November 2009
Volume 50, Issue 11
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Anatomy and Pathology/Oncology  |   November 2009
α11 Integrin in the Human Cornea: Importance in Development and Disease
Author Affiliations & Notes
  • Berit Byström
    From the Department of Clinical Sciences, Ophthalmology, and
    the Department of Integrative Medical Biology, Section for Anatomy, Umeå University, Umeå, Sweden; and
  • Sergio Carracedo
    the Department of Biomedicine, Section for Physiology, University of Bergen, Bergen, Norway.
  • Anders Behndig
    From the Department of Clinical Sciences, Ophthalmology, and
  • Donald Gullberg
    the Department of Biomedicine, Section for Physiology, University of Bergen, Bergen, Norway.
  • Fatima Pedrosa-Domellöf
    From the Department of Clinical Sciences, Ophthalmology, and
    the Department of Integrative Medical Biology, Section for Anatomy, Umeå University, Umeå, Sweden; and
Investigative Ophthalmology & Visual Science November 2009, Vol.50, 5044-5053. doi:10.1167/iovs.08-3261
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      Berit Byström, Sergio Carracedo, Anders Behndig, Donald Gullberg, Fatima Pedrosa-Domellöf; α11 Integrin in the Human Cornea: Importance in Development and Disease. Invest. Ophthalmol. Vis. Sci. 2009;50(11):5044-5053. doi: 10.1167/iovs.08-3261.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To examine the distribution of the α11 integrin chain in the human cornea during fetal development and in normal and diseased adult human corneas.

Methods.: Six fetal corneas, 10 to 20 weeks of gestation (wg), and 18 adult corneas including 3 normal, 7 with keratoconus, 5 with pseudophakic bullous keratopathy (PBK), 2 with Fuchs' corneal dystrophy, and 1 with a scar after deep lamellar keratoplasty (DLKP) were processed for immunohistochemistry with specific antibodies against the α11 integrin chain; collagen I, IV, and V; and α-smooth muscle actin (α-SMA). The cellular source of α11 integrin chain was further investigated in cell cultures.

Results.: At 10 to 17 wg, the α11 integrin chain was predominantly present in the anterior corneal stroma. At 20 wg, in normal adult corneas and in Fuchs' dystrophy corneas there was weak staining in the stroma. The PBK corneas showed variable and weak staining, generally accentuated in the posterior stroma near Descemet's membrane. In contrast, the anterior portion of the stroma in the keratoconus corneas was strongly stained in an irregular streaky pattern. Human corneal fibroblasts/myofibroblasts produced α11 integrin chain in culture. Cultures treated with TGF-β showed higher content of both α-SMA and the α11 integrin chain.

Conclusions.: The presence of the α11 integrin chain during early corneal development and the enhanced expression in scarred keratoconus corneas indicates that this integrin chain is likely to play an important role in collagen deposition during corneal development and in keratoconus with a scarring component and compromised basement membrane integrity.

Integrins are the major adhesion receptors connecting cells to components of the extracellular matrix (ECM), 1 and integrin signaling determines cell survival, proliferation, and differentiation. Integrins are heterodimeric transmembrane glycoproteins composed of noncovalently associated α and β chains. 2,3 Eighteen α and 8 β integrin subunits, that can assemble into 24 different known heterodimers, have been identified in the human genome. 2 In line with the important roles of integrins in cell adhesion, cell migration, and the formation of basement membrane (BM) during early development, most integrin mutations lead to embryonic or perinatal death. 4 Mutations in genes encoding the hemidesmosomal integrin α6β4 in humans lead to a skin-blistering disease, epidermolysis bullosa. 5 Mutant mice that lack the ubiquitously expressed β1 family of integrins die during the early stages of embryonic development. 69 In contrast, α1-null mice are viable, fertile, and apparently normal. 10  
The most recently described member of the integrin family is the α11 integrin chain, 11 which is closely related to the α10 integrin chain 12 and both α11β1 and α10β1 integrin are collagen receptors. α11β1 Integrin mediates cell adhesion to collagens I and IV in vitro, with preference for collagen I. 13,14 The α11 integrin chain has been detected in the human cornea at 8 wg and it has therefore been suggested that it is involved in the ordered collagen matrix organization of the cornea. 13 However, the spatial and temporal patterns of expression of the α11 integrin chain in the human eye have not been investigated. 
A minimum of 12 different integrin heterodimers have been reported to be expressed in the cornea. 15 These integrins are suggested to play an important role in keeping corneal clarity by organizing the interaction between the corneal cells and the ECM. 15 A clear cornea is a prerequisite for optimal vision, and the physical properties of the cornea are important for the mechanical stability of the deeper components of the eye. 
The corneal stroma, located underneath the epithelium and the Bowman's layer, constitutes 90% of the corneal thickness and is predominantly composed of collagen type I. Collagen type V molecules are also present in the corneal stroma, but they are less abundant and are buried within the collagen type I fibrils. Type V collagen molecules are considered to be a limiting factor for collagen fibril growth. 16 The tightly packed and highly organized fibrils of collagens type I and V contribute both to the strength and the clarity of the cornea. 17 Type IV collagen is a major element of BMs 18 and there is evidence of developmentally regulated expression of collagen type IV chains in the human central corneal epithelial BM. 19 There is a shift in collagen type IV isoforms during corneal maturation from those containing α1 and α2 chains in the infant to those containing α3 and α4 chains in the adult. 19,20  
Keratoconus is a noninflammatory corneal disease of unclear etiology, 21,22 characterized by progressive thinning and deformation of the central cornea and requiring corneal transplantation in advanced stages. 
Pseudophakic bullous keratopathy (PBK), a complication of ocular surgery, is characterized by chronic corneal edema and epithelial blisters (bullae). Rupture of the blisters causes pain and interferes with vision. 23 PBK and keratoconus are leading indications for penetrating keratoplasty. Fuchs' dystrophy, clinically similar to PBK, is also associated with corneal edema, but it additionally comprises corneal guttae, mushroomlike Descemet's membrane excrescences, together with pigment granules within or attached to the endothelial cells. 24 DLKP is a surgical procedure where a maximum of corneal stroma is replaced by donor corneal tissue, 2527 keeping the Descemet's membrane of the recipient patient intact. 
The spatial and temporal patterns of distribution of the α11 integrin chain in the human cornea during fetal development as well as in normal and diseased adult human corneas are unknown, although previous studies in vertebrates suggest an important role for integrin chains in the cornea. 69,15  
We report the distribution of the α11 integrin chain in human corneas from 10 weeks of gestation (wg) up to adulthood and in diseases treated with corneal transplantation. 
Materials and Methods
A total of six eyes were obtained from human fetuses at 10, 11, 14, 16, 17, and 20 wg, after legal interruptions of pregnancy. Gestational age was dated from the first day of the last menstrual period and was further confirmed by ultrasound before abortion in most cases. Furthermore, 18 adult human corneas were obtained (Table 1). All samples were collected with the approval of the Ethics Committee of the Medical Faculty, Umeå University, after informed consent and in accordance with the tenets of the Declaration of Helsinki of 1975. The normal corneas included both the central and peripheral part as they were obtained after evisceration, enucleation, or donation. The diseased corneas were obtained from patients undergoing penetrating keratoplasty and comprised the central button only. DLKP was performed manually; the primary indication for surgery was keratoconus. 
Table 1.
 
Patient Data
Table 1.
 
Patient Data
Age (y) Sex Diagnosis and Comments
40 M Normal, evisceration due to trauma*
86 M Normal, donation with retinitis pigmentosa*
87 M Normal, enucleation due to choroidal tumor*
23 M Keratoconus, with a central scar
29 M Keratoconus, with a central scar
38 M Keratoconus, with a central scar
48 M Keratoconus, with a central scar
21 M Keratoconus, with central striation†
25 M Keratoconus, with central striation†
50 M Keratoconus, retransplantation‡
65 M PBK after cataract surgery
72 F PBK after cataract surgery
74 F PBK+Fuchs' with cataract surgery
78 F PBK+Fuchs' with cataract surgery
84 M PBK+Fuchs' with cataract surgery
50 F Fuchs' without earlier surgery§
59 F Fuchs' without earlier surgery§
37 M Scar, post DLKP‖
The samples were mounted in embedding medium (Tissue-Tek OCT; Miles, Elkhart, IN), rapidly frozen in propane chilled with liquid nitrogen (−160°C), and stored at −80°C until use. Serial cross sections, 5 μm thick, were processed for immunohistochemistry with a previously characterized polyclonal antibody (Ab) specific for integrin α11 chain. 11 In the adult normal and diseased corneas, monoclonal Ab 1A4 against α-SMA (α-smooth muscle actin) 28 was used to detect myofibroblasts in especially scarred regions of the corneas. 2931 In vitro α11β1 integrin mediates cell adhesion to collagens I and IV (preferably collagen I). 13 To investigate whether the staining pattern for integrin α11 chain correlates with the presence of collagens I and IV, a polyclonal Ab specific for collagen I (Ab D9-R349; Southern Biotechnology Associates, Inc., Birmingham, AL) and a monoclonal Ab (Ab CIV22; Dako, Glostrup, Denmark) specific for the α1-α2 chain-containing collagen IV trimer 18 were used. As type V collagen molecules are buried within the collagen type I fibrils and are considered to be a limiting factor for collagen fibril growth, a monoclonal Ab (Ab 36382) specific for collagen V (Abcam, Cambridge, UK) was also used to reveal possible correlation to the staining pattern of integrin α11 chain. To visualize breaks in the epithelial BM, the monoclonal Ab 4C7 32,33 against laminin α5 chain was used. The bound Abs were visualized by using a standard indirect fluorescence technique, with the secondary Ab conjugated with a fluorochrome (Cy3; Jackson ImmunoResearch Laboratories, West Grove, PA, or Alexa 488; Molecular Probes, Leiden, The Netherlands). Nuclei were detected with DAPI staining (Vectashield; Vector Laboratory, Burlingame, CA). Control sections from which the primary Ab was omitted were completely negative. Control sections prepared with normal rabbit serum instead of the primary polyclonal Ab revealed the level of unspecific staining and served as reference for the evaluation of the level of specific staining in the other sections. Although the staining level observed in corneal epithelium was higher than that of background, this is unspecific as epithelium is known to be “sticky.” Furthermore, similar staining of skin epithelium was observed but no corresponding labeling was seen with in situ hybridization (Popova SN, Gullberg D, unpublished data, 2008). 
The tissue sections were studied by epifluorescence microscope (Eclipse E800; Nikon Inc., Melville, NY) equipped with a color camera (SPOT RT; Diagnostic Instruments Inc., Sterling Heights, MI) for image acquisition. Digital images were then processed (Photoshop; Adobe Systems Inc., Mountain View, CA). Exposure times were determined, and background-level adjustments to the photographs were made to truly reflect the staining observed under the microscope. 
Small pieces of corneal stroma were put in cell culture flasks in 50:50 Dulbecco's MEM (DMEM): Hams F12 (Invitrogen-Gibco, Inc., Grand Island, NY) supplemented with 10% fetal calf serum (FCS) and 72 μg benzylpenicillin/mL. The culture medium was changed twice weekly and the cells were grown to near confluence. The cells were subsequently kept at −80°C until further investigation or were immediately seeded either on collagen-coated chamber covered glass slides (100 μg of collagen/mL of PBS) or on collagen-coated Petri dishes (100 μg/mL in PBS). Five nanograms per milliliter of TGF-β (Prepotech, London, UK) was added the same day (day 0), whereas control cultures were untreated. At day 4, cells cultured on glass slides were fixed in methanol and processed for immunohistochemistry with Abs against integrin α11 chain, 11 α-SMA and vinculin (Sigma-Aldrich, St. Louis, MO). 
Cells grown on collagen coated Petri dishes were harvested at days 1, 3, and 6, and protein levels of the α11 integrin chain, α-SMA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA) were determined by Western blot analysis. 34 mRNA levels of α1-, α2-, α10-, and α11 integrins, in similar cell culture conditions at 3 days after TGF-β treatment, were determined by semiquantitative RT-PCR. Total RNA was isolated from corneal fibroblasts cultured for 3 days in the presence or absence of TGF-β (RNeasy Mini Kit; Qiagen, Oslo[b], Norway). cDNA was generated from 350 ng of total RNA using M-MulV reverse transcriptase (Fermentas, Helsingborg, Sweden) and oligo (dT)18 primer (Thermo Scientific, Oslo, Norway). One microliter of amplified cDNA was used as a template in the PCR reactions (Pac5000 DNA polymerase; Stratagene, Oslo, Norway). The primers used for detection of different integrin chains are listed in Table 2. Amplification of the β-actin gene was used for normalization. PCRs were performed for a number of cycles specified for each gene in Table 2 (within linear range of amplification): denaturation (94°C, 30 seconds); annealing (corresponding temperatures for each primer, 45 seconds), and extension (72°C, 1 minute). A final extension step was performed at 72°C for 10 minutes. The products of the PCR reactions were separated on 2% agarose gels. C2C12 cells transfected with α10 integrin cDNA 12 in pBJ-1 were used as a positive control in RT-PCR when assessing mRNA levels of this integrin subunit in corneal fibroblasts, as described. 35  
Table 2.
 
