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.

Methods

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.

TABLE 1

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.

TABLE 2

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

 

Keratoconus Patient Data
	Age (yrs)	Gender	Diagnosis	Keratometry (D)	Pachymetry (μm)	Severity
	26	Male	Keratoconus	65	411	Severe
	46	Female	Keratoconus	70	277	Severe
	26	Female	Keratoconus	63	370	Severe
	58	Female	Keratoconus	65	323	Severe
	23	Male	Keratoconus	64	381	Severe
Mean	36			65	352
SD	15			3	53

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