It is known that high-cholesterol diet impairs coronary vasodilatation in animal models of athe-rosclerosis irrespective of overt pathology. We evaluated the specific role of LDL and HDL on adenosine-elicited coronary vasodilatation after short time (10 weeks) high-cholesterol diet in pigs. Nineteen pigs on standard (C), atherogenic (HF) and alternate standard or atherogenic diet every other week (IHF) underwent left coronary angiography and flow (CFR) measurement during intracoronary adenosine injection. Total cholesterol, HDL, LDL, Apo lipoprotein A-1, IL-6, TNF-α and ICAM-1 were measured and histology of coronary samples was performed. IHF and HF show comparable intimal thickening of lesions, similar cholesterol (598.4 ± 198.2 and 633.2 ± 83.5 mg/dL) and LDL (502.6 ± 193.7 and 576.1 ± 83.2), while HDL is double in IHF group (88.3 ± 6.4 vs 46.4 ± 18.7 p < 0.0001). Vasodilation is reduced in HF (CFR = 1.6 ± 0.2, p < 0.001) as compared to C (2.6 ± 0.4), whilst it is preserved in IHF (2.7 ± 0.4). CFR and HDL values of all hypercholesterolemia cases are positively correlated (r = 0.88, p < 0.001). No relation with cytokines/cell adhesion markers is present. These findings suggest for the first time that elevation of plasma HDL level counteracts LDL related vasodilation impairment during coronary atherogenesis in swine.
The effect of hypercholesterolemia (total cholesterol and its lipoprotein fractions) on coronary microvascular function and anatomy has been investigated in many former [
In this study we aimed to evaluate the effect of plasma lipoprotein profile, modified by a different timing of cholesterol supplemented diet, on coronary vasodilator capability in-vivo, as assessed by coronary pressure-flow measurements during intracoronary adenosine injection and on the extent of early stage artery disease.
The protocol has been performed in 21 castrated male domesticpigs (3 months old, body weight 23 - 46 kg). The animals were allocated into three groups: controls on standard diet (C, n = 7, body weight 31 ± 2 kg); animals fed daily on cholesterol supplemented high fat diet (HF, n = 7, body weight 36 ± 1 kg); pigs on alternate diet, fed on either standard or cholesterol supplemented high fat diet every other week (IHF, n = 7, body weight 42 ± 2 kg) [
Standard diet (4.2% vegetable fat, 15% proteins, 54% carbohydrates, 4.8% fibres, 22% vitamins minerals moisture) has been administered for 10 weeks to control animals (C).
High lipid diet (27% fat - 4% cholesterol, 20% lard, 3% oil - 17.5% proteins, 33% carbohydrates, 7% fibres, 15.5% vitamins minerals and moisture, 1.5% Na Cholate) has been administered to HF animals for 10 weeks consecutively. In IHF animals, the same diet has been administered every other week for 10 weeks.
Total energy from standard diet was 3170 Kcal/kg with Fatty Energy of 378 Kcal, 12% of total energy. Total energy from hypercholesterolemic diet was 4450 Kcal/kg with Fatty Energy of 2430 Kcal, 54.6% of total energy.
The anesthesia has been induced by intramuscular administration of 10 mg/kg of Zoletil® and 0.05 mg/kg of atropine and maintained with gas (isofluorane, nitrous oxide and oxygen) together with 5 mg/kg/h of propofol intravenous infusion. Animals were mechanically ventilated (respiratory volume: 150 ml/kg/min, respiratory rate: 15 cycles/min). The last day of the last high-fat diet week corresponded to the final acute experiment.
Coronary angiography and coronary pressure-flow measurements were performed in all except two HF cases could not complete the hemodynamic study with pressure-flow assessment due to incurring fatal cardiac arrhythmias during angiography.
