Raghu Krishnamoorthy, Ph.D.
Permanent URI for this communityhttps://hdl.handle.net/20.500.12503/31800
Associate Professor, North Texas Eye Research Institute
Associate Professor, Pharmacology & Neuroscience
Email: Raghu.Krishnamoorthy@unthsc.edu
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Browsing Raghu Krishnamoorthy, Ph.D. by Subject "Animals"
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Item A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells(PLOS, 2017-09-22) Wang, Junming; Ma, Hai-Ying; Krishnamoorthy, Raghu R.; Yorio, Thomas; He, Shaoqingc-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein beta (C/EBPbeta) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPbeta in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPbeta as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPbeta augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPbeta was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPbeta and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPbeta appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.Item Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells(ARVO Journals, 2021-05-03) Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Vignesh R.; Krishnamoorthy, Raghu R.Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.Item Upregulation of the endothelin A (ETA) receptor and its association with neurodegeneration in a rodent model of glaucoma(BioMed Central Ltd., 2017-03-01) McGrady, Nolan R.; Minton, Alena Z.; Stankowska, Dorota L.; He, Shaoqing; Jefferies, Hayden B.; Krishnamoorthy, Raghu R.BACKGROUND: Primary open angle glaucoma is a heterogeneous group of optic neuropathies that results in optic nerve degeneration and a loss of retinal ganglion cells (RGCs) ultimately causing blindness if allowed to progress. Elevation of intraocular pressure (IOP) is the most attributable risk factor for developing glaucoma and lowering of IOP is currently the only available therapy. However, despite lowering IOP, neurodegenerative effects persist in some patients. Hence, it would be beneficial to develop approaches to promote neuroprotection of RGCs in addition to IOP lowering therapies. The endothelin system is a key target for intervention against glaucomatous neurodegeneration. The endothelin family of peptides and receptors, particularly endothelin-1 (ET-1) and endothelin B (ETB) receptor, has been shown to have neurodegenerative roles in glaucoma. The purpose of this study was to examine changes in endothelin A (ETA) receptor protein expression in the retinas of adult male Brown Norway rats following IOP elevation by the Morrison's model of ocular hypertension and the impact of ETA receptor overexpression on RGC viability in vitro. RESULTS: IOP elevation was carried out in one eye of Brown Norway rats by injection of hypertonic saline through episcleral veins. After 2 weeks of IOP elevation, immunohistochemical analysis of retinal sections from rat eyes showed an increasing trend in immunostaining for ETA receptors in multiple retinal layers including the inner plexiform layer, ganglion cell layer and outer plexiform layer. Following 4 weeks of IOP elevation, a significant increase in immunostaining for ETA receptor expression was found in the retina, primarily in the inner plexiform layer and ganglion cells. A modest increase in staining for ETA receptors was also found in the outer plexiform layer in the retina of rats with IOP elevation. Cell culture studies showed that overexpression of ETA receptors in 661W cells as well as primary RGCs decreases cell viability, compared to empty vector transfected cells. Adeno-associated virus mediated overexpression of the ETA receptor produced an increase in the ETB receptor in primary RGCs. CONCLUSIONS: Elevated IOP results in an appreciable change in ETA receptor expression in the retina. Overexpression of the ETA receptor results in an overall decrease in cell viability, accompanied by an increase in ETB receptor levels, suggesting the involvement of both ETA and ETB receptors in mediating cell death. These findings raise possibilities for the development of ETA/ETB dual receptor antagonists as neuroprotective treatments for glaucomatous neuropathy.