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(Investigative Ophthalmology and Visual Science. 2005;46:3502-3506.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0309
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Contribution of Bone Marrow–Derived Pericyte Precursor Cells to Corneal Vasculogenesis

Ugur Ozerdem,1 Kari Alitalo,2 Petri Salven,3 and Andrew Li4

1From the La Jolla Institute for Molecular Medicine, La Jolla, California; 2Biomedicum Helsinki, 3University of Helsinki, Helsinki, Finland; 4University of California San Diego, La Jolla, California.


   Abstract
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Methods
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 Discussion
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PURPOSE. Bone-marrow (BM)–derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization.

METHODS. A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)–positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software.

RESULTS. BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM.

CONCLUSIONS. Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.

The neovascularization of tissues is accomplished by two distinct processes: vasculogenesis (V) and angiogenesis (A).1 2 3 4 5 6 During angiogenesis, preexisting blood vessels form neovascular sprouts. In vasculogenesis, blood vessels develop from progenitor cells that coalesce and differentiate to form vessels. The walls of neovascular blood vessel capillaries are composed of two principal cell types: vascular endothelial cells (VECs), and Rouget cells.7 Rouget cells, also known as mural cells or pericytes, form an outer sheath6 8 9 surrounding the endothelial cells on the outer aspect of microvessels.10

Reports about pericyte or endothelial differentiation from bone-marrow hematopoietic cells in various neovascularization models11 12 13 14 15 16 17 18 , are controversial. Two reports have suggested that adult bone-marrow (BM)–derived precursors give rise to pericytes but not to VECs,11 12 and numerous other reports have suggested that BM-derived precursors give rise to VECs.14 15 16 17 18 Differences in tissues, disease models, and neovascularization models may be a reason for this controversy. Difficulty in identifying pericytes, which are in very close spatial proximity to endothelial cells, may be another factor resulting in this controversy.

The normal mammalian cornea is one of the few tissues that is devoid of preexisting lymphatics and blood vessels. Therefore, the presence of any blood and lymphatic vessels in the cornea always signifies a pathologic process. The actual in vivo differentiation capacity of adult BM-derived hematopoietic precursors cells into pericytes or endothelial cells in corneal neovascularization remains unclear, as do the contribution of vasculogenesis and angiogenesis of new vessels (i.e., the vessels formed by BM-derived precursor cells [vasculogenesis] and the vessels formed by the cells sprouting from preexisting vessels [angiogenesis]).

Whereas endothelial cells have been studied extensively, much less is known about pericytes. As the name indicates, pericytes surround the endothelial cells on the abluminal aspect of microvessels.10 A recent search of the PubMed database (http://www.ncbi.nlm.nih.gov/ National Institutes of Health, Bethesda, MD) revealed a 109-fold difference between the number of papers published on these two vascular cell types. Because the cellular processes and the role of circulating BM-derived progenitor cells underlying neovascular sprout formation remain incompletely understood,11 19 20 increased attention to pericytes and their interaction with endothelial cells will yield a better understanding of the role of BM in corneal disorders associated with neovascularization.

We used a BM-reconstituted mouse model in which the BM from enhanced green fluorescent protein (GFP)+ mice was transplanted into normal C57BL/6 mice. Using this mouse model, we set out to test the neovascular potential of adult BM-derived hematopoietic progenitor cells and their progeny in vivo. We used the basic fibroblast growth factor (bFGF)-induced mouse corneal neovascularization model to quantify vasculogenesis and angiogenesis. In addition, we differentiated quantitatively BM-derived pericytes from angiogenic pericytes derived from preexisting vessels.

We showed that BM-derived vascular precursors contributed only to the formation of pericytes but not VECs in corneal neovascularization. This investigation revealed that a significant fraction of neovascular pericytes in the cornea derive from BM-derived hematopoietic progenitor cells rather than from preexisting vessels, suggesting an extensive role for BM and vasculogenesis in corneal neovascularization. We also showed that a small fraction of lymphatic endothelial cells (LECs) in lymphangiogenic vessel walls in the cornea are from BM-derived hematopoietic precursor cells, whereas most lymphatic capillaries originate from preexisting limbal lymphatics.


   Methods
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All animal studies were performed in accordance with The National Institutes of Health Office of Laboratory Animal Welfare (OLAW) guidelines and the ARVO Statement for the Use of Animals in Research and were approved by the La Jolla Institute for Molecular Medicine animal research committee.

Animals and BM Transplantations
Mice reconstituted with enhanced GFP+ syngeneic BM cells were created to study the behavior of BM cells in vivo. Briefly, BM cells were collected by flushing the femurs and tibias of female C57BL/6 TgN(ACTbEGFP)1Osb mice (Jackson Laboratory, Bar Harbor, ME) with ice-cold phosphate-buffered saline (PBS) via a 25-gauge needle. After the lysis of red blood cells, BM progenitor cells were resuspended in PBS. After counting, the cells were spun down and resuspended at a concentration of 3 x 106 cells/100 µL. The recipient male mice (four mice) were irradiated by a lethal dosage of 1000 rads (10 Gy). Three days before the irradiation, the animals were housed in sterilized cages and fed standard chow that has been irradiated. The mice drank water containing the antibiotic sulfamethoxazole/trimethoprim to reduce the risk of opportunistic infections. The animals were housed in sterile cages and fed sterile food throughout the procedure. Three hours after irradiation, 3 x 106 cells were injected retro-orbitally via the ophthalmic venous plexus in the right orbit. We found previously that this procedure is much more effective than using the tail vein injections. It takes, on average, less than 30 seconds to inject 100 mL of cells versus several minutes to vasodilate the vessels in the mouse before trying to inject the cells via the tail vein, many times unsuccessfully. In addition, the animals are less stressed when this ophthalmic venous plexus injection procedure is used. Flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ) showed that peripheral blood cells of the recipients were reconstituted with GFP+ cells 4 weeks after transplantation. Peripheral blood smears stained with May-Grunwald revealed Barr body chromatin (the inactivated X chromosome seen in females) in recipient male mice corroborating the reconstitution of BM after transplantation (Fig. 1A) . More than 95% reconstitution of the BM by GFP+ donor-derived cells is previously reported with similar BM transplantation techniques.11 21



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  		FIGURE 1. (A) Peripheral blood smear stained with May-Grunwald shows a polymorphonuclear leukocyte with Barr body chromatin in the form of drumstick (arrow) in a BM recipient male mouse. Scale bar, 10 µm. (B) Polymer (hydron) pellet (P) containing 90 ng of bFGF implanted into the corneal stroma of BM-recipient mouse. Arrows: neovasculature penetrating the cornea from the corneoscleral limbus 7 days after pellet implantation. Scale bar, 300 µm. (C) Frozen section of neovascularized cornea (as seen in B) obtained from a C57BL/6 mouse in which the BM was reconstituted with enhanced GFP+ BM via transplantation. Blood vessel endothelial cells (red) were identified by using confocal microscopy in conjunction with combined immunohistochemical staining for CD31, CD105, and flk-1. Donor-derived GFP+ pericytes formed perivascular sleeves (green) around GFP-negative blood vessel endothelium (red). Scale bar, 10 µm. (D–G) Frozen section of neovascularized cornea stained using immunohistochemistry to identify pericytes (NG2, red in D) and blood vessel endothelium (combined CD31 + CD105 + flk-1; blue in E). (F, G) BM-derived cells expressing GFP (green). (G) Merger of images (D–F) shows both GFP+ and GFP– (limbus-derived) pericytes investing the GFP– blood vessel endothelium (blue). (H) Frozen section of neovascularized cornea stained using immunohistochemistry to identify lymphangiogenic endothelium with LYVE-1 (red). A lymphangiogenic sprout in the cornea shows both LYVE-1+ and GFP– cells (red) and LYVE-1+ and GFP+ cells (yellow) as indicated by arrows. Arrowhead: perivascular cell investing the lymphatic wall like a sleeve. (I–L) Frozen section of neovascularized cornea immunostained for LYVE-1 (I, red) and NG2 (J, blue) for identification of lymphatic endothelium and pericytes, respectively. Bone-marrow–derived cells are identified by their GFP expression (K, green). Perivascular cells (blue) show discontinuous investment of the lymphatic capillary wall (red). Both BM-derived (green) and non-BM–derived perivascular cells (blue) support the lymphatic capillary wall (images I, J, and K merged in L). Scale bar: (A, D–G, H) 10 µm; (B) 300 µm; (I–L) 5 µm.

