Combination human umbilical cord perivascular and endothelial colony forming cell therapy for ischemic cardiac injury.

Cell-based therapeutics are promising interventions to repair ischemic cardiac tissue. However, no single cell type has yet been found to be both specialized and versatile enough to heal the heart. The synergistic effects of two regenerative cell types including endothelial colony forming cells (ECFC) and first-trimester human umbilical cord perivascular cells (FTM HUCPVC) with endothelial cell and pericyte properties respectively, on angiogenic and regenerative properties were tested in a rat model of myocardial infarction (MI), in vitro tube formation and Matrigel plug assay. The combination of FTM HUCPVCs and ECFCs synergistically reduced fibrosis and cardiomyocyte apoptosis, while promoting favorable cardiac remodeling and contractility. These effects were in part mediated by ANGPT2, PDGF-ß, and VEGF-C. PDGF-ß signaling-dependent synergistic effects on angiogenesis were also observed in vitro and in vivo. FTM HUCPVCs and ECFCs represent a cell combination therapy for promoting and sustaining vascularization following ischemic cardiac injury.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

The follicular fluid adipocytokine milieu could serve as a prediction tool for fertility treatment outcomes.

RESEARCH QUESTION: Can the adipocytokine milieu of the follicular niche improve the ability to predict treatment outcomes in infertile patients? DESIGN: Follicular fluid samples from overweight patients were analysed and compared with samples from matched normal-weight patients. Concentrations of adiponectin, chemerin, C-reactive protein, interleukin-6 (IL-6), IL-10, IL-18, insulin, leptin, prolactin, resistin, tumour necrosis factor alpha (TNF-α) and bone morphogenetic protein-15 (BMP-15) were assessed by multiple magnetic bead immunoassay (MMBI) and enzyme-linked immunosorbent assay and correlated with fertility treatment outcomes. RESULTS: Analysis of samples from 22 overweight and 22 normal-weight patients demonstrated that TNF-α can predict oocyte maturation rate. When stratified by body mass index (BMI), IL-10 emerges as a better predictor of oocyte maturation in normal-weight patients. Prolactin was a negative predictor for fertilization rate in the full cohort, and this prediction power was lost upon stratification. No adipocytokines were predictive of blastulation rate, and only age remained predictive. BMP-15 was a strong predictor of high-quality blastulation in the full cohort, more so in the normal-weight population. CONCLUSIONS: The adipocytokine milieu of the follicular fluid provides a snapshot of the growing oocyte's environment and can help predict fertility treatment outcomes, fine-tuning understanding of the dysregulation caused by increasing BMI. Inflammatory cytokines can predict oocyte maturation; prolactin, oocyte competence; and BMP-15, high-quality blastulation. Further analysis of these findings with a larger sample size and assessing individual oocytes will help shed more light on the clinical significance of these findings.

Cannabis alters epigenetic integrity and endocannabinoid signalling in the human follicular niche.

STUDY QUESTION: Do phytocannabinoids (PCs) affect follicular endocannabinoid signalling and the epigenome in the surrounding granulosa cells (GCs)? SUMMARY ANSWER: Exposure to PCs increases the expression of endocannabinoid receptors and reduces DNA methylation enzyme expression and global DNA methylation in naïve GCs. WHAT IS KNOWN ALREADY: Cannabis plant derivatives, known as PCs, are used for medicinal and recreational purposes. The main PC, tetrahydrocannabinol (THC), is the third most commonly used substance by women of childbearing age, hence knowledge of the effect it has on reproduction is of utmost importance. THC exerts its effects via receptors of the endocannabinoid system (ECS) and can interfere with folliculogenesis, oocyte development and ovulation. Endocannabinoids have been measured in follicular fluid (FF) obtained during oocyte retrieval and are implicated in controlling folliculogenesis. It has been established that in the placenta, PCs disrupt endocannabinoid homeostasis via impairment of the synthetic and degrading enzymes, leading to a net increase of endocannabinoid levels. Finally, previous studies have shown that THC alters methylation and histone modifications in sperm, brain and blood cells. STUDY DESIGN, SIZE, DURATION: This study included an in vivo cohort assessment of cannabis exposure and its effects on the follicle and in vitro assays conducted to validate the in vivo findings and to explore possible mechanisms of action. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 318 FF samples, from 261 patients undergoing IVF treatment at a private fertility clinic who consented for biobanking biological waste material between January 2018 and July 2019, were included in this study. Concentrations of PCs and endocannabinoids were assessed in FF by liquid chromatography-mass spectrometry (LC-MS/MS). Exposure to PCs was determined based on these measured levels. Levels of both endocannabinoid receptors (CB1R, CB2R) and the de novo DNA methylating enzyme, DNMT3b, in GCs were assessed by flow cytometry both in vitro and in vivo and global DNA methylation was assessed in vitro by ELISA. In vivo effects were assessed by comparing samples positive for at least one PC, with samples negative for all measured PCs. In vitro effects were determined in naive GCs, obtained concurrently with FF samples that had tested negative for all PCs. These GCs were treated with different combinations of the main three PCs. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 17 patients (6.4%) were positive for cannabis consumption. Furthermore, the prevalence of cannabis positivity in the FF increased from 4% of the tested samples that were collected prior to national legalisation in October 2018 to 12% of those collected following legalisation. Of note, 59% of patients who tested positive for PCs (10 of 17) reported previous or ongoing exposure to cannabis upon their initial intake. Endocannabinoid levels were not affected by the presence of PCs. CB2R was more prevalent than CB1R in GCs and its expression increased following acute and chronic in vitro exposure to PCs. The expression of DNMT3b and global methylation decreased following exposure, suggesting that cannabis may affect the epigenome in the follicular niche. The acute changes were sustained throughout chronic treatment. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by lack of details regarding mode, frequency and timing of PC consumption. Moreover, we were not able to adequately assess the effect of PCs on immediate or long-term clinical outcomes, due to the small sample size and the lack of follow up. Future, large-scale studies should focus on assess the clinical implications of cannabis exposure, validate our findings, and determine to what extent cannabis affects the epigenome ovarian follicle and the developing oocyte. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study measuring PCs in FF by LC-MS/MS. We show that consuming cannabis alters the ECS in the developing follicle, and directly affects DNMT expression and global DNA methylation levels. Cannabis legalisation and use is increasing worldwide, therefore further understanding its role in female fertility and folliculogenesis is critical. STUDY FUNDING/COMPETING INTEREST(S): All funding was provided by CReATe Fertility Centre through the reinvestment of clinical earnings. The authors declare no competing interests.

Human umbilical cord perivascular cells maintain regenerative traits following exposure to cyclophosphamide.

Chemotherapies can cause germ cell depletion and gonadal failure. When injected post-chemotherapy, mesenchymal stromal cells (MSCs) from various sources have been shown to have regenerative effects in rodent models of chemotherapy-induced gonadal injury. Here, we evaluated two properties of a novel source of MSC, first trimester (FTM) human umbilical cord perivascular cells (HUCPVCs) (with increased regenerative potential compared to older sources), that may render them a promising candidate for chemotherapeutic gonadal injury prevention. Firstly, their ability to resist the cytotoxic effects of cyclophosphamide (CTX) in vitro, as compared to term HUCPVCs and bone marrow cells (BMSCs); and secondly, whether they prevent gonadal dysfunction if delivered prior to gonadotoxic therapy in vivo. BMSC, FTM HUCPVC, term HUCPVC, and control NTERA2 cells were treated with moderate (150 µmol/L) and high (300 µmol/L) doses of CTX in vitro. Viability, proliferative capacity, mesenchymal cell lineage markers and differentiation capacity, immunogenicity, and paracrine gene expression were assessed. CTX was administered to Wistar rats 2 days following an intra-ovarian injection of FTM HUCPVC. HUCPVC survival and ovarian follicle numbers were assessed using histological methods. We conclude that FTM HUCPVC maintain key regenerative properties following chemotherapy exposure and that pre-treatment with these cells may prevent CTX-induced ovarian damage in vivo. Therefore, HUCPVCs are promising candidates for fertility preservation.

Glucose withdrawal induces Endothelin 1 release with significant angiogenic effect from first trimester (FTM), but not term human umbilical cord perivascular cells (HUCPVC).

BACKGROUND: Perivascular cells (PVC) and their "progeny," mesenchymal stromal cells (MSC), have high therapeutic potential for ischemic diseases. While hypoxia can increase their angiogenic properties, the other aspect of ischemic conditions-glucose shortage-is deleterious for MSC and limits their therapeutic applicability. Regenerative cells in developing vascular tissues, however, can adapt to varying glucose environment and react in a tissue-protective manner. Placental development and fetal insulin production generate different glucose fluxes in early and late extraembryonic tissues. We hypothesized that FTM HUCPVC, which are isolated from a developing vascular tissue with varying glucose availability react to low-glucose conditions in a pro-angiogenic manner in vitro. METHODS: Xeno-free (Human Platelet Lysate 2.5%) expanded FTM (n = 3) and term (n = 3) HUCPVC lines were cultured in low (2 mM) and regular (4 mM) glucose conditions. After 72 h, the expression (Next Generation Sequencing) and secretion (Proteome Profiler) of angiogenic factors and the functional angiogenic effect (rat aortic ring assay and Matrigel™ plug) of the conditioned media were quantified and statistically compared between all cultures. RESULTS: Low-glucose conditions had a significant post-transcriptional inductive effect on FTM HUCPVC angiogenic factor secretion, resulting in significantly higher VEGFc and Endothelin 1 release in 3 days compared to term counterparts. Conditioned media from low-glucose FTM HUCPVC cultures had a significantly higher endothelial network enhancing effect compared to all other experimental groups both in vitro aortic ring assay and in subcutan Matrigel™ plugs. Endothelin 1 depletion of the low-glucose FTM HUCPVC conditioned media significantly diminished its angiogenic effect CONCLUSIONS: FTM HUCPVC isolated from an early extraembryonic tissue show significant pro-angiogenic paracrine reaction in low-glucose conditions at least in part through the excess release of Endothelin 1. This can be a substantial advantage in cell therapy applications for ischemic injuries.

A solution to prevent secondary flow in adherent cell cultures.

High quality cell cultures require reliable laboratory practices. Today's small-scale in vitro cell culture format is dominated by circular topology vessels, with the inherent disadvantage of secondary flow induced each time the cell cultures are repositioned. The secondary flow generates uneven sedimentation and adherence that negatively impacts cell culture quality. Here we show a modification of the circular culture vessel that abrogates these disturbances. Cell culture wells were augmented with a central column to diminish secondary flow. Human carcinoma cell lines (BeWo, JEG-3), mesenchymal stem cells [human umbilical cord perivascular cells (HUCPVC)] and mouse embryonic fibroblasts (MEF) were cultured in both column-augmented and regular culture wells. Human carcinoma cell cultures showed even cell densities and significantly more viable cells in column-augmented vessels. In FTM HUCPVC cultures, cell surface MSC marker (CD90, CD105) expression and cell differentiation-related gene expression patterns were significantly more homogeneous in column-augmented vessels. MEF cells in column-augmented culture vessels showed a more consistent expression of IGF-1. Column-augmented cell culture vessels significantly improve the homogeneity of adherent cell cultures by mitigating the adverse effect of the secondary flow.This article has an associated First Person interview with the first author of the paper.

Pericytes in the Umbilical Cord.

The structural components of the umbilical cord, including two arteries and one vein, the stromal region/Wharton's jelly, and amniotic epithelial membrane, are well described at various time points of gestation. Over the last two decades, evidence has emerged that multipotent cells sharing properties of mesenchymal stromal cell and pericytes/mural cells can be isolated from multiple regions of the umbilical cord, including the perivascular region of the umbilical cord arteries and vein, Wharton's jelly, and subamnion. These cells have increasingly gained interest for their potential use in regenerative and immunomodulatory medicine. Recent studies suggest that obstetrical complications including gestational diabetes mellitus and preeclampsia may alter the yield, properties, and potency of mesenchymal stromal cells isolated from the umbilical cord. The role that pericytes or pericyte-like cells play in the development of the human umbilical cord and associated pathologies, however, remains to be investigated.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature. Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and "translatable" pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection. We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

Angiogenic potency evaluation of cell therapy candidates by a novel application of the in vitro aortic ring assay.

BACKGROUND: Due to limitations of current angiogenesis assays, we aimed to develop a novel application of the rat aortic ring assay to assess the angiogenic potential of mesenchymal stromal cells (MSCs). First-trimester human umbilical cord-derived perivascular cells (FTM HUCPVCs) have multipotent characteristics and previously demonstrated angiogenic potential. We compared the effect of this young source of MSCs and adult bone marrow stromal cells (BMSCs) on ex vivo aortic endothelial network formation. METHODS: Thoracic segments of adult rat aortas were isolated, sectioned and embedded into Matrigel™. Fluorophore-labeled FTM HUCPVC lines and BMSCs (N = 3) were cocultured with developing endothelial networks (day 0). MSC integration, tube formation and endothelial network growth were monitored daily using phase-contrast and fluorescence microscopy. Quantification of endothelial networks was performed using ImageJ network analysis software on day 5 of coculture. RESULTS: FTM HUCPVCs from two umbilical cord samples migrated toward and integrated with developing aortic ring tubular networks while displaying elongated morphologies (day 1). In contrast, BMSCs did not show targeted migration and maintained spherical morphologies with limited physical interactions. Within 1 week of coculture, FTM HUCPVC lines contributed to significantly greater radial network growth and network loop formation when compared to BMSCs and untreated networks. CONCLUSIONS: We have developed a novel potency assay to assess the angiogenic potential of cell therapy candidates. Favorable properties of FTM HUCPVCs over BMSCs that we observed with this assay and which merit further study include chemotaxis, affinity for developing vasculature, and physical supportive interactions contributing to the development of endothelial networks.

In Vitro Differentiation of First Trimester Human Umbilical Cord Perivascular Cells into Contracting Cardiomyocyte-Like Cells.

Myocardial infarction (MI) causes an extensive loss of heart muscle cells and leads to congestive heart disease (CAD), the leading cause of mortality and morbidity worldwide. Mesenchymal stromal cell- (MSC-) based cell therapy is a promising option to replace invasive interventions. However the optimal cell type providing significant cardiac regeneration after MI is yet to be found. The aim of our study was to investigate the cardiomyogenic differentiation potential of first trimester human umbilical cord perivascular cells (FTM HUCPVCs), a novel, young source of immunoprivileged mesenchymal stromal cells. Based on the expression of cardiomyocyte markers (cTnT, MYH6, SIRPA, and CX43) FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to bone marrow MSCs, while their immunogenicity remained significantly lower as indicated by HLA-A and HLA-G expression and susceptibility to T cell mediated cytotoxicity. When applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells within 1 week of coculture, making them the first MSC type with this ability. Our results indicate that young FTM HUCPVCs have superior cardiomyogenic potential coupled with beneficial immunogenic properties when compared to MSCs of older tissue sources, suggesting that in vitro predifferentiation could be a potential strategy to increase their effectiveness in vivo.

On the role of 4-hydroxynonenal in health and disease.

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,ß-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1.

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cell-substratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell's underside by the thin lamellae and filopodia of a cell in close apposition.

Transient knockdown of presenilin-1 provokes endoplasmic reticulum stress related formation of autophagosomes in HepG2 cells.

The involvement of presenilins in the endoplasmic reticulum (ER) related autophagy was investigated by their transient knockdown in HepG2 cells. The silencing of PSEN1 but not of PSEN2 led to cell growth impairment and decreased viability. PSEN1 silencing resulted in ER stress response as evidenced by the elevated levels of glucose regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and CCAAT/enhancer-binding protein homologous protein (CHOP) and by the activation of activating transcription factor 6 (ATF6). The activation of autophagy was indicated by the increased procession of microtubule-associated light chain 3 protein isoform B (LC3B) and by decreased phosphorylation of mammalian target of rapamycin (mTOR) and 70kDa ribosomal protein S6 kinase (p70S6K). Formation of ER-related cytoplasmic vacuolization colocalizing with the autophagic marker LC3B was also observed. The morphological effects and LC3B activation in presenilin-1 knockdown cells could be prevented by using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin or by calcium chelation. The results show that presenilin-1 hampers the ER stress dependent initiation of macroautophagy.

Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.

Human umbilical cord-derived perivascular cells (PVCs) are a recently characterized source of mesenchymal stromal cells that has gained much interest in the field of cellular therapeutics. However, very little is known about the changes in fate potential and restrictions that these cells undergo during gestational development. This study is the first to examine the phenotypic, molecular, and functional properties of first trimester (FTM)-derived PVCs, outlining properties that are unique to this population when compared to term (TERM) counterparts. FTM- and TERM-PVCs displayed analogous mesenchymal, perivascular, and immunological immunophenotypes. Both PVCs could be maintained in culture without alteration to these phenotypes or mesenchymal lineage differentiation potential. Some unique features of FTM-PVCs were uncovered in this study: (1) while the gene signatures of FTM- and TERM-PVCs were similar, key differences were observed, namely, that the Oct4A and Sox17 proteins were detected in FTM-PVCs, but not in TERM counterparts; (2) FTM-PVCs exhibited a greater proliferative potential; and (3) FTM-PVCs were more efficient in their in vitro differentiation toward selective mesenchymal cell types, including the chondrogenic and adipogenic lineages, as well as toward neuronal- and hepatocyte-like lineages, when compared to TERM-PVCs. Both PVCs were able to generate osteocytes and cardiomyocyte-like cells with similar efficiencies in vitro. Overall, FTM-PVCs show more plasticity than TERM-PVCs with regard to fate acquisition, suggesting that a restriction in multipotentiality is imposed on PVCs as gestation progresses. Taken together, our findings support the idea that PVCs from earlier in gestation may be better than later sources of multipotent stromal cells (MSCs) for some regenerative medicine applications.

Inhibition of glycoprotein synthesis in the endoplasmic reticulum as a novel anticancer mechanism of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG) has been found to trigger the unfolded protein response (UPR) likely due to the inhibition of glucosidase II, a key enzyme of glycoprotein processing and quality control in the endoplasmic reticulum (ER). These findings strongly suggest that EGCG interferes with glycoprotein maturation and sorting in the ER. This hypothesis was tested in SK-Mel28 human melanoma cells by assessing the effect of EGCG and deoxynojirimycin (DNJ) on the synthesis of two endogenous glycoproteins. Both tyrosinase and vascular endothelial growth factor (VEGF) protein levels were remarkably reduced despite unaltered mRNA expression in EGCG- or DNJ-treated cells compared to control. The hindrance of tyrosinase and VEGF protein synthesis could be prevented by proteasome inhibitor, lactacystine. Collectively, our results support that glucosidase II inhibitor EGCG interferes with protein processing and quality control in the ER, which diverts tyrosinase, VEGF, and likely other glycoproteins towards proteasomal degradation. This mechanism provides a novel therapeutic approach in dermatology and might play an important role in the antitumor effect or hepatotoxicity of EGCG.

Altered redox state of luminal pyridine nucleotides facilitates the sensitivity towards oxidative injury and leads to endoplasmic reticulum stress dependent autophagy in HepG2 cells.

Maintenance of the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum - an important pro-survival factor in the cell - is ensured by the concerted action of glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase. The mechanism by which the redox imbalance leads to cell death was investigated in HepG2 cells. The chemical inhibition of the glucose-6-phosphate transporter, the silencing of hexose-6-phosphate dehydrogenase and/or the glucose-6-phosphate transporter, or the oxidation of luminal NADPH by themselves did not cause a significant loss of cell viability. However, these treatments caused ER calcium store depletion. If these treatments were supplemented with the administration of a subliminal dose of the oxidizing agent menadione, endoplasmic reticulum vacuolization and a loss of viability were observed. Combined treatments resulted in the activation of ATF6 and procaspase-4, and in the induction of Grp78 and CHOP. In spite of the presence of UPR markers and proapoptotic signaling the effector caspases - caspase-3 and caspase-7 - were not active. On the other hand, an elevation of the autophagy marker LC3B was observed. Immunohistochemistry revealed a punctuated distribution of LC3B II, coinciding with the vacuolization of the endoplasmic reticulum. The results suggest that altered redox state of endoplasmic reticulum luminal pyridine nucleotides sensitizes the cell to autophagy.

Changes of endoplasmic reticulum chaperone complexes, redox state, and impaired protein disulfide reductase activity in misfolding alpha1-antitrypsin transgenic mice.

Alpha1-antitrypsin (AAT) deficiency is characterized by the accumulation of the misfolded mutant, Z form of alpha1-antitrypsin (PiZ) inside the lumen of the hepatic endoplasmic reticulum (ER). Both human patients and PiZ transgenic mice have similar symptoms of hepatic failure culminating in cirrhosis and hepatocellular carcinoma. The involvement of molecular chaperones, as well as the relevance of oxidative stress in this disease is not characterized well yet. Here, we show that, in the PiZ transgenic mice, the 58-kDa protein disulfide isomerase (PDI), the most important oxidoreductase and chaperone of the endoplasmic reticulum, is in a complex with PiZ, which is accompanied by a decrease of protein disulfide reductase activity of the ER. PiZ transgenic mice have a shift toward a more reduced ER environment and an elevation of cytoplasmic chaperones and antioxidant enzymes. Our data suggest that lower availability of PDI and a decreased protein disulfide reductase activity of the ER along with a cytoplasmic stress may contribute to the toxic effects of PiZ aggregation.