Primer Sequences and PCR Conditions Used in the Semiquantitative RT-PCR Analysis
Table 2.
 
Primer Sequences and PCR Conditions Used in the Semiquantitative RT-PCR Analysis
Forward (5′-3′) Reverse (5′-3′) Annealing T (°C) Cycles Product (bp)
α1 CGA AGA ACC TCC TGA AAC CC CGA AAC ATT GAC TTG GCT GA 52 33 478
α2 AAA AAT AAA AGG GAA AGT GC CTT GTT TTC TTC TTG GCT TT 50 33 276
α10 TCT AGA AAC CTC CAC CTG G CTG GAA GGA GGG CTG AGA TGA TGA 65 33 438
α11 GAT TCA CCA ACA GAG CCG TA TCA AAA TCA AGA ACGG AAA GC 58 30 598
β-actin GTG TGA TGG TGG GAA TGG GT TCT GGG TCA TCT TTT CAC GGT TGG 58 30 240
Results
Fetal Human Corneas
At 10 wg there was strong staining in streaks of the Ab against the α11 integrin chain through the whole stroma (Figs. 1A–C). Between 11 and 17 wg, the α11 integrin chain was present in streaks in the whole stroma, but the staining was denser in the anterior stroma (Figs. 1D–F). Specific staining in very fine streaks was observed throughout the whole stroma with only a slightly denser stain anteriorly at 20 wg (Figs. 1G–I), resembling the pattern in normal adult corneas. 
Figure 1.
 
Cross sections of human fetal corneas at different gestational ages showing immunoreactivity for the α11 integrin chain in the corneal stroma. In all frames, the corneal epithelium is located in the top of the image. Fetal age is indicated in weeks of gestation (wg). (A, G) Low-power images of sections stained with hematoxylin eosin; the black boxes correspond to the areas shown in (B, C) and (H, I), respectively. The level of nonspecific staining is shown in sections treated with normal rabbit serum (pre, B, H) at 10 and 20 wg. Note the intense staining in streaks for the α11 integrin chain in the whole corneal stroma, accentuated in the anterior (top) part at 10 to 17 wg (C–F, I). (A–C, *) The space between the cornea and the overlying eyelid. Bar: (B–F, H, I) 50 μm; (A, G) 100 μm.
Figure 1.
 
Cross sections of human fetal corneas at different gestational ages showing immunoreactivity for the α11 integrin chain in the corneal stroma. In all frames, the corneal epithelium is located in the top of the image. Fetal age is indicated in weeks of gestation (wg). (A, G) Low-power images of sections stained with hematoxylin eosin; the black boxes correspond to the areas shown in (B, C) and (H, I), respectively. The level of nonspecific staining is shown in sections treated with normal rabbit serum (pre, B, H) at 10 and 20 wg. Note the intense staining in streaks for the α11 integrin chain in the whole corneal stroma, accentuated in the anterior (top) part at 10 to 17 wg (C–F, I). (A–C, *) The space between the cornea and the overlying eyelid. Bar: (B–F, H, I) 50 μm; (A, G) 100 μm.
Adult Human Corneas
Normal corneas showed specific staining with the α11 integrin Ab in very fine streaks distributed throughout the whole stroma. These streaks were clearly separated, although sparse and weak in the central part of the corneal stroma (Figs. 2A, B), whereas they were strongly stained and more densely packed in the periphery of the corneal stroma (Figs. 2C, 2D). 
Figure 2.
 
Cross sections of adult human corneas. Top row: images from the same normal cornea. (A, B) The anterior central and (C, D) anterior peripheral cornea. Note the more intense staining pattern in streaks in the periphery (D). (A, C) Sections treated with normal rabbit serum (called pre) and (B, D) anti-α11 integrin. Middle row: cross sections of corneas with Fuchs' dystrophy (E, F) and PBK (G, H). (E, F) Normal rabbit serum and α11 integrin staining in a cornea with Fuchs' dystrophy. (G, H) The anterior part of the cornea of two different patients with PBK. Note the irregular epithelium in (G) without any increased irregularities in the underlying stroma. Bottom row: the posterior part of a normal cornea (I), a cornea with Fuchs' dystrophy (J) and a PBK cornea (K, L). The staining with anti-α11 integrin is more intense in the posterior part of the PBK cornea (K, arrow) compared with the staining in the normal (I) and the Fuchs' dystrophy cornea (J). Increased cellularity visualized in (L) with DAPI staining corresponded to the more intense staining in (K). Bar, 50 μm.
Figure 2.
 
