Research Paper J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
Received: March 15, 2013 Accepted after revision: August 19, 2013 Published online: October 26, 2013
Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries Gongyong Peng a Xing Wen c Yu Shi d Yongliang Jiang a, e Guoping Hu a, b Yumin Zhou a Pixin Ran a a
Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Disease, The First Affiliated Hospital, and b Department of Respiratory Medicine, The Third Affiliated Hospital, Guangzhou Medical University, c Department of Acupuncture, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou University of Traditional Chinese Medicine, and d Centre for Reproductive Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China; e Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md., USA
Key Words Pulmonary hypertension · Cell culture · Endothelial cells · Rat pulmonary arteries
Abstract Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial
© 2013 S. Karger AG, Basel 1018–1172/13/0506–0468$38.00/0 E-Mail [email protected] www.karger.com/jvr
cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium. © 2013 S. Karger AG, Basel
Introduction
Pulmonary arterial endothelial cells (PAECs), forming the inner lining of a vast network of pulmonary arteries and arterioles, are involved in many physiological and pathophysiological processes in the lung such as the maintenance of vascular tone, the transduction of luminal signals to abluminal vascular tissues and the production of growth factors and cell signals with autocrine and paracrine effects. Although the pathogenesis of pulmonary hypertension involves a complex and multifactorial process, endothelial dysfunction seems to play an integral role in mediating the structural changes in the pulmonary vasculature. The isolation and culture of PAECs from pulmonary arteries, mainly from humans and large aniDr. Pixin Ran Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Disease The First Affiliated Hospital, Guangzhou Medical University 151 Yanjiang Road, Guangzhou, Guangdong 510120 (People’s Republic of China) E-Mail pxran @ vip.163.com
mals, has contributed to a better understanding of the pathogenesis and development of pulmonary hypertension [1–6]. However, it has proved difficult and inconvenient to obtain PAECs from humans and large animals. Previous studies have described the isolation of PAECs from small animals such as rats [7–13]. The rat pulmonary arteries in these reports, however, were mainly isolated from the pulmonary truncus [7–12]. In addition, some methods have limitations represented by the need for complex equipment, such as microcarrier beads [13]. The purpose of this study was to develop a new method to isolate pulmonary arteries, obtain cultures of PAECs and further characterize the phenotype of these cells. Such cells would be of great value for in vitro experiments to explore the mechanisms of pulmonary endothelial dysfunction and pulmonary hypertension. The method to isolate endothelial cells from human umbilical veins is commonly used for cardiovascular studies [14–20]. We applied a similar rationale and developed a simple and economical technique with Chinese acupuncture needles to isolate and culture PAECs from rat pulmonary arteries. Endothelial cell phenotype was verified by surveying the pattern of growth, morphology, immunohistological staining and ultrastructural characteristics. Methods Rats Male Wistar rats (body weight 250–350 g) were used in all experiments. The rats were housed in community cages with 12/12hour light-dark cycles and maintained on a standard laboratory rat diet with access to water. All animal care and experiments were approved and carried out in accordance with the Animal Care and Use Committee of Guangzhou Medical University and in accordance with the principles and guidelines of the National Institutes of Health.
goat IgG antibody were from Jackson ImmunoResearch (West Grove, Pa., USA) and YO-PRO-1 was from Invitrogen (Carlsbad, Calif., USA). As shown in figure 1, the micromanipulation instruments, including microforceps, microscissors, hemostats and scissors were from Jinzhong Surgical Instruments (Shanghai, China), the Chinese acupuncture needles were from Tianxie Acupuncture Instruments (size: 0.22 × 40 mm, Suzhou, Jiangsu, China) and the copper wires (0.13 mm diameter) were obtained from some old stock. Isolation of Pulmonary Arteries Rats were deeply anesthetized with pentobarbital sodium (65 mg · kg–1 i.p.), and anesthesia was confirmed by the lack of a withdrawal reflex from a toe pinch. Figure 2 shows the procedure used to isolate the pulmonary arteries. This was adapted from our previous description of the isolation of rat pulmonary veins [21]. Briefly, under sterile conditions, anesthetized rats were placed in the supine position and their chests were opened. The heart and lungs were removed en bloc and transferred to a petri dish with cold (4 ° C) physiological salt solution (PSS) that contained 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM HEPES and 10 mM glucose (fig. 2a). After being rinsed twice with cold PSS, the heart and lungs were placed in the arteries-up position and fixed in an isolation dish with acupuncture needles (fig. 2b). Under a stereomicroscope, we first identified the extrapulmonary arteries connected with the right cardiac ventricle. The right and left branches of the intrapulmonary arteries that followed from the extrapulmonary arteries were carefully dissected from the lungs within 20 min of the removal of the heart and lungs from the thoracic cavity (fig. 2b, c). Adipose and connective tissue were carefully removed in cold Ca2+-reduced PSS (20 μM CaCl2) with microdissection forceps and scissors (fig. 2c). The right and left branches of the pulmonary arteries were cut from the pulmonary truncus (fig. 2d).
Materials Unless otherwise specified, all reagents were obtained from Sigma Chemicals. The endothelial cell basal medium-2 (EBM-2), the EGM-2 MV SingleQuots, the smooth muscle cell basal medium (SmBM) and the SmGM-2 SingleQuots were from Clonetics (Walkersville, Md.), rat renal fibroblasts (NRK-49F) were from American Type Culture Collection (ATCC, Manassas, Va., USA), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), PBS and antibiotic-antimycotic were from GIBCO (Carlsbad, Calif., USA), rabbit polyclonal antibody to von Willebrand factor, mouse monoclonal antibody to CD31, mouse monoclonal antibody to smooth muscle myosin heavy chain and rabbit monoclonal antibody to CD90/Thy-1 were from Abcam (Cambridge, Mass., USA), goat polyclonal antibody to CD34 was from R&D Systems (Minneapolis, Minn., USA), Cy3-conjugated donkey anti-rabbit IgG antibody, HRP-conjugated donkey anti-rabbit IgG antibody, goat anti-mouse IgG antibody and donkey anti-
Cell Isolation and Culture Figure 3 shows the procedure for cell isolation. The blunt acupuncture needles were inserted into the right and left branches of the pulmonary arteries from the distal extremity to the proximal extremity (fig. 3a, b). The pulmonary arteries were reversed with microforceps along the acupuncture needle handles to expose their endothelium (fig. 3c–f). Both the distal and proximal extremities of the pulmonary arteries were ligated with copper wires using hemostats (fig. 3g, h). After the blunt acupuncture needles were removed from the handles, the reversed pulmonary arteries, with the exposed endothelium kept tightly around the handles, were placed in the 15-ml plastic conical tube containing 2 ml of Ca2+-reduced collagenase enzymatic solution (type I, 1,800 U/ml) and digested at 37 ° C for 8 min (fig. 3i). The digestion was terminated by adding 8 ml of EBM-2 supplemented with EGM-2 MV SingleQuots (EBM-2MV) and 10% FBS into the plastic conical tube. The cells were separated from the reversed arteries by gentle aspiration and repulsing about 10 times with a wide-bore transfer pipette set to 1 ml. After the reversed arteries were washed with 20 ml of 1 × PBS, the cell suspension was collected and centrifuged for 10 min at 400 g at room temperature (RT). The supernatant was carefully discarded. After resuspension of the cell pellet, the cells were plated onto 35-mm petri dishes precoated with human fibronectin. Coating of the dishes was accomplished by incubation with 4 ml of human fibronectin solution (10
Development of a New Method for the Culture of Rat PAECs
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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Fig. 1. a All the micromanipulation instruments used in the experiments for isolation of rat pulmonary arteries. b The enlarged image from the inset (a) showing the Chinese acupuncture needles and the copper wires. c The Chinese acupuncture needles in an open box.
Fig. 2. a The heart and lungs were removed
en bloc and placed in the arteries-up position in an isolation dish. b The heart and lungs were fixed with acupuncture needles and the right and left branches of the intrapulmonary arteries that followed the extrapulmonary arteries were carefully dissected from the lungs. c Adipose and connective tissues were carefully removed from the pulmonary arteries that connected with the heart. d The right and left branches of the pulmonary arteries were cut and used for the experiments.
