Oxidized LDL lipids as a risk factor for atherosclerosis

University of Turku, Faculty of Medicine, Department of Clinical Medicine, Department of Physical Activity and Health, Paavo Nurmi Centre, Doctoral Programme of Clinical Investigation, University of Turku, Turku, Finland. Annales Universitatis Turkuensis. Medica – Odontologica, Turku, Finland, 2014. Background: Atherosclerosis progression spans an entire lifetime and has a wide pool of risk factors. Oxidized LDL (oxLDL) is a crucial element in the progression of atherosclerosis. As a rather new member in the atherosclerosis risk factor family, its interaction with the traditional pro-atherogenic contributors that occur at different ages is poorly known. Aims: The aim of this study was to investigate oxLDL and its relation to major contributing risk factors in estimating atherosclerosis risk in data consisting mostly of adult men. The study subjects of this study consisted of four different sets of data, one of which contained also women. The age range of participants was 18-100 years and totaled 2337 participants (of whom 69% were men). Data on anthropometric and hormonal parameters, laboratory measures and medical records were assessed during 1998-2009. Results: Obesity was paralleled with high concentrations of oxLDL, which consequentially was reduced by weight reduction. Importantly, successful weight maintenance preserveed this benefit. A shift from insulin sensitivity to insulin resistance increased oxLDL. Smokers had more oxLDL than non-smokers. A combination of obesity and smoking, or smoking and low serum total testosterone,resulted in even higher levels of oxLDL than any of the three conditions alone. Proportioning oxLDL to HDL-c or apoA1 stood out as a risk factor of all-cause mortality in the elderly. Conclusions: OxLDL was associated with aging, androgens, smoking, obesity, insulin metabolism, weight balance and other circulating lipid classes. Through this variety of metabolic environments containing both constant conditions (aging and gender) as well as lifestyle issues, these findings supported an essential and multidimensional role that oxLDL plays in atherosclerosis pathogenesis.

Morphological changes of carotid bodies in acute respiratory distress syndrome: a morphometric study in humans

Carotid bodies are chemoreceptors sensitive to a fall of partial oxygen pressure in blood (hypoxia). The morphological alterations of these organs in patients with chronic obstructive pulmonary disease (COPD) and in people living at high altitude are well known. However, it is not known whether the histological profile of human carotid bodies is changed in acute clinical conditions such as acute respiratory distress syndrome (ARDS). The objective of the present study was to perform a quantitative analysis of the histology of carotid bodies collected from patients who died of ARDS. A morphometric study of carotid bodies collected during routine autopsies was carried out on three groups: patients that died of non-respiratory diseases (controls, N = 8), patients that presented COPD and died of its complications or associated diseases (N = 7), and patients that died of ARDS (N = 7). Morphometric measurements of the volume fraction of clusters of chief cells were performed in five fields on each slide at 40X magnification. The numerical proportion of the four main histological cell types (light, dark, progenitor and sustentacular cells) was determined analyzing 10 fields on each slide at 400X magnification. The proportion of dark cells was 0.22 in ARDS patients, 0.12 in controls (P<0.001), and 0.08 in the COPD group. The proportion of light cells was 0.33 (ARDS), 0.44 (controls) (P<0.001), and 0.36 (COPD). These findings suggest that chronic and acute hypoxia have different effects on the histology of glomic tissue.

