Fuchs endothelial corneal dystrophy (Fuchs dystrophy) is a primary disease of the corneal endothelium characterized by loss of endothelial cells and abnormalities of Descemet membrane. These pathologic changes lead progressively to corneal edema and loss of vision, occurring approximately 20 years after disease onset. Fuchs dystrophy recently has been characterized as having an early-onset form, with diagnosis as early as the first decade of life, and a late-onset form with diagnosis typically between the third and fourth decades of life. Previous studies of Fuchs dystrophy pathogenesis indicate increased endothelial cell apoptosis. However, early disease stages are asymptomatic, and studies of Fuchs dystrophy tissues are limited to failed patient corneas undergoing corneal transplant surgery. Thus, the cellular pathophysiologic features of Fuchs dystrophy remain poorly understood.

Protein folding is critical for cellular functioning, and all cells have mechanisms to ensure proper protein folding and disposal of irreversibly misfolded proteins. Accumulation of misfolded proteins can result in endoplasmic reticulum stress, a condition that is toxic to cells. To counteract this stress, cells initiate the unfolded protein response, which is a comprehensive program to reduce the accumulation of toxic unfolded proteins. Endoplasmic reticulum stress and unfolded protein response have been shown to play important roles in the pathogenesis of multiple human diseases, including diabetes, Alzheimer and Parkinson diseases, and atherosclerosis. In addition, endoplasmic reticulum stress and unfolded protein response have been implicated in a variety of ophthalmologic diseases, including cataract, retinitis pigmentosa, and glaucoma.

The unfolded protein response consists of 3 effector arms mediated by 3 endoplasmic reticulum transmembrane receptors: pancreatic endoplasmic reticulum kinase–like endoplasmic reticulum kinase, activating transcription factor 6, and inositol requiring enzyme 1. These effectors are maintained in an inactive state by binding to the intraluminal endoplasmic reticulum chaperone protein GRP78. Additional important markers of unfolded protein response activation include the α subunit of eukaryotic initiation factor 2 (phospho-eIF2α), which is phosphorylated by pancreatic endoplasmic reticulum kinase–like endoplasmic reticulum kinase under endoplasmic reticulum stress conditions and C/EBP homologous protein (CHOP). Levels of GRP78, phospho-eIF2α, and CHOP are increased in cells undergoing unfolded protein response. Failure to alleviate endoplasmic reticulum stress by unfolded protein response can lead to cellular apoptosis. The complex molecular mechanisms involved in the apoptotic death of endoplasmic reticulum–stressed cells have not been elucidated fully. However, downstream effectors of unfolded protein response–mediated apoptosis in humans may include caspase 4, caspase 3, and caspase 9 ( Figure 1 ).

Simplified diagram of the unfolded protein response. Three major effectors of unfolded protein response, IRE1, ATF6, and PERK, are maintained in an inactive, membrane bound form by binding with GRP78. Binding of unfolded proteins to GRP78 causes release of GRP78 from the 3 unfolded protein response effector proteins and results in their activation. Activated ATF6 and IRE1 lead to increased activity of CHOP protein, which stimulates unfolded protein response. Activated PERK also activates CHOP via phosphorylation of eIF2α. Depending on the magnitude and duration of unfolded protein stress, unfolded protein response results in either cell recovery or cell death via apoptosis mediated by caspase 3 and 9.

ATF6 = activating transcription factor 6; CHOP = C/EBP homologous protein; GRP78 = glucose response protein 78; IRE1 = inositol requiring enzyme 1; PERK = pancreatic ER kinase (PKR)-like ER kinase; phospho-eIF2α = phosphorylated eukaryotic initiation factor 2α.

We hypothesized that unfolded protein response is activated in Fuchs dystrophy by the abundant production of misfolded protein, as indicated by thickening of Descemet membrane and the accumulation of wide-spaced collagen aggregates, which are hallmark findings in Fuchs dystrophy. We tested this hypothesis by electron microscopic assessment of endoplasmic reticulum in Fuchs dystrophy patients to evaluate for structural evidence of unfolded protein response activation. In addition, we performed immunohistochemistry of Fuchs dystrophy corneal endothelium for 3 unfolded protein response markers (GRP78, phospho-eIF2α, and CHOP) and 2 apoptosis markers (caspase 3 and 9).

