Exploration of Molecular Factors Impairing Superoxide Dismutase Isoforms Activity in Human Senile Cataractous Lenses

PURPOSE. To explore different molecular factors impairing the activities of superoxide dismutase (SOD) isoforms in senile cataractous lenses. METHODS. Enzyme activity of SOD isoforms, levels of their corresponding cofactors copper (Cu), manganese (Mn), zinc (Zn), and expression of mRNA transcripts and proteins were determined in the lenses of human subjects with and without cataract. DNA from lens epithelium (LE) and peripheral blood was isolated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing was carried out to screen somatic mutations. The impact of intronic insertion/deletion (INDEL) variations on the splicing process and on the resultant transcript was evaluated. Genotyping of IVS4þ42delG polymorphism of SOD1 gene was done by PCR-restriction fragment length polymorphism (RFLP). RESULTS. A significant decrease in Cu/Zn-and Mn-SOD activity (P < 0.001) and in Cu/Zn-SOD transcript (P < 0.001) and its protein (P < 0.05) were found in cataractous lenses. No significant change in the level of copper (P ¼ 0.36) and an increase in the level of manganese (P ¼ 0.01) and zinc (P ¼ 0.02) were observed in cataractous lenses. A significant positive correlation between the level of Cu/Zn-SOD activity and the levels of Cu (P ¼ 0.003) and Zn (P ¼ 0.005) was found in the cataractous lenses. DNA sequencing revealed three intronic INDEL variations in exon4 of SOD1 gene. Splice-junction analysis showed the potential of IVS4þ42delG in creating a new cryptic acceptor site. If it is involved in alternate splicing, it could result in generation of SOD1 mRNA transcripts lacking exon4 region. Transcript analysis revealed the presence of complete SOD1 mRNA transcripts. Genotyping revealed the presence of IVS4þ42delG polymorphism in all subjects. CONCLUSIONS. The decrease in the activity of SOD1 isoform in cataractous lenses was associated with the decreased level of mRNA transcripts and their protein expression and was not associated with either modulation in the level of enzyme cofactors or with INDEL variations

Dysregulation of Mesenchymal Cell Survival Pathways in Severe Fibrotic Lung Disease: The Effect of Nintedanib Therapy

Impaired apoptotic clearance of myofibroblasts can result in the continuous expansion of scar tissue during the persistent injury in the lung. However, the molecular and cellular mechanisms underlying the apoptotic clearance of multiple mesenchymal cells including fibrocytes, fibroblasts and myofibroblasts in severe fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) remain largely unknown. We analyzed the apoptotic pathways activated in mesenchymal cells of IPF and in a mouse model of TGFα-induced pulmonary fibrosis. We found that fibrocytes and myofibroblasts in fibrotic lung lesions have acquired resistance to Fas-induced apoptosis, and an FDA-approved anti-fibrotic agent, nintedanib, effectively induced apoptotic cell death in both. In support, comparative gene expression analyses suggest that apoptosis-linked gene networks similarly dysregulated in both IPF and a mouse model of TGFα-induced pulmonary fibrosis. TGFα mice treated with nintedanib show increased active caspase 3-positive cells in fibrotic lesions and reduced fibroproliferation and collagen production. Further, the long-term nintedanib therapy attenuated fibrocyte accumulation, collagen deposition, and lung function decline during TGFα-induced pulmonary fibrosis. These results highlight the importance of inhibiting survival pathways and other pro-fibrotic processes in the various types of mesenchymal cells and suggest that the TGFα mouse model is relevant for testing of anti-fibrotic drugs either alone or in combination with nintedanib

Rescue of Photoreceptor Degeneration by Curcumin in Transgenic Rats with P23H Rhodopsin Mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects

Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis

Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro‐fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα‐driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof‐of‐concept that demonstrate barasertib as a possible intervention therapy for IPF.SynopsisFibroblast activation is central for the initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis. Our preclinical study describes the pathological role for AURKB in fibroblast activation and presents a potential therapy for the treatment of pulmonary fibrosis using barasertib.AURKB is upregulated in the lungs of IPF patients and mouse models of pulmonary fibrosis.WT1 binds directly to the promoter of AURKB to upregulates its expression.AURKB functions as a positive regulator of fibroproliferation, myofibroblast survival, and ECM production.In vivo barasertib therapy attenuates TGFα‐and bleomycin‐induced pulmonary fibrosis.Fibroblast activation is central for the initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis. Our preclinical study describes the pathological role for AURKB in fibroblast activation and presents a potential therapy for the treatment of pulmonary fibrosis using barasertib.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/7/emmm202012131-sup-0003-SDataFig1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/6/emmm202012131-sup-0002-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/5/emmm202012131-sup-0006-SDataFig6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/4/emmm202012131.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/3/emmm202012131-sup-0005-SDataFig4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/2/emmm202012131-sup-0001-Appendix.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/1/emmm202012131_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/9/emmm202012131.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162746/8/emmm202012131-sup-0004-SDataFig2.pd

Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis

Abstract Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro‐fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα‐driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof‐of‐concept that demonstrate barasertib as a possible intervention therapy for IPF

Improved retinal morphology and gene expression of P23H transgenic rats upon administration of curcumin.

<p>Light micrographs of retinal sections from untreated control (A) and curcumin treated (B) and wild type Wistar rat retina (C) P23H-R transgenic rats demonstrated a significant improvement in retinal morphology in curcumin administered rats compared to untreated controls. OS = outer segments; IS = inner segments; ONL = outer nuclear layer; OPL = outer plexiform layer and INL = inner nuclear layer; IPL = inner plexiform layer; GC: Ganglion cell layer. The thickness of the ONL and INL in curcumin treated rats were quantified using Aperio Image Scope software Ver. 10.2.2.2319 program (D). We found significant increase in the thickness of both ONL (p≤3.6∧-07) and INL (p≤2∧-05) of the retina of curcumin treated P23H rats when compared to vehicle treated P23H rats. The results are presented as Mean± SD with ** denoting p-values<0.005. Quantitative expression of rod photoreceptor specific markers <i>Rho and Rom-1</i> and cone specific makers <i>S-Opsin</i> and <i>M-Opsin</i>(E) demonstrated an increased expression in curcumin treated rat retinas (Gray bars) is compared to untreated controls (white bars). Gene expression data is calculated from at least 3 independent samples, each of which was analyzed at least in 3 replication reactions. The results are presented as Mean± SD with ** denoting p-values<0.005.</p

Decreased expression of ER stress markers in curcumin treated COS-7 cells expressing mutant rhodopsin.

<p>Quantitative expression of ER stress responsive genes <i>Grp78/Bip</i> and <i>Chop</i> were determined by qRT-PCR. Expression values were presented on an arbitrary scale (<i>y</i>-axis) after normalization with the house keeping gene RPL-19. Data represent the mean (±SD) on an arbitrary scale and were calculated from at least three independent observations. P<0.001 (**), and P<0.025(*).</p

Bioavailability of curcumin.

<p>Amount of curcumin detected by LC-MS/MS analysis in the eye and brain tissues of curcumin administered SD rats. (A) Representative LC-MS/MS chromatogram of curcumin (standard) and naringenin (internal standard). (B) Delivery of curcumin to ocular tissues and brain of SD rats following oral administration. Data are expressed as mean ± SD for n = 4.</p