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J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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Fig. 3. a, b The blunt acupuncture needles
were inserted into the right and left branches of the pulmonary arteries from the distal extremity to the proximal extremity. b Enlarged inset from a. c–f The pulmonary arteries were reversed along the acupuncture needle handles to expose the endothelium of the arteries. c One was reversed and the other was reversing. d Enlarged inset from c. e Two arteries were reversed. f Enlarged inset from e. g Both the distal and proximal extremities of the pulmonary arteries were ligated with copper wires. h Enlarged inset from g. i After the blunt acupuncture needles were removed, the reversed pulmonary arteries with the exposed endothelium maintained around the handles were placed in a plastic conical tube (containing 2 ml of collagenase enzymatic solution) and digested.
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μg/ml PBS) for 2 h in a tissue culture incubator at 37 ° C. Excess fluid was removed immediately before adding the cells. The cells were then incubated in EBM-2MV with 5% FBS in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C. The medium was removed and replaced every 2–3 days. Cell growth and cell morphology were examined daily under an inverted light microscope. The rat pulmonary arterial smooth muscle cells (PASMCs), isolated and cultured as described earlier [22, 23], were used as a morphological control. The cells were subcultured when they reached more than 80% confluence. Cultures were first washed twice with PBS, then treated with 0.25% trypsin and 0.01% EDTA, and when most cells became visibly rounded, they were neutralized with EBM-2MV with 5% FBS. Detached cells were centrifuged and the supernatant was removed. After resuspension of the cell pellet, the cells were passaged into fibronectin-coated flasks at a ratio of 1:2–1:3 and incubated at 37 ° C with 5% CO2 and 95% air.
Cell Identification Immunofluorescence The endothelial cells were identified by the presence of von Willebrand factor using a rabbit polyclonal antibody. The cells not treated with the primary antibody specific for von Willebrand factor were used as negative control. The endothelial cells were also identified by the absence of α-smooth muscle actin. Rat PASMCs were used as a positive control. The third-to-fourth passage cells were seeded and grown on fibronectin-coated coverslips in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C for 5–6 days. Cells were rinsed twice with PBS and fixed for 15 min with 2 ml of 4% paraformaldehyde in PBS at RT. Cells were rinsed 3 times with PBS and treated for 15 min with 2 ml of PBS containing 0.25% Triton X-100 at RT. The permeabilization buffer was aspirated, and cells were rinsed 3 times with 2 ml of PBS for 5 min each. Cells were incubated at RT for 30 min in PBS with 1% BSA and then for 60 min at RT with a rabbit
Development of a New Method for the Culture of Rat PAECs
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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polyclonal antibody to von Willebrand factor (1:200). To visualize the proteins, the cells were treated for 2 h at RT in the dark with a Cy3-conjugated secondary donkey anti-rabbit IgG (Fab-specific) antibody (1: 100), which produced red fluorescence. Cells were rinsed and incubated with YO-PRO-1 solution at 3.5 μl/ml for 5 min at RT and away from light. The coverslips were mounted with a drop of mounting medium. Immunocytochemistry Immunocytochemistry was performed on the subcultured cells (passages 8–10) grown to 40–50% confluence on fibronectin-coated coverslips according to the procedure described previously [24, 25]. Briefly, following extensive washes with PBS, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.25% Triton X-100 as described above in immunofluorescence. After blocking with 1% BSA for 30 min at RT, the cells were incubated with 200 μl of the following primary antibodies for 90 min at RT: rabbit polyclonal antibody to von Willebrand factor (1: 200), mouse monoclonal antibody to CD31 (1:200), goat polyclonal antibody to CD34 (1:100), mouse monoclonal antibody to α-smooth muscle actin (1:200), mouse monoclonal antibody to smooth muscle myosin heavy chain (1: 200) and mouse monoclonal antibody to CD90/Thy-1 (1: 100). After three washes in PBS, the cells were incubated with the appropriate HRP-conjugated secondary antibody [donkey anti-rabbit IgG (1: 100), goat anti-mouse IgG (1: 100) or donkey anti-goat IgG (1:100)] for 30 min at RT. Antibody complexes were visualized with 3,3′-diaminobenzidine tetrahydrochloride. The nuclei were counterstained with hematoxylin. Rat PASMCs were used as positive controls for α-smooth muscle actin and smooth muscle myosin heavy chain and NRK-49F cells were used as a positive control for CD90/Thy-1. Transmission Electron Microscopy As previously described [26, 27], cells grown to confluence on fibronectin-coated coverslips were fixed by immersion in 2.5% glutaraldehyde for 24 h, postfixed with 1% osmium tetroxide for 1 h and dehydrated through graded alcohols to be embedded in Spurr’s resin. Subsequently, thin sections (60 nm) were cut and stained with 2% uranyl acetate and 0.1% lead citrate. Sections were examined in a transmission electron microscope at 80 kV.
Results
Cell Morphology The vessels used in this study were isolated from the right and left branches of the pulmonary arteries (fig. 2, 3). The cultured cells were identified on the basis of their patterns of growth, morphology, immunohistological staining and ultrastructural characteristics. Observed with a phase-contrast microscope, both the endothelial cells and the smooth muscle cells started to divide and extend within 3 days. The endothelial cells were large, flat and polygonal and displayed a characteristic ovoid nucleus with 2–3 prominent nucleoli and peri472
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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Fig. 4. Phase-contrast microscopy of rat PAECs and PASMCs. a PAECs cultured for 3 days. b PASMCs cultured for 3 days. c PAECs cultured for 7 days. d PASMCs cultured for 7 days.
nuclear granules (fig. 4a). However, the smooth muscle cells assumed a spindle-shaped appearance with cytoplasmic projections extending from a larger central area that contained the nucleus (fig. 4b). As soon as the cells became confluent, the endothelial cells displayed typical ‘cobblestone’ morphology characteristic of endothelial cells (fig. 4c), whereas the smooth muscle cells grew in a ‘hill-and-valley’-like pattern (fig. 4d). Both the endothelial cells and the smooth muscle cells grew to confluence (95%) in 7–8 days. Immunofluorescence As shown in figure 5a, the cultured cells stained strongly for von Willebrand factor. However, the immunocytochemical fluorescence of von Willebrand factor was not observed in control cells, which did not receive the primary monoclonal antibody raised against von Willebrand factor, but were otherwise treated similarly (fig. 5b). The endothelial cells were also identified by the absence of α-smooth muscle actin. The staining of cultured endothelial cells for α-smooth muscle actin was similar to background levels (fig. 5c). However, α-smooth muscle actin was strongly positive in rat PASMCs (fig. 5d). Peng /Wen /Shi /Jiang /Hu /Zhou /Ran
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Fig. 5. a Fluorescence immunocytochemical identification of von Willebrand factor was shown in red in PAECs. The nuclear marker YO-PRO-1 was shown in green. b The immunocytochemical fluorescence of von Willebrand factor did not appear in control PAECs (which did not receive primary monoclonal antibody raised against von Willebrand factor, but were otherwise treated similarly). c The staining of PAECs for α-smooth muscle actin was similar to the background. d Rat PASMCs strongly expressed α-smooth muscle actin (in red).
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Immunocytochemistry To further confirm the purity of subcultured endothelial cells, after 8–10 passages, the cells were examined for the presence of various endothelial-cell-specific markers and the absence of smooth-muscle-cell or fibroblast-specific markers by immunocytochemical analysis. As shown in figure 6, all subcultured cells exhibited the presence of the endothelial cell markers von Willebrand factor (fig. 6a), CD31 (fig. 6b) and CD34 (fig. 6c). Negative controls for three markers had no staining (data not shown). The endothelial cell cultures did not stain for α-smooth muscle actin (fig. 6d) or smooth muscle myosin heavy chain (fig. 6e). These cells also did not stain for CD90/ Thy-1 (fig. 6f). However, rat PASMCs were positive for α-smooth muscle actin (fig. 6g) and smooth muscle myosin heavy chain (fig. 6h). NRK-49F cells showed positivity for CD90/Thy-1 (fig. 6i). Respective negative controls for α-smooth muscle actin (fig. 6j), smooth muscle myosin heavy chain (fig. 6k) and CD90/Thy-1 (fig. 6l), which were performed by omitting the primary antibody, had no staining.
bers and were localized in subplasmalemmal regions. These data demonstrate that our cultured cells were, in fact, vascular endothelial cells.
