Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we showed that Caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered Caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression, and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampens inflammatory gene expression and promotes cell resilience.
Soon-Gook Hong, Julianne W. Ashby, John P. Kennelly, Meigan Wu, Michelle Steel, Eesha Chattopadhyay, Rob Foreman, Peter Tontonoz, Elizabeth J. Tarling, Patric Turowski, Marcus Gallagher-Jones, Julia J. Mack
Diffuse midline glioma (DMG) H3K27-altered is one of the devastating childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation anti-tumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extra-terminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-seq and CUT & RUN showed that BET bromodomain inhibitors regulate the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation-response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.
Jun Watanabe, Matthew R. Clutter, Michael J. Gullette, Takahiro Sasaki, Eita Uchida, Savneet Kaur, Yan Mo, Kouki Abe, Yukitomo Ishi, Nozomu Takata, Manabu Natsumeda, Samantha Gadd, Zhiguo Zhang, Oren J. Becher, Rintaro Hashizume
Background: Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. Methods: To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. Results: At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Conclusions: Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.
Daniel F. Selgrade, Dominic E. Fullenkamp, Ivana A. Chychula, Binjie Li, Lisa Dellefave-Castillo, Adi D. Dubash, Joyce Ohiri, Tanner O. Monroe, Malorie Blancard, Garima Tomar, Cory Holgren, Paul W. Burridge, Alfred L. George Jr., Alexis R. Demonbreun, Megan. Puckelwartz, Sharon A. George, Igor R. Efimov, Kathleen J. Green, Elizabeth M. McNally
The diversity of structural variants (SVs) in melanoma and how they impact oncogenesis are incompletely known. We performed harmonized analysis of SVs across melanoma histological and genomic subtypes, and we identified distinct global properties between subtypes. These included the frequency and size of SVs and SV classes, their relation to chromothripsis events, and the role of topologically associated domain (TAD) boundary altering SVs on cancer-related genes. Following our prior identification of double-stranded break repair deficiency in a subset of triple wild-type cutaneous melanoma, we identified MRE11 and NBN loss-of-function SVs in melanomas with this mutational signature. Experimental knockouts of MRE11 and NBN, followed by olaparib cell viability assays in melanoma cells, indicated that dysregulation of each of these genes may cause sensitivity to PARPi in cutaneous melanomas. Broadly, harmonized analysis of melanoma SVs revealed distinct global genomic properties and molecular drivers, which may have biological and therapeutic impact.
Jake R. Conway, Riaz Gillani, Jett Crowdis, Brendan Reardon, Jihye Park, Seung Hun Han, Breanna M. Titchen, Mouadh Benamar, Rizwan Haq, Eliezer M. Van Allen
Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.
Dominik Saul, Madison L. Doolittle, Jennifer L. Rowsey, Mitchell N. Froemming, Robyn L. Kosinsky, Stephanie J. Vos, Ming Ruan, Nathan K. LeBrasseur, Abhishek Chandra, Robert J. Pignolo, João F. Passos, Joshua N. Farr, David G. Monroe, Sundeep Khosla
Given the global surge in autoimmune diseases, it is critical to evaluate emerging therapeutic interventions. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leveraged advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of autoantibody profiles, proteome-wide, in both health and disease. Using a custom set of over 730,000 human derived peptides, we demonstrated that each individual, regardless of disease state, possesses a distinct and complex constellation of autoreactive antibodies. For each individual, the set of resulting autoreactivites constituted a unique immunological fingerprint, or "autoreactome,” that was remarkably stable over years. Using the autoreactome as a primary output, we evaluated the relative effectiveness of various immunomodulatory therapies in altering autoantibody repertoires. We found that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly altered an individual’s autoreactome, while anti-CD19 and CD20 therapies had minimal effects. These data both confirm that the autoreactome is comprised of autoantibodies secreted by plasma cells, and strongly suggest that BCMA or other plasma cell targeting therapies may be highly effective in treating currently refractory autoantibody mediated diseases.
