Desmin Ensheathment Ratio as an Indicator of Vessel Stability
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63389-5
ISSN1525-2191
AutoresTailoi Chan‐Ling, Matthew P. Page, Tom A. Gardiner, Louise Baxter, Emilia Rosinova, Suzanne Hughes,
Tópico(s)Neonatal Respiratory Health Research
ResumoWe developed a measure of pericyte/endothelial interaction, the desmin ensheathment ratio (DER), using the intermediate filament desmin as an indicator of pericyte ensheathment and have examined the DER in normal retinal vascular development and in the kitten retinopathy of prematurity (ROP) model. We also examined the role of mural cells in the pathogenesis of ROP. Postnatal day 1 to 45 kitten retinae were labeled for desmin, α-smooth muscle actin (SMA), and isolectin-B4. Newborn kittens exposed to hyperoxia and then returned to room air for 0 to 40 days (dRA) were similarly labeled. The ratio of desmin to lectin labeling on confocal images yielded the DER. Ultrastructural studies showed that mural cells were present on even the most primitive vessels. During normal development, immature vascular beds had DERs of 0.3 to 0.6 whereas mature beds, which predominated by postnatal day 28, had DERs greater than 0.9. Immature pericytes and smooth muscle cells did not prevent hyperoxia-induced vessel regression. During the vasoproliferative stage of ROP, the DERs of intra- and preretinal vessels ranged between 0.2 and 0.5. In the recovery stage, the DER increased in parallel with regression of pathology, reaching 0.9 at 34 dRA. Stabilization of the DER by the fifth postnatal week was temporally coincident with the development of resistance to hyperoxia-induced vessel regression previously reported in the kitten. These observations lead us to suggest that a DER of 0.9 represents a vascular stability threshold and that a low DER observed during ROP raises the possibility that mural cell abnormalities play a key role in the pathogenesis of ROP. We developed a measure of pericyte/endothelial interaction, the desmin ensheathment ratio (DER), using the intermediate filament desmin as an indicator of pericyte ensheathment and have examined the DER in normal retinal vascular development and in the kitten retinopathy of prematurity (ROP) model. We also examined the role of mural cells in the pathogenesis of ROP. Postnatal day 1 to 45 kitten retinae were labeled for desmin, α-smooth muscle actin (SMA), and isolectin-B4. Newborn kittens exposed to hyperoxia and then returned to room air for 0 to 40 days (dRA) were similarly labeled. The ratio of desmin to lectin labeling on confocal images yielded the DER. Ultrastructural studies showed that mural cells were present on even the most primitive vessels. During normal development, immature vascular beds had DERs of 0.3 to 0.6 whereas mature beds, which predominated by postnatal day 28, had DERs greater than 0.9. Immature pericytes and smooth muscle cells did not prevent hyperoxia-induced vessel regression. During the vasoproliferative stage of ROP, the DERs of intra- and preretinal vessels ranged between 0.2 and 0.5. In the recovery stage, the DER increased in parallel with regression of pathology, reaching 0.9 at 34 dRA. Stabilization of the DER by the fifth postnatal week was temporally coincident with the development of resistance to hyperoxia-induced vessel regression previously reported in the kitten. These observations lead us to suggest that a DER of 0.9 represents a vascular stability threshold and that a low DER observed during ROP raises the possibility that mural cell abnormalities play a key role in the pathogenesis of ROP. Vessel stability has important implications for many disease processes including sight-threatening diseases of the retina, tumor biology, and diabetic nephropathy. The capillaries of mature vascular beds are considered stable when vascular cell proliferation and vessel regression are negligible and their endothelial cells do not require vascular endothelial growth factor for their survival and are ensheathed by mature mural cells.1Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1427) Google Scholar, 2Benjamin LE Keshet E Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.Proc Natl Acad Sci USA. 1997; 94: 8761-8766Crossref PubMed Scopus (444) Google Scholar, 3Hughes S Chan-Ling T Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.Invest Ophthalmol Vis Sci. 2004; 45: 2795-2806Crossref PubMed Scopus (160) Google Scholar In contrast, the capillaries of angiogenic plexuses are considered unstable and are characterized by significant endothelial proliferation, vessel regression in response to vascular endothelial growth factor withdrawal, and ensheathment by immature mural cells. The forming retinal vasculature