Increased ER Stress as a Mechanism of Retinal Neurovasculopathy in Mice with Severe Hyperhomocysteinemia

Research Article

Austin J Clin Ophthalmol. 2014;1(5): 1023.

Increased ER Stress as a Mechanism of Retinal Neurovasculopathy in Mice with Severe Hyperhomocysteinemia

Amany Tawfik1,2 and Sylvia B Smith1,2,3*

1Department of Cellular Biology and Anatomy, Georgia Regents University, USA

2James and Jean Culver Vision Discovery Institute, Georgia Regents University, USA

3Department of Ophthalmology, Medical College of Georgia, Georgia Regents University, Augusta, USA

*Corresponding author: Sylvia B Smith, Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Regents University, 1120 15th Street, CB 1114, Augusta, 30912-2000, GA, USA

Received: May 21, 2014; Accepted: June 13, 2014; Published: June 16, 2014


Hyperhomocysteinemia is implicated in retinal neurovascular diseases including arterial occlusive disease, venous occlusive disease and pseudoexfoliation glaucoma. The mechanism for these diseases is not known. Here we used hyperhomocysteinemic mice lacking the gene encoding cystathionine-beta-synthase (cbs -/-) to examine whether ER stress could be a mechanism for the retinal neurovasculopathy reported in these mice. Retinas of cbs+/+and cbs-/- mice (age: 3-5 wks) were used to investigate the expression of ER stress genes (BiP/GRP78, Perk, Atf6, Atf4, Ire1α,Chop) and the proteins they encode. The levels of poly (ADP-ribose) Polymerase (PARP) and cleaved cysteine-aspartic proteases-3 (caspase-3), proteins known to be involved in apoptosis, were also examined. Quantitative reverse transcription polymerase chain reaction and western blotting revealed an increase in BiP/ GRP78 and PERK in retinas of cbs -/-mice compared with cbs+/+mice. There was an elevation of CCAAT-enhancer-binding protein Homologous Protein (CHOP) in retinal cryosections of cbs -/- mice indicating apoptosis, which was confirmed by increased levels of PARP and cleaved caspase-3. The data suggest that the genes and proteins that are major players in the ER stress pathway, particularly the PERK pathway, are up regulated in retinas of cbs -/- mice. The data support a role for ER stress in the pathophysiology associated with the hyperhomocysteinemia-linked retinal disease.

Keywords : Homocysteine; Retinal degeneration; Retinal neurovasculopathy; Apoptosis; BiP/GRP78; Cystathionine-β-synthase


ER: Endoplasmic Reticulum; Hcy: Homocysteine; HHcy: Hyperhomocysteinemia; SAM: S-Adenosylmethionine; CBS: Cystathionine Beta Synthase; MTHFR: Methylene Tetrahydrofolate Reductase; NMDA: N-methyl-D-aspartate Receptor; VEGF: Vascular Endothelial Growth Factor, UPR: Unfolded Protein Response; BiP/ GRP78: Immunoglobulin Heavy Chain-binding Protein; CHOP: CCAAT-enhancer-binding Protein Homologous Protein; PERK: PKR-like Endoplasmic Reticulum Kinase; IRE1: Inositol-requiring Enzyme 1; ATF6: Activating Transcription Factor 6; ATF4: Activating Transcription Factor 4; qRT-PCR: Quantitative Reverse Transcription Polymerase Chain Reaction; HRP: Horseradish Peroxidase


This study investigated Endoplasmic Reticulum (ER) stress as a mechanism of homocysteine-linked retinopathy. Homocysteine (Hcy), a key intermediate in metabolism of methionine, is the direct precursor of S-Adenosyl Methionine (SAM), the most important methyl group donor in the body. Depending upon metabolic demands, Hcy is the substrate for regeneration of methionine (remethylation pathway) or is diverted from the methionine cycle to produce cystathionine and ultimately cysteine (transsulfuration pathway). Normal human fasting plasma levels of Hcy (total Hcy) are 5-15μmol/l; while Hyperhomocysteinemia (HHcy) is classified as: moderate (16-30μmol/l), intermediate (31-100μmol/l), severe (>100μmol/l) [1]. HHcy is an independent risk factor in cardiovascular diseases (stroke, venous thrombosis, peripheral arterial occlusive disease [1-4]) and neurodegenerative diseases (Alzheimer’s and Parkinson’s Diseases) [5,6]. Given that the retina is a neurovascular tissue, it is not surprising that many clinical studies have investigated levels of Hcy in retinopathies [7,8]. HHcy is a risk factor in retinal venous and arterial occlusions [9] including central retinal vein occlusion [10-14]. It has been linked to pseudoexfoliation glaucoma [15-19], macular degeneration [20-22] and diabetic retinopathy [23- 25].

