HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:135-140 (2008)
© 2008 The Gerontological Society of America

Normal Aging Involves Altered Expression of Growth Factors in the Rat Choroid

Jena J. Steinle, Sheena Sharma and Vannak C. Chin

1 Department of Ophthalmology, University of Tennessee Health Science Center, Memphis.
2 Department of Physiology, Southern Illinois University School of Medicine, Carbondale.

Address correspondence to Jena J. Steinle, PhD, Assistant Professor, Department of Ophthalmology, University of Tennessee Health Science Center, 930 Madison Ave., Suite 722A, Memphis, TN 38163. E-mail: jjsteinle{at}utmem.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Normal aging of the choroid results in morphological and physiological changes. The growth factors that mediate these changes are unclear. Vascular endothelial growth factor (VEGF) binds its receptor 2, VEGFR2, to mediate vascular remodeling. Pigment epithelium-derived factor (PEDF) is an inhibitor of angiogenesis produced by retinal pigmented epithelial (RPE) cells. Angiopoietin1 (Ang-1) binds its receptor Tie-2 to recruit mural cells to stabilize vessels. To investigate age-related changes in growth factor activities in aged choroidal vasculature, real-time polymerase chain reaction and protein analysis of VEGF, VEGFR2, PEDF, Ang-1, and Tie-2 were completed on rat choroid/RPE complexes at 8, 22, and 32 months. VEGF messenger RNA (mRNA) peaked at 22 months, whereas protein levels were significantly decreased by 32 months. Both mRNA and protein levels of PEDF were significantly decreased with age. Ang-1 protein levels were not altered, whereas Tie-2 had increased protein levels with age. These results indicate that normal aging involves temporal changes in many of the growth factors common in age-related disease.

Key Words: Aging • Choroid • Growth factors

AGE-RELATED macular degeneration (AMD) is the leading cause of blindness in the elderly (1). There are two forms of AMD: dry and wet. Dry macular degeneration occurs in a larger number of individuals and is characterized by the presence of an accumulation of drusen (2). Although both forms of macular degeneration can threaten vision, the "wet" form involves changes in angiogenic growth factors, as the choroidal blood vessels undergo extensive neovascularization (3). The stimulating factors for this vascular remodeling are relatively established, with evidence associating changes in vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) with macular degeneration (4,5).

Whereas some of the growth factors involved in macular degeneration are clear, the changes in growth factors within the choroid that occur during the normal aging process are much less so. Several structural changes occur in the choroid and retinal pigmented epithelium (RPE) with age. Data have shown that the total thickness of Bruch's membrane, the membrane that separates the choriocapillaris from the RPE, is substantially increased with age, whereas the total choroidal thickness was decreased by 57% in normal aged human samples (6). No differences in Bruch's membrane thickening and choroidal thickness were noted between normal aged human samples and samples from patients with advanced AMD (6). Substantial thickening of Bruch's membrane has also been reported in Fisher 344 rats (an aging rat colony) (7). In this same study, RPE cells were noted to have significantly more membrane inclusions, as well as reduced photoreceptor numbers (7), in 30-month-old as compared to 6-month-old rats. Using the senescence-accelerated mouse, Majji and colleagues (8) found that similar increases in Bruch's membrane thickness and choroid/RPE complex were observed as noted in human samples. These results all suggest that the morphological changes that occur during aging of the choroid/RPE complex in humans and rodents are similar, even though rodents do not possess a macula.

In addition to morphological changes in aging of the choroid, Ito and colleagues (9) found that the choroidal vasculature filled much more slowly with dye and noted that the number of choroidal arterioles and fluorescent intensity of the macula were reduced. Other groups have observed that this delayed perfusion of the choroid in humans is associated with discrete elevated scotopic threshold, suggesting that the altered blood flow may affect visual processing (10). In addition, laser Doppler flowmetry measurements from both humans and pigeons suggest that choroidal blood flow is substantially reduced with increasing age (11–13). Grunwald and colleagues (14) have also reported that choroidal blood flow is reduced in human eyes with increasing AMD severity. Thus, it appears that normal aging of the choroid/RPE complex is associated with a number of both physiological and morphological changes.