Discussion
Transmission Electron Microscopy The cultured cells were investigated by transmission electron microscopy. Electron microscopic analysis showed the presence of Weibel-Palade bodies (fig. 7a, b), which is a unique ultrastructural marker of vascular endothelium. Furthermore, the cells contained pinocytotic vesicles (fig. 7c, d), which were present in moderate num-
In this study, we describe a new method to acquire primary cultures of PAECs from rat pulmonary arterial branches for in vitro studies of pulmonary artery-related diseases. As mentioned above, it has proved difficult and inconvenient to obtain the pulmonary arteries from humans and large animals, and some methods for culture of rat PAECs obtain the cells mainly from the pulmonary truncus or else require complex equipment. In our experiments, we developed, for the first time, a simple and economical method with Chinese acupuncture needles to acquire rat pulmonary arteries and expose the vessel endothelium. The method for isolation of rat pulmonary arteries in our study was similar to that for isolation of rat distal pulmonary veins and arteries, used to obtain the pulmonary venous smooth muscle cells and PASMCs and described by us and other investigators [21, 28]. The technique to reverse the pulmonary arteries with Chinese acupuncture needles was a new method for exposure of the vessel endothelium. Although this method needs micromanipulation, it was really simple and economical. It has been suggested that all vascular endothelial cells contain characteristic structural similarities that can be
Development of a New Method for the Culture of Rat PAECs
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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Fig. 6. Immunocytochemical analysis showed PAECs were positive for von Willebrand factor (a), CD31 (b) and CD34 (c) and negative for α-smooth muscle actin (d), smooth muscle myosin heavy chain (e) and CD90/Thy-1 (f). Rat PASMCs were positive for α-smooth muscle actin (g) and smooth muscle myosin heavy chain (h). i NRK-49F cells were positive for CD90/ Thy-1. Respective negative controls for α-smooth muscle actin (j), smooth muscle myosin heavy chain (k) and CD90/Thy-1 (l) had no staining. The nuclei were counterstained with hematoxylin (blue).
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used to identify them. Therefore, in our subsequent experiments, the cell phenotype, immunological properties and ultrastructural characteristics were used to characterize the isolated cells as vascular endothelial cells. The rat PAECs that we obtained exhibited characteristic morphological features, immunoreactivity to von Willebrand factor, CD31 and CD34 as well as unique ultrastructural characteristics that were consistent with observations by other investigators [26, 29–34, 36, 41–48]. PAECs started to divide within 3 days, and the culture reached confluence in approximately 1 week. At low den474
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
sity, PAECs were flat and polygonal and displayed a characteristic ovoid nucleus. As soon as the cells became confluent, the primary culture of PAECs, like other vascular endothelial cells described by other investigators [26, 29, 30], grew in a typical ‘cobblestone’-like pattern that is characteristic of endothelium. PASMCs, however, showed a spindle-shaped appearance at low density and grew in a ‘hill-and-valley’-like pattern at a high density that was completely different from that of PAECs. To confirm the origin of the cultured cells, we used a sensitive and specific marker of vascular endothelial cells, Peng /Wen /Shi /Jiang /Hu /Zhou /Ran
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Fig. 7. a, b Electron microscopic analysis showed the presence of Weibel-Palade bodies in PAECs. b Enlarged inset from a. c The pinocytotic vesicles were present in moderate numbers and were localized in subplasmalemmal regions in PAECs. d Enlarged inset from (c). N = Nucleus.
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von Willebrand factor, to detect the immunofluorescence of early passage cells (passage 3–4). The presence of Willebrand factor is the most widely used criterion for identification of cells of endothelial origin. We found that von Willebrand factor was strongly positive in the cultures of PAECs, consistent with observations from other investigators [30–33]. However, PAECs did not express α-smooth muscle actin, whereas PASMCs expressed the protein. PAECs showed strong immunostaining for von Willebrand factor and a lack of α-smooth muscle actin, clearly suggesting that the cells that we acquired and cultured were indeed the vascular endothelial cells. To further confirm the purity of PAECs, we next used two additional endothelial cell markers, CD31 and CD34, as well as three other smooth muscle cell or fibroblast markers, α-smooth muscle actin, smooth muscle myosin heavy chain and CD90/Thy-1, in the immunocytochemical analysis of late passage cells (passage 8–10). CD31 is widely used and also known as platelet endothelial cell adhesion molecule, PECAM-1. It is strongly expressed by all endothelial cells and weakly expressed by several types of leukocytes [34]. CD34 is a cell surface protein implicated in cell migration and halting of differentiation that has been documented to be expressed in PAECs, and which has served most commonly as a hematopoietic stem cell marker but is also known as an endothelial cell
marker [35, 36]. All PAECs expressed Willebrand factor, CD31 and CD34 after 8–10 passages. Nevertheless, these cell cultures did not express α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Both α-smooth muscle actin and smooth muscle myosin heavy chain are smooth muscle cell myofilamentous proteins and are widely used for the characterization of cells of smooth muscle cell phenotype [21, 37–38]. CD90/Thy-1 is a glycophosphatidylinositol-anchored, strongly glycosylated protein that is expressed on the cell surface. CD90/ Thy-1 was originally identified as a thymocyte antigen and is also known as the fibroblast-specific antigen, shown to be expressed in fibroblasts [39, 40]. Hence, the cells we isolated from rat pulmonary arteries with Chinese acupuncture needles and subcultured up to passage 10 were certainly of endothelial origin and were not contaminated with smooth muscle cells or fibroblasts. Although the cell morphology and immunological properties indicated that the cultured cells were endothelial cells, ultrastructural examination was carried out to further determine whether the cells in culture exhibited ultrastructural features of vascular endothelial cells. Transmission electron micrographs showed that WeibelPalade bodies were present in the cultures of PAECs. Weibel-Palade bodies are characteristically found in vascular endothelial cells but not in smooth muscle cells or
Development of a New Method for the Culture of Rat PAECs
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in fibroblasts, and have been shown to be present in the cultured vascular endothelial cells of rat, bovine and human origin [41–44]. Furthermore, pinocytotic vesicles were found in moderate numbers and were present only at the periphery of the cells close to the membrane; this is consistent with observations from other investigators [45–48]. In summary, we have developed a simple and economical method for obtaining PAECs from rat pulmonary arteries. PAECs show the morphological characteristics of vascular endothelial cells, express von Willebrand factor, CD31 and CD34, lack α-smooth muscle actin, smooth muscle myosin heavy chain and CD90/Thy-1 and possess Weibel-Palade bodies consistent with their vascular en-
dothelial cell origin. PAECs could subsequently be used to further explore the pathophysiological mechanisms of pulmonary endothelial dysfunction and pulmonary hypertension. Acknowledgments This work was supported by the National Natural Science Foundation of China (81000020 and 81170043), the Specialized Research Fund for the Doctoral Program of Higher Education, China (20104423110001), the Guangdong Key Research Project grant (B30301), the research grants from Guangzhou Department of Education, China (10A025G and 10A276) and the Science Foundation of State Key Laboratory of Respiratory Diseases (2011011).
References 1 Teichert-Kuliszewska K, Kutryk MJ, Kuliszewski MA, Karoubi G, Courtman DW, Zucco L, Granton J, Stewart DJ: Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ Res 2006;98:209–217. 2 Ramos M, Lamé MW, Segall HJ, Wilson DW: Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells. Vascul Pharmacol 2007;46:439–448. 3 Mathew R, Huang J, Shah M, Patel K, Gewitz M, Sehgal PB: Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 2004; 110: 1499–1506. 4 Sehgal PB, Mukhopadhyay S, Xu F, Patel K, Shah M: Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1526– L1542. 5 Zharikov S, Krotova K, Hu H, Baylis C, Johnson RJ, Block ER, Patel J: Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells. Am J Physiol Cell Physiol 2008; 295:C1183–C1190. 6 Sud N, Sharma S, Wiseman DA, Harmon C, Kumar S, Venema RC, Fineman JR, Black SM: Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90. Am J Physiol Lung Cell Mol Physiol 2007; 93:L1444–L1453. 7 Zhu B, Strada S, Stevens T: Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 289:L196–L206.
