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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Invest Ophthalmol Vis Sci. Author manuscript; available in PMC 2010 June 1.
Published in final edited form as:
PMCID: PMC2754868

Biologically Active Fibronectin Fragments Stimulate Release of MCP-1 and Catabolic Cytokines from Murine Retinal Pigment Epithelium



High-temperature requirement serine protease (HTRA1) was identified as a candidate age-related macular degeneration gene in multiple genetic studies in humans. To date, no functional studies have shown a mechanism for HTRA1 to instigate ocular tissue abnormalities. In the present study, the authors focused on a substrate of HTRA1, fibronectin, because fibronectin fragments (Fnfs) stimulate biochemical events in other age-related degenerative diseases that are analogous to changes associated with age-related macular degeneration (AMD). The purpose of the study was to determine whether Fnfs stimulate the release of proinflammatory and catabolic cytokines from murine retinal pigment epithelium (RPE).


Fibronectin was purified from murine serum by gelatin cross-linked agarose chromatography and subsequently was enzymatically digested with α-chymotrypsin. The bioactivity of Fnfs was verified by measuring levels of IL-6 and TNF-α in Fnf-exposed murine splenocytes. To analyze the effect of Fnfs on RPE, cytokine and chemokine levels in RPE culture supernatants were assayed by ELISA.


IL-6 and TNF-α proinflammatory cytokines were released from primary murine splenocytes in proportion to the dose and length of Fnf treatment, indicating that α-chymotryptic digests of fibronectin are biologically active. Fnf treatment of murine RPE cells stimulated the release of microgram and nanogram levels of IL-6, MMP-3, MMP-9, and MCP-1, whereas only picogram levels were detected in untreated cells.


Fnfs stimulate the release of proinflammatory cytokines, matrix metalloproteinases, and monocyte chemoattractant protein from murine RPE cells. This observation indicated that Fnfs could contribute to ocular abnormalities by promoting inflammation, catabolism, and monocyte chemoattraction.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in patients older than 50.1 As the baby boomer generation ages, the incidence of AMD in the United States is expected to increase to 3 million.2 Despite knowledge about the clinical and pathologic features of AMD, little is known about its underlying cause or causes. Previous attempts to identify an underlying cause for AMD have included genetic studies. More specifically, multiple groups have screened humans with AMD for evidence of restriction fragment length polymorphisms.

High-temperature requirement serine protease (HTRA1) polymorphisms have been strongly associated with AMD in diverse human populations.3-30 In human eyes taken from persons with AMD, HTRA1 levels are upregulated in the macular region,6 yet no functional studies have mechanistically linked alterations in HTRA1 expression with ocular changes. Substrates of HTRA1 are extracellular matrix proteins, including fibronectin.

Fibronectin is present in Bruch’s membrane, underlying the retinal pigment epithelium (RPE), where it anchors the RPE to the basal lamina through α5β1 integrins.31 Fibronectin is more abundantly expressed by the RPE of donors with AMD than of age-matched controls,32 and fibronectin accumulates in basal linear deposits in the macula of elderly eyes.33 Furthermore, fibronectin is abundant in drusen,34 defined as the abnormal accumulation of extracellular deposits in the basal aspect of the RPE, which is a risk factor for AMD.35,36 We were intrigued by the observation that recombinant HTRA1 can directly digest fibronectin to produce fibronectin fragments (Fnfs)37,38 and reasoned that the Fnfs could be a downstream protagonist of intraocular inflammation and catabolism.

Fnfs have been studied extensively in age-related inflammatory diseases outside the eye. It is established that intact fibronectin is capable of binding to many different types of receptors on a diverse range of cells, including fibroblasts, epithelium, endothelium, neural crest, B and T lymphocytes, monocytes, and megakaryocytes. Furthermore, it is known that Fnfs are more potent stimulators of proinflammatory and catabolic cytokines than intact fibronectin. One of the best-studied inflammatory diseases of aging, in which Fnfs play a central role, is arthritis. Two well-studied examples are osteoarthritis and rheumatoid arthritis. Specifically, Fnfs accumulate in synovial fluid in arthritic joints.39-43 The accumulation of Fnfs causes pathology through multiple biochemical mechanisms, including increased release of matrix metalloproteinases44-49 and enhanced accumulation of proinflammatory cytokines, among them IL-6 and TNF-α.33,44,50,51 Given that AMD is also associated with increased levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines, Fnfs may contribute to these changes.