Under anesthesia, a guide catheter was advanced into the ostium of left main coronary artery (LMCA) through- out an introducer placed into the left carotid artery. Standardized projections have been used for image acquisitions, which have been set at 15 frames/sec with the magnification commonly used for coronary angiography (Cardio 945, SIAS, Bologna, Italy). The distance source of detector was 97 cm. 3 - 7 ml of contrast liquid was injected and images obtained with an X-ray system. Biplane X-ray coronary angiography (RAO 20˚, LAO 20˚) was performed to confirm catheter position in artery. Pressure and flow measurements have been obtained by coronary Doppler flow velocimetry catheter (ComboMap System, Volcano Corporation, San Diego, CA, USA) which can run the two values with a single probe. The catheter was positioned in the proximal portion of left anterior descending artery (LAD) upstream the first diagonal branch under angiography control, for monitoring and recording. To asses microcirculatory vasodilator function, a supramaximal dose of adenosine (3 mg) was administered as an intracoronary bolus of 1 ml, slowly injected through the Doppler flow-pressure catheter guide; 3 injections were repeated at 20 min interval to ensure maximal flow response assessment. Values of pressure and flow, under baseline condition and during adenosine injection, were recorded. Only stable recordings, free of ectopic beats and/or arrhythmias, with well-defined blood flow velocity pattern for at least 30 seconds, have been utilized for flow measurements. Coronary Flow Reserve (CFR), as an index of the vasodilator response of LAD-dependent territory to intracoronary adenosine injection, is calculated as the ratio between Average Peak Velocity (APV) recorded after adenosine injection and that of baseline pre-injection state. APV values are also normalized for inter-animal and inter-groups coronary pressure differences and an arbitrary coronary resistance index (CRI) was calculated, equal to Mean Coronary Pressure/APV (mmHg/cm/sec).
For heart rate data acquisition, signals were collected by a Gould amplifier (Gould TA 5000, Cleveland, OH, USA) and elaborated by Pulse Viewer, an in-house developed software. Controls and diet-treated animals have been sacrificed after completion of the hemodynamic study by KCl i.v. bolus injection.
Animal instrumentation and experimental protocol were approved by the Animal Care Committee of the Italian Ministry of Health (Protocol number: 06/2009-B-2009/01/26) according with the Italian Law (DL-116, Jan. 27, 1992) and National Institute of Health Guide for the Care and Use of Laboratory Animals.
At the end of the in-vivo study the entire heart was excised, washed in isotonic physiologic solution, examined macroscopically and immersed in 10% buffered formalin for tissue fixation (7 - 10 days). Thereafter, arterial samples were harvested from the fixed heart: transverse segments were obtained from left main coronary artery (1 segment of about 10 mm), LAD (6 - 7 segments of 10 mm) and left circumflex artery (3 segments of 10 mm) for routine histologic processing and paraffin embedding. After standard Haematoxylin and Eosin, Weigert van Gieson (elastic laminas) and Mallory trichrome (collagen) staining, consecutive sections from each coronary segment were examined under light microscopy (Olympus BX43, Olympus, Milan, Italy) from 2× to 40× original magnification and digitized by a video system (Olympus DP20 camera, Olympus, Milan, Italy) interfaced to a computer with dedicated software (CellSens Dimension, Olympus, Milan, Italy) for image acquisition and mo- rphometric analysis. Pathologic changes of the coronary wall were classified according to Stary stages of atherosclerotic lesions [
Total cholesterol, High Density Lipoprotein (HDL), triglycerides (TG), albumin, glucose, aspartate aminotransferase (ALT) and alanine aminotransferase (AST) were measured by standard enzymatic techniques (Synchron CX9 Pro, Beckman Coulter Inc., Fullerton, CA, USA); Low density lipoprotein (LDL) was calculated according to Friedewald et al. [
Analysis of total and fractional GGT was performed, as previously described [
IL-6, TNF-α and ICAM-1 were purchased by Abcam (Cambridge, UK), while oxidized LDL (oxLDL) was a product of Antibodies-Online (Atlanta, GA, USA).
End-diet measurements include 7 controls, 5 HF and 7 IHF pigs. Statistical comparison of the three groups has been performed using ANOVA with Fisher LSD test. Linear regression analysis was also used when appropriate.
Data are expressed as means ± SD (standard deviation) and p < 0.05 is considered as statistically significant.
Bio-humoral profile of all cases of the three experimental groups is summarized in
High fat diet-treated cases, besides markedly increased plasma lipid levels, show also significantly higher mean values of hematocrit (HF), albumin (HF and IHF) and AST (HF), though with a wide intra group variability. Greater intra group variations are also present in inflammatory markers and ICAM-1 average values, without statistically significant differences among groups.
Eight weeks high-fat diet induced significantly higher level of total cholesterol (p < 0.0001), LDL (p < 0.0001), Apo lipoprotein A1 (p < 0.0005) and oxLDL in HF and IHF as compared to C (
HDL, Total Cholesterol/HDL and LDL/HDL ratio were significantly higher in IHF and HF as compared to C group. The values of oxLDL comply with the marked increase of LDL, both in IHF and in HF diet-treated group. IHF cases showed a HDL concentration about double that of HF cases and a markedly lower Total Cholesterol/HDL ratio. Inflammatory markers and ICAM-1 displayed a marked intra-group variability with no statistical inter-group difference.
