Abstract
purpose. Dilated and tortuous vessels (plus disease) in ROP is a grim prognostic indicator of visual outcome. The purpose of this study was to determine whether alterations in pericytes and smooth muscle cells (SMCs), are associated with the pathogenesis of ROP, including plus disease.
methods. Kittens were exposed to either 4 (standard obliterative model) or 2 (modified model) days of hyperoxia, resulting in vaso-obliteration or localized vessel regression, respectively, and returned to room air. The modified model more closely resembles human ROP. Desmin and α-smooth muscle actin (SMA) immunohistochemistry and lectin labeling were used to label mural cells and vessels. The desmin ensheathment ratio (DER), a quantitative measure of vessel stability, was determined.
results. In the neovasculature of the standard model and surviving vasculature of the modified model, radial arterioles and venules were dilated and SMCs attenuated. SMA expression on venules was decreased, and the difference in desmin expression normally observed between arterioles and venules was lost, indicating altered SMC differentiation. The DER was reduced in both ROP models, consistent with highly unstable vascular plexuses, receptive to angiogenic and vascular regression signals.
conclusions. The results provide compelling evidence of significant changes in arteriolar and venular SMCs in both experimental models of ROP. The delayed differentiation and apparent dedifferentiation of SMCs during the hypoxic phases would result in an impaired ability to regulate blood flow, contributing to the vasodilation and tortuosity, hallmarks of plus disease. Vessel tortuosity was seen only in the nonobliterative model, suggesting that tortuosity may be due to increased capillary resistance resulting from capillary closure.
Retinopathy of prematurity (ROP) is a disease that affects premature infants, in which abnormal retinal vasoproliferation can be followed by extraretinal fibrovascular proliferation causing tractional detachment, which may lead to blindness. Risk factors for ROP include extreme prematurity, artificial ventilation, and very low birth weight,
1 2 and the incidence of the disease correlates with the degree of ocular immaturity at birth.
3 ROP is a major cause of childhood blindness in the developed world, where its incidence is increasing in line with the enhanced survival of premature infants under 1 kg and 26 weeks’ gestation and is an emerging cause of blindness in the developing world, due to improved neonatal care.
4 5 Depending on the level of oxygen administered to neonates, the resultant retinal hyperoxia can result in severe vessel constriction and delayed retinal vascularization. The consequent tissue hypoxia when oxygen administration is stopped and/or neuronal metabolic activity increases, results in VEGF-driven vasoproliferation and aberrant neovascularization.
6 7 8 9 10
ROP has been classified according to the location and extent of the abnormalities and the stage of the vasoproliferative response at the vascular periphery. The severity of the disease ranges from the least severe stage, characterized by a demarcation line between the outer edge of the retinal vasculature and the avascular zone at the retinal periphery, to a severe stage, characterized by extraretinal fibrovascular proliferation and tractional retinal detachment.
11
An additional risk factor, designated “plus disease” and characterized by tortuous arterioles and dilated veins in the posterior retina, may be superimposed on the other pathologic changes at any stage of ROP.
11 Plus disease may be accompanied by iris vessel engorgement, pupil rigidity, and vitreous haze; is a grim predictor of visual outcomes in ROP; and correlates strongly with retinal detachment.
12 13 14 15 16 Indeed, laser photocoagulation is now recommended in most cases in which plus disease, as defined by significant dilation and tortuosity of posterior retinal blood vessels, is present.
15 17 Even in milder cases in which posterior vessel dilation and tortuosity are less pronounced, a condition designated pre-plus disease,
18 the incidence of ROP progression is increased.
19 A rapidly progressive form of ROP, aggressive posterior ROP (AP-ROP), has now been recognized. It is characterized by posterior location, significant dilation and tortuosity of arteries and veins, and brushlike neovascularization at the vascular periphery.
18
Although features of plus disease’s have been recognized since 1949,
20 and its importance as a prognostic indicator is known, our understanding of the pathogenesis of the disease is still lacking. It is thought to arise as a result of altered hemodynamics due to shunting of blood at the vascular periphery combined with retention of blood producing engorgement and vasodilation. To date, no studies of the cellular events underlying the pathogenesis of plus disease have been undertaken, and its cause has been speculative. Fluorescein angiography has shown that in the acute proliferative stages of ROP, there is often a major arteriovenous shunt, which is extremely leaky,
21 and it is thought that plus disease signifies high blood flow as a result of these shunts. However, plus disease can occur in AP-ROP without any evidence of such peripheral shunts.
18
Aggressive posterior ROP is characterized by prominent plus disease in posterior retina in which both venules and arterioles become dilated and tortuous and thus difficult to differentiate from one another.
18
Vascular smooth muscle cells (SMCs) are the specialized contractile cells of the arterial wall and the major effectors in control of blood pressure and flow. Pericytes represent their counterpart in the capillaries and venules, and although they show less development in their contractile apparatus, they are thought to play a role in the regulation of blood flow and to stabilize vessels (reviewed by Hughes and Chan-Ling
22 ). In particular, they are thought to prevent hyperoxia-induced vessel regression and to regulate endothelial cell proliferation.
