Effect of a Blueberry-Derived Antioxidant Matrix on Infrared-A Induced Gene Expression in Human Dermal Fibroblasts.

There is compelling evidence that Infrared A (IRA) from natural sunlight contributes to photoaging of human skin by inducing the expression of matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Corresponding mechanistic studies have shown that IRA does so by increasing the production of reactive oxygen species in irradiated cells. In the present study, we therefore asked if treatment of primary human skin fibroblasts with a blueberry-derived antioxidant matrix (BerrimatrixTM), which is employed as an active ingredient in commercially available skin care products that are topically applied, can prevent IRA-induced MMP-1 expression in these cells. In this in vitro study, we have found that this antioxidant containing matrix is well tolerated by fibroblast over a broad concentration range and that it efficiently prevents IRA-induced MMP-1 mRNA expression. It may thus be speculated that topical application of this antioxidant containing matrix may be efficient in protecting human skin against IRA-induced wrinkle formation.

J Drugs Dermatol. 2017;16(8 Suppl 2):s125-128.

Integrative functional genomics of hepatitis C virus infection identifies host dependencies in complete viral replication cycle.

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

Laboratory intercomparison on the γ-H2AX foci assay.

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the γ-H2AX foci assay as a diagnostic triage tool for rapid individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-4 Gy) as well as blinded test samples (0.1-6.4 Gy) were incubated at 37°C for 2 and 24 h (repair time) and sent to the participants. The foci assay was performed according to protocols individually established in participating laboratories and therefore varied. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) of estimated doses relative to the actual doses was calculated and radiation doses were merged into four triage categories reflecting clinical relevance to calculate accuracy, sensitivity and specificity. First γ-H2AX based dose estimates were reported 7 h after sample receipt. Estimates were similarly accurate for 2 and 24 h repair times, providing scope for its use in the early phase of a radiation exposure incident. Equal accuracy was achieved by scoring 20, 30, 40 or 50 cells per sample. However, MAD values of 0.5-0.7 Gy and 1.3-1.7 Gy divided the data sets into two groups, driven mainly by the considerable differences in foci yields between calibration and blind samples. Foci yields also varied dramatically between laboratories, highlighting reproducibility issues as an important caveat of the foci assay. Nonetheless, foci counts could distinguish high- and low-dose samples in all data sets and binary dose categories of clinical significance could be discriminated with satisfactory accuracy (mean 84%, ±0.03 SEM). Overall, the results suggest that the γ-H2AX assay is a useful tool for rapidly screening individuals for significant exposures that occurred up to at least 24 h earlier, and may help to prioritize cytogenetic dosimetry follow-up.

Calcifying human aortic smooth muscle cells express different bone alkaline phosphatase isoforms, including the novel B1x isoform.

BACKGROUND: Vascular calcification, causing cardiovascular morbidity and mortality, is associated with hyperphosphatemia in chronic kidney disease (CKD). In vitro, phosphate induces transdifferentiation of vascular smooth muscle cells to osteoblast-like cells that express alkaline phosphatase (ALP). In vivo, raised serum ALP activities are associated with increased mortality. A new bone ALP isoform (B1x) has been identified in serum from CKD patients. The present study investigated the different ALP isoforms in calcifying human aortic smooth muscle cells (HAoSMCs). METHODS: HAoSMCs were cultured for 30 days in medium containing 5 or 10 mmol/l ß-glycerophosphate in the presence or absence of the ALP-specific inhibitor tetramisole. RESULTS: All known bone-specific ALP (BALP) isoforms (B/I, B1x, B1 and B2) were identified in HAoSMCs. ß-Glycerophosphate stimulated calcification of HAoSMCs, which was associated with increased BALP isoforms B/I, B1x and B2. Tetramisole inhibited the ß-glycerophosphate-induced HAoSMC calcification, which was paralleled by the inhibition of the B1x and B/I, but not the other isoforms. CONCLUSIONS: HAoSMCs express the four known BALP isoforms. B/I, B1x and B2 could be essential for soft tissue calcification. B/I and B1x were more affected by tetramisole than the other isoforms, which suggests different biological functions during calcification of HAoSMCs.

Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.

Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 µM) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 µM) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 µM) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons.

Differential expression of AURKA and AURKB genes in bone marrow stromal mesenchymal cells of myelodysplastic syndrome: correlation with G-banding analysis and FISH.

It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells.

AKT2 confers protection against aortic aneurysms and dissections.

RATIONALE: Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown. OBJECTIVE: In this study, we examined the role of AKT2 in protecting the aorta from developing AAD. METHODS AND RESULTS: AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II, these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. In addition, angiotensin II-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase-9 (MMP-9) and reduced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 to the MMP-9 and TIMP-1 promoters. CONCLUSIONS: Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.

Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase ß-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

Differential roles of JNK, ERK1/2, and p38 mitogen-activated protein kinases on endothelial cell tissue repair functions in response to tumor necrosis factor-α.

Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µM) or ERK1/2 (PD98059, 5 µM) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µM) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Does inhibiting Sur1 complement rt-PA in cerebral ischemia?

Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA-associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA-associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA-associated HT in stroke.

Antidepressants inhibit DNA methyltransferase 1 through reducing G9a levels.