is an unstable vascular bed and this instability underlies the initiating event in the pathogenesis of retinopathy of prematurity (ROP), the significant vaso-obliteration that occurs when the premature infant is exposed to therapeutic hyperoxia. Mural cells are thought to play a role in vessel stabilization. In vitro, close contact between mural cells and endothelial cells inhibited endothelial cell proliferation and migration.4Orlidge A D'Amore P Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells.J Cell Biol. 1987; 105: 1455-1462Crossref PubMed Scopus (528) Google Scholar, 5Sato Y Rifkin DB Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture.J Cell Biol. 1989; 109: 309-315Crossref PubMed Scopus (768) Google Scholar More recently impaired mural cell recruitment has been associated with aberrant angiogenesis and vessel regression.6Suri C Jones PF Patan S Bartunkova S Maisonpierre PC Davis S Sato TN Yancopoulos GD Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis.Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2419) Google Scholar, 7Carmeliet P Ferreira V Breier G Pollefeyt S Kieckens L Gertsenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Pawling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryo is lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3484) Google Scholar, 8Maisonpierre PC Suri C Jones PF Bartunkova S Wiegand SJ Radziejewski C Compton D McClain J Aldrich TH Papadopoulos N Daly TJ Davis S Sato TN Yancopoulos GD Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science. 1997; 277: 55-60Crossref PubMed Scopus (2995) Google Scholar, 9Enge M Bjarnegard M Gerhardt H Gustafsson E Kalen M Asker N Hammes HP Shani M Fassler R Betsholtz C Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy.EMBO J. 2002; 21: 4307-4316Crossref PubMed Scopus (314) Google Scholar Using retinal digest preparations it was noted that new vessel formation in retina ceased only when pericytes became discernable and neovascularization observed in diabetic retinopathy only occurred when pericytes were lost, leading the authors to suggest that pericytes may inhibit vessel formation.10Cogan DG Toussaint D Kuwabara T Retinal vascular patterns. IV. Diabetic retinopathy.Arch Ophthalmol. 1961; 66: 366-377Crossref PubMed Scopus (522) Google Scholar, 11Kuwabara T Cogan DG Retinal vascular patterns (VI). Mural cells of the retinal capillaries.Arch Ophthalmol. 1963; 69: 472-502Crossref Scopus (259) Google Scholar, 12Hammes HP Lin J Renner O Shani M Lundqvist A Betsholtz C Brownlee M Deutsch U Pericytes and the pathogenesis of diabetic retinopathy.Diabetes. 2002; 51: 3107-3112Crossref PubMed Scopus (461) Google Scholar The retinal digest preparation permitted a quantitative measure of the ratio between the numbers of endothelial cells and pericytes. In diabetic retinopathy, one of the earliest indicators of disease is pericyte dropout. This led to the idea that the absolute ratio of these two cell types is critical to normal retinal vascular function. However, the accuracy of the endothelial cell to pericyte (E/P) ratio is limited by the difficulty in distinguishing these cell types because of their often ambiguous nuclear morphology13Glatt HJ Henkind P Aging changes in the retinal capillary bed of the rat.Microvasc Res. 1979; 18: 1-17Crossref PubMed Scopus (15) Google Scholar, 14Cuthbertson RA Mandel TE Anatomy of the mouse retina. Endothelial cell-pericyte ratio and capillary distribution.Invest Ophthalmol Vis Sci. 1986; 27: 1659-1664PubMed Google Scholar and the possibility that the nuclei of perivascular glia may also be included.15Kuwabara T Cogan DG Studies of retinal vascular patterns. Part I. Normal architecture.Arch Ophthalmol. 1960; 64: 904-911Crossref PubMed Scopus (288) Google Scholar These limitations have led us to develop a new measure of pericyte/endothelial interaction using the intermediate filament desmin as an indicator of pericyte ensheathment. Desmin is expressed by mature and immature pericytes16Verhoeven D Buyssens N Desmin-positive stellate cells associated with angiogenesis in a tumour and non-tumour system.Virchows Arch B Cell Pathol. 1988; 54: 263-272Crossref Scopus (29) Google Scholar, 17Nehls V Denzer K Drenckhahn D Pericyte involvement in capillary sprouting during angiogenesis in situ.Cell Tissue Res. 1992; 270: 469-474Crossref PubMed Scopus (329) Google Scholar, 18Hellstrom M Kalen M Lindahl P Abramsson A Betsholtz C Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.Development. 1999; 126: 3047-3055Crossref PubMed Google Scholar and a subpopulation of smooth muscle cells (SMCs) on mature and developing arteries, arterioles, venules, and veins.19Osborn M Caselitz J Weber K Heterogeneity of intermediate filament expression in vascular smooth muscle: a gradient in desmin positive cells from the rat aortic arch to the level of the arteria iliaca communis.Differentiation. 