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From the Department of Anatomy and Histology and Institute for Biomedical Research, University of Sydney, Sydney, New South Wales, Australia.
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Abstract |
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METHODS. Pericyte and SMC differentiation was characterized by immunohistochemistry with antibodies to NG2, desmin, 
–smooth muscle actin (SMA), calponin, and caldesmon.
RESULTS. Immature mural precursor cells (MPCs) were scattered throughout the primitive capillary plexus in the rat retina at embryonic day (E)20. The postnatal differentiation of pericytes and arteriolar and venous SMCs followed with distinct intermediate phenotypes. SMC differentiation coincided with selection of major vessels from the primordial capillary bed. Maturation of radial arteriolar SMCs was indicated by the expression of calponin and caldesmon, proteins that play a role in the regulation of SMC contraction. The mere presence of immature mural cells did not confer vessel stability; rather vessel stability in the developing rat retina coincided with caldesmon and calponin expression in arteriolar SMCs.
CONCLUSIONS. This normative data and the identification of suitable in vivo markers of pericytes and SMCs will allow meaningful interpretation of the changes in these cell types. When examining the role of mural cells in developmental and pathologic vascularization, the results show that there is a need to use multiple-marker immunohistochemistry because of significant mural cell heterogeneity. The observation that the expression of caldesmon and calponin in arteriolar SMCs coincides with resistance to hyperoxia in the developing rat retina, lead us to suggest that maturation of SMCs and their consequent ability to regulate blood flow may play a key role in vessel stabilization.
Vessel stabilization has particular relevance to tumor biology, wound healing, and ocular angiogenesis. An unstable vascular bed is actively remodeling and is characterized by endothelial cell proliferation and migration, deposition of new basement membrane, expression of vß3 integrin4 and high levels of matrix metalloproteinases,5 6 vessel withdrawal in response to VEGF withdrawal and hyperoxia.7 Unstable vessels are found during normal retinal vascular development, ocular neovascularization, tumorigenesis, female reproductive tissue cycling, and wound healing. Vessel stabilization is associated with vascular maturation and loss of plasticity and is characterized in the retina by insignificant levels of endothelial cell proliferation and migration, low levels of expression of matrix metalloproteinases, increasing thickness of the basement membrane, an absence of vessel regression, and resistance to VEGF withdrawal and hyperoxia.3 8 9
Mural cells are associated with newly formed vessels.10 11 12 13 14 15 During development and angiogenesis, they are thought to play an important role in vascular remodeling and vessel stabilization. Impaired mural cell recruitment is associated with aberrant vascular remodeling and angiogenesis in mouse embryos deficient in Tie-2, angiopoietin-1, or tissue factor.16 17 18 19 Close contact between mural cells and endothelial cells inhibits endothelial cell proliferation and migration in vitro,20 21 whereas the absence of mural cells during vascular development results in endothelial hyperplasia, microaneurysms, vessel dilation, abnormal vascular remodeling, and edema.22 23 24 25 In addition, it has been suggested that the absence of -smooth muscle actin (SMA)–positive pericyte ensheathment provides a window of plasticity that permits vascular remodeling.8 9 26
Mural cell abnormalities have been implicated in the pathogenesis of various conditions, including diabetic retinopathy and atherosclerosis, and the proposed roles of mural cells in vessel growth and stabilization have important clinical ramifications for cancer treatment and angiogenic therapy.3 27
Studies of SMC differentiation have focused mainly on the SMCs of the developing aorta. Aortic SMCs are derived from the surrounding mesoderm.28 They initially express SMA and later synthesize smooth muscle myosin heavy chains and smooth muscle–specific regulatory proteins, such as calponin, caldesmon, and smooth muscle -tropomyosin.29 However, the SMCs of mature vessels are heterogeneous in both their morphology and the proteins they express, differing according to their location in the vascular tree and within a vascular segment. Arteriolar SMCs, unlike those of the aorta, do not always express calponin or adult smooth muscle myosin heavy chains.30 31 These cells play an important role in the regulation of blood flow, but little is known of their differentiation other than that they express SMA during the early stages.
