Hydroxychloroquine increased cholesterol transfer to high-density lipoprotein in systemic lupus erythematosus: A possible mechanism for the reversal of atherosclerosis in the disease.

INTRODUCTION: The beneficial effect of hydroxychloroquine (HCQ) in decreasing LDL levels on Systemic Lupus Erythematosus (SLE) is well defined. The influence of this drug on HDL levels is still under debate and information about its effect on cholesterol reverse transport is lacking. OBJECTIVE: To evaluate the effects of HCQ on HDL levels and the transfer of lipids to this lipoprotein in SLE. METHODS: Nineteen SLE patients using only HCQ (SLE WITH HCQ), 19 SLE patients without any therapy (SLE WITHOUT THERAPY), and 19 healthy controls (CONTROL) were included. All three groups were premenopausal women age- and gender-matched. Serum lipids and apolipoproteins were determined by commercial kits. An in vitro transfer of four lipids (14C-Phospolipid, 3H-Cholesteryl ester, 3H-Triglyceride, and 14C-Unesterified cholesterol) from a radioactively labeled nanoemulsion donor to HDL was performed in all participants. RESULTS: Groups had comparable mean age, weight, height, BMI(body mass index), and waist circumference (p > .05). Mean HDL levels were higher in SLE WITH HCQ group compared to SLE WITHOUT THERAPY(58.37 ± 14.04 vs 49.79 ± 8.0 mg/dL; p < .05) but lower than CONTROL (58.37 ± 14.04 vs 68.58 ± 9.99 mg/dL; p < .05). Total cholesterol (TC) and LDL levels were also significantly lower in SLE WITH HCQ compared SLE WITHOUT THERAPY(148.16 ± 16.43 vs 167.11 ± 30.18 mg/dL; p < .05, 75.05 ± 22.52 vs 96.05 ± 25.63 mg/dL; p < .05) and CONTROL (148.16 ± 16.43 vs 174.11 ± 23.70 mg/dL; p < .05, 75.05 ± 22.52 vs 88.53 ± 20.24 mg/dL; p < .05). The in vitro lipid transfer to HDL study revealed a significant difference among the three groups (p = .002) with a higher transfer of unesterified cholesterol(UC) in SLE WITH HCQ compared to SLE WITHOUT THERAPY(5.40 ± 1.05% vs. 4.44 ± 1.05%; p < .05). The latter was significantly decreased compared to CONTROL (5.40 ± 1.05% vs. 5.99 ± 1.71%; p < .05).The percentages of transfer of triacylglycerol (4.93 ± 0.69% vs. 4.50 ± 0.69% vs. 5.14 ± 1.01%; p = .054), esterified cholesterol (5.24 ± 0.70% vs. 4.96 ± 0.89% vs. 5.69 ± 1.27%; p = .079), and phospholipid (15.67 ± 1.03% vs. 15.34 ± 1.44% vs. 16.47 ± 1.89%; p = .066) were similar among groups. CONCLUSION: The present study is the first to demonstrate that HCQ promoted a higher transfer of unesterified cholesterol which may account for the increased HDL levels in lupus patients under HCQ. This desirable effect may underlie the reported reduced atherosclerosis in SLE.

Novel Approach for Bone Marrow Transplantation Conditioning in Acute Myelogenous Leukemia not Responding to the Induction Therapy Using Etoposide Carried in Lipid Core Nanoparticles: A Pilot Clinical Study.

Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for acute myelogenous leukemia (AML) not responding to induction therapy. It is a therapeutic choice for the blast phase of chronic myelogenous leukemia (CML-BP) in patients failing to respond to tyrosine kinase inhibitors (TKIs). Lipid core nanoparticles (LDEs) concentrate severalfold more in blast cells than in corresponding normal cells. Incorporation of anticancer drugs to LDE formulations increases the pharmacologic action and decreases the toxicity. We tested a drug-targeting system, LDE-etoposide plus total body irradiation (TBI; 1200 cGy dose), in 13 patients with AML not responding to the induction therapy and in 2 patients with CML-BP refractory to second-generation TKIs. The mean patient age was 46.7 years (range, 22 to 66 years). The LDE-etoposide dose was escalated at 20, 30, 40, 50, and 60 mg/kg. No patients developed grade 4 or 5 toxicity; however, mucositis grade 3 occurred in 6 patients, 3 patients experienced diarrhea, and 1 patient had an elevated total bilirubin level. No deaths were related to conditioning. All patients were successfully engrafted. The median times to neutrophil and platelet engraftment were 20 ± 5 days and 16 ± 4 days, respectively. Five patients (33.4%) had acute graft-versus-host-disease (GVHD), including 4 grade I, and 1 with grade II, and 8 patients (57.1%) had moderate-to-severe chronic GVHD. This pilot study shows the potential of LDE-etoposide plus TBI as an HCT conditioning regimen in AML patients not responding to the induction and refractory therapies for CML-BP patient. These findings pave the way for subsequent larger clinical trials.

