Correction: Suppression of choroidal neovascularization and epithelial-mesenchymal transition in retinal pigmented epithelium by adeno-associated virus-mediated overexpression of CCN5 in mice.

[This corrects the article DOI: 10.1371/journal.pone.0269937.].

Suppression of choroidal neovascularization and epithelial-mesenchymal transition in retinal pigmented epithelium by adeno-associated virus-mediated overexpression of CCN5 in mice.

Choroidal neovascularization (CNV) is a defining characteristic feature of neovascular age-related macular degeneration (nAMD) that frequently results in irreversible vision loss. The current strategies for the treatment of nAMD are mainly based on neutralizing vascular endothelial growth factor (VEGF). However, anti-VEGF therapies are often associated with subretinal fibrosis that eventually leads to damages in macula. In this study, we tested whether an anti-fibrotic and anti-angiogenic protein CCN5 can potentially be an effective and safe therapeutic modality in a mouse model of CNV. Laser photocoagulation was utilized to induce CNV, which was followed by intravitreal injection of recombinant adeno-associated virus serotype 2 encoding CCN5 (rAAV2-CCN5). Our data demonstrated that rAAV2-CCN5, but not a control viral vector, rAAV2-VLP, prominently attenuated both CNV lesions and angiogenesis. Aflibercept, which was utilized as a positive control, exhibited similar effects on CNV lesions and angiogenesis in our experimental settings. Upon laser photocoagulation, retinal pigmented epithelium (RPE) cells underwent significant morphological changes including cellular enlargement and loss of hexagonality. rAAV2-CCN5 significantly normalized these morphological defects. Laser photocoagulation also led to fibrotic deformation in RPE cells through inducing epithelial-mesenchymal transition (EMT), which was completely blocked by rAAV2-CCN5. In a striking contrast, aflibercept as well as rAAV2-VLP failed to exhibit any effects on EMT. Collectively, this study suggest that CCN5 might provide a potential novel strategy for the treatment of nAMD with a capability to inhibit CNV and fibrosis simaultaneously.

Cariporide Enhances the DNA Damage and Apoptosis in Acid-tolerable Malignant Mesothelioma H-2452 Cells.

The Na+/H+ exchanger is responsible for maintaining the acidic tumor microenvironment through its promotion of the reabsorption of extracellular Na+ and the extrusion of intracellular H+. The resultant increase in the extracellular acidity contributes to the chemoresistance of malignant tumors. In this study, the chemosensitizing effects of cariporide, a potent Na+/H+-exchange inhibitor, were evaluated in human malignant mesothelioma H-2452 cells preadapted with lactic acid. A higher basal level of phosphorylated (p)-AKT protein was found in the acid-tolerable H-2452AcT cells compared with their parental acid-sensitive H-2452 cells. When introduced in H-2452AcT cells with a concentration that shows only a slight toxicity in H-2452 cells, cariporide exhibited growth-suppressive and apoptosis-promoting activities, as demonstrated by an increase in the cells with pyknotic and fragmented nuclei, annexin V-PE(+) staining, a sub-G0/G1 peak, and a G2/M phase-transition delay in the cell cycle. Preceding these changes, a cariporide-induced p-AKT down-regulation, a p53 up-regulation, an ROS accumulation, and the depolarization of the mitochondrial-membrane potential were observed. A pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 markedly augmented the DNA damage caused by the cariporide, as indicated by a much greater extent of comet tails and a tail moment with increased levels of the p-histone H2A.X, p-ATMSer1981, p-ATRSer428, p-CHK1Ser345, and p-CHK2Thr68, as well as a series of pro-apoptotic events. The data suggest that an inhibition of the PI3K/AKT signaling is necessary to enhance the cytotoxicity toward the acid-tolerable H-2452AcT cells, and it underlines the significance of proton-pump targeting as a potential therapeutic strategy to overcome the acidic-microenvironment-associated chemotherapeutic resistance.

Nickel(II)-induced nasal epithelial toxicity and oxidative mitochondrial damage.

