The incidence of cardiovascular events is largely reduced in patients with maximally tolerated drug therapy and lipoprotein apheresis. A single-center experience.

AIM: Lipoprotein apheresis (LA) is the elective therapy for homozygous and other forms of familial hypercholesterolemia (FH) and familial combined hypercholesterolemia (FCH), resistant/intolerant to lipid lowering drugs, and hyperlipoproteinemia(a) for which drugs are not available. To assess the effect of LA on the incidence of adverse cardiac or vascular events (ACVE) at the time period of pre-initiation of apheresis and during the LA treatment. METHODS: We collected data of 30 patients (mean age 62 ± 8 years, males 73%), with FH, or FCH and cardiovascular disease on maximally tolerated lipid lowering therapy and LA treatment (median 5 years, interquartile range 3-8 years). Associated hyperlipoproteinemia(a) was present in 16/30 subjects. The LA treatment was performed biweekly as clinically indicated by dextran-sulfate or heparin-induced LDL precipitation apheresis. The ACVE incidence, before and after treatment, was evaluated by statistical analyses. RESULTS: The ACVE incidence occurred before and after the LA treatment inception, were 86 and 15 events respectively. Notably, 6/15 of ACVE were secondary to stent restenosis and 7/15 follow-up events occurred during the first 5 years. The AVCE rates/year were 0.58 and 0.13 respectively (p < 0.001). CONCLUSIONS: Our data confirm long-term efficacy and positive impact of LA on morbidity in patients with FH and FCH and atherosclerotic disease at maximally tolerated lipid lowering therapy.

Modulation of lipid homeostasis in response to continuous or intermittent high-fat diet in pigs.

A high-fat diet is known to induce atherosclerosis in animal models. Dietary factors and timing of atherogenic food delivery may affect plasma lipoprotein content composition and its potential atherogenic effect. Increasingly often, humans spend periods/days eating in a completely unregulated way, ingesting excessive amounts of food rich in oils and fats, alternating with periods/days when food intake is more or less correct. We investigate the effect on lipid homeostasis of a high-fat diet administered either continuously or intermittently. We investigated control pigs receiving standard diet (C, n=7), pigs receiving a high-fat diet every day for 10 weeks (CHF, n=5), and pigs receiving a high-fat diet every other week for 10 weeks (IHF, n=7). IHF animals were shown to have a different lipid profile compared with CHF animals, with a significant increase in high-density lipoproteins (HDL) levels with respect to C and CHF groups. CHF also showed significantly higher values of TC/HDL cholesterol compared with C and IHF. Hepatic expression analysis of genes involved in lipid homeostasis showed an increasing trend of nuclear receptor LXRα along with its target genes in the CHF group and in the IHF group, whereas SREBP2 and LDLr were significantly inhibited. A significant correlation was found between ABCA1 expression and circulating levels of HDL-C. Periodic withdrawals of a high-fat atherogenic diet compared with a regular administration results in a different adaptive response of lipoprotein metabolism, which leads to a significantly higher plasma level of HDL-C and lower TC/HDL-C.

Cellular cholesterol efflux and cholesterol loading capacity of serum: effects of LDL-apheresis.

High LDL-cholesterol (LDL-C) characterizes familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH). LDL-apheresis, used in these patients to reduce LDL-C levels, has been shown to also affect HDL levels and composition. We studied LDL-apheresis effects on six FH and nine FCH subjects' serum capacity to modulate cellular cholesterol efflux, an index of HDL functionality, and to load macrophages with cholesterol. Serum cholesterol efflux capacity (CEC) and macrophage cholesterol loading capacity (CLC) were measured before, immediately after, and two days after LDL-apheresis. The procedure reduced total cholesterol (TC), LDL-C, and apoB plasma levels (-69%, -80% and -74%, respectively), parameters only partially restored two days later. HDL-C and apoA-I plasma levels, reduced after LDL-apheresis (-27% and -16%, respectively), were restored to almost normal levels two days later. LDL-apheresis reduced serum aqueous diffusion (AD) CEC, SR-BI-CEC, and ABCA1-CEC. AD and SR-BI were fully restored whereas ABCA1-CEC remained low two days later. Sera immediately and two days after LDL-apheresis had a lower CLC than pre-LDL-apheresis sera. In conclusion, LDL-apheresis transiently reduces HDL-C levels and serum CEC, but it also reduces also serum capacity to deliver cholesterol to macrophages. Despite a potentially negative effect on HDL levels and composition, LDL-apheresis may counteract foam cells formation.