Aaron Bodansky, David J.L. Yu, Alysa N. Rallistan, Muge Kalaycioglu, Jim Boonyaratanakornkit, Damian J. Green, Jordan Gauthier, Cameron J. Turtle, Kelsey C. Zorn, Brian O'Donovan, Caleigh Mandel-Brehm, James Asaki, Hannah Kortbawi, Andrew F. Kung, Elze Rackaityte, Chung-Yu Wang, Aditi Saxena, Kimberly de Dios, Gianvito Masi, Richard J. Nowak, Kevin C. O'Connor, Hao Li, Valentina E. Diaz, Rowan Saloner, Kaitlin B. Casaletto, Eva Q. Gontrum, Brandon J. Chan, Joel H. Kramer, Michael R. Wilson, Paul J. Utz, Joshua A. Hill, Shaun W. Jackson, Mark S. Anderson, Joseph L. DeRisi
Jarmila Stremenova Spegarova, Praisoody Sinnappurajar, Dalila Al Julandani, Rokas Navickas, Helen Griffin, Manisha Ahuja, Angela Grainger, Katie Livingstone, Gillian I. Rice, Fraser Sutherland, Corinne Hayes, Simon Parke, Lewis Pang, Marion R. Roderick, Mary Slatter, Yanick Crow, Athimalaipet V. Ramanan, Sophie Hambleton
Recently developed anti-migraine therapeutics targeting calcitonin gene-related peptide (CGRP) signaling are effective, though their sites of activity remain elusive. Notably, the lymphatic vasculature is responsive to CGRP signaling, but whether meningeal lymphatic vessels (MLVs) contribute to migraine pathophysiology is unknown. Mice with lymphatic vasculature deficient in the CGRP receptor (CalcrliLEC mice) treated with nitroglycerin (NTG)-mediated chronic migraine exhibit reduced pain and light avoidance compared to NTG-treated littermate controls. Gene expression profiles of lymphatic endothelial cells (LECs) isolated from the meninges of Rpl22HA/+;Lyve1Cre RiboTag mice treated with NTG revealed increased MLV-immune interactions compared to cells from untreated mice. Interestingly, the relative abundance of mucosal vascular addressin cell adhesion molecule 1 (MAdCAM1)-interacting CD4+ T cells was increased in the deep cervical lymph nodes of NTG-treated control mice but not in NTG-treated CalcrliLEC mice. Treatment of cultured hLECs with CGRP peptide in vitro induced vascular endothelial (VE)-cadherin rearrangement and reduced functional permeability. Likewise, intra cisterna magna injection of CGRP caused rearrangement of VE-Cadherin, decreased MLV uptake of cerebrospinal fluid (CSF), and impaired CSF drainage in control mice, but not in CalcrliLEC mice. Collectively, these findings reveal a previously unrecognized role for lymphatics in chronic migraine, whereby CGRP signaling primes MLVs-immune interactions and reduces CSF efflux.
Nathan P. Nelson-Maney, Laszlo Balint, Anna L.S. Beeson, D. Stephen Serafin, Bryan M. Kistner, Elizabeth S. Douglas, Aisha H. Siddiqui, Alyssa M. Tauro, Kathleen M. Caron
There is increasing need to expand availability of donor liver grafts, including steatotic livers. However, the current use of steatotic grafts in liver transplantation is less acceptable due to their higher susceptibility to ischemia-reperfusion (I/R) injury. To investigate the mechanism underlying the susceptibility of steatotic liver to I/R injury, we detected cell death markers and inflammation in clinical donor livers and animal models. We found that caspase-8-mediated hepatic apoptosis is activated in steatotic liver I/R. However, ablation of caspase-8 only slightly mitigated steatotic liver I/R injury without affecting inflammation. We further demonstrated that RIPK1 kinase induces both caspase-8-mediated apoptosis and cell death-independent inflammation. Inhibition of RIPK1 kinase significantly protects against steatotic liver I/R injury by alleviating both hepatic apoptosis and inflammation. Additionally, we found that RIPK1 activation is induced by Z-DNA binding protein 1 (ZBP1) but not the canonical TNFα pathway during steatotic liver I/R. Deletion of ZBP1 substantially decreases the steatotic liver I/R injury. Mechanistically, ZBP1 is amplified by palmitic acid-activated JNK pathway in steatotic livers. Upon I/R, excessive reactive oxygen species trigger ZBP1 activation by inducing its aggregation independent of the Z-nucleic acids sensing action in steatotic livers, leading to the kinase activation of RIPK1 and the subsequent aggravation of liver injury. Thus, ZBP1-mediated RIPK1-driven apoptosis and inflammation exacerbate steatotic liver I/R injury, which could be targeted to protect steatotic donor livers during transplantation.
Ran Liu, Huan Cao, Shuhua Zhang, Mao Cai, Tianhao Zou, Guoliang Wang, Di Zhang, Xueling Wang, Jianjun Xu, Shenghe Deng, Tongxi Li, Daichao Xu, Jinyang Gu
Impairment of oligodendrocytes and myelin contributes to neurological disorders including multiple sclerosis (MS), stroke and Alzheimer's disease. Regeneration of myelin (remyelination) decreases the vulnerability of demyelinated axons, but this repair process commonly fails with disease progression. A contributor to inefficient remyelination is the altered extracellular matrix (ECM) in lesions that remains to be better defined. We have identified fibulin-2 (FBLN2) as a highly upregulated ECM component in lesions of MS and stroke, and in proteome databases of Alzheimer’s disease and traumatic brain injury. Focusing on MS, the inhibitory role of FBLN2 was suggested in the experimental autoimmune encephalomyelitis (EAE) model in which genetic FBLN2 deficiency improved behavioral recovery by promoting the maturation of oligodendrocytes and enhancing remyelination. Mechanistically, when oligodendrocyte progenitors were cultured in differentiation media, FBLN2 impeded their maturation into oligodendrocytes by engaging the Notch pathway, leading to cell death. Adeno-associated virus-deletion of FBLN2 in astrocytes improved oligodendrocyte numbers and functional recovery in EAE and generated new myelin profiles after lysolecithin-induced demyelination. Collectively, our findings implicate FBLN2 as a hitherto unrecognized injury-elevated ECM, and a therapeutic target, that impairs oligodendrocyte maturation and myelin repair.
Samira Ghorbani, Cenxiao Li, Brian M. Lozinski, Dorsa Moezzi, Charlotte D'Mello, Yifei Dong, Frank Visser, Hongmin Li, Claudia Silva, Mohammadparsa Khakpour, Colin J. Murray, Marie-Ève Tremblay, Mengzhou Xue, V. Wee Yong
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