In addition to the morphological and physiological changes that occur in both normal aging of the choroid/RPE complex and in macular degeneration, there are some known alterations of growth factor expression in normal aging of the eye and in AMD.

The growth factors and receptors to be investigated in this study are VEGF, VEGFR2, pigment epithelium-derived factor (PEDF), Ang-1, and Tie-2. VEGF is a potent angiogenic vasopermeability factor mitogen that is expressed by endothelial cells involved in promoting proliferation, migration, and vascular formation (15). VEGF shows preferential binding to flt-1 (VEGFR1) and flk-1/VEGFR2 (16). Binding of VEGF to one of its receptors will lead to the change of endothelial cells from a dormant to an active state. PEDF is produced by the RPE cells and is a well-known endogenous inhibitor of angiogenesis (17,18). In contrast to VEGF, VEGFR2, and PEDF, which are involved in modulating endothelial cell proliferation, Ang-1 is known to bind to its receptor, Tie-2, and primarily aid in new vessel stabilization (19,20). In normal human aged choroid/RPE complexes, intense staining for both VEGF and PEDF were observed. The staining for PEDF was reduced in samples from patients with AMD (21). Similarly, changes in gene expression for VEGF, VEGFR2, and PEDF are altered with age in human RPE and choroidal samples using microarray analysis (22). Others have also found PEDF levels to be decreased with normal aging in the aqueous humor of humans (23) and in human skin (24). Based on the use of anti-VEGF therapeutics for macular degeneration, it is clear that VEGF levels are elevated in AMD. Little is known about changes in VEGF levels with normal aging in the choroid. Previous work has shown that normal aging of mouse skeletal muscle (25), rat cardiac myocytes (26), and human skeletal muscle (27) is associated with reduced VEGF expression. Wagatsuma (25) also found that messenger RNA (mRNA) for Ang-1 was unchanged in mouse skeletal muscle, whereas mRNA for Tie-2 was decreased. Therefore, it is clear that growth factor expression can be substantially altered during aging; however, the time course of these changes is unknown.

The hypothesis of the present work was that normal aging of the choroid/RPE complex involves increases in VEGF and Ang-1 expression, with a concurrent decrease in expression of PEDF. The present work adds to current knowledge of the progression of growth factor changes in the choroid/RPE complex, using young, middle-aged, and aged rats.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Experimental Animals
Male Fisher 344 x Brown Norway F1 hybrid (F344xBN F1 hybrid) rats age 8 months, 22 months, and 32 months purchased from the National Institute on Aging (NIA) through Harlan were used to determine changes in growth factor expression in the choroid/RPE complex with age. These three ages were used to represent young, middle aged, and old, as determined by the age curves for these animals (28). Currently, the NIA recommends and maintains three rat strains for aging studies: the F344, the BN, and the F344xBN F1 hybrid (29). Males from the F344xBN F1 hybrid rat strain were used because they show fewer age-related pathologies and less biological variability (30). At the appropriate age, rats were anesthetized using pentobarbital (150 mg/kg), and the eyes were removed. The cornea was cut and the lens and vitreous removed. The choroid/RPE complex was separated from the retina and placed into separate tubes for RNA isolation and protein analysis. All surgical procedures were approved by the Institutional Animal Care and Use Committee at Southern Illinois University and conform to National Institutes of Health (NIH) guidelines.

RNA Isolation and Reverse Transcription
RNA was isolated from choroid/RPE complex samples from the rats at each age in TriReagent (Molecular Research Center, Inc., Cincinnati, OH). RNA was isolated using chloroform and isopropanol. RNA purity was detected by agarose gel electrophoresis, and RNA concentration was measured spectrophotometrically. RNA analysis, reverse transcription (RT), and real-time polymerase chain reaction (PCR) were done as described previously (31,32). Primer sequences for the growth factors investigated can be found in Table 1. All mRNA data are normalized to 18s ribosomal RNA.


