Investigation of Cellular Interactions of Lipid-Structured Nanoparticles With Oral Mucosal Epithelial Cells.

Lipid-based nanosystems enable intracellular delivery of drugs in the oral cavity for the treatment of local diseases. To rationally design such systems, suitable matrix compositions and particle properties need to be identified, and manufacturing technologies that allow reproducible production have to be applied. This is a prerequisite for the reliable and predictable performance of in-vitro biological studies. Here, we showed that solid lipid nanoparticles (SLN, palmitic acid) and nanostructured lipid carriers (NLC, palmitic acid and oleic acid in different ratios) with a size of 250 nm, a negative zeta potential, and a polydispersity index (PdI) of less than 0.3 can be reproducibly prepared by high-pressure homogenization using quality by design and a predictive model. SLN and NLC were colloidally stable after contact with physiological fluid and did not form agglomerates. The in-vitro studies clearly showed that besides particle size, surface charge and hydrophobicity, matrix composition had a significant effect. More specifically, the addition of the liquid lipid oleic acid increased the cellular uptake capacity without changing the underlying uptake mechanism. Regardless of the matrix composition, caveolin-mediated endocytosis was the major route of uptake, which was confirmed by particle localization in the endoplasmic reticulum. Thus, this work provides useful insights into the optimal composition of lipid carrier systems to enhance the intracellular uptake capacity of drugs into the oral mucosa.

Human evidence for the involvement of insulin-induced gene 1 in the regulation of plasma glucose concentration.

AIMS/HYPOTHESIS: Insulin-induced gene 1 (INSIG1) is a protein that blocks proteolytic activation of sterol regulatory element-binding proteins (SREBPs), transcription factors that activate genes regulating cholesterol and fatty acid metabolism and possibly genes involved in glucose homeostasis. In search of genetic regulation of these processes we examined human INSIG1 for common polymorphisms and analysed their associations with biochemical parameters related to lipid and glucose metabolism. METHODS: Associations between common polymorphisms in INSIG1 and several biochemical parameters were analysed in a group of 618 healthy, 50-year-old men. A replication analysis was performed in a cohort of 472 healthy, middle-aged men. The impact of one promoter polymorphism on oral glucose tolerance was analysed in a subset of 181 subjects. Small interfering RNA (siRNA) inhibition was used to test the significance of INSIG1 for gene expression in human Huh7 hepatoma cells. RESULTS: A potentially functional polymorphism, a C to T substitution at position -169, was discovered in a highly conserved section of the promoter. Significant relationships between the -169C>T polymorphism and plasma glucose concentration were found in two cohorts of healthy, middle-aged men (p < 0.01 and p < 0.02, respectively). The -169T allele was associated with significantly lower post-load plasma glucose concentrations. A significant (p = 0.02) reduction in expression of phosphoenolpyruvate carboxykinase (PCK2) was observed following siRNA inhibition of INSIG1 in human Huh7 hepatoma cells. CONCLUSIONS/INTERPRETATION: Population studies demonstrate that INSIG1 plays a role in glucose homeostasis. Experiments with siRNA suggest that this action of INSIG1 is related to SREBP-mediated regulation of PCK2.

Oestrogen receptor alpha gene polymorphism is related to aortic valve sclerosis in postmenopausal women.

OBJECTIVES: Aortic valvular sclerosis (AS) is an inflammatory process and not a result of normal ageing. The sclerotic process is accelerated by risk factors such as smoking and high cholesterol levels. The genetic factors for the development of AS are however unknown. Therefore the purpose of the present study was to investigate whether polymorphisms in the oestrogen receptor alpha (ORalpha) gene and in the transforming growth factor beta (TGF-beta1) gene were related to the presence of AS in postmenopausal women. DESIGN: Case-control study. SUBJECTS AND METHODS: Relationships were tested between polymorphisms in the ORalpha gene defined by the restriction enzymes PvuII and XbaI, and in the TGF-beta1 gene defined by AocI, and AS, lipid levels, and lipoprotein(a) [Lp(a)] in 41 postmenopausal female patients and 41 age- and sex-matched controls. These polymorphisms were also tested in relation to lipid levels and Lp(a), in 99 healthy Caucasian girls, aged 16.9 +/- 1.2 years. RESULTS: In the postmenopausal patients and age-matched controls, the PvuII polymorphism was independently associated with an increased risk of AS [odds ratio (OR) = 3.38; 95% confidence interval (CI) 1.13-10.09). A genotype defined by at least one restriction site in the PvuII polymorphism and two restriction sites in the TGF-beta1 polymorphism was related to a highly significantly increased risk of AS (OR = 4.58; 95% CI 1.68-12.51). In the adolescent female cohort, presence of two restriction sites in the PvuII polymorphism was associated with higher levels of total cholesterol (TC) (P = 0.02), and low-density lipoprotein cholesterol (LDL) (P = 0.04). CONCLUSIONS: We have demonstrated that the PvuII polymorphism in the ORalpha gene is related to both the presence of AS in postmenopausal women and to lipid levels in adolescent females, suggesting that this polymorphism may influence the risk of AS partly by affecting lipid levels.

Lipoprotein(a), Chlamydia pneumoniae, leptin and tissue plasminogen activator as risk markers for valvular aortic stenosis.

AIMS: The aim of the present study was to identify risk markers for the development of valvular aortic stenosis (AS). Lipoprotein(a) (Lp(a)) and Chlamydia pneumoniae IgG antibody titres in plasma and in circulating immune complexes as well as leptin and tissue plasminogen activator (t-PA) in plasma were studied. METHODS AND RESULTS: One hundred and one patients (41 women and 60 men, mean age 71+/-8 years) with significant AS and 101 age- and sex-matched controls were included in this study. All patients underwent aortic valve replacement at the University Hospital in Umeå, Sweden. The controls had no symptoms of cardiovascular disease and they were examined echocardiographically. An Lp(a) level >or=480 mg x l(-1), a C. pneumoniae-specific IgG titre >or=1/128, a high leptin level and a high t-PA mass concentration in plasma were identified as risk markers for AS. A strong synergism between Lp(a) and C. pneumoniae IgG antibodies in circulating immune complexes was found. CONCLUSION: Our data indicate that a chronic C. pneumoniae infection and a high plasma Lp(a) level might influence and aggravate aortic heart valve sclerosis via the formation of circulating immune complexes. The present study also strongly suggests an association between high plasma leptin, t-PA mass concentration and AS.

Is lipoprotein(a) a predictor for survival in patients with established coronary artery disease? Results from a prospective patient cohort study in northern Sweden.

OBJECTIVES: Lipoprotein(a) [Lp(a)] is a known risk factor for the development of atherosclerosis. The aim of the present study was to test the importance of Lp(a) as a predictor for the further prognosis in patients with established coronary artery disease. DESIGN: A prospective patient cohort study was carried out. SETTING AND SUBJECTS: The cohort consists of 1216 patients who were examined with coronary angiography at the University Hospital in Umeå, Sweden, because of stable effort angina. MAIN OUTCOME MEASURES: Lipids, Lp(a), fibrinogen, antithrombin III (AT III), sedimentation rate and clinical data were registered at angiography. After a mean follow-up time of 6.7 years information on survival was collected from the municipal census lists and death certificates were examined. Total mortality and mortality because of cardiovascular disease were both used as outcome variables in the survival analyses. RESULTS. The total mortality in the patient cohort was 16.4%. An Lp(a) level of 300 mg L-1 or more was found in 30% of the study population and was found to be an independent predictor for death. A high fibrinogen, a low AT III level, a depressed left ventricular function and a high coronary obstruction score were other significant independent predictors of death. Total cholesterol, HDL- and LDL-cholesterol were not related to survival in this study, but a substantial proportion of the population probably received lipid-lowering agents during the study period. CONCLUSIONS: An Lp(a) level exceeding 300 mg L-1 indicates a poor further prognosis and may help to identify patients who probably need powerful secondary prevention programmes to improve their prognosis.

The proatherogenic properties of lipoprotein(a) may be enhanced through the formation of circulating immune complexes containing Chlamydia pneumoniae-specific IgG antibodies.

AIMS: To investigate a possible relationship between the atherogenic properties of lipoprotein(a) (Lp(a)) and Chlamydia pneumoniae infections. METHODS AND RESULTS: The study population was nested within the Västerbotten Intervention Program or the WHO MONICA project. In this incident case-control study, 78 patients who had suffered acute myocardial infarction and 156 matched controls were included. The contents of circulating immune complexes were analysed for C. pneumoniae IgG antibodies and Lp(a). A significantly larger proportion of cases than controls had >/=13 mg. l(-1)Lp(a) and a C. pneumoniae specific IgG antibody titre >/=1/2 in circulating immune complexes (odds ratio=3.8). CONCLUSION: The proatherogenic effects of Lp(a) may be enhanced and/or partly mediated through the formation of circulating immune complexes containing C. pneumoniae -specific IgG antibodies. The connection between chronic C. pneumoniae infections and atherosclerosis may, at least in part, be explained by an interaction with Lp(a) through the formation of circulating immune complexes.

Chlamydia pneumoniae antibodies and high lipoprotein(a) levels do not predict ischemic cerebral infarctions. Results from a nested case-control study in Northern Sweden.

BACKGROUND AND PURPOSE: An association between high lipoprotein(a) [Lp(a)] levels and positive Chlamydia pneumoniae IgG titers in coronary artery disease has been described. The possibility of predicting ischemic stroke by measurements of plasma Lp(a) and C pneumoniae antibodies was investigated. METHODS: This incident case-control study included 101 case subjects (cases) who had suffered ischemic cerebral infarctions and 201 matched control subjects (controls). The study population was nested within the Västerbotten Intervention Program or the WHO MONICA project. Plasma samples were measured for C pneumoniae-specific IgG and IgA antibodies and Lp(a). RESULTS: A significantly higher mean Lp(a) level was found in female cases than in female controls. However, plasma Lp(a) was unable to predict ischemic cerebral infarctions in either women or men. The proportion of individuals with positive C pneumoniae-specific IgG or IgA titers did not differ between cases and controls. Antibody titers were unable to predict a future stroke. The proportion of individuals with a positive C pneumoniae IgG titer in combination with a high Lp(a) level did not differ significantly between cases and controls. CONCLUSIONS: These data suggest that there is no association between baseline plasma Lp(a) levels, presence of C pneumoniae antibodies, and future ischemic cerebral infarctions. Furthermore, no evidence of an interactive effect between high Lp(a) levels and C pneumoniae IgG titers was found. However, selection bias and a recent C pneumoniae epidemic may have influenced the results.