Impact of a Combined High Cholesterol Diet and High Glucose Environment on Vasculature

<div><p>Aims</p><p>Vascular complications are the leading cause of mortality and morbidity in patients with diabetes. However, proper animal models of diabetic vasculopathy that recapitulate the accelerated progression of vascular lesions in human are unavailable. In the present study, we developed a zebrafish model of diabetic vascular complications and the methodology for quantifying vascular lesion formation real-time in the living diabetic zebrafish.</p><p>Methods and Results</p><p>Wild type zebrafish (AB) and transgenic zebrafish lines of <i>fli1:EGFP</i>, <i>lyz:EGFP, gata1:dsRed</i>, double transgenic zebrafish of <i>gata1:dsRed/fli1:EGFP</i> were exposed to high cholesterol diet and 3% glucose (HCD-HG) for 10 days. The zebrafish model with HCD-HG treatment was characterized by significantly increased tissue levels of insulin, glucagon, glucose, total triglyceride and cholesterol. Confocal microscopic analysis further revealed that the diabetic larvae developed clearly thickened endothelial layers, distinct perivascular lipid depositions, substantial accumulations of inflammatory cells in the injured vasculature, and a decreased velocity of blood flow. Moreover, the vascular abnormalities were improved by the treatment of pioglitazone and metformin.</p><p>Conclusion</p><p>A combination of high cholesterol diet and high glucose exposure induces a rapid onset of vascular complications in zebrafish similar to the early atherosclerotic vascular injuries in mammalian diabetic models, suggesting that zebrafish may be used as a novel animal model for diabetic vasculopathy.</p></div

Endothelial layer thickening of optical vessels in HCD-HG treatment group.

<p>A: Different treatments led to changes of endothelial layer in optical arteries. Transgenic zebrafish (Fli1:EGFP) larvae were imaged in a lateral position by confocal microscope, and emission wavelength of 488 nm was detected. Scale bar  = 10 µm. B: Endothelial layer thickness (T) was measured by subtracting inner diameter (Di) from outer diameter (Do), T = (Do-Di)/2. Changes in the thickness of endothelial layer were quantified and presented as mean ±SD. Scale bar  = 20 µm. C: Pioglitazone and metformin effectively prevented the endothelial layer from becoming thick afer HCD-HG treatment. N = 11 in the control group, N = 24 in the HCD-HG group, N = 10 in HCD-HG+ pioglitazone group, N = 18 in HCD-HG+ metformin group. PGZ: pioglitazone; Met: metformin. Asterisk (*): Comparison of EC layer thickness between control group and HCD-HG group (p<0.05). Cross (†): Comparison of EC layer thickness between control and drugs treatment groups (p<0.05).</p

HCD-HG treatment led to reduced velocity of blood flow.

<p>A: Velocity of the blood flow in the caudal aorta (CA) was measured. An example of the red blood cell tracking was present in the CA. Each cell was tracked for more than 10 time points, and corresponding frames were produced; motion trajectory was estimated. Relative time and distance were used for the calculation of the velocity. Upper panel scale bar:40 µm; the beneath panel scale bar: 80 µm. B: The values of maximum, minimum, and mean velocities were compared between normal and HCD-HG treated group, HCD-HG treated group and HCD-HG+ pioglitazone or metformin group respectively by Student's t test (n>5 in each group). Asterisk (*): Comparison of flow velocity between control group and corresponding groups, p<0.05.</p

HCD-HG treatment induced inflammatory cells infiltration.

<p>A: HCD-HG treatment resulted in the increased number of GFP⁺ cells around the caudal vein. B: GFP⁺ cells that locate within 50 µm from the lumen of the caudal artery were counted. n>5 in each group and the experiment was repeated for three times. The results were compared between normal and HCD-HG treated group, HCD-HG treated group and HCD-HG+ pioglitazone or metformin group respectively compared between the four groups by Student t test, p<0.05. Scale bar  = 80 µm. Asterisk (*): Comparison of the number of GFP⁺ cells between control group and HCD-HG group. Cross(†): Comparison of the number of GFP⁺ cells between HCD-HG group and drug treatment groups, respectively.</p

Characteristic biochemical profiles of Type 2 diabetes in zebrafish larvae.

<p>The results are presented as mean ±SD. After 10 days of the HCD-HG treatment, the zebrafish displayed increased levels of glucose (A), total cholesterol (B), and total triglycerides (C). The mRNA levels of insulin, glucagon and PEPCK also increased (D). For the measurements of these biomarkers, 30 larvae were homogenized in each group, and we repeated for 3 times. Levels of glucose, TC, and TG were normalized by the total protein. Asterisk: Comparison of glucose, TC and TG respectively between control group and HCD-HG group, p<0.05.</p

Primers for RT-qPCR.

<p>Primers used for real time-PCR. We checked the expression levels of glucagon, insulin, PEPCK. All three genes in HCD-HG group were expressed higher than those in the control group, displaying some characteristics of type 2 diabetes. In zebrafish, there are two insulin isoforms named Insa and Insb. Insa functions similarly to the mammalian homologue in glucose regulation, whereas the Insb plays a more important role in development. Therefore, in our study we take Insa as the index of total insulin.</p>#<p>Pck1: phosphoenolpyruvate carboxykinase (PEPCK).</p

Lipid accumulation in vasculature.

<p>A: Apparent lipid accumulation (yellow) was observed. (a), (b), (c), (d) panels respectively show lipid deposits in different experimental groups. (a) Control group; (b) HCD-HG group; (c) Pioglitazone treatment group; (d) Metformin treatment group. White arrows point out the locations of lipid deposits. Scale bar  = 40 µm. B: Areas of the lipid accumulation were selected as the region of interest (ROI), and estimated by measuring the dimension of ROI. Using the Student t test, we found that the amount of lipid accumulation was significantly larger in the HCD-HG treated group than that in the control group (p<0.05). The addition of pioglitazone or metformin significantly reduced the lipid accumulation (p<0.05). Asterisk (*): Comparison of lipid accumulation between control group and HCD-HG group. Cross (†): Comparison of lipid accumulation between HCD-HG group and drug treatment groups, respectively.</p

Additional file 1: of Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16

The sequencing result of the coding region of RADA-16 in the C-terminus of 36R. This result indicated the coding sequence of RADA-16 in cloning vector pMD19-T-hELP36-RADA-16. (AB1 291 kb

Additional file 2: of Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16

The sequencing result of the coding region of RADA-16 in the C-terminus of 60R. This result indicated the coding sequence of RADA-16 in cloning vector pMD19-T-hELP60-RADA-16. (AB1 292 kb

Autophagy and mitophagy were increased in PA-treated cells.

<p><b>A.</b> Primary human aortic endothelial cells (HAECs) were treated 0 or 0.3mM palmitic acid (PA) for 24 hours. Treated cells were stained with mitochondria marker Mito, autophagy marker LC3, and lysosome marker Lamp1. The numbers of mitochondria co-localized with LC3 or with Lamp1 per cell were quantified. Representative immunostaining images and quantification of 3 independent experiments demonstrated increased co-localization of mitochondria with LC3 and Lamp1 in PA treated cells. At least 25–30 cells per experiment were analyzed. <b>B.</b> HAECs were treated with 0 or 0.3mM PA for 24h and 5nM bafilomycin A1 during the last 6h prior to fixation. Representative electron microscopy photomicrographs showed the formation of autophagosomes containing heterogeneous cytoplasmic materials (black arrows) and mitophagosomes containing mitochondria fragments (white arrows). Asterisk indicated normal mitochondria. At least 10–15 cells per condition were imaged. Quantification showed the number of autophagosome and mitophagosome was increased by PA treatment. <b>C.</b> HAECs were treated with 0 to 0.5mM PA for 24h. Representative images of western blot and quantification from 3 independent experiments showed dual effects of PA on PINK1 and Parkin expression. */#P< 0.001 vs. PA (0mM). <b>D.</b> HAECs were treated with 0 or 0.3mM PA for 24h. Representative immunostaining images of 3 independent experiments demonstrated co-localization of PINK1 and Parkin with mitochondria in perinuclear area in PA treated cells.</p