23 24 25 26 27 28 29 A decrease in the number of mural cells (SMCs and pericytes) during development and in diseases such as diabetic retinopathy is associated with aberrant neovascularization, vessel dilation, microaneurysm formation, vascular incompetence, and possible impaired perfusion.
30 31 32 33 Earlier reports have demonstrated the key role played by astrocytes, the glial cells associated with developing retinal vasculature,
34 and VEGF
8 35 in the pathogenesis of ROP, but little is known of the role of pericytes and SMCs in this disease.
The aberrant vasoproliferation of ROP and the ability of pericytes to regulate endothelial cell proliferation
27 suggest that these cells may play a role in the pathogenesis of ROP. We have shown that immature mural cells are present in the neovasculature in the kitten model of ROP and that these vessels have a decreased ratio of desmin filaments to endothelial cell area, or desmin ensheathment ratio (DER).
36 The low DER may reflect aberrant endothelial cell-pericyte interaction. Furthermore, the radial vessel tortuosity and vasodilation that characterizes pre-plus disease, plus disease, and AP-ROP suggests that SMCs and pericytes play a key role in these more severe manifestations of ROP, due to their functions, known and postulated, in regulating retinal blood flow.
The vascular events in ROP can be studied in a kitten model of the disease, in which hyperoxia-induced vaso-obliteration is followed by hypoxia-driven neovascularization in both the retina and vitreous; the neovasculature is subsequently remodeled, resulting in disease regression.
10 34 36 37 In this model of ROP, virtually all the retinal vessels are lost, whereas in the human disease there is a surviving, albeit altered, retinal vasculature consisting of congested peripheral capillary plexuses supplied by radial vessels that may also be altered. Shorter exposures to hyperoxia result in partial rather than total vaso-obliteration in the kitten.
38 The purpose of our study was to determine whether pericytes and/or SMCs are altered during the various phases of the standard obliterative model of ROP: the hyperoxic phase when existing retinal vessels regress, the hypoxic neovascular phase, and the remodeling-resolving phase. In addition, we set out to determine the changes in mural cells in a modified nonobliterative kitten model of ROP in which the vasculature survives, albeit with some vessel regression and delayed vascularization and thus more closely follows the postulated early events of human ROP.
Our findings led us to suggest that mural cell-endothelial cell relationships are severely compromised during hyperoxia and hypoxia and that changes in mural cells play a significant role in the pathogenesis of ROP—in particular, the potentially blinding form of ROP associated with plus disease. The vasculature of ROP was characterized by a massive increase in capillary density.
9 As a consequence, a higher blood pressure or greater vasoconstriction is necessary to push this larger blood volume through this immensely dense vasculature. Plus disease is a likely clinical manifestation of these changes.
Pericyte Changes on Nonperfused Capillary Segments after Hyperoxia in an Obliterative Kitten Model of ROP
Altered Pericyte Phenotype in Neovasculature during Hypoxic and Subsequent Vascular Remodeling Phases of an Obliterative Model of ROP
Altered Hemodynamics and Tissue Hypoxia in the Modified Nonobliterative Model of ROP
Altered Pericyte Phenotype and Reduced DER in Surviving Vasculature of the Nonobliterative Model of ROP
Alterations in SMCs on Radial Arterioles and Venules in Hypoxia-Induced Neovasculature in the Obliterative Model
Dedifferentiation of SMCs on Arterioles and Venules That Had Survived Exposure to Hyperoxia
As in previous studies, 4 days of hyperoxia resulted in vaso-obliteration, followed by aberrant vasoproliferation on return to room air, resulting in preretinal membranes and a multilayered dense retinal neovasculature, fed by an increased number of radial vessels, that was later remodeled. Our results showed that mural cell dedifferentiation and/or loss occurred in nonperfused vascular remnants. We modified this model of ROP by reducing the duration of hyperoxia exposure so that it more closely resembled the human condition of ROP. In this model, as in human ROP, retinal vascularization was retarded, with most existing vessels surviving the hyperoxic insult, in contrast to the established model in which the entire retinal vasculature is rendered nonfunctional by widespread vessel regression in response to prolonged hyperoxia. This new nonobliterative model of ROP displayed the radial vessel tortuosity and enlargement that characterizes plus disease and the newly described clinical entity of “pre-plus disease,” but does not mimic the end stages of human plus disease of extraretinal fibrovascular proliferation followed by tractional detachment.
Electron microscopy confirmed that mural cells were present on the vessels of the preretinal membranes. Desmin labeling revealed that the immature mural cells of the surviving capillary plexuses of the nonobliterative model bore a closer resemblance to those of the aberrant multilayered and remodeling capillary neovasculature of the obliterative model, than to mural cells of the immature capillary plexuses in control animals. As in the neovasculature of the obliterative model,
36 the DER of the surviving plexus of the nonobliterative model was lower than that of the control and remained well below the threshold DER for vascular stability of 0.9, even after 29 dRA, indicating that a comparatively brief exposure to hyperoxia results in increased vessel instability and prolongs retinal vascular instability observed during neonatal development.
During the hypoxic phase, in both the neovasculature of the obliterative model and the surviving vasculature of the nonobliterative model, the radial vessels were enlarged and their SMCs showed desmin morphology and electron microscopy features consistent with immaturity.
Increased VEGF expression cause arteriole and venule enlargement; an effect that appears to be dependent on the Ang-2 expression in the retina.
58 59 Thus, the radial vessel dilation in plus disease may be a sign of increased VEGF and Ang-2 levels in the posterior retina.
In both models of ROP, radial vessel dilation was accompanied by impaired SMC differentiation during the hypoxic phase; thus, an impaired ability to regulate blood flow may further contribute to radial vessel dilation in these models. In both models, arterioles could no longer be distinguished from venules on the basis of desmin filament ensheathment. Similarly, in AP-ROP the distinction between arteries and veins is lost
18 although radial vessel tortuosity, the hallmark of plus disease, was seen only in the nonobliterative model.
Increased VEGF levels cause retinal endothelial cell hypertrophy and capillary luminal narrowing which is likely to compromise blood flow.
60 In the nonobliterative model capillary bed, resistance is postulated to further increase with the decrease in connections between the capillary beds and the radial vessels, and with vasoproliferation resulting in increased capillary density, whereas in the obliterative model, although the capillary plexuses are very dense, they are fed by an increased number of radial vessels. Indeed, in the canine model of ROP where vessel regression is more widespread, radial tortuosity during the hypoxic phase is more pronounced,
61 raising the possibility that vessel tortuosity is the result of increased capillary bed resistance that may be due to either aberrant vasoproliferation or vessel withdrawal at the vascular periphery. The development of vascular shunts in human ROP could be an adaptation in an attempt to overcome pronounced capillary bed resistance and further support the similarity of the pathogenetic mechanisms underlying vessel tortuosity and dilation in human, feline, and canine ROP.
Our observations lead us to speculate that plus disease is indicative of a highly unstable vasculature with elevated VEGF and Ang-2 levels in the posterior retina, an impaired ability to regulate retinal blood flow and severely compromised blood flow at the vascular periphery resulting in altered hemodynamics in the radial vessels.
A remarkable finding in this study was the concordance between the clinical presentation of plus disease and the changes reported. The progression of human ROP is characterized by retinal vessel morphology, which clearly allows differentiation of artery and vein, but which becomes distorted over time by dilation and tortuosity to become plus disease. We report a similar dedifferentiation of SMCs on arterioles and venules.
Revised indications for the treatment of ROP now support ablative therapy for eyes at any stage of plus disease. Data from this study suggest a hypoxic environment and unstable vasculature in plus disease, thus providing a pathogenetic basis and scientific rationale for this recommendation.
Our data provide compelling evidence of the contribution of a hypoxia-induced, VEGF-mediated change to the retinal vasculature and phenotypic alterations in pericytes and SMCs in the pathogenesis of plus disease in ROP. Based on these observations, we suggest that therapy combining anti-VEGF antiangiogenic therapy, Ang-1 for vessel normalization, and PDGFβ for pericyte recruitment and vessel stabilization could offer an effective combinational form of intervention in cases of ROP where plus disease, as defined by significant dilation and tortuosity of posterior retinal vessels, is present.
Our observations in kitten models of ROP show that during the hypoxic phase, pericytes adopt a phenotype associated with increased vascular instability and that SMC differentiation is impaired, implying a decreased ability to regulate blood flow. Our findings are consistent with the proposal that plus disease is the manifestation of a highly unstable vasculature with impaired regulation of blood flow and highly compromised blood flow at the vascular periphery, resulting in a vascular bed that is more susceptible to fluctuations in oxygen levels and growth factors and thus prone to vicious cycles of increasing VEGF levels. Similar changes in the human condition could contribute to the pathogenesis of ROP and plus disease.
Further, vessel tortuosity was evident only in the nonobliterative model of kitten ROP, not in the obliterative model. Significant capillary closure evident around surviving large vessels in the nonobliterative model leads us to suggest that local mechanical forces due to the unusual nature of the remodeled vascular tree are associated with arteriole dilation.
W conclude that plus disease is associated with changes to pericytes and SMCs that may be associated with alterations in blood flow. More work is needed to verify this conclusion. Dedifferentiation of SMCs on retinal arterioles and venules, aberrant pericyte phenotypes, altered hemodynamics, and high VEGF and Ang-2 levels may all contribute to the arterial and venous dilation and tortuosity observed clinically.
Supported by grants from the Financial Markets Foundation for Children, the Rebecca Cooper Medical Research Foundation, and National Health and Medical Research Council of Australia Grants 402581, 153789, and 402824 (TCL).
Submitted for publication July 24, 2006; revised October 3, 2006; accepted December 21, 2007.
Disclosure:
S. Hughes, None;
T. Gardiner, None;
L. Baxter, 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;
[email protected].
The authors thank Ruth-Ann Sterling for assistance with digital imaging and artwork and John Flynn and Glen Gole for helpful comments on the manuscript.
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