View this table:
[in this window]
[in a new window]

  		Table 1. Primers Used in Real-Time Polymerase Chain Reaction Experiments.

 
Protein Electrophoresis and Western Blot Analysis
Eyes were extracted at the appropriate age. The lens, vitreous, and retina were removed, and the choroid/RPE complex was placed into cold lysis buffer (50 mM Tris–HCl, pH 7.4; 1% Nonidet P-40, 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM phenylmethanesulfonylfluoride; aprotinin, leupeptin, and pepstatin, each at 1 µg/mL; 1 mM Na3VO4; 1 mM NaF; 0.1% sodium dodecyl sulfate) for homogenization. Homogenization was done using a PowerGen Model 1000 mechanical homogenizer (Fisher Scientific, Pittsburgh, PA). Samples were homogenized at room temperature for 2–3 minutes. Following homogenization, samples were centrifuged at 10,000g for 5 minutes and the supernatant was collected, assayed for protein content, and then stored at –80°C. Equal protein was loaded into each well based on the results from the protein assay. Western blot analysis was done as described previously (31). Primary polyclonal antibodies to PEDF (diluted 1:1000; Bioproducts, Middletown, MD), VEGFR2 (diluted 1:500; Chemicon, Temecula, CA), Ang-1 (diluted 1:1000; Alpha Diagnostic, San Antonio, TX), and Tie-2 (diluted 1:1000; Chemicon) were applied for 2 hours at room temperature. Membranes were probed with horseradish peroxidase–conjugated anti-rabbit secondary antibodies (Promega, Madison, WI) applied at a 1:10,000 dilution at room temperature for 2 hours. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, U.K.) and analyzed using the Kodak 2000R image station. Mean densitometry was assessed using Kodak software, and results are expressed as a percentage of 8-month-old rats. A one-way analysis of variance was performed, and significance was accepted at p <.05. Equal lane loading was verified using Ponceau S staining.

Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) analysis for VEGF (RayBiotech, Norcross, GA) was done according to manufacturer's instructions, except that equal protein concentrations were loaded into each well. Analysis was done according to the optical density measurement obtained from the plate reader.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Protein Levels of VEGF Were Significantly Decreased in the Aged Choroid/RPE Complex
Steady-state mRNA expression for VEGF was significantly increased in the choroid/RPE complex of rats at age 22 months as compared to those at 8 months of age (Figure 1A, p <.05 vs 8-month-old rats). Gene expression returned to close to baseline levels by 32 months of age. Protein levels of VEGF were significantly reduced in the choroid/RPE complex at 32 months of age relative to 8 months, whereas a significant increase was observed at 22 months as compared to 8 months (Figure 1B, p <.05 vs 8-month-old rats). Five rats were used for RNA work, and four were used for ELISA analysis.


Figure 01
View larger version (38K):
[in this window]
[in a new window]

  		Figure 1. Steady-state messenger RNA (mRNA) (A) and protein (B) expression of vascular endothelial growth factor (VEGF) in the 8-, 22-, and 32-month-old rat choroid/retinal pigmented epithelium (RPE) complex. Gene expression was significantly increased at 22 months as compared to 8 months. Protein expression was significantly increased at 22 months, but significantly reduced at 32 months as compared to 8 months. n = 5 for gene expression experiments and n = 4 for enzyme-linked immunosorbent assay analysis, *p <.05. O.D. = optical density

 
Gene Expression of the Key Human VEGF Receptor, VEGFR2, Is Downregulated with Age
Significant reduction was noted in steady-state mRNA expression for VEGFR2 in the aged choroid/RPE complex (Figure 2A, p <.05 vs 8-month-old rats); however, there were no significant differences in the protein levels of VEGFR2 (Figure 2B). Four animals were used for Western blot analysis, and five were used for real-time PCR.


Figure 02
View larger version (29K):
[in this window]
[in a new window]

  		Figure 2. Gene (A) and protein (B) expression for VEGFR2 (human VEGF receptor 2) in 8-, 22-, and 32-month-old rat choroid/retinal pigmented epithelium (RPE) complex. Gene expression for VEGFR2 was significantly decreased with age, with no change in protein expression. n = 5 for real-time polymerase chain reaction and n = 4 for Western blot analysis, *p <.05. mRNA = messenger RNA

 
Gene Expression and Protein Levels for PEDF Are Reduced in Aging
Both gene expression and protein levels of PEDF were significantly reduced at 32 months as compared to the other age groups (p <.05, Figure 3A and B). These results suggest that normal aging may involve changes in angiostatic factors. Five animals were used for both real-time PCR and Western blot analysis.


Figure 03
View larger version (34K):
[in this window]
[in a new window]

  		Figure 3. Gene (A) and protein (B) expression of pigment epithelium-derived factor (PEDF) in 8-, 22-, and 32-month-old rat choroid/retinal pigmented epithelium (RPE) complex. Both gene expression and protein expression were significantly reduced with increasing age. n = 5 for both real-time polymerase chain reaction and Western blot analysis, *p <.05. mRNA = messenger RNA

 
Ang-1 Protein Levels Are Not Altered with Increasing Age
Normal aging did not appear to alter protein levels of Ang-1 (Figure 4B). However, there was a significant increase in steady-state mRNA expression for Ang-1 at 22 months of age (Figure 4A, p <.05 vs 8-month-old rats) that returned to baseline levels at 32 months of age. Tissues from five animals were used for gene studies, whereas four animals were used for protein work.


Figure 04
View larger version (36K):
[in this window]
[in a new window]

  		Figure 4. Gene (A) and protein (B) expression for angiopoietin 1 (Ang-1) in 8-, 22-, and 32-month-old rat choroid/retinal pigmented epithelium (RPE) complex. Gene expression for Ang-1 was increased at 22 months as compared to 8 months, but no differences in protein expression were noted. n = 5 for gene expression and n = 4 for protein analysis, *p <.05. mRNA = messenger RNA

 
Whereas No Changes in Ang-1 Protein Levels Were Noted, There Was a Significant Increase in Protein Levels for Tie-2
Gene expression for Tie-2 was not altered in the choroid/RPE complex with increasing age (Figure 5A). Protein levels, however, were significantly increased in the aged (22 and 32 month) choroid/RPE complex (Figure 5B, p <.05 vs 8-month-old rats). Four animals were used for Western blot analysis, and five were used for real-time PCR.


Figure 05
View larger version (33K):
[in this window]
[in a new window]

  		Figure 5. Gene (A) and protein (B) expression for Tie-2 in 8-, 22-, and 32-month-old rat choroid/retinal pigmented epithelium (RPE) complex. Protein levels of Tie-2 were significantly increased with age. n = 5 for gene expression analysis; n = 4 for protein analysis, *p <.05. mRNA = messenger RNA

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
With an ever-increasing aging population, understanding of the normal aging process in various organs becomes essential. Because normal aging of the eye can involve a loss of night vision and in some people can progress to macular degeneration, it is imperative to determine the factors that are present in normal aging, in order to distinguish normal aging from those factors that are part of a disease process.

One of the predominant factors observed in macular degeneration is an elevated VEGF level (33), so a number of current therapies are targeted at blocking VEGF activities in the choroid (34,35). Although it is clear that patients with AMD have increased VEGF expression, it is not known whether normal aging produces similar changes in VEGF in the choroid. Results from the present study suggest that VEGF protein levels were decreased in the choroid of rats with increasing age (Figure 1B), which is opposite of what is observed in macular degeneration. The observation that VEGF protein levels are reduced with age suggests that diseases such as macular degeneration may induce an increase in VEGF expression, potentially due to ischemia because normal aging reduces choroidal blood flow (11,12). In addition, others have reported that altered perimetry does occur due to loss of choroidal perfusion (10). In this manner, the loss of VEGF in response to the decreased choroidal blood flow may be a physiological response and may be an attempt to restore visual processing. These ocular findings for reduced VEGF protein levels in aging are in agreement with those reported for mouse skeletal muscle (25) and rat cardiac myocytes (26).