Methods

Patients and Controls

The diagnosis of Fuchs dystrophy and keratoconus was made by fellowship-trained corneal specialists. For Fuchs dystrophy specimens, diagnosis was based on the presence of confluent endothelial guttae in both eyes and presence of corneal edema (stromal, epithelial, or both) in the operative eye. Diagnosis of keratoconus was based on corneal topography along with the presence of standard clinical signs. Corneas from autopsy cases with no history of corneal disease or pathologic corneas with diagnoses other than Fuchs dystrophy served as controls. Patient and control characteristics are summarized in Table 1 (TEM experiments) and Table 2 (immunohistochemistry).

TABLE 1

Presence of Prominent Rough Endoplasmic Reticulum in the Corneal Endothelium of Fuchs Dystrophy and Control Corneas Assessed by Transmission Electron Microscopy

	Prominent RER +/−	Diagnosis
Controls
1	−	Granular dystrophy
2	−	Lattice dystrophy
3	+	Macular dystrophy
4	+/−	Lattice dystrophy
5	−	Macular dystrophy
6	−	Lattice dystrophy
7	−	Macular dystrophy
8	+/−	Granular dystrophy
9	−	Granular dystrophy
Fuchs dystrophy
1	+	Fuchs dystrophy
2	+	Fuchs dystrophy
3	+	Fuchs dystrophy
4	+	Fuchs dystrophy
5	+	Fuchs dystrophy
6	+	Fuchs dystrophy
7	+	Fuchs dystrophy
8	+	Fuchs dystrophy
9	+	Fuchs dystrophy
10	+	Fuchs dystrophy

 

RER = rough endoplasmic reticulum.

TABLE 2

Patient Information for Corneas Used for Immunohistochemistry

Autopsy Corneas
	Age (yrs)	Cause of Death	Corneal Findings	Gender	Death to Preservation Time (hours)
	19	Cystic fibrosis	Normal	Male	16
	54	Larynx carcinoma	Normal	Female	11
	70	Liver cirrhosis	Normal	Male	44
	92	Aspiration pneumonia	Normal	Male	49
	58	Human immunodeficiency virus	Normal	Male	13
	65	Pneumonia	Normal	Female	58
	59	Adenocarcinoma gall bladder	Normal	Female	15
	53	Multisystem organ failure	Normal	Male	21
	6	Cerebral infarctions	Normal	Male	23
	63	Metastatic carcinoma	Normal	Male	52
	75	Aneurysm rupture	Normal	Female	53
	80	Aspiration pneumonia	Normal	Male	46
	47	Lung cancer	Normal	Male	59
	60	Necrotizing pancreatitis	Normal	Male	7
	45	Pulmonary failure, liver transplantation rejection	Normal	Male	63
	51	Metastatic melanoma	Normal	Male	24
	71	Adenocarcinoma colon	Normal	Male	30
	67	Gastrointestinal hemorrhage	Normal	Male	43
Mean	58				35
SD	20				19

 

Fuchs Dystrophy Corneas
	Age (yrs)	Clinical Diagnosis	Pathologic Diagnosis	Gender
	38	Fuchs dystrophy	Fuchs dystrophy	Female
	60	Fuchs dystrophy	Fuchs dystrophy	Male
	89	Fuchs dystrophy	Fuchs dystrophy	Female
	68	Fuchs dystrophy	Fuchs dystrophy	Male
	34	Pseudophakic bullous keratopathy	Fuchs dystrophy	Male
	68	Fuchs dystrophy	Fuchs dystrophy	Male
	74	Fuchs dystrophy	Fuchs dystrophy	Female
	67	Fuchs dystrophy	Fuchs dystrophy	Male
Mean	62
SD	18

 

Keratoconus Corneas
	Age (yrs)	Clinical Diagnosis	Pathologic Diagnosis	Gender
	16	Keratoconus	Keratoconus	Male
	34	Keratoconus	Keratoconus	Female
	18	Keratoconus	Keratoconus	Female
	15	Keratoconus	Keratoconus	Male
	14	Keratoconus	Keratoconus	Male
Mean	19
SD	8

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