Studies of the developmental biology of mural cells have been hindered by a lack of pericyte-specific markers in vivo, unstable in vitro phenotypes, and the difficulty of identifying developing vessel types and of monitoring transitions in SMC phenotype along vessels in sectioned material. The retina is an ideal system with which to study mural cell development in the microcirculation, given that it contains a relatively high ratio of pericytes to vessels and a laminar vascular plexus in which arterioles, venules, capillaries, and postcapillary venules are readily visualized in situ. There is a proximal–distal gradient of vascular maturation in the retina. Thus, in any one static retinal wholemount preparation, it is possible to observe various stages of mural cell differentiation by following a vessel from the more mature central retina into the immature distal vascular plexus. Furthermore, in contrast to the adult retinal vasculature, the developing retinal vasculature is highly unstable, with active angiogenesis occurring in close proximity to vascular segments undergoing vessel retraction32 and with vessels regressing in response to hyperoxia-induced VEGF withdrawal.7 Studies have shown that mural cells are present on early retinal capillaries25 33 34 35 36 and that SMA expression becomes more widespread with maturation.8 36
With the use of wholemount preparations, we undertook a detailed marker analysis of mural cell differentiation in a growing vasculature. Our data showed a heterogeneous population of mural cells, during normal development of the retina. Our observations have lead us to propose a model of pericyte and SMC development in which a mural precursor cell evident at embryonic day (E)20 to E21 gives rise to both pericytes and SMCs. Further studies are needed to substantiate this model of mural cell differentiation.
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Materials and Methods |
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Immunohistochemistry
Multiple labeling was used to covisualize various antigens and the vasculature. A maximum of three fluorochromes were applied to each specimen. Thus, antigenic characterization of each stage of mural cell differentiation was performed with several marker combinations at each age. A minimum of four specimens were analyzed per described phenomenon, and the conclusions reached were representative of all specimens examined. Retinas were incubated overnight at 4°C with primary antibodies, washed with 0.1% Triton X-100 in PBS, incubated for 4 hours at room temperature with the appropriate secondary antibodies, and washed again. For double or triple labeling, this procedure was repeated with the different primary antibodies and appropriate secondary antibodies. Negative controls omitting a primary antibody were performed for each antibody and protocol. Vessels were labeled with biotinylated Griffonia simplicifolia (GS) lectin followed by streptavidin conjugated with either fluorescein isothiocyanate (FITC; Amersham Biosciences, Piscataway, NJ), Texas red (Amersham), or Cy5 (Jackson ImmunoResearch, West Grove, PA).37 All antibodies and streptavidin were diluted with 1% bovine serum albumin in PBS, and all washes were performed with 0.1% Triton X-100 in PBS. Retinal wholemounts were finally mounted, ganglion cell layer up, in glycerol:PBS (2:1, vol/vol) or antifade medium (Prolong Anti Fade; Molecular Probes, Eugene, OR).
Antibodies
To identify both mature and immature mural cells in the developing rat retina, we used antibodies to NG2, to desmin, and to SMA. NG2 is the rat homologue of HMW-MAA, a transmembrane chondroitin sulfate proteoglycan expressed by immature human SMCs and pericytes13 35 38 39 40 as well as by mature SMCs, but it is expressed only sporadically and at a low level by the capillaries of a quiescent vasculature.13 Desmin is expressed by immature and mature pericytes14 41 as well as by a subpopulation of SMCs associated with developing or mature arteries.42 43 SMA is expressed by immature and mature SMCs,29 as well as by a subpopulation of pericytes.44 Preliminary studies were performed to confirm that NG2 and desmin were mural-cell–specific markers in the developing retina. Double labeling with lectin and antibodies against desmin and NG2 showed that these antibodies labeled cells on the surface of developing retinal vessels (Figs. 1A 1B) . Double labeling with NG2 or desmin antibodies and antibodies against the astrocyte markers glial fibrillary acidic protein (GFAP) and S100 confirmed that astrocytes did not express NG2 or desmin (Figs. 1C 1D) .
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To detect anti-NG2, we used either Texas red– or FITC-conjugated donkey anti-rabbit Ig (Amersham) diluted 1:50 or biotinylated donkey anti-rabbit Ig (Amersham) diluted 1:50 followed by Cy5-conjugated streptavidin diluted 1:100. To detect anti-desmin, anti-calponin, or anti-caldesmon, we used Texas red– or FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL) diluted 1:60. To detect anti-SMA, we used either FITC-conjugated goat anti-mouse IgG2a (Southern Biotechnology Associates) diluted 1:60 or Texas red–conjugated sheep anti-mouse Ig (Amersham) diluted 1:50. To detect anti-S100, we used FITC-conjugated donkey anti-rabbit Ig diluted 1:50. To detect anti-GFAP, we used Texas red–conjugated sheep anti-mouse Ig diluted 1:50. Because our antigenic characterization was undertaken using a minimum of five markers at each age, the availability of only three fluorochrome channels per specimen necessitated the application of two markers plus the GS lectin per specimen on multiple specimens at each age.