Androgen deprivation therapy improves the in vitro capacity of high-density lipoprotein (HDL) to receive cholesterol and other lipids in patients with prostate carcinoma.

BACKGROUND: Androgen deprivation therapy (ADT) is widely used in the treatment of testosterone-dependent prostate carcinomas. ADT often increases plasma LDL and HDL cholesterol and triglycerides. The aim was to test whether ADT changes the transfer of lipids to HDL, an important aspect of this metabolism and HDL protective functions, and related parameters. METHODS: Sixteen volunteers with advanced prostate carcinoma submitted to pharmacological ADT or orchiectomy had plasma collected shortly before and after 6 months of ADT. In vitro transfer of lipids to HDL was performed by incubating plasma with donor emulsion containing radioactive lipids by 1 h at 37 °C. After chemical precipitation of apolipoprotein B-containing lipoprotein, the radioactivity of HDL fraction was counted. RESULTS: ADT reduced testosterone to nearly undetectable levels and markedly diminished PSA. ADT increased the body weight but glycemia, triglycerides, LDL and HDL cholesterol, HDL lipid composition and CETP concentration were unchanged. However, ADT increased the plasma unesterified cholesterol concentration (48 ± 12 vs 56 ± 12 mg/dL, p = 0.019) and LCAT concentration (7.15 ± 1.81 vs 8.01 ± 1.55µg/mL, p = 0.020). Transfer of unesterified (7.32 ± 1.09 vs 8.18 ± 1.52%, p < 0.05) and esterified cholesterol (6.15 ± 0.69 vs 6.94 ± 1.29%, p < 0.01) and of triglycerides (6.37 ± 0.43 vs 7.18 ± 0.91%, p < 0.001) to HDL were increased after ADT. Phospholipid transfer was unchanged. CONCLUSION: Increase in transfer of unesterified and esterified cholesterol protects against cardiovascular disease, as shown previously, and increased LCAT favors cholesterol esterification and facilitates the reverse cholesterol transport. Thus, our results suggest that ADT may offer anti-atherosclerosis protection by improving HDL functional properties. This could counteract, at least partially, the eventual worse effects on plasma lipids.

Obstructive sleep apnea and effects of continuous positive airway pressure on triglyceride-rich lipoprotein metabolism.

This study aimed to explore lipoprotein metabolism in obstructive sleep apnea (OSA) and the effects of continuous positive airway pressure (CPAP). We studied 15 men with severe OSA [apnea-hypopnea index (AHI) ≥30 events/hour] and 12 age-, BMI-, and waist circumference-matched volunteers without OSA (AHI <5 events/hour). Carotid intima-media thickness (CIMT) was determined by a blind examiner. After 12 h fasting, a triglyceride-rich chylomicron-like emulsion, labeled with [14C]cholesteryl oleate and [3H]triolein, was injected intravenously followed by blood sample collection at preestablished times. Fractional clearance rate (FCR) of the radiolabeled lipids was estimated by compartmental analysis of radioisotope decay curves. Compared with controls, patients with OSA showed a significant delay in both cholesteryl ester FCR (0.0126 ± 0.0187 vs. 0.0015 ± 0.0025 min-1; P = 0.0313) and triglycerides FCR (0.0334 ± 0.0390 vs. 0.0051 ± 0.0074 min-1; P = 0.0001). CIMT was higher in the OSA group: 620 ± 17 vs. 725 ± 29 µm; P = 0.004. Cholesteryl ester FCRs were inversely related to total sleep time <90% (r = -0.463; P = 0.029) and CIMT (r = -0.601; P = 0.022). The triglyceride FCR was inversely correlated with AHI (r = -0.537; P = 0.04). In a subgroup of patients treated with CPAP for 3 months (n = 7), triglyceride FCR increased 5-fold (P = 0.025), but the cholesteryl ester FCR was unchanged. In conclusion, severe OSA decreased lipolysis of triglyceride-rich lipoproteins and delayed removal of remnants. CPAP treatment may be effective to restore the lipolysis rates.

Cholesteryl ester transfer protein (CETP), HDL capacity of receiving cholesterol and status of inflammatory cytokines in patients with severe heart failure.