In probing the underlying mechanisms of nickel(II)-induced cytotoxicity on nasal epithelium, we investigated the effects of nickel(II) acetate on nasal epithelial RPMI-2650 cells. Nickel(II) elicited apoptosis, as signified by pyknotic and fragmented nuclei, increased caspase-3/7 activity, and an increase in annexin V binding, hypodiploid DNA, and Bax/Bcl-2 protein ratio. Nickel(II)-induced G2/M arrest was associated with up-regulation of p21(WAF1/CIP1) expression, decrease in phosphorylation at Thr(161) of Cdc2, and down-regulation of cyclin B1. Associated with these responses, ROS generation and mitochondrial depolarization increased in a nickel(II) concentration-dependent fashion. Pretreatment with N-acetylcysteine (NAC) attenuated these changes. p53 reporter gene assay and analyses of p53, Puma, Bax, and Bcl-2 protein levels indicated that NAC inhibited nickel(II)-induced activation of p53-mediated mitochondrial apoptotic pathway. Collectively, our study provides evidences that nickel(II) may induce oxidative damage on nasal epithelium in which antioxidant NAC protects cells against nickel(II)-induced apoptosis through the prevention of oxidative stress-mediated mitochondrial damage.

TPA-induced cell transformation provokes a complex formation between Pin1 and 90 kDa ribosomal protein S6 kinase 2.

Post-translational modification of peptidyl cis/trans prolyl isomerase Pin1 is crucial in regulation of gene stability. Pin1 phosphorylation at Ser(16) has been regarded as a marker for Pin1 isomerase activity and introduction of phosphorylation on Ser/Thr-Pro of substrate proteins is prerequisite for its binding activity with Pin1 and subsequent isomerization. Here, we found that 90 kDa ribosomal protein S6 kinase 2 (RSK2) could form a physical complex with Pin1, leading to phosphorylation of Pin1 at Ser(16) ex vivo and in vitro respectively. Intriguingly, Pin1(+/+) mouse embryonic fibroblasts (MEFs) exhibited significantly an increase in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced RSK2 phosphorylation with a marginal Pin1 phosphorylation compared with Pin1(-/-) MEFs. Moreover, TPA-induced Ser(16) Pin1 phosphorylation as well as RSK2 phosphorylation was considerably profound in RSK(+/+) MEFs but not in RSK(-/-) MEFs. Consequently, knockdown of Pin1 using shRNA-Pin1 suppressed TPA-induced cell transformation in JB6 CI41 cells. Overall, these results indicate that Pin1 plays a critical role in TPA-induced tumorigenesis plausibly via physical interaction with RSK2 and reciprocal phosphorylation, therefore suggesting a potential therapeutic target for cancer treatment.

Association of ADAMTS12 polymorphisms with rheumatoid arthritis.

a disintegrin and metalloproteinase (ADAM) with thrombospondin type 1 motif 12 (ADAMTS12) is a degradative enzyme that interacts with the degradable fragments of cartilage oligomeric matrix protein, which is a prominent non-collagenous matrix component in articular cartilage. ADAMTS12 has been observed in the cartilage, synovial fluid and serum of arthritic patients, and may play an important role in the pathogenesis of arthritis. In the present study, we investigated whether genetic polymorphisms of ADAMTS12 are associated with rheumatoid arthritis (RA). To observe the association between ADAMTS12 and RA, we genotyped three single nucleotide polymorphisms (SNPs) (rs1364044, intron C/T; rs10461703, intron C/T; rs25754, missense Thr1495Ile) of ADAMTS12 using a direct sequencing method in 303 RA patients and 495 control subjects. Multiple logistic regression models were performed to analyze the genetic data. SNPStats and SNPAnalyzer Pro programs were used to estimate the odds ratios, 95% confidence intervals and p-values. Bonferroni's correction (pc) was conducted to obtain a defined result. Of the three SNPs, the genotype frequency of rs10461703 was associated with the development of RA (pc=0.0024 in the co-dominant model; pc=0.0009 in the dominant model; pc=0.0006 in the log-additive model). The allele frequency of rs10461703 also showed a significant difference between RA and controls (pc<0.0001). The C allele frequency of rs10461703 was lower in the RA group (36.6%) compared to the control group (45.7%), whereas the T allele frequency of rs10461703 in the RA group (63.4%) was higher compared to that in the control group (54.3%). The other two SNPs (rs1364044 and rs25754) were not associated with the development of RA. However, we did not find any association between the three tested SNPs and RA patients according to clinical features, including erythrocyte sedimentation rate, C-reactive protein level, rheumatoid factor (+ and -) and bone erosion (+ and -). Our results suggest that ADAMTS12 may be a susceptibility gene for RA development.

Phosphoinositol 3-kinase, a novel target molecule for the inhibitory effects of juglone on TPA-induced cell transformation.

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 µM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.

Effect of exogenous surfactant therapy on levels of pulmonary surfactant proteins A and D in preterm infants with respiratory distress syndrome.

OBJECTIVES: To examine whether exogenous pulmonary surfactants (exPS) substitute for or merely supplement endogenous pulmonary surfactants (enPS) by looking at sequential changes in the surfactant proteins (SP) SP-A and SP-D in alveolar pools. METHODS: Fourteen preterm infants with RDS treated with an artificial surfactant were compared to five normal-term infants without RDS who were treated with artificial ventilation at birth. RESULTS: Immediately after birth, SP-A and SP-D were essentially absent in the alveolar pools of the RDS group, but were present at normal levels in the controls. Treatment with exPS apparently stimulated enPS production. CONCLUSIONS: In infants who responded well to exPS therapy, the SP concentration reached essentially normal levels within 48-72 h after birth.

Relationship between pulmonary surfactant protein and lipid peroxidation in lung injury due to paraquat intoxication in rats.

BACKGROUND: Pulmonary damage resulting from lipid peroxidation is a principal effect of paraquat intoxication. The host-defense functions of surfactant are known to be mediated by the surfactant proteins A and D (SP-A and SP-D, respectively). The primary objective of this study was to evaluate the variations over time in levels of surfactant protein and lipid peroxidation (LPO) in lung tissue following free-radical-induced injury. METHODS: 42 adult, male, Sprague-Dawley rats were administered intraperitoneal injections of paraquat (35 mg/kg body weight). SP-A and SP-D levels were determined via Western blot. LPO in the left lung homogenate was measured via analyses of the levels of thiobarbituric acid-reactive substances. RESULTS: LPO levels peaked at 6 hours, with no associated histological changes. SP-D levels increased until hour 12 and declined until hour 48; SP-D levels subsequently began to increase again, peaking at hour 72. SP-A levels peaked at hour 6, declining thereafter. CONCLUSIONS: We suggest that in the early phase of paraquat injury, SP-D levels reflect alveolar damage and that de novo synthesis of SP-D takes 72 hours. Levels of SP-A, on the other hand, reflect abnormalities in the surfactant system in the late stage of paraquat intoxication. Surfactant proteins may play a role in protecting the lungs from reactive oxygen injury. A time-dependent variation has been observed in the levels of surfactant proteins A and D following paraquat injury, and it has been suggested that these proteins play a role in the protection of lung tissue against ROS-induced injuries.

Aberrant splicing of FHIT transcripts in human gastric cancer cell lines.

Alterations of the FHIT gene occur as frequent events in several human cancers. Replacement of exogenous wild-type FHIT gene has been shown to induce suppression of tumorigenicity of human FHIT-negative cells in nude mice and aberrant FHIT transcripts have been observed in a variety of human solid tumors. In the presence study, we performed a nested reverse transcription-polymerase chain reaction (RT-PCR) analysis to identify aberrant FHIT transcripts in 6 gastric cancer cell lines. In addition to the wild-type FHIT transcript, small-sized transcripts with various number and lengths were observed in all of the cell lines examined. Sequence analysis confirmed that different types of truncated transcripts included exonic deletions, insertions of intron 5 sequences between exons, and combinations of both. Most of these transcripts lacked exon 5 in which translation initiation codon is located. Aberrant transcripts with partial exonic deletions, resulting from activation of cryptic splice sites, were also observed in 5 cell lines. Additionally, multi-step splice patterns, indicative of additional downstream processing, were observed in several cancer lines. Our results suggest that the aberrant FHIT transcripts in gastric cancer cell lines resulted from faulty splicing, including exon skipping, selection of cryptic splice site and additional downstream splice processing.

Expression of the fragile histidine triad gene in normal rat tissues and human kidney cancer cell lines.

The fragile histidine triad (FHIT) gene located in human chromosome 3p14.2 is a candidate tumor suppressor gene that has a diadenosine triphosphate (Ap3A) hydrolase activity, but its role in carcinogenesis remains uncertain. To investigate the role of FHIT in normal cells, specific polyclonal antibodies to recombinant rat FHIT protein were generated. Immunoblot analysis revealed the 17-kd FHIT protein was most abundantly expressed in kidney and liver, whereas heart, skeletal muscle, and adrenal gland expressed trace amounts. In kidney, immunohistochemical staining was strongly observed in distal convoluted tubule and collecting duct during postnatal growth period. By a nested reverse transcription-PCR analysis of FHIT from 2 human kidney cancer cell lines, four abnormal-sized FHIT transcripts, with deletion and/or insertion, were detected. These were derived from the results of exon skipping, and/or insertion of FHIT intron 5 sequence, or selection of cryptic splice site within the FHIT cDNA sequence 179-180. Taken together, our data indicate that FHIT expression is frequently altered in human kidney cancer cell lines by alternative splicing, and suggest that the FHIT protein may play a pivotal role in regulating intracellular metabolism of the distal convoluted tubule and collecting duct in maturity.