476
8 Wang Q, Pfeiffer GR 2nd, Stevens T, Doerschuk CM: Lung microvascular and arterial endothelial cells differ in their responses to intercellular adhesion molecule-1 ligation. Am J Respir Crit Care Med 2002; 166: 872– 877. 9 Zhu D, Zhang C, Medhora M, Jacobs ER: CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium. J Appl Physiol 2002;93:330–337. 10 Stevens T, Creighton J, Thompson WJ: Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function. Am J Physiol 1999;277:L119– L126. 11 Moore TM, Brough GH, Babal P, Kelly JJ, Li M, Stevens T: Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1. Am J Physiol 1998;275:L574–L582. 12 Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, Thompson WJ: Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol 1998;274:L810–L819. 13 Michael JR, Markewitz BA, Kohan DE: Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells. Am J Physiol 1997;273:L768– L774. 14 Baudin B, Bruneel A, Bosselut N, Vaubourdolle M: A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2007;2:481–485. 15 Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745–2756. 16 Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, Ziemann M, Helbing T, El-Osta A, Jowett JB, Peter K: Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc Res 2012;93:633–644.
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17 Nishiyama K, Takaji K, Kataoka K, Kurihara Y, Yoshimura M, Kato A, Ogawa H, Kurihara H: Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis. Circulation 2005;112:2840–2850. 18 Buerkle MA, Lehrer S, Sohn HY, Conzen P, Pohl U, Krötz F: Selective inhibition of cyclooxygenase-2 enhances platelet adhesion in hamster arterioles in vivo. Circulation 2004; 110:2053–2059. 19 Margariti A, Zampetaki A, Xiao Q, Zhou B, Karamariti E, Martin D, Yin X, Mayr M, Li H, Zhang Z, De Falco E, Hu Y, Cockerill G, Xu Q, Zeng L: Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin. Circ Res 2010;106:1202–1211. 20 Osanai T, Magota K, Tanaka M, Shimada M, Murakami R, Sasaki S, Tomita H, Maeda N, Okumura K: Intracellular signaling for vasoconstrictor coupling factor 6: novel function of beta-subunit of ATP synthase as receptor. Hypertension 2005;46:1140–1146. 21 Peng G, Wang J, Lu W, Ran P: Isolation and primary culture of rat distal pulmonary venous smooth muscle cells. Hypertens Res 2010;33:308–313. 22 Lu W, Wang J, Peng G, Shimoda LA, Sylvester JT: Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 2009;297:L17–L25. 23 Lu W, Ran P, Zhang D, Peng G, Li B, Zhong N, Wang J: Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle. Am J Physiol Cell Physiol 2010;298:C114–C123.
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24 Zhang Q, Cooper RK, Wolters WR, Tiersch TR: Isolation, culture and characterization of a primary fibroblast cell line from channel catfish. Cytotechnology 1998;26:83–90. 25 Straszewski-Chavez SL, Abrahams VM, Alvero AB, Aldo PB, Ma Y, Guller S, Romero R, Mor G: The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71. Placenta 2009;30:939–948. 26 Browning AC, Gray T, Amoaku WM: Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells. Br J Ophthalmol 2005; 89: 1343– 1347. 27 Wu Z, Hofman FM, Zlokovic BV: A simple method for isolation and characterization of mouse brain microvascular endothelial cells. J Neurosci Methods 2003;130:53–63. 28 Whitman EM, Pisarcik S, Luke T, Fallon M, Wang J, Sylvester JT, Semenza GL, Shimoda LA: Endothelin-1 mediates hypoxia-induced inhibition of voltage-gated K+ channel expression in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 2008;294:L309–L318. 29 Wang D, Lehman RE, Donner DB, Matli MR, Warren RS, Welton ML: Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells. Am J Physiol Gastrointest Liver Physiol 2002;282:G1088– G1096. 30 Banumathi E, Haribalaganesh R, Babu SS, Kumar NS, Sangiliyandi G: High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels. Microvasc Res 2009; 77: 377–381. 31 Thorin E, Shatos MA, Shreeve SM, Walters CL, Bevan JA: Human vascular endothelium heterogeneity. A comparative study of cerebral and peripheral cultured vascular endothelial cells. Stroke 1997;28:375–381.
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32 Argall KG, Armati PJ, Pollard JD: A method for the isolation and culture of rat peripheral nerve vascular endothelial cells. Mol Cell Neurosci 1994;5:413–417. 33 Yu SY, Song YM, Li AM, Yu XJ, Zhao G, Song MB, Lin CM, Tao CR, Huang L: Isolation and characterization of human coronary arteryderived endothelial cells in vivo from patients undergoing percutaneous coronary interventions. J Vasc Res 2009;46:487–494. 34 Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY: JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol 1990; 43:752–757. 35 Nielsen JS, McNagny KM: CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery. Microcirculation 2009;16:487–496. 36 Xiaozhuang Z, Xianqiong L, Jingbo J, Shuiqing H, Jie Y, Yunbin C: Isolation and characterization of fetus human retinal microvascular endothelial cells. Ophthalmic Res 2010; 44:125–130. 37 Teng B, Ansari HR, Oldenburg PJ, Schnermann J, Mustafa SJ: Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptorknockout mice. Am J Physiol Heart Circ Physiol 2006;290:H1713–H1720. 38 Cairrão E, Santos-Silva AJ, Alvarez E, Correia I, Verde I: Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels. In Vitro Cell Dev Biol Anim 2009;45:175–184. 39 Saalbach A, Aneregg U, Bruns M, Schnabel E, Herrmann K, Haustein UF: Novel fibroblastspecific monoclonal antibodies: properties and specificities. J Invest Dermatol 1996;106: 1314–1319.
40 Hagood JS, Prabhakaran P, Kumbla P, Salazar L, MacEwen MW, Barker TH, Ortiz LA, Schoeb T, Siegal GP, Alexander CB, Pardo A, Selman M: Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis. Am J Pathol 2005;167:365–379. 41 Sölder E, Böckle BC, Nguyen VA, Fürhapter C, Obexer P, Erdel M, Stössel H, Romani N, Sepp NT: Isolation and characterization of CD133+CD34+VEGFR-2+CD45– fetal endothelial cells from human term placenta. Microvasc Res 2012;84:65–73. 42 Green DF, Hwang KH, Ryan US, Bourgoignie JJ: Culture of endothelial cells from baboon and human glomeruli. Kidney Int 1992; 41: 1506–1516. 43 Okruhlicová L, Dlugosová K, Mitasíková M, Bernátová I: Ultrastructural characteristics of aortic endothelial cells in borderline hypertensive rats exposed to chronic social stress. Physiol Res 2008;57(suppl 2):S31–S37. 44 Ryan US, Ryan JW: Angiotensin-converting enzyme: II. Pulmonary endothelial cells in culture. Environ Health Perspect 1980; 35: 171–180. 45 McGuire PG, Twietmeyer TA: Morphology of rapidly frozen aortic endothelial cells. Glutaraldehyde fixation increases the number of caveolae. Circ Res 1983;53:424–429. 46 Sakamoto T, Sakamoto H, Hinton DR, Spee C, Ishibashi T, Ryan SJ: In vitro studies of human choroidal endothelial cells. Curr Eye Res 1995;14:621–627. 47 Tu J, Stoodley MA, Morgan MK, Storer KP: Ultrastructure of perinidal capillaries in cerebral arteriovenous malformations. Neurosurgery 2006;58:961–970. 48 Espina AI, Castellanos AV, Fereira JL: Agerelated changes in blood capillary endothelium of human dental pulp: an ultrastructural study. Int Endod J 2003;36:395–403.