BACKGROUND: Heart failure (HF) courses with chronic inflammatory process and alterations in lipid metabolism may aggravate the disease. The aim was to test whether the severity of HF, using brain natriuretic peptide (BNP) as a marker, is associated with alterations in functional aspects of HDL, such as lipid transfer, cholesterol ester transfer protein (CETP) and lecithin-cholesterol acyltransferase (LCAT) concentration. METHODS: Twenty-five HF patients in NYHA class I/II and 23 in class III/IV were enrolled. Plasma lipids, apolipoproteins, CETP, LCAT, oxidized-LDL (oxLDL) and paraoxonase-1 (PON-1) activity were determined. Lipid transfer from a donor artificial nanoparticle to HDL was measured by in vitro assay. RESULTS: Total cholesterol (p = 0.049), LDL-C (p = 0.023), non-HDL-C (p = 0.029) and CETP, that promotes lipid transfer among lipoproteins (p = 0.013), were lower in III/IV than in I/II group. Triglycerides, HDL-C, apo A-I, apo B, oxLDL, LCAT, enzyme that catalyzes serum cholesterol esterification, PON-1 activity, and in vitro transfers of cholesterol, triglycerides and phospholipids to HDL, important steps in HDL metabolism, were equal. IL-8 was higher in III/IV (p = 0.025), but TNFα, IL-1ß, IL-6 and MCP-1 were equal. BNP was negatively correlated with CETP (r = - 0.294; p = 0.042) and positively correlated with IL-8 (r = 0.299; p = 0.039). CONCLUSIONS: Our results disclosed the relationship between CETP levels and HF severity, by comparing two HF groups and by correlation analysis. Lower CETP levels may be a marker of HF aggravation and possibly of worse prognosis. Practical applications of this initial finding, as the issue whether CETP could be protective against HF aggravation, should be explored in larger experimental and clinical studies.

mRNA levels of low-density lipoprotein receptors are overexpressed in the foci of deep bowel endometriosis.

STUDY QUESTION: Is mRNA expression of LDL receptors altered in deep bowel endometriotic foci? SUMMARY ANSWER: mRNA expression of LDL receptors is up-regulated in deep bowel endometriotic foci of patients with endometriosis. WHAT IS KNOWN ALREADY: Several studies have demonstrated the overexpression of low-density lipoprotein receptors in various tumour cell lines and endometriosis has similar aspects to cancer, mainly concerning the pathogenesis of both diseases. This is the first study we know of to investigate lipoprotein receptors expression in deep endometriosis with bowel involvement. STUDY DESIGN, SIZE, DURATION: During 2014-2015, an exploratory case-control study was conducted with 39 patients, including 20 women with a histological diagnosis of deep endometriosis compromising the bowel and 19 women without endometriosis who underwent laparoscopic tubal ligation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood samples were collected on the day of surgery for lipid profile analysis, and samples of endometrial tissue and of bowel endometriotic lesions were also collected. The tissue samples were sent for histopathological analysis and for LDL-R and LRP-1 gene expression screening using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Patients with deep endometriosis had lower LDL-cholesterol than patients without the disease (119 ± 23 versus 156 ± 35; P = 0.001). Gene expression analysis of LDL receptors revealed that LDL-R was more highly expressed in endometriotic lesions when compared to the endometrium of the same patient but not more than in the endometrium of women without endometriosis (0.027 ± 0.022 versus 0.012 ± 0.009 versus 0.019 ± 0.01, respectively; P < 0.001). LRP-1 was more highly expressed in endometriotic lesions, both when compared with the endometrium of the same patient and when compared with the endometrium of patients without the disease (0.307 ± 0.207 versus 0.089 ± 0.076 and versus 0.126 ± 0.072, respectively; P < 0.001). The study also showed that LDL-R gene expression in the endometrium of women with endometriosis was higher during the secretory phase of the menstrual cycle (P = 0.001). LRP-1 gene expression was increased during the secretory phase in the endometrium of women without the disease (P = 0.008). LIMITATIONS, REASONS FOR CAUTION: In the endometriotic lesions, the presence of fibrosis is substantial, restricting access to the stromal and glandular components of the lesion. Despite that, we found that LDL receptor mRNA was overexpressed. Future studies may perform laser microdissection to isolate the area of interest in the target tissue, excluding fibrosis contamination. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the feasibility of LDL-R targeted therapy in the treatment of deep endometriosis. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2011/17245-0). The authors have no conflicts of interest to declare.

Reduction of Atherosclerotic Lesions by the Chemotherapeutic Agent Carmustine Associated to Lipid Nanoparticles.

