Organotypic Cells Research Articles

Dopamine internalization via Uptake2 and stimulation of intracellular D5-receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

Dopamine stimulates CDP-diacylglycerol biosynthesis through D1-like receptors, particularly the D5 subtype most of which is intracellularly localized. CDP-diacylglycerol regulates phosphatidylinositol-4,5-bisphosphate-dependent signaling cascades by serving as obligatory substrate for phosphatidylinositol biosynthesis. Here, we used acute and organotypic brain tissues and cultured cells to explore the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol. Dopamine stimulated CDP-diacylglycerol in organotypic and neural cells lacking the presynaptic dopamine transporter, and this action was selectively mimicked by D1-like receptor agonists SKF38393 and SKF83959. Dopaminergic CDP-diacylglycerol stimulation was blocked by decynium-22 which blocks Uptake2-like transporters and by anti-microtubule disrupters of cytoskeletal transport, suggesting transmembrane uptake and guided transport of the ligands to intracellular sites of CDP-diacylglycerol regulation. Fluorescent or radiolabeled dopamine was saturably transported into primary neurons or B35 neuroblastoma cells expressing the plasmamembrane monoamine transporter, PMAT. Microinjection of 10-nM final concentration of dopamine into human D5-receptor-transfected U2-OS cells rapidly and transiently increased cytosolic calcium concentrations by 316%, whereas non-D5-receptor-expressing U2-OS cells showed no response. Given that U2-OS cells natively express PMAT, bath application of 10 μM dopamine slowly increased cytosolic calcium in D5-expressing cells. These observations indicate that dopamine is actively transported by a PMAT-implicated Uptake2-like mechanism into postsynaptic-type dopaminoceptive cells where the monoamine stimulates its intracellular D5-type receptors to mobilize cytosolic calcium and promote CDP-diacylglycerol biosynthesis. This is probably the first demonstration of functional intracellular dopamine receptor coupling in neural tissue, thus challenging the conventional paradigm that postsynaptic dopamine uptake serves merely as a mechanism for deactivating spent or excessive synaptic transmitter.

Cobalt-induced apoptosis of cochlear organotypic cultures and HEI-OC1 cells is mediated by Dicer

Cobalt is widely used in the medical industry, mainly including cobalt alloy joint implants and cobalt-chromium porcelain crowns. However, unexplained ototoxicity and neurotoxicity often occur in the clinical use of cobalt agents at present, which limits the development of the cobalt industry. In this study, based on the clinical problem of cobalt ototoxicity, we first conducted an extensive search and collation of related theories, and on this basis, prepared an HEI-OC1 cell model and basilar membrane organotypic cultures after cobalt treatment. We used immunofluorescence staining, western blot, CCK8, and si-RNA to investigate the mechanism of cobalt ototoxicity, to discover its potential therapeutic targets. After comparing the reactive oxygen species, mitochondrial transmembrane potential, apoptosis-related protein expression, and cell viability of different treatment groups, the following conclusions were drawn: cobalt causes oxidative stress in the inner ear, which leads to apoptosis of inner ear cells; inhibition of oxidative stress and apoptosis can alleviate the damage of cobalt on inner ear cells; and the Dicer protein plays a role in the mechanism of inner ear damage and is a potential target for the treatment of cobalt-induced inner ear damage. Taken together, these results suggest that cobalt-induced ototoxicity triggered by oxidative stress activates a cascade of apoptotic events where cCaspase-3 decreases Dicer levels and amplifies this apoptotic pathway. It may be possible to prevent and treat cobalt ototoxicity by targeting this mechanism.

Melatonin Activates Anti-Inflammatory Features in Microglia in a Multicellular Context: Evidence from Organotypic Brain Slices and HMC3 Cells.

Melatonin (MEL) is a neurohormone endowed with neuroprotective activity, exerted both directly on neuronal cells and indirectly through modulation of responsive glial cells. In particular, MEL's effects on microglia are receptor-mediated and in part dependent on SIRT1 activation. In the present study, we exploited the highly preserved cytoarchitecture of organotypic brain cultures (OC) to explore the effects of MEL on hippocampal microglia in a 3D context as compared to a single cell type context represented by the human HMC3 cell line. We first evaluated the expression of MEL receptor MT1 and SIRT1 and then investigated MEL action against an inflammatory stimulation with LPS: OCs were cultured for a total of 2 weeks and during this time exposed to 0.1 μg/mL of LPS for 24 h either on day 1 (LPS 1°) or on day 11 (LPS 11°). MEL was added immediately after plating and kept for the entire experiment. Under these conditions, both MEL and LPS induced amoeboid microglia. However, the same round phenotype matched different polarization features. LPS increased the number of nuclear-NF-kB+ round cells and MEL alone or in combination with LPS increased BDNF+ round microglia. In addition, MEL contrasted LPS effects on NF-kB expression. Data from HMC3 microglia confirmed MEL's anti-inflammatory effects against LPS in terms of CASP1 induction and BDNF release, identifying SIRT1 as a mediator. However, no effects were evident for MEL alone on HMC3 microglia. Overall, our results point to the importance of the multicellular context for full MEL activity, especially in a preventive view, and support the use of OCs as a favorable model to explore inflammatory responses.

Organotypic stromal cells impact endothelial cell transcriptome in 3D microvessel networks

Endothelial cells line all major blood vessels and serve as integral regulators of many functions including vessel diameter, cellular trafficking, and transport of soluble mediators. Despite similar functions, the phenotype of endothelial cells is highly organ-specific, yet our understanding of the mechanisms leading to organ-level differentiation is incomplete. We generated 3D microvessel networks by combining a common naïve endothelial cell with six different stromal cells derived from the lung, skin, heart, bone marrow, pancreas, and pancreatic cancer. Single cell RNA-Seq analysis of the microvessel networks reveals five distinct endothelial cell populations, for which the relative proportion depends on the stromal cell population. Morphologic features of the organotypic vessel networks inversely correlate with a cluster of endothelial cells associated with protein synthesis. The organotypic stromal cells were each characterized by a unique subpopulation of cells dedicated to extracellular matrix organization and assembly. Finally, compared to cells in 2D monolayer, the endothelial cell transcriptome from the 3D in vitro heart, skin, lung, and pancreas microvessel networks are more similar to the in vivo endothelial cells from the respective organs. We conclude that stromal cells contribute to endothelial cell and microvessel network organ tropism, and create an endothelial cell phenotype that more closely resembles that present in vivo.

Studying Circadian Clock Entrainment by Hormonal Signals.

In mammals, molecular circadian clocks not only exist in the suprachiasmatic nucleus (SCN) but in almost all organ systems. Intriguingly, tissue clocks can operate in both isolated tissues and cell lines with endocrine signals mediating the circadian expression of local transcriptomes. This can be demonstrated by treating tissue explants with endocrine cues in a phase- and dose-dependent manner. In this chapter we provide an overview of methods to study the effects of candidate hormonal time cues on tissue clock resetting. We propose an experimental procedure based on an in vitro setup consisting of several consecutive steps in which organotypic tissue cultures or cells can be used. Our approach targets the potential resetting mechanism at three levels: the hormone, the direct clock gene target, and the tissue clock response.

From Stem Cells to Populations-Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects.

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype–phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development.

Architectural control of metabolic plasticity in epithelial cancer cells

Metabolic plasticity enables cancer cells to switch between glycolysis and oxidative phosphorylation to adapt to changing conditions during cancer progression, whereas metabolic dependencies limit plasticity. To understand a role for the architectural environment in these processes we examined metabolic dependencies of cancer cells cultured in flat (2D) and organotypic (3D) environments. Here we show that cancer cells in flat cultures exist in a high energy state (oxidative phosphorylation), are glycolytic, and depend on glucose and glutamine for growth. In contrast, cells in organotypic culture exhibit lower energy and glycolysis, with extensive metabolic plasticity to maintain growth during glucose or amino acid deprivation. Expression of KRASG12V in organotypic cells drives glucose dependence, however cells retain metabolic plasticity to glutamine deprivation. Finally, our data reveal that mechanical properties control metabolic plasticity, which correlates with canonical Wnt signaling. In summary, our work highlights that the architectural and mechanical properties influence cells to permit or restrict metabolic plasticity.

The Impact of Oxygen Availability and Multilineage Communication on Organoid Maturation.

Significance: An optimal supply with oxygen is of high importance during embryogenesis and a prerequisite for proper organ development. Different tissues require varying amounts of oxygen, and even within single organs, different phases of development go alongside with either physiological hypoxia or the need for sufficient oxygen supply. Recent Advances: Human induced pluripotent stem cell-derived organoid models are state of the art cell culture platforms for the investigation of developmental processes, disease modeling, and drug testing. Organoids modeling the development of multiple tissues were developed within the past years. Critical Issues: Until now, optimization of oxygen supply and its role during organoid growth, differentiation, and maturation have only rarely been addressed. Recent publications indicate that hypoxia-induced processes play an important role in three-dimensional tissue cultures, triggering multilineage communication between mesenchymal cells, the endothelium, as well as organotypic cells. Later in culture, a sufficient supply with oxygen is of high importance to allow larger organoid sizes. Moreover, cellular stress is reduced and tissue maturation is improved. Therefore, a functional blood vessel network is required. Future Directions: In this review, we will briefly summarize aspects of the role of oxygen during embryonic development and organogenesis, present an update on novel organoid models with a special focus on organoid vascularization, and discuss the importance of complex organoids involving parenchymal cells, mesenchymal cells, inflammatory cells, and functional blood vessels for the generation of mature and fully functional tissues in vitro. Antioxid. Redox Signal. 35, 217-233.

Scalable Generation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cells (hiPSCs) can be expanded at limitless scale in vitro and give rise to various organotypic cells, cardiomyocytes (CMs) among them. Advanced protocols shape the differentiation process of pluripotent stem cells by controlled growth factor application. Modulating the Wnt signaling pathway is effective to direct hiPSCs to CMs (hiPSC-CMs) and native growth factors were replaced by small chemical compounds. Here, we describe a refined protocol for scalable generation of hiPSC-CMs that manipulates porcupine and tankyrase sub-pathways of Wnt signaling for tight inhibition of non-canonical Wnt signaling. The approach results in a differentiation efficiency toward hiPSC-CMs of 87±0.9% in stirred bioreactor cultures and yields about 70 million hiPSC-CMs per 100mL serum free cardiac differentiation medium. The differentiation protocol is easily adapted from 3D to 2D culture and vice versa and has been demonstrated to work with different hiPSC lines.

Imidazoline ligand BU224 reverses cognitive deficits, reduces microgliosis and enhances synaptic connectivity in a mouse model of Alzheimer's disease.

Activation of type 2 imidazoline receptors has been shown to exhibit neuroprotective properties including anti-apoptotic and anti-inflammatory effects, suggesting a potential therapeutic value in Alzheimer's disease (AD). Here, we explored the effects of the imidazoline-2 ligand BU224 in a model of amyloidosis. Six-month-old female transgenic 5XFAD and wild-type (WT) mice were treated intraperitoneally with 5-mg·kg-1 BU224 or vehicle twice a day for 10 days. Behavioural tests were performed for cognitive functions and neuropathological changes were investigated by immunohistochemistry, Western blot, elisa and qPCR. Effects of BU224 on amyloid precursor protein (APP) processing, spine density and calcium imaging were analysed in brain organotypic cultures and N2a cells. BU224 treatment attenuated spatial and perirhinal cortex-dependent recognition memory deficits in 5XFAD mice. Fear-conditioning testing revealed that BU224 also improved both associative learning and hippocampal- and amygdala-dependent memory in transgenic but not in WT mice. In the brain, BU224 reduced levels of the microglial marker Iba1 and pro-inflammatory cytokines IL-1β and TNF-α and increased the expression of astrocytic marker GFAP in 5XFAD mice. These beneficial effects were not associated with changes in amyloid pathology, neuronal apoptosis, mitochondrial density, oxidative stress or autophagy markers. Interestingly, ex vivo and in vitro studies suggested that BU224 treatment increased the size of dendritic spines and induced a threefold reduction in amyloid-β (Aβ)-induced functional changes in NMDA receptors. Sub-chronic treatment with BU224 restores memory and reduces inflammation in transgenic AD mice, at stages when animals display severe pathology.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) “premalignant” keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call ‘Organ-On-VascularNet’. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Downregulated UCHL1 Accelerates Gentamicin-Induced Auditory Cell Death via Autophagy.

The clinical use of aminoglycoside antibiotics is partly limited by their ototoxicity. The pathogenesis of aminoglycoside-induced ototoxicity still remains unknown. Here, RNA-sequencing was conducted to identify differentially expressed genes in rat cochlear organotypic cultures treated with gentamicin (GM), and 232 and 43 genes were commonly up- and downregulated, respectively, at day 1 and 2 after exposure. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1) was one of the downregulated genes whose expression was prominent in spiral ganglion cells (SGCs), lateral walls, as well as efferent nerve terminal and nerve fibers. We further investigated if a deficit of Uchl1 in organotypic cochlea and the House Ear Institute-Organ of Corti 1 (HEI-OC1) cells accelerates ototoxicity. We found that a deficit in Uchl1 accelerated GM-induced ototoxicity by showing a decreased number of SGCs and nerve fibers in organotypic cochlear cultures and HEI-OC1 cells. Furthermore, Uchl1-depleted HEI-OC1 cells revealed an increased number of autophagosomes accompanied by decreased lysosomal fusion. These data indicate that the downregulation of Uchl1 following GM treatment is deleterious to auditory cell survival, which results from the impaired autophagic flux. Our results provide evidence that UCHL1-dependent autophagic flux may have a potential as an otoprotective target for the treatment of GM-induced auditory cell death.

Cardiomyocytes facing fibrotic conditions re-express extracellular matrix transcripts

Pathophysiological conditions, such as myocardial infarction and mechanical overload affect the mammalian heart integrity, leading to a stiffened fibrotic tissue. With respect to the pathophysiology of cardiac fibrosis but also in the limelight of upcoming approaches of cardiac cell therapy it is of interest to decipher the interaction of cardiomyocytes with fibrotic matrix. Therefore, we designed a hydrogel-based model to engineer fibrotic tissue in vitro as an approach to predict the behavior of cardiomyocytes facing increased matrix rigidity. Here, we generated pure induced pluripotent stem cell-derived cardiomyocytes and cultured them on engineered polyacrylamide hydrogels matching the elasticities of healthy as well as fibrotic cardiac tissue. Only in cardiomyocytes cultured on matrices with fibrotic-like elasticity, transcriptional profiling revealed a substantial up-regulation of a whole panel of cardiac fibrosis-associated transcripts, including collagen I and III, decorin, lumican, and periostin. In addition, matrix metalloproteinases and their inhibitors, known to be essential in cardiac remodeling, were found to be elevated as well as insulin-like growth factor 2. Control experiments with primary cardiac fibroblasts were analyzed and did not show comparable behavior. In conclusion, we do not only present a snapshot on the transcriptomic fingerprint alterations in cardiomyocytes under pathological conditions but also provide a new reproducible approach to study the effects of fibrotic environments to various cell types. Statement of SignificanceThe ageing population in many western countries is faced with an increasing burden of ageing-related diseases such as heart failure which is associated with cardiac fibrosis. A deeper understanding of the interaction of organotypic cells with altered extracellular matrix mechanical properties is of pivotal importance to understand the underlying mechanisms. Here, we present a strategy to combine hydrogel matrices with induced pluripotent stem cell derived cardiomyocytes to study the effect of matrix stiffening on these cells. Our findings suggest an active role of matrix stiffening on cardiomyocyte function and heart failure progression.

Isolation and Characterization of Mouse Organ-Specific Endothelial Transcriptomes.

Endothelial cells play an essential role in homeostasis and regeneration and their transcriptional profile has been found to be tissue specific. Here we describe the isolation of organ specific mouse endothelial cells using either magnetic beads or fluorescence-activated cell sorting. This efficient protocol should allow for the transcriptional characterization of organotypic endothelial cells.

A human population-based organotypic in vitro model for cardiotoxicity screening_suppl

Assessing inter-individual variability in responses to xenobiotics remains a substantial challenge, both in drug development with respect to pharmaceuticals and in public health with respect to environmental chemicals.  Although approaches exist to characterize pharmacokinetic variability, there are no methods to routinely address pharmacodynamic variability.  In this study, we aimed to demonstrate the feasibility of characterizing inter-individual variability in a human in vitro model. Specifically, we hypothesized that genetic variability across a population of iPSC-derived cardiomyocytes translates into reproducible variability in both baseline phenotypes and drug responses. We measured baseline and drug-related effects in iPSC-derived cardiomyocytes from 27 healthy donors on kinetic Ca2+ flux and high-content live cell imaging.  Cells were treated in concentration-response with cardiotoxic drugs: isoproterenol (β-adrenergic receptor agonist/positive inotrope), propranolol (β-adrenergic receptor antagonist/negative inotrope), and cisapride (hERG channel inhibitor/QT prolongation).  Cells from four of the 27 donors were further evaluated in terms of baseline and treatment-related gene expression. Reproducibility of phenotypic responses was evaluated across batches and time. iPSC-derived cardiomyocytes exhibited reproducible donor-specific differences in baseline function and drug-induced effects. We demonstrate the feasibility of using a panel of population-based organotypic cells from healthy donors as an animal replacement experimental model. This model can be used to rapidly screen drugs and chemicals for inter-individual variability in cardiotoxicity. This approach demonstrates the feasibility of quantifying inter-individual variability in xenobiotic responses, and can be expanded to other cell types for which in vitro populations can be derived from iPSCs. &nbsp

A human population-based organotypic in vitro model for cardiotoxicity screening.

Assessing inter-individual variability in responses to xenobiotics remains a substantial challenge, both in drug development with respect to pharmaceuticals and in public health with respect to environmental chemicals. Although approaches exist to characterize pharmacokinetic variability, there are no methods to routinely address pharmacodynamic variability. In this study, we aimed to demonstrate the feasibility of characterizing inter-individual variability in a human in vitro model. Specifically, we hypothesized that genetic variability across a population of iPSC-derived cardiomyocytes translates into reproducible variability in both baseline phenotypes and drug responses. We measured baseline and drug-related effects in iPSC-derived cardiomyocytes from 27 healthy donors on kinetic Ca2+ flux and high-content live cell imaging. Cells were treated in concentration-response with cardiotoxic drugs: isoproterenol (β-adrenergic receptor agonist/positive inotrope), propranolol (β-adrenergic receptor antagonist/negative inotrope), and cisapride (hERG channel inhibitor/QT prolongation). Cells from four of the 27 donors were further evaluated in terms of baseline and treatment-related gene expression. Reproducibility of phenotypic responses was evaluated across batches and time. iPSC-derived cardiomyocytes exhibited reproducible donor-specific differences in baseline function and drug-induced effects. We demonstrate the feasibility of using a panel of population-based organotypic cells from healthy donors as an animal replacement experimental model. This model can be used to rapidly screen drugs and chemicals for inter-individual variability in cardiotoxicity. This approach demonstrates the feasibility of quantifying inter-individual variability in xenobiotic responses and can be expanded to other cell types for which in vitro populations can be derived from iPSCs.

IL-36γ Augments Host Defense and Immune Responses in Human Female Reproductive Tract Epithelial Cells.

IL-36γ is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36γ in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36γ and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36γ in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36γ treatment resulted in self-amplification of IL-36γ and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36γ are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36γ is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.

Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model.

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas.

Abstract 126: Pregnancy Hormones Induce An Increase Of Capillary Density In The Mouse Heart

During pregnancy the maternal organism undergoes a variety of adaptive physiological changes. The cardiovascular system is strongly affected and its response is characterized by cardiac hypertrophy. The molecular mechanisms underlying these adaptations are still poorly understood. The recent postulation of resident organotypic stem cells in the cardiovascular system prompted us to explore the potential involvement of these cells and the related signaling cascades involved in this adaptation process in the heart. We have analyzed cell proliferation using immunostainings in mouse hearts at different stages during pregnancy. The average percentage of proliferating cells in cardiac sections increased from virgin controls to gestational day 3 (GD3), peaked at GD14, and immediately stopped after delivery. Co-staining revealed that the majority of proliferating cells were fibroblasts and endothelial cells (ECs). The lack of cardiomyocyte proliferation was corroborated using BrdU pulse chase experiments analyzed in single cells upon Langendorff perfusion. The proliferation of fibroblasts and ECs reflects angiogenesis, which was underscored by an increase of capillary density, and extracellular matrix remodeling known to occur during pregnancy induced hypertrophy. To mimic the action of pregnancy hormones we implanted pellets with constant release of hormone for 21 days subcutaneously in ovariectomized mice. Capillary density was significantly increased in mice receiving progesterone-pellets alone or in combination with estrogen-pellets without induction of hypertrophy. Thus, increase of capillary density is directly induced by pregnancy hormones. Assessment of differentially expressed genes after hormone treatment revealed induction of angiogenetic factors and their receptors. We are currently investigating the mechanisms underlying the pregnancy hormone-induced proliferation in cardiac ECs and fibroblasts.