In many tissues, changes in VEGF expression occur with concurrent changes in expression of PEDF (36,37). In the present study, this also occurs, because both gene expression and protein levels of PEDF were reduced in the choroid/RPE complex with age. Others have found that PEDF can alter VEGF expression or the effects of VEGF stimulation (38,39). Recently in the choroid, Bhutto and colleagues (21) found that normal aging was associated with high VEGF and PEDF staining in the RPE–Bruch's membrane–choriocapillaris complex in human samples. In the work by Bhutto and colleagues (21), no comparisons were made with samples from younger patients, which may explain the differences noted for PEDF protein levels between their study and ours. The normal aging process in the rat choroid seems to involve significant decreases in VEGF and PEDF protein levels. It may be that the decrease in PEDF or VEGF protein levels is the critical switch from normal aging to a disease of aging.

In addition to changes in PEDF and VEGF levels, the present work also investigated changes in Ang-1 and its receptor Tie-2. Previous reports have suggested that immunohistochemical staining of the choroid from macular degeneration patients shows expression of Ang-1 with little staining for Tie-2 (40). Others have shown that human choroidal neovascular membranes have intense staining for Tie-2 and Ang-1 (3). Reports from healing skin wounds and during hormonally stimulated follicular maturation indicate that Tie-2 expression is upregulated in cases of angiogenesis; however, Tie-2 expression can also be noted in normal quiescent adult tissues (41). Others have also reported that members of the Tie receptor family are important for adult hematopoiesis (42). Results from the present study suggest that normal aging of the choroid/RPE complex is associated with limited changes in Ang-1 protein expression, but a noted increase in Tie-2 expression, which matches well the results of mouse skeletal muscle (25). The potential reasons for the significantly increased Ang-1 mRNA expression with no changes in protein levels are unknown, but may suggest that the mRNA is unstable or needs additional post-transcription modifications that are not occurring. Alternatively, Ang-1 may be bound or located in the extracellular matrix, which may prevent detecting changes in protein levels by Western blot analysis. Nonetheless, the finding of increased Tie-2 protein with limited changes in Ang-1 protein may suggest that the normal aged choroidal vasculature is stable, but has the ability to remodel as needed, as noted by the increased expression of Tie-2 receptors.

Further investigations into the regulation of growth factor expression with age should be conducted to better understand the difference between normal aging of the choroid/RPE complex and disease states. One of the discrepancies noted in this work is the lack of correlation between mRNA expression and protein levels in the choroid with age. It is unclear whether the altered mRNA expression cannot be translated to protein or is degraded. Alternatively, it may be something in the cellular milieu that leads to increased protein levels without increased mRNA expression. In addition, although a protein assay was done for all samples to insure equal protein loading, it is possible that a given blot may have proteins from a different region of the choroid than another immunoblot has. Because the choroid does have some noted regional differences, this may explain some of the discrepancies. However, it would be very difficult to obtain enough protein samples from each region to be able to run multiple Western blot or ELISA analyses.

Overall, these findings suggest that Tie-2 levels are increased and PEDF and VEGF protein levels are decreased in the choroid/RPE complex during the normal aging process, with little change in Ang-1 levels. These findings suggest that diseases of aging may lead to an upregulation of VEGF, such that neovascularization in macular degeneration can proceed in the choroid. Expanding our knowledge of the temporal changes in the normal aging process of the choroid/RPE complex will greatly help in determining normal aging versus diseases of aging.


   Acknowledgments
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by National Institute on Aging grant R01AG027827 (to J.J.S.).


