Isolation and Culture of Arterial Smooth Muscle Cells From Human Placenta

Abstract

Abstract-A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed ␣-smooth muscle actin, ␤-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for ␣-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-␣, and interleukin-1␤ by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology. Recently, we demonstrated increased vascular smooth muscle expression of interleukin-8 (IL-8), a potent chemotactic agent for neutrophils, coincident with neutrophil transendothelial migration into the systemic vasculature of women with preeclampsia, a hypertensive disorder of pregnancy. 7 Increased vascular smooth muscle expression of IL-8 indicated inflammation and dysfunction of vascular smooth muscle in preeclampsia, but the cause of this dysfunction is unknown.The purpose of this study was to develop a simple and economical technique for the isolation and culture of human arterial smooth muscle cells and to characterize the phenotype of these cells. Such cells would be of value for in vitro experiments to explore mechanisms for vascular smooth muscle cell dysfunction. This has been performed for endothelial cells, which are commonly used for cardiovascular studies, by isolating endothelial cells from human umbilical veins. We applied a similar rationale and developed a simple and economical technique to isolate and culture arterial smooth muscle cells from placental chorionic plate arteries. Smooth muscle cell phenotype was verified by surveying expression of smooth muscle cell myofilamentous proteins and functionality of the cells by responsiveness to inflammatory mediators. Me...

Increased Expression of Matrix Metalloproteinase-1 in Systemic Vessels of Preeclamptic Women

“…To test this hypothesis, we examined the effects of neutrophils and their soluble products (ie, TNF-␣ and ROS) on extracellular matrix protein gene expression using primary cultures of human VSMCs, an in vitro model previously validated. 24 Analysis of the extracellular matrix gene array of VSMCs after overnight treatments showed a similar expression profile as that observed in blood vessels from preeclamptic women (see Supplemental Tables S2-S4 at http://ajp.amjpathol.org), including the upregulation of MMP1 and the down-regulation or no change of TIMP1 and COL1A1 gene expression. Realtime RT-PCR confirmed that neutrophils, TNF-␣, and ROS significantly up-regulated MMP-1 expression in VSMCs by 6-fold (P ϭ 0.05), 9-fold (P ϭ 0.028), and 19-fold (P ϭ 0.001), respectively, compared with controls.…”

“…24 The cells were treated for 24 hours with ROS generated by the oxidation of 0.05-mmol/L hypoxanthine (Sigma, St Louis, MO), 0.003-U/ml xanthine oxidase (Calbiochem, La Jolla, CA), 1-ng/ml TNF-␣ (R&D Systems, Minneapolis, MN), or neutrophils activated with 50-mol/L arachidonic acid (Cayman Chemical, Ann Arbor, MI) in a 1:16 ratio of neutrophils/VSMCs. Hypoxanthine plus xanthine oxidase generates superoxide that quickly dismutates to hydrogen peroxide, the physiological signaling molecule.…”

TMEM16A regulates portal vein smooth muscle cell proliferation in portal hypertension

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A preliminary proteomic evaluation of smooth muscle cells in thoracic aortic aneurysms

“…#03-031), and all samples were processed within a few hours of operation. SMCs were isolated using the explant method (Leik et al, 2004). Cells were isolated on the same day the surgery was performed.…”

“…SMCs were isolated from aortic tissue via the explant technique (Leik et al, 2004) and maintained under cell culture conditions until growth was observed ( Figure 1A). During initial experiments, cells were explanted in DMEM/F12 supplemented with 10% fetal bovine serum.…”