Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Summary

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Abstract

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.²⁶ that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

Introduction

Pulmonary hypertension is normal during intrauterine life since the placenta serves as the major organ of gas exchange and only 10% of the cardiac output is circulated through the pulmonary vascular bed. In utero, pulmonary pressures are similar to systemic pressures due to elevated pulmonary vascular resistance. As gestation progresses, there is rapid growth of the small PA within the lung, preparing the fetus for the dramatic increase in pulmonary blood flow that occurs at birth¹. When the normal perinatal transition fails in near-term and full term infants, the result is persistent pulmonary hypertension of the newborn (PPHN). PPHN is a clinical syndrome caused by many different underlying pathologies. However, all of these infants share common pathophysiologic features such as elevated pulmonary vascular resistance, hypoxemia, and right-to-left shunting of blood flow across persistent fetal connections such as the ductus arteriosus or foramen ovale. PPHN affects 2-6 per 1,000 live births and conveys an 8-10% risk of mortality as well as significant short-term and long-term morbidity². Additionally, very low birth weight premature infants may develop pulmonary hypertension as a result of their underlying lung disease. The most common underlying lung disease of premature infants is bronchopulmonary dysplasia (BPD). While the overall risk of BPD correlates with gestational age and birth weight, it remains unclear why a subset of these infants develops significant pulmonary hypertension and how to appropriately treat these infants. Poor outcomes, including prolonged hospital stays and increased mortality, are common^3-6.

Historically, ovine fetal PASMC or porcine fetal PASMC from healthy animals have been used to study the signaling pathways involved in the normal pulmonary vascular transition after birth. These are typically isolated from fifth generation resistance PA of an ovine or porcine fetus that is delivered and euthanized prior to any spontaneous respiration^7-9. Additionally, some investigators have isolated and utilized PASMC from slightly older and spontaneously breathing lambs and piglets at 3 days, 2 weeks, and 4 weeks^10-12. More recently, some groups have successfully isolated and utilized PASMC isolated from lambs with PPHN to examine the derangements in signaling pathways in the disease state^13-17. These cells have proved to be a valuable tool to examine which signaling pathways are crucial in both the normal and diseased near-term and term pulmonary vasculature. However, they do not give insight into the signaling pathways impacted in premature infants with pulmonary hypertension. Nor do they allow the possibilities of genetic manipulation seen in mouse models of disease.

Rat and mouse models have long been used to model BPD and more recently are being used to model pulmonary hypertension resulting from BPD^18-22. Neonatal rats are enticing to work with due to their larger size, but they also suffer from lack of potential for genetic modification. Genetically modified animals have been extensively used to investigate the effects of specific gene targets on whole animal physiology in neonatal mice, but to date no one has previously successfully isolated PASMC from these small mice. By isolating PASMC, greater information can be obtained about how pathways change in response to environmental stimuli and/or genetic modification specifically in the pulmonary artery smooth muscle. Additionally, live PASMC can be imaged in real time to examine rapid changes in key signaling molecules such as calcium and reactive oxygen species^23-25. We recently described the successful isolation of PASMC from adult mice using a variation of the technique of Marshall et al.²⁶ used to isolate rat PASMC^23,25,26. We now have adapted and extended this technique to small mice 7-21 days of age (P7, P14, and P21). The primary limitation to this new PASMC isolation technique is that it requires multiple mice to generate sufficient cells for experiments and that the cells grow very slowly, which is characteristic of primary smooth muscle cells. Despite these limitations, we believe this technique to isolate neonatal mouse PASMC will allow for the enhanced investigation of key signaling pathways involved in the development of pulmonary hypertension and represents a significant advance in this field.

Protocol

The Institutional Animal Care and Use Committee at Northwestern University approved this protocol. 1. Pulmonary Artery Isolation from Neonatal Mice – Day One Prepare the Complete M199 media – Mix 400 ml M199 with 100 ml (final concentration = 20%) heat inactivated FBS and 5 ml (final concentration = 1%) Penicillin/Streptomycin. Prepare the Serum Free M199 Media – Mix 500 ml M199 with 5 ml (final concentration = 1%) Penicill…

Representative Results

During and after isolation, PASMC are examined both by light microscopy and by immunostaining for smooth muscle cell markers. By light microscopy early in the protocol, PASMC are seen migrating onto the tissue culture dish from the small iron containing vessels (Figure 1A). After pooling plates one through three on day thirteen, then iron particles are no longer seen as those have been pulled out in the final pooling step. Instead, a population of PASMC is seen on the tissue culture dish (Figure …

Discussion

In this manuscript, we describe for the first time the isolation of PASMC from mice at P7, P14, and P21. In order to accomplish this, a slurry of agarose and 0.2 μM iron particles are infused through the RV into the PA. Due to the small size of the iron particles, they cannot pass through the pulmonary capillary bed and are thus deposited in the small PA. The lungs are inflated, removed and dissociated. The iron-containing vessels are pulled out of solution using a magnet. Ultimately, the vessels are plated into a t…

Materials

Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Bard Parker surgical blade handle	BD	371030
Stainless steel surgical blades #10 (sterile)	Miltex	4-310
Syringes (3 ml and 5 ml, sterile)	BD	309657 and 306646
Needles (27 G, sterile)	BD	305109
Angiocatheter (24 G, sterile)	BD	381412
Monoject blunt cannula (15 G)	Kendall	SWD202314Z
Sutures	Fisher Scientific	NC9782896
Dynal magnet particle collector	Invitrogen	120-01D	This is a critical tool for the protocol.
Tissue culture plates (35 mm, 60 mm, and 10 cm, sterile)	BD	353001, 353004, and 353003	Any brand of tissue culture plates will be fine.
Iris Scissors (4 ½ inch stainless steel)	American Diagnostic Corporation	3424
Forceps (4 inch stainless steel)	Quick Medical	L5-5004
D-PBS	Mediatech	21-031-CV
M199 media	Mediatech	10-060-CV
Penicillin/streptomycin	VWR	TX16777-164NWU
Fetal bovine serum	Hyclone/Thermo Scientific	SH3091003	Heat inactivate at 55 °C for 45 min. For consistency in results, lot match all serum and obtain from same vendor.
Iron particles (iron (II, III) oxide powder)	Aldrich Chemical Company	#31,006-9
Agarose	Sigma	A9539
Collagenase (made to 80 U/ml)	Sigma	C5138
Isoflurane	Butlet Schein	NDC 11695-6776-1
Nikon Eclipse TE2000-U with a Cascade Photometrics 12-bit camera	Nikon	TE2000-U	Any good light microscope will be fine to observe PASMC in culture.
Anti-desmin antibody	Sigma	D-8281	Use at 1:200 dilution for immunostaining.
Anti-smooth muscle myosin	Biomedical Technologies	BT-562	Use at 1:2,000 dilution for immunostaining.
Rhodamine-red anti-rabbit secondary	Molecular Probes/Invitrogen	R-6394	Use at 1:200 dilution for immunostaining.
Nikon Eclipse TE-300 fluorescent microscope and Cool Snap digital camera	Nikon	TE300	Any good epifluorescence microscope will be fine for immunostaining.
Cyclic nucleotide phosphodiesterase assay kit	Enzo Life Sciences	BML-AK800-0001	This is the only colorimetric PDE enzyme activity assay available.
Sildenafil	Sigma	PZ-0003	A PDE5-selective inhibitor is required for the PDE enzyme activity to determine specificity of cGMP hydrolysis.