 
Basic Fibroblast Growth Factor–Induced Corneal Neovascularization Model
Basic fibroblast growth (bFGF) is known to be a potent stimulus for angiogenesis and lymphangiogenesis.22 Implantation of hydron pellets containing bFGF (90 ng) induces both neovascularization and lymphangiogenesis in the mouse cornea.23 24 25 The mouse corneal micropocket assay, a noninflammatory neovascularization assay, was performed as previously described.23 Previous studies using this in vivo model have shown the contribution of pericytes coexpressing NG2 proteoglycan and platelet-derived growth factor (PDGF) ß-receptor to neovascular vessel walls as the new vessels spread into the cornea.26 27 Slow-release polyhydroxyethyl methacrylate (hydron) pellets (0.4 x 0.4 x 0.2 mm) were formulated to contain 90 ng recombinant basic fibroblast growth factor (bFGF; Invitrogen-Gibco, Grand Island, NY) and 45 µg sucrose aluminum sulfate (sucralfate; Sigma-Aldrich, St. Louis, MO). The mice reconstituted with enhanced GFP+ syngeneic BM cells were anesthetized with Avertin (0.015–0.017 mL/g body weight), and a single hydron pellet was surgically implanted into the corneal stroma of the left eye, 0.7 mm from the corneoscleral limbus (Fig. 1B) . On postoperative day 7, mice were killed and their eyes enucleated.

Immunohistochemistry, Confocal Microscopic Imaging, and Image Analysis
Enucleated eyes were fixed in 4% paraformaldehyde for 6 hours, cryoprotected in 20% sucrose overnight, and frozen in optimal cutting temperature (OCT) embedding compound (Miles, Inc., Elkhardt, IN). Cryostat sections (40 µm) were air-dried onto coated slides (Superfrost; Fisher Scientific, Pittsburgh, PA). Pericytes were identified by labeling with rabbit, or rat antibodies against the NG2 proteoglycan, or rabbit PDGF ß-receptor antibody.11 26 27 28 29 30 Both NG2 and PDGF ß-receptors are regarded as specific markers for pericytes.31 32

Lymphatic endothelium was identified by immunolabeling with rabbit anti-mouse LYVE-1 antibody, as described elsewhere.33 34 Blood vessel endothelial cells were identified with a cocktail of rat antibodies against mouse endoglin (CD105), platelet-endothelial cell adhesion molecule (PECAM-1; CD31), and VEGF receptor-2 (flk-1; BD-Pharmingen, San Diego, CA),27 29 35 a strategy that has been used to maximize labeling of all blood vessel endothelial cells. Immunohistochemical identification of hematopoietic cells was made using rat anti-mouse CD45 (LCA, Ly-5; BD-Pharmingen), and rat anti-mouse CD11b (Mac-1{alpha}) antibodies (BD-Pharmingen), as previously described.11

Alexa-647 and Alexa-594-labeled goat anti-rat secondary antibodies were obtained from Molecular Probes (Eugene, OR), and rhodamine red X (RRX)-labeled goat anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Slides were mounted with antifade medium (Vectashield; Vector Laboratories, Burlingame, CA).

After cryosectioning, sections representing the entire thickness of the cornea were selected from the numbered sections (180 sections from four mice) by using systematic random sampling.36 The sampled histologic sections (72 sections) were analyzed with a multichannel laser scanning confocal microscope. Briefly, optical sections were obtained from the specimens using the microscope (Fluoview 1000; Olympus USA, Melville, NY) in the three-channel sequential scanning mode. Serial optical sections (1 µm each) across the entire thickness (40 µm) of the histologic specimens were overlaid (z-stack) to provide reconstructions of entire vessels. This allowed unambiguous identification of the spatial relationship between pericytes and endothelial cells in the vessel wall.

