Serum-deprived Conditions Research Articles

O1-06-08: Caspase-6 activation disrupts the human neuronal cytoskeleton: Strong implication for an early cause of neuronal disruption in mild cognitively impaired and Alzheimer's disease individuals

The cystenyl Caspase-6 protease is highly active in the neurofibrillary tangles, neuropil threads and neuritic plaques of sporadic Alzheimer disease (AD) brains. The active Caspase-6 occurs in neurons that display neurodegeneration but do not have apoptotic morphology. Active Caspase-6 is present in pre-tangles and occurs pre-clinically in aged individuals that have normal but lower cognitive scores than their counterparts, suggesting that Caspase-6 could be an instigator of neurodegeneration. The objective of this study is to determine the effect of active Caspase-6 in the neurons of aged and AD individuals. Cytoskeleton proteins were assessed as substrates of active Caspase-6 in vitro. Caspase-6 was selectively activated by serum deprivation of human primary neurons in the absence or presence of Caspase-6 inhibitors. Cytoskeleton integrity was assessed by immunohistochemistry and western blotting against tubulin, MAP2, neurofilament, and Tau protein. The presence of Caspase-6-cleaved proteins in AD was assessed by immunohistochemistry with neoepitope antisera. Tubulin, MAP2, and Tau, but not Neurofilament proteins, are cleaved by Caspase-6. Whereas tubulin and MAP2 are homogenously distributed in the neurites of primary human neurons in normal culture conditions, they take a beaded appearance in serum-deprived conditions. Tau protein also appears beaded in serum deprivation and additionally redistributes around the nuclei. In contrast, the neurofilaments remain intact with serum deprivation. Caspase-6 inhibitors prevent the cytoskeleton beading and Tau redistribution. Tubulin and Tau cleaved by Caspase-6 are present in AD brains. These results show that the activation of Caspase-6 in human neurons disrupts the integrity of the cytoskeleton. Because of the importance of the cytoskeleton in proper vesicular and protein trafficking, it is likely that the activation of Caspase-6 not only disrupts the architecture but the function of neurons. Together, these results suggest that the activation of Caspase-6 disrupts cytoskeletal integrity and neuronal function in AD.

Kaposi's Sarcoma–Associated Herpesvirus Confers a Survival Advantage to Endothelial Cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different human malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. The KS lesion is of endothelial cell in origin and is highly dependent on autocrine and paracrine factors for survival and growth. In this study, we show that KSHV infection of endothelial cells induces the activation of the prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin pathway. KSHV infection of endothelial cells augmented cell survival in the presence of apoptotic inducers, including etoposide and staurosporine, and under conditions of serum deprivation. We found that KSHV infection of endothelial cells also increased the ability of these cells to form an in vitro tubular network under conditions of stress and growth factor deprivation. Finally, we show that the nuclear factor-kappaB and PI3K pathways are also required for endothelial tubular network formation. Collectively, these results suggest that KSHV infection of endothelial cells modulates cell signaling pathways and induces cell survival and angiogenesis, thereby contributing to the pathogenesis induced by KSHV.

Activation of autophagy in retinal ganglion cells

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell-protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy-related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was up-regulated 1.5- to 1.8-fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6-fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6-, 2.7-, and 1.7-fold increases in LC3-II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin-1 was 1.7-fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3-methyladenine, and Wortmannin in RGC-5 cells under serum-deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC-5 cells maintained under conditions of serum deprivation.