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Copyright: S. Karger AG, Basel 2013. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: March 15, 2013 Accepted after revision: August 19, 2013 Published online: October 26, 2013
Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries Gongyong Peng a Xing Wen c Yu Shi d Yongliang Jiang a, e Guoping Hu a, b Yumin Zhou a Pixin Ran a a
Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Disease, The First Affiliated Hospital, and b Department of Respiratory Medicine, The Third Affiliated Hospital, Guangzhou Medical University, c Department of Acupuncture, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou University of Traditional Chinese Medicine, and d Centre for Reproductive Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China; e Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md., USA
Key Words Pulmonary hypertension · Cell culture · Endothelial cells · Rat pulmonary arteries
Abstract Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial
© 2013 S. Karger AG, Basel 1018–1172/13/0506–0468$38.00/0 E-Mail [email protected] www.karger.com/jvr
cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium. © 2013 S. Karger AG, Basel
Introduction
Pulmonary arterial endothelial cells (PAECs), forming the inner lining of a vast network of pulmonary arteries and arterioles, are involved in many physiological and pathophysiological processes in the lung such as the maintenance of vascular tone, the transduction of luminal signals to abluminal vascular tissues and the production of growth factors and cell signals with autocrine and paracrine effects. Although the pathogenesis of pulmonary hypertension involves a complex and multifactorial process, endothelial dysfunction seems to play an integral role in mediating the structural changes in the pulmonary vasculature. The isolation and culture of PAECs from pulmonary arteries, mainly from humans and large aniDr. Pixin Ran Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Disease The First Affiliated Hospital, Guangzhou Medical University 151 Yanjiang Road, Guangzhou, Guangdong 510120 (People’s Republic of China) E-Mail pxran @ vip.163.com
mals, has contributed to a better understanding of the pathogenesis and development of pulmonary hypertension [1–6]. However, it has proved difficult and inconvenient to obtain PAECs from humans and large animals. Previous studies have described the isolation of PAECs from small animals such as rats [7–13]. The rat pulmonary arteries in these reports, however, were mainly isolated from the pulmonary truncus [7–12]. In addition, some methods have limitations represented by the need for complex equipment, such as microcarrier beads [13]. The purpose of this study was to develop a new method to isolate pulmonary arteries, obtain cultures of PAECs and further characterize the phenotype of these cells. Such cells would be of great value for in vitro experiments to explore the mechanisms of pulmonary endothelial dysfunction and pulmonary hypertension. The method to isolate endothelial cells from human umbilical veins is commonly used for cardiovascular studies [14–20]. We applied a similar rationale and developed a simple and economical technique with Chinese acupuncture needles to isolate and culture PAECs from rat pulmonary arteries. Endothelial cell phenotype was verified by surveying the pattern of growth, morphology, immunohistological staining and ultrastructural characteristics. Methods Rats Male Wistar rats (body weight 250–350 g) were used in all experiments. The rats were housed in community cages with 12/12hour light-dark cycles and maintained on a standard laboratory rat diet with access to water. All animal care and experiments were approved and carried out in accordance with the Animal Care and Use Committee of Guangzhou Medical University and in accordance with the principles and guidelines of the National Institutes of Health.
goat IgG antibody were from Jackson ImmunoResearch (West Grove, Pa., USA) and YO-PRO-1 was from Invitrogen (Carlsbad, Calif., USA). As shown in figure 1, the micromanipulation instruments, including microforceps, microscissors, hemostats and scissors were from Jinzhong Surgical Instruments (Shanghai, China), the Chinese acupuncture needles were from Tianxie Acupuncture Instruments (size: 0.22 × 40 mm, Suzhou, Jiangsu, China) and the copper wires (0.13 mm diameter) were obtained from some old stock. Isolation of Pulmonary Arteries Rats were deeply anesthetized with pentobarbital sodium (65 mg · kg–1 i.p.), and anesthesia was confirmed by the lack of a withdrawal reflex from a toe pinch. Figure 2 shows the procedure used to isolate the pulmonary arteries. This was adapted from our previous description of the isolation of rat pulmonary veins [21]. Briefly, under sterile conditions, anesthetized rats were placed in the supine position and their chests were opened. The heart and lungs were removed en bloc and transferred to a petri dish with cold (4 ° C) physiological salt solution (PSS) that contained 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM HEPES and 10 mM glucose (fig. 2a). After being rinsed twice with cold PSS, the heart and lungs were placed in the arteries-up position and fixed in an isolation dish with acupuncture needles (fig. 2b). Under a stereomicroscope, we first identified the extrapulmonary arteries connected with the right cardiac ventricle. The right and left branches of the intrapulmonary arteries that followed from the extrapulmonary arteries were carefully dissected from the lungs within 20 min of the removal of the heart and lungs from the thoracic cavity (fig. 2b, c). Adipose and connective tissue were carefully removed in cold Ca2+-reduced PSS (20 μM CaCl2) with microdissection forceps and scissors (fig. 2c). The right and left branches of the pulmonary arteries were cut from the pulmonary truncus (fig. 2d).
Materials Unless otherwise specified, all reagents were obtained from Sigma Chemicals. The endothelial cell basal medium-2 (EBM-2), the EGM-2 MV SingleQuots, the smooth muscle cell basal medium (SmBM) and the SmGM-2 SingleQuots were from Clonetics (Walkersville, Md.), rat renal fibroblasts (NRK-49F) were from American Type Culture Collection (ATCC, Manassas, Va., USA), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), PBS and antibiotic-antimycotic were from GIBCO (Carlsbad, Calif., USA), rabbit polyclonal antibody to von Willebrand factor, mouse monoclonal antibody to CD31, mouse monoclonal antibody to smooth muscle myosin heavy chain and rabbit monoclonal antibody to CD90/Thy-1 were from Abcam (Cambridge, Mass., USA), goat polyclonal antibody to CD34 was from R&D Systems (Minneapolis, Minn., USA), Cy3-conjugated donkey anti-rabbit IgG antibody, HRP-conjugated donkey anti-rabbit IgG antibody, goat anti-mouse IgG antibody and donkey anti-
Cell Isolation and Culture Figure 3 shows the procedure for cell isolation. The blunt acupuncture needles were inserted into the right and left branches of the pulmonary arteries from the distal extremity to the proximal extremity (fig. 3a, b). The pulmonary arteries were reversed with microforceps along the acupuncture needle handles to expose their endothelium (fig. 3c–f). Both the distal and proximal extremities of the pulmonary arteries were ligated with copper wires using hemostats (fig. 3g, h). After the blunt acupuncture needles were removed from the handles, the reversed pulmonary arteries, with the exposed endothelium kept tightly around the handles, were placed in the 15-ml plastic conical tube containing 2 ml of Ca2+-reduced collagenase enzymatic solution (type I, 1,800 U/ml) and digested at 37 ° C for 8 min (fig. 3i). The digestion was terminated by adding 8 ml of EBM-2 supplemented with EGM-2 MV SingleQuots (EBM-2MV) and 10% FBS into the plastic conical tube. The cells were separated from the reversed arteries by gentle aspiration and repulsing about 10 times with a wide-bore transfer pipette set to 1 ml. After the reversed arteries were washed with 20 ml of 1 × PBS, the cell suspension was collected and centrifuged for 10 min at 400 g at room temperature (RT). The supernatant was carefully discarded. After resuspension of the cell pellet, the cells were plated onto 35-mm petri dishes precoated with human fibronectin. Coating of the dishes was accomplished by incubation with 4 ml of human fibronectin solution (10
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Fig. 1. a All the micromanipulation instruments used in the experiments for isolation of rat pulmonary arteries. b The enlarged image from the inset (a) showing the Chinese acupuncture needles and the copper wires. c The Chinese acupuncture needles in an open box.
Fig. 2. a The heart and lungs were removed
en bloc and placed in the arteries-up position in an isolation dish. b The heart and lungs were fixed with acupuncture needles and the right and left branches of the intrapulmonary arteries that followed the extrapulmonary arteries were carefully dissected from the lungs. c Adipose and connective tissues were carefully removed from the pulmonary arteries that connected with the heart. d The right and left branches of the pulmonary arteries were cut and used for the experiments.