In both HF and IHF groups, initial coronary lesions Type I to III (according to Stary stages [
Frequency distribution and morphometric data (mean intimal thickness and mean intima to media thickness ratio) of all lesions are summarized in
Despite a comparable global incidence of lesions and despite similar average intimal thickness and intima to media thickness ratio, a relatively more advanced stage of coronary lesions is evidenced in HF relative to IHF cases (higher Stary type III and lower type I lesions incidence).
Coronary angiography in C group showed normal coronary vessel profile and diameter without any abnormalities. After 10 weeks diet, none of the 12 pigs treated by high fat diet showed LAD lumen reduction >10% at coronary angiography examination. Left coronary angiography evidenced mild irregularities of the coronary profile in 4/5 HF cases and a similar degree of lumen defects in all IHF cases (
A typical tracing of coronary pressure and flow recorded during intracoronary adenosine injection is shown in
As a consequence of these different effects on LAD flow velocity also CFR ratio, as an index of adenosine related increment of myocardial perfusion, is significantly lower in HF compared to IHF group (p < 0.001).
CRI and CFR values during adenosine injection were correlated to the corresponding HDL concentration of each IHF and HF case (
Moreover, a statistical inverse correlation between CFR and Total Cholesterol/HDL ratio was also evidenced
Biohumoral markers | Groups | p | ||||
---|---|---|---|---|---|---|
C (7) | HF (7) | IHF (7) | C vs HF | C vs IHF | HF vs IHF | |
Total Cholesterol (mg/dL) | 63.3 ± 4.2 | 633.2 ± 83.6 | 598.4 ± 198.2 | <0.0001 | <0.0001 | ns |
HDL Cholesterol (mg/dL) | 25.8 ± 3.8 | 46.4 ± 18.7 | 88.3 ± 6.4 | 0.016 | <0.0001 | <0.0001 |
LDL Cholesterol (mg/dL) | 27.5 ± 3.0 | 576.1 ± 83.2 | 502.6 ± 193.7 | <0.0001 | <0.0001 | ns |
Total Cholesterol/HDL ratio | 2.5 ± 0.3 | 15.2 ± 5.0 | 6.7 ± 2.0 | <0.0001 | 0.045 | 0.0003 |
LDL/HDL ratio | 1.1 ± 0.2 | 14.0 ± 5.0 | 5.6 ± 2.0 | <0.0001 | 0.032 | 0.0003 |
Apolipoprotein A1 (mg/dL) | 25.1 ± 5.0 | 61.7 ± 13.0 | 52.6 ± 4.7 | <0.0001 | <0.0001 | ns |
oxLDL (UI/L) | 5.3 ± 5.2 | 31.9 ± 4.2 | 28.9 ± 9.4 | 0.0002 | 0.0002 | ns |
Triglycerides (mg/dL) | 50.3 ± 10.5 | 54.0 ± 16.8 | 33.7 ± 12.7 | ns | ns | 0.023 |
Leukocytes (*1000 n/mL) | 12.9 ± 0.4 | 14.7 ± 2.4 | 15.9 ± 3.6 | ns | ns | ns |
Haematocrit (%) | 26.3 ± 2.5 | 31.6 ± 4.2 | 27.8 ± 1.7 | 0.013 | ns | 0.038 |
Glucose (mg/dL) | 72.0 ± 31.0 | 90.4 ± 38.2 | 65.8 ± 13.4 | ns | ns | ns |
Albumin (g/dL) | 2.0 ± 0.2 | 2.6 ± 0.4 | 2.5 ± 0.2 | 0.009 | 0.019 | ns |
AST1 (UI/L) | 29.7 ± 7.6 | 92.0 ± 36.3 | 47.4 ± 10.0 | <0.0001 | ns | 0.003 |
ALT2 (UI/L) | 51.2 ± 5.0 | 52.0 ± 7.0 | 53.6 ± 13.0 | ns | ns | ns |
GGT3 (UI/L) | 69.5 ± 27.3 | 81.6 ± 27.4 | 50.1 ± 33.7 | ns | ns | ns |
ICAM-1 (ng/mL) | 750.1 ± 314.5 | 812.1 ± 141.2 | 789.6 ± 236.7 | ns | ns | ns |
IL-6 (pg/mL) | 9.2 ± 18.4 | 71.4 ± 124.7 | 79.8 ± 110.6 | ns | ns | ns |
TNF-α (pg/mL) | 1251.1 ± 1320.2 | 622.0 ± 1181.9 | 566.6 ± 988.6 | ns | ns | ns |
1AST, aspartate amino transferase, 2ALT, alanine amino transferase, 3GGT, gamma-glutamyltransferase.
