insulin sensitivity with 76%. It had no effect on plasma cholesterol and triglycerides. RT-PCR analysis showed that the agonist increased the expression of fatty acid transport protein-4, fatty acid binding protein-4, glucose transporter-4, hormone-sensitive lipase, and adiponectin in white adipose tissue that was associated with the increase in insulin sensitivity. At 24 weeks, the shortening fraction (SF) of placebo DKO mice was 30% lower than that of C57BL6 mice. The PPAR agonist increased PPARgamma but not PPARalpha expression in the heart and prevented loss of left ventricular function. Adiponectin correlated positively with PPARgamma in the heart and with SF. The agonist had no effect on atherosclerosis in the aortic arch of DKO mice.CONCLUSIONSThe dual PPARalpha,gamma-agonist improved insulin sensitivity without affecting cholesterol and triglycerides. This was associated with induction of PPARgamma in the heart and prevention of loss of left ventricle function.

Objective: To study the effect of dietary cholesterol withdrawal on size and composition of LDL-hypercholesterolemia-induced coronary plaques in miniature pigs. Methods: Pigs were on normal chow (control group), on a cholesterol-rich diet for 37 weeks (hypercholesterolemic group) or on a cholesterol-rich diet followed by normal chow for 26 weeks (cholesterol withdrawal group). Endothelial function was assessed with quantitative angiography after intracoronary infusion of acetylcholine, plaque load with intra-coronary ultrasound and plaque composition with image analysis of cross-sections. The effect of porcine serum on coronary smooth muscle cell (SMC) function was studied in vitro. Results: Cholesterol-rich diet caused LDL-hypercholesterolemia, increased plasma levels of oxidized LDL (ox-LDL) and C-reactive protein (CRP), and induced endothelial dysfunction and coronary atherosclerosis. Dietary cholesterol withdrawal lowered LDL, ox-LDL and CRP. It restored endothelial function, did not affect plaque size but decreased lipid, ox-LDL and macrophage content. Smooth muscle cells and collagen accumulated within the plaque. Increased smoothelin-to-alpha-smooth muscle actin ratio indicated a more differentiated SMC phenotype. Cholesterol lowering reduced proliferation and apoptosis. In vitro, hypercholesterolemic serum increased SMC apoptosis and decreased SMC migration compared to non-hypercholesterolemic serum. Conclusions: Cholesterol lowering induced coronary plaque stabilization as evidenced by a decrease in lipids, ox-LDL, macrophages, apoptosis and cell proliferation, and an increase in differentiated SMC and collagen. Increased migration and decreased apoptosis of SMC may contribute to the disappearance of the a-cellular core after lipid lowering. (C) 2002 Elsevier Science B.V. All rights reserved.

OBJECTIVE:To assess the effect of hypercholesterolemia on neointima formation and vascular remodelling after porcine coronary angioplasty. METHODS:Left anterior descending coronary angioplasty was carried out in five control and 16 age-matched hypercholesterolemic miniature pigs. Vascular remodelling was measured by intravascular ultrasound. Neointima size and composition were assessed by quantitative image analysis. Coronary smooth muscle cells (SMC) from control and diet pigs were collected 1 h after angioplasty for in vitro study of the effect of hypercholesterolemic serum on SMC migration and of macrophage-induced matrix degradation on SMC adhesion. RESULTS:Twenty-eight days after angioplasty, lumen increase was 0.08+/-1.7 mm(2) in diet and 2.7+/-2.7 mm(2) (P=0.016) in control pigs. Lumen increase correlated with vascular remodelling (IEL(post)/IEL(pre); R(2)=0.59; P<0.001) and with the circumferential gain relative to the neointima (R(2)=0.32; P<0.01) but not with neointimal area that was similar in control and diet pigs. Circumferential gain correlated with VSMC deposition at the site of the injury (R(2)=0.28; P<0.01) that correlated with organized collagen (R(2)=0.34; P<0.01). The VSMC and collagen content of neointima in diet pigs was lower whereas the macrophage content was higher. Hypercholesterolemic serum and oxidised LDL reduced migration of VSMC from diet pigs. Macrophage-induced degradation of VSMC extracellular matrix reduced VSMC adhesion (P=0.015). CONCLUSION:Hypercholesterolemia impairs vascular remodelling of balloon-treated coronary arteries. It decreases VSMC and collagen accumulation at the site of injury. Our in vitro data suggest that this decrease can be due to macrophage-induced matrix degradation and reduced VSMC adhesion and to impaired VSMC migration. Oxidised LDL mimics the inhibitory effect of hypercholesterolemic serum.

Background-Hypercholesterolemia induces functional and structural changes of the microvasculature and reduces coronary flow reserve in humans and experimental animals. The effect of hypercholesterolemia on left ventricular (LV) function in the absence of coronary stenosis is, however, unknown. Our objective was therefore to assess the effect of hypercholesterolemia and cholesterol withdrawal on LV function in the presence of advanced coronary plaques that do not cause stenosis.Methods and Results-Twenty-eight minipigs on cholesterol diet for 34 weeks and 16 control pigs were studied. Seven hypercholesterolemic pigs were withdrawn from the diet for 26 weeks. LV function was assessed with cine-MRI, myocardial blood flow with colored microspheres, and capillary density with immunohistochemistry, and microvascular endothelial cell apoptosis with terminal dUTP nick-end labeling staining. Hypercholesterolemia (17 +/- 8 versus 268 +/- 150 versus 12 +/- 10 mg/dL LDL cholesterol, control versus hypercholesterolemic versus cholesterol withdrawal; P<0.001) induced atherosclerosis but not stenosis in the left coronary artery. Baseline cardiac output, ejection fraction, and stroke volume were similar in control and hypercholesterolemic pigs. In dobutamine stress test, cardiac output (P<0.05) and stroke volume (P<0.01) were lower in hypercholesterolemic pigs compared with controls. The impaired response to dobutamine was reversible by dietary cholesterol withdrawal. Hypercholesterolemia reduced endomyocardial coronary flow reserve (P<0.01) and capillary density (P<0.05) and induced capillary endothelial cell apoptosis. Hypercholesterolemic pigs failed to reduce vascular resistance in response to increased LV workload and pharmacological vasodilation.Conclusion-LDL hypercholesterolemia in minipigs impaired LV response to dobutamine stress in the absence of coronary stenosis.

Atherosclerosis was studied in apolipoprotein E (apoE) knockout mice expressing human apolipoprotein A-I (apoA-I) or an apoA-I/apolipoprotein A-II (apoA-II) chimera in which the Arg123-Tyr166 central domain of apoA-I was substituted with the Ser12-Ala75 segment of apoA-II. High density lipoprotein (HDL) cholesterol levels were identical in apoA-I and apoA-I/apoA-II mice, but at 4 months, plaques were 2.7-fold larger in the aortic root of the apoA-I/apoA-II mice (P<0.01). The macrophage-to-smooth muscle cell ratio of lesions was 2.1-fold higher in apo-I/apoA-II mice than in apoA-I mice (P<0.01). This was due to a 2.7-fold higher (P<0.001) in vivo macrophage homing in the aortic root of apoA-I/apoA-II mice. Plasma platelet-activating factor acetyl hydrolase activity was lower (P<0.01) in apoA-I/apoA-II mice, resulting in increased oxidative stress, as evidenced by the higher titer of antibodies against oxidized low density lipoprotein (P<0.01). Increased oxidative stress resulted in increased stimulation of ex vivo macrophage adhesion by apoA-I/apoA-II beta-very low density lipoprotein and decreased inhibition of beta-very low density lipoprotein-induced adhesion by HDL from apoA-I/apoA-II mice. The cellular cholesterol efflux capacity of HDL from apoA-I/apoA-II mice was very similar to that of apoA-I mice. Thus, the Arg123-Tyr166 central domain of apoA-I is critical for reducing oxidative stress, macrophage homing, and early atherosclerosis in apoE knockout mice independent of its role in HDL production and cholesterol efflux.

HDL metabolism and atherosclerosis were studied in apo E knockout (KO) mice overexpressing human apo AI, a des- (190-243)-apo AI carboxyl-terminal deletion mutant of human apo AI or an apo AI-(1-189)-apo AII-(12-77) chimera in which the carboxyl-terminal domain of apo AI was substituted with the pair of helices of apo AII. HDL cholesterol levels ranked: apo AI/apo E KO approximately apo AI-(1-189)-apo AII- (12-77)/apo E KO > > des-(190-243)-apo AI/apo E KO > apo E KO mice. Progression of atherosclerosis ranked: apo E KO > des-(190-243)-apo AI/apo E KO > > apo AI-(1-189)- apo AII-(12-77)/apo E KO approximately apo AI/apo E KO mice. Whereas the total capacity to induce cholesterol efflux from lipid-loaded THP-1 macrophages was higher for HDL of mice overexpressing human apo AI or the apo AI/apo AII chimera, the fractional cholesterol efflux rate, expressed in percent cholesterol efflux/microg apolipoprotein/h, for HDL of these mice was similar to that for HDL of mice overexpressing the deletion mutant and for HDL of apo E KO mice. This study demonstrates that the tertiary structure of apo AI, e.g., the number and organization of its helices, and not its amino sequence is essential for protection against atherosclerosis because it determines HDL cholesterol levels and not cholesterol efflux. Amino acid sequences of apo AII, which is considered to be less antiatherogenic, can be used to restore the structure of apo AI and thereby its antiatherogenicity.