Wine, alcohol and atherosclerosis: clinical evidences and mechanisms

Atherosclerosis is a chronic inflammatory disease which may cause obstructions of the coronary, cerebral and peripheral arteries. It is typically multifactorial, most often dependent on risk factors such as hypercholesterolemia, diabetes, smoking, hypertension, sedentarism, and obesity. It is the single main cause of death in most developed countries due to myocardial infarction, angina, sudden death, and heart failure. Several epidemiological studies suggest that moderate alcohol intake, especially red wine, decrease cardiac mortality due to atherosclerosis. The alcohol effect is described by a J curve, suggesting that moderate drinkers may benefit while abstainers and heavy drinkers are at higher risk. Experimental studies indicate that most beneficial effects of drinking are attributable to flavonoids that are present in red wine, purple grape juice and several fruits and vegetables. The mechanisms include antiplatelet actions, increases in high-density lipoprotein, antioxidation, reduced endothelin-1 production, and increased endothelial nitric oxide synthase expression which causes augmented nitric oxide production by endothelial cells. These findings lead to the concept that moderate red wine drinking, in the absence of contraindications, may be beneficial to patients who are at risk of atherosclerotic cardiovascular events. Moreover, a diet based on fruits and vegetables containing flavonoids may be even more beneficial.

Atherosclerosis in aged mice over-expressing the reverse cholesterol transport genes

We determined whether over-expression of one of the three genes involved in reverse cholesterol transport, apolipoprotein (apo) AI, lecithin-cholesterol acyl transferase (LCAT) and cholesteryl ester transfer protein (CETP), or of their combinations influenced the development of diet-induced atherosclerosis. Eight genotypic groups of mice were studied (AI, LCAT, CETP, LCAT/AI, CETP/AI, LCAT/CETP, LCAT/AI/CETP, and non-transgenic) after four months on an atherogenic diet. The extent of atherosclerosis was assessed by morphometric analysis of lipid-stained areas in the aortic roots. The relative influence (R²) of genotype, sex, total cholesterol, and its main sub-fraction levels on atherosclerotic lesion size was determined by multiple linear regression analysis. Whereas apo AI (R² = 0.22, P < 0.001) and CETP (R² = 0.13, P < 0.01) expression reduced lesion size, the LCAT (R² = 0.16, P < 0.005) and LCAT/AI (R² = 0.13, P < 0.003) genotypes had the opposite effect. Logistic regression analysis revealed that the risk of developing atherosclerotic lesions greater than the 50th percentile was 4.3-fold lower for the apo AI transgenic mice than for non-transgenic mice, and was 3.0-fold lower for male than for female mice. These results show that apo AI overexpression decreased the risk of developing large atherosclerotic lesions but was not sufficient to reduce the atherogenic effect of LCAT when both transgenes were co-expressed. On the other hand, CETP expression was sufficient to eliminate the deleterious effect of LCAT and LCAT/AI overexpression. Therefore, increasing each step of the reverse cholesterol transport per se does not necessarily imply protection against atherosclerosis while CETP expression can change specific athero genic scenarios.

Effect of aging on carotid artery stiffness and baroreflex sensitivity during head-out water immersion in man

To examine the possible age-related blood pressure (BP) deregulation in response to central hypervolemia, we measured spontaneous baroreflex sensitivity (SBRS), carotid arterial compliance (CC), and R-R interval coefficient of variation (RRICV) during basal and thermoneutral resting head-out-of-water immersion (HOWI) in 7 young (YG = 24.0 ± 0.8 years) and 6 middle-aged/older (OL = 59.3 ± 1.3 years) healthy men. Compared with basal conditions (YG = 19.6 ± 4.0 vs OL = 6.1 ± 1.5 ms/mmHg, P < 0.05), SBRS remained higher in YG than OL during rest HOWI (YG = 23.6 ± 6.6 vs OL = 9.3 ± 2.1 ms/mmHg, P < 0.05). The RRICV was significantly different between groups (YG = 6.5 ± 1.4 vs OL = 2.8 ± 0.4%, P < 0.05) under HOWI. The OL group had no increase in CC, but a significant increase in systolic BP (basal = 115.3 ± 4.4 vs water = 129.3 ± 5.3 mmHg, P < 0.05) under HOWI. In contrast, the YG group had a significant increase in CC (basal = 0.16 ± 0.01 vs water = 0.17 ± 0.02 mm²/mmHg, P < 0.05) with no changes in systolic BP. SBRS was positively related to CC (r = 0.58, P < 0.05 for basal vs r = 0.62, P < 0.05 for water). Our data suggest that age-related vagal dysfunction and reduced CC may be associated with SBRS differences between YG and OL groups, and with BP elevation during HOWI in healthy older men.