PURPOSE: After injection in the bloodstream, a lipid nanoparticle (LDE) resembling low-density lipoprotein (LDL) concentrates in atherosclerotic lesions of cholesterol-fed rabbits. Here, rabbits with atherosclerosis were treated with carmustine, an antiproliferative agent used in cancer chemotherapy, associated to LDE to investigate the effects on the lesions. METHODS: Twenty-seven male New Zealand rabbits were fed a 1 % cholesterol diet for 8 weeks. After 4 weeks nine animals were treated with intravenous saline solution, nine with intravenous LDE alone, and nine with intravenous LDE-carmustine (4 mg/kg, weekly for 4 weeks). RESULTS: LDE-carmustine reduced lesion size by 90 % compared to the controls. LDE-carmustine reduced the presence of macrophages, vascular smooth muscle cells, and regulatory T cells in the arterial intima, as well as the presence of matrix metallopeptidase-9, interleukin-1ß and TNF-α and lipoprotein receptors, namely LDL-receptor, LDL-related protein-1, scavenger receptor class B member 1. When injected alone, without association to carmustine, LDE was not different from injected saline solution. CONCLUSIONS: LDE-carmustine treatment resulted in marked reduction of lesion area, of the invasion of the arterial intima by macrophages and vascular smooth muscle cells and pro-inflammatory factors. Therefore, this new formulation shows great potential for therapy of atherosclerotic cardiovascular disease.

Alterations in lipid transfers to HDL associated with the presence of coronary artery disease in patients with type 2 diabetes mellitus.

BACKGROUND: We previously showed that unesterified-cholesterol transfer to high-density lipoprotein (HDL), a crucial step in cholesterol esterification and role in reverse cholesterol transport, was diminished in non-diabetic patients with coronary artery disease (CAD). The aim was to investigate whether, in patients with type 2 diabetes mellitus (T2DM), the occurrence of CAD was also associated with alterations in lipid transfers and other parameters of plasma lipid metabolism. METHODS: Seventy-nine T2DM with CAD and 76 T2DM without CAD, confirmed by cineangiography, paired for sex, age (40-80 years), BMI and without statin use, were studied. In vitro transfer of four lipids to HDL was performed by incubating plasma of each patient with a donor emulsion containing radioactive lipids during 1 h at 37 °C. Lipids transferred to HDL were measured after chemical precipitation of non-HDL fractions and the emulsion. Results are expressed as % of total radioactivity of each lipid in HDL. RESULTS: In T2DM + CAD, LDL-cholesterol and apo B were higher than in T2DM. T2DM + CAD also showed diminished transfer to HDL of unesterified cholesterol (T2DM + CAD = 7.6 ± 1.2; T2DM = 8.2 ± 1.5%, p < 0.01) and of cholesteryl-esters (4.0 ± 0.6 vs 4.3 ± 0.7, p < 0.01). Unesterified cholesterol in the non-HDL serum fraction was higher in T2DM + CAD (0.93 ± 0.20 vs 0.85 ± 0.15, p = 0.02) and CETP concentration was diminished (2.1 ± 1.0 vs 2.5 ± 1.1, p = 0.02). Lecithin-cholesterol acyltransferase activity, HDL size and lipid composition were equal. CONCLUSION: Reduction in T2DM + CAD of cholesterol transfer to HDL may impair cholesterol esterification and reverse cholesterol transport and altogether with simultaneous increased plasma unesterified cholesterol may facilitate CAD development in T2DM.

Use of combined chemotherapy with etoposide and methotrexate, both associated to lipid nanoemulsions for atherosclerosis treatment in cholesterol-fed rabbits.

PURPOSE: Treatment of atherosclerotic rabbits with intravenous methotrexate or etoposide carried in lipid nanoemulsions (LDE) markedly reduced the lesions in the aorta. Here, the combined treatment with LDE-methotrexate and LDE-etoposide was investigated aiming to increase the anti-atherosclerosis effect. METHODS: Thirty-six male rabbits received a diet with 1 % cholesterol for 2 months. After the first month, the animals received 4 weekly intravenous injections of LDE-methotrexate (4 mg/kg dose), LDE-etoposide (6 mg/kg), or a combination of those two drugs, while the control animals were injected with LDE (n = 9 for each group). RESULTS: LDE-methotrexate+LDE-etoposide reduced aortic lesion areas by 95 % compared with controls and the intima-media ratio was reduced five-fold, whereas LDE-methotrexate reduced the lesions by 81 % and LDE-etoposide by 83 %. Compared to controls, the positive area of macrophages and MMP-9 in the arterial intima was significantly reduced in all treated groups (p < 0.001), but the MMP9 reduction was greater with the combined chemotherapy than the reduction achieved by the isolated treatments. Presence of CD3 positive cells was equal in controls and LDE-methotrexate+LDE-etoposide treated animals. However, FOXP3 positive T lymphocytes in the intima were increased in the LDE-methotrexate+LDE-etoposide rabbits. Weight, food intake evolution and the hematologic parameters suggested that the treatment had very low toxicity. CONCLUSIONS: Compared to the single treatments with LDE-methotrexate and LDE-etoposide, the combined treatment was more effective in reducing the atherosclerotic lesions. Because the toxicity of the novel drug-target combined scheme was low, those results favor the possibility of future clinical studies in patients with cardiovascular disease.

