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

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.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.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

Secretoneurin Gene Therapy Improves Blood Flow in an Ischemia Model in Type 1 Diabetic Mice by Enhancing Therapeutic Neovascularization

<div><p>Deficient angiogenesis after ischemia may contribute to worse outcome of peripheral arterial disease in patients with diabetes mellitus. Based on our previous work where we demonstrated that Secretoneurin (SN) is up-regulated under hypoxic conditions and enhances angiogenesis, we analyzed the therapeutic potential of SN gene therapy using a model of severe hind limb ischemia in streptozotocin-induced diabetic mice (STZ-DM). After induction of hind limb ischemia, blood flow was assessed by means of laser Doppler perfusion imaging (LDPI) and increased blood perfusion in the SN-treated animal group was observed. These results were complemented by the clinical observation of reduced necrosis and by an increased number of capillaries and arterioles in the SN-treated animal group. <i>In vitro</i>, we found that SN is capable of promoting proliferation and chemotaxis and reduces apoptosis in HUVECs cultured under hyperglycemic conditions. Additionally, SN activated ERK, eNOS and especially AKT as well as EGF-receptor in hyperglycemic HUVECs. In conclusion, we show that SN gene therapy improves post-ischemic neovascularization in diabetic mice through stimulation of angiogenesis and arteriogenesis indicating a possible therapeutic role of this factor in ischemia-related diseases in diabetic patients.</p></div

Effects of SN on metabolic parameters.

<p>No significant changes in (A) blood glucose level and (B) body weight were found between p-SN and p-CTR treated animals after 28 days.</p

Secretoneurin gene therapy improves blood flow monitored <i>in vivo</i> by LDPI in diabetic mice.

<p>(A) Time course of hind limb blood flow measured by LDPI is expressed as ischemic-to-control leg ratio. Treatment with p-SN significantly improved perfusion 7, 14 and 28 days after induction of ischemia compared to p-CTR: #, p<0.05. (B) Representative pictures of the ischemic (left) and non ischemic (right) hind limbs on day 28 after ischemia induction. In color-coded images treatment with p-SN improves perfusion where red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb.</p

Secretoneurin gene therapy rescues ischemic tissue defects in a diabetic PAD model.

<p>(A) Necrosis score at day 28 is shown as % mice with saved limbs (white), % mice with skin necrosis (grey) and % mice with amputations (black). Treatment with p-SN significantly inhibited ischemic tissue loss (necrosis and amputation). p<0.05. (B) Representative pictures of the ischemic (left) and non ischemic (right) limbs on day 28 after ischemia induction and treatment with p-SN or p-CTR.</p

SN stimulates proliferation and reduces apoptosis of HUVECs under hyperglycemic conditions.

<p>(A) HUVECs were cultured with medium containing mannitol or glucose and stimulated with SN 10 ng/ml, SN 100 ng/ml and VEGF 50 ng/ml. Proliferation was determined using a BrdU assay. Proliferation was stimulated by SN and VEGF to a comparable extent even under hyperglycemic conditions. *, p<0.001 vs. mannitol or glucose respectively. (B) Apoptotic cells were identified as TUNEL positive cells and are shown as percentage of the total cell number (obtained by DAPI staining). SN and VEGF significantly inhibited apoptotic reactions even under hyperglycemic conditions. *, p<0.001; #, p<0.05; §, p<0.005 vs. mannitol or glucose respectively.</p

SN induces outgrowth of ECs from aortic rings.

<p>Incubation of aortic rings with SN resulted in a significant outgrowth of EC from explanted aortic rings under hyperglycemic conditions (A) and with mannitol as osmotic control (B). The observed effects in our study were comparable or even superior to VEGF used as positive control. +, p<0.01 and #, p<0.05 vs. CTR.</p

SN induces HUVEC migration and induces MAPK, AKT, eNOS and EGF receptor activation under hyperglycemic conditions.

<p>(A) HUVECs were cultured with medium containing mannitol or glucose and stimulated with SN 10 ng/ml and VEGF 50 ng/ml. Migration was determined as movement of cells within a membrane towards attracting substances and is given as relative (to control) chemotactic index. *, p<0.001 vs. mannitol or glucose respectively. (B) As shown in this representative western blot hyperglycemia didn't affect activation of the MAPK-ERK1/2 signaling pathway by SN. (C) HUVECs stimulation with SN 10 ng/ml for 40 minutes activated eNOS under high glucose as well as under mannitol control. AKT activation mediated by SN is shown after 5 and 10 minutes under diabetic and control conditions. Under high glucose, AKT stimulation by SN is even stronger compared to mannitol control. (D) SN 10 ng/ml stimulation for 20 minutes revealed phosphorylation of EGF receptor (EGFR) under high glucose as well as under mannitol osmotic control conditions.</p