Transforming Growth Factor-β Signaling Pathway Activation in Keratoconus


To assess the presence of transforming growth factor-β (TGFβ) pathway markers in the epithelium of keratoconus patient corneas.


Retrospective, comparative case series of laboratory specimens.


Immunohistochemistry results for TGFβ2, total TGFβ, mothers against decacentaplegic homolog (Smad) 2, and phosphorylated Smad2 was performed on formalin-fixed, paraffin-embedded sections of keratoconus patient corneas and normal corneas from human autopsy eyes. Keratoconus patient corneas were divided in two groups, depending on their severity based on keratometer readings and pachymetry. Autopsy controls were age-matched with the keratoconus cases. Immunohistochemistry signal quantification was performed using automated software. Real-time reverse-transcriptase polymerase chain reaction was performed on total ribonucleic acid of epithelium of keratoconus patient corneas and autopsy control corneas.


Immunohistochemistry quantification showed a significant increase in mean signal in the group of severe keratoconus cases compared with normal corneas for TGFβ2 and phosphorylated Smad2 ( P < .05). Immunohistochemistry analysis using antibodies against total TGFβ and Smad2 did not show any significant increase in the keratoconus cases versus the autopsy controls. Reverse-transcriptase polymerase chain reaction exhibited elevated messenger ribonucleic acid levels of Smad2 and TGFβ2 in severe keratoconus corneal epithelium.


This work shows increased TGFβ pathway markers in severe keratoconus cases and provides the rationale for investigating TGFβ signaling further in the pathophysiology of keratoconus.

Keratoconus is a bilateral progressive corneal disease, leading to thinning, scarring, and protrusion of the central cornea. The origin and the pathogenesis of this disorder are not well understood. Although most often an isolated disease, it has been associated with several accompanying factors such as Down syndrome, contact lens wear, connective tissue disease, atopy, and eye rubbing, and it can occur in a familial setting. Keratoconus most likely is caused by multiple genes and may result from complex interactions between genes and environmental factors. Therapeutic measures focus first on the correction of refractive errors. Although preliminary results on riboflavin/ultraviolet-A-induced collagen-crosslinking suggest a favorable outcome, in the advanced stages, corneal transplantation is still the most effective treatment to date. Keratoconus in fact is the most common indication for keratoplasty. Gaining more insight into the mechanisms of keratoconus to find ways to prevent disease progression or to discover new treatment options therefore would be an important accomplishment.

Histologically, in the course of the disease, breaks in Bowman membrane and subepithelial scarring can be observed. Furthermore, the affected areas have marked alterations in the components of the extracellular matrix and show apoptotic cells, which, along with the thinning of the corneal stroma, suggest an increased activation of degrading enzymes and cell death resulting from oxidative stress. However, the exact mechanisms of the tissue breakdown remain unclear.

The signaling pathway of transforming growth factor-β (TGFβ) is a complex, multibranched signal transduction cascade that may modulate ECM alterations in keratoconus. TGFβ, with its 3 isoforms, TGFβ1, TGFβ2, and TGFβ3, is only one of numerous ligands of the TGFβ superfamily that bind to the TGFβ receptors that exist in 3 different isoforms. Binding of ligands to the TGFβ2 receptor, which has an intrinsic serin/threonine kinase activity, leads to recruitment and phosphorylation of the TGFβ1 receptor, which subsequently phosphorylates the mothers against decacentaplegic homolog (Smad) 2 and Smad3 proteins intracellularly. The Smad proteins are homologs of the Drosophila protein mothers against decapentaplegic and the C. elegans protein SMA. Phosphorylated Smad2 (pSmad2) forms a complex with the mediator Smad4 and is translocated into the nucleus, where it acts as a transcription factor for multiple TGFβ-dependent genes. Smad2 and Smad3 can be activated as well by non-TGFβ growth factors, which are capable of activating mitogen-activated protein kinases. These multiple growth factors include fibroblast growth factor, insulin-like growth factor-1, hepatocyte growth factor, and endothelial growth factor. Many of the cellular effects of the TGFβ pathway have in common their involvement in the restoration of normal tissue after injury by induction of both extracellular matrix and matrix-degrading enzymes. The involvement of the TGFβ pathway in the modulation and production of extracellular matrix suggests involvement in the pathogenesis of keratoconus, either in a causative role or a secondary repair response leading to structural changes in keratoconus. However, previous reports linking the TGFβ pathway with the pathogenesis of keratoconus have been inconclusive. Although Maier and associates found TGFβ2 levels to be elevated in the aqueous humor in keratoconus cases, immunofluorescence studies on TGFβ2 in patients with keratoconus did not show an increase in staining as compared with normal controls. This work attempts to elucidate the role of the TGFβ signaling pathway in keratoconus by focusing on the extracellular receptor ligands TGFβ and its isoforms, as well as the intracellular activation of Smad2, by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) of keratoconus epithelium.


