1Department of Pathology, University of Washington; 2Division of Pathology and Laboratory Medicine, Veterans Administration Puget Sound Health Care System, Seattle, WA; and 3Laboratory of Neurobiology and Neuroregenerative Therapy, "Carlo Besta" Institute, Milan, Italy
Submitted 21 April 2005 ; accepted in final form 28 July 2005
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ABSTRACT |
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angiogenesis; stem cells; smooth muscle; mural cells; collagen
Angiogenic sprouting in the adult was initially considered an exclusive feature of terminally differentiated endothelial cells, which have the capacity to form vascular tubes and recruit mural cells (pericytes or smooth muscle cells). Later studies, however, demonstrated that the angiogenic process might also involve bone marrow-derived vascular progenitor cells (3, 44). These immature cells circulate in the peripheral blood and colonize angiogenic sites, where they differentiate into endothelial cells or mural cells, becoming incorporated into the developing neovasculature (10, 46). Immature mesenchymal cells with vascular progenitor features have also been shown to reside in peripheral tissues such as skeletal muscle (30), where they participate in the angiogenic response to injury.
Studies conducted at our laboratory and by others have demonstrated that angiogenesis can be reproduced ex vivo by culturing explants of adult rat aorta or other blood vessel types in three-dimensional (3D) biomatrices (24, 36, 38, 39). Neovessels formed in this model are composed of a luminal layer of endothelial cells and surrounding pericytes. The origin of these neovessels has been attributed to differentiated cells of the vessel wall. However, there is evidence from experiments with fetal aortas that neovessels may also derive from primitive CD34-positive/CD31-negative cells (1), raising the possibility that immature mesenchymal cells contribute to the postnatal angiogenic response of the vessel wall. Despite the many studies of circulating vascular progenitor cells that have been performed, the involvement in postnatal angiogenesis of progenitor cells that reside in the vessel wall has not been evaluated.
The purpose of this study was to investigate the hypothesis that the postnatal aorta contains vascular progenitor cells and to develop culture conditions for their isolation and long-term maintenance in an undifferentiated state. To identify and isolate these cells, we adapted a method originally described for neural stem cells (42, 47). Isolated cells were characterized by performing immunofluorescent staining, RT-PCR, and electron microscopy as well as functional studies with 3D models of angiogenesis (35, 59).
In this report, we describe how morphologically primitive, CD34-positive/-smooth muscle actin (
-SMA)-negative nonendothelial cells can be isolated from the mature rat aorta and propagated in suspension culture, where they grow as spheroid structures. These cells express markers of early pericyte lineage, can be induced to differentiate into CD34-negative/
-SMA-positive adherent cells, and behave functionally as pericytes when cocultured in collagen gels with vasoformative endothelial outgrowths. These immature mesenchymal cells may be an important source of mural cells in physiological or pathological angiogenic responses of the aortic wall.
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MATERIALS AND METHODS |
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Isolation and culture of rat aortic endothelial cells. Rat aortic endothelial cells (RAECs) were isolated from collagenase-digested aortic tissue, using anti-CD31 antibody-coated magnetic beads. CD31-positive RAECs were plated onto dishes coated with collagen and fibronectin (1 µg/cm2; Invitrogen). Cells were grown in EBM containing 10% FBS, 10 ng/ml EGF, 20 ng/ml bFGF, 10 ng/ml VEGF, and 10 µg/ml heparin.
Coculture of pericyte progenitor cells with aortic rings.
The aortic ring assay of angiogenesis (59) was modified to include SFACs. Briefly, individual aortic rings were embedded in a 40-µl drop of 1 mg/ml rat tail collagen containing 30 spheroids. After collagen gelation, cultures were grown in EBM containing 10 ng/ml VEGF. Pericyte transformation of SFACs was evaluated using phase-contrast microscopy and immunocytochemistry, followed by confocal microscopy.
Coculture of pericyte progenitor cells with endothelial cells. A capillary tube formation assay (33, 37, 52) was used to study interactions between SFACs and isolated RAECs in a 3D collagen matrix. SFACs and RAECs were suspended in rat tail collagen (4 x 105 of each cell type/ml) and plated in 16-mm wells (200 µl/well). After collagen gelation, cultures were incubated in EBM containing 10% FBS, 10 ng/ml EGF, 50 ng/ml bFGF, 50 ng/ml VEGF, and 10 µg/ml heparin. Control cultures consisted of RAECs embedded in collagen gels without SFACs. Pericyte transformation of SFACs was evaluated as described above.