Upregulation of the immune system in primary hypercholesterolaemia: effect of atorvastatin therapy.

OBJECTIVES: High levels of plasma high sensitivity C-reactive protein (CRP), sensitive to therapy with statins, have been described in hypercholesterolaemia. In vitro evidence shows that CRP activates the complement system, which, in turn, leads to an increased expression of ICAM-1. Our objectives were to verify whether primary hypercholesterolaemia (PHC) is associated with an upregulation of the inflammatory/immune response, and whether this is sensitive to atorvastatin. METHODS AND RESULTS: We examined the levels of sICAM-1, C3, C4 complement fractions in 48 patients with PHC, with (CAD group) or without (No-CAD group) coronary artery disease (CAD) in comparison with a group of 48 healthy controls. The two patient groups were studied before and after atorvastatin therapy. Both hypercholesterolaemic groups showed higher mean values of sICAM-1, C3 and C4 (P < 0.0001) when compared with the controls. The two groups of patients responded differently to atorvastatin therapy. After 3 months, the C3 levels normalized in both groups of patients (P < 0.02 compared with basal values); C4 was greatly reduced only in the CAD group (P < 0.01). After 12 months of therapy, in CAD group C3 mean levels were still significantly lower than baseline values (P < 0.01); a further decrease in the C4 values (P < 0.05 with respect to levels after 3 months of therapy) and also a substantial reduction in sICAM-1 values (P < 0.001 with respect to basal values) were observed. CONCLUSIONS: High plasma values of C3 and C4 in PHC cluster with high values of sICAM-1, distinguish subjects with CAD and could be used to monitor the anti-inflammatory effect of statin therapy in these patients.

Up regulation of C3, C4, and soluble intercellular adhesion molecule-1 co-expresses with high sensitivity C reactive protein in familial hypoalphalipoproteinaemia: further evidence of inflammatory activation.

OBJECTIVE: To test the working hypothesis that inflammation underlying precocious and severe coronary atherosclerotic disease in familial hypoalphalipoproteinaemia (FH) can be mediated by up regulation of the innate immune response. METHODS AND RESULTS: 52 patients with FH were compared with 52 healthy controls with regard to immune system markers such as C reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), C3c, and C4. Patients differed from controls in their significantly lower concentrations of high density lipoprotein cholesterol (30.2 (4.0) v 50.5 (13.6) mg/dl, p < 0.0001) and apolipoprotein A I (113.2 (19.9) v 148.7 (25.1) mg/dl, p < 0.0001) and their higher triglyceride (139.3 (63.2) v 81.4 (41.7) mg/dl, p < 0.0001) and CRP plasma concentrations (median 0.33 mg/dl, range 0.02-4.66 mg/dl v median 0.07 mg/dl, range 0.02-0.85 mg/dl, p < 0.0001), but not in their total cholesterol and low density lipoprotein cholesterol concentrations. Concentrations of protein complement were higher in patients (C3: 150.8 (42.3) v 101.9 (17.4) mg/dl, p < 0.0001; C4: 35.5 (13.6) v 22.8 (6.4) mg/dl, p < 0.0001) and sICAM-1 concentrations were more than double those found in the controls (335.1 (107.5) v 159.5 (78.2) mg/dl, p < 0.0001). CONCLUSIONS: Increased concentrations of sICAM-1, C3c, and C4 co-express with high concentrations of CRP in FH. The lack of signs and symptoms of inflammation in these patients may suggest that the immune response is up regulated as part of the pro-inflammatory mechanisms that are activated in this atherogenic condition.

Plasma cholesterol regulates soluble cell adhesion molecule expression in familial hypercholesterolemia.

