Absence Of Growth Factors Research Articles

Molecular mechanisms of oogenesis

This literature review is devoted to the molecular mechanisms of oogenesis and depletion of the ovarian reserve. One of the factors in this process is constantly changing environment of the ovaries, both during intrauterine development and the postnatal period. Numerous mechanisms and factors affecting the internal environment of the female gonad are described, such as stem cell factor (SCF), which regulates migration of primordial germ cells and survival of early oocytes, insulin-like growth factor I (IGF-I), and leukocyte migration inhibitory factor (LMIF). The capabilities of the endocrine system, namely sex steroids, which can both replenish the number of germ cells and deplete the ovarian reserve through the expression of apoptotic markers, were shown. Apoptosis causes degeneration of most of the germ cells formed during oogenesis. The molecular mechanisms and factors involved in this process are numerous. Pathways mediated by mitochondria of germ cells and external pathways mediated by receptors of the cell surface were described. A mediator between two apoptotic pathways was established – the Bid protein (BH3-interacting domain death agonist), the activation of which triggers the apoptosis mechanism of the intrafollicular microenvironment. Some other factors were identified that mediate programmed germ cell death and result in diminished ovarian reserve: eukaryotic elongation factor 2 kinase (eEF-2 K), PUMA and NOXA genes, the absence of growth factors and members of tumor necrosis factor (TNF) family. Changes in the epigenetic modification of chromatin in the follicular and germ cells, oxidative stress, decreased DNA repair, and the involvement of the genes BRAC1, RAD51, ERCC2, and H2AX associated with this process can also affect reproductive health and the ovarian reserve. A significant role of mitochondrial dysfunction of follicular cells in depletion of the ovarian reserve is of great interest, which leads to impaired oocyte competence, deteriorates the gamete quality, and depletes the ovarian reserve. Therefore, oogenesis depends on a huge number of factors and the internal environment of the ovaries, the knowledge of which can maintain the stability of the reproductive function and preserve the quality of the ovarian reserve.

A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity

Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Growth factors improve the proliferation of Jeju black pig muscle cells by regulating myogenic differentiation 1 and growth-related genes.

ObjectiveThe growth rate of pigs is related to differentiation and proliferation of muscle cells, which are regulated by growth factors and expression of growth-related genes. Thus, the objective of this study was to establish optimal culture conditions for Jeju black pig (JBP) muscle cells and determine the relationship of various factors involved in muscle growth with the proliferation of JBP muscle cells.MethodsMuscles were taken from the femur skeletal muscle of JBP embryos. After isolation of the muscle cells, cells were cultured in a 6-well plate under four different culture conditions to optimize culture conditions for JBP muscle cells. To analyze proliferation rate of JBP muscle cells, these muscle cells were seeded into 6-well plates at a density of 1.5×105 cells per well and cultured for 3 days. Western blot and quantitative real-time polymerase chain reaction were applied to verify the myogenic differentiation 1 (MyoD) expression and growth-related gene expression in JBP muscle cells, respectively.ResultsWe established a muscle cell line from JBP embryos and optimized its culture conditions. These muscle cells were positive for MyoD, but not for paired box 7. The proliferation rate of these muscle cells was significantly higher in a culture medium containing bFGF and epidermal growth factor + basic fibroblast growth factor (EGF+bFGF) than that without a growth factor or containing EGF alone. Treatment with EGF and bFGF significantly induced the expression of MyoD protein, an important transcription factor in muscle cells. Moreover, we checked the changes of expression of growth-related genes in JBP muscle cells by presence or absence of growth factors. Expression level of collagen type XXI alpha 1 gene was changed only when EGF and bFGF were added together to culture media for JBP muscle cells.ConclusionConcurrent use of EGF and bFGF increased the expression of MyoD protein, thus regulating the proliferation of JBP muscle cells and the expression of growth-related genes.

Skin Resident γδ T Cell Function and Regulation in Wound Repair.

The skin is a critical barrier that protects against damage and infection. Within the epidermis and dermis reside γδ T cells that play a variety of key roles in wound healing and tissue homeostasis. Skin-resident γδ T cells require T cell receptor (TCR) ligation, costimulation, and cytokine reception to mediate keratinocyte activity and inflammatory responses at the wound site for proper wound repair. While both epidermal and dermal γδ T cells regulate inflammatory responses in wound healing, the timing and factors produced are distinct. In the absence of growth factors, cytokines, and chemokines produced by γδ T cells, wound repair is negatively impacted. This disruption in γδ T cell function is apparent in metabolic diseases such as obesity and type 2 diabetes. This review provides the current state of knowledge on skin γδ T cell activation, regulation, and function in skin homeostasis and repair in mice and humans. As we uncover more about the complex roles played by γδ T cells in wound healing, novel targets can be discovered for future clinical therapies.

Abstract 1447: Elucidating the mechanism of Dock7-mediated cellular transformation

Abstract Akt is a serine/threonine protein kinase that receives signals from a plethora of external and internal stimuli in order to promote survival and regulate cellular growth and metabolism. Akt is part of the highly conserved PI3K/Akt signaling axis and is classically regulated by its interaction with the phosphoinositide, PIP3, and its subsequent phosphorylation at S473 and S308 by the mTOR complex 2 (mTORC2) and 3-Phosphoinositide-dependent protein kinase 1 (PDK1), respectively. Altered Akt activity has been linked to numerous diseases, such as cancer, diabetes and neurodegenerative diseases. Specifically, PI3K/Akt deregulation occurs in approximately one third of human cancers and ~40% of breast cancers. My research focuses on Dock7, a Guanine Nucleotide Exchange Factor (GEF) for the small GTPases Cdc42 and Rac1, and a novel player in the regulation of Akt activity and stability. Dock7 belongs to the Dock family of atypical GEFs and contains two evolutionarily conserved domains, DHR1 (a putative lipid binding domain) and DHR2 (the catalytic GEF domain). The expression level of Dock7 correlates with an unfavorable prognosis in liver cancer patients, and it is highly expressed in triple-negative breast cancers. Studies in our laboratory showed Dock7 is essential for the transformed properties of several cancer cell lines, and that it interacts with Akt in co-immunoprecipitation assays. In order to decipher the mechanism by which Dock7 mediates transformation, I created a Crispr-Cas9 Dock7 Knock-out HeLa cell line. I proceeded to confirm that these cells are not able to grow in an anchorage independent manner nor in serum free conditions, two hallmarks of transformation. Furthermore, in comparison to the phosphorylation status of Akt in parental HeLa cells, I found that in Dock7 KO HeLa cells, Akt is not phosphorylated at the S473 site in the absence of growth factors, indicative of lower basal activity. Accordingly, phosphorylation of downstream effectors of Akt is also downregulated. Besides promoting the phosphorylation state of Akt, Dock7 plays a role in the stability of the Akt protein during certain cellular stresses, highlighting a versatile and important role for Dock7 in Akt regulation and a potential target for therapeutic intervention in tumors where Akt is deregulated. Citation Format: Oriana Y. Teran Pumar, Kristin F. Wilson, Richard A. Cerione. Elucidating the mechanism of Dock7-mediated cellular transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1447.

Albumin promotes the progression of fibroblasts through late G1 into S-phase in the absence of growth factors

ABSTRACT G1 cell cycle progression is controlled largely by growth factors in early G1 indicating that it is appropriate to divide and by nutrients in late G1 indicating sufficient raw material for cell division. We previously mapped a late G1 cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G1 checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G1 into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [3 H]-thymidine deoxyribose ([3 H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase – the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [3 H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G1. Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin – the most abundant protein in serum – reflects a broader availability of essential amino acids needed for cell growth.

Robust phenotypic maintenance of limb cells during heterogeneous culture in a physiologically relevant polymeric-based constructed graft system

A major challenge during the simultaneous regeneration of multiple tissues is the ability to maintain the phenotypic characteristics of distinct cell populations on one construct, especially in the presence of different exogenous soluble cues such as growth factors. Therefore, in this study, we questioned whether phenotypic maintenance over a distinct population of cells can be achieved by providing biomimetic structural cues relevant to each cell phenotype into the construct’s design and controlling the presentation of growth factors in a region-specific manner. To address this question, we developed a polymeric-based constructed graft system (CGS) as a physiologically relevant model that consists of three combined regions with distinct microstructures and growth factor types. Regions A and B of the CGS exhibited similar microstructures to the skin and soft tissues and contained rhPDGF-BB and rhIGF-I, while region C exhibited a similar microstructure to the bone tissue and contained rhBMP-2. Primary rat skin fibroblasts, soft tissue fibroblasts, and osteoblasts were then cultured on regions A, B, and C of the CGS, respectively and their phenotypic characteristics were evaluated in this heterogenous environment. In the absence of growth factors, we found that the structural cues presented in every region played a key role in maintaining the region-specific cell functions and heterogeneity during a heterogeneous culture. In the presence of growth factors, we found that spatially localizing the growth factors at their respective regions resulted in enhanced region-specific cell functions and maintained region-specific cell heterogeneity compared to supplementation, which resulted in a significant reduction of cell growth and loss of phenotype. Our data suggest that providing biomimetic structural cues relevant to each cell phenotype and controlling the presentation of growth factors play a crucial role in ensuring heterogeneity maintenance of distinct cell populations during a heterogeneous culture. The presented CGS herein provides a reliable platform for investigating different cells responses to heterogeneous culture in a physiologically relevant microenvironment. In addition, the model provides a unique platform for evaluating the feasibility and efficacy of different approaches for simultaneously delivering multiple growth factors or molecules from a single construct to achieve enhanced cell response while maintaining cellular heterogeneity during a heterogenous culture.

Osteogenic potential of Wharton's jelly mesenchymal stromal cells cultured on a biomimetic scaffold

Background & Aim In regenerative medicine, Wharton's jelly mesenchymal stromal cells (WJ-MSCs) have a growing interest in its properties similar to those of bone marrow cells and the immunomodulatory potential. Tissue engineering combines the design of biomaterials and the potential of cells implanted in the scaffold that interacts with the added growth factors, and its main objective is to return morphology and function by providing a biological mechanically stable niche. This evidence has directed the approach of tissue regeneration towards the use of decellularized native cell matrices. The mature organs and tissues adequately decellularized, can be used as scaffolds, because the immunogenic determinants present in the cells are eliminated, conserving a native MEC, in which its structure, architecture and mechanical properties are preserved, along with the bioavailability of proteins and growth factors necessary for tissue regeneration. We aimed is provide a temporary bone biomimetic scaffold capable of inducing osteogenic differentiation of Wharton's jelly mesenchymal stromal cells. Methods, Results & Conclusion We decellularized, demineralized, and hydrolyzed the collagen protein of the bovine bone. Then we evaluate the structure and decellularization of the scaffolds. We performed scanning electron microscopy and chemical analysis by EDS. Raman Spectroscopy and FTIR techniques determined the chemical composition by identifying functional groups and the fingerprint of type I collagen protein. We aisled WJ-MSCs and characterize surface antigens profiles of WJ-MSCs; we kept the cells in culture for 3,7,14,21 and 28 days to evaluate the biocompatibility of the scaffolds and the cellular response in vitro. On histology sections, the scaffolds showed the conservation of the structure of the ECM and cell removal. We determined that the main component is collagen type I with different degrees of denaturation depending on the concentration of hydrochloric acid (HCl) by Raman and FTIR. We eliminate the mineral phase according to the guidelines established for the FDA for demineralized scaffolds. The scaffolds also showed excellently swelling and mechanical stability. In the in vitro cell culture, the scaffolds have no cytotoxic effect and mineralized matrix deposit after 28 days in culture in the absence of growth factors or inducers of mineralization. Future studies using animal subjects are required to evaluate the degree of mineralization and scaffold response.

Gamma-treated placental amniotic membrane allograft as the adjuvant treatment of unresponsive diabetic ulcer of the foot

IntroductionDiabetic ulcer of the foot is a major cause of morbidity and is a leading cause of hospitalization in patients with diabetes, and causes productivity and financial losses that lower the quality of life of the patient. The wound is categorized as responsive and unresponsive wound, which occurs in debilitated patients as seen in diabetes mellitus. The delay in wound repair can be caused by senescent cells, absence of growth factors, and other cellular abnormalities. MethodThis is a retrospective, single-centre case series with non-consecutive cases. Patients with diabetic ulcer of the foot managed using gamma-treated placental amniotic membrane, with the minimum follow up of 1 month in academic practice setting were recruited. ResultThree patients with 4 weeks period of oozing ulcers and signs of inflammation were included in this case series. Two layers of amniotic membrane dressing was applied weekly after cleaning and debridement for 3 weeks. Wound size and secretion were documented by taking photographs every week. At the end of the third week, the wound healed. DiscussionPlacental amniotic membranes are composed of cells, extra-cellular matrix (ECM), and a complex of regulatory cytokines which promote cell proliferation, cell modulation, and cytokine secretion by variety of cell types involved in wound healing. Our study showed that the treatment of diabetic ulcer wound using amniotic membrane was successful in achieving wound healing in unresponsive, chronic wound of diabetic ulcer of the foot. ConclusionOur results clearly indicated the usefulness of the application of amniotic membranes in treatment of diabetic ulcer of the foot. Amniotic membrane favoured healing of unresponsive and non-healing ulcers.

The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3.

Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1 This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD.This article has an associated First Person interview with the first author of the paper.

Efficacy of mechanical vibration in regulating mesenchymal stem cells gene expression.

This study aimed at investigating the expression of osteoblast and chondrocyte-related genes in mesenchymal stem cells (MSCs), derived from rabbit adipose tissue, under mechanical vibration. The cells were placed securely on a vibrator's platform and subjected to 300Hz of sinusoidal vibration, with a maximum amplitude of 10μm, for 45min per day, and for 14 consequent days, in the absence of biochemical reagents. The negative control group was placed in the conventional culture medium with no mechanical loading. The expression of osteoblast and chondrocyte-related genes was investigated using real-time polymerase chain reaction (real-time PCR). In addition, F-actin fiber structure and alignment with the help of actin filament fluorescence staining were evaluated, and the level of metabolic activity of MSCs was determined by the methyl thiazolyl tetrazolium assay. The real-time PCR study showed a significant increase of bone gene expression in differentiated cells, compared with MSCs (P < 0.05). On the other hand, the level of chondrocyte gene expression was not remarkable. Applying mechanical vibration enhanced F-actin fiber structure and made them aligned in a specific direction. It was also found that during the differentiation process, the metabolic activity of the cells increased (P < 0.05). The results of this work are in agreement with the well-accepted fact that the MSCs, in the absence of growth factors, are sensitive to low-amplitude, high-frequency vibration. Outcomes of this work can be applied in cell therapy and tissue engineering, when regulation of stem cells is required.

Capillary Lymphatic Venous Malformations are caused by Endothelial‐Specific Gain‐of‐Function Mutations in the PIK3CA Gene

Capillary lymphatic venous malformations (CLVM) are complex congenital vascular malformations characterized by aberrant vessels and soft tissue overgrowth (figure 1). CLVM are first detected in utero during late embryonic development and enlarge with time. Previous studies show that somatic, mosaic mutations in the 110‐kD catalytic □‐subunit of phosphoinositide‐3 kinase (PIK3CA) are present in CLVM lesions. These hotspot mutations are known to promote cell proliferation and angiogenesis in cancer. Since targeted molecular therapies for CLVM are lacking, patients require repeated sessions of sclerotherapy and surgical debulking over their lifetime. Therefore, there is a significant need to understand the pathogenesis of CLVM in order to develop therapies that target etiology rather than manage symptoms.This study uses patient‐derived cell lines from affected tissue to accomplish two aims: 1) Characterize disease pathogenesis by studying proliferation and signaling in the cell type expressing PIK3CA mutations and 2) Generate a patient‐derived xenograft model which recapitulates the histology of patient lesions.We isolated cells from the resected CLVM tissue of nine patients and cultured them in endothelial growth medium. Cells were separated into CD31+ (endothelial) and CD31‐ (non‐endothelial) populations via immunomagnetic bead sorting. By Sanger sequencing for PIK3CA hotspot mutations, we found that mutations are localized to endothelial cells (EC) in 8/9 patient lesions. This novel finding suggests that PIK3CA‐mutated EC drive CLVM pathogenesis and therefore are the cell‐of‐interest in therapeutic studies.To test this hypothesis, we evaluated AKT, a downstream effector of PI3K, via immunoblotting. We found that CLVM‐EC exhibit constitutive and significantly elevated phosphorylation of AKT (p&lt;0.05) compared to healthy donor EC. Additionally, proliferation studies revealed that CLVM‐EC proliferate 5‐fold faster compared to donor EC, even in the absence of growth factors (p&lt;0.05).Finally, we generated a xenograft murine model of CLVM by injecting patient‐derived CLVM‐EC (suspended in Matrigel□) subcutaneously into the flanks of immunocompromised mice. Healthy donor EC served as a control. Lesions were dissected at day 10, fixed, embedded in paraffin, and stained with hematoxylin and eosin. We found that patient‐derived CLVM‐EC form enlarged vascular and lymphatic channels in the xenograft model, and xenograft lesions resemble the histology of patient lesions.This pre‐clinical xenograft model will be important in identifying FDA‐approved inhibitors that selectively attenuate CLVM growth and may be advanced to clinical trial.Support or Funding InformationNIH‐R01HL117952, Ohio Physiological SocietyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phenotypic characterization of malignant progenitor cells in patients with idiopathic myelofibrosis

Objective/BackgroundIdiopathic myelofibrosis (IM) is a clonal hematological malignancy originating from pluripotent hematopoietic stem cells (HSC). HSC are very rare potent cells that reside in the bone marrow (BM) and at a lower level in peripheral blood (PB). Previous studies showed that IM PB CD34+ cells contain not only BM repopulating cells belonging to the malignant clone but also residual normal HSC. MethodsIn the current study, we separated the subpopulations of IM PB CD34+ cells using IL-3Rα/CD123 labeling and further characterized them by genetic and functional analyses. ResultsWe differentiated IM PB CD34+ cells into three subpopulations (IL-3Rαhigh, IL-3Rαlow, and IL-3Rαnegative). IL-3Rαhigh CD34+ cell subgroup represents a small population in IM PB CD34+ cells which was not seen in normal G-CSF mobilized CD34+ cells. IM IL-3Rαhigh CD34+ cells contained significant higher percentage of cells bearing marker chromosome detected by fluorescence in situ hybridization (FISH) analysis. In the absence of growth factors, IM IL-3Rαhigh CD34+ cells exhibited abnormal colony forming ability and carried greater percentage of JAK2V617F mutant allele compared with IL-3Rαlow and IL-3Rαnegative CD34+ cells. ConclusionThese data indicate that IL-3Rαhigh CD34+ cells from IM enriched for the malignant progenitor cells and IL-3Rα/CD123 may be a potential biomarker and therapeutic target for IM. Our findings will be further validated in future studies with a larger sample size and serial transplant in murine models.

Molecular Mechanisms of Tryptophan-Tyrosine Nanostructures Formation and their Influence on PC-12 Cells.

The discovery of self-assembling peptides, which can form well-ordered structures, has opened a realm of opportunity for the design of tailored short peptide-based nanostructures. In this study, a combined experimental and computational approach was utilized to understand the intramolecular and intermolecular interactions contributing to the self-assembly of linear and cyclic tryptophan-tyrosine (WY) dipeptides. The density functional tight binding (DFTB) calculations with empirical dispersive corrections assisted the identification of the lowest energy conformers. Conformer analysis and the prediction of the electronic structure for the monomeric, dimeric, and hexameric forms of the cyclic and linear WY confirmed the contributions of hydrogen bonding, π-π stacking, and CH-π interactions in the stability of the self-assembled nanotubes. The influence of the processing conditions on the morphological and thermal characteristics, as well as the secondary structures of the synthesized nanostructures, were analyzed. Preliminary studies of the influence of the nanotubes on the fate of neuronalcell lines such as, PC-12cells indicate that the nanotubes promote cellular proliferation, and differentiation in the absence of growth factors. The aspect ratio of the nanotubes played an essential role in cellular interactions where a higher cellular uptake was observed in nanotubes of lower aspect ratios. These results provide insight for future applications of such nanotubes as scaffolds for tissue engineering and nerve regeneration and in drug delivery.

Roles of microRNA in prostate cancer cell metabolism.

MicroRNAs are non-coding RNA which functions as regulators of genes expression. MicroRNAs have shown their biological functions in cell proliferation, cell cycle, cell metabolism, apoptosis, invasion and metastasis. Cancer cells have the ability to grow in the absence of growth factors by increased metabolic activity. MicroRNAs regulate cell metabolic processes by targeting the key enzymes or transporters and change the metabolic activities by interfering with oncogenes/tumor suppressors, hypoxia, signalling pathways and cell adhesion. This review mainly explains the roles of microRNAs in prostate cancer cell metabolism, such as glucose uptake, glycolysis and lactate secretion, lipid metabolism and interaction with signalling pathways. The relation of microRNAs with hypoxia and cell adhesion in cell metabolism is also highlighted. Therefore, miRNAs help in regulating the metabolism of survived tumor cells, understanding such miRNA-mediated interaction could lead to new avenues in therapeutic application to treat PCa.

Thyroid hormones act as mitogenic and pro survival factors in rat ovarian follicles.

Thyroid disorders are clinically associated with impaired fertility in women, and these abnormalities can be improved by restoring the euthyroid state. The exact mechanisms of thyroid effect on female fertility are not well known; however, it is conceivable that thyroid hormones (THs) might act on ovarian physiology via receptors in granulosa cells. This work is aimed at evaluating the effects of THs on non-tumoral granulosa cells and follicles. Freshly isolated rat ovarian follicles and granulosa cells were exposed to T3 or T4 (THs). Cell growth and viability were evaluated by cell counting and the MTT assay, respectively, follicle growth was evaluated by volume measurements. Apoptosis was evaluated by the TUNEL assay and active Caspase 3 staining. rGROV cells were exposed to T3, and apoptosis was induced by serum deprivation. Bcl2, Bcl-2-associated X protein (BAX), Akt and pAkt expression were evaluated by western blot. T3 induced a 40% increase in follicle volume (after 7days). This increase was presumably due to the observed decrease (33%) in the apoptotic rate of the granulosa cell population. Both T3 and T4 caused a dose-dependent increase in rat granulosa cell number and viability. In addition, THs decreased the cell apoptotic rate in a dose-dependent manner. In both conditions, T3 appeared to be more efficient. In rGROV cells, 100nM T3 induced cell growth and, in the absence of growth factors, reduced cell apoptosis by 40%, downregulating Caspase 3 and BAX. This effect was associated with an increase in pAkt levels. The involvement of the PI3K pathway was confirmed by the ability of the PI3K specific inhibitor (LY-294,002) to abolish T3 pro-survival action. THs influence cell survival of ovarian granulosa cells. This effect likely contributes to the TH-induced follicle volume increase.

The Feasibility of Encapsulated Embryonic Medullary Reticular Cells to Grow and Differentiate Into Neurons in Functionalized Gelatin-Based Hydrogels.

The study of the behavior of embryonic neurons in controlled in vitro conditions require methodologies that take advantage of advanced tissue engineering approaches to replicate elements of the developing brain extracellular matrix. We report here a series of experiments that explore the potential of photo-polymerized gelatin hydrogels to culture primary embryonic neurons. We employed large medullary reticular neurons whose activity is essential for brain arousal as well as a library of gelatin hydrogels that span a range of mechanical properties, inclusion of brain-mimetic hyaluronic acid, and adhesion peptides. These hydrogel platforms showed inherent capabilities to sustain neuronal viability and were permissive for neuronal differentiation, resulting in the development of neurite outgrowth under specific conditions. The maturation of embryonic medullary reticular cells took place in the absence of growth factors or other exogenous bioactive molecules. Immunocytochemistry labeling of neuron-specific tubulin confirmed the initiation of neural differentiation. Thus, this methodology provides an important validation for future studies of nerve cell growth and maintenance.

Overexpressing TPTE2 (TPIP), a homolog of the human tumor suppressor gene PTEN, rescues the abnormal phenotype of the PTEN-/- mutant.

One possible approach to normalize mutant cells that are metastatic and tumorigenic, is to upregulate a functionally similar homolog of the mutated gene. Here we have explored this hypothesis by generating an overexpressor of TPTE2 (TPIP), a homolog of PTEN, in PTEN−/− mutants, the latter generated by targeted mutagenesis of a human epithelial cell line. Overexpression of TPTE2 normalized phenotypic changes associated with the PTEN mutation. The PTEN−/−-associated changes rescued by overexpressing TPTE2 included 1) accelerated wound healing in the presence or absence of added growth factors (GFs), 2) increased division rates on a 2D substrate in the presence of GFs, 3) adhesion and viability on a 2D substrate in the absence of GFs, 4) viability in a 3D Matrigel model in the absence of GFs and substrate adhesion 5) loss of apoptosis-associated annexin V cell surface binding sites. The results justify further exploration into the possibility that upregulating TPTE2 by a drug may reverse metastatic and tumorigenic phenotypes mediated in part by a mutation in PTEN. This strategy may also be applicable to other tumorigenic mutations in which a homolog to the mutated gene is present and can substitute functionally.

Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells.

Fascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors.