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.
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.
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 common3-6.
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 respiration7-9. Additionally, some investigators have isolated and utilized PASMC from slightly older and spontaneously breathing lambs and piglets at 3 days, 2 weeks, and 4 weeks10-12. 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 state13-17. 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 BPD18-22. 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 species23-25. 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 PASMC23,25,26. 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.
The Institutional Animal Care and Use Committee at Northwestern University approved this protocol.
1. Pulmonary Artery Isolation from Neonatal Mice – Day One
2. Pulmonary Artery Isolation from Neonatal Mice – Day Two
3. Pulmonary Artery Isolation from Neonatal Mice – Day Six
4. Pulmonary Artery Isolation from Neonatal Mice – Day Nine
5. Pulmonary Artery Isolation from Neonatal Mice – Day Thirteen
6. Pulmonary Artery Isolation from Neonatal Mice – Day Fourteen
7. Routine Care
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 1B).
Based on immunostaining, the initial cells that migrate off the iron-containing vessels are approximately 50% fibroblasts and 50% PASMC (data not shown). Since fibroblasts have a significant growth advantage, the fibroblasts will over time overgrow the PASMC on plate zero. For this reason, plate zero is discarded once there are clearly isolated PASMC on the subsequent plates. After pooling, the population of PASMC is >90% PASMC which stain positive for both α-smooth muscle myosin and desmin (Figure 2). When imaged at 20X, multiple spindle-shaped cells are seen consistent with a smooth muscle cell phenotype (Figure 2A). When imaged at higher magnification (40X), the lamellopodia of the leading edge of single cells are visualized as they migrate and grow towards other smooth muscle cells on the dish (Figure 2B).
Phosphodiesterase 5 (PDE5) is expressed in PASMC and hydrolyzes cGMP, a key mediator of vascular tone, into inactive GMP. Decrease in PDE5 plays a critical role in the normal pulmonary vascular transition after birth. In large animal models, PDE5 is developmentally regulated across gestation, and its expression and activity fall dramatically after birth27. In order to confirm that these isolated PASMC retain a phenotype consistent with their developmental stage, we examined PDE5 enzyme activity in mouse PASMC isolated from P7, P14, and P21 mice, as well as adult mice. We see the highest levels of PDE5 activity in the PASMC isolated from P7 mice. These levels fall in the PASMC isolated from P14 and P21 mice. The lowest levels of PDE5 activity are noted in the PASMC isolated from adult mice (Figure 3).
Figure 1. Neonatal PASMC Visualized By Light Microscopy. PASMC are visualized using a light microscope at 20X. A) On day three of the protocol, spindle-shaped PASMC can be seen migrating from the black iron-filled small PA onto the tissue culture dish. B) On day fourteen after pooling, a population of PASMC can be seen on the tissue culture dish.
Figure 2. Neonatal PASMC Stain Positively for Smooth Muscle Markers. PASMC were plated onto collagen-treated glass coverslips, fixed in 4% formaldehyde, and permeabilized with 0.2% Triton-X as previously described7,23. A) PASMC from P7, P14, and P21 mice were stained with anti-desmin (1:200 dilution) or anti-smooth muscle myosin (1:2,000 dilution) in 5% BSA, followed by rhodamine-red anti-rabbit secondary at a 1:200 dilution. Fluorescence was visualized with an epifluorescence microscope at 20X. B) PASMC from P14, and P21 mice were stained with anti-smooth muscle myosin or desmin as above, and fluorescence was visualized as above at 40X.
Figure 3. Phosphodiesterase 5 (PDE5) Activity is Developmentally Regulated in PASMC. PASMC were harvested for total protein and assayed for PDE5 enzymatic activity as previously described using a commercially available colorimetric cyclic nucleotide phosphodiesterase assay kit7. Each sample was read ± sildenafil (100 nM). The difference between the pmol cGMP hydrolyzed per mg total protein per minute ± sildenafil represents the PDE5-specific cGMP-hydrolytic activity. N=4 for P7 PASMC, n=10 for P14 PASMC, n=4 for P21 PASMC, and n=8 for adult PASMC. * p<0.05 versus P7 PASMC, # p<0.05 versus P14 PASMC.
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 tissue culture dish, and cells migrate off the vessels and onto the culture plate. The initial plate of cells is a mixture of fibroblasts and PASMC, and the pull down procedure is repeated multiple times to derive an enriched PASMC population. With prolonged exposure at high levels, there is a theoretical risk that residual iron may impact the redox balance within the isolated cells. Thus, care is taken in the last step to remove all residual iron prior to pooling and propagating the cells. Immunostaining for α-smooth muscle myosin and desmin is done to confirm that the enriched population is >90% PASMC. These cells have PDE5 enzymatic activity, consistent with a vascular smooth muscle cell phenotype. Interestingly, the PDE5 enzyme activity is different in PASMC isolated from mice of different ages, suggesting continued developmental regulation within the PASMC after isolation.
While this technique is intellectually very straightforward, a common problem is low yield of PASMC. There can be multiple reasons for this problem, even when 2-3 mice are pooled together. First, the best cell yield is achieved if the isolation procedure is initiated immediately after the mouse succumbs to the isoflurane overdose. Once death has occurred, micro-clots form throughout the vascular bed. These clots in the pulmonary vasculature will prevent the iron particles from getting into as many small PA, thereby decreasing the yield. Also, the viability of the isolated PASMC becomes of greater concern the longer the mouse is dead prior to isolation. The absolute key step that impacts PASMC yield is the iron infusion. If the lungs are not noticeably grey after the iron infusion, the possible reasons are: 1) the iron is going retrograde into the right atrium and liver, 2) there is hole or tear in the RV, or 3) there are micro-clots in the pulmonary circulation somewhere preventing good iron infusion. Lastly, we have noticed that PASMC isolated from different genetically modified strains of mice exhibit strikingly different growth rates in culture after isolation. Therefore, when first attempting this technique, it is best to use a wild-type strain of mice.
Obviously, contamination is a concern any time one is isolating cells from a whole animal. All instruments are sterilized and wiped with alcohol prior to the procedure, and then once the heart-lung bloc is removed, all of the subsequent steps are done in a tissue culture hood with sterile technique. Using these precautions together with antibiotic-containing media has virtually eliminated any contamination.
With the isolation of any primary cell line, there is a concern about how long a cell will maintain its phenotype in culture. Ovine FPASMC isolated from control and PPHN lambs maintain their smooth muscle phenotype for 8 and 5 passages, respectively7,14. For the mouse PASMC, the cells begin to lose signaling responses by passage 5 and smooth muscle markers by passage 7 (data not shown). This highlights one of the limitations of this technique. Multiple mice are required to achieve small amounts of cells, and the time from isolation to enough cells for experiments is long; 2-4 weeks depending on the number of cells needed. Finally, once cells are isolated, one only has three passages to work with them before they lose their smooth muscle cell signaling responses. One potential area for improvement would be to develop a method whereby these cells could be frozen and recovered successfully from liquid nitrogen. This would allow a lab to regularly make and freeze cells as mice are available, and then investigators could thaw and utilize the cells for experiments as needed.
Despite the technical challenge of performing the isolation and the long timeline for isolation, these PASMC represent a novel and invaluable tool for the study of signaling pathways in the developing murine pulmonary vasculature. 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, which will lead to better understanding of the disease pathogenesis and allow for development of new treatments.
The authors have nothing to disclose.
This work was supported by NIH HL109478 (KNF). The authors acknowledge and thank Gina Kim and Joann Taylor for their assistance in isolating and maintaining the PASMC in culture.
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. |