Incubation In Serum-free Medium Research Articles

Evaluating the Reparative Potential of Secretome from Patient-Derived Induced Pluripotent Stem Cells during Ischemia–Reperfusion Injury in Human Cardiomyocytes

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

Abstract 14110: Inhibition of Lpl and CD36 Does Not Prevent Lipid Accumulation in the Hearts of Adipose Triglyceride Lipase (ATGL) Deficient Mice

We tested whether murine ATGL deficiency-induced cardiotoxicity would be prevented by reducing uptake of circulating lipids through inhibition of lipoprotein lipase (LpL) or the fatty acid transporter CD36. Mice were created with floxed alleles for Atgl and Lpl and expression of MHC-Cre. Triglyceride content in the heart was 10x greater in the Atgl KO mice compared to control, and concurrent Lpl deletion did not change that. The Lpl Atgl DKO hearts had a comparable gross appearance and similar weights to the Atgl KO mice. The mice also had a similar survival rate with early death beginning at ~12 weeks. We then hypothesized that non-esterified fatty acids rather than triglyceride-derived fatty acids were the source of lipids in Atgl deficient hearts. To test this, we reduced CD36 expression using an antisense oligonucleotide (ASO) and also by creating heart specific combined Atgl and Cd36 knockout mice. The ASO knocked down CD36 expression in both CD31 (endothelial cells) and non-CD31 cells from the heart. It also reduced starvation lipid droplet formation and accumulated heart lipids after 3 weeks of a high fat diet. Surprisingly, neither the ASO nor genetic knockout rescued the Atgl deficient hearts from lipid accumulation or prevented early mortality. Placing mice on high fat (60%) or high sucrose (40%) diets also did not alter the development of heart dysfunction. We then hypothesized that the lipid accumulation with ATGL deficiency could occur independent of access to exogenous lipids. We tested this in vitro using H9c2(2-1) rat cardiomyocytes. After an overnight incubation in serum-free medium to deplete lipid droplets, inhibition of ATGL with Atglistatin led to cardiomyocyte accumulation of lipid droplets in the absence of a lipid source in the media. Concurrent inhibition of fatty acid synthase (FASN) with C75, cerulenin, or platensimycin prevented lipid droplet accumulation. Together, our data suggest that ATGL deficient lipid accumulation and toxicity can occur exclusive of lipid uptake.

Telomerase Does Not Improve DNA Repair in Mitochondria upon Stress but Increases MnSOD Protein under Serum-Free Conditions.

Telomerase is best known for its function in maintaining telomeres but has also multiple additional, non-canonical functions. One of these functions is the decrease of oxidative stress and DNA damage due to localisation of the telomerase protein TERT into mitochondria under oxidative stress. However, the exact molecular mechanisms behind these protective effects are still not well understood. We had shown previously that overexpression of human telomerase reverse transcriptase (hTERT) in human fibroblasts results in a decrease of mitochondrial DNA (mtDNA) damage after oxidative stress. MtDNA damage caused by oxidative stress is removed via the base excision repair (BER) pathway. Therefore we aimed to analyse whether telomerase is able to improve this pathway. We applied different types of DNA damaging agents such as irradiation, arsenite treatment (NaAsO2) and treatment with hydrogen peroxide (H2O2). Using a PCR-based assay to evaluate mtDNA damage, we demonstrate that overexpression of hTERT in MRC-5 fibroblasts protects mtDNA from H2O2 and NaAsO2 induced damage, compared with their isogenic telomerase-negative counterparts. However, overexpression of hTERT did not seem to increase repair of mtDNA after oxidative stress, but promoted increased levels of manganese superoxide dismutase (MnSOD) and forkhead-box-protein O3 (FoxO3a) proteins during incubation in serum free medium as well as under oxidative stress, while no differences were found in protein levels of catalase. Together, our results suggest that rather than interfering with mitochondrial DNA repair mechanisms, such as BER, telomerase seems to increase antioxidant defence mechanisms to prevent mtDNA damage and to increase cellular resistance to oxidative stress. However, the result has to be reproduced in additional cellular systems in order to generalise our findings.

Transforming Growth Factor-Beta Produced by Non-small Cell Lung Cancer Cells Contributes to Lung Fibroblast Contractile Phenotype.

Fibroblasts can alter the extracellular matrix (ECM), contributing to cancer progression by providing a scaffold for cancer cells. The influence of lung cancer cells (LCCs) on lung fibroblast-mediated ECM alteration is not well understood. After incubation in serum-free medium, LCC- or fibroblast-conditioned media were collected. The ECM alteration was assessed by collagen gel contraction assay. Both LCC-conditioned medium and exogenous transforming growth factor (TGF)-β1 increased collagen gel contraction by lung fibroblasts. TGF-β1 was produced in LCC-conditioned media at approximately 2 ng/ml. SB431542, a specific TGF-β receptor kinase inhibitor, partially inhibited the collagen gel contraction that had been increased by LCC-conditioned media. Lung fibroblast-conditioned medium stimulated TGF-β1 production from LCCs, whereas LCC-conditioned medium decreased fibroblast survival and α-smooth muscle actin expression by fibroblasts. Interaction between LCCs and lung fibroblasts through TGF-β signaling induces fibroblasts to assume the contractile phenotype and may contribute to cancer progression.

Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis

BackgroundThe therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo.MethodsMSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model.ResultsPlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model.ConclusionsPlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.

SiRNA Loaded Nanoparticles: Serum Stability and In Vitro Release Studies

Objective: The objective of the present study is to examine the serum stability of siRNA loaded nanoparticles that were prepared with certain polymer and evaluate the influence of different pH values on the in vitro release of siRNA from these particles. Methods: Serum stability of siRNA loaded nanoparticles was studied by incubation in serum free medium and in presence of 10 %, and 25 % serum at 37oC for 6 h. The unshielded fraction (liable to nucleases) of siRNA was measured by Fluoroskan microplate reader at lex = 485 nm, and lem = 518 nm using fluorescence PicoGreen dye. The in vitro release of siRNA from nanoparticles, measured by electrophoresis, was studied in DMEM at two different pH values of 3.5 and 7.4. Results: The results of serum stability showed that 8.6 %, 24.4 %, and 23.5 % siRNA was unshielded from the nanoparticles in the presence of 0 %, 10 %, and 25 % serum, respectively. After 12 h, the released fraction of siRNA was 17.8 % and 95.2 % at pH 7.4 and pH 3.5, respectively. Conclusion: The obtained results revealed that the nanoparticles were considered as a reasonable delivery system that could protect siRNA from degradation by nucleases enzymes upon systemic injection and able to release the loaded siRNA in the acidic pH inside the required cells.

Abstract 314: Evidence for the Expression of Renin Angiotensin System (RAS) and a Disintegrin and Metalloproteinase (ADAM) 17-mediated shedding of ACE2 in COS-7 cells

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal system. COS-7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aim of this study was to 1) demonstrate the presence of an endogenous and functional RAS in COS7 cells and 2) to investigate the role of ADAM17 in the ectodomain shedding of ACE2. COS-7 cells were grown to 90% confluency followed by incubation in serum-free media for 24 hours. Western blot, immunohistochemistry and mass spectrometry (MS)-based enzyme assays were used to study RAS protein expression and activity in COS7 cells. Western blot and immunostaining confirmed endogenous expression of ACE (195 kDa), ACE2 (75-70 kDa), AT1R (43 kDa), renin (41 kDa) and ADAM17 (130 kDa) in COS7 cells. A sensitive and selective MS approach was used to determine endogenous RAS enzymatic activity in COS-7 lysate and media using the natural RAS substrate Ang II (m/z 1046). MS analysis detected Ang-(1-7) formation (m/z 899) in lysate and media of COS7 cells. While Ang-(1-7) formation in media was completely blocked by the ACE2-specific inhibitor MLN-4760, Ang-(1-7) formation in the lysate was significantly inhibited by the prolyl carboxypeptidase-specific inhibitor Z-pro-prolinal. Using short-hairpin (sh) RNA-mediated technology, ADAM17 protein expression and activity was significantly reduced in silenced cells compared to normal cells. Western blot analysis showed a significant increase of ACE2 protein expression in the lysate of silenced cells compared to normal cell and ACE2 shedding into the media was significantly reduced in silenced cells. This is the first study to demonstrate endogenous expression of the RAS and ADAM17 in COS-7 cells. Use of COS-7 could enhance our understanding of the cross talk between different arms of the RAS and dissect the direct effects of ACE inhibitors and Ang II receptor blockers. The transfectable nature of this cell line makes it an attractive in vitro cell model for studying the molecular, functional and pharmacological properties of the renal RAS. The data support the utility of COS-7 for the study of ectodomain shedding of ACE2 in vitro.

Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5 days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6 days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3 days, but decreased slightly after 5 days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3–5 days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Effect of Stressed and Unstressed Cell Culture Environments on the Viability of MC3T3 Cells with Calcium Phosphates

The effect of Fetal Bovine Serum (FBS) deprivation on survival and apoptosis of osteoblasts cultured on various calcium phosphates was studied. Test materials were two calcium alkali orthophosphates (materials denominated: GB9 and GB14), which were compared to β-tricalcium phosphate (TCP). Tissue culture polystyrene (PS) served as control. Test materials were characterized by X-Ray Diffraction and scanning electron microscopy. An apoptotic challenge assay entailing serum withdrawal was applied: MC3T3-E1 osteoblasts were cultured for 72h on the test materials in serum containing medium, followed by incubation in serum free medium for another 24h. Serum withdrawal is an apoptotic challenge, which creates a stressed environment. Cells cultured on the test specimens in serum containing medium served as control. The TUNEL Assay was employed to quantify the percentage of apoptotic cells. GB9 and GB14 displayed a significantly lower percentage of apoptotic cells than TCP. TCP had significantly fewer apoptotic cells than PS. The percentage of apoptotic cells on GB9 and GB14 was less than 10%, while the number of apoptotic cells found on the untreated control specimens ranged between 5 and 7%. These findings indicate that GB9 and GB14 endow osteoblasts cultured on them with a decreased sensitivity to apoptosis, which corresponds well with the results of previous in vitro and in vivo studies.

Cell‐Penetrating Peptides as Delivery Vehicles for a Protein‐Targeted Terbium Complex

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake. CPP conjugation can be used to deliver otherwise cell-impermeable, ligand-fluorophore conjugates.

PWE-007 Paracrine prostaglandin-E signalling modulates canine gastric epithelial cell migration

IntroductionGastric ulceration is a limiting complication of therapy with cyclooxygenase (COX) antagonists, widely used anti-inflammatory/analgesic drugs in both humans and dogs. COX-derived prostaglandin E (PGE) has an important role in...

Abstract 1414: Lysophosphatidic acid-induced Cyr61 is associated with prostate cancer progression

Abstract Lysophosphatidic acid (LPA) is a serum phospholipid that has been associated with progression in several types of cancer. Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. We have reported that Cyr61 expression in benign prostate cells is regulated by LPA (Endocrinology 2004). Furthermore that Cyr61 expression is significantly up-regulated in prostate cancer (PCa) and that the expression levels correlate with Gleason score (J Urol 2010). The aim of this study was to determine if LPA-induced Cyr61 up-regulation is associated with PCa progression. BPH1 was utilized as a benign prostate cell line and PC3 as a PCa cell line. In real-time PCR of untreated BPH1 and PC3 cells, Cyr61 expression ratios to TBP were 0.5 and 5.1, and these ratios after 2 hours of 1uM LPA under serum-free medium treatment were 1.8 and 44.3, respectively. These results indicated that the Cyr61 expression levels and their increase by LPA stimulation were higher in PCa cells than in benign prostate cells. To examine whether LPA promotes cell invasion, Matrigel invasion assays were performed for 72 hours with or without 1uM of LPA in the lower chamber. LPA stimulation increased the invaded cell number from 0.3±0.5 to 12.6±2.5 (p<0.001) in BPH1 cells and from 31.9±20.9 to 55.2±14.8 (p=0.005) in PC3 cells. These results indicate that LPA promotes cell invasion in both benign prostate and PCa cells. To examine whether LPA-induced Cyr61 is associated with cell invasion, the Cyr61 was knocked down by siRNA in PC3 cells. Invaded cell numbers were changed by 48 hours of LPA stimulation after scramble siRNA from 2.2±1.3 to 12.3±10.2 (p=0.002) and after Cyr61 targeting siRNA from 1.8±2.6 to 2.7±2.7 (p=0.235), respectively, revealing that Cyr61 down-regulation suppresses LPA induced cell invasion. To investigate the association between Cyr61 up-regulation and prostate carcinogenesis, Cyr61 over-expressing BPH1 cells (BPH1-Cyr61) were established and their cell proliferation was compared with vector only transfected BPH1 cells (BPH1-mock) by MTS assays. The cell proliferation ratio of BPH1-mock and BPH1-Cyr61 cells after 48 hours of incubation in serum-free medium was 1.24±0.05 and 1.40±0.02 (p=0.043), respectively, indicating that Cyr61 over-expression promoted cell proliferation of BPH1 in vitro. In conclusion, LPA induced Cyr61 up-regulation was suggested to be associated with PCa progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2011-1414

Microvesicle-mediated RNA Molecule Delivery System Using Monocytes/Macrophages

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

New Potential Anticancer Agent of Carborane Derivatives: Selective Cellular Interaction and Activity of Ferrocene-Substituted Dithio-o-carborane Conjugates

The large diversity of structures and unique bonding modes of organometallic complexes make them possible to act as promising candidate therapeutic agents. In this study, the new type of ferrocene-substituted dithio-o-carborane conjugates (FcSB1, FcSB2, and FcSBCO) has been synthesized, and their in vitro antineoplastic activities have been explored by means of the electrochemical study, the real time cell electronic sensing (RT-CES) system, and biological assays. The conjugate-cell interactions were first monitored by electrochemistry, and the results show different cell uptake efficiency for FcSB1, FcSB2, and FcSBCO toward target cells. Both the highly hydrophobic ferrocenyl and carboranyl groups render the conjugates able to rapidly enter cells and exert acute cytotoxicity after 4 h incubation in serum-free media. However, FcSB1, FcSB2, and FcSBCO display different inhibition efficiencies toward SMMC-7721 and HepG2 cancer cells via the G(0)/G(1) arrest mechanism in a physiological environment. The anticancer activity is in the order FcSB2 > FcSB1 > FcSBCO, which is parallel to the order of the redox potentials of the ferrocenyl groups in the three complexes. In particular, FcSB1 and FcSB2 display a potent selective inhibition effect on the proliferation of the cancer cell lines SMMC-7721 and HepG2, but almost no effect on the normal cell line, the human embryonic lung fibroblast (HELF) cells. Thus, these results may provide some clues for use of the ferrocene-carborane conjugates in developing anticancer drugs.

Autocrine production of TGF-beta1 promotes myofibroblastic differentiation of neonatal lung mesenchymal stem cells.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extracellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-beta1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-beta1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of alpha-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-beta1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-beta1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the "default program" for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair.

Activation of EphA receptors on CD4+CD45RO+ memory cells stimulates migration

We have demonstrated previously that binding of ephrin-A1 to EphA receptors on human CD4(+) and CD8(+) T cells stimulates migration. Two EphA receptors have been reported in T cells: EphA1 at the protein level and EphA4 at the mRNA level. In this study, we wanted to investigate the expression profile of these receptors in T cell subpopulations and to test if expression differences would affect the potential of cells to migrate upon ephrin-A1 binding. We have generated an anti-EphA4 mAb for expression analysis. Our data show that functional EphA4 is expressed on the cell surface of CD4(+) and CD8(+) T cells. In addition, EphA4 receptor expression is induced after overnight incubation in serum-free medium, in particular, on CD4(+)CD45RO(+) T cells. Migration of CD4(+) T cells in response to ephrin-A1 is observed for memory cells (CD45RO(+)) and much weaker for naïve cells (CD45RA(+)). A signaling complex associated with the EphA4 receptor has also been isolated and includes EphA1, the Src family kinases Fyn and Lck, Slp76, and Vav1. To conclude, T cells express EphA1 and EphA4 receptors. Expression differences of EphA4 are observed in subpopulations of CD4(+) T cells. This is related to the cell migration potential after ephrin-A1 binding.

A role for the lysosomal protease cathepsin B in zebrafish follicular apoptosis

This study presents evidence that cathepsin B, a lysosomal protease, may be involved in the regulation of apoptosis during serum-starvation in teleost follicles. Zebrafish vitellogenic follicles were isolated, incubated under serum-free conditions and homogenized. The follicle extracts demonstrated caspase-3-like activity using the fluorogenic substrate DEVD-AMC, indicating the onset of apoptosis. Cathepsin B activity as measured using the fluorogenic cathepsin B substrate, Z-Arg-Arg-AMC was elevated within the first 6h of incubation in serum-free media and coincided with the onset of apoptosis. This increase in cathepsin B activity was sensitive to the cathepsin B inhibitor, CA-074-ME. Furthermore, adding CA-074-ME to the follicle incubation blocked caspase-3-like activation, suggesting that cathepsin B activity is a positive regulator of the apoptotic cascade during serum-starvation. Interestingly, the increase in cathepsin-B-like activity was not preceded by an increase in cathepsin B mRNA transcription, suggesting that regulation of this enzyme is at a level other than of the gene. These results suggest a regulatory role for cathepsin B during follicular apoptosis in zebrafish ovarian follicles.

Interaction of Double-stranded RNA-dependent Protein Kinase (PKR) with the Death Receptor Signaling Pathway in Amyloid β (Aβ)-treated Cells and in APPSLPS1 Knock-in Mice

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-beta peptide (Abeta) neurotoxicity and in APP(SL)PS1 KI transgenic mice. In SH-SY5Y cells, results showed that Abeta42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP(SL)PS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT(451)-PKR and pS(194)-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.