Dissecting aortic aneurysm induced by N-(2-aminoethyl) ethanolamine in rat: Role of defective collagen during development.

Dissecting aortic aneurysm (DAA) is a tear in the wall of the aorta that causes blood to flow, or "dissect," between the medial layers of the media. Pregnant rats (dams) were treated with the industrial chemical n-(2-aminoethyl) ethanolamine (AEEA) by intraperitoneal injection or gavage. The histology and pathology of aorta in the thorax from newborn pups were examined. Aortas of fetuses of gestational day 20 from dams exposed to AEEA were harvested for immunohistochemical staining and native Western blot to study the changes of collagen type 1 and type 3 in aorta. Dissecting aortic aneurysm of newborn rats was induced by treating with AEEA through intraperitoneal injection or gavage. The incidence of DAA reached 100% in live pups at the high dose by means of gavage of AEEA, but without lethality compared with intraperitoneal injection. A grading system for the dose-response of DAA lesions associated with AEEA by gavage was established. Gestational day 20 fetuses from treated dams showed a decreased content and altered distribution of medial and adventitial collagen type 1 and 3 in aorta by immunohistochemistry; this decrease was confirmed by native Western blot. This in vivo model of spontaneous aortic dissection bears striking similarities histologically to human aortic dissection. As such, the model conceivably could contribute to elucidating the mechanisms of DAA formation and to exploring diagnostic and therapeutic strategies. The pathogenesis of AEEA-induced DAA may be related to defects in the normal developmental progression of collagen types 1 and 3 in the vascular wall.

Xu Y ，Treumann S ，Rossbacher R ，Schneider S ，Boor PJ ... -  《-》

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

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.

Chen Z ，Xu Y ，Bujalowski P ，Oberhauser AF ，Boor PJ ... -  《-》

Nonlinear imaging study of extracellular matrix in chemical-induced, developmental dissecting aortic aneurysm: evidence for defective collagen type III.

Using a recent model of dissecting aortic aneurysm (DAA) caused by in utero exposure to semicarbazide, we examined the elastin and collagen using standard methods and two nonlinear imaging techniques, multiphoton fluorescence (MPF) and second harmonic generation (SHG) microscopy. Sprague-Dawley rat dams were given semicarbazide (6.13 mg/kg/day i.p.) from gestational days 14 to 20 (GD14-20). Fetuses were harvested on GD20 and pups on postnatal day 1 (PND1), PND7, and PND28; matched controls were from dams treated with saline. Aortic immunohistopathology and collagen/elastin signal intensity via MPF and SHG microscopy at an excitation wavelength of 800 nm were studied. Massive DAA of the aortic arch occurred in nearly 100% of pups at birth (i.e., no GD20 fetuses showed lesions). MPF and SHG demonstrated that collagen was significantly degraded at GD20 and in newborns, but normalized by PND28. GD20 fetuses and newborn pups showed a decreased content of medial and adventitial collagen type III in pooled aortas by Western blot and immunohistochemistry. In 7- and 28-day-old pups resolution of DAA blood in vascular media and a recovery of stainable collagen type III was found. Elastin in healed DAA (PND28 pups) was focally disorganized. MPF and SHG microscopy provide sensitive and high-resolution information on aortic elastin and collagen. In this model of DAA, collagen displays aberrant imaging quality likely linked to a marked decrease in collagen type III in the developing extracellular matrix. Birth Defects Research (Part A) 2008.

Gong B ，Sun J ，Vargas G ，Chang Q ，Xu Y ，Srivastava D ，Boor PJ ... -  《-》

Chemical-induced, nonlethal, developmental model of dissecting aortic aneurysm.

A chemical-induced, nonlethal, dissecting aortic aneurysm (DAA) is described following in utero exposure to semicarbazide, an inhibitor of the vascular enzyme semicarbazide sensitive amine oxidase (SSAO). Sprague-Dawley rat dams were given semicarbazide (0.096-49.000 mg/kg/day) by IP injection on gestation days (GDs) 14-20, a period of rapid aortic development. Newborn rats (day 1) were killed and their thoracic organs were removed en bloc for near-serial cross sections and routine histopathology, Movat stain for elastin, and immunohistochemistry to differentiate cells involved in the evolution of the DAA. In subsequent experiments, pups from treated dams (0.096-6.125 mg/kg/day) were allowed to survive for 7 or 28 days. DAA occurred in nearly 100% of the rats at all doses except the lowest tested (1.530, 0.096 mg/kg/day). Dissections frequently extended to the carotids and, less frequently, to the abdominal aorta. Remodeling of vascular lesions proceeded by organization of collections of blood in vascular media (the "false lumen"), proliferation of vascular smooth muscle cells, fibrosis, and formation of irregular frayed elastic lamellae in healed vascular media. Biochemical quantitation and Western blot analysis of main extracellular matrix proteins on GD 20 showed no overt difference in expression of collagen type I, fibrillin-1, or elastin. This developmental model provides investigators an opportunity to explore the pathologic mechanisms of DAA and to examine the potential long-term effects of vascular remodeling of DAA.

Gong B ，Trent MB ，Srivastava D ，Boor PJ ... -  《birth defects research part a-clinical and molecular teratology》

Toxicokinetic profile of N-(2-aminoethyl)ethanolamine in the female Wistar rat and distribution into the late gestation fetus and milk.

N-(2-aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t(1/2) = 0.1-0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t(1/2 α) = 1.6-1.8 hr) followed by a slower phase (t(1/2 β) = 16.7-17.3 hr). Following repeated gavage administration during gestation day 17 to 19, (14) C-AEEA-derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, (14) C-AEEA-derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6- and 2.5-fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.

Moore NP ，Saghir SA ，Clark AJ ，Hansen SC ，Carney EW ，Marshall VA ，Rasoulpour RJ ，Bartels MJ ... -  《-》