Gunter Almer

Inflammation, Adiponectin, Obesity and Cardiovascular Risk

The development of atherosclerotic lesions leading to myocardial infarction (MI) or stroke encompasses a cascade of cellular and molecular events that can well be characterized as a chronic immune-mediated inflammation occurring preferentially in the biologic surrounding of the so called metabolic syndrome. Adipokines, chemokines, cytokines, and their receptors are critically involved in the initiation and perpetuation of atherosclerosis, and they play important roles at all levels in the pathogenesis of this disease. Metabolic risk profiles associated with sedentary lifestyle, obesity, especially intra-abdominal fat accumulation, insulin resistance, and dyslipidemia pave the way for a chronic, immune-mediated vascular inflammation around vascular lipid deposits. In the present article, the impact of adiponectin, monocyte and T-cell associated cytokines (with emphasis on Neopterin), individual adipose tissue - distribution and pleiotropic drug effects on the individual course of atherosclerosis and associated cardiovascular disease are reviewed.

Obesity-related dysregulation of the tryptophan-kynurenine metabolism: role of age and parameters of the metabolic syndrome.

Obesity‐related immune mediated systemic inflammation was associated with the development of the metabolic syndrome by induction of the tryptophan (TRP)–kynurenine (KYN) pathway. The study aimed to assess whether this holds true across the lifespan from juvenility to adulthood.

Preatherosclerosis and Adiponectin Subfractions in Obese Adolescents

We evaluated total adiponectin, high‐molecular weight (HMW), medium‐molecular weight (MMW), low‐molecular weight (LMW) adiponectin subfractions, clinical parameters, routine lab parameters, lipids, metabolic, inflammatory biomarkers, and intima‐media thickness (IMT) of common carotid arteries in 70 obese juveniles and adolescents with preatherosclerosis and 55 normal weight controls of similar age and gender distribution. Compared with the controls, the obese probands had a significantly increased IMT (P < 0.001) and elevated ultra‐sensitive C‐reactive protein (P < 0.001) indicating early vascular burden. Total and HMW adiponectin were significantly decreased in the obese cohort. The ratio between HMW and total adiponectin was significantly decreased in obese probands whereas the LMW/total adiponectin ratio was increased. Overall, total‐, HMW, and MMW adiponectin were significantly negatively correlated with carotid IMT. The HMW/total adiponectin ratio correlated significantly negatively, and the LMW/total adiponectin ratio significantly positively with the IMT. Furthermore, HMW adiponectin was significantly positively correlated with high‐density lipoprotein (HDL)‐cholesterol and serum apolipoprotein A1, and negatively with BMI, triglycerides, homeostatic model assessment (HOMA)‐index, leptin, liver transaminases, and uric acid. This remained stable after controlling for gender. Multiple regression analysis of body measures and all other lab parameters showed the strongest correlation between HMW adiponectin and carotid IMT (β = −0.35, P < 0.001). Taken together, our study provides the first evidence that preatherosclerosis in obese juveniles and adolescents is associated with altered subfractions of adiponectin, whereas after multiple testing the HMW subfraction showed a better correlation to IMT compared with total adiponectin.

Ultrasmall superparamagnetic iron oxide (USPIO)-based liposomes as magnetic resonance imaging probes

Background Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast. Methods Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice. Results DLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes. Conclusion Our results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.

Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats

The aim of the present study was the in vivo evaluation of thiomer-coated liposomes for an oral application of peptides. For this purpose, salmon calcitonin was chosen as a model drug and encapsulated within liposomes. Subsequently, the drug loaded liposomes were coated with either chitosan–thioglycolic acid (CS–TGA) or an S-protected version of the same polymer (CS–TGA–MNA), leading to an increase in the particle size of about 500 nm and an increase in the zeta potential from approximately − 40 mV to a maximum value of about + 44 mV, depending on the polymer. Coated liposomes were demonstrated to effectively penetrate the intestinal mucus layer where they came in close contact with the underlying epithelium. To investigate the permeation enhancing properties of the coated liposomes ex vivo, we monitored the transport of fluoresceinisothiocyanate-labeled salmon calcitonin (FITC-sCT) through rat small intestine. Liposomes coated with CS–TGA–MNA showed the highest effect, leading to a 3.8-fold increase in the uptake of FITC-sCT versus the buffer control. In vivo evaluation of the different formulations was carried out by the oral application of 40 μg of sCT per rat, either encapsulated within uncoated liposomes, CS–TGA-coated liposomes or CS–TGA–MNA-coated liposomes, or given as a solution serving as negative control. The blood calcium level was monitored over a time period of 24 h. The highest reduction in the blood calcium level, to a minimum of 65% of the initial value after 6 h, was achieved for CS–TGA–MNA-coated liposomes. Comparing the areas above curves (AAC) of the blood calcium levels, CS–TGA–MNA-coated liposomes led to an 8.2-fold increase compared to the free sCT solution if applied orally in the same concentration. According to these results, liposomes coated with S-protected thiomers have demonstrated to be highly valuable carriers for enhancing the oral bioavailability of salmon calcitonin.

Interleukin-10: An Anti-Inflammatory Marker To Target Atherosclerotic Lesions via PEGylated Liposomes

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF−) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Nuchal thickness of subcutaneous adipose tissue is tightly associated with an increased LMW/total adiponectin ratio in obese juveniles

Subcutaneous adipose tissue (SAT) topography contributes significantly to metabolic risk profiles and atherosclerotic vascular burden in obese adults. However, little information exists concerning individual risk profiles in early phases of obesity found in childhood and adolescence. Thus, the rationale of this study was to evaluate possible impacts of SAT topography in obese juveniles on adiponectin subfractions, with special emphasis on low molecular weight (LMW) adiponectin. To address this, we analysed associations between lipometry, early metabolic and preatherosclerotic symptoms and adiponectin subfractions in 71 obese juveniles and 75 normal weight controls of similar age and gender distribution. Compared to the controls, obese juveniles had a significantly decreased ratio between high molecular weight (HMW) and total adiponectin whereas the LMW/total adiponectin ratio was increased. The LMW/total adiponectin ratio correlated significantly positively with the SAT thickness of trunk-located lipometer measure points neck, biceps, upper back, lower back, body mass index (BMI), and waist circumference. Further significant positive correlations were seen with systolic blood pressure, intima media thickness (IMT) of common carotid arteries, and metabolic parameters such as HOMA-index, leptin, oxidized LDL (oxLDL), liver transaminases, and HDL-triglycerides. This remained stable after controlling for gender. A stepwise multiple regression analysis encompassing all these variables revealed a robust positive association between LMW/total adiponectin ratio and nuchal SAT thickness defined by the lipometer measure point neck. Taken together, our data provide the first evidence that nuchal SAT thickness is tightly positively associated with an increased LMW/total adiponectin ratio.

Antioxidant food supplements and obesity-related inflammation.

The obesity prevalence is growing worldwide and largely responsible for the increased incidence of cardiovascular disease, the most common cause of death in the western world. Excessive food intake along with insufficient physical exercise is the basic impetus for this development. The obese state is commonly associated with an increase in leptin levels and chronic immune-mediated inflammation. Despite high leptin levels, the leptin response, normally associated with satiety and satiation, seems to be impaired and individuals continue to consume calorie-rich food. Antioxidant food additives such as sodium sulphite, sodium benzoate and curcumin were shown to suppress the leptin release in lipopolysaccharide- treated murine adipocytes. Based on this, we hypothesize that the insufficient leptin release, caused by excessive consumption of food additives, may lead to a reduced exposure of the central nervous system to leptin and ultimately propagate obesity. On the other hand, leptin has been shown to favor Th1-type activity, which ultimately decreases tryptophan levels. Tryptophan derivatives, serotonin and melatonin, induce satiety/satiation through several mechanisms. In this context, the antioxidant suppression of leptin release and Th1-type activity is beneficial to increase serotonin and melatonin levels. The molecules in the mechanism described in this review are highly integrated in the reward system, and have been implicated in the addiction behavior of obesity. Based on these facts, the involvement of antioxidant food supplements in the mechanisms of the reward-deficiency syndrome which perpetuates obesity will be discussed.

Trunk weighted obesity, cholesterol levels and low grade inflammation are main determinants for enhanced thrombin generation

OBJECTIVE Endogenous thrombin generation (ETP) may be critically involved in obesity associated thromboembolism. METHODS Three hundred and one participants of the STyrian Juvenile OBesity (STYJOBS)/Early DEteCTion of Atherosclerosis (EDECTA) study cohort (age, 16-58years) were analysed. ETP was measured by the new CE-IVD marked Siemens-Innovance(®) ETP test on a BCS-XP analyser, and correlated to clinical findings and extended lipometry-based anthropometric data, biomarkers, and coagulation parameters. RESULTS In the overweight/obese study group, ETP and fibrinogen levels were significantly higher compared to controls (p<0.001). In a multiple stepwise regression including all subjects, subcutaneous adipose tissue thickness of upper back, cholesterol and ultrasensitive C-reactive protein were the best predictors for ETP. CONCLUSION Trunk weighted obesity together with low grade inflammation and hypercholesterolemia enhance thrombin generation.

Adiponectin-coated nanoparticles for enhanced imaging of atherosclerotic plaques

Background: Atherosclerosis is a leading cause of mortality in the Western world, and plaque diagnosis is still a challenge in cardiovascular medicine. The main focus of this study was to make atherosclerotic plaques visible using targeted nanoparticles for improved imaging. Today various biomarkers are known to be involved in the pathophysiologic scenario of atherosclerotic plaques. One promising new candidate is the globular domain of the adipocytokine adiponectin (gAd), which was used as a targeting sequence in this study. Methods: gAd was coupled to two different types of nanoparticles, namely protamine-oligonucleotide nanoparticles, known as proticles, and sterically stabilized liposomes. Both gAd-targeted nanoparticles were investigated for their potency to characterize critical scenarios within early and advanced atherosclerotic plaque lesions using an atherosclerotic mouse model. Aortic tissue from wild type and apolipoprotein E-deficient mice, both fed a high-fat diet, were stained with either fluorescent-labeled gAd or gAd-coupled nanoparticles. Ex vivo imaging was performed using confocal laser scanning microscopy. Results: gAd-targeted sterically stabilized liposomes generated a strong signal by accumulating at the surface of atherosclerotic plaques, while gAd-targeted proticles became internalized and showed more spotted plaque staining. Conclusion: Our results offer a promising perspective for enhanced in vivo imaging using gAd-targeted nanoparticles. By means of nanoparticles, a higher payload of signal emitting molecules could be transported to atherosclerotic plaques. Additionally, the opportunity is opened up to visualize different regions in the plaque scenario, depending on the nature of the nanoparticle used.