Ursula Stanzl

Potential role of regulatory T cells in reversing obesity-linked insulin resistance and diabetic nephropathy

OBJECTIVE To assess the potential role of FoxP3-expressing regulatory T cells (Tregs) in reversing obesity-linked insulin resistance and diabetic nephropathy in rodent models and humans. RESEARCH DESIGN AND METHODS To characterize the role of Tregs in insulin resistance, human visceral adipose tissue was first evaluated for Treg infiltration and second, the db/db mouse model was evaluated. RESULTS Obese patients with insulin resistance displayed significantly decreased natural Tregs but an increase in adaptive Tregs in their visceral adipose tissue as compared with lean control subjects. To further evaluate the pathogenic role of Tregs in insulin resistance, the db/db mouse model was used. Treg depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney. Conversely, adoptive transfer of CD4+FoxP3+ Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant proinflammatory CD8+CD69+ T cells in visceral adipose tissue and kidneys of Treg-treated animals. CONCLUSIONS Data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

The Angiogenic Factor Secretoneurin Induces Coronary Angiogenesis in a Model of Myocardial Infarction by Stimulation of Vascular Endothelial Growth Factor Signaling in Endothelial Cells

Background—Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated by hypoxia, and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. Methods and Results—In vivo secretoneurin improved left ventricular function, inhibited remodeling, and reduced scar formation. In the infarct border zone, secretoneurin induced coronary angiogenesis, as shown by increased density of capillaries and arteries. In vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis, and activated Akt and extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (VEGF) antibody, and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells, and binding was blocked by heparinase, indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger RNA and protein. Conclusions—Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low-affinity binding sites and neuropilin-1 and stimulates further growth factor receptors like fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.

The Liver-Selective Thyromimetic T-0681 Influences Reverse Cholesterol Transport and Atherosclerosis Development in Mice

Background Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice. Methodology/Principal Findings T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls. Conclusions/Significance The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans.

The Arachidonic Acid Metabolome Serves as a Conserved Regulator of Cholesterol Metabolism

Summary Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolome as conserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.

Leoligin, the major lignan from Edelweiss, activates cholesteryl ester transfer protein

Highlights ► Leoligin significantly activated CETP in human plasma at 100 pM. ► Leoligin concentrations of 1 mM inhibited CETP activity. ► There was no short-term toxicity apparent in mice treated with leoligin.

Increased plasma levels of LDL cholesterol in rabbits after adenoviral overexpression of human scavenger receptor class B type I

Scavenger receptor class B type I (SR-BI), a CD36 family member, plays a key role in high-density lipoprotein (HDL) metabolism, reverse cholesterol transport, and whole body cholesterol homeostasis, and is shown to be involved in the development of atherosclerosis in mice. In this report, we describe the effects of the adenoviral overexpression of human SR-BI (hSR-BI) in New Zealand White (NZW) rabbits, a wild-type animal model that expresses cholesteryl ester transfer protein (CETP) in plasma, displays a manlike lipoprotein profile, and is susceptible to atherosclerosis. A total of 1×1012 adenoviral particles containing either hSR-BI or lacZ complementary deoxyribonucleic acid (control) were infused into the ear vein of NZW rabbits. Transgene expression was ascertained by TaqMan Real Time polymerase chain reaction measurements. Rabbits infected with Ad/hSR-BI (adenoviral plasmids containing hSR-BI) showed a faster clearance of administered [3H]HDL cholesterol and significantly decreased apolipoprotein (apo) A-I levels when compared to control rabbits, respectively. Interestingly, we found markedly increased levels of low-density lipoprotein (LDL) cholesterol exclusively in SR-BI-overexpressing rabbits. These changes were not accompanied by alterations in LDL receptor expression but by increased levels of CE transfer in these animals. By lowering HDL cholesterol and increasing plasma apoB-containing lipoprotein levels, the overexpression of SR-BI leads to a lipoprotein pattern, which is believed to enhance the development of atherosclerosis. The role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits—a CETP-expressing animal model displaying a manlike lipoprotein profile—may therefore be different from the one found in rodents.

