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1.  Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases 
Biochimica et biophysica acta  2013;1842(2):208-219.
The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.
PMCID: PMC4326177  PMID: 24200652
Mitochondrial haplogroups; transmitochondrial cybrids; inflammation; complement; mitochondria; complement activation; innate immunity; haplogroups; cybrids; retina
2.  Steroid Differentiation: The Safety Profile of Various Steroids on Retinal Cells in Vitro and their Implications for Clinical Use (An American Ophthalmological Society Thesis) 
To determine if potentially viable alternatives to the clinical use of intravitreal triamcinolone acetonide should be considered based on a comparative assessment of the in vitro effects of five commercially available corticosteroids. We hypothesized that dexamethasone, betamethasone, methylprednisolone, loteprednol etabonate, and fluocinolone acetonide, at clinically relevant doses, may show different levels of in vitro cytotoxicity to retinal cells.
Cultures of human retinal pigment epithelial cells (ARPE-19) and rat embryonal neurosensory precursor retinal cells (R28) were treated with dexamethasone, betamethasone, methylprednisolone, loteprednol, or fluocinolone acetonide. Cell viability as a measure of cell death was determined by trypan blue dye exclusion assay. The mechanical effect of drug crystals was evaluated by solubilizing the steroid formulations. Mitochondrial dehydrogenase and membrane potential were assessed to measure cell damage.
Betamethasone, loteprednol, and methylprednisolone, in commercially available forms, caused significant cytotoxic changes to retinal cells in vitro at clinically relevant doses. This effect was less pronounced with solubilized betamethasone. Dexamethasone at concentrations up to 5 times the clinical dose of free drug injections and 1000 times greater than a drug implant did not cause decreased cell viability. Fluocinolone acetonide at doses 1000 times higher than observed with drug delivery systems showed no cytotoxic effect.
Betamethasone, loteprednol, and methylprednisolone exhibited cytotoxicity at clinically relevant doses and do not appear to be good therapeutic options for intravitreal use. In comparison, dexamethasone and fluocinolone acetonide, which exhibited fewer cytotoxic effects than other steroids, may be potentially viable alternatives to triamcinolone acetonide for clinical use.
PMCID: PMC4311675  PMID: 25646032
3.  Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases 
PLoS ONE  2014;9(6):e99003.
It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation.
Methodology/Principal Findings
Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids.
In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.
PMCID: PMC4053329  PMID: 24919117
4.  Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro 
Indian Journal of Ophthalmology  2014;62(4):429-436.
To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells.
Materials and Methods:
HTM cells were cultured and treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II.
Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 μg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 μg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 μg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S.
Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 μg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.
PMCID: PMC4064217  PMID: 24817746
Triamcinolone acetonide; human trabecular meshwork cells; in vitro
5.  Effect of bevacizumab (Avastin™) on mitochondrial function of in vitro retinal pigment epithelial, neurosensory retinal and microvascular endothelial cells 
Indian Journal of Ophthalmology  2013;61(12):705-710.
To evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial (HMVEC) cells in culture.
Materials and Methods:
ARPE-19 and R28 cells were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab. The HMVEC cultures were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab or 1 mg/ml of immunoglobulin G (control). Mitochondrial function assessed by mitochondrial dehydrogenase activity (MDA) was determined using the WST-1 assay.
Bevacizumab doses of 0.125 to 1 mg/ml for 5 days did not significantly affect the MDA of ARPE-19 cells. Bevacizumab treatment at 0.125 and 0.25 mg/ml (clinical dose) did not significantly affect the MDA of R28 cells; however, 0.50 and 1 mg/ml doses significantly reduced the R28 cell mitochondrial function. All doses of bevacizumab significantly reduced the MDA of proliferating and non-proliferating HMVEC.
Bevacizumab exposure for 5 days was safe at clinical doses in both ARPE-19 and R28 retinal neurosensory cells in culture. By contrast, bevacizumab exposure at all doses show a significant dose-dependent decrease in mitochondrial activity in both the proliferating and non-proliferating HMVEC in vitro. This suggests a selective action of bevacizumab on endothelial cells at clinical doses.
PMCID: PMC3917387  PMID: 24413824
Bevacizumab; mitochondrial function; microvascular endothelial cells; neurosensory retinal cells; retinal pigment epithelial cells; tissue culture
6.  Effects of a superoxide dismutase mimetic on biomarkers of lung angiogenesis and alveolarization during hyperoxia with intermittent hypoxia 
Extremely premature neonates requiring oxygen therapy develop an accumulation of reactive oxygen species (ROS), impaired alveolarization and dysmorphic pulmonary vasculature. Regulators of ROS (i.e. antioxidants), alveolarization (i.e. matrix metalloproteinases - MMPs) and microvascular maturation (i.e. vascular endothelial growth factor - VEGF) are altered in bronchopulmonary dysplasia (BPD). We tested the hypothesis that early treatment with MnTBAP, a superoxide dismutase mimetic and superoxide anion and peroxynitrite scavenger, alters lung biomarkers of angiogenesis and alveolarization during hyperoxia with intermittent hypoxia (IH) in neonatal rats. Neonatal rats were exposed to 50% O2 with brief IH episodes (12% O2) from P0 to P14, or to room air (RA). On P0, P1 & P2, the pups received a daily IP injection of 1, 5, or 10 mg/kg MnTBAP, or saline. At P14, the pups were either euthanized, or allowed to recover in RA until P21. RA littermates were similarly treated. Lung VEGF, sVEGFR-1, MMP-2, MMP-9 and TIMP-1 were determined. Low-dose MnTBAP (1 mg/kg) prevented the increase in lung VEGF induced by intermittent hypoxia noted in the control group. This dose was also effective for decreasing MMP-9 and MMP-9/TIMP-1 ratio suggesting an anti-inflammatory effect for MnTBAP. IH decreased MMP-2 with no ameliorating effect by MnTBAP. Our data demonstrate that brief, repeated intermittent hypoxia during hyperoxia can alter biomarkers responsible for normal microvascular and alveolar development. In addition to prevention of hypoxic events, the use of antioxidants needs to be explored as a possible therapeutic intervention in neonates at risk for the development of oxidative lung injury.
PMCID: PMC3786267  PMID: 24093057
Antioxidants; hyperoxia; intermittent hypoxia; matrix metalloproteinases; tissue inhibitor of metalloproteinase
7.  Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration 
PLoS ONE  2013;8(1):e54339.
Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).
Methodology/Principal Findings
Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.
Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.
PMCID: PMC3554762  PMID: 23365660
8.  Mitochondrial DNA haplogroups confer differences in risk for age-related macular degeneration: a case control study 
BMC Medical Genetics  2013;14:4.
Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170).
Total DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene.
The JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background.
There is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.
PMCID: PMC3566905  PMID: 23302509
Age-related macular degeneration; Mitochondrial haplogroups; mtDNA; CFH; ARMS2
9.  Valganciclovir in the treatment of cytomegalovirus retinitis in HIV-infected patients 
Oral valganciclovir is a new and highly efficacious alternative to the chronic administration of ganciclovir in the treatment of cytomegalovirus (CMV) retinitis in HIV-infected patients. In addition to its excellent bioavailability and favorable pharmacokinetic profile, valganciclovir has also proved cost effective and is the most widely used drug in the armamentarium for the treatment of CMV retinitis. Valganciclovir is a prodrug of ganciclovir, the erstwhile commonly used therapy. In March 2001, the US Food and Drug Administration approved valganciclovir for the induction and maintenance treatment of CMV disease, including CMV retinitis. Valganciclvoir has compared favorably with both oral and intravenous treatments for induction and maintenance therapy with ganciclovir. The reduced pill burden and the ease of oral administration has helped avoid the risks associated with intravenous therapy. The most serious adverse event is neutropenia, which makes the patient susceptible to infections. In the current review, we have compiled all the available evidence-based information on valganciclovir.
PMCID: PMC2835533  PMID: 20234777
CMV retinitis; ganciclovir; valganciclovir
10.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
11.  Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals 
To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization.
The Eye Institute, University of California, Irvine, California, USA.
Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed.
Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman’s layer, suggesting an anchoring function.
Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.
PMCID: PMC1855208  PMID: 17081858
12.  Use of Intravitreal Triamcinolone in the Treatment of Macular Edema Related to Retinal Vein Occlusion 
To analyze the increasing trend of intravitreal triamcinolone (IVTA) use in the treatment of retinal vein occlusion-related macular edema.
We performed MEDLINE/PUBMED searches (September 1984 - December 2007) to identify articles containing the keywords macular edema and triamcinolone. Case reports, reviews and abstracts were identified from references in the reviewed literature. This review focuses on literature published during the past 7 years with more than two-thirds of the articles that we reviewed being printed during the past 5 years. These reports analyzed the success of IVTA in the treatment of macular edema over a 12 month course of time.
The majority of studies suggested promising results for short time periods (4-6 months) after IVTA treatments. However, long term results were not encouraging.
The success of IVTA therapy for short durations has been the impetus for development of sustained release devices to be used in the treatment of macular edema associated with various retinal diseases including edema related to retinal vein occlusion.
PMCID: PMC2694595  PMID: 19517029
Intravitreal triamcinolone; macular edema; retinal vein occlusion.
13.  Second-Harmonic Imaging Microscopy of Normal Human and Keratoconus Cornea 
The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas.
Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae.
Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman’s layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 μm. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman’s layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman’s layer or scarring.
Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.
PMCID: PMC1894888  PMID: 17325150
14.  Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture 
The British Journal of Ophthalmology  2014;98(Suppl 1):i11-i16.
To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture.
Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death.
Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses.
At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses.
PMCID: PMC4033208  PMID: 24836865
Macula; Treatment Medical; Angiogenesis

Results 1-14 (14)