Patients and Controls

The clinical diagnosis of keratoconus was made by fellowship-trained corneal specialists. Diagnosis of keratoconus was based on corneal topography along with the presence of standard clinical signs. Cases for immunohistochemistry experiments were divided in two groups depending on disease severity. Severe cases were regarded as those with mean keratometry readings K ≥ 50 diopters (D) or a pachymetry reading of ≤ 400 μm, and mild cases were categorized as those with both K < 50 D and a pachymetry reading of > 400 μm. If both readings were not available, cases were classified on available data. Keratometry readings were obtained using the Pentacam (Oculus, Wetzlar, Germany) or Orbscan II (Bausch & Lomb, Rochester, New York, USA) devices, pachymetry was obtained using ultrasound (DGH Technologies, Exton, Pennsylvania, USA). Corneas from autopsy cases with no history of corneal disease served as controls. Patient and control characteristics are summarized in Table 1 (immunohistochemistry) and Table 2 (RT-PCR). No patients in this study were wearing contact lenses at the time of keratoplasty.


Autopsy Control and Keratoconus Patient Demographic Data for Immunohistochemistry Results

Autopsy Patient Data
Age (yrs) Gender Corneal Status Cause of Death Death to Preservation Time (hrs)
46 Male Normal Multiple organ failure 19
40 Male Normal Non-Hodgkin lymphoma 21
48 female Normal Multiple organ failure 6
33 female Normal Acquired immunodeficiency syndrome 13
48 Male Normal Lung failure 22
Mean 43 16
SD 6 7

Keratoconus Patient Data
Age (yrs) Gender Diagnosis Keratometry (D) Pachymetry (μm) Severity
25 Male Keratoconus 44 624 Mild
35 Female Keratoconus Unknown 443 Mild
27 Female Keratoconus 43 Unknown Mild
39 Female Keratoconus Unknown 427 Mild
42 Female Keratoconus Unknown 565 Mild
16 Male Keratoconus 42 538 Mild
Mean 31 43 519
SD 10 1 83
54 Female Keratoconus 63 321 Severe
28 Male Keratoconus 41 380 Severe
68 Female Keratoconus 63 420 Severe
35 Female Keratoconus 56 445 Severe
17 Male Keratoconus Unknown 400 Severe
26 Female Keratoconus 51 275 Severe
75 Male Keratoconus 89 359 Severe
Mean 43 61 371
SD 22 15 59

D = diopters; hrs = hours; SD = standard deviation; yrs = years.


Autopsy Control and Keratoconus Patient Demographic Data for Quantitative Reverse-Transcriptase Polymerase Chain Reaction

Autopsy Patient Data
Age (yrs) Gender Corneal Status Cause of Death Death to Preservation Time (hrs)
50 Male Normal Hypertrophic cardiomyopathy 26
29 Female Normal Metastatic gall bladder carcinoma 15
84 Male Normal Coronary artery disease 36
58 Female Normal Metastatic gastric carcinoma 10
Mean 55 22
SD 23 12

Jan 16, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Transforming Growth Factor-β Signaling Pathway Activation in Keratoconus
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