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1.  Low-Density Lipoprotein Apheresis 
Executive Summary
Objective
To assess the effectiveness and safety of low-density lipoprotein (LDL) apheresis performed with the heparin-induced extracorporeal LDL precipitation (HELP) system for the treatment of patients with refractory homozygous (HMZ) and heterozygous (HTZ) familial hypercholesterolemia (FH).
Background on Familial Hypercholesterolemia
Familial hypercholesterolemia is a genetic autosomal dominant disorder that is caused by several mutations in the LDL-receptor gene. The reduced number or absence of functional LDL receptors results in impaired hepatic clearance of circulating low-density lipoprotein cholesterol (LDL-C) particles, which results in extremely high levels of LDL-C in the bloodstream. Familial hypercholesterolemia is characterized by excess LDL-C deposits in tendons and arterial walls, early onset of atherosclerotic disease, and premature cardiac death.
Familial hypercholesterolemia occurs in both HTZ and HMZ forms.
Heterozygous FH is one of the most common monogenic metabolic disorders in the general population, occurring in approximately 1 in 500 individuals1. Nevertheless, HTZ FH is largely undiagnosed and an accurate diagnosis occurs in only about 15% of affected patients in Canada. Thus, it is estimated that there are approximately 3,800 diagnosed and 21,680 undiagnosed cases of HTZ FH in Ontario.
In HTZ FH patients, half of the LDL receptors do not work properly or are absent, resulting in plasma LDL-C levels 2- to 3-fold higher than normal (range 7-15mmol/L or 300-500mg/dL). Most HTZ FH patients are not diagnosed until middle age when either they or one of their siblings present with symptomatic coronary artery disease (CAD). Without lipid-lowering treatment, 50% of males die before the age of 50 and 25% of females die before the age of 60, from myocardial infarction or sudden death.
In contrast to the HTZ form, HMZ FH is rare (occurring in 1 case per million persons) and more severe, with a 6- to 8-fold elevation in plasma LDL-C levels (range 15-25mmol/L or 500-1000mg/dL). Homozygous FH patients are typically diagnosed in infancy, usually due to the presence of cholesterol deposits in the skin and tendons. The main complication of HMZ FH is supravalvular aortic stenosis, which is caused by cholesterol deposits on the aortic valve and in the ascending aorta. The average life expectancy of affected individuals is 23 to 25 years. In Ontario, it is estimated that there are 13 to 15 cases of HMZ FH. An Ontario clinical expert confirmed that 9 HMZ FH patients have been identified to date.
Diagnosis
There are 2 accepted clinical diagnostic criterion for the diagnosis of FH: the Simon Broome FH Register criteria from the United Kingdom and the Dutch Lipid Network criteria from the Netherlands. The criterion supplement cholesterol levels with clinical history, physical signs and family history. DNA-based-mutation-screening methods permit a definitive diagnosis of HTZ FH to be made. However, given that there are over 1000 identified mutations in the LDL receptor gene and that the detection rates of current techniques are low, genetic testing becomes problematic in countries with high genetic heterogeneity, such as Canada.
Treatment
The primary aim of treatment in both HTZ and HMZ FH is to reduce plasma LDL-C levels in order to reduce the risk of developing atherosclerosis and CAD.
The first line of treatment is dietary intervention, however it alone is rarely sufficient for the treatment of FH patients. Patients are frequently treated with lipid-lowering drugs such as resins, fibrates, niacin, statins and cholesterol absorption-inhibiting drugs (ezetimibe). Most HTZ FH patients require a combination of drugs to achieve or approach target cholesterol levels.
A small number of HTZ FH patients are refractory to treatment or intolerant to lipid-lowering medication. According to clinical experts, the prevalence of refractory HTZ FH in Ontario is between 1 to 5%. Using the mean of 3%, it is estimated that there are approximately 765 refractory HTZ FH patients in Ontario, of which 115 are diagnosed and 650 are undiagnosed.
Drug therapy is less effective in HMZ FH patients since the effects of the majority of cholesterol-lowering drugs are mediated by the upregulation of LDL receptors, which are often absent or function poorly in HMZ FH patients. Some HMZ FH patients may still benefit from drug therapy, however this rarely reduces LDL-C levels to targeted levels.
Existing Technology: Plasma Exchange
An option currently available in Ontario for FH patients who do not respond to standard diet and drug therapy is plasma exchange (PE). Patients are treated with this lifelong therapy on a weekly or biweekly basis with concomitant drug therapy.
Plasma exchange is nonspecific and eliminates virtually all plasma proteins such as albumin, immunoglobulins, coagulation factors, fibrinolytic factors and HDL-C, in addition to acutely lowering LDL-C by about 50%. Blood is removed from the patient, plasma is isolated, discarded and replaced with a substitution fluid. The substitution fluid and the remaining cellular components of the blood are then returned to the patient.
The major limitation of PE is its nonspecificity. The removal of HDL-C prevents successful vascular remodeling of the areas stenosed by atherosclerosis. In addition, there is an increased susceptibility to infections, and costs are incurred by the need for replacement fluid. Adverse events can be expected to occur in 12% of procedures.
Other Alternatives
Surgical alternatives for FH patients include portocaval shunt, ileal bypass and liver transplantation. However, these are risky procedures and are associated with a high morbidity rate. Results with gene therapy are not convincing to date.
The Technology Being Reviewed: LDL Apheresis
An alternative to PE is LDL apheresis. Unlike PE, LDL apheresis is a selective treatment that removes LDL-C and other atherogenic lipoproteins from the blood while minimally impacting other plasma components such as HDL-C, total serum protein, albumin and immunoglobulins. As with PE, FH patients require lifelong therapy with LDL apheresis on a weekly/biweekly basis with concomitant drug therapy.
Heparin-Induced Extracorporeal LDL Precipitation
Heparin-induced extracorporeal LDL precipitation (HELP) is one of the most widely used methods of LDL apheresis. It is a continuous closed-loop system that processes blood extracorporeally. It operates on the principle that at a low pH, LDL and lipoprotein (a) [Lp(a)] bind to heparin and fibrinogen to form a precipitate which is then removed by filtration. In general, the total duration of treatment is approximately 2 to 3 hours.
Results from early trials indicate that LDL-C concentration is reduced by 65% to 70% immediately following treatment in both HMZ and HTZ FH and then rapidly begins to rise. Typically patients with HTZ FH are treated every 2 weeks while patients with HMZ FH require weekly therapy. Heparin-induced extracorporeal LDL precipitation also produces small transient decreases in HDL-C, however levels generally return to baseline within 2 days. After several months of therapy, long-term reductions in LDL-C and increases in HDL-C have been reported.
In addition to having an impact on plasma cholesterol concentrations, HELP lowers plasma fibrinogen, a risk factor for atherosclerosis, and reduces concentrations of cellular adhesion molecules, which play a role in early atherogenesis.
In comparison with PE, HELP LDL apheresis does not have major effects on essential plasma proteins and does not require replacement fluid, thus decreasing susceptibility to infections. One study noted that adverse events were documented in 2.9% of LDL apheresis treatments using the HELP system compared with 12% using PE. As per the manufacturer, patients must weigh at least 30kgs to be eligible for treatment with HELP.