Cross sections of adult human corneas. Top row: images from the same normal cornea. (A, B) The anterior central and (C, D) anterior peripheral cornea. Note the more intense staining pattern in streaks in the periphery (D). (A, C) Sections treated with normal rabbit serum (called pre) and (B, D) anti-α11 integrin. Middle row: cross sections of corneas with Fuchs' dystrophy (E, F) and PBK (G, H). (E, F) Normal rabbit serum and α11 integrin staining in a cornea with Fuchs' dystrophy. (G, H) The anterior part of the cornea of two different patients with PBK. Note the irregular epithelium in (G) without any increased irregularities in the underlying stroma. Bottom row: the posterior part of a normal cornea (I), a cornea with Fuchs' dystrophy (J) and a PBK cornea (K, L). The staining with anti-α11 integrin is more intense in the posterior part of the PBK cornea (K, arrow) compared with the staining in the normal (I) and the Fuchs' dystrophy cornea (J). Increased cellularity visualized in (L) with DAPI staining corresponded to the more intense staining in (K). Bar, 50 μm.
The Fuchs' dystrophy corneas displayed a pattern of staining identical with that of the central part of the normal corneas (Figs. 2E, 2F). The PBK corneas were generally labeled with the Ab against integrin α11 chain through the whole stroma in fine streaks (Figs. 2G, 2H). In some PBK cases, the epithelium was typically very irregular, forming blisters (Fig. 2G), but no differences in the labeling with Ab to α11 integrin was detected in the underlying stroma (Fig. 2G). The irregular epithelium and its BM did not correspond to any interruptions of the BM or Bowman's layer (see Figs. 4C, 4D). In many PBK cases, a denser staining pattern with longer streaks was observed in the posterior stroma near Descemet's membrane (Fig. 2K), a feature not present in the normal (Fig. 2I) and Fuchs' dystrophy corneas (Fig. 2J). 
The scarred keratoconus corneas showed an increased and irregular staining pattern with the Ab to the α11 integrin chain in the anterior portion of the stroma (Figs. 3B, 3I), whereas the staining in the deeper stroma (not shown) was similar to that observed in normal corneas. 
Figure 3.
 
Staining patterns of keratoconus corneas treated with normal rabbit serum (A, H, N, pre), Abs to α11 integrin (B, I, O), α-SMA (D, J, P), collagen I (E, K, Q), the α1 and α2 chains of collagen IV (F, L, R), and collagen V (G, M, S). Each row represents a single keratoconus case. (A–G) A case with clinically central scarring of the cornea. Note the intense staining in the same region with different Abs (B–G, arrows). DAPI staining revealed increased cellularity in this region (C). Note also the very thick and irregular epithelium. There was staining of the epithelial BM by the Ab to the α1 and α2 chains of collagen IV (F, arrowhead). (H–M) Another scarred keratoconus cornea with intense staining in streaks by the Abs (I–M, arrows) and staining of the epithelial BM by the Abs to collagen IV and V (L, M, arrowheads). (N–S) In contrast to (H–M), a keratoconus cornea with very weak staining with antibodies to α11 integrin, α-SMA, the α1 and α2 chains of collagen IV and V, resembling the section treated with normal rabbit serum (N). Bar, 50 μm.
Figure 3.
 
Staining patterns of keratoconus corneas treated with normal rabbit serum (A, H, N, pre), Abs to α11 integrin (B, I, O), α-SMA (D, J, P), collagen I (E, K, Q), the α1 and α2 chains of collagen IV (F, L, R), and collagen V (G, M, S). Each row represents a single keratoconus case. (A–G) A case with clinically central scarring of the cornea. Note the intense staining in the same region with different Abs (B–G, arrows). DAPI staining revealed increased cellularity in this region (C). Note also the very thick and irregular epithelium. There was staining of the epithelial BM by the Ab to the α1 and α2 chains of collagen IV (F, arrowhead). (H–M) Another scarred keratoconus cornea with intense staining in streaks by the Abs (I–M, arrows) and staining of the epithelial BM by the Abs to collagen IV and V (L, M, arrowheads). (N–S) In contrast to (H–M), a keratoconus cornea with very weak staining with antibodies to α11 integrin, α-SMA, the α1 and α2 chains of collagen IV and V, resembling the section treated with normal rabbit serum (N). Bar, 50 μm.
There was a clear correlation between labeling with the Abs against α-SMA and the α1 and α2 chains of collagen IV and collagen V and the presence of the α11 integrin chain, especially with respect to the irregular staining in streaks in the anterior stroma beneath Bowman's layer in scarred keratoconus corneas (Figs. 3B, 3D, 3F, 3G, 3I, 3J, 3L, 3M). In the keratoconus cases with little or no labeling with Ab to α11-integrin chain (Fig. 3O), there was also little or no presence of α-SMA or α1 and α2 chains of collagen IV or V (Figs. 3P, 3R, 3S). The more intense and irregular staining pattern in Figures 3A–M (see also Figs. 4A, 4B, compared with 4E, 4F) corresponded to cases with damaged epithelial BM and Bowman's layer in contrast to a case with an intact BM (Figs. 3N–S). 
Figure 4.
 
Staining patterns in a case of keratoconus (A, B), a case of PBK (C, D) and a normal cornea (E, F). Left column: sections stained with hematoxylin and eosin; right column: sections treated with the Ab to the α11 integrin chain. (B, inset) Laminin (LM) α5 chain staining of the epithelial BM in the same region of an adjacent section; arrowheads: breaks in the BM. Note the correspondence between the break in the Bowman's layer (A, B, arrows), epithelial BM (B), and increased staining with the Ab against the α11 integrin chain in the same area, in the keratoconus case. In the PBK case and the normal cornea, the BM and Bowman's layer are regular and uninterrupted, and there is no increase in α11 integrin chain labeling (C–F). Bar, 50 μm.
Figure 4.
 