The release of fibroblast growth factor-1 from melanoma cells requires copper ions and is mediated by phosphatidylinositol 3-kinase/Akt intracellular signaling pathway

Melanoma is a highly invasive tumor with elevated mortality rates. Progression and aggressiveness appear related to the achievement of an angiogenic phenotype. Melanoma cells express several angiogenic factors, including fibroblast growth factor (FGF)-1 and FGF-2. The autocrine production and release of FGFs and the subsequent activation of FGF receptors, have a central role in melanoma tumor progression. We demonstrated that FGF-1 is secreted from a human melanoma cell line, A375, under conditions of serum deprivation. The release of FGF-1 is inhibited by the copper chelator ammonium tetrathiomolybdate, suggesting a role of copper in the secretory pathway, and is triggered by activation of phosphatidylinositol 3-kinase (PI3K)/Akt intracellular signaling. Interestingly, overexpression or activation of Akt has been correlated with poor prognosis in melanoma patients. Our data indicate a novel role for Akt in supporting the progression of human melanomas and advocate the need for new treatments targeting PI3K/Akt signaling pathway, to control tumor development and progression.

Serum Deprivation Enhances Apoptotic Cell Death by Increasing Mitochondrial Enzyme Activity

Mitochondria are important sensor of apoptosis. <TEX>$H_2O_2-induced$</TEX> cell death rate was enhanced by serum deprivation. In this study, we investigated whether serum deprivation using 0.5 or 3 % FBS induces apoptotic cell death through mitochondrial enzyme activation as compared to 10 % FBS. Apoptotic cell death was observed by chromosome condensation and the increase of sub-G0/G1 population. Serum deprivation reduced cell growth rate, which was confirmed by the decrease of S-phase population in cell cycle. Serum deprivation significantly increased caspase-9 activity and cytochrome c release from mitochondria into cytosol. Serum deprivation-induced mitochondrial changes were also indicated by the increase of ROS production and the activation of mitochondrial enzyme, succinate dehydrogenase. Mitochondrial enzyme activity increased by serum deprivation was reduced by the treatment with rotenone, mitochondrial electron transport inhibitor. In conclusion, serum deprivation induced mitochondrial apoptotic cell death through the elevation of mitochondrial changes such as ROS production, cytochrome c release and caspase-9 activation. It suggests that drug sensitivity could be enhanced by the increase of mitochondrial enzyme activity in serum-deprived condition.

Nuclear localization of TRK-A in liver cells.

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.

Morphological and Functional Features of Hepatic Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease

We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [(3)H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17beta-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17beta-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism.

IN VITRO ASSESSMENT OF MESENCHYMAL STROMAL CELL CHARACTERISTICS: IMPLICATIONS FOR THEIR CLINICAL USE

INTRODUCTION: Bone marrow (BM) stroma represents a source of progenitor stromal cells, termed mesenchymal stromal cells (MSCs), which are multipotent and can differentiate into cartilage, bone, and adipose tissue. Several questions have arisen regarding their long-term expansion and their safety before use. OBJECTIVE: Our goal was to assess the long-term expansion and safety of MSCs in clinical practice. METHODS: MSCs from BM of children with benign hematologic disorders and solid tumors without BM involvement were isolated and cultured for 10 consecutive passages (P). Immunophenotypic and functional characteristics, apoptosis, and the expression of cell cycle regulatory genes (p53, p16, and Rb) and signal transduction genes (H-Ras) involved in oncogenesis were assessed. RESULTS: MSCs expressed mesenchymal-related surface antigens, &amp;gt;85% from P1. They had the ability to differentiate into osteocytes, adipocytes, and chondrocytes (reverse-transcription polymerase chain reaction). Colony forming units (fibroblast) ranged from 40.71 ± 4.3 at P1 to 15.5 ± 6.7 at P10. Their doubling time was 2.01 ± 0.14 days at P1 and 3.5 ± 1.19 days at P9. A low percentage of apoptotic cells was detected (7-amino-actinomycin D [7AAD]) at P2 until P10. MSCs were resistant to apoptosis under serum-deprivation conditions. The expression of the cell cycle genes studied was not statistically different compared with controls, and cells did not grow on soft agar. CONCLUSIONS: MSCs isolated from BM of children retain their characteristics for a serial number of passages and survive under serum-deprivation conditions, a necessary process in a transplantation setting. The cells do not have oncogenic properties, as shown by normal expression levels of oncogenes and tumor suppressor genes, and no growth on soft agar. These findings enhance the use of MSCs in clinical applications.

Expression of heregulin and ErbB receptors in mesenchymal stem cells

Mesenchymal stem cells are a promising cell type for cell transplantation in myocardial infarction. Type I neuregulins-1, also known as heregulin, can promote the survival of cardiomyocytes and stimulate angiogenesis. The purpose of this study was to investigate the expression of heregulin and ErbB receptors in mesenchymal stem cells, then further detect the secretion of heregulin and the changes in expression of heregulin and ErbB receptors under conditions of serum deprivation and hypoxia. Mesenchymal stem cells isolated from bone marrow of 180 g male Sprague-Dawley rats were cultured. Passage 3 cells were detected experimentally by regular reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real time PCR and Western blotting. Heregulin and ErbB receptors were expressed in mesenchymal stem cells, and all three ErbB receptors mRNA expressions were significantly down-regulated by serum deprivation and hypoxia, but serum deprivation and hypoxia significantly increased the protein expression of heregulin. Serum deprivation and hypoxia more than 12 hours could induce the secretion of heregulin. Mesenchymal stem cells can express all three ErbB receptors and heregulin. Serum deprivation and hypoxia decrease the mRNA expression of ErbB receptors, increase the expression of heregulin, and activate the secretion of heregulin.

C-reactive protein augments hypoxia-induced apoptosis through mitochondrion-dependent pathway in cardiac myocytes

C-reactive protein (CRP) is an important predictive factor for cardiac disorders including acute myocardial infarction. Therapeutic inhibition of CRP has been shown to be a promising new approach to cardioprotection in acute myocardial infarction in rat models, but the direct effects of CRP on cardiac myocytes are poorly defined. In this study, we investigated the effects of CRP on cardiac myocytes and its molecular mechanism involved. Neonatal rat cardiac myocytes were exposed to hypoxia for 8 h. Hypoxia induced myocyte apoptosis under serum-deprived conditions, which was accompanied by cytochrome c release from mitochondria into cytosol, as well as activation of Caspase-9, Caspase-3. Hypoxia also increased Bax and decreased Bcl-2 mRNA and protein expression, thereby significantly increasing Bax/Bcl-2 ratio. Cotreatment of CRP (100 mug/ml) under hypoxia significantly increased the percentage of apoptotic myocytes, translocation of cytochrome c, Bax/Bcl-2 ratio, and the activity of Caspase-9 and Caspase-3. However, no effects were observed on myocyte apoptosis when cotreatment of CRP under normoxia. Furthermore, Bcl-2 overexpression significantly improved cellular viability through inhibition of hypoxia or cotreatment with CRP induced Bax/Bcl-2 ratio changes and cytochrome c release from mitochondria to cytosol, and significantly blocked the activity of Caspase-9 and Caspase-3. The present study demonstrates that CRP could enhance apoptosis in hypoxia-stimulated myocytes through the mitochondrion-dependent pathway but CRP alone has no effects on neonatal rat cardiac myocytes under normoxia. Bcl-2 overexpression might prevent CRP-induced apoptosis by inhibiting cytochrome c release from the mitochondria and block activation of Caspase-9 and Caspase-3.

Obestatin Promotes Survival of Pancreatic β-Cells and Human Islets and Induces Expression of Genes Involved in the Regulation of β-Cell Mass and Function

Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of beta-cells and human pancreatic islets and the underlying signaling pathways. beta-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. Obestatin showed specific binding on HIT-T15 and INS-1E beta-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-gamma/tumor necrosis factor-alpha/interleukin-1 beta treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys(3)]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in beta-cells and human islets. beta-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased beta-cell cAMP and activated extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on beta-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element-binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. These results indicate that obestatin promotes beta-cell and human islet cell survival and stimulates the expression of main regulatory beta-cell genes, identifying a new role for this peptide within the endocrine pancreas.