HHcy is caused by genetic deficiencies in enzymes responsible for transsulfuration or remethylation of Hcy or by nutritional deficiencies in vitamins serving as cofactors for these enzymes (folate/B12 /B6). The most frequent genetic deficiencies are associated with the enzymes Cystathionine Beta Synthase (CBS) and Methylene Tetrahydrofolate Reductase (MTHFR). There are more than 150 disease-associated CBS mutations, making this the most common genetic cause of severely elevated Hcy [26,27]. The I278T mutation accounts for 25% of all homocystinuric alleles, and is a common cause of Homocystinuria (severe HHcy), a genetically inherited, autosomal recessive inborn error of metabolism (1:200,000 live U.S. births) [28]. The cornerstone clinical features of these patients are mental retardation, ectopia lentis, visual deficits and skeletal abnormalities; premature death istypically due to thromboembolic events [29].

Our lab and others have performed in vitro and in vivo studies to elucidate mechanisms of HHcy-linked retinal disease. A clinically relevant experimental system is the mouse deficient or lacking the gene encoding CBS allowing studies of the effects of mild to severe endogenous elevation of Hcy [30]. In previous studies, we examined consequences on retina structure and function using either cbs-/-mice, which have a 30-40 fold increase in plasma Hcy and a shortened life-span of 3-5 weeks; or cbs+/-mice, which have a much milder HHcy with ~4-7 fold increase in plasma Hcy (and a 2-fold increase in retinal Hcy) and a normal lifespan. Our work has shown that both cbs-/-and cbs+/-mice have retinal neuronal involvement and disruption of the retinal vasculature [31-36].

To understand mechanisms for HHcy-induced retinal neuronal death we previously investigated the role of excitotoxicity and oxidative stress using perforated patch clamp analysis and fluorescent detection of intracellular Ca2+ in primary mouse retinal ganglion cells and found that Hcy-induced cell death, which was blocked partially by MK-801, an N-methyl-D-aspartate receptor (NMDA) receptor antagonist [36]. Hcy increased intracellular Ca2+ 7-fold. Additionally exposure of ganglion cells to 50 μM Hcy increased levels of superoxide, nitric oxide and peroxynitrite levels by 40%, 90% and 85%, respectively. We also investigated retinal vasculature in mice with HHcy and observed a marked vasculopathy developing very early in cbs-/- mice associated with increased levels of Vascular Endothelial Growth Factor (VEGF) [33]. Interestingly, VEGF has been linked to ER stress in various systems including retina [37-39], prompting this investigation of well-known ER stress markers. We were interested in determining whether ER stress plays a role in Hcy-induced retinal neurovascular pathology observed incbs-/- mice.

Materials and Methods


The generation of mice deficient in cbs has been reported [30]. Breeding pairs of cbs+/- mice (B6.129P2-Cbstm1Unc/J; Jackson Laboratories, Bar Harbor, ME) were used to establish our mouse colony. Genotyping, husbandry and housing conditions for the mice have been described [33]. Wild type (cbs+/+, n = 17) and homozygous mutant (cbs-/-, n = 18) mice were used in this study at ~3 weeks. Mean body weight for cbs+/+ mice (6.84 ± 0.2 g) was significantly greater than age-matched cbs-/- mice (4.53 ± 0.2 g).Experiments adhered to the Association for Research in Vision and Ophthalmology Statement forthe Use of Animals in Ophthalmic and Vision Research and followed our animal use protocol approved by the Institutional Animal Care and Use Committee of Georgia Regents University.

Analysis of VEGF levels in retinas of severely hyperhomocysteinemic mice

Elevation of VEGF has been linked to increased ER stress in various tissues, this observation, along with other evidence suggesting ER stress as a mechanism of HHcy-induced pathology, prompted the current experiments. We performed immunohistochemical studies in cryosections using mouse monoclonal anti-VEGF (IgM) (1:250, Abcam Corp., Cambridge, MA) and detected the protein using Alexafluor 488 (donkey anti-mouse antibody, Invitrogen, Eugene, OR).The Metamorph analysis system was used to quantify immunofluorescence levels.

Real time quantitative RT-PCR (RT-q PCR) analysis of genes in the ER stress pathways

Expression levels of mRNA transcripts specific for several key genes involved in ER stress pathways (BiP/GRP78, Perk, Atf6, Ire1α, Atf4, Chop) were examined in neural retinas isolated from cbs+/+ and cbs-/- mice per our method [40]. Total RNA was isolated using TRIzolTM Reagent (Invitrogen, Carlsbad, CA) and quantified. 2 μg of RNA was reverse transcribed using iScript™ Synthesis kit (BioRad Laboratories, Hercules, CA). cDNAs were amplified for 45 cycles using Absolute QPCR SYBR Green Fluorescein (AB gene, Surrey, UK) and gene specific primers (Table 1) using the Bio-Rad icycler (Hercules, CA). Expression levels were calculated by comparison of Ct values (delta-delta Ct).PCR was performed (40 cycles: 95°C for 30 s; 60°C for 30 s; 72°C for 30 s); melt curve analysis confirmed end product purity. Resulting CT values were normalized to 18s and analyzed using the comparative CT method to obtain fold-changes in gene expression.