Topical secretoneurin gene therapy accelerates diabetic wound healing by interaction between heparan-sulfate proteoglycans and basic FGF

Diabetic foot ulcers represent a therapeutic problem of high clinical relevance. Reduced vascular supply, neuropathy and diminished expression of growth factors strongly contribute to wound healing impairment in diabetes. Secretoneurin, an angiogenic neuropeptide, has been shown to improve tissue perfusion in different animal models by increasing the amount of vessels in affected areas. Therefore, topical secretoneurin gene therapy was tested in a full thickness wound healing model in diabetic db/db mice. Secretoneurin significantly accelerated wound closure in these mice and immunohistochemistry revealed higher capillary and arteriole density in the wounded area compared to control mice. In-vitro, the mechanism of action of secretoneurin on human dermal microvascular endothelial cells was evaluated in normal and diabetic cells. Secretoneurin shows positive effects on in vitro angiogenesis, proliferation and apoptosis of these cells in a basic fibroblast growth factor dependent manner. A small molecular weight inhibitor revealed fibroblast growth factor receptor 3 as the main receptor for secretoneurin mediated effects. Additionally, we could identify heparan-sulfates as important co-factor of secretoneurin induced binding of basic fibroblast growth factor to human dermal endothelial cells. We suggest topical secretoneurin plasmid therapy as new tool for delayed wound healing in patients suffering from diabetes.

Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification.

Abstract Cholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), bothknown to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective KD values of 4.36×10−9 M and 4.64×10−9 M as determined by affinity sensor technology. Amino acid sequence comparison indicated the complementarity-determining regions of the respective heavy chains to be responsible for CETP high affinity binding. Fragment 1CL8 was successfully employed in clinical chemical quantification systems that uncovered an association in humans between plasma CETP concentration and total body fat mass (r=0.50, p<0.002). Because of the demonstrated superb CETP capturing capacity, combined withh igh binding affinity to CETP, ready access and unlimited supply, 1CL8 and 1CL10 are expected to prove powerful tools for studies on the role of CETP in atherogenesis.

A Murine Model of Phosphate Nephropathy

We established a murine model of phosphate nephropathy with secondary hyperparathyroidism. db/db mice, which develop obesity and type 2 diabetes mellitus, were uninephrectomized at the age of 6 weeks and were fed either standard chow or a phosphorus-rich diet during the next 8 weeks. Thereafter, renal cryosections showed abundant tubular casts with a strong histochemical von Kossa reaction in all db/db mice on the phosphorus-rich diet but none in the controls. X-ray diffraction and Raman spectroscopy proved that these tubular casts consist mostly of hydroxyapatite Ca₅(PO₄)₃(OH). These intraluminal precipitations were located in distal tubuli and collecting ducts and were associated with degenerative tubular changes and peritubular infiltration of T cells and macrophages. In line, kidneys of db/db mice on the phosphorus-rich diet displayed significantly increased mRNA expression of the T(H)1 cytokines interferon γ, IL-6, and tumor necrosis factor α. In addition, mice developed signs of secondary hyperparathyroidism as shown by elevated serum phosphate, decreased serum calcium, and increased parathyroid hormone, osteopontin, and fibroblast growth factor 23 levels. db/db mice on the phosphorus-rich diet also presented with significantly lower body weight, lower homeostasis model assessment of insulin resistance index, and hypertrophic cardiomyopathy. Thus, we provide a murine model of phosphate nephropathy and secondary hyperparathyroidism, which can be used for future pharmacologic and pathophysiologic studies to analyze the effect of hyperphosphatemia on renal, metabolic, and cardiovascular phenotypes.

Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits

Highlights ► Small hairpin RNA specific for SR-BI nucleotides 214-232 reduces SR-BI expression in vitro. ► SiRNA treatment reduces liver expression of SR-BI in New Zealand White rabbits. ► Liver specific inhibition of SR-BI leads to a 50% reduction of atherosclerosis in cholesterol fed rabbits. ► The role of SR-BI in rabbits may be different from the one found in rodents.