Staining patterns in a case of keratoconus (A, B), a case of PBK (C, D) and a normal cornea (E, F). Left column: sections stained with hematoxylin and eosin; right column: sections treated with the Ab to the α11 integrin chain. (B, inset) Laminin (LM) α5 chain staining of the epithelial BM in the same region of an adjacent section; arrowheads: breaks in the BM. Note the correspondence between the break in the Bowman's layer (A, B, arrows), epithelial BM (B), and increased staining with the Ab against the α11 integrin chain in the same area, in the keratoconus case. In the PBK case and the normal cornea, the BM and Bowman's layer are regular and uninterrupted, and there is no increase in α11 integrin chain labeling (C–F). Bar, 50 μm.
The corneal lamellar morphology revealed with the Ab against collagen I was more irregular in most cases of keratoconus (Figs. 3E, 2K, 2Q) than in all other corneas (not shown). However, no differences in staining intensity with this Ab were observed. 
The DLKP cornea was only weakly stained by the Ab against the α11 integrin chain in the anterior stroma (Figs. 5A, 5B), as in the normal cornea (Figs. 2A, 2B). In contrast, the presence of the α11 integrin chain was obvious in the posterior stroma—that is, in the portion belonging to the recipient (Fig. 5D). There was a clear correlation between labeling with the Ab to collagen V and the presence of the α11 integrin chain in the posterior stroma (Figs. 5D, 5E), whereas there was no staining with anti-α-SMA (Fig. 5F). The interface between the donor and host cornea did not reveal any novel staining with α11 integrin, collagen V, or α-SMA Abs (Figs. 5D–F). 
Figure 5.
 
Cross sections of a DLKP cornea, where (A) and (B) are from the anterior part of the cornea and (C–F) show the posterior part. (C–F) (*) Interface between the donor and host cornea. Note the streaky staining with anti-α11 integrin chain and anti-collagen V in the recipient cornea (the posterior part, D, E, arrows) and the correspondence with increased cellularity shown with DAPI staining of the nuclei (C). Bar, 50 μm.
Figure 5.
 
Cross sections of a DLKP cornea, where (A) and (B) are from the anterior part of the cornea and (C–F) show the posterior part. (C–F) (*) Interface between the donor and host cornea. Note the streaky staining with anti-α11 integrin chain and anti-collagen V in the recipient cornea (the posterior part, D, E, arrows) and the correspondence with increased cellularity shown with DAPI staining of the nuclei (C). Bar, 50 μm.
In all cases, increased cellularity was noted in the same regions where the intense staining in streaks was present (Figs. 2L, 3C, 5C). 
Cell Culture
Human corneal fibroblasts treated with TGF-β were strongly labeled with Abs against the α11 integrin chain and α-SMA (Figs. 6A–D) at 4 days. In untreated cells, α11 integrin and α-SMA was barely detected (Fig. 6E). Double staining with anti-α11 integrin chain and anti-vinculin showed co-distribution of these two epitopes in focal adhesions in cultured corneal fibroblasts (Figs. 6F–K). 
Figure 6.
 
Cultured cells at 4 days were labeled with Abs against α-SMA (A) and the α11 integrin chain (B) after treatment with TGF-β. Note the coexistence of α-SMA and α11 integrin staining in (C) suggesting myofibroblasts as a source for the α11 integrin chain. Single cell α-SMA upregulation by TGF-β is obvious in (D) compared with untreated cells in (E). Double-staining with anti-α11 integrin and anti-vinculin 4 days after treatment with TGF-β (F–H) and untreated controls (I–K) revealed co-localization of upregulated α11 integrin with vinculin in focal adhesions.
Figure 6.
 
Cultured cells at 4 days were labeled with Abs against α-SMA (A) and the α11 integrin chain (B) after treatment with TGF-β. Note the coexistence of α-SMA and α11 integrin staining in (C) suggesting myofibroblasts as a source for the α11 integrin chain. Single cell α-SMA upregulation by TGF-β is obvious in (D) compared with untreated cells in (E). Double-staining with anti-α11 integrin and anti-vinculin 4 days after treatment with TGF-β (F–H) and untreated controls (I–K) revealed co-localization of upregulated α11 integrin with vinculin in focal adhesions.
SDS-PAGE revealed that cells stimulated by TGF-β produced more α11 integrin and α-SMA at day 1 than did untreated cells. This production increased over time, but at 6 days the levels of α11 integrin and α-SMA were similar in the TGF-β-treated and untreated cell cultures (Fig. 7A). Upregulation of α1 and α2 but not α10 integrin chains was also observed for both TGF-β-treated and control cells. Note that upregulation of the α11 integrin chain was independent of that of the other integrins tested (Fig. 7B). 
Figure 7.
 
(A) Corneal fibroblasts plated on collagen I were treated with TGF-β and protein levels of α11 integrin and α-SMA were analyzed at indicated time points. Note higher levels of α11 integrin and α-SMA after 1 and 3 days of treatment. Signal for α11 integrin and α-SMA after 6 days of treatment were similar in the TGF-β-treated and control cell cultures. mRNA levels for collagen receptors (α1-, α2-, α10-, and α11 integrin) in corneal fibroblasts treated with TGF-β for 3 days (+) and control cells (−) are shown in (B). A reference positive control (Ref) obtained from transfected C2C12 cells was used for α10 integrin. Note the independent regulation of α11 integrin chain.
Figure 7.
 