Regulatory Status
The H.E.L.P.® System (B.Braun Medizintechnologie GmbH, Germany) has been licensed by Health Canada since December 2000 as a Class 3 medical device (Licence # 26023) for performing LDL apheresis to acutely remove LDL from the plasma of 3 high-risk patient populations for whom diet has been ineffective and maximum drug therapy has either been ineffective or not tolerated. The 3 patient groups are as follows:
Functional hypercholesterolemic homozygotes with LDL-C >500 mg/dL (>13mmol/L);
Functional hypercholesterolemic heterozygotes with LDL-C >300 mg/dL (>7.8mmol/L);
Functional hypercholesterolemic heterozygotes with LDL-C >200 mg/dL (>5.2mmol/L) and documented CAD
No other LDL apheresis system is currently licensed in Canada.
Review Strategy
The Medical Advisory Secretariat systematically reviewed the literature to assess the effectiveness and safety of LDL apheresis performed with the HELP system for the treatment of patients with refractory HMZ and HTZ FH. A standard search methodology was used to retrieve international health technology assessments and English-language journal articles from selected databases.
The GRADE approach was used to systematically and explicitly make judgments about the quality of evidence and strength of recommendations.
Summary of Findings
The search identified 398 articles published from January 1, 1998 to May 30, 2007. Eight studies met the inclusion criteria. Five case series, 2 case series nested within comparative studies, and one retrospective review, were included in the analysis. A health technology assessment conducted by the Alberta Heritage Foundation for Medical Research, and a review by the United States Food and Drug Administration were also included.
Large heterogeneity among the studies was observed. Studies varied in inclusion criteria, baseline patient characteristics and methodology.
Overall, the mean acute1 relative decrease in LDL-C with HELP LDL apheresis ranged from 53 to 77%. The mean acute relative reductions ranged as follows: total cholesterol (TC) 47 to 64%, HDL-C +0.4 to -29%, triglycerides (TG) 33 to 62%, Lp(a) 55 to 68% and fibrinogen 56 to 65%.
The mean chronic2 relative decreases in LDL-C and TC with HELP LDL apheresis ranged from 9 to 46% and 5 to 34%, respectively. Familial hypercholesterolemia patients treated with HELP did not achieve the target LDL-C value set by international guidelines (LDL-C < 2.5mmol/L, 100mg/dL). The chronic mean relative increase in HDL-C ranged from 12 to 27%. The ratio of LDL:HDL and the ratio of TC:HDL are 2 measures that have been shown to be important risk factors for cardiac events. In high-risk patients, the recommended target LDL:HDL ratio is less than or equal to 2, and the target TC:HDL ratio is less than 4. In the studies that reported chronic lipid changes, the LDL:HDL and TC:HDL ratios exceeded targeted values.
Three studies investigated the effects of HELP on coronary outcomes and atherosclerotic changes. One noted that twice as many lesions displayed regression in comparison to those displaying progression. The second study found that there was a decrease in Agatston scores3 and in the volume of coronary calcium. The last study noted that 2 of 5 patients showed regression of coronary atherosclerosis, and 3 of the 5 patients showed no change as assessed by a global change score.
Adverse effects were typically mild and transient, and the majority of events were related to problems with vascular access. Of the 3 studies that provided quantitative information, the proportion of adverse events ranged from 2.9 to 5.1%.
GRADE Quality of Evidence
In general, studies were of low quality, i.e., case series studies (Tables 1-3). No controlled studies were identified and no studies directly compared the effectiveness of the HELP system with PE or with diet and drug therapy. Conducting trials with a sufficiently large control group would not have been feasible or acceptable given that HELP represents a last alternative in these patients who are resistant to conventional therapeutic strategies.
A major limitation is that there is limited evidence on the effectiveness and safety of HELP apheresis in HMZ FH patients. However, it is unlikely that better-quality evidence will become available, given that HMZ FH is rare and LDL apheresis is a last therapeutic option for these patients.
Lastly, there is limited data on the long-term effects of LDL apheresis in FH patients. No studies with HELP were identified that examined long-term outcomes such as survival and cardiovascular events. The absence of this data may be attributed to the rarity of the condition, and the large number of subjects and long duration of follow-up that would be needed to conduct such trials.
Homozygous Familial Hypercholesterolemia - Lipid Outcomes
Heterozygous Familial Hypercholesterolemia - Lipid Outcomes
Heterozygous Familial Hypercholesterolemia - Coronary Artery Disease Outcomes
Economic Analysis
A budget-impact analysis was conducted to forecast future costs for PE and HELP apheresis in FH patients. All costs are reported in Canadian dollars. Based on epidemiological data of 13 HMZ, 115 diagnosed HTZ and 765 cases of all HTZ patients (diagnosed + undiagnosed), the annual cost of weekly treatment was estimated to be $488,025, $4,332,227 and $24,758,556 respectively for PE. For HELP apheresis, the annual cost of weekly treatment was estimated to be $1,025,338, $9,156,209 and $60,982,579 respectively. Costs for PE and HELP apheresis were halved with a biweekly treatment schedule.
The cost per coronary artery disease death avoided over a 10-year period in HTZ FH-diagnosed patients was also calculated and estimated to be $37.5 million and $18.7 million for weekly and biweekly treatment respectively, when comparing HELP apheresis with PE and with no intervention. Although HELP apheresis costs twice as much as PE, it helped to avoid 12 deaths compared with PE and 22 deaths compared with no intervention, over a period of 10 years.
Ontario Health System Impact Analysis
Low-density lipoprotein apheresis using the HELP system is currently being funded by the provinces of Quebec and Alberta. The program in Quebec has been in operation since 2001 and is limited to the treatment of HMZ FH patients. The Alberta program is relatively new and is currently treating HMZ FH patients, but it is expanding to include refractory HTZ FH patients.
Low-density lipoprotein apheresis is a lifelong treatment and requires considerable commitment on the part of the patient, and the patient’s family and physician. In addition, the management of FH continues to evolve. With the advent of new more powerful cholesterol-lowering drugs, some HTZ patients may be able to sufficiently control their hypercholesterolemia. Nevertheless, according to clinical experts, HMZ patients will likely always require LDL apheresis.
Given the substantial costs associated with LDL apheresis, treatment has been limited to HMZ FH patients. However, LDL apheresis could be applied to a much larger population, which would include HTZ FH patients who are refractory to diet and drug therapy. HTZ FH patients are generally recruited in a more advanced state, demonstrate a longer natural survival than HMZ FH patients and are older.
Conclusions
For HMZ FH patients, the benefits of LDL apheresis clearly outweigh the risks and burdens. According to GRADE, the recommendation would be graded as strong, with low- to very low-quality evidence (Table 4).