Investigators have recently started to consider that drusen accumulation may be secondary to RPE abnormalities involving chronic inflammation.52-57 Therefore, we designed our study to examine the impact of Fnfs on the secretion of proinflammatory and catabolic cytokines from the RPE by testing the hypothesis that exposure of murine RPE cells to high levels of Fnfs stimulates the RPE to release catabolic and proinflammatory cytokines. We show that Fnf treatment of murine RPE cells causes enhanced secretion of IL-6, MCP-1, MMP-3, and MMP-9.


Primary Cell Culture

To establish primary cell cultures, C57BL/6 mice (age range, 3-6 months) were euthanatized by CO2 asphyxiation. Animal care was conducted in compliance with National Institutes of Health guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Unless specified otherwise, culture reagents were from Invitrogen/Gibco (Carlsbad, CA). Primary cell cultures were incubated at 37°C in 5% CO2.


Spleens were removed from C57BL/6 mice after euthanatization. The connective tissue capsule was removed, and cells were gently suspended in medium with a Pasteur pipette. The cell suspension was pushed through a 70-μM cell strainer (BD Biosciences, Bedford, MA). Cells were clarified by centrifugation (300 rcf [relative centrifugal force] × 5 minutes). Pelleted cells were resuspended in lysing buffer (Ack; Quality Biological, Inc., Gaithersburg, MD) and incubated for 5 minutes at room temperature. Cells were washed in 10 vol medium. Then cells were plated (1 × 106 cells/mL in a 24-well plate) in RPMI supplemented with 10% fetal bovine serum, 1% glutamine, and a 1% antibiotic solution containing penicillin, streptomycin, and glutamine (catalog no. 10378-016; Gibco).

Retinal Pigment Epithelium

Eyes were removed from C57BL/6 mice and transferred to a Petri dish containing supplemented RPMI (10% fetal bovine serum, 1% glutamine, and a 1% antibiotic solution containing penicillin, streptomycin, and glutamine [catalog no. 10378-016; Gibco], and 1× N1 medium supplement for neural cell cultures [N6530; Sigma, St. Louis, MO]). Corneas, lenses, vitreous, and retinas were dissected away from the posterior globe. RPE cells were loosened from the posterior eyecup by digestion in 0.25% trypsin-EDTA (30-40 minutes at 37°C). Cells were later suspended in medium by triturating complete medium in the digested posterior globe. Cells were then washed once and were plated (60,000-100,000 cells/well) in a 96-well plate.

Fibronectin Fragments

Fibronectin was purified from normal mouse serum (Biodesign International, Saco, ME) by binding to gelatin cross-linked agarose (Sepharose 4B; Amersham Biosciences, Piscataway, NJ) and subsequent elution with 4 M urea. Protein elution was monitored by measuring spectrophotometric absorbance at 280 nm. Eluted protein was dialyzed against 1× PBS. Purified fibronectin was digested overnight at 37°C with the following enzymes: 1 U/mL thrombin, 0.3 U/mL cathepsin-D, and 1 U/mL α-chymotrypsin (all from Calbiochem, La Jolla, CA). Because α-chymotrypsin provided the most effective enzyme digestion (Fig. 1A), it was used in later studies. Overnight digestion with α-chymotrypsin was found to be too long and did not produce a broad spectrum of fragments (Fig. 1A); therefore, time-course enzymatic digestion was subsequently performed with α-chymotrypsin to optimize the methodology. Ten-minute digestions were found to be optimal (Fig. 1B).

Figure 1
Optimal enzymatic digestion of fibronectin, based on number of fragments, lasted 10 minutes with α-chymotrypsin. Stained gels of electrophoresed Fnfs are depicted. After enzymatic digestion, protease inhibitor (pi) was added. (A) Murine serum ...


Digestion of fibronectin was verified by electrophoresis into 4%-20% Bis-Tris gels (Invitrogen, Carlsbad, CA), followed by staining (SimplyBlue SafeStain; Invitrogen). Parallel Western blot detection of electrophoresed Fnfs, with rabbit anti-fibronectin antibodies (AbD Serotec, Raleigh, NC), produced the same band pattern as the stained gels (not shown).