What is new in familial hypercholesterolemia?

PURPOSE OF REVIEW: The purpose of this review is to describe advances in the diagnosis, cause, metabolism, risk factors for atherosclerosis, and treatment of familial hypercholesterolemia. RECENT FINDINGS: Heterozygous familial hypercholesterolemia is almost four-fold more frequent than previously thought and is associated with 10-fold to 13-fold risk of cardiovascular disease comparing with normolipidemics. LDL receptor (LDLR) dysfunction and LDL-cholesterol (LDL-C) accumulation disturb the metabolism of other lipoprotein classes, such as chylomicrons and remnants and HDL. Next-generation sequencing can improve familial hypercholesterolemia molecular diagnosis due to its better performance and lower costs than usual techniques. Despite this, roughly 40% of familial hypercholesterolemia patients do not present mutations on the LDLR, apolipoprotein B, or proprotein convertase subtilisin/kexin type 9 genes. Many individuals with familial hypercholesterolemia phenotype have polygenic instead of monogenic cause of their elevated LDL-C concentrations. Individuals with familial hypercholesterolemia show elevated burden of subclinical atherosclerosis. The intensity of atherosclerosis burden is associated with the severity of LDLR mutation rather than maternal or paternal heritability. Newer-approved and on-development medications that reduce LDL-C hold promise for preventing cardiovascular disease in familial hypercholesterolemia. SUMMARY: Familial hypercholesterolemia is frequent and currently underdiagnosed and undertreated, but effective cascade screening programs and early and intensive LDL-C lowering can change this picture and the natural history of the disease.

Plasma kinetics of chylomicron-like emulsion and lipid transfers to high-density lipoprotein (HDL) in lacto-ovo vegetarian and in omnivorous subjects.

PURPOSE: Previously, it was showed that vegan diet improves the metabolism of triglyceride-rich lipoproteins by increasing the plasma clearance of atherogenic remnants. The aim of the current study was to investigate this metabolism in lacto-ovo vegetarians whose diet is less strict, allowing the ingestion of eggs and milk. Transfer of lipids to HDL, an important step in HDL metabolism, was tested in vitro. METHODS: Eighteen lacto-ovo vegetarians and 29 omnivorous subjects, all eutrophic and normolipidemic, were intravenously injected with triglyceride-rich emulsions labeled with ¹4C-cholesterol oleate and ³H-triolein. Fractional clearance rates (FCR, in min⁻¹) were calculated from samples collected during 60 min. Lipid transfer to HDL was assayed by incubating plasma samples with a donor nanoemulsion labeled with radioactive lipids. RESULTS: LDL cholesterol was lower in vegetarians than in omnivores (2.1 ± 0.8 and 2.7 ± 0.7 mmol/L, respectively, p < 0.05), but HDL cholesterol and triglycerides were equal. Cholesteryl ester FCR was greater in vegetarians than in omnivores (0.016 ± 0.012, 0.003 ± 0.003, p < 0.01), whereas triglyceride FCR was equal. Cholesteryl ester transfer to HDL was lower in vegetarians than in omnivores (2.7 ± 0.6, 3.5 ± 1.5 %, p < 0.05), but free cholesterol, triglyceride and phospholipid transfers and HDL size were equal. CONCLUSION: Similarly to vegans, lacto-ovo vegetarian diet increases remnant removal, as indicated by cholesteryl oleate FCR, which may favor atherosclerosis prevention, and has the ability to change lipid transfer to HDL.

Structural characterization of cholesterol-rich nanoemulsion (LDE).

Cholesterol-rich nanoemulsion (LDE) can carry chemotherapeutic agents in the circulation and can concentrate those agents in the neoplastic and inflammatory tissues. This method improves the biodistribution of the drug and reduces toxicity. However, the structural stability of LDE particles, without or with associated drugs, has not been extensively investigated. The aim of the present study is to investigate the structural stability of LDE and LDE associated to paclitaxel, etoposide or methotrexate in aqueous solution over time by small-angle X-ray scattering (SAXS and Ultra SAXS) and dynamic light scattering (DLS). The results show that LDE and LDE associated with those chemotherapeutic agents had reproducible and stable particle diameter, physical structure, and aggregation behavior over 3-month observation period. As estimated from both DLS and Ultra-SAXS methods, performed at pre-established intervals, the average particle diameter of LDE alone was approx. 32 nm, of LDE-paclitaxel was 31 nm, of LDE-methotrexate was 35 nm and of LDE-etoposide was 36 nm. Ultra-SAXS analysis showed that LDE nanoparticles were quasi-spherical, and SAXS showed that drug molecules inside the particles showed a layered-like organization. Formulations of LDE with associated PTX, ETO or MTX were successfully tested in animal experiments and in patients with cancer or with cardiovascular disease, showing markedly low toxicity, good tolerability and possible superior pharmacological action. Our results may be useful for ensuing clinical trials of this novel Nanomedicine tool, by strengthening the knowledge of the structural aspects of those LDE formulations.

Anti-atherogenic effects of methotrexate carried by a lipid nanoemulsion that binds to LDL receptors in cholesterol-fed rabbits.

PURPOSE: Nanoemulsions (LDE) with a lipid composition resembling that of LDL can concentrate in aortic lesions and when associated with anti-blastic agents, such as paclitaxel or etoposide, decrease atherosclerotic lesions induced in rabbits. Our aim was to test the association of a lipophilic derivative of methotrexate, didodecyl-methotrexate (ddMTX) to LDE on the lesions and on the expression of pro-inflammatory and anti-inflammatory genes. METHODS: Twenty male New Zealand rabbits were fed 1 % cholesterol diet for 60 days. Starting from day 30, 10 animals were treated with 4 weekly LDE-ddMTX injections (4 mg/kg, I.V.) and 10 with LDE injections (20 mg LDE total lipid mass/kg). RESULTS: LDE-ddMTX reduced the size of the lesion areas by 65 % and the intima-media ratio by 2-fold. Reduction of intimal macrophage was 67 % and of apoptotic cells was 88 %. Smooth muscle cells migration into the intima was unaffected. LDE-ddMTX treatment diminished metalloproteinase-9 in the intima. In aortas of atherosclerotic rabbits, downregulation of 6 pro-inflammatory genes, TNF-α, MCP-1, IL-1ß, IL-18, MMP-9, MMP-12 and upregulation of the anti-inflammatory IL-10 gene were observed. Incubation of LDE-ddMTX with HUVEC cells led to downregulation of TNF-α IL1-ß VAP-1, TLR2 and CXCL2. CONCLUSIONS: LDE-ddMTX is potentially useful to threat atherosclerosis by acting on inflammatory processes which are instrumental in the development of the disease.

Lipoprotein metabolism in patients with type 1 diabetes under intensive insulin treatment.

BACKGROUND: Type 1 diabetes (T1DM) is frequently accompanied by dyslipidemia related with insulin-dependent steps of the intravascular lipoprotein metabolism. T1DM dyslipidemia may predispose to precocious cardiovascular disease and the lipid status in T1DM under intensive insulin treatment has not been sufficiently explored. The aim was to investigate the plasma lipids and the metabolism of LDL and HDL in insulin-treated T1DM patients with high glycemic levels. METHODS: Sixteen male patients with T1DM (26 ± 7 yrs) with glycated hemoglobin >7%, and 15 control subjects (28 ± 6 yrs) were injected with a lipid nanoemulsion (LDE) resembling LDL and labeled with (14)C-cholesteryl ester and (3)H-free-cholesterol for determination of fractional clearance rates (FCR, in h-1) and cholesterol esterification kinetics. Transfer of labeled lipids from LDE to HDL was assayed in vitro. RESULTS: LDL-cholesterol (83 ± 15 vs 100 ± 29 mg/dl, p=0.08) tended to be lower in T1DM than in controls; HDL-cholesterol and triglycerides were equal. LDE marker 14C-cholesteryl ester was removed faster from plasma in T1DM patients than in controls (FCR=0.059 ± 0.022 vs 0.039 ± 0.022h-1, p=0.019), which may account for their lower LDL-cholesterol levels. Cholesterol esterification kinetics and transfer of non-esterified and esterified cholesterol, phospholipids and triglycerides from LDE to HDL were also equal. CONCLUSION: T1DM patients under intensive insulin treatment but with poor glycemic control had lower LDL-cholesterol with higher LDE plasma clearance, indicating that LDL plasma removal was even more efficient than in controls. Furthermore, HDL-cholesterol and triglycerides, cholesterol esterification and transfer of lipids to HDL, an important step in reverse cholesterol transport, were all normal. Coexistence of high glycemia levels with normal intravascular lipid metabolism may be related to differences in exogenous insulin bioavailabity and different insulin mechanisms of action on glucose and lipids. Those findings may have important implications for prevention of macrovascular disease by intensive insulin treatment.