In both HMZ and HTZ FH patients, there is evidence of overall clinical benefit of LDL apheresis from case series studies. Low-density lipoprotein apheresis has several advantages over the current treatment of PE, including decreased exposure to blood products, decreased risk of adverse events, conservation of nonatherogenic and athero-protective components, such as HDL-C and lowering of other atherogenic components, such as fibrinogen.
In contrast to HMZ FH patients, there remains a lot of uncertainty in the social/ethical acceptance of this technology for the treatment of refractory HTZ FH patients. In addition to the substantial costs, it is unknown whether the current health care system could cope with the additional demand. There is uncertainty in the estimates of benefits, risks and burdens. According to GRADE, the recommendation would be graded as weak with low- to very-low-quality evidence (Table 5).
GRADE Recommendation - Homozygous Patients
GRADE of recommendation: Strong recommendation, low-quality or very-low-quality evidence
Benefits clearly outweigh risk and burdens
Case series study designs
Strong, but may change when higher-quality evidence becomes available
GRADE Recommendation - Heterozygous Patients
GRADE of recommendation: Weak recommendation, low-quality or very-low-quality evidence
Uncertainty in the estimates of benefits, risks and burden, which these may be closely balanced
Case series study designs
Very weak; other alternatives may be equally reasonable
PMCID: PMC3377562  PMID: 23074505
2.  Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. 
Journal of Clinical Investigation  1989;84(4):1086-1095.
Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.
Images
PMCID: PMC329764  PMID: 2794046
3.  C-Reactive Protein-Bound Enzymatically Modified Low-Density Lipoprotein Does Not Transform Macrophages into Foam Cells1 
The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.
PMCID: PMC2698222  PMID: 18322245
4.  Imbalance in superoxide dismutase/thioredoxin reductase activities in hypercholesterolemic subjects: relationship with low density lipoprotein oxidation 
Background
There is a relationship among hypercholesterolemia, oxidative stress and inflammation in the atherogenesis. Thus, the objective of the present study was to assess paraoxonase (PON1), superoxide dismutase (SOD) and thioredoxin reductase (TrxR-1) activities and their relationship with lipids, oxidative stress and inflammation in subjects with different low density lipoprotein-cholesterol (LDL) levels.
Methods
Serum lipids, highly sensitive C-reactive protein (hs-CRP), lipid and protein oxidation, oxidized LDL (LDLox) and LDLox autoantibodies (LDLoxAB) levels and enzymes activities were measured in a total of 116 subjects that were divided into the following groups according to their LDL levels: low-LDL group (LDL < 100 mg/dL, n = 23), intermediate-LDL group (LDL 100–160 mg/dL, n = 50) and high-LDL group (LDL > 160 mg/dL, n = 43).
Results
The LDLox and hs-CRP levels increased in the high-LDL group (2.7- and 3.7- fold, respectively), whereas the intermediate and high-LDL groups had higher LDLoxAB (2.2- and 3.1-fold) when compared to low-LDL group (p < 0.05). Similarly, SOD activity, the atherogenic index (AI) and protein oxidation were also higher in the intermediate (1.3-, 1.3- and 1.2-fold) and high-LDL (1.6-, 2.3- and 1.6-fold) groups when compared to the low-LDL group (p < 0.05). Lipid oxidation and SOD/TrxR-1 ratio increased only in the high-LDL group (1.3- and 1.6-fold) when compared to the low-LDL group (p < 0.05). The SOD/TrxR-1 ratio was positively correlated to TBARS (r = 0.23, p < 0.05), LDLox (r = 0.18, p < 0.05), LDLoxAB (r = 0.21, p < 0.05), LDL (r = 0.19, p < 0.05) and AI (r = 0.22, p < 0.05). PON1 and TrxR-1 activities were similar among groups.
Conclusions
Some oxidative events initiate when LDL levels are clinically acceptable. Moreover, hypercholesterolemic patients have an imbalance in SOD and TrxR-1 activities that is positively associated to LDL oxidation.
doi:10.1186/1476-511X-11-79
PMCID: PMC3490833  PMID: 22721254
Atherogenic index; Hypercholesterolemia; Oxidized low density lipoprotein; Superoxide dismutase; Thioredoxin reductase
5.  Probucol inhibits oxidative modification of low density lipoprotein. 
Journal of Clinical Investigation  1986;77(2):641-644.
Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 microM) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.
PMCID: PMC423406  PMID: 3944273
6.  Effect of rosuvastatin on concentrations of plasma lipids, urine and plasma oxidative stress markers, and plasma high-sensitivity C-reactive protein in hypercholesterolemic patients with and without type 2 diabetes mellitus: A 12-week, open-label, pilot study 
Background: Oxidative stress and inflammation of the arterial wall are now recognized as important factors in the progression of atherosclerosis. C-reactive protein (CRP) has been defined as a sensitive but not specific marker of inflammation. Statin therapy has been reported to decrease plasma high-sensitivity CRP (hs-CRP) concentration in hypercholesterolemic patients.
Objective: The aim of this study was to examine the effect of rosuvastatin on concentrations of plasma lipids, urine and plasma oxidative stress markers, and plasma hs-CRP in hypercholesterolemic patients with and without type 2 diabetes mellitus.
Methods: Patients with hypercholesterolemia with and without type 2 diabetes mellitus were enrolled in this pilot study after written informed consent was given. At baseline and after 12 weeks of open-label treatment with rosuvastatin 2.5 mg/d, concentrations of plasma lipids, urine and plasma oxidative stress markers, and plasma hs-CRP were measured. Urine 8-iso-prostaglandin F2α (8-iso-PGF2α) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentrations were also measured to asess whole-body oxidative stress. Plasma free-radical generation was estimated using a total reactive oxygen species (TROS) assay system. Adverse effects were assessed at each study visit (4-week intervals) through patient interviews and laboratory testing.
Results: Thirty-five patients were enrolled with 1 dropping out prior to study completion; therefore, 34 patients (19 women, 15 men; mean [SE] age, 55.4 [13.6] years; range, 30–78 years) completed the study. Compared with baseline, significant decreases were found in serum concentrations of total cholesterol (TC) (252.3 [39.3] vs 187.8 [30.1] mg/dL; P < 0.001; Δ = 24.5%), LDL-C (162.0 [44.3] vs 98.5 [31.9] mg/dL; P < 0.001; Δ = 38.7%), and triglycerides (TG) (157.2 [93.6] vs 124.4 [69.9] mg/dL; P < 0.05; Δ = 11.7%) after 12 weeks of treatment with rosuvastatin. Serum HDL-C concentration did not change significantly from baseline (59.7 [20.5] vs 63.7 [19.3] mg/dL; Δ = 9.4%). The plasma LDL-C/HDL-C ratio decreased significantly after rosuvastatin treatment (3.03 [1.33] vs 1.72 [0.83]; P < 0.001; Δ = 43.2%). Compared with baseline, significant decreases were observed in urine concentrations of the oxidative stress markers after 12 weeks of rosuvastatin treatment: 8-iso-PGF2α (342.8 [154.3] vs 300.6 [101.2] pg/mg; P < 0.05) and 8-OHdG (11.1 [4.53] vs 8.1 [2.7] ng/mg; P < 0.01). TROS decreased significantly (182.3 [29.0] vs 157.6 [17.3] U; P < 0.001), and plasma hs-CRP concentration also decreased significantly (0.107 [0.100] vs 0.054 [0.033] mg/dL; P < 0.05). When the patients' results were assessed according to the presence or absence of type 2 diabetes mellitus, urine 8-iso-PGF2α concentration was significantly decreased from baseline only in the nondiabetic group. No adverse events were reported or observed during the course of the study.