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Fig. 3. a, b The blunt acupuncture needles
were inserted into the right and left branches of the pulmonary arteries from the distal extremity to the proximal extremity. b Enlarged inset from a. c–f The pulmonary arteries were reversed along the acupuncture needle handles to expose the endothelium of the arteries. c One was reversed and the other was reversing. d Enlarged inset from c. e Two arteries were reversed. f Enlarged inset from e. g Both the distal and proximal extremities of the pulmonary arteries were ligated with copper wires. h Enlarged inset from g. i After the blunt acupuncture needles were removed, the reversed pulmonary arteries with the exposed endothelium maintained around the handles were placed in a plastic conical tube (containing 2 ml of collagenase enzymatic solution) and digested.
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μg/ml PBS) for 2 h in a tissue culture incubator at 37 ° C. Excess fluid was removed immediately before adding the cells. The cells were then incubated in EBM-2MV with 5% FBS in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C. The medium was removed and replaced every 2–3 days. Cell growth and cell morphology were examined daily under an inverted light microscope. The rat pulmonary arterial smooth muscle cells (PASMCs), isolated and cultured as described earlier [22, 23], were used as a morphological control. The cells were subcultured when they reached more than 80% confluence. Cultures were first washed twice with PBS, then treated with 0.25% trypsin and 0.01% EDTA, and when most cells became visibly rounded, they were neutralized with EBM-2MV with 5% FBS. Detached cells were centrifuged and the supernatant was removed. After resuspension of the cell pellet, the cells were passaged into fibronectin-coated flasks at a ratio of 1:2–1:3 and incubated at 37 ° C with 5% CO2 and 95% air.
Cell Identification Immunofluorescence The endothelial cells were identified by the presence of von Willebrand factor using a rabbit polyclonal antibody. The cells not treated with the primary antibody specific for von Willebrand factor were used as negative control. The endothelial cells were also identified by the absence of α-smooth muscle actin. Rat PASMCs were used as a positive control. The third-to-fourth passage cells were seeded and grown on fibronectin-coated coverslips in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C for 5–6 days. Cells were rinsed twice with PBS and fixed for 15 min with 2 ml of 4% paraformaldehyde in PBS at RT. Cells were rinsed 3 times with PBS and treated for 15 min with 2 ml of PBS containing 0.25% Triton X-100 at RT. The permeabilization buffer was aspirated, and cells were rinsed 3 times with 2 ml of PBS for 5 min each. Cells were incubated at RT for 30 min in PBS with 1% BSA and then for 60 min at RT with a rabbit
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polyclonal antibody to von Willebrand factor (1:200). To visualize the proteins, the cells were treated for 2 h at RT in the dark with a Cy3-conjugated secondary donkey anti-rabbit IgG (Fab-specific) antibody (1: 100), which produced red fluorescence. Cells were rinsed and incubated with YO-PRO-1 solution at 3.5 μl/ml for 5 min at RT and away from light. The coverslips were mounted with a drop of mounting medium. Immunocytochemistry Immunocytochemistry was performed on the subcultured cells (passages 8–10) grown to 40–50% confluence on fibronectin-coated coverslips according to the procedure described previously [24, 25]. Briefly, following extensive washes with PBS, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.25% Triton X-100 as described above in immunofluorescence. After blocking with 1% BSA for 30 min at RT, the cells were incubated with 200 μl of the following primary antibodies for 90 min at RT: rabbit polyclonal antibody to von Willebrand factor (1: 200), mouse monoclonal antibody to CD31 (1:200), goat polyclonal antibody to CD34 (1:100), mouse monoclonal antibody to α-smooth muscle actin (1:200), mouse monoclonal antibody to smooth muscle myosin heavy chain (1: 200) and mouse monoclonal antibody to CD90/Thy-1 (1: 100). After three washes in PBS, the cells were incubated with the appropriate HRP-conjugated secondary antibody [donkey anti-rabbit IgG (1: 100), goat anti-mouse IgG (1: 100) or donkey anti-goat IgG (1:100)] for 30 min at RT. Antibody complexes were visualized with 3,3′-diaminobenzidine tetrahydrochloride. The nuclei were counterstained with hematoxylin. Rat PASMCs were used as positive controls for α-smooth muscle actin and smooth muscle myosin heavy chain and NRK-49F cells were used as a positive control for CD90/Thy-1. Transmission Electron Microscopy As previously described [26, 27], cells grown to confluence on fibronectin-coated coverslips were fixed by immersion in 2.5% glutaraldehyde for 24 h, postfixed with 1% osmium tetroxide for 1 h and dehydrated through graded alcohols to be embedded in Spurr’s resin. Subsequently, thin sections (60 nm) were cut and stained with 2% uranyl acetate and 0.1% lead citrate. Sections were examined in a transmission electron microscope at 80 kV.
Results
Cell Morphology The vessels used in this study were isolated from the right and left branches of the pulmonary arteries (fig. 2, 3). The cultured cells were identified on the basis of their patterns of growth, morphology, immunohistological staining and ultrastructural characteristics. Observed with a phase-contrast microscope, both the endothelial cells and the smooth muscle cells started to divide and extend within 3 days. The endothelial cells were large, flat and polygonal and displayed a characteristic ovoid nucleus with 2–3 prominent nucleoli and peri472
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Fig. 4. Phase-contrast microscopy of rat PAECs and PASMCs. a PAECs cultured for 3 days. b PASMCs cultured for 3 days. c PAECs cultured for 7 days. d PASMCs cultured for 7 days.
nuclear granules (fig. 4a). However, the smooth muscle cells assumed a spindle-shaped appearance with cytoplasmic projections extending from a larger central area that contained the nucleus (fig. 4b). As soon as the cells became confluent, the endothelial cells displayed typical ‘cobblestone’ morphology characteristic of endothelial cells (fig. 4c), whereas the smooth muscle cells grew in a ‘hill-and-valley’-like pattern (fig. 4d). Both the endothelial cells and the smooth muscle cells grew to confluence (95%) in 7–8 days. Immunofluorescence As shown in figure 5a, the cultured cells stained strongly for von Willebrand factor. However, the immunocytochemical fluorescence of von Willebrand factor was not observed in control cells, which did not receive the primary monoclonal antibody raised against von Willebrand factor, but were otherwise treated similarly (fig. 5b). The endothelial cells were also identified by the absence of α-smooth muscle actin. The staining of cultured endothelial cells for α-smooth muscle actin was similar to background levels (fig. 5c). However, α-smooth muscle actin was strongly positive in rat PASMCs (fig. 5d). Peng /Wen /Shi /Jiang /Hu /Zhou /Ran
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Fig. 5. a Fluorescence immunocytochemical identification of von Willebrand factor was shown in red in PAECs. The nuclear marker YO-PRO-1 was shown in green. b The immunocytochemical fluorescence of von Willebrand factor did not appear in control PAECs (which did not receive primary monoclonal antibody raised against von Willebrand factor, but were otherwise treated similarly). c The staining of PAECs for α-smooth muscle actin was similar to the background. d Rat PASMCs strongly expressed α-smooth muscle actin (in red).
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Immunocytochemistry To further confirm the purity of subcultured endothelial cells, after 8–10 passages, the cells were examined for the presence of various endothelial-cell-specific markers and the absence of smooth-muscle-cell or fibroblast-specific markers by immunocytochemical analysis. As shown in figure 6, all subcultured cells exhibited the presence of the endothelial cell markers von Willebrand factor (fig. 6a), CD31 (fig. 6b) and CD34 (fig. 6c). Negative controls for three markers had no staining (data not shown). The endothelial cell cultures did not stain for α-smooth muscle actin (fig. 6d) or smooth muscle myosin heavy chain (fig. 6e). These cells also did not stain for CD90/ Thy-1 (fig. 6f). However, rat PASMCs were positive for α-smooth muscle actin (fig. 6g) and smooth muscle myosin heavy chain (fig. 6h). NRK-49F cells showed positivity for CD90/Thy-1 (fig. 6i). Respective negative controls for α-smooth muscle actin (fig. 6j), smooth muscle myosin heavy chain (fig. 6k) and CD90/Thy-1 (fig. 6l), which were performed by omitting the primary antibody, had no staining.
bers and were localized in subplasmalemmal regions. These data demonstrate that our cultured cells were, in fact, vascular endothelial cells.