   Footnotes
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received April 20, 2007

Accepted October 15, 2007


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. O'Shea JG. Age-related macular degeneration. Postgrad Med J. 1998;74:203-207.[Abstract/Free Full Text]
  2. Anderson DH, Ozaki S, Nealon M, et al. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol. 2001;131:767-781.[Medline]
  3. Otani A, Takagi H, Oh H, Koyama S, Matsumura M, Honda Y. Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1999;40:1912-1920.[Abstract/Free Full Text]
  4. Holz FG, Pauleikhoff D, Klein R, Bird AC. Pathogenesis of lesions in late age-related macular disease. Am J Ophthalmol. 2004;137:504-510.[Medline]
  5. Nowak JZ. Age-related macular degeneration (AMD): pathogenesis and therapy. Pharmacol Rep. 2006;58:353-363.[Medline]
  6. Ramrattan RS, van der Schaft TL, Mooy CM, de Bruijn WC, Mulder PG, de Jong PT. Morphometric analysis of Bruch's membrane, the choriocapillaris, and the choroid in aging. Invest Ophthalmol Vis Sci. 1994;35:2857-2864.[Abstract/Free Full Text]
  7. Fan W, Lin N, Sheedlo HJ, Turner JE. Muller and RPE cell response to photoreceptor cell degeneration in aging Fischer rats. Exp Eye Res. 1996;63:9-18.[Medline]
  8. Majji AB, Cao J, Chang KY, et al. Age-related retinal pigment epithelium and Bruch's membrane degeneration in senescence-accelerated mouse. Invest Ophthalmol Vis Sci. 2000;41:3936-3942.[Abstract/Free Full Text]
  9. Ito YN, Mori K, Young-Duvall J, Yoneya S. Aging changes of the choroidal dye filling pattern in indocyanine green angiography of normal subjects. Retina. 2001;21:237-242.[Medline]
  10. Chen JC, Fitzke FW, Pauleikhoff D, Bird AC. Functional loss in age-related Bruch's membrane change with choroidal perfusion defect. Invest Ophthalmol Vis Sci. 1992;33:334-340.[Abstract/Free Full Text]
  11. Fitzgerald ME, Tolley E, Frase S, et al. Functional and morphological assessment of age-related changes in the choroid and outer retina in pigeons. Vis Neurosci. 2001;18:299-317.[Medline]
  12. Grunwald JE, Hariprasad SM, DuPont J. Effect of aging on foveolar choroidal circulation. Arch Ophthalmol. 1998;116:150-154.[Abstract/Free Full Text]
  13. Dallinger S, Findl O, Strenn K, Eichler HG, Wolzt M, Schmetterer L. Age dependence of choroidal blood flow. J Am Geriatr Soc. 1998;46:484-487.[Medline]
  14. Grunwald JE, Metelitsina TI, Dupont JC, Ying GS, Maguire MG. Reduced foveolar choroidal blood flow in eyes with increasing AMD severity. Invest Ophthalmol Vis Sci. 2005;46:1033-1038.[Abstract/Free Full Text]
  15. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci. 2001;22:201-207.[Medline]
  16. Joussen AM, Lemmen KD, Kirchhof B. Diabetic maculopathy. Etiological mechanisms and possible treatment approaches. Ophthalmologe. 2001;98:908-918; quiz 919, 921 [In German].[Medline]
  17. Holekamp NM, Bouck N, Volpert O. Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to age-related macular degeneration. Am J Ophthalmol. 2002;134:220-227.[Medline]
  18. Ogata N, Wada M, Otsuji T, Jo N, Tombran-Tink J, Matsumura M. Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;43:1168-1175.[Abstract/Free Full Text]
  19. Hayes AJ, Huang WQ, Mallah J, Yang D, Lippman ME, Li LY. Angiopoietin-1 and its receptor Tie-2 participate in the regulation of capillary-like tubule formation and survival of endothelial cells. Microvasc.Res. 1999;58:224-237.
  20. Papapetropoulos A, Garcia-Cardena G, Dengler TJ, Maisonpierre PC, Yancopoulos GD, Sessa WC. Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest. 1999;79:213-223.[Medline]
  21. Bhutto IA, McLeod DS, Hasegawa T, et al. Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration. Exp Eye Res. 2006;82:99-110.[Medline]