Cardiovascular health from childhood to adulthood – with special reference to early vascular changes. The Cardiovascular Risk in Young Finns Study

Background: Recent recommendations aim to improve cardiovascular health (CVH) by encouraging the general population to meet positive and modifiable ideal CVH metrics: not smoking, being physically active, and maintaining normal weight, blood pressure, blood glucose and total cholesterol levels and a healthy diet. Aims: The aim of the present study was to report the prevalence of ideal CVH in children and young adults and study the associations of CVH metrics with markers of subclinical atherosclerosis. Participants and methods: The present thesis is part of the Cardiovascular Risk in Young Finns Study (Young Finns Study). Data on associations of CVH metrics and subclinical atherosclerosis were available from 1,898 Young Finns Study participants. In addition, joint analyses were performed combining data from the International Childhood Cardiovascular Cohort (i3C) Consortium member studies from Australia and the USA. Results: None of the participants met all 7 CVH metrics and thus had ideal CVH in childhood and only 1% had ideal CVH as young adults. The number of CVH metrics present in childhood and adulthood predicted lower carotid artery intima-media thickness, improved carotid artery distensibility and lower risk of coronary artery calcification. Those who improved their CVH status from childhood to adulthood had a comparable risk of subclinical atherosclerosis to participants who had always had a high CVH status. Conclusions: Ideal CVH proved to be rare among children and young adults. A higher number of ideal CVH metrics and improvement of CVH status between childhood and adulthood predicted a lower risk of subclinical atherosclerosis.

Glutamic acid decarboxylase antibodies are indicators of the course, but not of the onset, of diabetes in middle-aged adults: the Atherosclerosis Risk in Communities Study

To efficiently examine the association of glutamic acid decarboxylase antibody (GADA) positivity with the onset and progression of diabetes in middle-aged adults, we performed a case-cohort study representing the ~9-year experience of 10,275 Atherosclerosis Risk in Communities Study participants, initially aged 45-64 years. Antibodies to glutamic acid decarboxylase (GAD65) were measured by radioimmunoassay in 580 incident diabetes cases and 544 non-cases. The overall weighted prevalence of GADA positivity (³1 U/mL) was 7.3%. Baseline risk factors, with the exception of smoking and interleukin-6 (P £ 0.02), were generally similar between GADA-positive and -negative individuals. GADA positivity did not predict incident diabetes in multiply adjusted (HR = 1.04; 95%CI = 0.55, 1.96) proportional hazard analyses. However, a small non-significant adjusted risk (HR = 1.29; 95%CI = 0.58, 2.88) was seen for those in the highest tertile (³2.38 U/mL) of positivity. GADA-positive and GADA-negative non-diabetic individuals had similar risk profiles for diabetes, with central obesity and elevated inflammation markers, aside from glucose, being the main predictors. Among diabetes cases at study's end, progression to insulin treatment increased monotonically as a function of baseline GADA level. Overall, being GADA positive increased risk of progression to insulin use almost 10 times (HR = 9.9; 95%CI = 3.4, 28.5). In conclusion, in initially non-diabetic middle-aged adults, GADA positivity did not increase diabetes risk, and the overall baseline profile of risk factors was similar for positive and negative individuals. Among middle-aged adults, with the possible exception of those with the highest GADA levels, autoimmune pathophysiology reflected by GADA may become clinically relevant only after diabetes onset.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 µg DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Coronary fat content evaluated by morphometry in patients with severe atherosclerosis has no relation with serum lipid levels

The relationship between lipid serum levels and coronary atherosclerotic plaque fat content was studied in 51 necropsy patients. Serum lipids were measured by standard techniques, during life, in the absence of lipid-lowering drugs. Intima, intimal fat and media areas were measured using a computerized system in cryosections of the odd segments of the right, anterior descending and circumflex coronary arteries stained with Sudan-IV. Mean intimal and lipid areas were 5.74 ± 1.98 and 1.22 ± 0.55 mm² (22.12 ± 8.48%) in 26 cases with high cholesterol (³200 mg/dL) and 4.98 ± 1.94 and 1.16 ± 0.66 mm² (22.75 ± 9.06%) in 25 cases with normal cholesterol (<200 mg/dL; P > 0.05). Patients with high levels of low-density lipoprotein (³130 mg/dL, N = 15) had a higher intima/media area ratio than those with normal levels of low-density lipoprotein (<130 mg/dL, N = 13, P < 0.01). No significant difference in the morphometrical variables was found in groups with high or low serum levels of triglycerides (³200 mg/dL, N = 13 vs <200 mg/dL, N = 36) or high-density lipoprotein (³35 mg/dL, N = 11 vs <35 mg/dL, N = 17). The association between the morphological measurements and serum levels of cholesterol, its fractions, and triglycerides was also tested and the correlation coefficients were low. Although high cholesterol is a risk factor, we show here that in patients with severe atherosclerosis blood cholesterol and triglyceride levels seem to have little influence on coronary lipid content, indicating that other factors may contribute to arterial lipid deposition and plaque formation.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Normal flow-mediated vasodilatation of the brachial artery and carotid artery intima-media thickness in subclinical hypothyroidism

Subclinical hypothyroidism (SHT) is a disease for which exact therapeutic approaches have not yet been established. Previous studies have suggested an association between SHT and coronary heart disease. Whether this association is related to SHT-induced changes in serum lipid levels or to endothelial dysfunction is unclear. The aim of this study was to determine endothelial function measured by the flow-mediated vasodilatation of the brachial artery and the carotid artery intima-media thickness (IMT) in a group of women with SHT compared with euthyroid subjects. Triglycerides, total cholesterol, HDL-C, LDL-C, apoprotein A (apo A), apo B, and lipoprotein(a) were also determined. Twenty-one patients with SHT (mean age: 42.4 ± 10.8 years and mean thyroid-stimulating hormone (TSH) levels: 8.2 ± 2.7 µIU/mL) and 21 euthyroid controls matched for body mass index, age and atherosclerotic risk factors (mean age: 44.2 ± 8.5 years and mean TSH levels: 1.4 ± 0.6 µIU/mL) participated in the study. Lipid parameters (except HDL-C and apo A, which were lower) and IMT values were higher in the common carotid and carotid bifurcation of SHT patients with positive serum thyroid peroxidase antibodies (TPO-Ab) (0.62 ± 0.2 and 0.62 ± 0.16 mm for the common carotid and carotid bifurcation, respectively) when compared with the negative TPO-Ab group (0.55 ± 0.24 and 0.58 ± 0.13 mm, for common carotid and carotid bifurcation, respectively). The difference was not statistically significant. We conclude that minimal thyroid dysfunction had no adverse effects on endothelial function in the population studied. Further investigation is warranted to assess whether subclinical hypothyroidism, with and without TPO-Ab-positive serology, has any effect on endothelial function.

Pitfalls in the assessment of murine atherosclerosis

This review provides examples of the fact that different procedures for the measurement of atherosclerosis in mice may lead to interpretation of the extent of atherosclerosis having markedly different biological and clinical significance for humans: 1) aortic cholesterol measurement is highly sensitive for the detection of early and advanced atherosclerosis lesions, but misses the identification of the location and complexity of these lesions that are so critical for humans; 2) the histological analysis of the aortic root lesions in simvastatin-treated and control mice reveals similar lesion morphology in spite of the remarkable simvastatin-induced reduction of the aortic cholesteryl ester content; 3) in histological analyses, chemical fixation and inclusion may extract the tissue fat and also shrink and distort tissue structures. Thus, the method may be less sensitive for the detection of slight differences among the experimental groups, unless a more suitable procedure employing physical fixation with histological sample freezing using optimal cutting temperature and liquid nitrogen is employed. Thus, when measuring experimental atherosclerosis in mice, investigators should be aware of several previously unreported pitfalls regarding the extent, location and complexity of the arterial lesion that may not be suitable for extrapolation to human pathology.

Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice

The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune®, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg·kg-1·day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 ± 4 vs 124 ± 10 mmHg, respectively) or ACh- (maximal response: 51 ± 8 vs 53 ± 5%, respectively) and SNP-induced vasorelaxation (maximal response: 73 ± 6 vs 74 ± 6%, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 ± 59 vs 722 ± 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 ± 12 vs 68 ± 8 µm² x 10³). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.

Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event

Patients with metabolic syndrome are at high-risk for development of atherosclerosis and cardiovascular events. The objective of this study was to examine the major determinants of coronary disease severity, including those coronary risk factors associated with metabolic syndrome, during the early period after an acute coronary episode. We tested the hypothesis that inflammatory markers, especially highly sensitive C-reactive protein (hsCRP), are related to coronary atherosclerosis, in addition to traditional coronary risk factors. Subjects of both genders aged 30 to 75 years (N = 116) were prospectively included if they had suffered a recent acute coronary syndrome (acute myocardial infarction or unstable angina pectoris requiring hospitalization) and if they had metabolic syndrome diagnosed according to the National Cholesterol Education Program/Adult Treatment Panel III. Patients were submitted to a coronary angiography and the burden of atherosclerosis was estimated by the Gensini score. The severity of coronary disease was correlated (Spearman’s or Pearson’s coefficient) with gender (r = 0.291, P = 0.008), age (r = 0.218, P = 0.048), hsCRP (r = 0.256, P = 0.020), ApoB/ApoA ratio (r = 0.233, P = 0.041), and carotid intima-media thickness (r = 0.236, P = 0.041). After multiple linear regression, only male gender (P = 0.046) and hsCRP (P = 0.012) remained independently associated with the Gensini score. In this high-risk population, male gender and high levels of hsCRP, two variables that can be easily obtained, were associated with more extensive coronary disease, identifying patients with the highest potential of developing new coronary events.

Effect of carotid and aortic baroreceptors on cardiopulmonary reflex: the role of autonomic function

We determined the sympathetic and parasympathetic control of heart rate (HR) and the sensitivity of the cardiopulmonary receptors after selective carotid and aortic denervation. We also investigated the participation of the autonomic nervous system in the Bezold-Jarish reflex after selective removal of aortic and carotid baroreceptors. Male Wistar rats (220-270 g) were divided into three groups: control (CG, N = 8), aortic denervation (AG, N = 5) and carotid denervation (CAG, N = 9). AG animals presented increased arterial pressure (12%) and HR (11%) compared with CG, while CAG animals presented a reduction in arterial pressure (16%) and unchanged HR compared with CG. The sequential blockade of autonomic effects by atropine and propranolol indicated a reduction in vagal function in CAG (a 50 and 62% reduction in vagal effect and tonus, respectively) while AG showed an increase of more than 100% in sympathetic control of HR. The Bezold-Jarish reflex was evaluated using serotonin, which induced increased bradycardia and hypotension in AG and CAG, suggesting that the sensitivity of the cardiopulmonary reflex is augmented after selective denervation. Atropine administration abolished the bradycardic responses induced by serotonin in all groups; however, the hypotensive response was still increased in AG. Although the responses after atropine were lower than the responses before the drug, indicating a reduction in vagal outflow after selective denervation, our data suggest that both denervation procedures are associated with an increase in sympathetic modulation of the vessels, indicating that the sensitivity of the cardiopulmonary receptors was modulated by baroreceptor fibers.