The murine monoclonal antibody mAb-1H11 raised against malondialdehyde (MDA)-modified LDL, was used to detect cross-reacting material in human atheromatous tissue and in plasma. MDA-modified LDL levels in plasma were 0.19 +/- 0.02 mg/dl (mean +/- SEM) in 44 control subjects, 0.24 +/- 0.02 mg/dl in 15 patients with chronic stable angina pectoris (P = NS vs LDL cholesterol matched controls), 1.4 +/- 0.1 mg/dl in 60 patients with acute myocardial infarction (P < 0.001 vs controls), and 0.86 +/- 0.11 mg/dl in 22 patients with carotid atherosclerosis (P < 0.001 vs controls). Modified LDL, isolated from pooled LDL of 10 patients, showed a higher electrophoretic mobility on agarose gels, a higher content of thiobarbituric acid reactive substances, and a higher cholesterol/protein ratio than native LDL and had a similar reactivity (antigen/protein ratio) in the assay as the in vitro MDA-modified LDL used for calibration. Its apo B-100 moiety was not fragmented. Uptake of this modified LDL by macrophages resulted in foam cell generation. In conclusion, elevated plasma levels of atherogenic MDA-modified LDL may be a marker for unstable atherosclerotic cardiovascular disease.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.

A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.

Fibrin-directed monoclonal antibodies may be clinically useful for in vitro thrombus imaging and for the targeting of fibrinolytic agents to blood clots. One such murine monoclonal antibody, (mAb-15C5), raised against the fragment-D dimer epitope of cross-linked human fibrin, was previously characterized [Holvoet, P., Stassen, J. M., Hashimoto, Y., Spriggs, D., Devos, P. & Collen, D. (1989) Thromb. Haemostasis 61, 307-313] has recently been cloned and expressed [Vandamme, A.-M., Bulens, F., Bernar, H., Nelles, L., Lijnen, H. R. & Collen, D. (1990) Eur. J. Biochem. 192, 767-775]. In order to reduce the immunogenicity of the murine mAb-15C5 in man, we have now constructed a murine--human chimera of mAb-15C5, by substituting the cDNA sequences encoding the constant regions of the murine kappa light chain and gamma 1 heavy chain by the corresponding human genomic sequences. Both chimeric murine--human Ig chains were cloned into two separately selectable expression vectors, which were contransfected into Chinese hamster ovary (CHO) cells. Murine--human chimeric mAb-15C5 (mAb-15C5Hu) was purified from the conditioned medium of selected cell lines by chromatography on Zn-chelating Sepharose, protein-A-Sepharose and on insolubilized antigen (fragment-D dimer), with a final yield of 29 micrograms/l and a recovery of 33%. SDS/PAGE without reduction revealed a homogeneous band with a mobility similar to that of natural mAb-15C5, whereas after reduction, both the heavy and the light chains had slightly slower mobilities than their natural counterparts. Expression in the presence of tunicamycin suggested that the differences in gamma 1-chain mobility were due to different N-glycosylation patterns. Immunoblotting of proteins from SDS gels showed immunological reactivity of recombinant mAb-15C5Hu with goat anti-(human IgG) IgG and of recombinant and natural murine mAb-15C5 with goat anti-(mouse IgG) IgG. Competitive binding revealed a comparable affinity of recombinant murine mAb-15C5, recombinant mAb-15C5Hu and natural mAb-15C5, for fragment-D dimer, indicating that recombinant mAb-15C5Hu was obtained in a functionally intact form. Thus, mAb-15C5Hu may constitute a useful alternative to mAb-15C5 for in vivo use in man.

cDNA libraries in λ phage were generated from the murine hybridoma secreting mAb‐15C5, a monoclonal antibody directed against fragment‐D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemo‐stasis 61, 307–313], and clones encoding fragments of the heavy (γ1) and the light (k) chain were isolated. The κ‐chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The γ1‐chain cDNA was reconstructed from the mAb‐15C5 κ‐chain signal sequence, the mAb‐15C5 γ1 variable‐domain coding sequence and murine γ1‐gene and γ1‐chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb‐15C5 was purified from the conditoned medium by chromatography on Zn‐chelate–Sepharose, protein‐A–Sepharose and insolubilized fragment‐D dimer, with a yield of 50 μg/1 and a recovery of 20%. SDS‐gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light‐chain band with identical and a heavy‐chain band with a somewhat slower mobility than that of the natural mAb‐15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb‐15C5, for fibrin fragment‐D dimer.Thus recombinant mAb‐15C5, obtained by co‐expression of the reconstructed cDNAs of the k and γ1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb‐15C5. These recombinant mAb‐15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.