Regular Practice of Physical Activity Improves Cholesterol Transfers to High-Density Lipoprotein (HDL) and Other HDL Metabolic Parameters in Older Adults.

The effects of regular physical activity on two important anti-atherosclerosis functions of high-density lipoprotein (HDL), namely its capacity to receive both forms of cholesterol and its anti-oxidant function, were investigated in this study comparing older adults with young individuals. One-hundred and eight healthy adult individuals were enrolled and separated into the following groups: active older (60-80 yrs, n = 24); inactive older (60-79 yrs, n = 21); active young (20-34 yrs, n = 39); and inactive young (20-35 yrs, n = 24). All performed cardiopulmonary tests. Blood samples were collected in order to assess the following measures: lipid profile, HDL anti-oxidant capacity, paraoxonase-1 activity, HDL subfractions, and lipid transfer to HDL. Comparing active older and active young groups with inactive older and inactive young groups, respectively, the active groups presented higher HDL-C levels (p < 0.01 for both comparisons), unesterified cholesterol transfer (p < 0.01, p < 0.05), and intermediate and larger HDL subfractions (p < 0.001, p < 0.01) than the respective inactive groups. In addition, the active young group showed higher esterified cholesterol transfer than the inactive young group (p < 0.05). As expected, the two active groups had higher VO2peak than the inactive groups; VO2peak was higher in the two younger than in the two older groups (p < 0.05). No differences in unesterified and esterified cholesterol transfers and HDL subfractions were found between active young and active older groups. HDL anti-oxidant capacity and paraoxonase-1 activity were equal in all four study groups. Our data highlight and strengthen the benefits of regular practice of physical activity on an important HDL function, the capacity of HDL to receive cholesterol, despite the age-dependent decrease in VO2peak.

Removal from the plasma of the free and esterified forms of cholesterol and transfer of lipids to HDL in type 2 diabetes mellitus patients.

BACKGROUND: The aim was to investigate new markers for type 2 diabetes (T2DM) dyslipidemia related with LDL and HDL metabolism. Removal from plasma of free and esterified cholesterol transported in LDL and the transfer of lipids to HDL are important aspects of the lipoprotein intravascular metabolism. The plasma kinetics (fractional clearance rate, FCR) and transfers of lipids to HDL were explored in T2DM patients and controls, using as tool a nanoemulsion that mimics LDL lipid structure (LDE). RESULTS: 14C- cholesteryl ester FCR of the nanoemulsion was greater in T2DM than in controls (0.07 ± 0.02 vs. 0.05 ± 0.01 h-1, p = 0.02) indicating that LDE was removed faster, but FCR 3 H- cholesterol was equal in both groups. Esterification rates of LDE free-cholesterol were equal. Cholesteryl ester and triglyceride transfer from LDE to HDL was greater in T2DM (4.2 ± 0.8 vs. 3.5 ± 0.7%, p = 0.03 and 6.8 ± 1.6% vs. 5.0 ± 1.1, p = 0.03, respectively). Phospholipid and free cholesterol transfers were not different. CONCLUSIONS: The kinetics of free and esterified cholesterol tended to be independent in T2DM patients and the lipid transfers to HDL were also disturbed. These novel findings may be related with pathophysiological mechanisms of diabetic macrovascular disease.

Use of paclitaxel carried in lipid core nanoparticles in patients with late-stage solid cancers with bone metastases: Lack of toxicity and therapeutic benefits.