DNA synthesis studies. Proliferating cells were identified by visualizing DNA synthesis in SFACs cultured for 24 h in H medium supplemented with 10 µM bromodeoxyuridine (BrdU; Sigma). Labeled cells were cytospun onto glass slides, fixed in ice-cold methanol, incubated with anti-BrdU antibody (Roche), and identified using the avidin-biotin complex immunoperoxidase method (Vector Laboratories, Burlingame, CA).
Cell culture photography. All images were captured with a 35-mm film camera or an Olympus MagnaFire S99806 digital camera (Olympus, Melville, NY). Both cameras were mounted on an Olympus IMT2 inverted microscope.
Electron microscopy. SFACs were fixed with 2.5% glutaraldehyde in 0.1% sodium cacodylate buffer at pH 7.4. After fixation, they were washed in 0.1% sodium cacodylate buffer pH 7.4, postfixed in 1% osmium tetroxide, and processed for Epon 812 (Ted Pella, Redding, CA) embedding. Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL transmission electron microscope.
Immunofluorescence staining.
Before staining, nonadherent, undifferentiated SFACs were cytospun onto glass slides and fixed with 10% buffered formalin (Fisher Scientific, Middletown, VA). Adherent cells were fixed directly in 10% buffered formalin. Nonspecific antibody binding was blocked by incubating cells in PBS-0.1% Tween 20 containing 5% goat serum. Primary antibodies directed against CD34, Tie-2, PDGF receptor (PDGFR)-, PDGFR-
(Santa Cruz Biotechnology, Santa Cruz, CA),
-SMA, calponin, desmin (NeoMarkers, Fremont, CA), NG2 (Chemicon International), and CD31 were used. Parallel cultures were incubated with negative control IgG. Secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 568 (Molecular Probes, Eugene, OR). Cells were mounted in Gelvatol (Monsanto, St. Louis, MO), and images were obtained with either a Leica TCS-SP laser-scanning confocal microscope or an Olympus BX41 fluorescence microscope equipped with an Optronics MicroFire SE digital camera.
RT-PCR. Total RNA was extracted from cells with the RNeasy Micro kit (Qiagen, Germany) and examined for both quality and quantity with a BioAnalyzer 2100 (Agilent, Palo Alto, CA). Random primed reverse transcription (RT) was performed with 200 ng of RNA and Superscript III reverse transcriptase (Invitrogen). Reactions lacking enzyme were performed in tandem for each RNA sample to act as negative controls.
For each sample, 1/20 RT reaction was used as a template for PCR. Reaction conditions were 10 mM Tris·HCl, 50 mM KCl, 0.1% Triton X-100, 250 µM 2-deoxynucleotide 5'-triphosphate, 1.5 mM (or 1 mM for CD34) MgCl2; 1 U of Taq DNA polymerase (Promega); and 0.5 µM each of forward and reverse primers (Table 1) in a 20-µl volume. PCR was performed at 95°C for 3 min, followed by 30 cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 40 s. PCR products were separated by performing electrophoresis in agarose gels containing ethidium bromide and visualized under UV light. All PCR products were sequenced to verify their identity.
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RESULTS |
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SFACs express a protein repertoire indicative of an immature mesenchymal phenotype.
Protein expression by SFACs was studied using immunofluorescent staining followed by confocal microscopy. SFACs expressed CD34 and Tie-2 (Fig. 2, A and B) but were negative for -SMA and CD31 (Fig. 2, C and D), indicating an immature cell phenotype. SFACs were also positive for NG2, nestin, PDGFR-
, and PDGFR-
(Fig. 2, EH).
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DISCUSSION |
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CD34-positive cells capable of smooth muscle cell differentiation have been identified in postnatal peripheral blood (58), small intestine (53), and skeletal muscle (22). There is also evidence that the embryonal aorta contains an abundant population of CD34+/CD31 cells (1), of which only a fraction differentiate into mature endothelial cells. The remaining cells may represent the embryonal lineage of the CD34-positive/CD31-negative SFACs that were isolated in this study from the postnatal rat aorta.
