Isolation and Culture of Fibroblasts, Vascular Smooth Muscle, and Endothelial Cells From the Fetal Rat Ductus Arteriosus

Abstract

ABSTRACT:The ductus arteriosus (DA), a fetal arterial shunt vessel between the proximal descending aorta and the pulmonary artery, closes shortly after birth. Initial functional closure as a result of the DA's smooth muscle contraction is followed by definite anatomical closure. The latter involves several complex mechanisms like endothelial cushion formation and smooth muscle cell migration resulting in fibrosis and sealing of the vessel. These complex steps indicate highly specialized functions of the DA vascular smooth muscle cells (VSMCs), endothelial cells, and fibroblasts. Herein, we describe a new reproducible method for isolating VSMCs, endothelial cells, and fibroblasts of high viability from fetal rat DA using immunomagnetic cell sorting. Purity of the different cell cultures was assessed by immunohistochemistry and flow cytometry and ranged between 85 and 94%. The capability of the VSMCs to react to hypoxic stimuli was assessed by intracellular calcium and ATP measurements and by VEGF mRNA expression analysis. VSMCs respond to hypoxia with decreases in intracellular calcium concentrations and ATP levels, whereas VEGF mRNA expression increased 3.2-fold. The purified vessel-specific different cell types are suitable for subsequent gene expression profiling and functional studies and provide important tools for improving our understanding of the complex processes involved in the closure of the DA. T he ductus arteriosus (DA) is a shunt which connects the proximal descending aorta and the main pulmonary artery (PA). During fetal life, it serves to bypass the pulmonary circulation. The DA closes shortly after birth, and pulmonary blood flow increases during late gestation (1). After birth, an abrupt increase in oxygen tension results in ductal constriction, which is further promoted by falling levels of prostaglandin E2 because of an increased pulmonary metabolism and the elimination of placental prostaglandin. DA medial smooth muscle contraction leads to wall thickening, luminal obliteration, and shortening of the vessel. Permanent sealing of the DA is a process generated by the infolding of the endothelium, neointima formation, subintimal disruption, and vasa vasorum ingrowth resulting in subsequent vessel fibrosis. Several major molecular pathways are known to be involved in this programmed proliferation during permanent closure of the DA, e.g. NO signaling, the system of VEGF-promoted angiogenesis, and the cyclooxygenase/prostaglandin system (2-5).However, many questions concerning the exact function and cross-talk of these signaling pathways and the molecular basis for failed DA closure still remain unanswered (6). Previous studies investigating molecular events in the duct were in part hampered by the lack of an in vitro model that allows to study the processes in question separately in each of the different cell types of the DA instead of using the whole vessel (7). Herein, we describe an improved and reproducible method to culture organotypic vascular smooth muscle cells (VSMCs), fibroblasts, and e...

Transcription Profiles of Endothelial Cells in the Rat Ductus Arteriosus during a Perinatal Period

“…We hypothesized that the ECs of the DA exhibit a unique gene profile involved in DA-specific vasoconstriction and vascular remodeling. Recently, Weber et al reported that they successfully isolated ECs from fetal rat DA using the immunomagnetic cell separation method and harvested the isolated ECs to further confirm their purity by flow cytometry analysis [12]. In the present study, we investigated gene expression differences in ECs between the DA and the aorta by using a combination of fluorescence-activated cell sorter (FACS) and DNA microarray experiments followed by further enrichment analysis using MetaCore GeneGo software.…”

Current Perspectives on Pathobiology of the Ductus Arteriosus

“…These microarray studies looked at a mixed cell population. In the future, a more refined approach may be to perform transcriptional profiling using specific cell populations such as smooth muscle cells, endothelial cells and fibroblasts [71] to gain insight into cell-specific mechanisms of DA development and function.…”

Thrombocytopenia in the First 24 Hours After Birth and Incidence of Patent Ductus Arteriosus

“…Although several molecular pathways (eg, nitric oxide, vascular endothelial growth factor, the cyclooxygenase [COX]/prostaglandin system [8][9][10][11] ) are known to be involved in permanent DA sealing, the complex cellular and molecular mechanisms contributing to DA closure remain poorly understood. 12 Intriguingly, Echtler et al 13 reported that platelet-triggered ductal sealing (with subsequent vascular remodeling) seems to be an important mechanism for definite DA closure. In their mouse models, platelets were recruited to the endothelium of the DA soon after birth, and induced platelet dysfunction or defective platelet formation resulted in persistent PDA.…”