Patients with heavily pretreated, late-stage cancer and bone metastasis are usually poor candidates for further chemotherapy. Previously, we showed that association to lipid nanoparticles (LDE) drastically decreases the toxicity of anti-cancer drugs. Here, we tested the hypothesis that paclitaxel (PTX) carried in LDE could benefit end-of-life patients with painful bone metastases that had been previously treated with conventional PTX. Methods: Eighteen consecutive patients with late-stage cancer, 8 with breast, 5 with prostate and 5 with lung carcinoma, aged 59±9 years, were included in this study. All were receiving opioid medication. LDE-PTX was administered at 175 mg/m 2 every 3 weeks until disease progression. Clinical imaging examinations and serum biochemistry determinations were performed to monitor disease progression. Intensity of bone pain, use of opioid medications and occurrence of pathological bone fractures were also evaluated. Results: In total, 104 chemotherapy cycles were performed and none of the patients showed clinical and laboratorial toxicities or pathological bone fractures. In all patients, pain was reduced so as to allow substitution of non-opioid for opioid medication. Median progression-free survival (PFS) was four months (95% CI 2.4-5.5), but in five patients PFS was longer than 6 months. Conclusions: Absence of observable clinical and laboratorial toxicities from LDE-PTX treatment, improvement of bone pain and the possible effect on PFS in some patients, despite previous use of conventional PTX, suggest that LDEPTX merits further clinical investigation .

Use of paclitaxel carried in solid lipid nanoparticles to prevent peritoneal fibrosis in rats.

BACKGROUND: Progressive fibrous thickening of peritoneal membrane (PM) is a major complication of long-term peritoneal dialysis. TGF-ß/SMAD pathway activation, inflammation and neoangiogenesis have an important role in PM changes induced by peritoneal dialysis. Here, we investigated the effects of paclitaxel (PTX) carried in lipid core nanoparticles (LDE) on the development of peritoneal fibrosis (PF) in rats. METHODS: To induce PF, 21 male Wistar rats (300-350g) were injected with chlorhexidine gluconate for 15 consecutive days and randomly assigned to three groups: 1)PF, n = 5: no treatment; 2)LDE, n = 8: treated with LDE only, 3/3 days during 15 days; 3)LDE-PTX, n = 8: treated with PTX (4mg/kg) associated with LDE, 3/3 days during 15 days. A Control group without PF induction (n = 5) was designed, received saline solution, 3/3 days. Peritoneum function tests were performed, and anterior abdominal wall samples of the PM were collected for analyses of peritoneal thickness, immunohistochemitry, and gene expression. RESULTS: LDE-PTX treatment preserved the membrane function, maintaining the ultrafiltration rate and mass transfer of glucose at normal levels. LDE-PTX also prevented PM thickening induced by chlorhexidine gluconate injections. LDE-PTX treatment reduced the number of myofibroblasts infiltrating PM and inhibited the cell proliferation. Gene expression of fibronectin, FSP-1, VEGF, TGF-ß, and SMAD3 were reduced by LDE-PTX. CONCLUSIONS: LDE-PTX was effective to prevent development of PF and preserve the PM filtration capacity in this rat model, with clear-cut actions on pro-fibrotic mechanisms. Thus, LDE-PTX can be candidate for future clinical trials as adjuvant to peritoneal dialysis to prevent PF development, since this preparation is devoid of toxicity as shown previously.

Use of paclitaxel carried in lipid nanoparticles to treat aortic allograft transplantation in rats.

OBJECTIVES: The aim of this study was to test whether lipid core nanoparticles loaded with paclitaxel (LDE-PTX) protect rat aortic allograft from immunological damage. METHODS: Fisher and Lewis rats were used differing in minor histocompatibility loci. Sixteen Lewis rats were allocated to four-animal groups: SYNG (syngeneic), Lewis rats receiving aorta grafts from Lewis rats; ALLO (allogeneic), Lewis rats receiving aortas from Fisher rats; ALLO+LDE (allogeneic transplant treated with LDE), Lewis rats receiving aortas from Fisher rats, treated with LDE (weekly injection for 3 weeks); ALLO+LDE-PTX (allogeneic transplant treated with LDE-PTX), Lewis rats receiving aortas from Fisher rats treated with LDE-PTX (4 mg/kg weekly for 3 weeks). Treatments began on transplantation day. RESULTS: Thirty days post-transplantation, SYNG showed intact aortas. ALLO and ALLO+LDE presented intense neointimal formation. In ALLO+LDE-PTX, treatment inhibited neointimal formation; narrowing of aortic lumen was prevented in ALLO and ALLO+LDE. LDE-PTX strongly inhibited proliferation and intimal invasion by smooth muscle cells, diminished 4-fold presence of apoptotic/dead cells in the intima, reduced the invasion of aorta by macrophages and T-cells and gene expression of pro-inflammatory tumour necrosis factor-alpha (TNFα), interferon gamma (IFNγ) and interleukin-1 beta (IL-1ß). CONCLUSIONS: LDE-PTX was effective in preventing the vasculopathy associated with rejection and may offer a potent therapeutic tool for post-transplantation.