Entamoeba lysyl-tRNA synthetase contains a cytokine-like fomain with chemokine activity towards human endothelial cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

Vitamin E but not 17B-estradiol protect against vascular toxicity induced by B-amyloid wild type and the Dutch amyploid variant

Amyloid β-peptide (Aβ) fibril deposition on cerebral vessels produces cerebral amyloid angiopathy that appears in the majority of Alzheimer's disease patients. An early onset of a cerebral amyloid angiopathy variant called hereditary cerebral hemorrhage with amyloidosis of the Dutch type is caused by a point mutation in Aβ yielding AβGlu22→Gln. The present study addresses the effect of amyloid fibrils from both wild-type and mutated Aβ on vascular cells, as well as the putative protective role of antioxidants on amyloid angiopathy. For this purpose, we studied the cytotoxicity induced by Aβ1–40 Glu22→Gln and Aβ1–40 wild-type fibrils on human venule endothelial cells and rat aorta smooth muscle cells. We observed that AβGlu22→Gln fibrils are more toxic for vascular cells than the wild-type fibrils. We also evaluated the cytotoxicity of Aβ fibrils bound with acetylcholinesterase (AChE), a common component of amyloid deposits. Aβ1–40 wild-type–AChE fibrillar complexes, similar to neuronal cells, resulted in an increased toxicity on vascular cells. Previous reports showing that antioxidants are able to reduce the toxicity of Aβ fibrils on neuronal cells prompted us to test the effect of vitamin E, vitamin C, and 17β-estradiol on vascular damage induced by Aβwild-type and AβGlu22→Gln. Our data indicate that vitamin E attenuated significantly the Aβ-mediated cytotoxicity on vascular cells, although 17β-estradiol and vitamin C failed to inhibit the cytotoxicity induced by Aβ fibrils.

Derivation of human embryonic stem cells at the center for regenerative medicine in Barcelona

We report here the legislative issues related toembryo research and human embryonic stem cell (hESC)research in Spain and the derivation of nine hESC lines atthe Center of Regenerative Medicine in Barcelona. You canfind the information for obtaining our lines for researchpurposes at blc@cmrb.eu.

Generation of induced pluripotent stem cells from human cord blood cells with only two factors:OCT4 and SOX2

Induced pluripotent stem cells (iPSC ) provide an invaluable resource for regenerative medicine as they allow the generationof patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach isdesirable, such as for cell therapy of acute conditions or when the patient’s somatic cells are altered as a consequence of a chronicdisease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carryminimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousandimmunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for thederivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors(OCT 4 and SO X2) in 2 weeks and without the need for additional chemical compounds.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats.

Background and aims: The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with powerful vascular permeability effects. We assessed regional blood flow, vascular leakage, mRNA and tissular expression of VEGF-A and Ang-2 and vascular permeability following VEGF receptor 2 blockade in control and cirrhotic rats to define the vascular territories showing altered vascular permeability in cirrhosis and to determine whether VEGF-A and Ang-2 are involved in this phenomenon. Methods: Arterial blood flow was analysed with the coloured microsphere method. Vascular leakage was measured and visualised with the dye Evan¿s Blue and colloidal carbon techniques, respectively. VEGF-A and Ang-2 expression were determined by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. The effect on vascular permeability induced by VEGFR2 blockade was assessed by administration of the receptor inhibitor SU11248. Results: Arterial blood flow was increased in the mesentery, pancreas and small intestine but not in the kidney and spleen of cirrhotic rats as compared to controls. Increased vascular leakage was observed in the mesentery and liver, where colloidal carbon spread from microvessels to the adjacent fibrotic tracts. Increased hepatic and mesenteric expression of VEGF-A and Ang-2 was found in cirrhotic rats as compared to controls. Blockade of VEGFR2 markedly reduced hepatic and mesenteric vascular leakage in cirrhotic rats. Conclusions: Enhanced endothelial permeability is restricted to the hepatic and mesenteric vascular beds in cirrhotic rats with ascites and VEGF-A and Ang-2 are key factors in the signalling pathways regulating this dysfunction.

PGC-1α induces mitochondrial and myokine transcriptional programs and lipid droplet and glycogen accumulation in cultured human skeletal muscle cells

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a chief activator of mitochondrial and metabolic programs and protects against atrophy in skeletal muscle (skm). Here we tested whether PGC-1α overexpression could restructure the transcriptome and metabolism of primary cultured human skm cells, which display a phenotype that resembles the atrophic phenotype. An oligonucleotide microarray analysis was used to reveal the effects of PGC-1α on the whole transcriptome. Fifty-three different genes showed altered expression in response to PGC-1α: 42 upregulated and 11 downregulated. The main gene ontologies (GO) associated with the upregulated genes were mitochondrial components and processes and this was linked with an increase in COX activity, an indicator of mitochondrial content. Furthermore, PGC-1α enhanced mitochondrial oxidation of palmitate and lactate to CO2, but not glucose oxidation. The other most significantly associated GOs for the upregulated genes were chemotaxis and cytokine activity, and several cytokines, including IL-8/CXCL8, CXCL6, CCL5 and CCL8, were within the most highly induced genes. Indeed, PGC-1α highly increased IL-8 cell protein content. The most upregulated gene was PVALB, which is related to calcium signaling. Potential metabolic regulators of fatty acid and glucose storage were among mainly regulated genes. The mRNA and protein level of FITM1/FIT1, which enhances the formation of lipid droplets, was raised by PGC-1α, while in oleate-incubated cells PGC-1α increased the number of smaller lipid droplets and modestly triglyceride levels, compared to controls. CALM1, the calcium-modulated δ subunit of phosphorylase kinase, was downregulated by PGC-1α, while glycogen phosphorylase was inactivated and glycogen storage was increased by PGC-1α. In conclusion, of the metabolic transcriptome deficiencies of cultured skm cells, PGC-1α rescued the expression of genes encoding mitochondrial proteins and FITM1. Several myokine genes, including IL-8 and CCL5, which are known to be constitutively expressed in human skm cells, were induced by PGC-1α.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion

Barrier-protective effects of activated protein C in human alveolar epithelial cells

Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 mg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

The availability of induced pluripotent stem cells (iPSCs)has created extraordinary opportunities for modeling andperhaps treating human disease. However, all reprogrammingprotocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirelydevoid of xenobiotics. We first developed a xeno-free cellculture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derivedprimary cultures of human dermal fibroblasts under strictxeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free humaniPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colonymorphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiationability in vitro and in teratoma assays. Overall, the resultspresented here demonstrate that human iPSCs can be generatedand maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Therapeutic targeting of tumor growth and angiogenesis with a novel anti-S100A4 monoclonal antibody

S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.

Obstructive apneas induce early activation of mesenchymal stem cells and enhancement of endothelial wound healing

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +- 0.58 (m +- SE) and 1.00 +- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.