both in all high-cholesterol diet treated cases as well as in all pigs, controls included (
A significant correlation with HDL values is also present in diet-treated pooled cases when APV values instead of CFR values are considered, suggesting a direct effect of HDL concentration also on absolute coronary flow velocity levels reached during adenosine administration (
On the other hand, no significant correlations between any hemodynamic parameter (CRI, CFR and APV) and inflammatory markers (IL-6, TNF-α) or ICAM-1 could be evidenced.
Groups | ||||
---|---|---|---|---|
HF (7) | IHF (7) | |||
Total segments (n°) | 72 | 98 | ||
Nr. | % | Nr. | % | |
Segments with lesions | 24 | 33,3 | 34 | 35 |
Type I | 7 | 29 | 18 | 53* |
Type II | 13 | 54 | 16 | 47 |
Type III | 4 | 17 | 0 | 0* |
Mean Intimal Thickness (IT) (µm) | 205 ± 120 | 206 ± 170 | ||
Mean Intima/Media Thickness (IMT) ratio | 0.6 ± 0.44 | 0.5 ± 0.38 |
Cholesterol supplemented high fat diet significantly increases plasma lipids and modifies the concentration of some biohumoral and inflammatory plasma factors.
The different timing of high cholesterol diet supply, either continuous or alternate, has a significant impact on
Hemodynamic parameters | Groups | p | ||||
---|---|---|---|---|---|---|
C (7) | HF (5) | IHF (7) | C vs HF | C vs IHF | HF vs IHF | |
Baseline SP4 (mmHg) | 110 ± 9 | 120 ± 11 | 120 ± 17 | ns | ns | ns |
Adenosine SP (mmHg) | 104 ± 9 | 113 ± 7 | 118 ± 17 | ns | ns | ns |
Baseline DP5 (mmHg) | 89 ± 15 | 102 ± 9 | 95 ± 16 | ns | ns | ns |
Adenosine DP (mmHg) | 83 ± 11 | 95 ± 5 | 91 ± 15 | ns | ns | ns |
Baseline MP6 (mmHg) | 96 ± 12 | 108 ± 8 | 103 ± 16 | ns | ns | ns |
Adenosine MP (mmHg) | 90 ± 9 | 101 ± 5 | 100 ± 16 | ns | ns | ns |
Baseline IPV7 (cm/sec) | 46 ± 16 | 29 ± 4 | 33 ± 9 | 0.004 | ns | ns |
Adenosine IPV (cm/sec) | 89 ± 29 | 44 ± 12 | 72 ± 11 | 0.001 | ns | 0.028 |
Baseline APV8 (cm/sec) | 18 ± 6 | 15 ± 2 | 15 ± 3 | ns | ns | ns |
Adenosine APV (cm/sec) | 47 ± 17 | 24 ± 4 | 41 ± 9 | 0.002 | ns | 0.023 |
Baseline HR9 (bpm) | 93 ± 18 | 105 ± 15 | 100 ± 21 | ns | ns | ns |
Adenosine HR (bpm) | 91 ± 20 | 105 ± 14 | 97 ± 18 | ns | ns | ns |
Baseline CRI10 (mmHg/cm/sec) | 5.9 ± 2.4 | 7.4 ± 1.2 | 7.0 ± 1.6 | ns | ns | ns |
Adenosine CRI (mmHg/cm/sec) | 2.2 ± 1.0 | 4.4 ± 0.8 | 2.5 ± 0.5 | <0.001 | ns | <0.001 |
CFR11 | 2.6 ± 0.4 | 1.6 ± 0.2 | 2.7 ± 0.4 | <0.001 | ns | <0.001 |
4SP, Systolic Pressure; 5DP, Diastolic Pressure; 6MP, Mean Pressure; 7IPV, Instantaneous Peak Velocity; 8APV, Average Peak Velocity; 9HR, Heart Rate; 10CRI, Coronary Resistance Index (calculated as MP/APV ratio); 11CFR, Coronary Flow Reserve (calculated as Adenosine APV/Baseline APV ratio).
HDL concentration level (about double in IHF, compared to HF cases), on Total Cholesterol/HDL ratio (more than double in HF compared to IHF group) as well as on TG concentration, significantly lower in IHF group.