1981; 20: 196-202Crossref PubMed Scopus (119) Google Scholar, 20Kacem K Seylaz J Aubineau P Differential processes of vascular smooth muscle cell differentiation within elastic and muscular arteries of rats and rabbits: an immunofluorescence study of desmin and vimentin distribution.Histochem J. 1996; 28: 53-61Crossref PubMed Scopus (25) Google Scholar In this study, we introduce the desmin ensheathment ratio (DER), which is the relative occurrence of desmin to lectin labeling as a measure of vessel stability. Changes in the DER were determined during postnatal maturation of the cat retinal vasculature to test whether the DER correlated with attainment of vessel stability. To further validate the applicability of the DER as a measure of vascular stability, we applied it to a neovascularizing disease of the retina: ROP. In the kitten model of ROP, kittens are exposed to hyperoxia to produce vaso-obliteration. When the kittens are returned to room air, the absence of retinal vasculature produces massive tissue hypoxia resulting in aberrant neovascularization both within the retina and in the vitreous chamber (the vasoproliferative phase of kitten ROP).21Chan-Ling T Tout S Hollander H Stone J Vascular changes and their mechanisms in the feline model of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 1992; 33: 2128-2147PubMed Google Scholar, 22Stone J Chan-Ling T Pe'er J Itin A Gnessin H Keshet E Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 1996; 37: 290-299PubMed Google Scholar This neovasculature later recovers with substantial vessel regression and the re-establishment of the blood-retinal barrier coinciding with close ensheathment of the vessels by astrocytes.23Chan-Ling T Stone J Degeneration of astrocytes in feline retinopathy of prematurity causes failure of the blood-retinal barrier.Invest Ophthalmol Vis Sci. 1992; 33: 2148-2159PubMed Google Scholar DER was determined during the vasoproliferative and recovery phases of the kitten model of ROP as examples of vascular beds undergoing active angiogenesis and remodeling, respectively. Mural cell abnormalities have been implicated in the pathogenesis of diabetic retinopathy and atherosclerosis. We postulated that mural cells might also play a role in the pathogenesis of ROP. During normal formation of the retinal vasculature under the influence of physiological hypoxia,24Chan-Ling T Gock B Stone J The effect of oxygen on vasoformative cell division. Evidence that “physiological hypoxia” is the stimulus for normal retinal vasculogenesis.Invest Ophthalmol Vis Sci. 1995; 36: 1201-1214PubMed Google Scholar, 25Stone J Itin A Alon T Pe'er J Gnessin H Chan-Ling T Keshet E Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia.J Neurosci. 1995; 15: 4738-4747Crossref PubMed Google Scholar the newly formed vessels are closely ensheathed by astrocytes26Ling TL Stone J The development of astrocytes in the cat retina: evidence of migration from the optic nerve.Brain Res Dev Brain Res. 1988; 44: 73-85Crossref PubMed Scopus (103) Google Scholar and pericytes.27Penfold PL Provis JM Madigan MC van Driel D Billson FA Angiogenesis in normal human retinal development: the involvement of astrocytes and macrophages.Graefe's Arch Clin Exp Ophthalmol. 1990; 228: 255-263Crossref PubMed Scopus (64) Google Scholar, 28Ozerdem U Grako KA Dahlin-Huppe K Monosov E Stallcup WB NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.Dev Dyn. 2001; 222: 218-227Crossref PubMed Scopus (476) Google Scholar, 29Fruttiger M Development of the mouse retinal vasculature: angiogenesis versus vasculogenesis.Invest Ophthalmol Vis Sci. 2002; 43: 522-527PubMed Google Scholar Our earlier studies have demonstrated a significant role played by astrocytes in the pathogenesis of ROP,23Chan-Ling T Stone J Degeneration of astrocytes in feline retinopathy of prematurity causes failure of the blood-retinal barrier.Invest Ophthalmol Vis Sci. 1992; 33: 2148-2159PubMed Google Scholar but no previous studies of mural cell changes have been reported in ROP. In the retina, extensive SMA expression by mural cells has been shown to be associated with vessel stability.30Benjamin LE Hemo I Keshet E A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development. 1998; 125: 1591-1598Crossref PubMed Google Scholar, 31Benjamin LE Golijanin D Itin A Pode D Keshet E Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1057) Google Scholar The authors concluded that there was a window of vascular plasticity characterized by the lack of ensheathment by SMA+ pericytes. However more recent reports have shown SMA+ pericytes are also evident on unstable vessels in tumors32Morikawa S Baluk P Kaidoh T Haskell A Jain RK McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2002; 160: 985-1000Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, 33Abramsson A Berlin O Papayan H Paulin D Shani M Betsholtz C Analysis of mural cell recruitment to tumor vessels.Circulation. 2002; 105: 112-117Crossref PubMed Scopus (151) Google Scholar indicating that SMA expression alone is not an ideal indicator of vessel stability. Our recent results suggest that mural cells differentiate from a common precursor through a number of immature phenotypes to give rise to pericytes and SMCs in the developing rat retina. Immature mural cells were observed on angiogenic and regressing vessels, leading us to conclude that vessel stability is not conferred by the mere presence of immature mural cells but requires ensheathment by mature mural cells.3Hughes S Chan-Ling T Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.Invest Ophthalmol Vis Sci. 2004; 45: 2795-2806Crossref PubMed Scopus (160) Google Scholar We examined the mural cells during various stages of ROP to further our understanding of the role of mural cells in the pathogenesis of ROP. Mural cells also control blood flow. Clinically, this has implications for ROP with plus-disease in which retinal vessels are dilated and tortuous and are associated with a worse prognosis.34Anonymous Supplemental therapeutic oxygen for prethreshold retinopathy of prematurity (STOP-ROP), a randomized, controlled trial. I: primary outcomes.Pediatrics. 2000; 105: 295-310Crossref PubMed Scopus (708) Google Scholar To induce experimental hyperoxia, posnatal day (P) 1 kittens were placed with a lactating mother in a hyperoxic chamber (70 to 80% oxygen in air) for 4 days35Chan-Ling T Gock B Stone J Supplemental oxygen therapy. Basis for noninvasive treatment of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 1995; 36: 1215-1229PubMed Google Scholar and then returned to room air for 0, 3, 7, 10, 14, 23, 27, 34, or 40 days (dRA) until sacrifice. Littermate controls were raised in room air from birth for 1, 3, 6, 17, 28, 32, or 45 postnatal days until sacrifice. Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (33 mg/kg) and xylazine (1 mg/kg), perfused transcardially with 0.1 mol/L phosphate-buffered saline (PBS), pH 7.4 and 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Retinal whole mounts were prepared as described previously.36Chan-Ling T Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.Microsc Res Tech. 1997; 36: 1-16Crossref PubMed Scopus (98) Google Scholar Retinae for immunohistochemistry were immersion-fixed in 4% paraformaldehyde in phosphate buffer for 30 to 60 minutes at 4°C, permeabilized with 1% (v/v) Triton X-100 in PBS for 30 minutes, and then incubated for 30 minutes at room temperature with 1% bovine serum albumin in PBS. Dual labeling was used to co-visualize mural cells and the vasculature. Retinae were incubated overnight at 4°C with primary antibody, washed with 0.1% Triton X-100 in PBS, incubated for 4 hours at room temperature with the appropriate secondary antibody and washed again. Retinae were then incubated with biotinylated Griffonia simplicifolia lectin followed by labeled streptavidin. All antibodies were diluted with 1% bovine serum albumin in PBS, and 0.1% Triton X-100 in 0.1 mol/L PBS was used for all washes. Washed retinal whole mounts were mounted ganglion cell layer up in glycerol: PBS (2:1, v/v) or Prolong Anti Fade (Molecular Probes, Eugene, OR). To identify both mature and immature mural cells, antibodies against desmin and SMA were used. For desmin immunohistochemistry, we used mouse IgG1 monoclonal antibody (clone D33; DAKO, Carpinteria, CA) diluted 1 in 75. For SMA, we used a mouse IgG2a monoclonal (clone 1A4, Sigma-Aldrich, St. Louis, MO) diluted 1 in 75. To detect desmin labeling we used Texas Red-conjugated (rabbit) anti-mouse IgG1 secondary antibody (Southern Biotechnology Associates, Birmingham, AL) diluted 1 in 60. To detect SMA labeling we used a Texas Red (rabbit)-conjugated anti-mouse Ig secondary antibody (Amersham-Biosciences, Piscataway, NJ) diluted 1 in 50. Labeling with antibodies against desmin and S100 (an astrocyte-specific marker) confirmed that desmin+ cells are a distinct population to astrocytes (data not shown). The endothelium was labeled using biotinylated Griffonia simplicifolia lectin followed by streptavidin conjugated with fluorescein isothiocyanate (Amersham).36Chan-Ling T Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.Microsc Res Tech. 1997; 36: 1-16Crossref PubMed Scopus (98) Google Scholar, 37Chan-Ling T Halasz P Stone J Development of retinal vasculature in the cat: processes and mechanisms.Curr Eye Res. 1990; 9: 459-478Crossref PubMed Scopus (105) Google Scholar Fluorescently labeled retinal whole mounts were examined by confocal microscopy with a Leica argon-krypton laser mounted on a Leica Axiophot epifluorescence photomicroscope. Fluorescein isothiocyanate and Texas Red fluorescence were excited sequentially at 488 and 588 nm, respectively. The retina was divided arbitrarily into 12 sectors, akin to the 12 hours of a clock. Images were taken in 10 of the 12 sectors. In control cat retinae, regions of mature, remodeled vasculature were selected in the central retina, where the capillary plexus displayed an open capillary mesh with low capillary density and small capillary caliber (Figure 1A; Figure 2G, bottom box). Regions of immature vascular beds with high capillary density and large vessel caliber were captured, just proximal to the leading edge (Figure 1B; Figure 2G, top box). In ROP retinae, regions immediately proximal to the leading edge were selected for analysis. For each field of view selected for analysis, a desmin/G. simplicifolia lectin pair of images was generated. To preserve objectivity, areas captured were selected using the lectin (fluorescein isothiocyanate) channel only, with a ×40 objective. Further, the sequence of analysis was randomized. Each confocal image was overlaid with a 10 × 10 equally spaced grid using Adobe Photoshop V5.0. Figure 3 shows representative fields of view during normal development and ROP. The actual grid has been superimposed onto each image and the actual intersections with lectin and desmin present are shown with a white dot. Although the micrographs show a high resolution, the actual resolution obtained on screen was even higher as a 23-inch Apple studio display with a screen resolution of 1920 × 1200 pixels was used and each half of a field of view filled one entire screen during the actual counting process. The occurrence of desmin labeling relative to lectin labeling at the 100 intersection points yielded the DER. The DER as a function of postnatal age and recovery period in room air were analyzed and plotted using SigmaPlot.Figure 2A–F: Retinal vessels double labeled with lectin (green) and antibodies against SMA (red) showing maturation of SMCs on inner vascular plexus during normal development. The intensity, extent, and organization of the SMA filaments increased with maturation. Kittens were aged P3 (A, B), P17 (C, D), and P45 (E, F). A, C, and E: Central radial vessels. B: Immature capillaries at the leading edge of vessel formation. D and F: Peripheral plexuses. G and H: Schematic representation of SMA distribution in the retinal vascular plexus at P3 (G) and P17 (H) in the kitten. Typical locations for image capture are shown for actively angiogenic (white box in peripheral retina) and remodeled, mature (white box in central retina) plexuses. Scale bars: 100 μm (A); 500 μm (G, H).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Determination of the DER. The figure shows representative fields of view during normal development and ROP. The actual grid with 100 intersection points has been superimposed onto each image and the actual intersections with lectin and desmin present are shown with a white dot. The numbers on each field of view indicate the intersection points with positive label. A and B: An area just proximal to the leading edge of vessel formation in a P6 kitten retina representative of an immature vasculature in which the DER = 0.23. C and D: A central area in a P6 kitten retina representative of a remodeled, mature vasculature where the DER = 0.89. E and F: An area just proximal to the leading edge of the intraretinal neovasculature in a kitten subjected to 4 days of hyperoxia from birth followed by return to room air for 3 days (4dO23dRA) in which the DER = 0.5.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Retinae from control kittens aged P1, 3, 6, 32, and 45 and from ROP kittens at 0, 23, 34, and 40 dRA were examined with transmission electron microscopy. One preretinal membrane obtained during the recovery phase of ROP at 23 dRA was also examined. A sector including the optic nerve head was fixed by immersion in 4% paraformaldehyde at room temperature for 24 hours then transferred to 2% paraformaldehyde at 4°C for storage and transport. Paraformaldehyde-fixed retinas were postfixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2. Tissue blocks were treated with in 1% osmium tetroxide, dehydrated in ethanol, and embedded in Spurr's resin. Mural cells encompass a continuum of phenotypes from SMCs to pericytes that are characterized by a combination of cell-specific markers, morphology, and location on the vascular tree. By definition, pericytes were found on capillaries, whereas SMCs were found on arteries, arterioles, venules, and veins. In the kitten, pericytes were desmin +/SMA− whereas SMCs were desmin+/−/SMA+. Figure 1, A to H, shows the changes in desmin ensheathment of retinal vessels and vessel morphology in the central retina (Figure 1; A, C, E, G) and at the leading edge of vessel formation (Figure 1; B, D, F, H). Desmin+ pericytes were found from birth throughout the vascular plexus, including newly formed vessels at the leading edge of vessel formation as well as on major vessels. Their processes ranged from bipolar through to stellate in appearance. SMA+ SMCs were evident on radial arteries and veins, arterioles, and venules at P3 (Figure 2; A, B, G). At this stage arteries were narrower and more strongly labeled with SMA than were veins, and SMA staining was amorphous, lacking filamentous structure. With maturation, SMA labeling became more widespread, evident on secondary and tertiary arterioles (Figure 2H). From P17 to P45, SMA labeling increased in intensity and revealed organized circumferentially oriented filaments on arteries and distinct stellate processes on veins (Figure 2; C to F) indicative of arterial and venous SMC maturation. SMC differentiation began on the radial arteries and veins, spread to the arterioles and venules, and matured with a proximal-distal topography. Our determination of the DER based on the relative occurrence of desmin and G. simplicifolia lectin labeling was examined on both immature actively angiogenic and mature, remodeled vascular beds during retinal development in the kitten. In the immature vascular beds at the leading edge of vessel formation, the DER was between 0.3 and 0.6 until P17, before increasing between P17 and P28, reaching 0.97 at P28, and remained greater than 0.9 thereafter (Figure 4A, filled circles). In contrast, in the central regions of the retina where the vascular bed had already undergone substantial remodeling, the DER was 0.85 at P6, reached 0.97 at P28, and remained greater than 0.9 until the last measurement at P45 (Figure 4A, open circles). By the end of the fourth postnatal week the peripheral DER reached that of the central vasculature. The convergence of central and peripheral DER at P28 was consistent with the morphological appearance of vascular maturity at this age. Before kittens were introduced into hyperoxia, desmin+ filaments were already present on virtually all vessels including capillaries at the leading edge of vessel formation and immature SMA+ SMCs were evident on radial arteries and veins (Figure 4, B and C). Despite this extensive ensheathment by mural cells, exposure to 70 to 80% oxygen for 4 days resulted in obliteration of the retinal vasculature including radial vessels. Thus, immature pericytes and SMCs did not prevent hyperoxia-induced vaso-obliteration. After 4 days of exposure to hyperoxia from P1, extensive vascular obliteration occurred, and lectin-labeled vascular remnants were scattered in the central retina (Figure 4D). Weakly labeled desmin processes persisted on some vascular remnants, but SMA was not detected (Figure 4D). On return to room air, a circular multilayered neovascular front formed, centered on the optic disk. Formation of the neovasculature took place in predominantly two layers: a dense superficial capillary layer and a deeper radial plexus with major vessels apparent after 3 dRA. During the early vasoproliferative phase the endothelial cells were characterized by an abnormal rounded morphology (Figure 4, E and F). Desmin+ pericytes were present throughout the neovascular plexus; however, their occurrence was decreased relative to the abnormally dense endothelium seen in the vasoproliferative phase of ROP (Figure 4; E to G). During the recovery phase between 27 dRA and 34 dRA vascular density decreased, the endothelial cells acquired a more elongated morphology and endothelial and mural cells adopted their normal relationship as seen in remodeled vasculature during normal development (Figure 4H). Large numbers of radial vessels were seen in the deep layers of the retinal neovasculature. Despite the aberrant nature of the neovasculature, SMA immunoreactivity was similar to that observed during normal development, revealing a distinction in labeling between arteries and veins (Figure 5, A–B). As the leading edge of neovascularization approached the edge of the retina at 10 dRA, a lag of ∼300 to 400 μm between the neovascularization front and the appearance of SMA immunoreactivity was observed on radial vessels. The magnitude of this lag was similar to that observed during normal development (Figure 5B and Figure 2H). During the recovery phase, the proximal-dis
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