Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

TGF-ß2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.

Purpose: The multifunctional profibrotic cytokine TGF-ß2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-ß2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-ß2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-ß2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-ß2 profibrotic responses in cultured primary human TM cells. Methods: Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-ß2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining. Results: Quiescent primary human TM cells cultured in the presence of TGF-ß2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-ß2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-ß2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-ß2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation. Conclusions: TGF-ß2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-ß2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-ß2 elicited profibrotic responses within the TM.

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants.

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Mitochondrial-Targeted Antioxidants Attenuate TGF-ß2 Signaling in Human Trabecular Meshwork Cells.

Purpose: POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-ß2-mediated remodeling of the ECM. Methods: Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-ß2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-ß2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Results: Primary or transformed human TM cells cultured in the presence of TGF-ß2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-ß2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 µM) or MitoQ (10 nM) attenuated TGF-ß2-mediated changes in Smad-dependent transcriptional activity. Conclusions: The multifunctional profibrotic cytokine TGF-ß2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-ß2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-ß2-mediated remodeling of the ECM within the TM.

Critical role of endoplasmic reticulum stress in chronic endothelial activation-induced visual deficits in tie2-tumor necrosis factor mice.

Diabetic retinopathy (DR) is the leading cause of vision loss among working-age adults. The interplay between hyperglycemia and endothelial activation in inducing endoplasmic reticulum (ER) stress pathways and visual deficits in DR is not fully understood. To address this, we used a mouse model of chronic vascular activation using endothelial-specific tumor necrosis factor-α (TNF-α)-expressing (tie2-TNF) mice to induce diabetes with streptozotocin. At 4 weeks post streptozotocin, a significant 2-fold to 10-fold increase in retinal neurovascular inflammatory gene transcript response in tie2-TNF mice was further increased in diabetic tie2-TNF mice. A decrease in visual acuity and scotopic b-wave amplitude in tie2-TNF mice was further accentuated in diabetic tie2-TNF mice and these changes correlated with a multi-fold increase in retinal ER stress markers and a reduction in adherens junctions. Cultured retinal endothelial cells showed a significant decrease in trans-endothelial resistance as well as VE-cadherin expression under TNF-α and high glucose stress. These changes were partly rescued by tauroursodeoxycholic acid, a potent ER stress inhibitor. Taken together, constant endothelial activation induced by TNF-α further exacerbated by hyperglycemia results in activation of ER stress and chronic proinflammation in a feed forward loop ultimately resulting in endothelial junction protein alterations leading to visual deficits in the retina. Inhibition of ER stress and endothelial activation may prove to be a novel therapeutic target in DR.

Lymphoblastoid Cell Lines as a Tool to Study Inter-Individual Differences in the Response to Glucose.

BACKGROUND: White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy. METHODS: Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species. RESULTS: Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001). CONCLUSIONS: Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.

hERG1/Kv11.1 activation stimulates transcription of p21waf/cip in breast cancer cells via a calcineurin-dependent mechanism.

The function of Kv11.1 is emerging in breast cancer biology, as a growing body of evidence indicates that the hERG1/Kv11.1 potassium channel is aberrantly expressed in several cancer types including breast cancers.The biological effects of Kv11.1 channel blockers and their associated side effects are very well known but the potential use of Kv11.1 activators as an anticancer strategy are still unexplored. In our previous work, we have established that stimulation of the Kv11.1 potassium channel activates a senescent-like program that is characterized by a significant increase in tumor suppressor protein levels, such as p21waf/cip and p16INK4A. In this study we investigated the mechanism linking Kv11.1 stimulation to augmentation of p21waf/cip protein level. We have demonstrated that the Kv11.1 channel activator NS1643 activates a calcineurin-dependent transcription of p21waf/cip and that this event is fundamental for the inhibitory effect of NS1643 on cell proliferation. Our results reveal a novel mechanism by which stimulation of Kv11.1 channel leads to transcription of a potent tumor suppressor and suggest a potential therapeutic use for Kv11.1 channel activators.

Voltage-gated ion channels in cancer cell proliferation.

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl.

Role of FAM18B in diabetic retinopathy.

PURPOSE: Genome-wide association studies have suggested an association between a previously uncharacterized gene, FAM18B, and diabetic retinopathy. This study explores the role of FAM18B in diabetic retinopathy. An improved understanding of FAM18B could yield important insights into the pathogenesis of this sight-threatening complication of diabetes mellitus. METHODS: Postmortem human eyes were examined with immunohistochemistry and immunofluorescence for the presence of FAM18B. Expression of FAM18B in primary human retinal microvascular endothelial cells (HRMECs) exposed to hyperglycemia, vascular endothelial growth factor (VEGF), or advanced glycation end products (AGEs) was determined with quantitative reverse-transcription PCR (qRT-PCR) and/or western blot. The role of FAM18B in regulating human retinal microvascular endothelial cell viability, migration, and endothelial tube formation was determined following RNAi-mediated knockdown of FAM18B. The presence of FAM18B was determined with qRT-PCR in CD34+/VEGFR2+ mononuclear cells isolated from a cohort of 17 diabetic subjects with and without diabetic retinopathy. RESULTS: Immunohistochemistry and immunofluorescence demonstrated the presence of FAM18B in the human retina with prominent vascular staining. Hyperglycemia, VEGF, and AGEs downregulated the expression of FAM18B in HRMECs. RNAi-mediated knockdown of FAM18B in HRMECs contributed to enhanced migration and tube formation as well as exacerbating the hyperglycemia-induced decrease in HRMEC viability. The enhanced migration, tube formation, and decrease in the viability of HRMECs as a result of FAM18B downregulation was reversed with pyrrolidine dithiocarbamate (PDTC), a specific nuclear factor-kappa B (NF-κB) inhibitor. CD34+/VEGFR2+ mononuclear cells from subjects with proliferative diabetic retinopathy demonstrated significantly reduced mRNA expression of FAM18B compared to diabetic subjects without retinopathy. CONCLUSIONS: FAM18B is expressed in the retina. Diabetic culture conditions decrease the expression of FAM18B in HRMECs. The downregulation of FAM18B by siRNA in HRMECs results in enhanced migration and tube formation, but also exacerbates the hyperglycemia-induced decrease in HRMEC viability. The pathogenic changes observed in HRMECs as a result of FAM18B downregulation were reversed with PDTC, a specific NF-κB inhibitor. This study is the first to demonstrate a potential role for FAM18B in the pathogenesis of diabetic retinopathy.

pH shift assembly of adenoviral serotype 5 capsid protein nanosystems for enhanced delivery of nanoparticles, proteins and nucleic acids.

Empty adenovirus serotype 5 (Ad5) capsids devoid of viral genome were developed as a novel delivery system for nanoparticles, proteins, and nucleic acids. Ad5 capsids of 110 nm diameter undergo an increase in particle size to 1637 nm in 1mM acetic acid at pH4.0 and then shrink to 60 nm, following pH reversal to 7.4. These pH shifts induced reversible changes in capsid zeta potential and secondary structure and irreversible changes in tertiary structure of capsid proteins. Using pH shift dependent changes in capsid size and structure, 20 nm fluorescent nanoparticles, FITC-BSA, and Alexa Fluor® 488 conjugated siRNA were encapsulated with high efficiency in Ad5 capsids, as confirmed by electron microscopy and/or flow cytometry. HEK cell uptake with capsid delivery system was 7.8-, 7.4-, and 2.9-fold greater for nanoparticles, FITC-BSA, and Alexa-siRNA, respectively, when compared to plain solutes. Physical mixtures of capsids and fluorescent solutes exhibited less capsid associated fluorescence intensity and cell uptake. Further, unlike physical mixture, pH shift assembled Ad5 capsids protected siRNA from RNase degradation. Ad5 capsids before and after pH shift exhibited endolysosomal escape. Thus, empty Ad5 capsids can encapsulate a variety of solutes based on pH shift assembly, resulting in enhanced cellular delivery.

Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage.

OBJECTIVE: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.

Intravitreal Poly(L-lactide) Microparticles Sustain Retinal and Choroidal Delivery of TG-0054, a Hydrophilic Drug Intended for Neovascular Diseases.

While poorly soluble drugs such as corticosteroids sustain drug delivery in the vitreous humor by virtue of slow dissolution, macromolecules such as antibodies and their fragments sustain their levels due to their slow clearance. However, currently there are no approaches to sustain the delivery of well water soluble small molecule drugs in the vitreous. In this study we optimized a PLA microparticle formulation for sustained intravitreal delivery of TG-0054, a well water soluble anti-angiogenic drug that is of potential value in treating choroid neovascularization. After determining the influence of process parameters on particle size and drug loading, spherical microparticles syringeable through a 27 G needle, with a mean diameter of 7.6 µm, 10% w/w TG-0054 loading, sustained in vitro drug release for at least 6 months, and low residual organic solvent content (~ 1 ppb/mg) were prepared. Microparticles as well as drug solution were assessed for their in vivo drug delivery over 3 months following intravitreal injection in New Zealand white rabbits. Drug levels in the microparticle dosed eyes at 3 months were 43.7 ± 16.2, 243 ± 42.6, 62.8 ± 22.6 µg/g vitreous, retina, and choroid-RPE, respectively, and similar to levels at one month. Intravitreal injection of plain drug solution resulted in significantly lower amounts of drug in the dosed eye, with the levels being 0.8 ± 0.5, 2.7 ± 2.8, and 4.9± 4.2 µg/g in vitreous, retina, and choroid-RPE, respectively, at one month, with no detectable drug at three months. Although surface degradation was evident, microparticles maintained their spherical structure during the 6 months in vitro study and the 3 months in vivo study, with the vitreal particle retention at 1 and 3 months being 60% and 27%, respectively. Thus, PLA microparticles capable of sustaining retinal and choroidal delivery of TG-0054 for three to six months were developed.

