Practical assessment of the quantification of atherosclerotic lesions in apoE⁻/⁻ mice.

Genetic manipulations have enabled the mouse to be widely used as an animal model for investigating the mechanisms of human atherosclerotic disease. However, there is no standard method for quantifying atherosclerotic lesions among different laboratories. The present study introduces a thorough and precise quantitative assessment of atherosclerotic lesions in mice. In the present study, 6­week­old apoE­/­ mice were fed either a chow diet or a high­fat diet (HFD) for 12 weeks. Plasma lipid levels were measured every four weeks. Aortic atherosclerotic lesions were quantified and analyzed using an image analysis system. The aortic tree was isolated and stained with Oil Red O to measure the gross lesion area. The heart was removed and divided into sequential cross sections, which were then assessed for microscopic intimal lesions in the aortic root as follows: (1) Elastic van Gieson staining was performed to determine the area of the atherosclerotic lesion; (2) cross sections were stained with hematoxylin and eosin for histological analysis; and (3) cross sections were stained with Oil Red O and immunohistochemical staining for quantitative analysis of the cellular components within the lesions. ApoE­/­ mice fed with either the chow diet or HFD developed severe atherosclerosis in the aortic root, however, there were few lesions in the remainder of the aortic tree. Compared with the control group, the HFD apoE­/­ mice had increased plasma lipid levels and increases in the gross lesion area in the aortic tree, the microscopic lesion area in the aortic root and the number of macrophages, vascular smooth muscle cells and neutral lipids present within the lesions. HFD feeding in the apoE­/­ mice accelerated the development of atherosclerosis. The quantitative method described in the present study may be used to assist in future investigations of atherosclerosis in mice.

Modulation, bioinformatic screening, and assessment of small molecular peptides targeting the vascular endothelial growth factor receptor.

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1-Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF125-136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of (125)I-VEGF to VEGFR in a dose-dependent manner. The IC50 values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF125-136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF125-136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.