Microscopy and Mapping
Retinal wholemounts were examined by both conventional and confocal microscopy. For conventional fluorescence microscopy and photography, we used a microscope (model AH BT, attachment AH2-RFL) and camera (both from Leica, Wetzlar, Germany). To map the expression of SMA, desmin, calponin, and caldesmon, we created and traced a photographic montage of the GS lectin–labeled vasculature in a retinal segment also labeled with antibodies specific for these mural cell markers. The tracing was compared with the original preparation, and the location of marker protein expression was then recorded on the tracing. The resultant diagram was scanned with a flatbed scanner (XRS-OmniMedia-3cx; Argosy OmniMedia, North Bethesda, MD) and processed with software (Photoshop, ver. 5.0; Adobe Systems, Mountain View, CA).
Confocal microscopy was performed with an argon-krypton laser (Leica) mounted on an epifluorescence photomicroscope (DMRBE; Leica). FITC, Texas red, and Cy5 fluorescence was excited sequentially at 488, 588, and 665 nm, respectively. Images were processed on computer (Photoshop, ver. 5.0; Adobe Systems).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
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In the embryonic retina desmin filaments were associated with a small subpopulation of MPCs (Fig. 1H) . By postnatal day (P)0, ensheathing MPCs with desmin filaments were more common (Figs. 1N 1O) and in the following postnatal days (P1–P2) most ensheathing MPCs acquired desmin filaments (Table 1) . The timing and pattern of desmin filament acquisition are depicted in Figures 2A 2B 2C 2D . Note that desmin filament acquisition occurred throughout the vascular plexus including at the vascular periphery where vessels were forming.
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Various stages of SMC differentiation were identifiable on central radial arterioles. At P5, SMA expression (Fig. 3A) was diffuse, with stellate aggregations of desmin filaments on only some NG2+ and SMA+ SMCs (Figs. 3B 3C) . By P7, SMA had aggregated into concentric filaments and low levels of diffuse calponin expression were detected in the cells (Figs. 3D 3E) . This stage of SMC differentiation was designated an immature radial arteriolar SMC. Further SMC maturation on the central radial arterioles was characterized at P13 to P15 by lengthening of SMA filaments, increased SMA filament density, concentric alignment of the cells (as revealed by NG2 expression), the appearance of concentric desmin filaments (Figs. 3F 3G) , increased calponin expression, and the onset of caldesmon expression. These cells were designated juvenile radial arteriolar SMCs. The adult radial arteriolar SMC phenotype, characterized by tightly packed, concentric SMA and desmin filaments, weak NG2 immunoreactivity, and calponin and caldesmon expression, was evident from P17 (Figs. 3H 3I 3J 3K , Table 1 ).
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Differentiation of SMCs of Primary and Secondary Arterioles
Primary arterioles branch from the radial arterioles giving rise to secondary arterioles. Like the radial arterioles in the peripheral retina, these vessels were selected from undifferentiated vessels with ensheathing MPCs, and the differentiation of SMCs of the two types of vessels showed similar changes. Juvenile arteriolar SMCs with diffuse SMA expression and stellate desmin filaments differentiated on primary arterioles from P13 to P15 (Figs. 3N 3O) . These cells matured to the adult phenotype in the third postnatal week, as evidenced by the appearance of concentric SMA (Fig. 3P) and desmin filaments (Fig. 3Q) , weak NG2 immunoreactivity, and the expression of caldesmon (Fig. 3I) and calponin. The differentiation of the more distal arteriolar SMCs on secondary and tertiary arterioles occurred from P15 to P21.
In the adult rat retina, a gradual transition from arteriolar SMCs to pericytes was associated with distal progression along arterioles. Calponin expression was lost during the transition from arteries to primary arterioles (Figs. 4D 4E 4F) . SMA filaments became shorter, and desmin filaments became less numerous and acquired a stellate configuration (Figs. 4G 4H) . More distally, SMA expression was diffuse rather than filamentous and petered out on a subpopulation of capillaries (Fig. 4I) , caldesmon expression was undetectable (Figs. 4C 4F) and some desmin filaments now ran parallel with the vessels (Fig. 4J) . These changes were accompanied by the progressive restriction of NG2 labeling to protruding abluminal somas (Figs. 4K 4L 4M) culminating in pericytes on capillaries with NG2+cell bodies and desmin filaments coursing parallel to the vessel (Fig. 4N) . These observations are consistent with previous ultrastructural and immunohistochemical data from other tissues1 30 45 46 showing a continuum in morphology and contractile protein expression from SMCs on arterioles, to pericytes on capillaries, to SMCs on veins.
Differentiation of Venous SMCs
Venous SMCs exhibited a distinct pattern of differentiation. At P2, when radial venules could be distinguished from radial arterioles, the venous mural cells had only low levels of SMA immunoreactivity (Fig. 2H) . At P5, the mural cells of radial venules contained stellate desmin filaments and SMA labeling was weak or undetectable (Fig. 3R) . With further postnatal development, diffuse SMA immunoreactivity gradually increased but remained patchy (Fig. 3S) . Short SMA filaments were detectable at P21 (Fig. 3T) . Venous SMC maturation thus lagged behind the selection of a vessel as a venule. In adult venous SMCs, SMA was not organized into obvious bundles of filaments and immunoreactivity, although substantial, was patchy (Fig. 3U) . Desmin filaments appeared randomly aligned, and neither calponin nor caldesmon was detected (Figs. 4A 4B 4C 4D 4E 4F ; Table 1 ). As with the more distal arterioles, the radial venules in the peripheral retina are derived from immature vascular segments, which already have desmin+/NG2+ensheathing MPCs, suggesting that venous SMCs can also be derived from these cells.
Differentiation of Pericytes
At P0, desmin+/NG2+ ensheathing MPCs were scattered throughout the entire vascular tree (Fig. 2B) . During the first postnatal week the immature inner vascular plexus spread to the retinal periphery. Desmin+/NG2+ ensheathing MPCs (Fig. 5A) were present throughout the vascular plexus at P5. Furthermore, they were present on the forming vessels at the vascular front (Figs. 5B 5C 5D) as well as on the vascular sprouts that descended into the inner nuclear layer (Figs. 5E 5F) and on the newly formed vessels of the outer plexus.
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Our results are consistent with the conclusion that SMCs and pericytes are derived from a common precursor, the desmin+/NG2+ ensheathing MPC present on undifferentiated vessels.
Mural Cells and Vessel Stability
To assess the role of mural cells in vessel stabilization, we examined vascular regression during normal retinal development. As demonstrated previously, the rat retinal vasculature underwent substantial remodeling during normal development.32 Vessel regression was most pronounced along the radial arterioles during the first postnatal week. Subsequently, the remaining immature vasculature was remodeled into arteriolar trees and a sparser capillary plexus by vessel selection and regression; this process began centrally by P10 and was complete by P21. At P5, desmin+/NG2+ ensheathing MPCs were present on all vessels, including those undergoing regression in the vicinity of radial arteries (Figs. 5K 5L 5M 5N) and in remodeling plexuses (Fig. 5O) . In the second postnatal week, desmin+/NG2+/SMA– juvenile pericytes were associated with vessels undergoing regression (Figs. 5G 5H) . Thus, the mere presence of ensheathing MPCs and juvenile pericytes did not prevent vessel regression during normal retinal development.
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Discussion |
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The desmin+/NG2+ ensheathing MPCs were associated with the undifferentiated vascular plexus of the early postnatal retinas. As the vasculature matures, these immature vascular segments give rise to arterioles, venules, or capillaries. Multimarker immunohistochemistry showed that vessel differentiation was accompanied by SMC and pericyte differentiation and revealed a phenotypic continuum between the desmin+/NG2+ ensheathing MPCs of the immature vascular segments and the SMCs of the differentiating arterioles and the pericytes of the maturing capillaries. These observations strongly suggest that the NG2+/desmin+ ensheathing MPC is a pluripotent cell capable of giving rise to both pericytes and SMCs. A schematic representation of pericyte and SMC differentiation based on these observations is shown in Figure 6 .
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Our observations of mural cell differentiation are consistent with previous results suggesting that SMA is expressed early in SMC development28 and later by both mature SMCs and a subset of mature pericytes.44 The extent of SMA expression by SMCs on arterioles and venules increased markedly with maturation. SMC coverage and SMA expression decreased with distal progression along the arteriolar tree. Given that SMA is a major contractile protein, the number of SMA filaments in a cell is thought to be proportional to the magnitude of the force the cell is able to generate. The final stage of arteriolar SMC differentiation was characterized by the expression of the regulatory proteins calponin and caldesmon and reflects the potential to modulate a contractile response.29 The observed SMC heterogeneity in the mature retinal plexus is consistent both with previous studies that found a morphologic and biochemical continuum from arterial SMCs to pericytes along arterioles12 30 31 46 and with the established plasticity of SMCs.29 This heterogeneity probably reflects the various roles that these cells play in the regulation of blood flow.
Expression of Functional Markers with SMC Maturation
We have shown that calponin expression precedes caldesmon expression on radial arteries by 
1 week. Whereas calponin expression stopped abruptly at the transition from a radial arteriole to a primary arteriole, caldesmon expression declined more gradually, extending into primary and secondary arterioles.
Calponin and caldesmon are expressed relatively late in SMC differentiation.47 They are associated with the thin filaments of SMCs and are thought to regulate SMC tone.29 48 49 50 Calponin is thought to serve three functions: to maintain relaxation at resting cytosolic calcium concentrations, to preserve energy during prolonged contraction, and, as a result of its phosphorylation by protein kinase C-
, to contribute to calcium-independent contraction.51 Furthermore, given that calponin interacts with desmin with an affinity similar to that with which it interacts with tropomyosin and myosin, calponin may also play a role in cytoskeletal organization.52
The intimate association of SMCs with vascular endothelial cells is essential for induction of functional markers indicative of SMC maturation, including calponin, caldesmon, and smooth muscle myosin heavy chains. Close contact between mesenchymal cells (SMC precursors) and vascular endothelial cells is thought to result in the activation of transforming growth factor-ß, which in turn promotes the expression of markers indicative of smooth muscle differentiation and maturation.53 In vitro the loss of endothelial cell contact results in the "dedifferentiation" of SMCs, as evidenced by downregulation of the expression of calponin, caldesmon, and smooth muscle myosin.54 55 Calponin expression is upregulated by the vasoconstrictor angiotensin II56 57 and is downregulated by nitric oxide.58 In contrast, the expression of caldesmon is upregulated in response to cyclic stretching.59
The timing and topography of expression of calponin and caldesmon during development of the rat retinal vasculature suggest that SMC maturation, as indicated by the expression of these two regulatory proteins and the resultant improved capacity to fine-tune autoregulatory responses to changes in tissue oxygen levels, may play an important role in the development of resistance to the hyperoxia associated with vessel stabilization.
Role of SMC Maturation in Vessel Stabilization
Both pericytes and SMCs have been implicated in vessel stabilization, limiting endothelial cell proliferation and preventing vessel regression. The present study shows that ensheathing mural precursor cells are present on newly formed and withdrawing vessels. Thus, the mere presence of immature mural cells does not confer vessel stability.
Previous studies have demonstrated that resistance of the retinal plexus to VEGF withdrawal coincides with widespread SMA expression.8 Our study has shown that increased SMA expression is due to the differentiation of SMCs on arterioles rather than SMA expression by pericytes on capillaries, as was previously concluded.8
Further, our results showed that the maturation of SMCs as indicated by the expression of caldesmon and calponin, proteins that regulate SMC contraction, occurs in the third postnatal week. Other studies have shown that the rat retinal vasculature also becomes resistant to hyperoxia in the third postnatal week.8 60 This leads us to suggest that SMC maturation as evidenced by calponin and caldesmon expression results in an improved capacity to regulate blood flow and therefore in a lack of susceptibility to hyperoxia-induced vessel regression.
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Acknowledgements |
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Footnotes |
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Submitted for publication December 4, 2003; revised February 24 and March 30, 2004; accepted April 12, 2004.
Disclosure: S. Hughes, None; T. Chan-Ling, 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: Tailoi Chan-Ling, Department of Anatomy and Histology (F13), University of Sydney, Sydney, NSW 2006, Australia; tailoi{at}anatomy.usyd.edu.au.
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