Serum-treated SFACs became CD34-negative and gained expression of mural cell markers but not CD31, eNOS, or Flk-1. This finding indicates that the SFACs were able to differentiate into mural cells but not into endothelial cells. Although the mechanisms regulating SFAC differentiation remain to be elucidated, it is possible that serum-mediated SFAC transformation into a mural cell phenotype is linked to the serum response factor, a serum-induced transcription factor that has been implicated in smooth muscle and skeletal muscle differentiation (27, 29).
Several observations in our study indicate that rat aorta-derived SFACs are pericyte progenitor cells. First, these cells proliferated in suspension-forming spheroids, a growth pattern associated with stem and/or progenitor cells (42, 49). Second, the rate of SFAC proliferation was relatively slow as is typically observed in progenitor cell populations. Third, the ultrastructural morphology of SFACs showed no evidence of features characteristic of a differentiated phenotype. Fourth, SFACs expressed CD34, a protein characteristically associated with stem cells (45). Finally, when cocultured with aorta-derived neovessels, SFACs gave rise to a multilayered coating of pericytes, which was much more complex than the single discontinuous layer of pericytes of control microvessels. The pericyte progenitor nature of SFACs was confirmed with an in vitro assay system in which capillary tubes formed by isolated endothelial cells became surrounded by SFAC-derived pericytes.
SFACs expressed both PDGF receptors and
and responded to PDGF-BB, which can bind and activate both receptors (15). PDGF-BB has been shown to play a critical role in pericyte recruitment during angiogenesis (8, 17, 20, 28). PDGFR-
appears to be particularly important for smooth muscle cells of large vessels (50), whereas PDGFR-
is essential for the differentiation of microvascular pericytes (21, 54). Interestingly, NG2, which is strongly expressed in SFACs, binds to PDGF-AA and functions as a requisite coreceptor for the action of this ligand on PDGFR-
(13, 14).
The capacity of SFACs to grow in suspension and their ability to form spheroids was strictly dependent on bFGF. When bFGF was omitted from the medium, SFACs attached to the culture surface and appeared to differentiate. These findings are consistent with the observation that bFGF is often required to maintain stem cells in culture (34). It is noteworthy that NG2 acts as an auxiliary bFGF receptor and potentiates the ability of bFGF to interact with and activate its tyrosine kinase receptors (13).
SFACs are positive for Tie-2, which was originally reported as an endothelial cell-specific receptor (51). However, there is evidence that Tie-2 also can be expressed in mural cell lineages (23, 43, 55) and by nonendothelial mesenchymal cells during angiogenesis in vivo (32).
Of particular interest is that the isolation and propagation of SFACs was made possible by the use of a serum-free medium originally introduced for the culture of neural stem cells (42, 47). Our finding that gene expression profiles are shared by neural cells and SFACs points to molecular similarities between these cell types. For example, NG2 and nestin, which are both expressed in SFACs, were originally described as neural markers but were later identified in immature mesenchymal cells and mural cells (2, 40, 41, 57). The observation that expression of genes associated with neural cells are also found in SFACs suggest that there are developmental overlaps in the differentiation of these distinct cell types. The finding that avian cranial neuroectoderm can generate pericytes when transplanted in quail chicks (26) supports this concept. Moreover, although most vascular mural cells are of mesodermal origin, the smooth muscle cells originate in the aortic arch, and proximal great vessels originate from the neural crest (7, 19, 31).
In addition to providing a model of mural cell differentiation, SFACs represent a valuable alternative to conventional pericyte cell strains and lines. SFACs are relatively easy to isolate and can be passaged and grown in culture for several months. In contrast, mature pericytes are difficult to isolate and fail to grow beyond a few passages unless immortalized (4, 5, 25).
In summary, our studies have indicated that the postnatal rat aorta contains a population of immature mesenchymal cells with progenitor cell features. The suspension culture method described in this article can be used to isolate, maintain, and propagate these cells in an undifferentiated state for several months. Serum, exposure to PDGF-BB, or coculture with endothelial cells induces transformation of these cells into pericytes. These cells lend themselves to studies of mural cell differentiation because of their characteristic immature phenotype. They also represent a useful source of mural cells for vascular bioengineering applications. Finally, because of their ability to survive in suspension and interact with angiogenic endothelial cells, these cells may prove to be a valuable tool for in vivo homing studies designed to target peripheral tissues undergoing physiological or pathological angiogenic responses.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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