The discovery of epigenetic processes as possible pivotal regulatory mechanisms in psychiatric diseases raised the question of how psychoactive drugs may impact the epigenetic machinery. In the present study we set out to explore the specificity and the mode of action of the reported inhibitory effect of the TCA (tricyclic antidepressant) amitriptyline on DNMT (DNA methyltransferase) activity in primary astrocytes from the rat cortex. We found that the impact on DNMT was shared by another TCA, imipramine, and by paroxetine, but not by venlafaxine or the mood stabilizers carbamazepine and valproic acid. DNMT activity in subventricular neural stem cells was refractory to the action of ADs (antidepressants). Among the established DNMTs, ADs primarily targeted DNMT1. The reduction of enzymatic DNMT1 activity was neither due to reduced DNMT1 expression nor due to direct drug interference. We tested putative DNMT1-inhibitory mechanisms and discovered that a known stimulator of DNMT1, the histone methyltransferase G9a, exhibited decreased protein levels and interactions with DNMT1 upon AD exposure. Adding recombinant G9a completely reversed the AD repressive effect on DNMT1 function. In conclusion, the present study presents a model where distinct ADs affect DNMT1 activity via G9a with important repercussions for possible novel treatment regimes.

SHP-1 as a critical regulator of Mycoplasma pneumoniae-induced inflammation in human asthmatic airway epithelial cells.

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.

Immune complexes and late complement proteins trigger activation of Syk tyrosine kinase in human CD4(+) T cells.

In systemic lupus erythematosus (SLE), the autoantibodies that form immune complexes (ICs) trigger activation of the complement system. This results in the formation of membrane attack complex (MAC) on cell membrane and the soluble terminal complement complex (TCC). Hyperactive T cell responses are hallmark of SLE pathogenesis. How complement activation influences the T cell responses in SLE is not fully understood. We observed that aggregated human γ-globulin (AHG) bound to a subset of CD4(+) T cells in peripheral blood mononuclear cells and this population increased in the SLE patients. Human naive CD4(+) T cells, when treated with purified ICs and TCC, triggered recruitment of the FcRγ chain with the membrane receptor and co-localized with phosphorylated Syk. These events were also associated with aggregation of membrane rafts. Thus, results presented suggest a role for ICs and complement in the activation of Syk in CD4(+) T cells. Thus, we propose that the shift in signalling from ζ-chain-ZAP70 to FcRγ chain-Syk observed in T cells of SLE patients is triggered by ICs and complement. These results demonstrate a link among ICs, complement activation and phosphorylation of Syk in CD4(+) T cells.

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification.

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.

Effects of nitric oxide on the survival and neuritogenesis of cerebellar Purkinje neurons.

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Cytotoxicity of gamma-ray in rat immature hippocampal neurons.

This in vitro study evaluated the detrimental effect of acute gamma (γ)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy γ-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after γ-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked γ-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of γ-rays in immature hippocampal cultured neurons may be caused by oxidative stress.

Stem cell based cancer gene therapy.

The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Suicide gene therapy using genetically engineered mesenchymal stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. This review provides an explanation of the stem cell-targeted prodrug cancer gene therapy principle, with focus on the choice of prodrug, properties of bone marrow and adipose tissue-derived mesenchymal stem and neural stem cells as well as the mechanisms of their tumor homing ability. Therapeutic achievements of the cytosine deaminase/5-fluorocytosine prodrug system and Herpes simplex virus thymidine kinase/ganciclovir are discussed. In addition, delivery of immunostimulatory cytokines, apoptosis inducing genes, nanoparticles and antiangiogenic proteins by stem cells to tumors and metastases is discussed as a promising approach for antitumor therapy. Combinations of traditional, targeted and stem cell-directed gene therapy could significantly advance the treatment of cancer.

NADPH oxidase 4 promotes endothelial angiogenesis through endothelial nitric oxide synthase activation.

UNLABELLED: BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia. METHODS AND RESULTS: Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia. CONCLUSIONS: Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.

Caspase 3 blocking avoids the expression of autoantigens triggered by apoptosis in neonatal Balb/c mice skin.

OBJECTIVE: To assess the effect of caspase 3 inhibition, in the expression of intracellular antigens induced by apoptosis. MATERIAL AND METHODS: Skin explants of neonatal Balb/c mice were used to assess the autoantigen expression. Skin was obtained by punch biopsies, tissues were cultured in DMEM; cell death was induced by chemicals and assessed by TUNEL. The expression of La, Ro, Sm, RNP, Cajal Bodies and NuMa antigens were monitored by immunohistochemistry using autoantibodies or monoclonal antibodies against these antigens. RESULTS: Chemicals used to induce cell death, successfully produced apoptosis or necrosis in more than 60% of keratinocytes, and viability was significantly decreased when it was compared with those in controls. An increased expression of all skin intracellular antigens in skin biopsies treated with chemicals, major antigenic expression was detected with anti-La and anti-Ro antibodies. The caspase 3 inhibitor DEVD-CMK significantly decreased the expression of antigens induced by chemicals. CONCLUSION: By this result we can infer that caspase inhibitors modify apoptosis and decrease the autoantigens associated to cell death.