Conclusion: Rosuvastatin treatment was associated with significant reductions in plasma concentrations of TC, LDL-C, and TG, urine and plasma oxidative stress markers, and plasma hs-CRP in these hypercholesterolemic patients.
doi:10.1016/j.curtheres.2009.12.003
PMCID: PMC3969978  PMID: 24692836
rosuvastatin; hypercholesterolemia; type 2 diabetes; hs-CRP; oxidative stress
7.  Human macrophages limit oxidation products in low density lipoprotein 
This study tested the hypothesis that human macrophages have the ability to modify oxidation products in LDL and oxidized LDL (oxLDL) via a cellular antioxidant defence system. While many studies have focused on macrophage LDL oxidation in atherosclerosis development, less attention has been given to the cellular antioxidant capacity of these cells.
Compared to cell-free controls (6.2 ± 0.7 nmol/mg LDL), macrophages reduced TBARS to 4.42 ± 0.4 nmol/mg LDL after 24 h incubation with LDL (P = 0.022). After 2 h incubation with oxLDL, TBARS were 3.69 ± 0.5 nmol/mg LDL in cell-free media, and 2.48 ± 0.9 nmol/mg LDL in the presence of macrophages (P = 0.034). A reduction of lipid peroxides in LDL (33.7 ± 6.6 nmol/mg LDL) was found in the presence of cells after 24 h compared to cell-free incubation (105.0 ± 14.1 nmol/mg LDL) (P = 0.005). The levels of lipid peroxides in oxLDL were 137.9 ± 59.9 nmol/mg LDL and in cell-free media 242 ± 60.0 nmol/mg LDL (P = 0.012). Similar results were obtained for hydrogen peroxide. Reactive oxygen species were detected in LDL, acetylated LDL, and oxLDL by isoluminol-enhanced chemiluminescence (CL). Interestingly, oxLDL alone gives a high CL signal. Macrophages reduced the CL response in oxLDL by 45% (P = 0.0016). The increased levels of glutathione in oxLDL-treated macrophages were accompanied by enhanced catalase and glutathione peroxidase activities.
Our results suggest that macrophages respond to oxidative stress by endogenous antioxidant activity, which is sufficient to decrease reactive oxygen species both in LDL and oxLDL. This may suggest that the antioxidant activity is insufficient during atherosclerosis development. Thus, macrophages may play a dual role in atherogenesis, i.e. both by promoting and limiting LDL-oxidation.
doi:10.1186/1476-511X-4-6
PMCID: PMC555960  PMID: 15745457
Macrophages; LDL; lipid peroxides; antioxidant enzymes
8.  Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta. 
British Journal of Pharmacology  1992;105(1):216-222.
1. The mechanism by which Cu(2+)-oxidized low-density lipoproteins (oxLDL) inhibit acetylcholine (ACh)-evoked relaxations mediated by endothelium-derived nitric oxide (EDRF) in rabbit aortic rings was investigated. The proposed role of lysophosphatidylcholine (LPC) in the inhibition was also studied. 2. The kinetics of lipid peroxidation of native low-density lipoproteins (LDL) from individual donors, as measured by changes in conjugated diene concentration, were related to the inhibitory effects of the resultant oxLDL. It was found that the more susceptible LDL was to oxidation, the greater the inhibition. 3. No correlation was found between the inhibitory effects of oxLDL and LPC content. 4. Synthetic 1-palmitoyl LPC produced an inhibition of ACh-induced relaxations and when added to precontracted rings evoked nitric oxide-mediated endothelium-dependent relaxation. This latter effect was not elicited by oxLDL. 5. Synthetic 1-palmitoyl (10 microM) had no effect on relaxations evoked by glyceryl trinitrate in endothelium-denuded aortic rings in contrast to the inhibition found previously for oxLDL. 6. Concentrations of oxLDL and phospholipase A2-treated LDL which inhibited relaxation contained very different LPC concentrations. Unlike oxLDL, the inhibitory effects of phospholipase A2-treated LDL preparations were independent of the donors and showed no lag period. 7. We suggest that there are differences in the mechanisms by which oxLDL and 1-palmitoyl LPC exert their inhibitory effects on relaxation. 8. The inhibition of relaxation by oxLDL (1-2 mg protein ml-1) was prevented by the presence of high-density lipoproteins (HDL; 1-2 mg protein ml-1).(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1908615  PMID: 1596684
9.  Oxidized LDL: Diversity, Patterns of Recognition, and Pathophysiology 
Antioxidants & Redox Signaling  2010;13(1):39-75.
Abstract
Oxidative modification of LDL is known to elicit an array of pro-atherogenic responses, but it is generally underappreciated that oxidized LDL (OxLDL) exists in multiple forms, characterized by different degrees of oxidation and different mixtures of bioactive components. The variable effects of OxLDL reported in the literature can be attributed in large part to the heterogeneous nature of the preparations employed. In this review, we first describe the various subclasses and molecular composition of OxLDL, including the variety of minimally modified LDL preparations. We then describe multiple receptors that recognize various species of OxLDL and discuss the mechanisms responsible for the recognition by specific receptors. Furthermore, we discuss the contentious issues such as the nature of OxLDL in vivo and the physiological oxidizing agents, whether oxidation of LDL is a prerequisite for atherogenesis, whether OxLDL is the major source of lipids in foam cells, whether in some cases it actually induces cholesterol depletion, and finally the Janus-like nature of OxLDL in having both pro- and anti-inflammatory effects. Lastly, we extend our review to discuss the role of LDL oxidation in diseases other than atherosclerosis, including diabetes mellitus, and several autoimmune diseases, such as lupus erythematosus, anti-phospholipid syndrome, and rheumatoid arthritis. Antioxid. Redox Signal. 13, 39–75.
Introduction
Definitions, Biochemistry, and Composition
Minimally modified LDL
Extensively oxidized LDL
What Is the Nature of Oxidized LDL That Occurs In Vivoο
What Are the Physiological Oxidizing Agents In Vivoο
Bioactive Compounds in OxLDL
Phospholipid products
Lysophospholipid products
sn-2 short chain PCs
PAF-like products
sn-2 epoxy PCs
Sphingolipid products
Ceramide
Sphingosine 1-phospate
Free fatty acid products
Oxysterols
Cholesteryl ester products
Hydroxynonenal and malondialdehyde
Products of Apo B modification
OxLDL–Cellular Interactions: Patterns of OxLDL Recognition
Class A scavenger receptors: Extensively oxidized LDL
Class B scavenger receptors: Extensively and moderately oxidized LDL
Class E scavenger receptors: Mildly oxidized LDL
FCγ receptor: OxLDL immune complexes
Other scavenger receptors
Alternative pathways for minimally-oxidized LDL
OxLDL, the Janus-Faced Particle: Pro- and Anti-Inflammatory Properties
Reactive Oxygen Species and OxLDL Effects
OxLDL Uptake in Foam Cell Formation and Lesion Development
OxLDL-induced cholesterol loading of macrophages in vitro
OxLDL-induced cholesterol loading in vivo
Disruption of OxLDL uptake pathways in lesion formation
OxLDL in Endothelial Cells: Cholesterol, Caveolae, and Lesion Formation
OxLDL-induced impact on endothelial cholesterol in vitro: Loading or depletionο
Dyslipidemia-induced disruption of endothelial caveolae in vivo
Is Oxidation of LDL Necessary for the Development of Atherosclerosisο
Evidence supporting the hypothesis
Evidence inconsistent with the hypothesis
OxLDL in Diabetes Mellitus
OxLDL in Autoimmune Diseases
Systemic lupus erythematosus and antiphospholipid syndrome
Rheumatoid arthritis
Concluding Remarks
doi:10.1089/ars.2009.2733
PMCID: PMC2877120  PMID: 19888833
10.  Antioxidant Effects of Vitamins C and E on the Low-Density Lipoprotein Oxidation Mediated by Myeloperoxidase 
Iranian Biomedical Journal  2013;17(1):22-28.
Background: Oxidative modification of low-density lipoprotein (LDL) appears to be an early step in the pathogenesis of atherosclerosis. Meanwhile, myeloperoxidase (MPO)-catalyzed reaction is one of the potent pathways for LDL oxidation in vivo. The aim of this study was to evaluate in vitro antioxidant effects of vitamins C and E on LDL oxidation mediated by MPO. Methods: MPO was isolated from fresh plasma by sequential centrifugation using density ultracentrifugation. It was incubated with LDL and the LDL oxidation level was determined spectrophotometrically by measuring conjugated diene absorbance at 234 nm. Furthermore, vitamin C (50-200 mM) and vitamin E (10-40 mM) were added and the LDL oxidation level was determined. Results: The purity index of MPO and its enzymatic activity were 0.69 and 1127 U/mg protein, respectively. It was demonstrated that vitamin C in vitro inhibited LDL oxidation mediated by MPO; however, vitamin E was unable to act in the same way. The protection by vitamin C was concentration dependent and maximum protective effect of vitamin C was observed at 150 mM, where about 64% of the LDL oxidation was inhibited. Vitamin C increased lag time of LDL oxidation mediated by MPO up to 2.4 times. Conclusion: It can be concluded from our results that vitamin C is able to improve LDL resistance to oxidative modification in vitro. In addition, vitamin C might be effective in LDL oxidation mediated by MPO in vivo, resulting in reduction of atherosclerosis process rate.
doi:10.6091/ibj.1092.2012
PMCID: PMC3600973  PMID: 23279831
Antioxidant; Myeloperoxidase (MPO); Low-density lipoprotein (LDL); Vitamin E
11.  Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats 
Journal of lipid research  2008;49(5):1015-1023.
C-reactive protein (CRP) is present in the atherosclerotic plaques and appears to promote atherogenesis. Intraplaque CRP colocalizes with oxidized low density lipoprotein (OxLDL) and macrophages in human atherosclerotic lesions. Matrix metalloproteinase-9 (MMP-9) has been implicated in plaque rupture. CRP promotes OxLDL uptake and MMP induction in vitro; however, these have not been investigated in vivo. We examined the effect of CRP on OxLDL uptake and MMP-9 production in vivo in Wistar rats. CRP significantly increased OxLDL uptake in the peritoneal and sterile pouch macrophages compared with human serum albumin (huSA). CRP also significantly increased intracellular cholesteryl ester accumulation compared with huSA. The increased uptake of OxLDL by CRP was inhibited by pretreatment with antibodies to CD32, CD64, CD36, and fucoidin, suggesting uptake by both scavenger receptors and Fc-γ receptors. Furthermore, CRP treatment increased MMP-9 activity in macrophages compared with huSA, which was abrogated by inhibitors to p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and nuclear factor (NF)-κB but not Jun N-terminal kinase (JNK) before human CRP treatment. Because OxLDL uptake by macrophages contributes to foam cell formation and MMP release contributes to plaque instability, this study provides novel in vivo evidence for the role of CRP in atherosclerosis.
doi:10.1194/jlr.M700535-JLR200
PMCID: PMC2311439  PMID: 18245817
mechanistic insights; macrophages; sterile pouch
12.  Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor 
PLoS ONE  2013;8(8):e70533.
Objectives
Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1.
Methods and Results
Plasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; n = 7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; n = 7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5]  =  L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (P = 0.011; n = 5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine.
Conclusions
Our results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5.
doi:10.1371/journal.pone.0070533
PMCID: PMC3738565  PMID: 23950953
13.  The Influence of L-Carnitine on Oxidative Modification of LDL In Vitro 
Toxicology Mechanisms and Methods  2008;18(6):455-462.
Owing to their structure and function, low-density lipoproteins (LDLs) are particularly susceptible to the oxidative modifications. To prevent against oxidative modification of LDL, L-carnitine, with endogenous small water-soluble quaternary amine possessing antioxidative properties, was used. The aim of this paper was to prove the in vitro influence of L-carnitine on the degree of oxidative modification of the lipid part (estimated by conjugated dienes, lipid hydroperoxides, and malondialdehyde levels) and the protein part (estimated by dityrosine and tryptophan levels) of LDL native and oxidized by cooper ions. The level of lipophylic LDL antioxidant—α-tocopherol was also measured.
Oxidation of LDL by Cu2+ enhanced lipid peroxidation. That was manifested by a statistically significant increase in the content of malondialdehyde (threefold), conjugated dienes (up to about 30%), and lipid hydroperoxides (up to about 50%). Cu2+ ions were also the cause of oxidative modifications of the protein part of LDLs. It was manifested by a significant increase in dityrosine (by about 50%), whereas the level of tryptophan was significantly decreased threefold in relation to native LDL. Incubation of LDL with Cu2+ ions also caused a significant sixfold decrease of α-tocopherol content in oxidized LDL. However, L-carnitine caused a decrease in the level of conjugated dienes, lipid hydroperoxide, malondialdehyde, and dityrosine by about 20% to 30%, and a significant increase (by about 50%) in the content of tryptophan in comparison with oxidative LDL and in a smaller degree significant changes with native LDL. Additionally, L-carnitine caused a significant twofold increase in α-tocopherol content in oxidized LDL.
The above results indicate that L-carnitine protects the lipid as well as protein part of LDL particles against oxidative modifications, and this natural antioxidant might be used to prevent against diseases of oxidative origin.
doi:10.1080/15376510701623508
PMCID: PMC2728756  PMID: 19696940
L-Carnitine; LDL; Lipid Peroxidation; Oxidative Stress; Protein Oxidative Modifications; α-Tocopherol
14.  C-Reactive Protein Stimulates Myeloperoxidase Release from Polymorphonuclear Cells and Monocytes: Implications for Acute Coronary Syndromes 
Clinical chemistry  2008;55(2):361-364.
BACKGROUND
C-reactive protein (CRP), the prototypic marker of inflammation, is present in atherosclerotic plaques and appears to promote atherogenesis. Also, CRP has been localized to monocytes and tissue macrophages, which are present in the necrotic core of lesions prone to plaque rupture. Leukocyte-derived myeloperoxidase (MPO), primarily hosted in human polymorphonuclear cells (PMNs), has also been shown to be present in human atherosclerotic lesions. Because MPO and CRP concentrations are increased in acute coronary syndrome (ACS) patients and predict poor outcomes, we tested the effect of CRP on MPO release from PMNs and monocytes.
METHODS
We treated human PMNs and monocytes with CRP (25 and 50 mg/L for 6 h) and measured MPO release as total mass and activity in culture supernatants. We also measured nitro-tyrosinylation (NO2-Tyr) of LDL as an indicator of biological activity of CRP-mediated MPO release. Furthermore, we explored the effect of human CRP on MPO release in the rat sterile pouch model.
RESULTS
CRP treatment significantly increased release of MPO (both mass and activity) from human PMNs as well as monocytes (P < 0.05) and caused NO2-Tyr of LDL. Human CRP injection in rats resulted in increased concentrations of MPO in pouch exudates (P < 0.05), thus confirming our in vitro data.
CONCLUSIONS
CRP stimulates MPO release both in vitro and in vivo, providing further cogent data for the proinflammatory effect of CRP. These results might further support the role of CRP in ACS.
doi:10.1373/clinchem.2008.109207
PMCID: PMC2662851  PMID: 19074520
15.  Binding of the monomeric form of C-reactive protein to enzymatically-modified low-density lipoprotein: effects of phosphoethanolamine 
Background
The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored.
Methods
We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL).
Results
mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37°C, the binding of mCRP to E-LDL was enhanced <2-fold, while the binding of CRP to E-LDL was enhanced >10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37°C.
Conclusions
Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL.
doi:10.1016/j.cca.2009.06.018
PMCID: PMC2739981  PMID: 19545552
C-reactive protein; Monomeric C-reactive protein; Phosphoethanolamine; Pneumococcal C-polysaccharide; Enzymatically-modified low-density lipoprotein
16.  Effects of Fluvastatin on Plasma Levels of Low-Density Lipoprotein Subfractions, Oxidized Low-Density Lipoprotein, and Soluble Adhesion Molecules: A Twenty-Four–Week, Open-Label, Dose-Increasing Study 
Background: Statins not only lower low-density lipoprotein (LDL) levels, but also have several antiarteriosclerotic effects (eg, decreasing arterial inflammation and arterial smooth muscle cell proliferation, as well as antioxidant effects). The relationship between the dose of statin and its effects on plasma LDL levels and other arteriosclerosis-related effects remains to be clarified.
Objective: We investigated the effect of a statin, fluvastatin, on plasma levels of lipoprotein subfractions, oxidized LDL (Ox-LDL), Ox-LDL immunoglobulin G (IgG), soluble adhesion molecules, reverse cholesterol transport (ie, transport of esterified high-density lipoprotein cholesterol [HDL-C] to triglyceride [TG]-rich lipoproteins by cholesteryl ester transfer protein [CETP] and reduction of plasma HDL-C levels), and on the intima-medial thickness (IMT) of the common carotid arteries.
Methods: Patients with nonfamilial type 2 hyperlipoproteinemia were eligible for this open-label, dose-increasing study. Fluvastatin 20 mg/d was administered for the first 12 weeks, and the daily dose was increased to 40 mg for the subsequent 12 weeks. Patients were examined at baseline and after 12 and 24 weeks of treatment. Plasma lipoprotein subfractions were determined using sequential ultracentrifugation at 100,000g. The plasma levels of Ox-LDL, Ox-LDL-IgG, CETP, and soluble adhesion molecules were measured using sandwich enzyme-linked immunosorbent assay. The maximum IMT of the common carotid arteries was measured using sonography.
Results: The plasma levels of LDL cholesterol (LDL-C) and apolipoprotein (apo) B were reduced by 25% and 17%, respectively (P<0.001 for both), after 12 weeks of treatment with fluvastatin 20 mg/d; no further significant reductions in LDL were observed after increasing the daily dose to 40 mg. Fluvastatin 20 mg/d for 12 weeks decreased plasma levels of intermediate-density lipoprotein cholesterol, LDL-I-C, LDL-II-C, and LDL-III-C by 25% (P<0.01), 30% (P<0.001), 23% (P<0.01), and 20% (P = 0.02), respectively. No further significant reductions in these levels were observed after increasing the daily dose to 40 mg. The plasma levels of Ox-LDL decreased in a similar fashion to the plasma levels of LDL-C (P<0.001). However, plasma levels of Ox-LDL-IgG and soluble P-selectin did not decrease after 12 weeks of fluvastatin 20 mg/d, but did decrease significantly (both 22%) after the next 12 weeks of treatment with fluvastatin 40 mg/d (P<0.05). Plasma levels of intercellular adhesion molecule 1and vascular cell adhesion molecule 1 and CETP mass were not altered by fluvastatin treatment. Significant changes in maximum IMT of the common carotid arteries were not seen throughout 24 weeks of fluvastatin treatment.
Conclusions: In this patient population, fluvastatin 20 mg/d was sufficient to significantly reduce plasma levels of LDL, the 3 LDL subfractions, and Ox-LDL, but was not sufficient to reduce plasma levels of Ox-LDL-IgG and soluble P-selectin. It is important to check not only plasma lipoprotein levels but also other factors relating to arteriosclerosis during treatment with statins for the prevention and treatment of arteriosclerosis.
doi:10.1016/S0011-393X(03)00060-2
PMCID: PMC4052990  PMID: 24944371
fluvastatin; lipoprotein subfractions; oxidized LDL; adhesion molecules; reverse cholesterol transport; carotid arteriosclerosis
17.  Recognition Functions of Pentameric C-Reactive Protein in Cardiovascular Disease 
Mediators of Inflammation  2014;2014:319215.
C-reactive protein (CRP) performs two recognition functions that are relevant to cardiovascular disease. First, in its native pentameric conformation, CRP recognizes molecules and cells with exposed phosphocholine (PCh) groups, such as microbial pathogens and damaged cells. PCh-containing ligand-bound CRP activates the complement system to destroy the ligand. Thus, the PCh-binding function of CRP is defensive if it occurs on foreign pathogens because it results in the killing of the pathogen via complement activation. On the other hand, the PCh-binding function of CRP is detrimental if it occurs on injured host cells because it causes more damage to the tissue via complement activation; this is how CRP worsens acute myocardial infarction and ischemia/reperfusion injury. Second, in its nonnative pentameric conformation, CRP also recognizes atherogenic low-density lipoprotein (LDL). Recent data suggest that the LDL-binding function of CRP is beneficial because it prevents formation of macrophage foam cells, attenuates inflammatory effects of LDL, inhibits LDL oxidation, and reduces proatherogenic effects of macrophages, raising the possibility that nonnative CRP may show atheroprotective effects in experimental animals. In conclusion, temporarily inhibiting the PCh-binding function of CRP along with facilitating localized presence of nonnative pentameric CRP could be a promising approach to treat atherosclerosis and myocardial infarction. There is no need to stop the biosynthesis of CRP.
doi:10.1155/2014/319215
PMCID: PMC4052174  PMID: 24948846
18.  Atherosclerosis-related functions of C-reactive protein 
C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into monomers. Accordingly, the monomeric CRP found in atherosclerotic lesions may be a stationary, ligand-bound, by-product of a ligand-binding function of CRP. CRP binds to modified forms of low-density lipoprotein (LDL). The binding of CRP to oxidized LDL requires acidic pH conditions; the binding at physiological pH is controversial. The binding of CRP to enzymatically-modified LDL occurs at physiological pH; however, the binding is enhanced at acidic pH. Using enzymatically-modified LDL, CRP has been shown to prevent the formation of enzymatically-modified LDL-loaded macrophage foam cells. CRP is neither pro-atherogenic nor atheroprotective in ApoE−/− and ApoB100/100Ldlr −/− murine models of atherosclerosis, except in one study where CRP was found to be slightly atheroprotective in ApoB100/100Ldlr −/− mice. The reasons for the ineffectiveness of human CRP in murine models of atherosclerosis are not defined. It is possible that an inflammatory environment, such as those characterized by acidic pH, is needed for efficient interaction between CRP and atherogenic LDL during the development of atherosclerosis and to observe the possible atheroprotective function of CRP in animal models.
PMCID: PMC3125067  PMID: 20932269
Atherosclerosis; C-reactive protein; Enzymatically-modified low-density lipoprotein; Foam cells; Oxidized low-density lipoprotein
19.  Role of endogenous ceruloplasmin in low density lipoprotein oxidation by human U937 monocytic cells. 
Journal of Clinical Investigation  1996;97(3):884-890.
Oxidation of lipids and lipoproteins by macrophages is an important event during atherogenesis. Activation of monocytic cells by zymosan and other agonists results in the release of multiple oxidant species and consequent oxidation of LDL. We now show evidence that ceruloplasmin, a copper-containing acute phase reactant, is secreted by zymosan-activated U937 monocytic cells, and that the protein has an important role in LDL oxidation by these cells. In one approach, ceruloplasmin has been shown to exhibit oxidant activity under the appropriate conditions. Exogenous addition of purified human ceruloplasmin stimulates U937 cell oxidation of LDL to nearly the same extent as activation by zymosan. In contrast to previous cell-free experiments (Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493-1501.) in which ceruloplasmin by itself (in PBS) oxidizes LDL, under the conditions of the current experiments (in RPMI 1640 medium) ceruloplasmin only oxidizes LDL in the presence of cells; the mechanism by which cells overcome the inhibition by medium components has not been ascertained. As further evidence for a role of ceruloplasmin, activation of U937 cells with zymosan induces ceruloplasmin mRNA and ceruloplasmin protein synthesis after a 5-6 h lag that is consistent with that preceding LDL oxidation. Finally, neutralization by a highly specific polyclonal antibody to human ceruloplasmin inhibits LDL oxidation by at least 65%. Moreover, multiple antisense oligodeoxynucleotides targeted to different regions of the ceruloplasmin mRNA block LDL oxidation by up to 95%. The specific action of the antisense oligonucleotides has been verified by showing inhibition of ceruloplasmin synthesis and by the ability of exogenous ceruloplasmin to overcome the inhibition. In summary, these results are consistent with a mechanism in which cell-derived ceruloplasmin participates in oxidation of LDL by U937 monocytic cells. The data also show that cellular factors in addition to ceruloplasmin, possibly active oxygen species and/or lipoxygenases, are essential and act synergistically with ceruloplasmin to oxidize LDL.
PMCID: PMC507130  PMID: 8609249
20.  3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. 
Oxidation of LDL may be of pivotal importance in atherogenesis, but the mechanisms that promote oxidation in vivo remain poorly understood. We have explored the possibility that one pathway involves myeloperoxidase, a heme protein secreted by phagocytes. Myeloperoxidase is the only human enzyme known to generate hypochlorous acid (HOCl), a potent oxidizing agent, at physiological halide concentrations. LDL exposed to the complete myeloperoxidase-H2O2-Cl- system underwent chlorination of its protein tyrosyl residues. Treatment of LDL with reagent HOCl resulted in 3-chlorotyrosine formation, implicating HOCl as an intermediate in the enzymatic reaction pathway. In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, hemin, glucose, peroxynitrite, horseradish peroxidase, lactoperoxidase, or lipoxygenase. These results indicate that 3-chlorotyrosine is a specific marker for LDL oxidation by myeloperoxidase. To address the role of myeloperoxidase in promoting LDL oxidation in vivo, we used stable isotope dilution gas chromatography-mass spectrometry to quantify 3-chlorotyrosine in human aortic tissue and in LDL isolated from atherosclerotic lesions. The level of 3-chlorotyrosine in atherosclerotic tissue obtained during vascular surgery was sixfold higher than that of normal aortic intima. Moreover, the level of 3-chlorotyrosine was 30-fold higher in LDL isolated from atherosclerotic intima compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic lesions indicates that halogenation reactions catalyzed by the myeloperoxidase system of phagocytes constitute one pathway for protein oxidation in vivo. These findings raise the possibility that the myeloperoxidase-H2O2-Cl- system plays a critical role in converting LDL into an atherogenic form.
PMCID: PMC508036  PMID: 9151778
21.  Associations between Oxidized LDL to LDL Ratio, HDL and Vascular Calcification in the Feet of Hemodialysis Patients 
Journal of Korean Medical Science  2009;24(Suppl 1):S115-S120.
Cardiovascular mortality is associated with vascular calcification (VC) in hemodialysis (HD) patients. The present study was designed to find factors related with medial artery calcification on the plain radiography of feet by comparing C-reactive protein (CRP), plasminogen activator inhibitor type 1 (PAI-1) and lipid profile including oxidized low density lipoprotein (ox-LDL) and to elucidate associations among these factors in HD patients. Forty-eight HD patients were recruited for this study. VC in the feet was detected in 18 patients (37.5%) among total patients and 12 patients (85.7%) among diabetic patients. Diabetes, cardiovascular disease (CVD), pulse pressure, ox-LDL/LDL were higher and high density lipoprotein (HDL) was lower in patients with VC than in patients without VC. Negative associations were found between HDL and CRP, PAI-1. PAI-1 had positive association with ox-LDL/LDL. History of CVD was the only determinant of vascular calcification on the plain radiography of feet. Ox-LDL/LDL, HDL, CRP, and PAI-1 were closely related with one another in HD patients. History of CVD is the most important factor associated with the presence of VC and low HDL and relatively high oxidized LDL/LDL ratio may affect VC formation on the plain radiography in the feet of HD patients.
doi:10.3346/jkms.2009.24.S1.S115
PMCID: PMC2633196  PMID: 19194540
Vascular Calcification; Oxidized Low Density Lipoprotein; High Density Lipoproteins; Hemodialysis
22.  Pathophysiological concentrations of glucose promote oxidative modification of low density lipoprotein by a superoxide-dependent pathway. 