(A) Corneal fibroblasts plated on collagen I were treated with TGF-β and protein levels of α11 integrin and α-SMA were analyzed at indicated time points. Note higher levels of α11 integrin and α-SMA after 1 and 3 days of treatment. Signal for α11 integrin and α-SMA after 6 days of treatment were similar in the TGF-β-treated and control cell cultures. mRNA levels for collagen receptors (α1-, α2-, α10-, and α11 integrin) in corneal fibroblasts treated with TGF-β for 3 days (+) and control cells (−) are shown in (B). A reference positive control (Ref) obtained from transfected C2C12 cells was used for α10 integrin. Note the independent regulation of α11 integrin chain.
Discussion
The organization of collagen fibrils largely determines corneal transparency and refraction. 17 α11β1 Integrin has been shown to take part in such collagen-mediated events as cell migration, collagen deposition, and collagen reorganization and has been suggested to play an important role during corneal organogenesis. 13,34,36 The predominance of the α11 integrin chain during early corneal development and the enhanced expression pattern in scarred keratoconus corneas in our study strengthens the hypothesis that the α11 integrin chain probably plays an important role in collagen deposition during corneal development and in disease with an ongoing wound-healing process, as confirmed by the presence of α-SMA, a marker for myofibroblasts. 29,37  
At 10 wg α11 integrin was homogenously distributed throughout the corneal stroma. The denser staining of the α11 integrin chain seen in the anterior stroma from 11 wg may reflect the fact that the collagen fibril formation has then reached this portion of the cornea. During fetal development collagen fibril formation in the corneal stroma starts in the posterior layers and proceeds to the anterior portion of the cornea. 38 α11β1 Integrin has been shown to be a receptor for collagen I, 34,36 and its preferred location in the anterior stroma fits the fact that collagen bundles are more compactly packed in this region of the adult cornea. 39  
In this study, the staining patterns for the α11 integrin chain and collagen V were very similar, especially in the scarred keratoconus cases, in the periphery of normal corneas and in the recipient part of the DLKP cornea. Of note, collagen V stimulates cell migration in an α11 integrin chain–dependent manner 34 and collagen V might therefore be an important ligand for α11β1 in the cornea. Although a similar disturbance in the lamellar morphology was noted in the scarred keratoconus cases regarding collagen I, there were no apparent differences in the staining intensity of this collagen. 
The weaker immunoreactivity observed in the older fetal corneas probably reflects a developmental downregulation of the α11 integrin chain, suggesting that it has an initial role organizing the deposition of the collagen fibrils. 
As the staining in normal adult corneas was not completely absent, we suggest that there is a maintenance level of expression of this integrin in the normal adult cornea. 
The enhanced expression of the α11 integrin chain in the adult corneas with evidence of scarring suggests an upregulation of this fetal integrin in repairing events with collagen deposition and collagen reorganization, as suggested earlier for the α9 integrin chain after debridement in the cornea. 40 Co-detection of α11 integrin and α-SMA in cultured myofibroblasts further supports the hypothesis that integrin is upregulated during the wound-healing process. 
The increased presence of the α11 integrin chain in the anterior stroma of the scarred keratoconus corneas correlated with a damaged BM. Novel expression of (e.g., laminin chains α3, α5, β3, and γ2) in streaks in the anterior stroma/Bowman's layer of keratoconus cases with scarring where there was disarray of the epithelial BM has been previously reported). 41,42 Earlier data show that the epithelial BM plays an important role in maintaining corneal homeostasis and minimizing the fibrotic response. TGF-β is a cytokine and a potent inducer of myofibroblast transformation. 43 A break in the BM causes a release of TGF-β by the corneal epithelial cells to the stroma and induces the transformation of the keratocytes into myofibroblasts, whereas a competent BM inhibits this transformation. 44 Our data confirm that TGF-β enhances the transformation into myofibroblasts containing α-SMA and upregulates the production of the α11 integrin chain. In contrast the PBK corneas with very irregular epithelium but intact BM and Bowman's layer did not show increased staining with the α11 integrin chain. 
The scarring process involves cell migration, collagen deposition, and collagen reorganization, 17,29 and the observed presence of the α11 integrin chain in the scarred keratoconus corneas, resembling the staining pattern for α-SMA, α1-α2 chains of collagen IV and collagen V, suggests a role for this integrin chain in collagen remodeling in adult corneas. 
The chronic edema of PBK corneas is usually attributed to decreased ability of endothelial cells to remove fluid from the corneal stroma, 45,46 although epithelial processes also may play a role. 23 In advanced cases of PBK a posterior collagenous layer (retrocorneal fibrous membrane) may form. 23,45 The fact that the α11 integrin chain was more abundant in the posterior part of the stroma in the PBK corneas could reflect an upregulation of this chain related to abnormal collagen deposition (Fig. 2K). These findings further suggest a role for the α11 integrin chain in corneal repair. α11β1 has also been suggested a role in cartilage repair. 13  
To date, efforts to pharmacologically control the corneal wound-healing process, have limited success and corneal scarring is an important cause of vision impairment and blindness. Understanding how the ECM is affected in corneal disease opens new possibilities for the development of topical therapeutic agents for conditions that significantly reduce vision and, presently, require corneal transplantation. The patients with keratoconus and those with scars after trauma, infection, or refractive surgery are often younger, and thereby a long recovery period with low vision has significant impact on their working capacity, with important costs for society. Further studies are under way to elucidate the role of the α11 integrin chain in the pathogenesis of corneal scarring in a knock-out animal model. 
Footnotes
 Supported by Grants 63x-20399 from the Swedish Research Council, 183258/S10–Helse Vest from the Research Council of Norway, MEST-CT-2004-514483 from the European Union Marie Curie Early Stage Training Contract, and by funds from Stiftelsen KMA, Synfrämjandet, Margit Thyselius Fond, and Lennanders Stiftelse.
Footnotes
 Disclosure: B. Byström, None; S. Carracedo, None; A. Behndig, None; D. Gullberg, None; F. Pedrosa-Domellöf, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Margaretha Enerstedt and Karin Hjertkvist for excellent technical assistance. 
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Figure 1.
 
Cross sections of human fetal corneas at different gestational ages showing immunoreactivity for the α11 integrin chain in the corneal stroma. In all frames, the corneal epithelium is located in the top of the image. Fetal age is indicated in weeks of gestation (wg). (A, G) Low-power images of sections stained with hematoxylin eosin; the black boxes correspond to the areas shown in (B, C) and (H, I), respectively. The level of nonspecific staining is shown in sections treated with normal rabbit serum (pre, B, H) at 10 and 20 wg. Note the intense staining in streaks for the α11 integrin chain in the whole corneal stroma, accentuated in the anterior (top) part at 10 to 17 wg (C–F, I). (A–C, *) The space between the cornea and the overlying eyelid. Bar: (B–F, H, I) 50 μm; (A, G) 100 μm.
Figure 1.
 