Delivery of SAR 1118 to the retina via ophthalmic drops and its effectiveness in a rat streptozotocin (STZ) model of diabetic retinopathy (DR).

PURPOSE: To determine the pharmacokinetics of SAR 1118, a small-molecule antagonist of leukocyte function-associated antigen (LFA)-1, after administration of ophthalmic drops in normal rats, and to determine its pharmacologic activity by assessing the inhibition of retinal leukostasis and vascular leakiness in a streptozotocin (STZ)-induced diabetic retinopathy model. METHODS: The ocular pharmacokinetics of SAR 1118 were studied in rats after a single topical dose of (14)C-SAR 1118 (1 mg/eye; 40 µCi; 15.5 µL). SAR 1118 concentration time profiles in plasma and ocular tissues were quantified by liquid scintillation counting (LSC). The pharmacologic activity of SAR 1118 eye drops administered thrice daily for 2 months at 1% (0.3 mg/eye/d) and 5% (1.5 mg/eye/d) was assessed in an STZ-induced diabetic rat model by determining retinal leukostasis and blood-retinal barrier breakdown. Diabetic rats treated with periocularly administered celecoxib microparticles served as the positive control, and vehicle-treated rats served as the negative control. RESULTS: A single dose of 6.5% (14)C-radiolabeled SAR 1118 ophthalmic drops delivered retinal drug levels greater than 1 µM in less than 30 minutes and sustained levels greater than 100 nM for 8 hours. SAR 1118 eye drops significantly reduced leukostasis and blood-retinal barrier breakdown in a dose-dependent manner. CONCLUSIONS: SAR 1118 ophthalmic drops administered thrice daily deliver therapeutic levels of SAR 1118 in the retina and can alleviate the retinal complications associated with diabetes.

Role of the ETB receptor in retinal ganglion cell death in glaucoma.

Recent observations suggest that the vasoactive peptide endothelin-1 (ET-1) may be an important contributor to the etiology of glaucoma. ET-1 administration has been shown to produce optic nerve axonal loss and apoptosis of retinal ganglion cells. Ocular ET-1 levels are elevated in aqueous humor in response to elevated intraocular pressure both in glaucoma patients and in animal models of glaucoma; however, the precise mechanisms by which ET-1 mediates glaucomatous optic neuropathy are not clear. Presently we report that ET-1-mediated apoptosis was markedly attenuated in ETB receptor-deficient rats, suggesting a key role for ETB receptors in apoptosis of retinal ganglion cells by ET-1 treatment. Using virally transformed rat retinal ganglion cells (RGC-5 cells), we found that ET-1 (100 nmol/L) treatment produced apoptotic changes in these cells that was determined by flow cytometric analyses, release of mitochondrial cytochrome c to the cytosol, and increased phosphorylation of c-Jun N-terminal kinase. Pretreatment with the ETB-receptor antagonist BQ788 (1 micromol/L) was able to significantly attenuate ET-1-mediated apoptosis in RGC-5 cells. ET-1-mediated apoptotic changes in RGC-5 cells were associated with ETB-receptor activation and were accompanied by a significant upregulation of ETB-receptor expression. These studies suggest that ocular ET-1 acts through ETB receptors to mediate apoptosis of retinal ganglion cells, a key event in glaucoma and related optic neuropathies.

Endothelin-1 mediated regulation of extracellular matrix collagens in cells of human lamina cribrosa.

Endothelin-1 (ET-1), a potent vaso-active peptide, mediates extracellular matrix regulation resulting in an increase in collagen deposition in various cell types and tissues and has been proposed to play a key role in glaucoma pathology. The role of ET-1 in the regulation of extracellular matrix collagens at the level of optic nerve head is not known. In this study we have examined the role of ET-1 in extracellular matrix collagen regulation in primary cultures of human lamina cribrosa cells. Our hypothesis is that ET-1 increases remodeling of the ECM of cells of the lamina cribrosa. Such actions could contribute to the development of optic neuropathy. QPCR analysis revealed that ET-1 mediated an increase in mRNA levels of collagen type I alpha1 and collagen type VI alpha1 chains at all doses of ET-1 with a significant increase at 1nM and 10nM concentration in LC cells. A dose-dependent increase in collagen type I and type VI protein deposition and secretion was also observed by Western blot in response to ET-1 and was significant at 10nM and 100nM concentrations of ET-1. ET-1 increased the [3H] proline uptake in LC cells suggesting that ET-1 contributed to an increase in total collagen synthesis in LC cells. ET-1-mediated increase in collagen type I, type VI and total collagen synthesis was significantly blocked by the ET(A) receptor antagonist, BQ610, as well as with the ET(B) receptor antagonist, BQ788, suggesting the involvement of both receptor subtypes in ET-1 mediated collagen synthesis in LC cells. These results suggest that ET-1 regulates extracellular matrix collagen synthesis in LC cells and may contribute to ECM remodeling at the level of LC of POAG subjects who have elevated plasma and aqueous humor levels of endothelin-1.

Changes in opioid receptor proteins during mitochondrial impairment in differentiated SK-N-SH cells.

Aging and neurodegenerative diseases are associated with oxidative damage that may contribute to changes in neurosensory processing, including pain. The effects of neuronal oxidation on the opioid receptor system are poorly understood. Earlier, we have reported that 3-nitroproprionic acid (3-NPA)-induced oxidative stress and impairment of mitochondrial energy metabolism significantly reduced the function of mu but not delta opioid receptors [A. Raut, M. Iglewski, A. Ratka, Differential effects of impaired mitochondrial energy production on the function of mu and delta opioid receptors in neuronal SK-N-SH cells, Neurosci. Lett. 404 (2006) 242-246]. In the present study, we studied the effects of 3-NPA-induced oxidative stress on protein levels of the mu, delta, and kappa opioid receptors (MOR, DOR, and KOR, respectively). The opioid-responsive differentiated SK-N-SH neuronal cells were used as an in vitro model. Cells were exposed to 0, 5, 10, and 20mM of 3-NPA for 0, 1, 2, 12, and 24h. After the 3-NPA treatments, plasma membrane preparations were made and used for the Western blot assay. There was a significant reduction in the level of the MOR protein while levels of DOR and KOR proteins remained unaffected after exposure to 3-NPA. These findings demonstrate for the first time that there is a selective impairment of the MOR protein under conditions of mitochondrial oxidative damage at the neuronal level. The reduction in the level of the MOR protein may contribute to the impairment of MOR function under oxidative damage conditions shown in our previous study.

Endothelin-1, endothelin A and B receptor expression and their pharmacological properties in GFAP negative human lamina cribrosa cells.

Primary open angle glaucoma (POAG) is a progressive optic neuropathy, characterized, in part by extensive extra cellular matrix remodeling and collapse of the lamina cribrosa (LC). Endothelin-1 (ET-1), a potent vasoactive peptide and its receptors, endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)), have been implicated in glaucomatous optic neuropathy. In this study we examined the expression of ET-1 and its receptors in GFAP negative LC cells. RT-PCR analysis revealed that LC cells express both ET(A), ET(B) receptors and prepro- ET-1, the primary gene transcript of ET-1. A dose-dependent increase in intra-cellular calcium concentrations was observed in the presence of 1, 10 and 100nM ET-1. Increased intracellular calcium concentrations were blocked by the ET(A) selective antagonist BQ610 but not by the ET(B) specific antagonist BQ788. Desensitization to ET(A)-mediated increase in intracellular calcium was observed in LC cells following pre-treatment with ET-1 for 24h. Western blot analysis of LC cells treated with ET-1 for 24h revealed a decreased expression of ET(A) receptor protein at 1, 10 and 100nM concentrations, while a dose dependent increase in the ET(B) receptor was observed with a significant increase at 100nM. Quantitative PCR showed a dose-dependent decrease in ET(A) receptor mRNA levels and an increase in the mRNA levels of ET(B) receptors. A Griess colorimetric assay was used to measure the NO released from LC cells and ET-1 induced a dose-dependent increase in NO release which was significant at 100nM concentration. ET-1 induced NO release was significantly blocked by BQ788, an ET(B) selective antagonist, and as well as BQ610, an ET(A) selective antagonist. These results suggested that human lamina cribrosa cells expressed functional ET(A) and ET(B) receptors and their expression and function was altered in response to prolong exposure to ET-1. This may have an implication in the normal physiology of LC cells and in POAG subjects where elevated levels of ET-1 could impact LC function.