Journal of Clinical Investigation  1994;94(2):771-778.
Oxidized lipoproteins may be important in the pathogenesis of atherosclerosis. Because diabetic subjects are particularly prone to vascular disease, and glucose autoxidation and protein glycation generate reactive oxygen species, we explored the role of glucose in lipoprotein oxidation. Glucose enhanced low density lipoprotein (LDL) oxidation at concentrations seen in the diabetic state. Conjugated dienes, thiobarbituric acid reactive substances, electrophoretic mobility, and degradation by macrophages were increased when LDL was modified in the presence of glucose. In contrast, free lysine groups and fibroblast degradation were reduced. Although loss of reactive lysine groups could be due to either oxidative modification or nonenzymatic glycation of apolipoprotein B-100, inhibition of lipid peroxidation by the metal chelator, diethylenetriamine pentaacetic acid, blocked the changes in free lysines. Thus, glycation of lysine residues is unlikely to account for the alterations in macrophage and fibroblast uptake of LDL modified in the presence of glucose. Glucose-mediated enhancement of LDL oxidation was partially blocked by superoxide dismutase and nearly completely inhibited by butylated hydroxytoluene. These findings indicate that glucose enhances LDL lipid peroxidation by an oxidative pathway involving superoxide and raise the possibility that the chronic hyperglycemia of diabetes accelerates lipoprotein oxidation, thereby promoting diabetic vascular disease.
PMCID: PMC296157  PMID: 8040332
23.  Increased Serum ox-LDL Levels Correlated with Lung Function, Inflammation, and Oxidative Stress in COPD 
Mediators of Inflammation  2013;2013:972347.
Chronic obstructive pulmonary disease (COPD) is associated with abnormal inflammation and high oxidative stress. Studies suggest that oxidized low density lipoprotein (ox-LDL) is involved in diseases associated with oxidative stress and inflammation. However, no data on the possible relationship between COPD and ox-LDL are available. This study compared serum levels of ox-LDL in 48 COPD patients and 32 health controls and correlated them with lung function, systematic inflammation, and oxidative stress. Serum levels of ox-LDL, C-reactive protein (CRP), and oxidative stress (measured by reactive oxygen species, ROS) were analyzed using commercial kits. Mean levels of serum ox-LDL were significantly higher in COPD patients than in controls (18.62 ± 7.56 versus 12.57 ± 5.90 mU/L, P < 0.05). Serum levels of CRP and ROS were also significantly higher in COPD patients. Serum levels of ox-LDL in COPD patients correlated inversely with FEV1% predicted, an index of lung function (r = −0.347, P = 0.016), while they correlated positively with CRP and ROS levels. These results suggest that serum levels of ox-LDL are increased in COPD patients and that these levels are associated with lung function, inflammation, and oxidative stress in COPD. Future studies are needed to determine whether and how ox-LDL plays a role in COPD.
doi:10.1155/2013/972347
PMCID: PMC3774040  PMID: 24078777
24.  A comparison of the antiatherogenic effects of probucol and of a structural analogue of probucol in low density lipoprotein receptor-deficient rabbits. 
Journal of Clinical Investigation  1994;94(1):392-398.
The efficacies of probucol and a close structural analogue as antioxidants in the prevention of atherogenesis in LDL receptor-deficient rabbits were compared. The antioxidant potency of the analogue in vitro was equal to that of probucol. Its biological availability was much greater: almost comparable concentrations in total plasma were achieved by feeding 1% probucol (wt/wt) and 0.05% analogue (wt/wt). Total plasma concentrations were comparable, but the concentration of probucol within the LDL fraction was about twice that of the analogue. Probucol slowed lesion progression by almost 50%, confirming earlier reports; the analogue, however, showed no detectable inhibitory effect on atherogenesis. Resistance of LDL to oxidation was measured at the end of the study by incubating it with Cu2+ and measuring the rate of diene conjugation. Probucol prolonged diene conjugation lag time from the control value of 130 min to values > 1,000 min. The analogue approximately tripled the lag time (mean, 410 min) and yet failed to slow the atherogenic process. The results suggest that LDL resistance to oxidation must reach some threshold level before there is significant protection against atherogenesis. However, probucol has additional biological effects, possibly not shared by the analogue, that could contribute to its antiatherogenic potential.
Images
PMCID: PMC296321  PMID: 8040279
25.  Effect of atorvastatin on C-reactive protein and benefits for cardiovascular disease in patients with type 2 diabetes: analyses from the Collaborative Atorvastatin Diabetes Trial 
Diabetologia  2015;58(7):1494-1502.
Aims/hypothesis
We investigated whether atorvastatin 10 mg daily lowered C-reactive protein (CRP) and whether the effects of atorvastatin on cardiovascular disease (CVD) varied by achieved levels of CRP and LDL-cholesterol.
Methods
CRP levels were measured at baseline and 1 year after randomisation to atorvastatin in 2,322 patients with type 2 diabetes (40–75 years, 69% males) in a secondary analysis of the Collaborative Atorvastatin Diabetes Study, a randomised placebo-controlled trial. We used Cox regression models to test the effects on subsequent CVD events (n = 147) of CRP and LDL-cholesterol lowering at 1 year.
Results
After 1 year, the atorvastatin arm showed a net CRP lowering of 32% (95% CI −40%, −22%) compared with placebo. The CRP response was highly variable, with 45% of those on atorvastatin having no decrease in CRP (median [interquartile range, IQR] per cent change −9.8% [−57%, 115%]). The LDL-cholesterol response was less variable, with a median (IQR) within-person per cent change of −41% (−51%, −31%). Baseline CRP did not predict CVD over 3.8 years of follow-up (HRper SD log 0.89 [95% CI 0.75, 1.06]), whereas baseline LDL-cholesterol predicted CVD (HRper SD 1.21 [95% CI 1.02, 1.44]), as did on-treatment LDL-cholesterol. There was no significant difference in the reduction in CVD by atorvastatin, with above median (HR 0.57) or below median (HR 0.52) change in CRP or change in LDL-cholesterol (HR 0.61 vs 0.50).
Conclusions/interpretation
CRP was not a strong predictor of CVD. Statin efficacy did not vary with achieved CRP despite considerable variability in CRP response. The use of CRP as an indicator of efficacy of statin therapy on CVD risk in patients with type 2 diabetes is not supported by these data.
Trial registration NCT00327418
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-015-3586-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
doi:10.1007/s00125-015-3586-8
PMCID: PMC4472939  PMID: 25899452
Atorvastatin; Cardiovascular disease; CARDS; C-reactive protein; LDL-cholesterol; Statins; Type 2 diabetes

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