Cross sections of human fetal corneas at different gestational ages showing immunoreactivity for the α11 integrin chain in the corneal stroma. In all frames, the corneal epithelium is located in the top of the image. Fetal age is indicated in weeks of gestation (wg). (A, G) Low-power images of sections stained with hematoxylin eosin; the black boxes correspond to the areas shown in (B, C) and (H, I), respectively. The level of nonspecific staining is shown in sections treated with normal rabbit serum (pre, B, H) at 10 and 20 wg. Note the intense staining in streaks for the α11 integrin chain in the whole corneal stroma, accentuated in the anterior (top) part at 10 to 17 wg (C–F, I). (A–C, *) The space between the cornea and the overlying eyelid. Bar: (B–F, H, I) 50 μm; (A, G) 100 μm.
Figure 2.
 
Cross sections of adult human corneas. Top row: images from the same normal cornea. (A, B) The anterior central and (C, D) anterior peripheral cornea. Note the more intense staining pattern in streaks in the periphery (D). (A, C) Sections treated with normal rabbit serum (called pre) and (B, D) anti-α11 integrin. Middle row: cross sections of corneas with Fuchs' dystrophy (E, F) and PBK (G, H). (E, F) Normal rabbit serum and α11 integrin staining in a cornea with Fuchs' dystrophy. (G, H) The anterior part of the cornea of two different patients with PBK. Note the irregular epithelium in (G) without any increased irregularities in the underlying stroma. Bottom row: the posterior part of a normal cornea (I), a cornea with Fuchs' dystrophy (J) and a PBK cornea (K, L). The staining with anti-α11 integrin is more intense in the posterior part of the PBK cornea (K, arrow) compared with the staining in the normal (I) and the Fuchs' dystrophy cornea (J). Increased cellularity visualized in (L) with DAPI staining corresponded to the more intense staining in (K). Bar, 50 μm.
Figure 2.
 
Cross sections of adult human corneas. Top row: images from the same normal cornea. (A, B) The anterior central and (C, D) anterior peripheral cornea. Note the more intense staining pattern in streaks in the periphery (D). (A, C) Sections treated with normal rabbit serum (called pre) and (B, D) anti-α11 integrin. Middle row: cross sections of corneas with Fuchs' dystrophy (E, F) and PBK (G, H). (E, F) Normal rabbit serum and α11 integrin staining in a cornea with Fuchs' dystrophy. (G, H) The anterior part of the cornea of two different patients with PBK. Note the irregular epithelium in (G) without any increased irregularities in the underlying stroma. Bottom row: the posterior part of a normal cornea (I), a cornea with Fuchs' dystrophy (J) and a PBK cornea (K, L). The staining with anti-α11 integrin is more intense in the posterior part of the PBK cornea (K, arrow) compared with the staining in the normal (I) and the Fuchs' dystrophy cornea (J). Increased cellularity visualized in (L) with DAPI staining corresponded to the more intense staining in (K). Bar, 50 μm.
Figure 3.
 
Staining patterns of keratoconus corneas treated with normal rabbit serum (A, H, N, pre), Abs to α11 integrin (B, I, O), α-SMA (D, J, P), collagen I (E, K, Q), the α1 and α2 chains of collagen IV (F, L, R), and collagen V (G, M, S). Each row represents a single keratoconus case. (A–G) A case with clinically central scarring of the cornea. Note the intense staining in the same region with different Abs (B–G, arrows). DAPI staining revealed increased cellularity in this region (C). Note also the very thick and irregular epithelium. There was staining of the epithelial BM by the Ab to the α1 and α2 chains of collagen IV (F, arrowhead). (H–M) Another scarred keratoconus cornea with intense staining in streaks by the Abs (I–M, arrows) and staining of the epithelial BM by the Abs to collagen IV and V (L, M, arrowheads). (N–S) In contrast to (H–M), a keratoconus cornea with very weak staining with antibodies to α11 integrin, α-SMA, the α1 and α2 chains of collagen IV and V, resembling the section treated with normal rabbit serum (N). Bar, 50 μm.
Figure 3.
 
Staining patterns of keratoconus corneas treated with normal rabbit serum (A, H, N, pre), Abs to α11 integrin (B, I, O), α-SMA (D, J, P), collagen I (E, K, Q), the α1 and α2 chains of collagen IV (F, L, R), and collagen V (G, M, S). Each row represents a single keratoconus case. (A–G) A case with clinically central scarring of the cornea. Note the intense staining in the same region with different Abs (B–G, arrows). DAPI staining revealed increased cellularity in this region (C). Note also the very thick and irregular epithelium. There was staining of the epithelial BM by the Ab to the α1 and α2 chains of collagen IV (F, arrowhead). (H–M) Another scarred keratoconus cornea with intense staining in streaks by the Abs (I–M, arrows) and staining of the epithelial BM by the Abs to collagen IV and V (L, M, arrowheads). (N–S) In contrast to (H–M), a keratoconus cornea with very weak staining with antibodies to α11 integrin, α-SMA, the α1 and α2 chains of collagen IV and V, resembling the section treated with normal rabbit serum (N). Bar, 50 μm.
Figure 4.
 
Staining patterns in a case of keratoconus (A, B), a case of PBK (C, D) and a normal cornea (E, F). Left column: sections stained with hematoxylin and eosin; right column: sections treated with the Ab to the α11 integrin chain. (B, inset) Laminin (LM) α5 chain staining of the epithelial BM in the same region of an adjacent section; arrowheads: breaks in the BM. Note the correspondence between the break in the Bowman's layer (A, B, arrows), epithelial BM (B), and increased staining with the Ab against the α11 integrin chain in the same area, in the keratoconus case. In the PBK case and the normal cornea, the BM and Bowman's layer are regular and uninterrupted, and there is no increase in α11 integrin chain labeling (C–F). Bar, 50 μm.
Figure 4.
 
