Explant Experiments Research Articles

GLP-1 Receptor Signaling Has Different Effects on the Perikarya and Axons of the Hypophysiotropic Thyrotropin-Releasing Hormone Synthesizing Neurons in Male Mice.

Background: Glucagon-like peptide 1 (GLP-1) is involved in the regulation of energy and glucose homeostasis. As GLP-1 has similar effects on the energy homeostasis as the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons that regulate the hypothalamic-pituitary-thyroid (HPT) axis, we raised the possibility that the TRH neurons are involved in the mediation of the effects of GLP-1. Therefore, the relationship and interaction of the GLP-1 system and the TRH neurons of the hypothalamic paraventricular nucleus (PVN) were studied. Methods: To examine the anatomical and functional relationship of TRH neurons and the GLP-1 system in the PVN, immunocytochemistry, in situ hybridization, in vitro patch-clamp electrophysiology, metabolic phenotyping, and explant experiments were performed. Results: Our data demonstrate that the TRH neurons of the PVN are innervated by GLP-1 producing neurons and express the GLP-1 receptor (GLP-1R). However, not only do the GLP-1-innervated TRH neurons express GLP-1R but the receptor is also present in the axons of the hypophysiotropic TRH neurons in the blood-brain barrier free median eminence (ME) suggesting that peripherally derived GLP-1 may also influence the TRH neurons. In vitro, GLP-1 increased the firing rate of TRH neurons and depolarized them. In addition, GLP-1 directly stimulated the GABAergic input of a population of TRH neurons. Furthermore, GLP-1 inhibited the release of TRH from the hypophysiotropic axons in the ME. In vivo, peripheral GLP-1R agonist administration markedly inhibited the food intake and the energy expenditure, but had no effect on the TRH expression in the PVN and resulted in lower circulating free T4 levels. Conclusions: Our results indicate that GLP-1R activation has a direct stimulatory effect on TRH neurons in the PVN, but the activation of GLP-1R may also inhibit TRH neurons by facilitating their inhibitory inputs or by inhibiting the axon terminals of these cells in the ME. The innervation of TRH neurons by GLP-1 neurons suggests that TRH neurons might be influenced by both circulating GLP-1 and by GLP-1 neurons of the nucleus tractus solitarii. The lack of GLP-1R agonist-induced regulation of TRH neurons in vivo suggests that the HPT axis does not mediate the GLP-1R agonist-induced weight loss.

Targeting PELP1 oncogenic signaling in TNBC with the small molecule inhibitor SMIP34

PurposeTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Oncogenic PELP1 is frequently overexpressed in TNBC, and it has been demonstrated that PELP1 signaling is essential for TNBC progression. The therapeutic utility of targeting PELP1 in TNBC, however, remains unknown. In this study, we investigated the effectiveness of SMIP34, a recently developed PELP1 inhibitor for the treatment of TNBC.MethodsTo ascertain the impact of SMIP34 treatment, we used seven different TNBC models for testing cell viability, colony formation, invasion, apoptosis, and cell cycle analysis. Western blotting and RT-qPCR were used to determine the mechanistic insights of SMIP34 action. Using xenograft and PDX tumors, the ability of SMIP34 in suppressing proliferation was examined both ex vivo and in vivo.ResultsTNBC cells’ viability, colony formation, and invasiveness were all decreased by SMIP34 in in vitro cell-based assays, while apoptosis was increased. SMIP34 treatment promoted the degradation of PELP1 through the proteasome pathway. RT-qPCR analyses confirmed that SMIP34 treatment downregulated PELP1 target genes. Further, SMIP34 treatment substantially downregulated PELP1 mediated extranuclear signaling including ERK, mTOR, S6 and 4EBP1. Mechanistic studies confirmed downregulation of PELP1 mediated ribosomal biogenesis functions including downregulation of cMyc and Rix complex proteins LAS1L, TEX-10, and SENP3. The proliferation of TNBC tumor tissues was decreased in explant experiments by SMIP34. Additionally, SMIP34 treatment markedly decreased tumor progression in both TNBC xenograft and PDX models.ConclusionsTogether, these findings from in vitro, ex vivo, and in vivo models show that SMIP34 may be a useful therapeutic agent for inhibiting PELP1 signaling in TNBC.

The developing murine kidney actively negotiates geometric packing conflicts to avoid defects.

The physiological functions of several organs rely on branched epithelial tubule networks bearing specialized structures for secretion, gas exchange, or filtration. Little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between increasing tubule tip numbers through branching and limited organ surface area. Through imaging of whole kidney explants, combined with computational and soft material modeling of tubule families, we identify six possible geometric packing phases, including two defective ones. Experiments in explants show that a radially oriented tension on tubule families is necessary and sufficient for them to switch to a vertical packing arrangement that increases surface tip density while avoiding defects. These results reveal developmental contingencies in response to physical limitations and create a framework for classifying congenital kidney defects.

Dynamic MicroRNA Expression Profiles During Embryonic Development Provide Novel Insights Into Cardiac Sinus Venosus/Inflow Tract Differentiation.

MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount in situ hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the sinus venosus/inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through in silico analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the sinus venosus at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and sinus venosus explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.

Signs of life: Oxygen sensors confirm viability, measure oxygen consumption and provide rapid, effective contamination monitoring for field‐based tissue culture

Abstract Understanding the ecology and evolution of wildlife and domesticated species requires knowledge of their physiological responses to environmental change and the constraints under which they operate. However, whole animal experiments are often limited in sample size and can be logistically and ethically challenging. Culture techniques represent a powerful approach, but are used infrequently in field research due to practical constraints. We used minimal tissue culture equipment in a remote field site for in vitro explant experiments using blubber from wild grey seals Halichoerus grypus. Assessing explant viability and detecting microbial contamination in remote field sites, where facilities are often small, unspecialised and more vulnerable to bacterial or fungal infection, present major challenges. We investigated whether oxygen‐sensitive planar optodes (OSPO) in closed system respirometry could be used to assess oxygen consumption by blubber explants from suckling and fasting wild seal pups as a proxy for viability. We also explored whether OSPOs could provide rapid information on whole animal relevant physiological metrics by determining whether explant oxygen consumption correlated with the nutritional state of the animal, blubber depth and other tissue metabolic properties, including glucose uptake, lactate production and lipolysis. Vials containing blubber explants consumed significant amounts of oxygen compared to controls, showing tissues were metabolically active. Oxygen consumption differed between nutritional states and blubber tissue depth. These differences were reflected in other tissue metabolic properties. Dissolved oxygen levels remained consistent over 24 hr in 94% of control vials containing only culture media. In 6% of control vials extremely rapid oxygen consumption preceded, by 2–3 days, colour changes in the phenol‐red containing media that indicate lactic acidosis from microbial metabolic activity. Oxygen use in control vials was, therefore, an effective monitoring system that provided vital early warning of media contamination, allowing stocks to be discarded, which prevented erroneous results and avoided waste of valuable field time and irreplaceable samples. OSPO are thus a useful tool for simultaneously assessing tissue oxygen consumption, investigating functional physiological differences and monitoring microbiological contamination in culture experiments, particularly in field laboratories studying live tissues from wildlife.

Activation of Endogenous H2S Biosynthesis or Supplementation with Exogenous H2S Enhances Adipose Tissue Adipogenesis and Preserves Adipocyte Physiology in Humans.

Aims: To investigate the impact of exogenous hydrogen sulfide (H2S) and its endogenous biosynthesis on human adipocytes and adipose tissue in the context of obesity and insulin resistance. Results: Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H2S or the activation of endogenous H2S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas chemical inhibition and gene knockdown of each enzyme generating H2S (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In agreement with these experimental data, visceral and subcutaneous adipose tissue expression of H2S-synthesising enzymes was significantly reduced in morbidly obese subjects in association with attenuated adipogenesis and increased markers of adipose tissue inflammation and senescence. Interestingly, weight-loss interventions (including bariatric surgery or diet/exercise) improved the expression of H2S biosynthesis-related genes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and H2S production were dramatically increased during adipocyte differentiation. More importantly, the adipocyte proteome exhibiting persulfidation was characterized, disclosing that different proteins involved in fatty acid and lipid metabolism, the citrate cycle, insulin signaling, several adipokines, and PPAR, experienced the most dramatic persulfidation (85-98%). Innovation: No previous studies investigated the impact of H2S on human adipose tissue. This study suggests that the potentiation of adipose tissue H2S biosynthesis is a possible therapeutic approach to improve adipose tissue dysfunction in patients with obesity and insulin resistance. Conclusion: Altogether, these data supported the relevance of H2S biosynthesis in the modulation of human adipocyte physiology. Antioxid. Redox Signal. 35, 319-340.

Rapid Jetlag Resetting of Behavioral, Physiological, and Molecular Rhythms in Proestrous Female Mice.

A gradual adaptation to a shifted light-dark (LD) cycle is a key element of the circadian clock system and believed to be controlled by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Endocrine factors have a strong influence on the regulation of the circadian clock network and alter acute photic responses of the SCN clock. In females, endocrine function depends on the stage of the ovarian cycle. So far, however, little is known about the effect of the estrous cycle on behavioral and molecular responses to shifts in the LD rhythm. Based on this, we investigated whether estrous state affects the kinetics of phase shift during jetlag in behavior, physiology, and molecular clock rhythms in the SCN and in peripheral tissues. Female mice exposed to an advanced LD phase at proestrous or metestrous showed different phase-shift kinetics, with proestrous females displaying accelerated adaptation in behavior and physiology. Constant darkness release experiments suggest that these fast phase shifts do not reflect resetting of the SCN pacemaker. Explant experiments on SCN, adrenal gland, and uterus confirmed this finding with proestrous females showing significantly faster clock phase shifts in peripheral tissues compared with the SCN. Together, these findings provide strong evidence for an accelerated adaptation of proestrous compared with metestrous females to new LD conditions that is accompanied by rapid behavioral, physiological, and molecular rhythm resetting. Not only do these findings open up a new avenue to understand the effect of estrous cycle on the clock network under changing environmental conditions but also imply a greater susceptibility in proestrous females.

MiR-22-3p inhibits fibrotic cataract through inactivation of HDAC6 and increase of α-tubulin acetylation.

ObjectivesFibrotic cataract, including posterior capsule opacification (PCO) and anterior subcapsular cataract (ASC), renders millions of people visually impaired worldwide. However, the underlying mechanism remains poorly understood. Here, we report a miRNA‐based regulatory pathway that controls pathological fibrosis of lens epithelium.Materials and methodsExpression of miR‐22‐3p and histone deacetylase 6 (HDAC6) in normal and PCO patient samples were measured by qPCR. Human lens epithelial explants were treated with TGF‐β2 in the presence or absence of miR‐22‐3p mimics or inhibitor. Cell proliferation was determined by MTS assay, and migration was tested by transwell assay. Expression of HDAC6 and EMT‐related molecules were analysed by Western blot, qPCR and immunocytochemical experiments.ResultsWe identify miR‐22‐3p as a downregulated miRNA targeting HDAC6 in LECs during lens fibrosis and TGF‐β2 treatment. Mechanistically, gain‐ and loss‐of‐function experiments in human LECs and lens epithelial explants reveal that miR‐22‐3p prevents proliferation, migration and TGF‐β2 induced EMT of LECs via targeting HDAC6 and thereby promoting α‐tubulin acetylation. Moreover, pharmacological targeting of HDAC6 deacetylase with Tubacin prevents fibrotic opaque formation through increasing α‐tubulin acetylation under TGF‐β2 stimulated conditions in both human lens epithelial explants and the whole rat lenses.ConclusionsThese findings suggest that miR‐22‐3p prevents lens fibrotic progression by targeting HDAC6 thereby promoting α‐tubulin acetylation. The ‘miR‐22‐HDAC6‐α‐tubulin (de)acetylation’ signalling axis may be therapeutic targets for the treatment of fibrotic cataract.

Transcriptome and physiological effects of toxaphene on the liver-gonad reproductive axis in male and female largemouth bass (Micropterus salmoides)

Toxaphene is an organochlorine pesticide and environmental contaminant that is concerning due to its atmospheric transport and persistence in soil. In Florida, toxaphene and other organochlorine pesticides were used heavily in agriculture on the north shore of Lake Apopka and they are still detectable in soil. Wild largemouth bass that inhabit the lake and the marshes along the north shore have been exposed to a variety of organochlorine pesticides including dieldrin, methoxychlor, and p,p′-DDE, among others. While these other organochlorine pesticides have been studied for their endocrine disrupting effects in largemouth bass, there is little information for toxaphene. In this study, male and female largemouth bass were given food containing 50 mg/kg toxaphene for almost 3 months, to achieve tissue levels similar to those found in fish at Lake Apopka. Sex-specific toxicity was then evaluated by measuring various reproductive endpoints and transcriptomic changes. In females, gonadosomatic index showed a trend towards reduction (p = 0.051) and plasma vitellogenin was reduced by ~40% relative to controls. However plasma levels of 17β-estradiol and testosterone were not perturbed by toxaphene exposure. These data suggest that toxaphene does not act as a weak estrogen as many other organochlorine pesticides do, but rather appears to be acting as an antiestrogen in female fish. There were no obvious changes in the gonadosomatic index and plasma hormones in male bass. However, ex vivo explant experiments revealed that toxaphene prevented human chorionic gonadotropin-stimulated testosterone production in the testis. This suggested that toxaphene had anti-androgenic effects in males. Subsequent transcriptomic analyses of the testis revealed that androgen receptor/beta-2-microglobulin signaling was up-regulated while insulin-related pathways were suppressed with toxaphene, which could be interpreted as a compensatory response to androgen suppression. In the male liver, the transcriptome analysis revealed an overwhelming suppression in immune-related signaling cascades (e.g. lectin-like receptor and ITSM-Containing Receptor signaling, CD16/CD14 Proinflammatory Monocyte Activation, and CD38/CD3-JUN/FOS/NF-kB Signaling in T-cell Proliferation). Overall, this study showed that toxaphene induced sex-specific effects. The transcriptomic and physiological responses observed can contribute to the development of adverse outcome pathways for toxaphene exposure in fish.

The secreted BMP antagonist ERFE is required for the development of a functional circulatory system in Xenopus

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

10 A VARIANT OF CHOLERA TOXIN B SUBUNIT ENHANCES COLON EPITHELIAL WOUND HEALING VIA AN UNFOLDED PROTEIN RESPONSE

Cholera toxin B subunit (CTB) exhibits multifaceted biological activity in the mucosa via binding to GM1 ganglioside on intestinal epithelial cells. We recently found that a recombinant variant of CTB, which contains an artificial KDEL endoplasmic reticulum (ER) retention signal (CTB-KDEL), induces mucosal healing in a mouse dextran sodium sulfate (DSS) acute colitis model. The objectives of this study are to investigate CTB-KDEL’s epithelial wound repair mechanism and characterize its efficacy in preclinical models. We hypothesize that CTB-KDEL’s wound healing effect is mediated through ER retention within epithelial cells, thereby facilitating mucosal healing in IBD. The effects of CTB-KDEL, CTB and G33D-CTB-KDEL (a non-GM1-binding variant) were analyzed for cell migration, subcellular localization, an unfolded protein response (UPR) and TGFβ expression in Caco2 cells. In a DSS acute colitis model, C57BL/6J mice were gavaged with a single dose of CTB-KDEL, CTB or PBS at the end of 7-day DSS exposure. An optimal dosing regimen of CTB-KDEL was evaluated in mice exposed to repeated DSS cycles. Efficacy was measured on the basis of colon length, Disease Activity Index, histopathological scoring, E-cadherin immunohistochemistry and wound healing gene/cytokine expression in the colon. For ex vivohuman colonic explant experiments, colon tissues were obtained from consenting IBD patients who underwent total colectomy, which were cultured with CTB-KDEL, CTB or a PBS for 24 hours. Caco2 cell migration was enhanced by CTB-KDEL at concentrations of 0.3 – 3 μM, while CTB and G33D-CTB-KDEL failed to show such an effect. CTB-KDEL showed longer residence in the ER than CTB, leading to the activation of UPRs, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm, and subsequent TGFβ signaling in Caco2 cells. In the acute colitis model, CTB-KDEL, but not CTB, facilitated recovery from colonic damage as evident in macroscopic and histological evaluations, with significant upregulation of wound healing genes and E-cadherin positive staining in the colon (Fig. 1). Weekly oral administration of 3 μg CTB-KDEL showed significant efficacy against established colitis in the chronic DSS model. In human IBD colon explants, CTB-KDEL at 1 μM induced significant upregulation of the IRE1/Xbp1 pathway as well as wound healing genes including TGFB1and the E-cadherin gene CDH1, along with the retention of viable crypts in the colon. The results demonstrated CTB-KDEL’s unique colon epithelial wound healing activity via a UPR, providing implications for new IBD treatment strategies.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Abstract LB-169: Asbestos and anthelmintics: Repurposing drugs to prevent mesothelioma in high-risk populations

Abstract The purpose of this study is to address the rising mortality rates in mesothelioma by using repurposed drugs in a chemoprevention setting. An initial screen of 100 repurposed agents at clinically relevant concentrations was conducted across three mesothelioma cell lines using AlamarBlue assays. Having identified the top three individual and combinatorial treatments, analysis of cell-cycle and apoptosis response to these drugs was used to corroborate the findings of the initial screen. As a more representative disease model a mouse PDX was developed from a human biphasic mesothelioma, and furthermore, primary tissue obtained, both of which we have used for explant experiments to assess the efficacy of the top treatments in a more realistic and translational model. As BAP-1 mutations are highly frequent in both acquired and spontaneous mesotheliomas, a CRISPR generated knock-out cell-line will model early-stage disease, and MET-5A mesothelial cells to assess normal cell response. The three top agents, comprising zinc acetate, niclosamide, and colchicine, have proven consistently efficacious in reducing mesothelioma cell-line viability by up to 72.59% individually and 93.55% in combination. Congruent results are provided by annexin V-FITC and FACS experiments which indicate treatments induce cell cycle alterations and apoptosis. Furthermore, FACS analysis of PDX explants also shows increased apoptosis of up to 27.0% in response to 24-hour treatment of two combined drugs. Importantly, the PDX still retains original histopathological features of the human tumour, making it an important foundation for further ex vivo and ultimately in vivo study. Initial results are promising and indicate that several repurposed agents significantly reduce survival and increase death of mesothelioma both in vitro and ex vivo. Mesothelioma, being predominantly caused by long-term asbestos exposure, has been under-researched due to an overreliance on the disuse of asbestos. As such, there is no effectual treatment and average survival remain static at under one year. However, following initial asbestos exposure, a 50-year latency precedes diagnosis, which offers an opportune window for a prevention strategy. Using the CRISPR-BAP-1-knockout cell-line to model early-stage disease, we hope to translate the top candidate into a primary prevention setting. However given the increasing frequency of late stage mesothelioma diagnoses, there is also a tangible need for secondary and tertiary prevention. Repurposed drugs, which offer long term safety and side effect profiles, as well as requiring less temporal and financial investment, allow expedited translation of any potential top candidate into clinic. Through further mechanistic analyses in in vitro and ex vivo cancer models, we hope to identify a single agent or combination that could significantly impede mesothelioma progression and ultimately reduce mortality. Citation Format: Bethan G. Rogoyski, Ankur Karmokar, Sameena Khan, Hong Cai, Farhat L. Khanim, Sara Busacca, Lynne Howells, Dean A. Fennell, Anne L. Thomas, Karen Brown. Asbestos and anthelmintics: Repurposing drugs to prevent mesothelioma in high-risk populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-169.

Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh−/− and Gli3Xt−J/Xt−J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt−J/Xt−J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh−/− mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh−/−;Gli3Xt−J/Xt−J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt−J/Xt−J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets.

The importance of being a macrophage during hematopoietic stem cell generation

The first hematopoietic stem cells (HSCs) arise in the mouse aorta-gonad-mesonephros (AGM) at E10.5 but little is known about the microenvironment supporting their generation. Interestingly, macrophages (Mo) are present in the embryo before the production of HSCs, leading to the hypothesis that yolk sac-derived cells could be involved in the HSC niche. Recent studies in zebrafish revealed that Mo have a role in hematopoiesis by affecting the migration of hematopoietic progenitors from the aorta to the caudal hematopoietic tissue. To investigate whether this is conserved in mice, we used the MacGreen mouse model that expresses EGFP under the control of the Csf1r promoter. Flow cytometry confirmed that all EGFP+ cells in the embryo (E8-11) are phenotypic Mo (CD45+CD11b+F4/80+MerTK+CD16/32+Ly6C-Ly6G-). Time-lapse imaging on AGM sections showed that embryonic Mo are highly motile cells that undergo mitosis, intravasate into the aorta and interact with CD31+cKit+ cells. Mo numbers in the AGM increase over time, peaking at E10-10.5 just before the production of the first HSCs. We tested whether chemokine receptors/ligands may affect Mo recruitment to the AGM by flow cytometry. AGM Mo were positive for CCR2, CCR5, CXCR4 and CX3CR1 expression. qrtPCR on sorted AGM endothelial cells, showed CX3CL1 and CXCL12 expression, suggesting that these chemokines can mediate Mo recruitment to the aorta. CX3CR1 deficient embryos showed a decreased number of Mo in the AGM confirming the involvement of the CX3CL1/CX3CR1 axis in Mo migration to the AGM. Moreover, AGM explant experiments revealed a 50% decrease in the number of CFU-GEMMs upon treatment with clodronate liposomes that ablate 80% of AGM Mo. CSF-1RCre:RosaDTA embryos, in which more than 95% of Mo are depleted, showed the absence of CFU-GEMMs. Two populations of AGM Mo differentially expressing CD206 were found. CD206+ Mo expressed higher levels of metalloproteinases and inflammatory cytokines. Together, these results support a role for Mo in regulating hematopoiesis in the mouse embryo and future experiments will focus on the mechanisms by which the interacting cells affect the establishment of definitive hematopoiesis.

Pasireotide prevents nuclear factor of activated T cells nuclear translocation and acts as a protective agent in aminoglycoside-induced auditory hair cell loss.

Hearing impairment is a global health problem with a high socioeconomic impact. Damage to auditory hair cells (HCs) in the inner ear as a result of aging, disease, trauma, or toxicity, underlies the majority of cases of sensorineural hearing loss. Previously we demonstrated that the Ca2+ -sensitive neuropeptide, somatostatin (SST), and an analog, octreotide, protect HCs from gentamicin-induced cell death invitro. Aminoglycosides such as gentamicin trigger a calcium ion influx (Ca2+ ) that activates pro-apoptotic signaling cascades in HCs. SST binding to the G-protein-coupled receptors (SSTR1-SSTR5) that are directly linked to voltage-dependent Ca2+ channels inhibits Ca2+ channel activity and associated downstream events. Here, we report that the SST analog pasireotide, a high affinity ligand to SSTRs 1-3, and 5, with a longer half-life than octreotide, prevents gentamicin-induced HC death in the mouse organ of Corti (OC). Explant experiments using OCs derived from SSTR1 and SSTR1and 2 knockout mice, revealed that SSTR2 mediates pasireotide's anti-apoptotic effects. Mechanistically, pasireotide prevented a nuclear translocation of the Ca2+ -sensitive transcription factor, nuclear factor of activated T cells (NFAT), which is ordinarily provoked by gentamicin in OC explants. Direct inhibition of NFAT with 11R-VIVIT also prevented the gentamicin-dependent nuclear translocation of NFAT and apoptosis. Both pasireotide and 11R-VIVIT partially reversed the effects of gentamicin on the expression of downstream survival targets (NMDA receptor and the regulatory subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase, PI3K). These data suggest that SST analogs antagonize aminoglycoside-induced cell death in an NFAT-dependent fashion. SST analogs and NFAT inhibitors may therefore offer new therapeutic possibilities for the treatment of hearing loss.

High Mobility Group-Box 1 (HMGB1) levels are increased in amniotic fluid of women with intra-amniotic inflammation-determined preterm birth, and the source may be the damaged fetal membranes

BackgroundHigh Mobility Group Box-1 (HMGB1) is considered a prototype alarmin molecule. Upon its extracellular release, HMGB1 engages pattern recognition receptors and the Receptor for Advanced Glycation End-products (RAGE) followed by an outpouring of inflammatory cytokines, including interleukin (IL)-6. MethodsWe assayed the amniotic fluid (AF) levels of HMGB1 and IL-6 in 255 women that either had a normal pregnancy outcome or delivered preterm. Immunohistochemistry on fetal membranes was used for cellular localization and validation of immunoassay findings. HMGB1 also was analyzed in amniochorion tissue explants subjected to endotoxin. ResultsAF HMGB1 levels are not gestational age regulated but are increased in women with intra-amniotic inflammation and preterm birth. The likely source is the damaged amniochorion, as demonstrated by immunohistochemistry and explant experiments. ConclusionsOur research supports a role for HMGB1 in the inflammatory response leading to preterm birth. As a delayed phase cytokine, in utero exposure to elevated AF HMGB1 levels may have an impact on the newborn beyond the time of birth.

Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells.

Vascular endothelial growth factor (VEGF)-A is a well-known major chemoattractant driver of angiogenesis--the formation of new blood vessels from pre-existing ones. However, the repellent factors that fine-tune this angiogenic process remain poorly characterized. We investigated the expression and functional role of endothelial cell-derived semaphorin 3A (Sema3A) in retinal angiogenesis, using genetic mouse models. We found Sema3a mRNA expression in the ganglion cell layer and the presence of Sema3A protein on larger blood vessels and at the growing front of blood vessels in neonatal retinas. The Sema3A receptors neuropilin-1 and plexin-A1 were expressed by retinal blood vessels. To study the endothelial cell-specific role of Sema3A, we generated endothelial cell-specific Sema3A knockout mouse strains by constitutive or inducible vascular endothelial cadherin-Cre-mediated gene disruption. We found that in neonatal retinas of these mice, both the number and the length of tip cell filopodia were significantly increased and the leading edge growth pattern was irregular. Retinal explant experiments showed that recombinant Sema3A significantly decreased VEGF-A-induced filopodia formation. Endothelial cell-specific knockout of Sema3A had no impact on blood vessel density or skin vascular leakage in adult mice. These findings indicate that endothelial cell-derived Sema3A exerts repelling functions on VEGF-A-induced tip cell filopodia and that a lack of this signaling cannot be rescued by paracrine sources of Sema3A.

Genome-wide meta-analysis uncovers novel loci influencing circulating leptin levels.

Leptin is an adipocyte-secreted hormone, the circulating levels of which correlate closely with overall adiposity. Although rare mutations in the leptin (LEP) gene are well known to cause leptin deficiency and severe obesity, no common loci regulating circulating leptin levels have been uncovered. Therefore, we performed a genome-wide association study (GWAS) of circulating leptin levels from 32,161 individuals and followed up loci reaching P<10−6 in 19,979 additional individuals. We identify five loci robustly associated (P<5 × 10−8) with leptin levels in/near LEP, SLC32A1, GCKR, CCNL1 and FTO. Although the association of the FTO obesity locus with leptin levels is abolished by adjustment for BMI, associations of the four other loci are independent of adiposity. The GCKR locus was found associated with multiple metabolic traits in previous GWAS and the CCNL1 locus with birth weight. Knockdown experiments in mouse adipose tissue explants show convincing evidence for adipogenin, a regulator of adipocyte differentiation, as the novel causal gene in the SLC32A1 locus influencing leptin levels. Our findings provide novel insights into the regulation of leptin production by adipose tissue and open new avenues for examining the influence of variation in leptin levels on adiposity and metabolic health.