Dominant-Negative Inhibition of MCT-1 Oncogene Attenuates a Malignant Phenotype through Translational Repression.

Background: Gene expression is controlled at multiple levels. Translation initiation is a critical checkpoint for regulating levels of protein synthesis. The MCT-1 (Multiple copies in T-cell lymphoma 1) oncogene product has been shown to bind to the cap complex through its PUA domain and recruit DENR, a SUI1 motif containing protein that can increase translation initiation of target mRNAs by scanning and recognition of the initiation codon. An important function of MCT-1 is its modulation of a subset of cancer related mRNAs in human tumors. Levels of MCT-1 protein are increased in a number of non-Hodgkin's lymphoma cell lines and diffuse large B-cell lymphoma. The abnormal regulation of protein synthesis in lymphoma cells has the potential to be exploited for cancer therapy. We have shown that an MCT-1 deletion mutant, containing only the PUA domain interacts with the cap-complex but does not promote translation. We hypothesized that a PUA-domain mutant acting as dominant-negative would interfere with MCT-1 function through translational repression and modify the malignant phenotype.Method: Using a retroviral vector we established stable Jurkat cell lines expressing either PUA (Jurkat-PUA) or empty vector (Jurkat-V) in order to investigate the feasibility of targeting MCT-1 using a dominant-negative approach and its impact on the transformed phenotype. Multiple clones were plated in soft agar to determine anchorage-independent growth capacity. We also examined growth under reduced serum conditions to evaluate growth kinetics under stress conditions. Jurkat cell lines expressing either PUA or empty vector were exposed to either doxorubicin or gamma-irradiation and cell viability was assessed using both trypan blue exclusion and TUNEL assay. Effects on translation were assayed employing a combination of Western blotting and in-vivo translation assays.Results: There was a greater than three-fold difference in colony formation comparing Jurkat-V with Jurkat-PUA cells. Under serum deprivation conditions Jurkat-PUA grew much slower than Jurkat-V, and cell cycle analysis demonstrated Jurkat-PUA clones progressing through the cell cycle significantly slower than Jurkat-V clones. Sensitivity to both doxorubicin and gamma-radiation was increased at least 2-fold in cells expressing the PUA deletion mutant. Quantitative real-time PCR performed in Jurkat-V and Jurkat-PUA cells demonstrated equivalent levels of selected target mRNAs including Dp-1 and Cyclin D1 however, there were lower protein levels in the Jurkat-PUA clones. Finally, Jurkat-PUA cells displayed reduced in-vivo translation.Conclusion: We have shown that Jurkat cells retrovirally transduced with a dominant-negative MCT-1 mutant can interfere with protein synthesis and modify the malignant phenotype of a highly aggressive lymphoma. As proof of principle, we have established the utility of targeting MCT-1 and the translation initiation complex in lymphoma cells as a potentially useful therapeutic approach.

VEGF receptor Flk-1 plays an important role in c-kit expression in adipose tissue derived stem cells

It is known that c-kit + cells are increased in heart after infarction. The exact origins of the cardiac c-kit + cells remain to be determined. We asked whether adipose tissue could be a potential source of c-kit + cells. Our data show that the number of c-kit + cells increased in adipose tissue derived stem cells when cultured with conditioned medium from neonatal cardiomyocytes grown under serum deprivation and hypoxia condition. We also found that VEGF receptor Flk-1 is involved in c-kit up regulation via ERK-mediated pathway.

Dyrk1A overexpression in immortalized hippocampal cells produces the neuropathological features of Down syndrome

Down syndrome (DS) is the most common genetic disorder, characterized by mental retardation, congenital heart abnormalities, and susceptibility to Alzheimer's disease (AD). Brain development of DS patients is associated with elevated apoptosis and abnormal neuronal differentiation. Those key features are closely associated with many genes mapped within Down syndrome critical region (DSCR) on human chromosome 21. Proline-directed serine/threonine kinase, Dyrk1A, is mapped within DSCR, and involved in the control of cell growth and postembryonic neurogenesis. Despite the potential involvement of Dyrk1A in neurodegeneration, its links to AD susceptibility and the neuropathology of DS patients are not yet clearly understood. Here, we report evidence supporting the correlation between Dyrk1A and neuropathology of DS. Our results show that Dyrk1A interacts with and directly phosphorylates tau and amyloid precursor protein in immortalized hippocampal progenitor H19-7 cells. In addition, the formation of tau inclusion and the enhanced generation of β-amyloid fragment were detected in H19-7 cells that overexpressed Dyrk1A. Furthermore, these cells show a marked increase in apoptotic cell death under conditions of serum deprivation and also exhibit defects in neuronal differentiation. These results suggest that up-regulation of Dyrk1A may cause AD-like pathogenesis and abnormal neurobiological features in DS patients.

Heregulin protects mesenchymal stem cells from serum deprivation and hypoxia-induced apoptosis

Heregulin can regulate the survival of cardiomyocytes, epithelial cells, neuron, glial cells, and other cell types through binding with the ErbB receptors. The aim of this study is to investigate the effects of heregulin (HRG) on the apoptosis of Bone marrow Mesenchymal stem cells (MSCs). We used the MSCs from adult Sprague-Dawley rats and the model of serum deprivation (SD) and hypoxia-induced apoptosis. The apoptosis was detected by TUNEL method. The apoptosis of MSCs significantly increased 12 h or 18 h after SD and hypoxia, but treatment with HRG significantly decreased the apoptosis induced by SD and hypoxia. Tyrphostin AG1478 (ErbB3/4 inhibitor) or Tyrphostin AG825 (ErbB2 inhibitor) could block this effects of HRG. Akt and ERK were activated by HRG under SD and hypoxia conditions, but HRG had no effects on the activation of JNK and p38. HRG also increased the ratio of Bcl-2/Bax and decreased the activation of caspase3 induced by SD and hypoxia. These results suggested HRG could decrease the apoptosis of MSCs induced by SD and hypoxia through the activation of Akt and ERK, the increase of Bcl-2/Bax ratio and the inhibition of caspase3 activation.

Activation of Wnt/β‐catenin pathway mediates growth and survival in B‐cell progenitor acute lymphoblastic leukaemia

This study investigated the response of acute lymphoblastic leukaemia (ALL) cells to Wnt proteins. Accumulation of beta-catenin was measured by Western blotting and immunofluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis of B-cell progenitor acute lymphoblastic leukaemia (ALL) cells revealed expression of Wnt genes, including WNT2B in 33%, WNT5A in 42%, WNT10B in 58% and WNT16B in 25% of cases. The Wnt receptors, (Frizzled) FZD7 and FZD8 were also expressed in most cases while FZD3, FZD4 and FZD9 were occasionally detected. Stimulation of ALL cells with Wnt-3a activated canonical Wnt signalling with increased expression and nuclear translocation of beta-catenin. This resulted in a 1.7- to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. A significant increase in the survival of ALL cells under conditions of serum deprivation was also observed. Microarray analysis and quantitative RT-PCR revealed that activation of the Wnt/beta-catenin pathway led to altered expression of genes involved in cell cycle regulation and apoptosis in normal and leukaemic B-cell progenitors. Our results demonstrate that Wnt-3a provides proliferative and survival cues in ALL cells. This data suggests that targeting the Wnt signalling pathway may be a useful therapeutic strategy in ALL.

In Vitro Angiogenesis Properties of Endothelial Progenitor Cells: A Promising Tool for Vascularization of Ex Vivo Engineered Tissues

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at the improvement of neovascularization of engineered tissues are a key issue. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft before transplantation by the use of cellular-based concepts. To explore the utility of endothelial progenitor cells (EPCs) for the ex vivo vascularization of tissue engineered grafts, we analyzed the in vitro angiogenic properties of this cell type in two different angiogenesis models: the 3-dimensional spheroid sprouting assay and the 2-dimensional matrigel assay. In both assays, EPCs were able to form tubelike structures, resembling early capillaries. This process was significantly enhanced by the addition of angiogenic growth factors. Direct comparison between EPCs and mature endothelial cells, represented by human umbilical vein endothelial cells (HUVECs), revealed that both cell types displayed an almost identical angiogenic potential. Other functional in vitro parameters such as angiogenic growth factor induced cell proliferation and cell survival were investigated as well, revealing a significantly decreased level of apoptosis of EPCs in relation to HUVECs under serum-deprived conditions. The observed survival advantage of EPCs along with the observation that EPCs perform very well in the above mentioned in vitro angiogenesis assays, make them an ideal autologous cell source for vascularization of ex vivo generated tissues. The attractiveness of this cell type for tissue engineering applications is strengthened further by the fact that these cells can be easily isolated from the peripheral blood of patients, thereby eliminating donor site morbidity.

Survivin mediates the anti-apoptotic effect of δ-opioid receptor stimulation in cardiomyocytes

Survivin is known to be essential for cell division and to inhibit apoptosis during embryonic development and in adult cancerous tissues. However, the cardiovascular role of survivin is unknown. We observed that in cardiomyocytes cultured under conditions of serum and glucose deprivation (DEPV), the levels of survivin, Bcl-2 and extracellular signal-regulated kinase (ERK) were positively correlated with the anti-apoptotic action of a delta-opioid receptor agonist, [D-Ala2, D-Leu5]-enkephalin acetate (DADLE). By contrast, Bax translocation, mitochondrial membrane damage and reactive oxygen species (ROS) production were inversely correlated with the changes of survivin and Bcl-2. The use of RNA interference (RNAi) targeting survivin increased DEPV-induced cardiomyocyte apoptosis, whereas the anti-apoptotic effect of DADLE was blunted by survivin RNAi. Moreover, survivin transfection and overexpression provided protection against DEPV-induced cardiomyocyte apoptosis. Inhibition of ERK prevented the DADLE-induced decrease in apoptosis and Bax translocation, and increase in survivin and Bcl-2. DADLE-induced increase in survivin was also blunted by phosphoinositol 3-kinase (PI 3-kinase) inhibition. In conclusion, the present study provides the first direct evidence of an anti-apoptotic role of survivin mediating the anti-apoptotic effect of delta-opioid receptor activation in cardiomyocytes. ERK and PI 3-kinase were found to be upstream regulators of survivin. Mitochondrial membranes as well as ROS, Bcl-2 and Bax were also involved in this anti-apoptotic action.

FUNCTIONAL CHARACTERIZATION OF THE SERINE/THREONINE PROTEIN PHOSPHATASE 5 FROM TRYPANOSOMA BRUCEI

PP5 is a member of the PPP family of serine/threonine protein phosphatases and is present in all eukaryotes. We previously cloned and characterized a PP5 homologue from Trypanosoma brucei. Here, we synchronized the T. brucei procyclic form by hydroxyurea treatment and showed that TbPP5 expression is regulated during cell cycle progression. TbPP5 transcript and protein levels were maximal in the G1 phase of the cell cycle, and reduced about 3-fold in the G2/M phase. To further evaluate its function, TbPP5 expression was depleted in both procyclic and bloodstream forms of T. brucei by RNA interference. In the procyclic form, TbPP5 knockdown resulted in a moderate reduction in cell growth. However, in the bloodstream form, ablation of TbPP5 caused an 8-fold decrease in cell growth. Furthermore, TbPP5 overexpression conferred the ability of procyclic cells to grow in serum-deprived conditions suggesting that TbPP5 acts downstream of serum factor-induced growth in T. brucei. Taken together; these findings suggest that a serum factor (or factors) induces up-regulation of TbPP5 expression during the G1 phase, which is required for proper cell growth.

The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells.

Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.