Ghosh A, Syed SM, Tanwar PS.
PMID: 28743800 | DOI: 10.1242/dev.149989
The epithelial lining of the Fallopian tube is vital for fertility, providing nutrition to gametes, and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions are primarily consisting of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing Fallopian tube epithelial homoeostasis are currently unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and different Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.
Wnts produced by Osterix-expressing osteolineage cells regulate their proliferation and differentiation.
Proc Natl Acad Sci U S A. 2014 Dec 9;111(49):E5262-71.
Tan SH, Senarath-Yapa K, Chung MT, Longaker MT, Wu JY, Nusse R.
Wnt signaling is a critical regulator of bone development, but the identity and role of the Wnt-producing cells are still unclear. We addressed these questions through in situ hybridization, lineage tracing, and genetic experiments. First, we surveyed the expression of all 19 Wnt genes and Wnt target gene Axin2 in the neonatal mouse bone by in situ hybridization, and demonstrated--to our knowledge for the first time--that Osterix-expressing cells coexpress Wnt and Axin2. To track the behavior and cell fate of Axin2-expressing osteolineage cells, we performed lineage tracing and showed that they sustain bone formation over the long term. Finally, to examine the role of Wnts produced by Osterix-expressing cells, we inhibited Wnt secretion in vivo, and observed inappropriate differentiation, impaired proliferation, and diminished Wnt signaling response. Therefore, Osterix-expressing cells produce their own Wnts that in turn induce Wnt signaling response, thereby regulating their proliferation and differentiation.
Tang, WC;Tsao, SW;Jones, GE;Liu, X;Tsai, MH;Delecluse, HJ;Dai, W;You, C;Zhang, J;Huang, SCM;Leung, MM;Liu, T;Ching, YP;Chen, H;Lo, KW;Li, X;Tsang, CM;
PMID: 36420735 | DOI: 10.1002/path.6036
Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they were visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. This article is protected by
Pathology - Research and Practice
Schwab, C;Domke, L;Rose, F;Hausser, I;Schirmacher, P;Longerich, T;
| DOI: 10.1016/j.prp.2022.154000
Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.
Bockmayr, M;Harnisch, K;Pohl, L;Schweizer, L;Mohme, T;Körner, M;Alawi, M;Suwala, A;Dorostkar, M;Monoranu, C;Hasselblatt, M;Wefers, A;Capper, D;Hench, J;Frank, S;Richardson, T;Tran, I;Liu, E;Snuderl, M;Engertsberger, L;Benesch, M;von Deimling, A;Obrecht, D;Mynarek, M;Rutkowski, S;Glatzel, M;Neumann, J;Schüller, U;
| DOI: 10.1093/neuonc/noac079.143
Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p<0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progression-free survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.
Broeckel, RM;Feldmann, F;McNally, KL;Chiramel, AI;Sturdevant, GL;Leung, JM;Hanley, PW;Lovaglio, J;Rosenke, R;Scott, DP;Saturday, G;Bouamr, F;Rasmussen, AL;Robertson, SJ;Best, SM;
PMID: 34855915 | DOI: 10.1371/journal.ppat.1009678
Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
bioRxiv : the preprint server for biology
Payne, LB;Abdelazim, H;Hoque, M;Barnes, A;Mironovova, Z;Willi, CE;Darden, J;Jenkins-Houk, C;Sedovy, MW;Johnstone, SR;Chappell, JC;
PMID: 36778261 | DOI: 10.1101/2023.02.03.527005
The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.
Am J Respir Crit Care Med. 2018 Dec 15.
Reyfman PA, Walter JM, Joshi N, Anekalla KR, McQuattie-Pimentel AC, Chiu S, Fernandez R, Akbarpour M, Chen CI, Ren Z, Verma R, Abdala-Valencia H, Nam K, Chi M, Han S, Gonzalez-Gonzalez FJ, Soberanes S, Watanabe S, Williams KJN, Flozak AS, Nicholson TT, Morgan VK, Winter DR, Hinchcliff M, Hrusch CL, Guzy RD, Bonham CA, Sperling AI, Bag R, Hamanaka RB, Mutlu GM, Yeldandi AV, Marshall SA, Shilatifard A, Amaral LAN, Perlman H, Sznajder JI, Argento AC, Gillespie CT, Dematte J, Jain M, Singer BD, Ridge KM, Lam AP, Bharat A, Bhorade SM, Gottardi CJ, Budinger GRS, Misharin AV.
PMID: 30554520 | DOI: 10.1164/rccm.201712-2410OC
Abstract RATIONALE: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. OBJECTIVES: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells or other cell types in lung tissue from subjects with pulmonary fibrosis compared with controls. METHODS: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data in using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. MEASUREMENTS AND MAIN RESULTS: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to non-overlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. CONCLUSIONS: We generated a single cell atlas of pulmonary fibrosis. Using this atlas we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
Simonson, B;Chaffin, M;Hill, MC;Atwa, O;Guedira, Y;Bhasin, H;Hall, AW;Hayat, S;Baumgart, S;Bedi, KC;Margulies, KB;Klattenhoff, CA;Ellinor, PT;
PMID: 36790929 | DOI: 10.1016/j.celrep.2023.112086
Ischemic cardiomyopathy (ICM) is the leading cause of heart failure worldwide, yet the cellular and molecular signature of this disease is largely unclear. Using single-nucleus RNA sequencing (snRNA-seq) and integrated computational analyses, we profile the transcriptomes of over 99,000 human cardiac nuclei from the non-infarct region of the left ventricle of 7 ICM transplant recipients and 8 non-failing (NF) controls. We find the cellular composition of the ischemic heart is significantly altered, with decreased cardiomyocytes and increased proportions of lymphatic, angiogenic, and arterial endothelial cells in patients with ICM. We show that there is increased LAMININ signaling from endothelial cells to other cell types in ICM compared with NF. Finally, we find that the transcriptional changes that occur in ICM are similar to those in hypertrophic and dilated cardiomyopathies and that the mining of these combined datasets can identify druggable genes that could be used to target end-stage heart failure.
Khan, M;Clijsters, M;Choi, S;Backaert, W;Claerhout, M;Couvreur, F;Van Breda, L;Bourgeois, F;Speleman, K;Klein, S;Van Laethem, J;Verstappen, G;Dereli, AS;Yoo, SJ;Zhou, H;Dan Do, TN;Jochmans, D;Laenen, L;Debaveye, Y;De Munter, P;Gunst, J;Jorissen, M;Lagrou, K;Meersseman, P;Neyts, J;Thal, DR;Topsakal, V;Vandenbriele, C;Wauters, J;Mombaerts, P;Van Gerven, L;
PMID: 36446381 | DOI: 10.1016/j.neuron.2022.11.007
Can SARS-CoV-2 hitchhike on the olfactory projection and take a direct and short route from the nose into the brain? We reasoned that the neurotropic or neuroinvasive capacity of the virus, if it exists, should be most easily detectable in individuals who died in an acute phase of the infection. Here, we applied a postmortem bedside surgical procedure for the rapid procurement of tissue, blood, and cerebrospinal fluid samples from deceased COVID-19 patients infected with the Delta, Omicron BA.1, or Omicron BA.2 variants. Confocal imaging of sections stained with fluorescence RNAscope and immunohistochemistry afforded the light-microscopic visualization of extracellular SARS-CoV-2 virions in tissues. We failed to find evidence for viral invasion of the parenchyma of the olfactory bulb and the frontal lobe of the brain. Instead, we identified anatomical barriers at vulnerable interfaces, exemplified by perineurial olfactory nerve fibroblasts enwrapping olfactory axon fascicles in the lamina propria of the olfactory mucosa.
He, S;Bhatt, R;Brown, C;Brown, EA;Buhr, DL;Chantranuvatana, K;Danaher, P;Dunaway, D;Garrison, RG;Geiss, G;Gregory, MT;Hoang, ML;Khafizov, R;Killingbeck, EE;Kim, D;Kim, TK;Kim, Y;Klock, A;Korukonda, M;Kutchma, A;Lewis, ZR;Liang, Y;Nelson, JS;Ong, GT;Perillo, EP;Phan, JC;Phan-Everson, T;Piazza, E;Rane, T;Reitz, Z;Rhodes, M;Rosenbloom, A;Ross, D;Sato, H;Wardhani, AW;Williams-Wietzikoski, CA;Wu, L;Beechem, JM;
PMID: 36203011 | DOI: 10.1038/s41587-022-01483-z
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Lovatt, D;Tamburino, A;Krasowska-Zoladek, A;Sanoja, R;Li, L;Peterson, V;Wang, X;Uslaner, J;
PMID: 36261573 | DOI: 10.1038/s42003-022-03970-0
Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.