Measurement of Cytokine Levels

IL-6 and TNF-α in splenocyte cell culture supernatants were analyzed with commercially available ELISA in accordance with the manufacturer’s instructions (BD Biosciences, San Jose, CA). Supernatants from primary murine RPE cultures were submitted to ThermoFisher Scientific (Woburn, MA) for cytokine testing services (SearchLight; Pierce, Rockford, IL).

Statistical Analysis

Data were analyzed and graphed (GraphPad Prism 5; La Jolla, CA). Specifically, either t-tests or Kruskal-Wallis ANOVA, followed by Dunn’s multiple comparison tests to identify differences among groups, were performed.


Fnfs Prepared by α-Chymotrypsin Digestion Are Biologically Active

Fibronectin is a dimeric protein composed of two subunits (220-250 kDa). Because thrombin, cathepsin-D, and α-chymotrypsin have previously been used to produce biologically active Fnfs, gelatin-purified murine serum fibronectin was digested with each enzyme (Fig. 1A). In our hands, digestion of murine fibronectin with α-chymotrypsin was more efficient than digestion with thrombin or cathepsin-D (Fig. 1A). Time-course enzymatic digestion was subsequently performed with α-chymotrypsin to optimize the methodology. The 10-minute digestion produced 11 distinct Fnfs (Fig. 1B). The same pattern of Fnfs shown in the representative image (Fig. 1B) was found in three separate 10-minute α-chymotrypsin enzyme digests, performed at different times (not shown).

To verify that the Fnfs were biologically active, murine splenocytes were treated with Fnfs for 1 to 24 hours. Levels of IL-6 and TNF-α were used as indicators of Fnf bioactivity. IL-6 and TNF-α in splenocytes exposed to Fnfs increased with time (Figs. 2A, B), whereas only basal levels of expression were found in splenocytes treated with vehicle (PBS) or enzyme alone (boiled α-chymotrypsin). Splenocytes exposed to the protease inhibitor used to stop the fibronectin digestion did not release significantly different levels of IL-6 or TNF-α compared with vehicle (not shown).

Figure 2
Splenocytes exposed to Fnfs secrete IL-6 and TNF-alpha in a time- and dose-dependent fashion, indicating that Fnfs prepared by α-chymotrypsin digestion are biologically active. Murine splenocytes (1 × 106) were treated with heat-inactivated ...

Murine RPE Cells Exposed to Fnfs Release Proinflammatory and Catabolic Cytokines and MCP-1

Proinflammatory Cytokines

Very low levels of IL-6 were detected in supernatants from murine RPE cells exposed to vehicle or enzyme only (Fig. 3A). Murine RPE cells exposed to low-dose Fnfs (10 μg) secreted more IL-6 than vehicle or enzyme-only treated cells, but the levels did not reach significance (Fig. 3A). Significantly more IL-6 was detected in supernatants from murine RPE cells exposed to the highest dose of Fnfs (50 μg) than from vehicle or enzyme-only treatments (Fig. 3A). Fnfs did not induce the secretion of IL-6 from murine RPE cells when the Fnfs were first boiled for 10 minutes (Fig. 3A). We also verified the observed differences in Figure 3A by standard ELISA and found significant differences (P < 0.05) for IL-6 levels (not shown). TNF-α levels also increased in response to the amount of Fnf treatment; however, the levels were not significantly different from vehicle or enzyme-only treatments (Fig. 3B).

Figure 3
Murine RPE cells exposed to Fnfs secrete IL-6. C57BL/6 primary murine RPE cells (60,000-100,000) were plated in 96-well plates. Cells were exposed to heat-inactivated enzyme (Δ enz), vehicle (serum-free media), 10 μg Fnf, or 50 μ ...

Levels of anti-inflammatory cytokines (IL-4 and IL-13) were also analyzed but were not found to be significant (not shown). IL-4 levels on day 3 were as follows: vehicle, 6.6 ± 3.6 pg/mL; 10 μg Fnf, 9 ± 2 pg/mL; 50 μg Fnf, 0 ± 0 pg/mL. IL-13 levels on day 3 were as follows: vehicle, 31.3 ± 11.5 pg/mL; 10 μg Fnf, 101.4 ± 4.6 pg/mL; 50 μg Fnf, 125.2 ± 29.8 pg/mL.


Levels of MCP-1 in RPE culture supernatants from cells treated with 10 and 50 μg Fnf were significantly higher the day after treatment than were supernatants from cells treated with enzyme only or vehicle (Fig. 4). After 3 days, cells exposed to 50 μg Fnf secreted significantly more MCP-1 than all other treatment groups (Fig. 4). The effectiveness of Fnfs to induce the secretion of MCP-1 was lost when the Fnfs were boiled before the RPE cells were treated (Fig. 4). Although the levels of MCP-1 detected by antibody array were variable, mean values changed by more than 100-fold when 50-μg Fnf treatment was compared with vehicle treatment (Fig. 4).

Figure 4
Murine RPE cells exposed to Fnfs secrete MCP-1. C57BL/6 primary murine RPE cells (60,000-100,000) were plated in 96-well plates. Cells were exposed to heat-inactivated enzyme (Δ enz), vehicle (serum-free media), 10 μg Fnf, or 50 μ ...

Catabolic Cytokines

To determine whether Fnfs are capable of increasing catabolism, we first analyzed the ability of supernatants from Fnf-treated primary murine RPE cells to digest fibronectin, casein, and gelatin (not shown). Enzymography using each substrate showed enhanced activation of MMP activity with respect to the dose of Fnf treatment (not shown). Based on molecular weight and substrate digestion, we decided to further examine levels of MMP-2, MMP-3, and MMP-9 in RPE culture supernatants by ELISA.

Fnf treatment of murine RPE cells did not affect the secretion of MMP-2 (Fig. 5A). MMP-3 levels were significantly elevated in RPE cells exposed to 50 μg Fnf for 3 days (Fig. 5B) compared with all other treatment groups. The effectiveness of Fnfs to induce MMP-3 secretion from murine RPE cells was lost when the Fnfs were boiled (Fig 5B). MMP-9 levels were significantly elevated in murine RPE culture supernatants from cells exposed to 50 μg Fnf after 1 day and 3 days compared with other treatment groups (Fig. 5C). The effectiveness of Fnfs to induce MMP-9 secretion from murine RPE cells was lost when the Fnfs were boiled (Fig. 5C).

Figure 5
Murine RPE cells exposed to Fnfs secrete MMP-3 and MMP-9. C57BL/6 primary murine RPE cells (60,000-100,000) were plated in 96-well plates. Cells were exposed to heat-inactivated enzyme (Δ enz), vehicle (serum-free media), 10 μg Fnf, or ...


The RPE is a monolayer of cuboidal cells located between the retina and the underlying choroidal vasculature. Proper functioning of the RPE is critical to the maintenance of ocular homeostasis and vision because the RPE has diverse functions, including phagocytosis of rods and cones, processing of vitamin A, and limiting transport of materials from the choroid to the retina.58 The dysfunction of RPE is implicated in AMD. Although changes in photoreceptors, RPE, Bruch’s membrane, and choroid are all seen in human patients with AMD, changes in RPE function are thought to be early events in the mechanism leading to the development of clinical AMD changes that cause photoreceptor loss.36,59

Inflammatory mediators and inflammatory cells are increasingly implicated in the progression of AMD,53,60 and the role of the RPE in determining whether the eye has a downimmuno-regulatory environment becomes increasingly more important over time.54,61 It has been suggested that a chronic inflammatory environment in the eye can lead to drusen biogenesis,57,62 one of the clinically visible signs associated with AMD progression. Similarly, drusen are thought to be biomarkers of an immune-mediated process at the RPE-Bruch’s membrane interface.55

In our study, we found that Fnf treatment of RPE cells enhanced the secretion of proinflammatory cytokines (Fig. 3), whereas differences in anti-inflammatory cytokine (IL-4, IL-13) secretion did not reach significance (data not shown). IL-6 release from RPE cells is an important observation because IL-6 has previously been shown to enhance choroidal neovascularization (CNV) through the induction of STAT3, whereas antibody neutralization of IL-6 suppressed CNV and resulted in reduced secretion of MCP-1 and macrophage infiltration into the area of CNV in a mouse model of laser-induced CNV.63 We were unable to show that Fnf treatment leads to enhanced Stat3 phosphorylation in murine RPE cells because it was difficult to obtain enough cells to detect a change. However, we did find that Fnfs affect Stat3 phosphorylation in human peripheral blood mononuclear cells, but the results were highly variable for different donors (data not shown).