The amount of circulating LDL is reflected by a higher plasma concentration of oxLDL, with about sixfold increase compared to controls [
A higher elevation (about tenfold) of total cholesterol and its fractions was observed in our experiments compared to other studies where hypercholesterolemic diets of similar fat and cholesterol composition were used in swine models of atherogenesis [
The short duration of high cholesterol diet can also account for the minor atheroprotective effect of elevated HDL in IHF group where no a significant difference was observed in overall coronary morph metric indexes despite relatively milder lesion stage.
Although the protective effect of HDL levels on endothelial-dependent vasodilation in hypercholesterolemia has been reported in humans and animal models [
It is known that the major vasodilator effect of adenosine is mediated by direct stimulation of A2-adenosine receptors on vascular smooth muscle cells, through cyclic adenosine monophosphate increase. Although adenosine has been used frequently in animal as well as in human studies to evaluate endothelium-independent vasodilation, it acts also as an endothelial-dependent vasodilator, both through flow-mediated dilation [
In our study, high fat diet is able to modulate the haemodynamic parameters. Mild increases of coronary pressure and heart rate are observed both at baseline and during adenosine administration, whilst a markedly lower coronary blood flow velocity is measured in the LAD of HF cases, in response to adenosine, indicating a consistent arteriolar dysfunction. These findings suggest a vasoconstricting effect generated by elevated LDL and oxLDL plasma levels, formerly attributed to abolished Kv channel contribution to adenosine-mediated relaxation due to its impaired activation [
A generalized vasoconstricting effect of elevated LDL and oxLDL plasma levels could not be excluded and can lead to an increased coronary perfusion pressure and an incorrect estimation of coronary vasodilator response to adenosine when absolute (APV) and relative (CFR ratio) flow velocity measurements are considered. For this reason, a coronary resistance index, i.e. the ratio of mean coronary perfusion pressure to mean coronary flow velocity was adopted as a more adequate estimate of coronary vasodilator function. A significant decrease of CRI is elicited by adenosine injection in IHF, where average HDL concentration is double, but not in HF group, despite similar LDL and oxLDL plasma levels.
Within the limits of the animal model, such as the duration of atherogenic diet, our data support the hypothesis that HDL plasma elevation is able to attenuate hypercholesterolemia related impairment of adenosine mediated metabolic vasodilation, despite supramaximal total, LDL and oxLDL cholesterol plasma levels. This effect is independent of preatherosclerotic changes observed in the epicardial coronary arteries, as HF and IHF cases have comparable histomorphometric features of left coronary artery lesions.
Moreover, HDL plasma concentration seems also able to modulate the extent of coronary arteriolar vasodilatation as suggested by the direct and strong relation between CRI and HDL plasma values within a wide range (from 20 to 100 mg/dL) in all high fat diet treated animals. In a previous report, CFR measured after a moderate dose of intracoronary adenosine (1 mcg/Kg) in hypercholesterolemic Ossabaw pigs resulted unchanged and arteriolar A2B receptors were found reduced, suggesting that the mechanisms mediating coronary arteriolar dilation to adenosine are altered in early-stage metabolic syndrome prior to overt decrements in coronary vasodilator reserve [
Moreover, the inverse relation between coronary vasodilator response and Total Cholesterol/HDL ratio in high fat diet treated animals suggests that the relative amount of LDL has a negative impact on adenosine-me- diated vasodilation, as reported in previous clinical studies [
Our observational study supports the view that circulating HDL lipoprotein fraction is a major protective factor of coronary microcirculatory function in hypercholesterolemia.
The protective effect of HDL on both endothelial function and global arteriolar vasodilator capacity is a relevant clinical issue in cardiovascular disease since its potential therapeutic application widens from atherosclerosis [
These observations underscore the role of HDL raising approaches as effective intervention for slowing progression or favoring regression of atherosclerosis.
In conclusion, key results of this study are:
1) Coronary vasodilator response to adenosine is not impaired despite markedly elevated total cholesterol, LDL and oxLDL plasma levels when a concomitant and disproportionate elevation of the HDL fraction is induced by diet manipulation, in absence of significant inflammation marker changes and at equivalent coronary pathology stage.
2) A strong inverse correlation between adenosine-induced coronary resistance decrease and HDL cholesterol fraction, over a wide range of plasma values, is evidenced in all hypercholesterolemic pigs, suggesting that HDL protection of coronary vasodilator function may be considered concentration-dependent.
The study was funded by a grant from ARTreat project, FP7-ICT-2007 (grant agreement 224297).
A special thanks goes to Dr. Daniele Panetta for helpful discussion of the results.