Image analysis software (Volocity; Openlab-Improvision, Inc., Lexington, MA) was used for differential quantification of pericytes, VECs, and LECs for GFP, CD45, and CD11b expression.


   Results
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Contribution of Bone-Marrow–Derived Pericyte Precursors to Neovascularization
Confocal microscopic examination of histologic sections (Figs. 1C 1D 1E 1F 1G) revealed evidence of donor-derived (GFP+) and host (limbal capillary)-derived (GFP-negative) NG2+ and PDGF ß-receptor+ periendothelial cells (pericytes) forming sleeves around GFP-negative blood vessel endothelium. We were unable to find evidence of BM-derived blood vessel endothelial cells in corneal neovasculature. This suggests that neovascular blood vessel endothelium in cornea originates from corneoscleral limbal blood vessel capillaries, whereas pericytes have a dual origin: corneoscleral limbus vasculature and bone-marrow hematopoietic cells. In 22 histologic slides from four mice, 53% of all neovascular pericytes (NG2+ periendothelial cells) were GFP+ and 47% ± 3.7% (SEM) of them were GFP negative. Quantification based on 40 histologic slides revealed that 96% ± 0.7% (SEM) of GFP+/NG2+ pericytes also expressed CD45, whereas 92% ± 1.7% of GFP+/NG2+ pericytes expressed CD11b. This suggests a hematopoietic origin from BM of these pericytes.

Contribution of Bone Marrow to Lymphangiogenic Endothelium
Lymphangiogenic sprouts in the cornea showed both LYVE-1+ and GFP–, and LYVE+ and GFP+ endothelium (Fig. 1H , arrows). Of 10 histologic slides examined, eight percent of LYVE-1+ cells were GFP+, whereas 92% of LYVE-1+ cells were GFP–, suggesting that BM-derived progenitors contribute minimally to lymphangiogenesis in cornea. Lymphangiogenic vessel walls in the cornea showed discontinuous investment of lymphatic endothelium by NG2+ and PDGFß-receptor–positive periendothelial cells (which are GFP+) in the form of incomplete sleeves (Fig. 1H 1I 1J 1K 1L) . Quiescent lymphatic capillaries in the corneoscleral limbus where no intervention was performed revealed no pericytes on the lymphatic capillary wall.


   Discussion
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Three observations presented in our study seem especially noteworthy. First, pericytes of the corneal new vessels have a dual source: BM (vasculogenesis) and preexisting limbal capillaries (angiogenesis). More pericytes originate from BM than preexisting limbal capillaries by a factor of 1.12. Our results also suggest that although most BM-derived pericytes in corneal neovascularization derive from hematopoietic precursor cells, a small fraction (4%–8%) of BM-derived pericytes in the cornea derive from nonhematopoietic BM precursor cells. These nonhematopoietic progenitor cells may represent BM stromal or mesenchymal stem cells.37 38 39 40 41 These findings establish BM with its hematopoietic and nonhematopoietic elements as a significant effector organ in corneal disorders associated with neovascularization.

