Hyperglycemia with or without insulin resistance triggers different structural changes in brain microcirculation and perivascular matrix.

Both type-1 and type-2 DM are related to an increased risk of cognitive impairment, neurovascular complications, and dementia. The primary triggers for complications are hyperglycemia and concomitant insulin resistance in type-2 DM. However, the diverse mechanisms in the pathogenesis of diabetes-related neurovascular complications and extracellular matrix (ECM) remodeling in type-1 and 2 have not been elucidated yet. Here, we investigated the high fat-high sucrose (HFHS) feeding model and streptozotocin-induced type-1 DM model to study the early effects of hyperglycemia with or without insulin resistance to demonstrate the brain microcirculatory changes, perivascular ECM alterations in histological sections and 3D-reconstructed cleared brain tissues. One of the main findings of this study was robust rarefaction in brain microvessels in both models. Interestingly, the HFHS model leads to widespread non-functional angiogenesis, but the type-1 DM model predominantly in the rostral brain. Rarefaction was accompanied by basement membrane thickening and perivascular collagen accumulation in type-1 DM; more severe blood-brain barrier leakage, and disruption of perivascular ECM organization, mainly of elastin and collagen fibers' structural integrity in the HFHS model. Our results point out that the downstream mechanisms of the long-term vascular complications of hyperglycemia models are structurally distinctive and may have implications for appropriate treatment options.

Three-dimensional neuron-astrocyte construction on matrigel enhances establishment of functional voltage-gated sodium channels.

This study aimed to investigate and compare cell growth manners and functional differences of primary cortical neurons cultured on either poly-d-lysine (PDL) and or Matrigel, to delineate the role of extracellular matrix on providing resemblance to in vivo cellular interactions in nervous tissue. Primary cortical neurons, obtained from embryonic day 15 mice pups, seeded either on PDL- or Matrigel-coated culture ware were investigated by DIC/bright field and fluorescence/confocal microscopy for their morphology, 2D and 3D structure, and distribution patterns. Patch clamp, western blot, and RT-PCR studies were performed to investigate neuronal firing thresholds and sodium channel subtypes Nav1.2 and Nav1.6 expression. Cortical neurons cultured on PDL coating possessed a 2D structure composed of a few numbers of branched and tortuous neurites that contacted with each other in one to one manner, however, neurons on Matrigel coating showed a more complicated dimensional network that depicted tight, linear axonal bundles forming a 3D interacted neuron-astrocyte construction. This difference in growth patterns also showed a significant alteration in neuronal firing threshold which was recorded between 80 < Iinj > 120 pA on PDL and 2 < Iinj > 160 pA on Matrigel. Neurons grown up on Matrigel showed increased levels of sodium channel protein expression of Nav1.2 and Nav1.6 compared to neurons on PDL. These results have demonstrated that a 3D interacted neuron-astrocyte construction on Matrigel enhances the development of Nav1.2 and Nav1.6 in vitro and decreases neuronal firing threshold by 40 times compared to conventional PDL, resembling in vivo neuronal networks and hence would be a better in vitro model of adult neurons.

Blood-brain barrier leakage and perivascular collagen accumulation precede microvessel rarefaction and memory impairment in a chronic hypertension animal model.

Hypertension (HT) is one of the main causes of vascular dementia, lead to cognitive decline. Here, we investigated the relationship between cerebral microvessels, pericytes, extracellular matrix (ECM) accumulation, blood-brain barrier (BBB) breakdown, and memory impairment at mid-life in a chronic hypertension animal model. Spontaneously hypertensive rats (SHRs) (n = 20) are chosen for the model and age matched Wistar rats (n = 16) as controls. Changes in brain microvasculature and in vitro experiments are shown with immunofluorescence studies and cognition with open field, novel object recognition, and Y maze tests. There was a significant reduction in pericyte coverage in SHRs (p = 0.021), while the quantitative parameters of the cerebral microvascular network were not different between groups. On the other hand, parenchymal albumin leakage, as a Blood-brain barrier (BBB) breakdown marker, was prominent in SHRs (p = 0.023). Extracellular matrix (ECM) components, collagen type 1, 3 and 4 were significantly increased (accumulated) around microvasculature in SHRs (p = 0.011, p = 0.013, p = 0.037, respectively). Furthermore, in vitro experiments demonstrated that human brain vascular pericytes but not astrocytes and endothelial cells secreted type I collagen upon TGFß1 exposure pointing out a possible role of pericytes in increased collagen accumulation around cerebral microvasculature due to HT. Furthermore, valsartan treatment decreased the amount of collagen type 1 secreted by pericytes after TGFß1 exposure. At the time of evaluation, SHRs did not demonstrate cognitive decline and memory impairments. Our results showed that chronic HT causes ECM accumulation and BBB leakage before leading to memory impairments and therefore, pericytes could be a novel target for preventing vascular dementia.

Microstructure of early embryonic aortic arch and its reversibility following mechanically altered hemodynamic load release.

In the embryonic heart, blood flow is distributed through a bilaterally paired artery system composed of the aortic arches (AAs). The purpose of this study is to establish an understanding of the governing mechanism of microstructural maturation of the AA matrix and its reversibility, toward the desired macroscopic vessel lumen diameter and thickness for healthy, abnormal, and in ovo repaired abnormal mechanical loading. While matrix-remodeling mechanisms were significantly different for normal versus conotruncal banding (CTB), both led to an increase in vessel lumen. Correlated with right-sided flow increase at Hamburger & Hamilton stages 21, intermittent load switching between collagen I and III with elastin and collagen-IV defines the normal process. However, decreases in collagen I, elastin, vascular endothelial growth factor-A, and fibrillin-1 in CTB were recovered almost fully following the CTB-release model, primarily because of the pressure load changes. The complex temporal changes in matrix proteins are illustrated through a predictive finite-element model based on elastin and collagen load-sharing mechanism to achieve lumen area increase and thickness increase resulting from wall shear stress and tissue strain, respectively. The effect of embryonic timing in cardiac interventions on AA microstructure was established where abnormal mechanical loading was selectively restored at the key stage of development. Recovery of the normal mechanical loading via early fetal intervention resulted in delayed microstructural maturation. Temporal elastin increase, correlated with wall shear stress, is required for continuous lumen area growth.NEW & NOTEWORTHY The present study undertakes comparative analyses of the mechanistic differences of the arterial matrix microstructure and dynamics in the three fundamental processes of control, conotruncal banded, and released conotruncal band in avian embryo. Among other findings, this study provides specific evidence on the restorative role of elastin during the early lumen growth process. During vascular development, a novel intermittent load-switching mechanism between elastin and collagen, triggered by a step increase in wall shear stress, governs the chronic vessel lumen cross-sectional area increase. Mimicking the fetal cardiovascular interventions currently performed in humans, the early release of the abnormal mechanical load rescues the arterial microstructure with time lag.

The role of ovarian reserve markers in prediction of clinical pregnancy.

To evaluate the role of ovarian reserve markers in the prediction of clinical pregnancy and embryo transfer accomplishment among poor responder IVF applicants. 304 female poor responder IVF applicants were included in this prospective cohort study conducted at the IVF-unit. Antral follicle count, FSH, LH, E2, AMH and IVF outcomes were compared in pregnant and non-pregnant groups as well as in ET vs. non-ET groups. The number of retrieved oocytes was significantly correlated positively with AMH and AFC, and negatively with FSH and age. Quartiles of FSH and AFC were similar to the rate of pregnancy. Quartiles of AMH (<25%/25-75% and <25%/>75%) were statistically significant. Mean serum levels for AMH were significantly lower in the non-ET group. Our findings seem to indicate that day 3 AMH values can predict ET accomplishment with a sensitivity of 96% and a specificity of 35%. Quartiles of AMH <25% (< 0.21 ng/mL) can predict the IVF results among poor responder IVF applicants. Impact statement Various cut-off values have been determined for day 3 serum AMH values. These values help to determine the groups that are expected to give normal, high or low response to stimulation and decide the treatment options. In contrast to other groups of patients, poor responders cannot reach the embryo transfer stage for several reasons. These are; absence of a mature oocyte after oocyte pick-up, fertilisation failure without male factor or poor embryo quality. In the present study; a cut-off value of 0.33 ng/mL for the prediction of ET accomplishment in poor responder patients was determined with a sensitivity of 96%. Additionally, clinical pregnancy could not be achieved under the value of 0.21 ng/mL day 3 AMH values. It is important to clarify the embryo transfer success of poor responder patients prior to expected treatment success. Pre-treatment counselling for these patients would lessen the disappointment that may develop after treatment. The cost-effectiveness of treatments below these AMH values can be determined by further studies.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

STUDY QUESTION: Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER: The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY: Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION: This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE: The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION: Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS: GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

Comparison of estradiol and progesterone priming/antagonist/letrozole and microdose flare-up protocols for poor responders undergoing intracytoplasmic sperm injection.

BACKGROUND: To compare the effect of the GnRH antagonist/letrozole/gonadotropin protocol with the microdose GnRH agonist flare-up protocol in poor ovarian responders for intracytoplasmic sperm injection. MATERIALS AND METHODS: One hundred twenty-one consecutive patients suspected of having or with a history of poor ovarian response between January 2009 and June 2010, who were undergoing ICSI were enrolled. The microdose flareup (MF) protocol was used in 79 patients and the estradiol + progesterone/letrozole + gonadotropin and GnRH antagonist (EP/ALG) protocol was used in 42 patients. RESULTS: Age of the patients, duration of infertility, basal FSH, the total gonadotropin consumption, duration of stimulation, E2 level on the day of hCG administration, the number of embryo transferred, the fertilization rate, implantation rate, clinical pregnancy rate and the live birth rate were not statistically different (p > 0.05). Only the number of oocytes retrieved was significantly higher in the EP/LGA group (1.7 ± 0.7 versus 2.6 ± 0.6). CONCLUSION: The EP/LGA protocol has no significant improvement against the microdose flare-up protocol in poor responder patients.

Splicing variants of versican in CD133+/CD44+ prostate cancer stem cells.

A cancer mass is composed of a heterogeneous group of cells, a small part of which constitutes the cancer stem cells since they are less differentiated and have a high capacity to develop cancer. Versican is an extracellular matrix protein located in many human tissues. The mRNA of versican has been shown to have "splicing patterns" as detected by RT-PCR, northern blot analysis, and cDNA sequencing. Based on this knowledge this study aims to reveal the splice variants of versican molecules, which are thought to be involved in the pathogenesis of the DU-145 human prostatic carcinoma cell line and prostatic cancer stem cells isolated from this cell line. In this study, RWPE-1 normal prostatic and DU-145 human prostate cancer cell lines have been used. Prostatic cancer stem cells and the remaining group of non-prostatic-cancer stem cells (bulk population) were isolated according to their CD133+/CD44+. RNA was isolated in all groups, and sequence analysis was accomplished for splicing variants by Illumina NextSeq 500 sequencing system. The results were analyzed by bioinformatic evaluation. As five isoforms of the versican gene in the differential transcript expression are analyzed, it was observed that a significant change was only found in the isoforms Versican 0 and Versican 1. In this study, we explored the function of this molecule which we think to be effective in cancer progression, and suggested that more valuable results can be obtained after the accomplishment of in vivo experiments.

Protein Scaffold-Based Multimerization of Soluble ACE2 Efficiently Blocks SARS-CoV-2 Infection In Vitro and In Vivo.

Soluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50 , comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections.

The Rhythmicity of Life: A Review of the Circadian Clocks.

Physiology of the mammalian body has been adapted to diurnal cycles of around 24 h, an evolutionary situation that affects a wide spectrum of biological events including sleep-to-wake transitions, feeding/fasting, body temperature, and hormonal regulations. The patterns of the diurnal cycle occur due to rhythmic oscillations that arise from the suprachiasmatic nucleus of hypothalamus, which also can be defined as the pacemaker of the system. The clock can be defined as a molecular machinery driven by the core clock genes that encode clock proteins in a rhythmic oscillatory fashion maintained by the light/dark cycles of the environment. Although the well-established knowledge refers to the function of the circadian rhythm as maintenance of the normal physiology, growing evidence shows that disruptions in the system usually caused by genetic and/or epigenetic misregulations may have a direct effect to lead major pathological conditions, such as carcinogenesis. This review outlines the main molecular aspects of circadian physiology, and reveals the reasons for and results of the circadian disruptions at different levels. In spite of the fact that more proof is needed for a direct correlation between circadian disruptions and oncogenesis and other pathological events, data obtained from current research supports the role of circadian rhythms in malfunctioning of the normal cellular metabolism.

The effects of oral mucosa-derived heterotopic fibroblasts on cutaneous wound healing.

An intriguing observation that has recently found support through clinical and experimental studies is that wounds of the oral mucosa tend to display faster healing and result in less scarring than in the skin. We aimed to investigate the potential of heterotopic oral mucosal fibroblasts in cutaneous wounds while determining the main differences between wounds conditioned with either the oral mucosa or dermis-derived human fibroblasts. A total of 48 nude mice were divided into four groups: control, sham, dermal fibroblast (DF), and oral fibroblast (OF). Fibroblasts were isolated, cultured, and seeded onto fibrin scaffolds for transfer to full-thickness dorsal wounds. Cell viability, wound area, healing rate, vascularization, cellular proliferation, dermal thickness, collagen architecture, and subtypes were evaluated. Both cell groups had a viability of 95% in fibrin gel prior to transfer. None of the wounds fully epithelialized on day 10, while all were epithelialized by day 21, which resulted in scars of different sizes and quality. Healing rate and scars were similar between the control and sham groups, whereas fastest healing and least scarring were noted in the OF group. Dermal thickness was highest in the DF group, which was also supported by highest levels of collagen types I and III. Proliferative cells and vascular density were highest in the OF group. DF result in healing through a thick dermal component, while oral fibroblasts result in faster healing and less scarring through potentially privileged angiogenic and regenerative gene expression.

Microrna analysis of human decidua mesenchymal stromal cells from preeclampsia patients.

INTRODUCTION: In preeclampsia (PE), human decidua mesenchymal stromal cells (hDMSCs) are exposed to abnormally high levels of oxidative stress and inflammatory factors circulating in the maternal blood. MicroRNAs (miRNAs) have been shown to have a significant impact on the differentiation, maturation and function of mesenchymal stromal cells (MSCs). Our aim in the present study is firstly to investigate differentially expressed miRNA levels to be used as a biomarker in the early detection of PE and secondly to investigate whether those differentially expressed miRNAs in hDMSCs have an effect on the pathogenesis of PE. METHODS: This study covers miRNA expression analysis of hDMSCs from 7 PE patient and 7 healthy pregnant women and is a preliminary study to investigate putative biomarkers. After cell culture and cell sorting, total RNA including miRNAs were isolated from hDMSCs. Let-7b-3p, let-7f-1-3p, miR-191-3p, miR-550a-5p, miR-33b-3p and miR-425-3p were used for miRNA analysis and U6 snRNA was used for normalization of the samples. MiRNA analysis was performed by droplet digital polymerase chain reaction (ddPCR) method and obtained results were evaluated statistically. RESULTS: As a result of the analysis, it was observed that the levels of hsa-miR-33b-3p significantly (AUC: 0.93, p = 0.04, fold change: 4.5) increased in hDMSC of PE patients compared to healthy controls. However, let-7b-3p, let-7f-1-3p, miR-191-3p, miR-550a-5p, and miR-425-3p were not considered as significant because they did not meet the p < 0,05 requirement. DISCUSSION: Within the scope of the study, it is predicted that miR-33b-3p (p = 0.004, AUC = 0.93) can be used as a biomarker in detecting PE.

Virulence Determinants of Colistin-Resistant K. pneumoniae High-Risk Clones.

We proposed the hypothesis that high-risk clones of colistin-resistant K. pneumoniae (ColR-Kp) possesses a high number of virulence factors and has enhanced survival capacity against the neutrophil activity. We studied virulence genes of ColR-Kp isolates and neutrophil response in 142 patients with invasive ColR-Kp infections. The ST101 and ST395 ColR-Kp infections had higher 30-day mortality (58%, p = 0.005 and 75%, p = 0.003). The presence of yersiniabactin biosynthesis gene (ybtS) and ferric uptake operon associated gene (kfu) were significantly higher in ST101 (99%, p ≤ 0.001) and ST395 (94%, p < 0.012). Being in ICU (OR: 7.9; CI: 1.43-55.98; p = 0.024), kfu (OR:27.0; CI: 5.67-179.65; p < 0.001) and ST101 (OR: 17.2; CI: 2.45-350.40; p = 0.01) were found to be predictors of 30-day mortality. Even the neutrophil uptake of kfu+-ybtS+ ColR-Kp was significantly higher than kfu--ybtS- ColR-Kp (phagocytosis rate: 78% vs. 65%, p < 0.001), and the kfu+-ybtS+ ColR-Kp survived more than kfu--ybtS- ColR-Kp (median survival index: 7.90 vs. 4.22; p = 0.001). The kfu+-ybtS+ ColR-Kp stimulated excessive NET formation. Iron uptake systems in high-risk clones of colistin-resistant K. pneumoniae enhance the success of survival against the neutrophil phagocytic defense and stimulate excessive NET formation. The drugs targeted to iron uptake systems would be a promising approach for the treatment of colistin-resistant high-risk clones of K. pneumoniae infections.

Terminal differentiation of human granulosa cells as luteinization is reversed by activin-A through silencing of Jnk pathway.

Molecular mechanisms underlying luteinization (terminal differentiation of granulosa and theca cells after ovulation) and luteolysis (demise of corpus luteum) are poorly understood in human ovary. Here we report that activin-A, after binding to its cognate receptors induces a functional luteolytic state and reverses luteinization phenotype by downregulating the expression of the steroidogenic enzymes, LH receptor and VEGF and reducing estradiol (E2) progesterone (P4) production and upregulating FSH receptor and cyclin D1 expression in human primary luteinized granulosa cells. Further, this action of activin-A involves downregulation of JNK signaling pathway and is opposite to that of human chorionic gonadotropin (hCG), which acts as a luteotropic hormone and improves luteal function through the activation of JNK pathway in the same cell type. Reversal of luteinization phenotype in luteal granulosa cells by activin-A potentially makes this hormone an attractive candidate for use under certain clinical situations, where induction of luteolysis and rapid reduction of endogenous sex steroid levels are beneficial such as ovarian hyperstimulation syndrome (OHSS), in which the ovaries hyper-respond to gonadotropin stimulation by producing too many growing follicles along with development of ascites, pleural effusion, and hemo-concentrations as a result of increased vascular permeability and leakage of intravascular volume into third spaces. Our work unveils a previously undefined role for activin-A and JNK signaling pathway in human corpus luteum biology, that might have a direct clinical impact in assisted reproductive technologies.

Functional structure of adipocytes differentiated from human umbilical cord stroma-derived stem cells.

It has been previously demonstrated that human umbilical cord stroma-derived stem cells (HUCSCs) are competent to differentiate into adipocytes. However, controversies have arisen as to whether HUCSCs can become mature adipocytes or not, and to what extent these cells can be induced in adipogenic pathway. Here, we extensively analyzed their adipogenic potency with a structural and functional approach by determining lipid formation dynamics in concordance to adipocyte-specific markers. During a 35-day period, HUCSCs respond to adipogenic induction, at which point 88% of cells exhibited multilocular lipid granules (LGs) having a mean diameter of 3 mum in round-shaped, F-actin-poor cells. Although the 1st week of induction did not generally display typical lipidogenic phenotypes, the degree of adipogenesis was dissected and confirmed by mRNA expressions of peroxisome proliferator-activated receptor gamma, C/EBP-beta, sterol regulatory element-binding transcription factor 1, adipophilin, stearoyl-CoA desaturase, glycerol 3-phosphate dehydrogenase 1, LIPE, adiponectin, and leptin. All markers tested were found elevated in various amounts (3-70-fold) around day 7 and reached a plateau after day 14 or 21 (5-335-fold). Perilipin as a surface protein around the LGs was confined exclusively to the enlarging LGs. Conclusively, we propose that after the termination of proliferation, HUCSCs possess the biochemical and cellular machinery to successfully differentiate into maturing adipocytes under adipogenic conditions, and this feature will ultimately allow these fetus-derived stem cells to be used for various therapeutic or esthetic purposes.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation.

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.

Effects of methyl-beta-cyclodextrin on blood-brain barrier permeability in angiotensin II-induced hypertensive rats.

The blood-brain barrier (BBB) permeability primarily increases in cerebral venules during acute hypertension. Methyl-ß-cyclodextrin (MßCD), a cholesterol-depleting agent, decreases the expression of caveolins disrupting caveolar structures. We aimed to determine the effects of MßCD on the BBB permeability of angiotensin (ANG) II-induced hypertensive rats. Three minutes after MßCD administration (5 mg/kg), acute hypertension was induced by ANG II (60 µg/kg). Evans blue (EB) and horseradish peroxidase (HRP) tracers were used to assess BBB permeability. Immunohistochemistry for caveolin (Cav)-1 and tight junction protein claudin-5 was performed. EB dye content significantly increased in both cerebral cortices and left hippocampus in MßCD (P < 0.05), right cerebral cortex and both hippocampi in ANG II (P < 0.05; P < 0.01), and both cerebral cortices and hippocampi in MßCD plus ANG II groups compared with controls (P < 0.05; P < 0.01). A significant decrease in claudin-5 immunostaining intensity was observed in animals treated with MßCD compared with controls (P < 0.05). Cav-1 immunostaining intensity increased in ANG II group (P < 0.05). Ultrastructurally, HRP reaction products were observed in endothelial cells of the microvessels in the hippocampus region in MßCD group while the tracer was mainly localized in astrocytes and neurons in ANG II, and MßCD plus ANG II groups. The endothelial cells of the venules in the cerebral cortex of the animals in the latter experimental groups also showed an abundance of caveolar vesicles devoid of HRP reaction products. Our results revealed that MßCD did not provide overall protective effects on BBB integrity in acute hypertension and even led to BBB disruption in normotensive animals.

The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme.

Harakiri (HRK) is a BH3-only protein of the Bcl-2 family and regulates apoptosis by interfering with anti-apoptotic Bcl-2 and Bcl-xL proteins. While its function is mainly characterized in the nervous system, its role in tumors is ill-defined with few studies demonstrating HRK silencing in tumors. In this study, we investigated the role of HRK in the most aggressive primary brain tumor, glioblastoma multiforme (GBM). We showed that HRK is differentially expressed among established GBM cell lines and that HRK overexpression can induce apoptosis in GBM cells at different levels. This phenotype can be blocked by forced expression of Bcl-2 and Bcl-xL, suggesting the functional interaction of Bcl-2/Bcl-xL and HRK in tumor cells. Moreover, HRK overexpression cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a known tumor-specific pro-apoptotic agent. Besides, secondary agents that augment TRAIL response, such as the histone deacetylase inhibitor MS-275, significantly increases HRK expression. In addition, GBM cell response to TRAIL and MS-275 can be partly abolished by HRK silencing. Finally, we showed that HRK induction suppresses tumor growth in orthotopic GBM models in vivo, leading to increased survival. Taken together, our results suggest that HRK expression is associated with GBM cell apoptosis and increasing HRK activity in GBM tumors might offer new therapeutic approaches.

Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells.

Human umbilical cord (UC) has been a tissue of increasing interest in recent years. Many groups have shown the stem cell potency of stromal cells isolated from the human UC mesenchymal tissue, namely, Wharton's jelly. Since UC is a postnatal organ discarded after birth, the collection of cells does not require an invasive procedure with ethical concerns. Stromal cells, as the dominant cells of this fetus-derived tissue, possess multipotent properties between embryonic stem cells and adult stem cells. They bear a relatively higher proliferation rate and self-renewal capacity. Although they share common surface markers with bone marrow-derived MSCs, they also express certain embryonic stem cell markers, albeit in low levels. Without any spontaneous differentiation, they can be successfully differentiated into mature adipocytes, osteoblasts, chondrocytes, skeletal myocytes, cardiomyocytes, neurons, and endothelial cells. While causing no immunorejection reaction, they effectively function in vivo as dopaminergic neurons, myocytes, and endothelial cells. Given these characteristics, particularly the plasticity and developmental flexibility, UC stromal cells are now considered an alternative source of stem cells and deserve to be examined in long-term clinical trials. This review first aims to document the published findings so far regarding the nature of human UC stroma with special emphasis on the spatial distribution and functional structure of stromal cells and matrix, which serves as a niche for residing cells, and, secondly, to assess the in vitro and in vivo experiments in which differential stem cell potencies were evaluated.

C-Abl is not activated in DNA damage-induced and Tap63-mediated oocyte apoptosis in human ovary.

There is a controversy in literature as to whether c-Abl is crucial for the induction of TAp63-mediated apoptosis and whether that inhibition of c-Abl with imatinib, which was designed to inhibit the oncogenic kinase BCR-ABL and c-kit, protects oocytes from chemotherapy-induced apoptosis in mice. No human data are available on this issue. We therefore aimed to explore whether genomic damage induced by chemotherapy drug cisplatin activates c-Abl along with TAp63 and the inhibition of c-Abl with imatinib prevents cisplatin-induced oocyte death and follicle loss in human ovary. Exposure to cisplatin induced DNA damage, activated TAp63 and SAPK/JNK pathway, and triggered apoptosis in the oocytes and granulosa cells. However, TAp63 activation after cisplatin was not associated with any increase in the expression of c-Abl. Imatinib did not prevent cisplatin-induced apoptosis of the granulosa cells or oocytes. Moreover, treatment with this drug resulted in the formation of bizarre shaped follicles lacking oocytes and increased follicular atresia by inducing apoptosis of granulosa cells and oocytes. Similar toxic effects were observed when ovarian tissue samples were incubated with a c-kit antagonist drug anti-CD117, but not with another c-Abl tyrosine kinase inhibitor GNF-2, which lacks an inhibitory action on c-kit. Intraperitoneal administration of imatinib to the xenografted animals produced similar histomorphological abnormalities in the follicles in human ovarian grafts and did not prevent cisplatin-induced follicle loss when co-administered with cisplatin. Our findings provide, for the first time, a molecular evidence for ovarian toxicity of this drug in human. Furthermore, this study together with two previous case reports of a severely compromised ovarian response to gonadotropin stimulation and premature ovarian failure in patients, while receiving imatinib, further heighten the concerns about its potential gonadotoxicity on human ovary and urge caution in its use in young female patients.