Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts.

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis

A Single-Arm, Prospective, Exploratory Study to Preliminarily Test Effectiveness and Safety of Skin Electrical Stimulation for Leber Hereditary Optic Neuropathy

Leber hereditary optic neuropathy (LHON) is an intractable disease associated with mitochondrial DNA (mtDNA) mutations. In this preliminary, single-arm, prospective, open-label exploratory trial, we investigated the effectiveness and safety of skin electrical stimulation (SES) for cases of LHON harboring the mtDNA 11,778 mutation. Of the 11 enrolled patients, 10 completed six sessions of SES once every two weeks over a 10-week period. The primary outcome measure was the change in logarithm of the minimum angle of resolution (logMAR)-converted best-corrected visual acuity (BCVA) at one week after the last session of SES. The main secondary outcome measures were the logMAR BCVA at four and eight weeks and Humphrey visual field test sensitivities at one, four, and eight weeks. At all follow-up points, the logMAR BCVA had improved significantly from baseline, [1.80 (1.70-1.80) at baseline, 1.75 (1.52-1.80) at one week, 1.75 (1.50-1.80) at four weeks, and 1.75 (1.52-1.80) at eight weeks; p 2-fold increase in the summed sensitivity at 52 measurement points from baseline. No adverse effects were observed. In conclusion, SES could be a viable treatment option for patients with LHON in the chronic phase harboring the mtDNA 11,778 mutation

Pharmacokinetics and Efficacy of Topically Applied Nonsteroidal Anti-Inflammatory Drugs in Retinochoroidal Tissues in Rabbits

<div><p>Purpose</p><p>To evaluate the pharmacokinetics and efficacy of topically applied nonsteroidal anti-inflammatory drugs (NSAIDs) in the retinochoroidal tissues of rabbits.</p><p>Methods</p><p>The cyclooxygenase (COX) inhibitory activity of diclofenac, bromfenac, and amfenac, an active metabolite of nepafenac, were determined using human-derived COX-1 and COX-2. Each of the three NSAIDs was applied topically to rabbits, and after 0.5 to 8 hrs, the concentration of each drug in the aqueous humor and the retinochoroidal tissues was measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetics of the drugs in the tissues after repeated doses as is done on patients was calculated by a simulation software. The inhibitory effect of each NSAID on the breakdown of the blood-retinal barrier was assessed by the vitreous protein concentration on concanavalin A-induced retinochoroidal inflammation in rabbits.</p><p>Results</p><p>The half-maximal inhibitory concentration (IC₅₀) of diclofenac, bromfenac, and amfenac was 55.5, 5.56, and 15.3 nM for human COX-1, and 30.7, 7.45, and 20.4 nM for human COX-2, respectively. The three NSAIDs were detected in the aqueous humor and the retinochoroidal tissue at all-time points. Simulated pharmacokinetics showed that the levels of the three NSAIDs were continuously higher than the IC₅₀ of COX-2, as an index of efficacy, in the aqueous humor, whereas only the bromfenac concentration was continuously higher than the IC₅₀ at its trough level in the retinochoroidal tissues. The intravitreous concentration of proteins was significantly reduced in rabbits that received topical bromfenac (<i>P</i> = 0.026) but not the other two NSAIDs.</p><p>Conclusions</p><p>Topical bromfenac can penetrate into the retinochoroidal tissues in high enough concentrations to inhibit COX-2 and exerts its inhibitory effect on the blood-retinal barrier breakdown in an experimental retinochoroidal inflammation in rabbits. Topical bromfenac may have a better therapeutic benefit than diclofenac and nepafenac for retinochoroidal inflammatory diseases.</p></div