Second, the diverse origin of pericytes and their spatial proximity to each other in the neovascular sprouts raise the possibility of a neovascularization mechanism composed of both angiogenesis and vasculogenesis. By definition, angiogenesis and vasculogenesis refer to the formation of blood vessels from prexisting blood vessels and stem cells, respectively. This suggests the possibility of a synergy or overlap of two mechanisms operating simultaneously in the same neovascular sprouts, rather than independent progression of angiogenesis from vasculogenesis. Further studies are warranted to elucidate the homing mechanisms of circulating BM-derived pericyte precursors through the preexisting endothelium and pericyte layer (barrier) at neovascular sprouts. This homing process may involve extensive pericyte–endothelium and pericyte–pericyte adhesion and extravasation (emigration) processes, along with weakening of the junctional complexes between these cell types. Our findings, coupled with our previous reports,11 26 28 29 also corroborate the findings of other investigators, revealing the participation of nascent pericytes during the earliest stages of neovascularization.9 31 42 43 44 45 46 Although pericytes are widely regarded to be the microvascular equivalent of smooth muscle cells, the origin, development, and function of these cells appear to be variable and complex.47 48 49 Our ability to detect the precocious contribution of pericytes to microvascular development depends heavily on the use of NG2 proteoglycan and PDGF ß-receptor as markers for these cells at an early stage of their development.31 32 NG2 proteoglycan on pericytes is a functional player in angiogenesis, and its extrinsic (pharmacological) or intrinsic (genetic) inhibition is associated with a significant decrease in angiogenesis and decreased pericyte-endothelium investment ratios in neovascularization.27 30 NG2, a membrane-spanning chondroitin sulfate proteoglycan associated with mitotically active, nascent pericytes, exhibits several properties that suggest it is a functional player in neovascularization.26 27 28 29 30 NG2 appears to serve as a coreceptor for both bFGF and PDGF.50 51 Pericyte-NG2 chondroitin sulfate proteoglycan binds to extracellular matrix components such as types V, VI, and II collagen; tenascin; and laminin.52 53 Biochemical data also demonstrate the involvement of both galectin-3 and {alpha}3ß1 integrin in the VEC response to pericyte-NG2 and show that NG2, galectin-3, and {alpha}3ß1 form a complex on the cell surface promoting cell motility.54 Our recent findings revealed decreased neovascularization after intrinsic (NG2 knockout mice) or extrinsic (hydron polymer pellets containing NG2 neutralizing antibody) targeting of NG2 proteoglycan in ischemia, neurofibromatosis type 1 (NF1), and the corneal bFGF-induced neovascularization model.27 30 In the retinal vessels of NG2-knockout mice, proliferation of both pericytes and endothelial cells after ischemia is significantly reduced, and the pericyte-endothelial cell ratio declines from the wild-type value of 0.86 to 0.24.27 When pericyte NG2 proteoglycan is targeted, a 53% reduction in tumor neovascularization is possible in NF1-derived malignant peripheral nerve sheath tumor (MPNST).30 One of the traditional markers for pericyte identification has been the expression of {alpha}-SMA. However, a growing body of evidence suggests that {alpha}-SMA is a late marker for differentiated pericytes and therefore may be poorly expressed in developing angiogenic microvasculature.31 32 Because only a fraction of developing pericytes can be identified on the basis of {alpha}-SMA expression,9 32 55 56 57 58 we used NG2 and PDGF ß-receptor immunohistochemistry to identify pericytes.29 31 32 59

Third, our investigations also identified discontinuous investment of lymphangiogenic capillary walls by BM-derived periendothelial cells in lymphangiogenesis induced by bFGF. These pericytes may provide the capillary wall with some temporary physical support until the lymphatic endothelium establishes direct connection with the extracellular matrix. The same cells may also represent a transitional stage of BM-derived precursors before they transdifferentiate into lymphatic endothelium. In our studies so far, we have not identified pericytes in quiescent lymphatic capillary walls.

Recent studies suggest that dual targeting of pericytes and endothelial cells improves the efficacy of treatments aimed at inhibiting neovascular vessels.27 30 60 61 62 Clearly, the design of improved targeting strategies aimed at pericytes and lymphatic endothelial cells that derive from hematopoietic precursors depends on further understanding of the role of pericytes in neovascularization. Our study provides evidence that BM-derived pericytes contribute to the early phases of neovascular sprout formation during pathologic neovascularization and lymphangiogenesis. As significant players in neovascularization, BM-derived precursors represent an additional target for treatments designed to downregulate neovascularization in cornea.


   Footnotes
 
Supported by Grant R03 HD044783 from the National Institute of Child Health and Human Development, a U.S. Department of Defense Prostate Cancer Research Program New Investigator Award PC020822, and Grant TRDRP 13IT-0067 from the Tobacco-Related Disease Research Program of the University of California Idea Award (UO).

Submitted for publication March 11, 2005; revised April 22, 2005; accepted July 29, 2005.

Disclosure: U. Ozerdem, None; K. Alitalo, None; P. Salven, None; A. Li, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Ugur Ozerdem, Assistant Professor, La Jolla Institute for Molecular Medicine, 4570 Executive Drive, Suite 100, San Diego, CA 92121; ozerdem{at}ljimm.org.


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