Along with proinflammatory cytokines, we also found that Fnfs greatly enhanced the release of MCP-1 (also known as CCL2) from the murine RPE. MCP-1 is known to be constitutively released from the RPE and is enhanced by proinflammatory cytokines.64,65 It is theoretically possible that this enhanced release of MCP-1 could attract resident ocular macrophages (microglia) and could contribute to digestion of the RPE and Bruch’s membrane. Evidence is found in histologic specimens from humans with AMD, in whom macrophages are physically located in areas of RPE atrophy, Bruch’s membrane breakdown, and choroidal neovascularization.66-70 In addition to affecting the RPE and the Bruch’s membrane, such high levels of MCP-1 could negatively impact the retina because retinal detachment-induced photoreceptor apoptosis has been associated with MCP-1.71

Another way in which Fnfs can contribute to retinal degeneration is through the induction of MMPs. Matrix metalloproteinases are involved in normal tissue remodeling and repair. However, the dysregulation of MMP levels is associated with macular degenerative changes, such as CNV and basal laminar deposits in humans.72-75 MMP-3/stromelysin and MMP-2 and MMP-9 (gelatinases A and B) are localized to the ventral aspect of the RPE in the interphotoreceptor matrix and vitreous.76 Only the gelatinases are located in the basal aspect of RPE (in basal laminar deposits)72 and in CNV membranes.72,73,75 Our observation that Fnfs induced increased levels of MMP-3 secretion from the RPE is potentially relevant to an ongoing disease process. One substrate of MMP-3 is fibronectin; hence, it is conceivable that increased levels of Fnfs could serve as a protagonist of a chronic catabolic condition in the eye by which Fnfs initiate a positive feedback pathway for the release of catabolic cytokines from the RPE. Fnfs increase the release of MMP-3, which can act back on fibronectin to promote more Fnf accumulation.

Of all our observations, the ability of Fnfs to stimulate such high levels of MCP-1 warrants further investigation. Similarly, though fibronectin is known to accumulate in diffuse drusen (based on immunohistochemical staining),34 there is no easy way to determine whether the fibronectin is fragmented and serves as a nidus for inflammation and catabolism in ocular tissue sections. Nevertheless, the present study is the first in which the role of Fnfs has been studied in ocular tissues. The ability of Fnfs to stimulate the secretion of such high levels of IL-6, MCP-1, and MMPs from the RPE underscores the importance of Fnfs in modulating acute inflammation in the eye. Their role in chronic ocular disease remains to be determined, and a method to stimulate the long-term accumulation of Fnfs in the eye remains to be developed.


Supported by a National Eye Institute Intramural Research Training Award.


Disclosure: B.A. Austin, None; B. Liu, None; Z. Li, None; R.B. Nussenblatt, None


1. Parier V, Soubrane G. Age-related macular degeneration. Rev Med Interne. 2008;29:215–223. [PubMed]
2. Friedman DS, O’Colmain BJ, Munoz B, et al. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol. 2004;122:564–572. [PubMed]
3. Cameron DJ, Yang Z, Gibbs D, et al. HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration. Cell Cycle. 2007;6:1122–1125. [PubMed]
4. Cameron DJ, Yang Z, Tong Z, et al. 10q26 is associated with increased risk of age-related macular degeneration in the Utah population. Adv Exp Med Biol. 2008;613:253–258. [PubMed]
5. Canfield AE, Hadfield KD, Rock CF, Wylie EC, Wilkinson FL. HtrA1: a novel regulator of physiological and pathological matrix mineralization? Biochem Soc Trans. 2007;35:669–671. [PubMed]
6. Chan CC, Shen D, Zhou M, et al. Human HtrA1 in the archived eyes with age-related macular degeneration. Trans Am Ophthalmol Soc. 2007;105:92–97. discussion 97-98. [PMC free article] [PubMed]
7. Chen H, Yang Z, Gibbs D, et al. Association of HTRA1 polymorphism and bilaterality in advanced age-related macular degeneration. Vision Res. 2008;48:690–694. [PubMed]
8. Deangelis MM, Ji F, Adams S, et al. Alleles in the HtrA serine peptidase 1 gene alter the risk of neovascular age-related macular degeneration. Ophthalmology. 2008;115:1209–1215. [PMC free article] [PubMed]
9. DeWan A, Bracken MB, Hoh J. Two genetic pathways for age-related macular degeneration. Curr Opin Genet Dev. 2007;17:228–233. [PubMed]
10. Dewan A, Liu M, Hartman S, et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science. 2006;314:989–992. [PubMed]
11. Gibbs D, Yang Z, Constantine R, et al. Further mapping of 10q26 supports strong association of HTRA1 polymorphisms with age-related macular degeneration. Vision Res. 2008;48:685–689. [PubMed]
12. Hughes AE, Orr N, Patterson C, et al. Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking. PLoS Med. 2007;4:e355. [PMC free article] [PubMed]
13. Kanda A, Chen W, Othman M, et al. A variant of mitochondrial protein LOC387715/ARMS2, not HTRA1, is strongly associated with age-related macular degeneration. Proc Natl Acad Sci U S A. 2007;104:16227–16232. [PubMed]
14. Kaur I, Katta S, Hussain A, et al. Variants in the 10q26 gene cluster (LOC387715 and HTRA1) exhibit enhanced risk of age-related macular degeneration along with CFH in Indian patients. Invest Ophthalmol Vis Sci. 2008;49:1771–1776. [PubMed]
15. Kondo N, Honda S, Ishibashi K, Tsukahara Y, Negi A. LOC387715/HTRA1 variants in polypoidal choroidal vasculopathy and age-related macular degeneration in a Japanese population. Am J Ophthalmol. 2007;144:608–612. [PubMed]
16. Leveziel N, Souied EH, Richard F, et al. PLEKHA1-LOC387715-HTRA1 polymorphisms and exudative age-related macular degeneration in the French population. Mol Vis. 2007;13:2153–2159. [PubMed]
17. Leveziel N, Zerbib J, Richard F, et al. Genotype-phenotype correlations for exudative age-related macular degeneration associated with homozygous HTRA1 and CFH genotypes. Invest Ophthalmol Vis Sci. 2008;49:3090–3094. [PubMed]
18. Lin JM, Wan L, Tsai YY, et al. HTRA1 polymorphism in dry and wet age-related macular degeneration. Retina. 2008;28:309–313. [PubMed]
19. Lotery A, Trump D. Progress in defining the molecular biology of age related macular degeneration. Hum Genet. 2007;122:219–236. [PubMed]
20. Lu F, Hu J, Zhao P, et al. HTRA1 variant increases risk to neovascular age-related macular degeneration in Chinese population. Vision Res. 2007;47:3120–3123. [PubMed]
21. Montes T, Goicoechea de Jorge E, Ramos R, et al. Genetic deficiency of complement factor H in a patient with age-related macular degeneration and membranoproliferative glomerulonephritis. Mol Immunol. 2008;45:2897–2904. [PubMed]
22. Montezuma SR, Sobrin L, Seddon JM. Review of genetics in age related macular degeneration. Semin Ophthalmol. 2007;22:229–240. [PubMed]
23. Mori K, Horie-Inoue K, Kohda M, et al. Association of the HTRA1 gene variant with age-related macular degeneration in the Japanese population. J Hum Genet. 2007;52:636–641. [PubMed]
24. Pulido JS, Peterson LM, Mutapcic L, Bryant S, Highsmith WE. LOC387715/HTRA1 and complement factor H variants in patients with age-related macular degeneration seen at the mayo clinic. Ophthalmic Genet. 2007;28:203–207. [PubMed]
25. Ross RJ, Verma V, Rosenberg KI, Chan CC, Tuo J. Genetic markers and biomarkers for age-related macular degeneration. Expert Rev Ophthalmol. 2007;2:443–457. [PMC free article] [PubMed]
26. Scholl HP, Fleckenstein M, Charbel Issa P, Keilhauer C, Holz FG, Weber BH. An update on the genetics of age-related macular degeneration. Mol Vis. 2007;13:196–205. [PMC free article] [PubMed]
27. Tam PO, Ng TK, Liu DT, et al. HTRA1 variants in exudative age-related macular degeneration and interactions with smoking and CFH. Invest Ophthalmol Vis Sci. 2008;49:2357–2365. [PMC free article] [PubMed]
28. Weger M, Renner W, Steinbrugger I, et al. Association of the HTRA1-625G>A promoter gene polymorphism with exudative age-related macular degeneration in a Central European population. Mol Vis. 2007;13:1274–1279. [PubMed]
29. Yang Z, Camp NJ, Sun H, et al. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science. 2006;314:992–993. [PubMed]
30. Yoshida T, DeWan A, Zhang H, et al. HTRA1 promoter polymorphism predisposes Japanese to age-related macular degeneration. Mol Vis. 2007;13:545–548. [PMC free article] [PubMed]
31. Mousa SA, Lorelli W, Campochiaro PA. Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells. J Cell Biochem. 1999;74:135–143. [PubMed]
32. An E, Lu X, Flippin J, et al. Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors. J Proteome Res. 2006;5:2599–2610. [PubMed]
33. Loffler KU, Lee WR. Basal linear deposit in the human macula. Graefes Arch Clin Exp Ophthalmol. 1986;224:493–501. [PubMed]
34. Newsome DA, Hewitt AT, Huh W, Robey PG, Hassell JR. Detection of specific extracellular matrix molecules in drusen, Bruch’s membrane, and ciliary body. Am J Ophthalmol. 1987;104:373–381. [PubMed]
35. Abdelsalam A, Del Priore L, Zarbin MA. Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression. Surv Ophthalmol. 1999;44:1–29. [PubMed]
36. Zarbin MA. Age-related macular degeneration: review of pathogenesis. Eur J Ophthalmol. 1998;8:199–206. [PubMed]
37. Grau S, Richards PJ, Kerr B, et al. The role of human HtrA1 in arthritic disease. J Biol Chem. 2006;281:6124–6129. [PubMed]
38. Hadfield KD, Rock CF, Inkson CA, et al. HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains. J Biol Chem. 2008;283:5928–5938. [PubMed]
39. Przybysz M, Borysewicz K, Szechinski J, Katnik-Prastowska I. Synovial fibronectin fragmentation and domain expressions in relation to rheumatoid arthritis progression. Rheumatology. 2007;46:1071–1075. [PubMed]
40. Barilla ML, Carsons SE. Fibronectin fragments and their role in inflammatory arthritis. Semin Arthritis Rheum. 2000;29:252–265. [PubMed]
41. Homandberg GA. Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments. Front Biosci. 1999;4:D713–D730. [PubMed]
42. Peters JH, Carsons S, Yoshida M, et al. Electrophoretic characterization of species of fibronectin bearing sequences from the N-terminal heparin-binding domain in synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis. Arthritis Res Ther. 2003;5:R329–R339. [PMC free article] [PubMed]
43. Xie DL, Meyers R, Homandberg GA. Fibronectin fragments in osteoarthritic synovial fluid. J Rheumatol. 1992;19:1448–1452. [PubMed]
44. Homandberg GA, Hui F. Association of proteoglycan degradation with catabolic cytokine and stromelysin release from cartilage cultured with fibronectin fragments. Arch Biochem Biophys. 1996;334:325–331. [PubMed]
45. Kong W, Longaker MT, Lorenz HP. Cyclophilin C-associated protein is a mediator for fibronectin fragment-induced matrix metalloproteinase-13 expression. J Biol Chem. 2004;279:55334–55340. [PubMed]
46. Saito S, Yamaji N, Yasunaga K, et al. The fibronectin extra domain A activates matrix metalloproteinase gene expression by an interleukin-1-dependent mechanism. J Biol Chem. 1999;274:30756–30763. [PubMed]
47. Stanton H, Ung L, Fosang AJ. The 45 kDa collagen-binding fragment of fibronectin induces matrix metalloproteinase-13 synthesis by chondrocytes and aggrecan degradation by aggrecanases. Biochem J. 2002;364:181–190. [PubMed]
48. Yasuda T, Poole AR. A fibronectin fragment induces type II collagen degradation by collagenase through an interleukin-1-mediated pathway. Arthritis Rheum. 2002;46:138–148. [PubMed]
49. Yasuda T, Shimizu M, Nakagawa T, Julovi SM, Nakamura T. Matrix metalloproteinase production by COOH-terminal heparin-binding fibronectin fragment in rheumatoid synovial cells. Lab Invest. 2003;83:153–162. [PubMed]
50. Homandberg GA, Hui F, Wen C, et al. Fibronectin-fragment-induced cartilage chondrolysis is associated with release of catabolic cytokines. Biochem J. 1997;321(pt 3):751–757. [PubMed]
51. Homandberg GA, Wen C. Exposure of cartilage to a fibronectin fragment amplifies catabolic processes while also enhancing anabolic processes to limit damage. J Orthop Res. 1998;16:237–246. [PubMed]
52. Johnson LV, Leitner WP, Staples MK, Anderson DH. Complement activation and inflammatory processes in drusen formation and age related macular degeneration. Exp Eye Res. 2001;73:887–896. [PubMed]
53. Kijlstra A, La Heij E, Hendrikse F. Immunological factors in the pathogenesis and treatment of age-related macular degeneration. Ocul Immunol Inflamm. 2005;13:3–11. [PubMed]
54. Nussenblatt RB, Ferris F., 3rd Age-related macular degeneration and the immune response: implications for therapy. Am J Ophthalmol. 2007;144:618–626. [PMC free article] [PubMed]
55. Hageman GS, Luthert PJ, Victor Chong NH, Johnson LV, Anderson DH, Mullins RF. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–732. [PubMed]
56. Mullins RF, Aptsiauri N, Hageman GS. Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis. Eye. 2001;15:390–395. [PubMed]
57. Johnson LV, Ozaki S, Staples MK, Erickson PA, Anderson DH. A potential role for immune complex pathogenesis in drusen formation. Exp Eye Res. 2000;70:441–449. [PubMed]
58. Bok D. The retinal pigment epithelium: a versatile partner in vision. J Cell Sci Suppl. 1993;17:189–195. [PubMed]
59. Nowak JZ. Age-related macular degeneration (AMD): pathogenesis and therapy. Pharmacol Rep. 2006;58:353–363. [PubMed]
60. Rodrigues EB. Inflammation in dry age-related macular degeneration. Ophthalmologica. 2007;221:143–152. [PubMed]
61. Zamiri P, Sugita S, Streilein JW. Immunosuppressive properties of the pigmented epithelial cells and the subretinal space. Chem Immunol Allergy. 2007;92:86–93. [PubMed]
62. Umeda S, Suzuki MT, Okamoto H, et al. Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis) FASEB J. 2005;19:1683–1685. [PubMed]
63. Izumi-Nagai K, Nagai N, Ozawa Y, et al. Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization. Am J Pathol. 2007;170:2149–2158. [PubMed]
64. Crane IJ, Wallace CA, McKillop-Smith S, Forrester JV. Control of chemokine production at the blood-retina barrier. Immunology. 2000;101:426–433. [PubMed]
65. Forrester JV. Macrophages eyed in macular degeneration. Nat Med. 2003;9:1350–1351. [PubMed]
66. Patel M, Chan CC. Immunopathological aspects of age-related macular degeneration. Semin Immunopathol. 2008;30:97–110. [PMC free article] [PubMed]
67. Dastgheib K, Green WR. Granulomatous reaction to Bruch’s membrane in age-related macular degeneration. Arch Ophthalmol. 1994;112:813–818. [PubMed]
68. Grossniklaus HE, Ling JX, Wallace TM, et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis. 2002;8:119–126. [PubMed]
69. Penfold PL, Madigan MC, Gillies MC, Provis JM. Immunological and aetiological aspects of macular degeneration. Prog Retin Eye Res. 2001;20:385–414. [PubMed]
70. Lopez PF, Grossniklaus HE, Lambert HM, et al. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration. Am J Ophthalmol. 1991;112:647–656. [PubMed]
71. Nakazawa T, Hisatomi T, Nakazawa C, et al. Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis. Proc Natl Acad SciUSA. 2007;104:2425–2430. [PubMed]
72. Lommatzsch A, Hermans P, Muller KD, Bornfeld N, Bird AC, Pauleikhoff D. Are low inflammatory reactions involved in exudative age-related macular degeneration? Morphological and immunohistochemical analysis of AMD associated with basal deposits. Graefes Arch Clin Exp Ophthalmol. 2008;246:803–810. [PubMed]
73. Zeng J, Jiang D, Liu X, Zhu X, Tang L. Matrix metalloproteinases expression in choroidal neovascular membranes. Yan Ke Xue Bao. 2004;20:191–193. [PubMed]
74. Tatar O, Adam A, Shinoda K, et al. Matrix metalloproteinases in human choroidal neovascular membranes excised following verteporfin photodynamic therapy. Br J Ophthalmol. 2007;91:1183–1189. [PMC free article] [PubMed]
75. Steen B, Sejersen S, Berglin L, Seregard S, Kvanta A. Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1998;39:2194–2200. [PubMed]
76. Plantner JJ, Smine A, Quinn TA. Matrix metalloproteinases and metalloproteinase inhibitors in human interphotoreceptor matrix and vitreous. Curr Eye Res. 1998;17:132–140. [PubMed]