Discussion
Transmission Electron Microscopy The cultured cells were investigated by transmission electron microscopy. Electron microscopic analysis showed the presence of Weibel-Palade bodies (fig. 7a, b), which is a unique ultrastructural marker of vascular endothelium. Furthermore, the cells contained pinocytotic vesicles (fig. 7c, d), which were present in moderate num-
In this study, we describe a new method to acquire primary cultures of PAECs from rat pulmonary arterial branches for in vitro studies of pulmonary artery-related diseases. As mentioned above, it has proved difficult and inconvenient to obtain the pulmonary arteries from humans and large animals, and some methods for culture of rat PAECs obtain the cells mainly from the pulmonary truncus or else require complex equipment. In our experiments, we developed, for the first time, a simple and economical method with Chinese acupuncture needles to acquire rat pulmonary arteries and expose the vessel endothelium. The method for isolation of rat pulmonary arteries in our study was similar to that for isolation of rat distal pulmonary veins and arteries, used to obtain the pulmonary venous smooth muscle cells and PASMCs and described by us and other investigators [21, 28]. The technique to reverse the pulmonary arteries with Chinese acupuncture needles was a new method for exposure of the vessel endothelium. Although this method needs micromanipulation, it was really simple and economical. It has been suggested that all vascular endothelial cells contain characteristic structural similarities that can be
Development of a New Method for the Culture of Rat PAECs
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Fig. 6. Immunocytochemical analysis showed PAECs were positive for von Willebrand factor (a), CD31 (b) and CD34 (c) and negative for α-smooth muscle actin (d), smooth muscle myosin heavy chain (e) and CD90/Thy-1 (f). Rat PASMCs were positive for α-smooth muscle actin (g) and smooth muscle myosin heavy chain (h). i NRK-49F cells were positive for CD90/ Thy-1. Respective negative controls for α-smooth muscle actin (j), smooth muscle myosin heavy chain (k) and CD90/Thy-1 (l) had no staining. The nuclei were counterstained with hematoxylin (blue).
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used to identify them. Therefore, in our subsequent experiments, the cell phenotype, immunological properties and ultrastructural characteristics were used to characterize the isolated cells as vascular endothelial cells. The rat PAECs that we obtained exhibited characteristic morphological features, immunoreactivity to von Willebrand factor, CD31 and CD34 as well as unique ultrastructural characteristics that were consistent with observations by other investigators [26, 29–34, 36, 41–48]. PAECs started to divide within 3 days, and the culture reached confluence in approximately 1 week. At low den474
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sity, PAECs were flat and polygonal and displayed a characteristic ovoid nucleus. As soon as the cells became confluent, the primary culture of PAECs, like other vascular endothelial cells described by other investigators [26, 29, 30], grew in a typical ‘cobblestone’-like pattern that is characteristic of endothelium. PASMCs, however, showed a spindle-shaped appearance at low density and grew in a ‘hill-and-valley’-like pattern at a high density that was completely different from that of PAECs. To confirm the origin of the cultured cells, we used a sensitive and specific marker of vascular endothelial cells, Peng /Wen /Shi /Jiang /Hu /Zhou /Ran
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Fig. 7. a, b Electron microscopic analysis showed the presence of Weibel-Palade bodies in PAECs. b Enlarged inset from a. c The pinocytotic vesicles were present in moderate numbers and were localized in subplasmalemmal regions in PAECs. d Enlarged inset from (c). N = Nucleus.
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von Willebrand factor, to detect the immunofluorescence of early passage cells (passage 3–4). The presence of Willebrand factor is the most widely used criterion for identification of cells of endothelial origin. We found that von Willebrand factor was strongly positive in the cultures of PAECs, consistent with observations from other investigators [30–33]. However, PAECs did not express α-smooth muscle actin, whereas PASMCs expressed the protein. PAECs showed strong immunostaining for von Willebrand factor and a lack of α-smooth muscle actin, clearly suggesting that the cells that we acquired and cultured were indeed the vascular endothelial cells. To further confirm the purity of PAECs, we next used two additional endothelial cell markers, CD31 and CD34, as well as three other smooth muscle cell or fibroblast markers, α-smooth muscle actin, smooth muscle myosin heavy chain and CD90/Thy-1, in the immunocytochemical analysis of late passage cells (passage 8–10). CD31 is widely used and also known as platelet endothelial cell adhesion molecule, PECAM-1. It is strongly expressed by all endothelial cells and weakly expressed by several types of leukocytes [34]. CD34 is a cell surface protein implicated in cell migration and halting of differentiation that has been documented to be expressed in PAECs, and which has served most commonly as a hematopoietic stem cell marker but is also known as an endothelial cell
marker [35, 36]. All PAECs expressed Willebrand factor, CD31 and CD34 after 8–10 passages. Nevertheless, these cell cultures did not express α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Both α-smooth muscle actin and smooth muscle myosin heavy chain are smooth muscle cell myofilamentous proteins and are widely used for the characterization of cells of smooth muscle cell phenotype [21, 37–38]. CD90/Thy-1 is a glycophosphatidylinositol-anchored, strongly glycosylated protein that is expressed on the cell surface. CD90/ Thy-1 was originally identified as a thymocyte antigen and is also known as the fibroblast-specific antigen, shown to be expressed in fibroblasts [39, 40]. Hence, the cells we isolated from rat pulmonary arteries with Chinese acupuncture needles and subcultured up to passage 10 were certainly of endothelial origin and were not contaminated with smooth muscle cells or fibroblasts. Although the cell morphology and immunological properties indicated that the cultured cells were endothelial cells, ultrastructural examination was carried out to further determine whether the cells in culture exhibited ultrastructural features of vascular endothelial cells. Transmission electron micrographs showed that WeibelPalade bodies were present in the cultures of PAECs. Weibel-Palade bodies are characteristically found in vascular endothelial cells but not in smooth muscle cells or
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in fibroblasts, and have been shown to be present in the cultured vascular endothelial cells of rat, bovine and human origin [41–44]. Furthermore, pinocytotic vesicles were found in moderate numbers and were present only at the periphery of the cells close to the membrane; this is consistent with observations from other investigators [45–48]. In summary, we have developed a simple and economical method for obtaining PAECs from rat pulmonary arteries. PAECs show the morphological characteristics of vascular endothelial cells, express von Willebrand factor, CD31 and CD34, lack α-smooth muscle actin, smooth muscle myosin heavy chain and CD90/Thy-1 and possess Weibel-Palade bodies consistent with their vascular en-
dothelial cell origin. PAECs could subsequently be used to further explore the pathophysiological mechanisms of pulmonary endothelial dysfunction and pulmonary hypertension. Acknowledgments This work was supported by the National Natural Science Foundation of China (81000020 and 81170043), the Specialized Research Fund for the Doctoral Program of Higher Education, China (20104423110001), the Guangdong Key Research Project grant (B30301), the research grants from Guangzhou Department of Education, China (10A025G and 10A276) and the Science Foundation of State Key Laboratory of Respiratory Diseases (2011011).
References 1 Teichert-Kuliszewska K, Kutryk MJ, Kuliszewski MA, Karoubi G, Courtman DW, Zucco L, Granton J, Stewart DJ: Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ Res 2006;98:209–217. 2 Ramos M, Lamé MW, Segall HJ, Wilson DW: Monocrotaline pyrrole induces Smad nuclear accumulation and altered signaling expression in human pulmonary arterial endothelial cells. Vascul Pharmacol 2007;46:439–448. 3 Mathew R, Huang J, Shah M, Patel K, Gewitz M, Sehgal PB: Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 2004; 110: 1499–1506. 4 Sehgal PB, Mukhopadhyay S, Xu F, Patel K, Shah M: Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2007; 292:L1526– L1542. 5 Zharikov S, Krotova K, Hu H, Baylis C, Johnson RJ, Block ER, Patel J: Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells. Am J Physiol Cell Physiol 2008; 295:C1183–C1190. 6 Sud N, Sharma S, Wiseman DA, Harmon C, Kumar S, Venema RC, Fineman JR, Black SM: Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90. Am J Physiol Lung Cell Mol Physiol 2007; 93:L1444–L1453. 7 Zhu B, Strada S, Stevens T: Cyclic GMP-specific phosphodiesterase 5 regulates growth and apoptosis in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 289:L196–L206.