  22. Kociok N, Joussen AM. Varied expression of functionally important genes of RPE and choroid in the macula and in the periphery of normal human eyes. Graefes Arch Clin Exp Ophthalmol. 2007;245:101-113.[Medline]
  23. Ogata N, Matsuoka M, Imaizumi M, Arichi M, Matsumura M. Decrease of pigment epithelium-derived factor in aqueous humor with increasing age. Am J Ophthalmol. 2004;137:935-936.[Medline]
  24. Francis MK, Appel S, Meyer C, Balin SJ, Balin AK, Cristofalo VJ. Loss of EPC-1/PEDF expression during skin aging in vivo. J Invest Dermatol. 2004;122:1096-1105.[Medline]
  25. Wagatsuma A. Effect of aging on expression of angiogenesis-related factors in mouse skeletal muscle. Exp Gerontol. 2006;41:49-54.[Medline]
  26. Iemitsu M, Maeda S, Jesmin S, Otsuki T, Miyauchi T. Exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in hearts. Am J Physiol Heart Circ Physiol. 2006;291:H1290-H1298.[Abstract/Free Full Text]
  27. Croley AN, Zwetsloot KA, Westerkamp LM, et al. Lower capillarization, VEGF protein, and VEGF mRNA response to acute exercise in the vastus lateralis muscle of aged vs. young women. J Appl Physiol. 2005;99:1872-1879.[Abstract/Free Full Text]
  28. Miller RA, Nadon NL. Principles of animal use for gerontological research. J Gerontol Biol Sci. 2000;55A:B117-B123.[Abstract/Free Full Text]
  29. DiLoreto D, Jr, Cox C, Grover DA, Lazar E, del Cerro C, del Cerro M. The influences of age, retinal topography, and gender on retinal degeneration in the Fischer 344 rat. Brain Res. 1994;647:181-191.[Medline]
  30. Phelan JP, Austad SN. Selecting animal models of human aging: inbred strains often exhibit less biological uniformity than F1 hybrids. J Gerontol. 1994;49:B1-B11.
  31. Smith CP, Sharma S, Steinle JJ. Age-related changes in sympathetic neurotransmission in rat retina and choroid. Exp Eye Res. 2007;84:75-81.[Medline]
  32. Wiley LA, Berkowitz BA, Steinle JJ. Superior cervical ganglionectomy induces changes in growth factor expression in the rat retina. Invest Ophthalmol Vis Sci. 2006;47:439-443.[Abstract/Free Full Text]
  33. Matsuoka M, Ogata N, Otsuji T, Nishimura T, Takahashi K, Matsumura M. Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy. Br J Ophthalmol. 2004;88:809-815.[Abstract/Free Full Text]
  34. Spaide RF. Rationale for combination therapies for choroidal neovascularization. Am J Ophthalmol. 2006;141:149-156.[Medline]
  35. D'Amico DJ, Masonson HN, Patel M, et al. Pegaptanib sodium for neovascular age-related macular degeneration: two-year safety results of the two prospective, multicenter, controlled clinical trials. Ophthalmology. 2006;113:992-1001.[Medline]
  36. Kim SY, Mocanu C, McLeod DS, et al. Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid. Exp Eye Res. 2003;77:433-445.[Medline]
  37. Ohno-Matsui K, Yoshida T, Uetama T, Mochizuki M, Morita I. Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells. Biochem Biophys Res Commun. 2003;303:962-967.[Medline]
  38. Takenaka K, Yamagishi S, Jinnouchi Y, Nakamura K, Matsui T, Imaizumi T. Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells. Life Sci. 2005;77:3231-3241.[Medline]
  39. Duh EJ, Yang HS, Suzuma I, et al. Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth. Invest Ophthalmol Vis Sci. 2002;43:821-829.[Abstract/Free Full Text]
  40. Hera R, Keramidas M, Peoc'h M, Mouillon M, Romanet JP, Feige JJ. Expression of VEGF and angiopoietins in subfoveal membranes from patients with age-related macular degeneration. Am J Ophthalmol. 2005;139:589-596.[Medline]
  41. Wong AL, Haroon ZA, Werner S, Dewhirst MW, Greenberg CS, Peters KG. Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues. Circ Res. 1997;81:567-574.[Abstract/Free Full Text]
  42. Puri MC, Bernstein A. Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis. Proc Natl Acad Sci U S A. 2003;100:12753-12758.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS