Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis

Background: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and two lysosomal proteins, the Niemann-Pick C1 (NPC1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. Methods: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells (VSMCs). To establish whether Wnt5a also protects against cholesterol accumulation in human VSMCs, we used a CRISPR/Cas9 guided nuclease approach to generate human VSMCs knockout for Wnt5a. Results: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum (ER). Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the ER, and promoted atherosclerosis. Conclusions: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

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MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽

10.3390/biomedicines9060630 ◽

2021 ◽

Vol 9 (6) ◽

pp. 630

Author(s):

Huili Lyu ◽

Cody M. Elkins ◽

Jessica L. Pierce ◽

C. Henrique Serezani ◽

Daniel S. Perrien

Keyword(s):

Heterotopic Ossification ◽

Muscle Injury ◽

Cell Types ◽

Fibrodysplasia Ossificans Progressiva ◽

Inflammatory Signaling ◽

Conditional Deletion ◽

Pathway Crosstalk ◽

Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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Single-cell mapping of focused ultrasound-transfected brain

Gene Therapy ◽

10.1038/s41434-021-00226-0 ◽

2021 ◽

Author(s):

A. S. Mathew ◽

C. M. Gorick ◽

R. J. Price

Keyword(s):

Single Cell ◽

Focused Ultrasound ◽

Cell Types ◽

Brain Cell ◽

Therapeutic Modality ◽

Full Potential ◽

Stress Genes ◽

Multiple Cell ◽

Differential Gene

AbstractGene delivery via focused ultrasound (FUS) mediated blood-brain barrier (BBB) opening is a disruptive therapeutic modality. Unlocking its full potential will require an understanding of how FUS parameters (e.g., peak-negative pressure (PNP)) affect transfected cell populations. Following plasmid (mRuby) delivery across the BBB with 1 MHz FUS, we used single-cell RNA-sequencing to ascertain that distributions of transfected cell types were highly dependent on PNP. Cells of the BBB (i.e., endothelial cells, pericytes, and astrocytes) were enriched at 0.2 MPa PNP, while transfection of cells distal to the BBB (i.e., neurons, oligodendrocytes, and microglia) was augmented at 0.4 MPa PNP. PNP-dependent differential gene expression was observed for multiple cell types. Cell stress genes were upregulated proportional to PNP, independent of cell type. Our results underscore how FUS may be tuned to bias transfection toward specific brain cell types in vivo and predict how those cells will respond to transfection.

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Regulation of Fertility, Survival, and Cuticle Collagen Function by the Caenorhabditis elegans eaf-1 and ell-1 Genes

Journal of Biological Chemistry ◽

10.1074/jbc.m111.270454 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35915-35921 ◽

Cited By ~ 23

Author(s):

Liquan Cai ◽

Binh L. Phong ◽

Alfred L. Fisher ◽

Zhou Wang

Keyword(s):

Caenorhabditis Elegans ◽

Cell Types ◽

Transcriptional Elongation ◽

Human Prostate Cancer ◽

Prostate Cancer Cells ◽

Transcription Factor Family ◽

Collagen Expression ◽

Extracellular Matrix Components ◽

Multiple Cell

EAF2, an androgen-regulated protein, interacts with members of the ELL (eleven-nineteen lysine-rich leukemia) transcription factor family and also acts as a tumor suppressor. Although these proteins control transcriptional elongation and perhaps modulate the effects of other transcription factors, the mechanisms of their actions remain largely unknown. To gain new insights into the biology of the EAF2 and ELL family proteins, we used Caenorhabditis elegans as a model to explore the in vivo roles of their worm orthologs. Through the use of transgenic worms, RNAi, and an eaf-1 mutant, we found that both genes are expressed in multiple cell types throughout the worm life cycle and that they play important roles in fertility, survival, and body size regulation. ELL-1 and EAF-1 likely contribute to these activities in part through modulating cuticle synthesis, given that we observed a disrupted cuticle structure in ell-1 RNAi-treated or eaf-1 mutant worms. Consistent with disruption of cuticle structure, loss of either ELL-1 or EAF-1 suppressed the rol phenotype of specific collagen mutants, possibly through the control of dpy-3, dpy-13, and sqt-3 collagen gene expression. Furthermore, we also noted the regulation of collagen expression by ELL overexpression in PC3 human prostate cancer cells. Together, these results reveal important roles for the eaf-1 and ell-1 genes in the regulation of extracellular matrix components.

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Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

Biomolecules ◽

10.3390/biom10091306 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1306

Author(s):

Ann-Kristin Afflerbach ◽

Mark D. Kiri ◽

Tahir Detinis ◽

Ben M. Maoz

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Types ◽

In Vitro Models ◽

Cell Source ◽

Fundamental Research ◽

Multiple Cell ◽

Vitro Model

The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

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Sertoli cell ablation and replacement of the spermatogonial niche in mouse

Nature Communications ◽

10.1038/s41467-019-13879-8 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Tetsuhiro Yokonishi ◽

Jennifer McKey ◽

Shintaro Ide ◽

Blanche Capel

Keyword(s):

Sertoli Cells ◽

Cell Types ◽

Supporting Cell ◽

Idiopathic Male Infertility ◽

Testicular Cells ◽

Cell Ablation ◽

Toxic Drug ◽

Donor Cells ◽

Multiple Cell

AbstractSpermatogonia, which produce sperm throughout the male lifetime, are regulated inside a niche composed of Sertoli cells, and other testis cell types. Defects in Sertoli cells often lead to infertility, but replacement of defective cells has been limited by the inability to deplete the existing population. Here, we use an FDA-approved non-toxic drug, benzalkonium chloride (BC), to deplete testis cell types in vivo. Four days after BC administration, Sertoli cells are preferentially depleted, and can be replaced to promote spermatogenesis from surviving (host) spermatogonia. Seven days after BC treatment, multiple cell types can be engrafted from fresh or cryopreserved testicular cells, leading to complete spermatogenesis from donor cells. These methods will be valuable for investigation of niche-supporting cell interactions, have the potential to lead to a therapy for idiopathic male infertility in the clinic, and could open the door to production of sperm from other species in the mouse.

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Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00262.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. L476-L486 ◽

Cited By ~ 60

Author(s):

Nicole M. White ◽

Dechen Jiang ◽

James D. Burgess ◽

Ilya R. Bederman ◽

Stephen F. Previs ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cholesterol Synthesis ◽

De Novo ◽

Cholesterol Content ◽

Cholesterol Homeostasis ◽

Lysosomal Storage ◽

In Vivo Models ◽

Cellular Processes ◽

Nasal Tissue

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr −/− mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr −/− mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

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Merging Microfluidics with Microtitre Technology for More Efficient Drug Discovery

JALA Journal of the Association for Laboratory Automation ◽

10.1016/j.jala.2008.05.002 ◽

2008 ◽

Vol 13 (5) ◽

pp. 275-279 ◽

Cited By ~ 2

Author(s):

Nicole V. Tolan ◽

Luiza I. Genes ◽

Dana M. Spence

Keyword(s):

Cellular Response ◽

Simultaneous Detection ◽

Drug Efficacy ◽

Cell Types ◽

Microtitre Plate ◽

Multiple Components ◽

Multiple Cell ◽

Improve Blood Flow

Detecting multiple components from a single red blood cell (RBC) sample within a flow-based system in less than 20 min will enable improved in vitro determinations of drug efficacy and cellular response to administered drugs. Here, an example of an improved in vitro measurement involving iloprost, a pharmaceutical reported to improve blood flow, has been determined by incorporating multiple cell types onto a single device. The method allows fluid flow to address individual rows of wells contained within an 18-well microfluidic array that serves as a precursor to a 96-well microtitre plate device. The ability to better mimic the in vivo circulation by incorporating the flow of blood components, coupled with simultaneous detection and laboratory automation in place for microtitre plates, suggests that the microfluidic array presented here will allow for improved mechanistic drug research studies. Using fluorescence microscopy, concentrations of multiple metabolites present within the RBC can also be determined using the microfluidic array. The current progress toward using this device for personalized medicine is presented here.

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Adipose-tissue derived signals control bone remodelling

10.1101/2020.03.02.972711 ◽

2020 ◽

Author(s):

Maria-Bernadette Madel ◽

He Fu ◽

Dominique D. Pierroz ◽

Mariano Schiffrin ◽

Carine Winkler ◽

...

Keyword(s):

Bone Erosion ◽

Cell Formation ◽

Cell Types ◽

Whole Body ◽

Long Bone ◽

Bone Homeostasis ◽

Multiple Cell ◽

The One

SummaryLong bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlight the dual bone phenotype of PPARγ null mice: on the one hand the increase bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explore the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibits mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis.

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Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.760667 ◽

2022 ◽

Vol 9 ◽

Author(s):

Maria Veronica Lipreri ◽

Nicola Baldini ◽

Gabriela Graziani ◽

Sofia Avnet

Keyword(s):

Extracellular Matrix ◽

Bone Structure ◽

Dynamic Network ◽

Cell Types ◽

New Drugs ◽

Culture Methods ◽

Increased Risk ◽

Multiple Cell

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.

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Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids

10.1101/2020.05.02.073320 ◽

2020 ◽

Cited By ~ 4

Author(s):

Xiaohua Duan ◽

Yuling Han ◽

Liuliu Yang ◽

Benjamin E. Nilsson-Payant ◽

Pengfei Wang ◽

...

Keyword(s):

Stem Cell ◽

Viral Entry ◽

Pluripotent Stem Cell ◽

Cell Types ◽

Human Pluripotent Stem Cell ◽

Humanized Mouse Model ◽

Multiple Cell ◽

Approved Drugs

Summary ParagraphThe current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo, and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.

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