476
8 Wang Q, Pfeiffer GR 2nd, Stevens T, Doerschuk CM: Lung microvascular and arterial endothelial cells differ in their responses to intercellular adhesion molecule-1 ligation. Am J Respir Crit Care Med 2002; 166: 872– 877. 9 Zhu D, Zhang C, Medhora M, Jacobs ER: CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium. J Appl Physiol 2002;93:330–337. 10 Stevens T, Creighton J, Thompson WJ: Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function. Am J Physiol 1999;277:L119– L126. 11 Moore TM, Brough GH, Babal P, Kelly JJ, Li M, Stevens T: Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1. Am J Physiol 1998;275:L574–L582. 12 Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, Thompson WJ: Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol 1998;274:L810–L819. 13 Michael JR, Markewitz BA, Kohan DE: Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells. Am J Physiol 1997;273:L768– L774. 14 Baudin B, Bruneel A, Bosselut N, Vaubourdolle M: A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2007;2:481–485. 15 Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973;52:2745–2756. 16 Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, Ziemann M, Helbing T, El-Osta A, Jowett JB, Peter K: Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc Res 2012;93:633–644.
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
17 Nishiyama K, Takaji K, Kataoka K, Kurihara Y, Yoshimura M, Kato A, Ogawa H, Kurihara H: Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis. Circulation 2005;112:2840–2850. 18 Buerkle MA, Lehrer S, Sohn HY, Conzen P, Pohl U, Krötz F: Selective inhibition of cyclooxygenase-2 enhances platelet adhesion in hamster arterioles in vivo. Circulation 2004; 110:2053–2059. 19 Margariti A, Zampetaki A, Xiao Q, Zhou B, Karamariti E, Martin D, Yin X, Mayr M, Li H, Zhang Z, De Falco E, Hu Y, Cockerill G, Xu Q, Zeng L: Histone deacetylase 7 controls endothelial cell growth through modulation of beta-catenin. Circ Res 2010;106:1202–1211. 20 Osanai T, Magota K, Tanaka M, Shimada M, Murakami R, Sasaki S, Tomita H, Maeda N, Okumura K: Intracellular signaling for vasoconstrictor coupling factor 6: novel function of beta-subunit of ATP synthase as receptor. Hypertension 2005;46:1140–1146. 21 Peng G, Wang J, Lu W, Ran P: Isolation and primary culture of rat distal pulmonary venous smooth muscle cells. Hypertens Res 2010;33:308–313. 22 Lu W, Wang J, Peng G, Shimoda LA, Sylvester JT: Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 2009;297:L17–L25. 23 Lu W, Ran P, Zhang D, Peng G, Li B, Zhong N, Wang J: Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle. Am J Physiol Cell Physiol 2010;298:C114–C123.
Peng /Wen /Shi /Jiang /Hu /Zhou /Ran
24 Zhang Q, Cooper RK, Wolters WR, Tiersch TR: Isolation, culture and characterization of a primary fibroblast cell line from channel catfish. Cytotechnology 1998;26:83–90. 25 Straszewski-Chavez SL, Abrahams VM, Alvero AB, Aldo PB, Ma Y, Guller S, Romero R, Mor G: The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71. Placenta 2009;30:939–948. 26 Browning AC, Gray T, Amoaku WM: Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells. Br J Ophthalmol 2005; 89: 1343– 1347. 27 Wu Z, Hofman FM, Zlokovic BV: A simple method for isolation and characterization of mouse brain microvascular endothelial cells. J Neurosci Methods 2003;130:53–63. 28 Whitman EM, Pisarcik S, Luke T, Fallon M, Wang J, Sylvester JT, Semenza GL, Shimoda LA: Endothelin-1 mediates hypoxia-induced inhibition of voltage-gated K+ channel expression in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 2008;294:L309–L318. 29 Wang D, Lehman RE, Donner DB, Matli MR, Warren RS, Welton ML: Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells. Am J Physiol Gastrointest Liver Physiol 2002;282:G1088– G1096. 30 Banumathi E, Haribalaganesh R, Babu SS, Kumar NS, Sangiliyandi G: High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels. Microvasc Res 2009; 77: 377–381. 31 Thorin E, Shatos MA, Shreeve SM, Walters CL, Bevan JA: Human vascular endothelium heterogeneity. A comparative study of cerebral and peripheral cultured vascular endothelial cells. Stroke 1997;28:375–381.
Development of a New Method for the Culture of Rat PAECs
32 Argall KG, Armati PJ, Pollard JD: A method for the isolation and culture of rat peripheral nerve vascular endothelial cells. Mol Cell Neurosci 1994;5:413–417. 33 Yu SY, Song YM, Li AM, Yu XJ, Zhao G, Song MB, Lin CM, Tao CR, Huang L: Isolation and characterization of human coronary arteryderived endothelial cells in vivo from patients undergoing percutaneous coronary interventions. J Vasc Res 2009;46:487–494. 34 Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY: JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol 1990; 43:752–757. 35 Nielsen JS, McNagny KM: CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery. Microcirculation 2009;16:487–496. 36 Xiaozhuang Z, Xianqiong L, Jingbo J, Shuiqing H, Jie Y, Yunbin C: Isolation and characterization of fetus human retinal microvascular endothelial cells. Ophthalmic Res 2010; 44:125–130. 37 Teng B, Ansari HR, Oldenburg PJ, Schnermann J, Mustafa SJ: Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptorknockout mice. Am J Physiol Heart Circ Physiol 2006;290:H1713–H1720. 38 Cairrão E, Santos-Silva AJ, Alvarez E, Correia I, Verde I: Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels. In Vitro Cell Dev Biol Anim 2009;45:175–184. 39 Saalbach A, Aneregg U, Bruns M, Schnabel E, Herrmann K, Haustein UF: Novel fibroblastspecific monoclonal antibodies: properties and specificities. J Invest Dermatol 1996;106: 1314–1319.
40 Hagood JS, Prabhakaran P, Kumbla P, Salazar L, MacEwen MW, Barker TH, Ortiz LA, Schoeb T, Siegal GP, Alexander CB, Pardo A, Selman M: Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis. Am J Pathol 2005;167:365–379. 41 Sölder E, Böckle BC, Nguyen VA, Fürhapter C, Obexer P, Erdel M, Stössel H, Romani N, Sepp NT: Isolation and characterization of CD133+CD34+VEGFR-2+CD45– fetal endothelial cells from human term placenta. Microvasc Res 2012;84:65–73. 42 Green DF, Hwang KH, Ryan US, Bourgoignie JJ: Culture of endothelial cells from baboon and human glomeruli. Kidney Int 1992; 41: 1506–1516. 43 Okruhlicová L, Dlugosová K, Mitasíková M, Bernátová I: Ultrastructural characteristics of aortic endothelial cells in borderline hypertensive rats exposed to chronic social stress. Physiol Res 2008;57(suppl 2):S31–S37. 44 Ryan US, Ryan JW: Angiotensin-converting enzyme: II. Pulmonary endothelial cells in culture. Environ Health Perspect 1980; 35: 171–180. 45 McGuire PG, Twietmeyer TA: Morphology of rapidly frozen aortic endothelial cells. Glutaraldehyde fixation increases the number of caveolae. Circ Res 1983;53:424–429. 46 Sakamoto T, Sakamoto H, Hinton DR, Spee C, Ishibashi T, Ryan SJ: In vitro studies of human choroidal endothelial cells. Curr Eye Res 1995;14:621–627. 47 Tu J, Stoodley MA, Morgan MK, Storer KP: Ultrastructure of perinidal capillaries in cerebral arteriovenous malformations. Neurosurgery 2006;58:961–970. 48 Espina AI, Castellanos AV, Fereira JL: Agerelated changes in blood capillary endothelium of human dental pulp: an ultrastructural study. Int Endod J 2003;36:395–403.
J Vasc Res 2013;50:468–477 DOI: 10.1159/000355271
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