N-(2-Aminoethyl) Ethanolamine-Induced Morphological, Biochemical, and Biophysical Alterations in Vascular Matrix Associated With Dissecting Aortic Aneurysm

Abstract

Dissecting aortic aneurysm (DAA) is an extended tear in the wall of the aorta along the plane of the vascular media. Our previous studies indicated in a developmental animal model, that DAA was related to pathological alteration in collagen, especially collagen type III. Accordingly, in the present studies, neonatal aortic vascular smooth muscle cells (VSMC) and timed pregnant Sprague-Dawley rat dams were treated with N-(2-aminoethyl) ethanolamine (AEEA), which, as shown previously, causes DAA in offspring. Morphological changes in extracellular matrix (ECM) produced by VSMC in vitro were detailed with scanning electron microscopy (SEM), and biochemical changes in cells and ECM produced by VSMCs were defined by Western blotting. Biophysical changes of the collagen extracted from both the ECM produced by VSMC and extracted from fetal rat aortas were studied with atomic force microscopy (AFM). ECM disruption and irregularities were observed in VSMCs treated with AEEA by SEM. Western blotting showed that collagen type I was much more extractable, accompanied by a decrease of the pellet size after urea buffer extraction in the AEEA-treated VSMC when compared with the control. AFM found that collagen samples extracted from the fetal rat aortas of the AEEA-treated dam, and in the in vitro formed ECM prepared by decellularization, became stiffer, or more brittle, indicating that the 3D organization associated with elasticity was altered by AEEA exposure. Our results show that AEEA causes significant morphological, biochemical, and biomechanical alterations in the ECM. These in vitro and in vivo strategies are advantageous in elucidating the underlying mechanisms of DAA.

Calpain-mediated cleavage of p53 in human cytomegalovirus-infected lung fibroblasts

“…45,51 Antibodies specific for p53, namely DO-1 (sc-126), Bp53-12 (sc-263), Pab240 (sc-99), C-19 (sc-1311-R), and FL393 (sc-6243), and antibodies against actin (sc-7210), PCNA (sc-56), and α-tubulin (sc-5546) were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX). Amersham™ ECL™ Rainbow™ Marker (RPN800E, GE Healthcare Bio-Sciences, Pittsburgh, PA) was loaded in a lane in each gel and separated with the protein samples simultaneously as a reference for estimating the molecular weights of protein bands.…”

“…The polypeptides were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA), as previously described. 45,51 Antibodies specific for p53, namely DO-1 (sc-126), Bp53-12 (sc-263), Pab240 (sc-99), C-19 (sc-1311-R), and FL393 (sc-6243), and antibodies against actin (sc-7210), PCNA (sc-56), and α-tubulin (sc-5546) were obtained from Santa Cruz Biotechnology, Inc (Dallas, TX). Antibody against calpain was a gift of Dr. R. L. Mellgren.…”

“…8 p53(ΔCp44) appears to be predominately located in the nuclei of HCMV-infected cells in these studies and appears to be tightly associated with a chromatin-rich fraction (see Figure 6), because it was difficult to extract with Buffer C, but could be extracted using the much stronger SUMO-1modified protein extraction buffer. 56 Protein extractability may depend on the strength of detergents and/or denaturing reagents in the lysis buffers 51,101 and intermolecular protein interactions, which are associated with post-translational modification. When the Buffer C-insoluble pellets were carefully resuspended in SUMO-1-modified protein extraction buffer, which contains a much higher concentration of SDS (1.275%), they became so sticky that no new pellet could be obtained after centrifugation.…”

Effect of N-(2-aminoethyl) ethanolamine on hypertrophic scarring changes in vitro: Finding novel anti-fibrotic therapies

“…Cells were cultured in Dulbecco's Modified Eagle’s Medium containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate (catalog number 10-013-CV, Coming Life Sciences, Manassas, VA) and supplemented with 15% fetal calf serum (catalog # 10437028) and 1% 100 × Antibiotic-Antimycotic (catalog # 15240062) obtained from Gibco, Thermo Fisher Scientific (Waltham, MA). Cells were treated with AEEA as described previously (Chen et al, 2015). Sodium ascorbate (50 μg/ml), which is a crucial cofactor for lysyl oxidase, lysyl hydroxylase, and proline hydroxylase, was added daily for 5 days a week to stimulate ECM assembly (Pinnell, 1985; Pinnel et al, 1987; Davidson et al, 1997).…”

“…AEEA induces aortic dissecting aneurysm in the offspring of rats exposed to the chemical during gestation (Moore et al, 2012; Schneider et al, 2012b). During studies of this developmental model of dissecting aortic aneurysm in the rat, our laboratory found that AEEA markedly alters the development of the extracellular matrix (ECM) both in vivo and in isolated rat aortic vascular smooth muscle cells in vitro (Xu et al, 2014; Chen et al, 2015).…”