Staining patterns in a case of keratoconus (A, B), a case of PBK (C, D) and a normal cornea (E, F). Left column: sections stained with hematoxylin and eosin; right column: sections treated with the Ab to the α11 integrin chain. (B, inset) Laminin (LM) α5 chain staining of the epithelial BM in the same region of an adjacent section; arrowheads: breaks in the BM. Note the correspondence between the break in the Bowman's layer (A, B, arrows), epithelial BM (B), and increased staining with the Ab against the α11 integrin chain in the same area, in the keratoconus case. In the PBK case and the normal cornea, the BM and Bowman's layer are regular and uninterrupted, and there is no increase in α11 integrin chain labeling (C–F). Bar, 50 μm.
Figure 5.
 
Cross sections of a DLKP cornea, where (A) and (B) are from the anterior part of the cornea and (C–F) show the posterior part. (C–F) (*) Interface between the donor and host cornea. Note the streaky staining with anti-α11 integrin chain and anti-collagen V in the recipient cornea (the posterior part, D, E, arrows) and the correspondence with increased cellularity shown with DAPI staining of the nuclei (C). Bar, 50 μm.
Figure 5.
 
Cross sections of a DLKP cornea, where (A) and (B) are from the anterior part of the cornea and (C–F) show the posterior part. (C–F) (*) Interface between the donor and host cornea. Note the streaky staining with anti-α11 integrin chain and anti-collagen V in the recipient cornea (the posterior part, D, E, arrows) and the correspondence with increased cellularity shown with DAPI staining of the nuclei (C). Bar, 50 μm.
Figure 6.
 
Cultured cells at 4 days were labeled with Abs against α-SMA (A) and the α11 integrin chain (B) after treatment with TGF-β. Note the coexistence of α-SMA and α11 integrin staining in (C) suggesting myofibroblasts as a source for the α11 integrin chain. Single cell α-SMA upregulation by TGF-β is obvious in (D) compared with untreated cells in (E). Double-staining with anti-α11 integrin and anti-vinculin 4 days after treatment with TGF-β (F–H) and untreated controls (I–K) revealed co-localization of upregulated α11 integrin with vinculin in focal adhesions.
Figure 6.
 
Cultured cells at 4 days were labeled with Abs against α-SMA (A) and the α11 integrin chain (B) after treatment with TGF-β. Note the coexistence of α-SMA and α11 integrin staining in (C) suggesting myofibroblasts as a source for the α11 integrin chain. Single cell α-SMA upregulation by TGF-β is obvious in (D) compared with untreated cells in (E). Double-staining with anti-α11 integrin and anti-vinculin 4 days after treatment with TGF-β (F–H) and untreated controls (I–K) revealed co-localization of upregulated α11 integrin with vinculin in focal adhesions.
Figure 7.
 
(A) Corneal fibroblasts plated on collagen I were treated with TGF-β and protein levels of α11 integrin and α-SMA were analyzed at indicated time points. Note higher levels of α11 integrin and α-SMA after 1 and 3 days of treatment. Signal for α11 integrin and α-SMA after 6 days of treatment were similar in the TGF-β-treated and control cell cultures. mRNA levels for collagen receptors (α1-, α2-, α10-, and α11 integrin) in corneal fibroblasts treated with TGF-β for 3 days (+) and control cells (−) are shown in (B). A reference positive control (Ref) obtained from transfected C2C12 cells was used for α10 integrin. Note the independent regulation of α11 integrin chain.
Figure 7.
 
(A) Corneal fibroblasts plated on collagen I were treated with TGF-β and protein levels of α11 integrin and α-SMA were analyzed at indicated time points. Note higher levels of α11 integrin and α-SMA after 1 and 3 days of treatment. Signal for α11 integrin and α-SMA after 6 days of treatment were similar in the TGF-β-treated and control cell cultures. mRNA levels for collagen receptors (α1-, α2-, α10-, and α11 integrin) in corneal fibroblasts treated with TGF-β for 3 days (+) and control cells (−) are shown in (B). A reference positive control (Ref) obtained from transfected C2C12 cells was used for α10 integrin. Note the independent regulation of α11 integrin chain.
Table 1.
 
Patient Data
Table 1.
 
Patient Data
Age (y) Sex Diagnosis and Comments
40 M Normal, evisceration due to trauma*
86 M Normal, donation with retinitis pigmentosa*
87 M Normal, enucleation due to choroidal tumor*
23 M Keratoconus, with a central scar
29 M Keratoconus, with a central scar
38 M Keratoconus, with a central scar
48 M Keratoconus, with a central scar
21 M Keratoconus, with central striation†
25 M Keratoconus, with central striation†
50 M Keratoconus, retransplantation‡
65 M PBK after cataract surgery
72 F PBK after cataract surgery
74 F PBK+Fuchs' with cataract surgery
78 F PBK+Fuchs' with cataract surgery
84 M PBK+Fuchs' with cataract surgery
50 F Fuchs' without earlier surgery§
59 F Fuchs' without earlier surgery§
37 M Scar, post DLKP‖
Table 2.
 
Primer Sequences and PCR Conditions Used in the Semiquantitative RT-PCR Analysis
Table 2.
 
Primer Sequences and PCR Conditions Used in the Semiquantitative RT-PCR Analysis
Forward (5′-3′) Reverse (5′-3′) Annealing T (°C) Cycles Product (bp)
α1 CGA AGA ACC TCC TGA AAC CC CGA AAC ATT GAC TTG GCT GA 52 33 478
α2 AAA AAT AAA AGG GAA AGT GC CTT GTT TTC TTC TTG GCT TT 50 33 276
α10 TCT AGA AAC CTC CAC CTG G CTG GAA GGA GGG CTG AGA TGA TGA 65 33 438
α11 GAT TCA CCA ACA GAG CCG TA TCA AAA TCA AGA ACGG AAA GC 58 30 598
β-actin GTG TGA TGG TGG GAA TGG GT TCT GGG TCA TCT TTT CAC GGT TGG 58 30 240
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