BACKGROUND: Hypercholesterolemia is associated with endothelial dysfunction. On the basis of the hypothesis that high plasma cholesterol per se may be a sufficient stimulus to upregulate endothelial adhesiveness and that this phenomenon might be reversible, soluble endothelial leukocyte adhesion molecules (sELAMs) were studied in patients with familial hypercholesterolemia undergoing LDL apheresis. METHODS AND RESULTS: Selective LDL absorption by dextran sulfate columns was used to treat plasma volumes of 6.5 to 9.2 L; after LDL apheresis, total cholesterol, LDL cholesterol, apolipoprotein B, triglycerides, and lipoprotein(a) levels were reduced by 74%, 82%, 79%, 56%, and 86%, respectively. Soluble intercellular adhesion molecule-1 (sICAM-1) and sELAM- were measured before, immediately after, and 2 and 6 days after LDL apheresis. Basal sICAM-1 and sELAM-1 values were higher than in healthy control subjects. After LDL apheresis, they decreased (P<.0001 and P<.0004, respectively); their removal by extracorporeal circulation components was excluded. Individual pretreatment and posttreatment values of sICAM-1 and sELAM-1 were positively correlated (P<.0001 and P<.001, respectively) with total cholesterol; their rebound curves showed patterns similar to the total cholesterol rebound curve but not to the triglyceride and lipoprotein(a) curves. CONCLUSIONS: In the absence of changes in clinical chemical parameters, tumor necrosis factor-alpha, interleukin-6, and acute-phase reactant proteins, these results confirm in a clinical setting the upregulation of endothelial adhesiveness observed in experimental hypercholesterolemia and suggest a direct role for cholesterol in regulating this phenomenon, at least in familial hypercholesterolemia.

Effect of a reduced-fat diet with or without pravastatin on glucose tolerance and insulin sensitivity in patients with primary hypercholesterolemia.

Pharmacological treatment of hyperlipidemia may be associated with deterioration of glucose tolerance. We randomized 20 nonobese patients with primary familial hypercholesterolemia (serum total cholesterol 7.8 +/- 0.4 mM, triglycerides 1.4 +/- 0.2 mM) to an isocaloric, reduced fat (< 30%) low-cholesterol (200 mg/day) diet with placebo or pravastatin (40 mg/day). Oral glucose tolerance, endogenous insulin response to glucose, insulin sensitivity (determined by the euglycemic insulin clamp technique), hepatic glucose production (by the tritiated glucose technique), and substrate utilization (by indirect calorimetry) were measured at baseline and after 8 weeks of treatment. Ten normocholesterolemic healthy subjects, matched to the patients by age, sex, and body weight, served as the control group. Diet alone (with no change in body weight) was associated with a significant 15% decrease in both serum low density lipoprotein (LDL)-cholesterol and triglycerides (p < 0.001 for both), and a slight decrease in high density lipoprotein (HDL)-cholesterol concentrations, paralleled by reductions in Apo B, C2, C3, and E levels (p < 0.05 or less). The addition of pravastatin led to a significantly larger reduction in LDL-cholesterol (30%, p < 0.05) and an 8% increase (p < 0.02) in total HDL-cholesterol concentrations. Accordingly, the ratio of LDL:HDL cholesterol (which was 60% higher than in controls at baseline) remained unchanged in the placebo-diet group whereas it was restored to normal in the pravastatin-diet group. Glucose tolerance, insulin response, insulin-induced inhibition of hepatic glucose production and lipolysis, and insulin-mediated glucose uptake and oxidation were all slightly but not significantly improved after treatment, with no significant differences between pravastatin and placebo. In nonobese patients with primary hypercholesterolemia, pravastatin treatment in combination with an isocaloric, reduced-fat diet leads to a marked reduction in LDL-cholesterol and triglycerides levels and a normalization of the LDL:HDL ratio without affecting glucose tolerance or insulin sensitivity.

Behavior of Lp(a) and apoproteins (A1, B, C2, C3, E) during and after therapy with simvastatin.

The efficacy of simvastatin in reducing plasma cholesterol is well documented. Other molecules within the apo lipoprotein family, particularly lipoprotein (a) Lp(a), have recently been found to have a predictive and/or causative role in atherosclerosis. Based on these considerations, we studied 15 patients affected by primary hypercholesterolemia to evaluate the effect of simvastatin in Lp(a) and apoprotein plasma levels (A1, B, C2, C3, E), in addition to the classic lipid parameters. Clinical and laboratory parameters were evaluated before therapy, after 12 weeks of therapy, and after 5 weeks of drug withdrawal. Simvastatin therapy produced a significant reduction in total cholesterol (CH) and LDL-CH (p < 0.0001), and a significant increase in HDL-CH (p < 0.01); no variation was observed in triglyceride (TG) levels. Simvastatin therapy further showed a significant decrease in apoC2 (p < 0.05), the apo C2/C3 ratio (p < 0.01), and apoE (p < 0.01), as well as a significant increase in Lp(a) plasma levels (p < 0.05). All of the parameters studied returned to pretreatment values 5 weeks after drug withdrawal; only HDL-CH persisted above the values reached during therapy. Our data agree with those documenting the beneficial effect of simvastatin in greatly decreasing CH and LDL-CH, but point out the need for further studies on the long-term effect of simvastatin on apoprotein molecules, such as on Lp(a), in order to fully establish its role in the secondary prevention of atherosclerosis.

[Effects of simvastatin on blood levels of lipoprotein (a)]. / Effetti della simvastatina sui livelli plasmatici della lipoproteina (a).

The efficacy of Simvastatin to reduce plasma cholesterol is well documented. Other molecule within the lipo-lipoprotein family, such as, particularly, lipoprotein (a) -Lp(a)-, have been recently found to have a predictive and/or causative role in atherosclerosis. Based on the above consideration, we studied 20 patients (7 females and 13 males), mean age 52.4 +/- 14.2 years, affected by primary hypercholesterolemia to evaluate the effect of simvastatin on Lp(a), in addition to the classic lipidic parameters. Five weeks after suspension of lipid-lowering drugs and on a normal caloric-fat diet, were given 20 mg simvastatin/day for 12 months. Clinical and laboratory parameters, cholesterol (CH), triglycerides (TG), high density and low density lipoprotein cholesterol (HDL-CH and LDH-CH) measured enzymatically, apoproteins A1, B measured radial immunodiffusion technique and Lp(a) measured as apoprotein(a) with immunoradiometric assay and were evaluated before therapy and after 12 months of therapy. Simvastatin determined a significant reduction in total cholesterol and cholesterol-LDL (CH 327.7 +/- 44.4 vs 255.5 +/- 37.3, p < 0.0001; LDL-CH 257.1 +/- 60.9 vs 183.8 +/- 46.9, p < 0.0001) and a significant increase in HDL-CH (36.7 +/- 5.9 vs 40.2 +/- 5.7, p < 0.005); no variation was observed in triglycerides (TG) levels. Simvastatin therapy further determined a significant increase in Lp(a) plasma levels (43.8 +/- 25.6 vs 50.5 +/- 28.0, p < 0.02). The our data, in agreement with those documenting the beneficial effect of Simvastatin in greatly decreasing CH and LDL-CH, but point out the need for further studies concerning the long-ter effect of simvastatin on Lp(a), in order to fully establish its role in the secondary prevention of atherosclerosis.

[Effects of pravastatin on serum lipids, apoproteins, and lipoprotein (a) in primary hypercholesterolemia]. / Effetti della pravastatina sui lipidi, apoproteine e lipoproteina (a) sierici nella ipercolesterolemia primitiva.

The efficacy of pravastatin as reducing plasma cholesterol, LDL-CH and Apo B is widely proved. Other molecules within the Apolipoprotein family are recently emerging to have a predictive and/or causative role in atherosclerosis such as particularly Lp(a). The aim of this study was to evaluate the effects of pravastatin therapy in patients affected by primary hypoercholesterolemia on apoprotein and Lp(a) plasma levels. We investigated the effects of pravastatin on 15 patients, seven female and eight male patients, mean age 50.23 +/- 17.2 (range 21-71 years) with primary hypercholesterolemia, of which 7 patients affected by familial hypercholesterolemia and 8 patients by polygenic hypercholesterolemia, were selected. Five weeks after suspension of lipid-lowering drugs and on a normocaloric-fat diet, were given 20 mg pravastatin/day for 12 weeks. The following parameters were measured basally, on the 6th week and the 12th week on pravastatin therapy and after five weeks from drug withdrawal: cholesterol (CH), triglicerides (TG), high density and low density lipoprotein cholesterol (HDL-CH and LDH-CH) measured enzymatically, apoproteins A1, B, C2, C3, E measured radial immunodiffusion technique (RID) and Lp(a), measured as apoprotein(a) with immunoradiometric assay (RIA). Our data confirm pravastatin efficacy in decreasing CH (from 305.6 +/- 43.4 mg/dl to 266.2 +/- 47.7 mg/dl, p < 0.01) LDL-CH (from 223.9 +/- 56.4 mg/dl to 187.2 +/- 59.8 mg/dl, p < 0.01) and Apo B (from 170.4 +/- 27.5 to 152.4 +/- 25.2, p < 0.02); non influence was observed on HDL-CH and apoproteins A1, C2, E and Lp(a). Pravastatin determined a significant increase only on Apo C3 (from 8.35 +/- 2.7 to 10.3 +/- 3.1, p < 0.04). The above data confirm the beneficial effect of pravastatin in greatly decreasing CH and LDL-CH considered as major risk factors for coronary artery disease, but also point to a role of pravastatin in regulating the apoproteins equilibrium, an aspect that surely merits further studies.

Insulin sensitivity in familial hypercholesterolemia.

Insulin resistance is found in association with obesity, non-insulin-dependent diabetes mellitus, and essential hypertension, which are all risk factors for atherosclerotic cardiovascular disease. Furthermore, hyperinsulinemia has been reported in familial combined hyperlipoproteinemia and endogenous hypertriglyceridemia. Finally, relatively high serum triglyceride and low high-density lipoprotein (HDL) cholesterol concentrations invariably accompany hyperinsulinemia. Whether insulin sensitivity is affected by the isolated presence of high levels of serum low-density lipoprotein (LDL) cholesterol has not been clearly established. We studied 13 subjects with heterozygous familial hypercholesterolemia (FHC) and 15 normocholesterolemic subjects selected to be free of any other known cause of insulin resistance. Thus FHC patients and controls had normal body weight and fat distribution, glucose tolerance, blood pressure, and serum triglyceride and HDL cholesterol concentrations, but were completely separated on plasma LDL cholesterol concentrations (6.05 +/- 0.38 v 3.27 +/- 0.15 mmol/L, P < .0001). Fasting plasma levels of glucose, insulin, free fatty acids (FFA), and potassium and fasting rates of net carbohydrate and lipid oxidation were superimposable in the two study groups. During a 2-hour euglycemic (approximately 5 mmol/L) hyperinsulinemic (approximately 340 pmol/L) clamp, whole-body glucose disposal rates averaged 30.4 +/- 2.3 and 31.1 +/- 3.0 mumol.kg-1 x min-1 in FHC and control subjects, respectively (P = 0.88). The ability of exogenous hyperinsulinemia to stimulate carbohydrate oxidation and energy expenditure and suppress lipid oxidation and plasma FFA and potassium levels was equivalent in FHC and control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased permeability of hamster microcirculation to glycosylated albumin.

The permeability of the microcirculation to native and glycosylated albumin was tested in 25 non-diabetic Syrian hamsters. The microvasculature of the cheek pouch was studied by a fluorescent video-microscopy technique after the animals had been injected with fluorescent native or glycosylated albumin or with both. Native albumin remained in the cheek pouch microvasculature, whereas glycosylated albumin leaked out of the microvascular bed along the postcapillary and collecting venules. The extravascular leakage of glycosylated albumin, probably due to its electrical or conformational change, may represent the initial event in the development of diabetic microangiopathy.

The clinical usefulness of glycated hemoglobin in monitoring diabetes mellitus: a long-term study.

To assess the long-term clinical usefulness of measuring glycated hemoglobin (Hb A1), we carried out a two-year longitudinal study involving 234 diabetic patients (116 males, 118 females; 139 with type I diabetes mellitus, 95 with type II). Hb A1 values correlated significantly (p less than 0.001) with a score index based on plasma glucose in a specimen collected after overnight fasting, and urinary glucose, and ketones in a 24-h specimen. However, we found that one of every three well-controlled patients (both type I and II subjects) had high values for Hb A1. Among poorly controlled patients, only those with "brittle" diabetes had good values for Hb A1.

A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins.

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.