Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by two oxidation-sensitive methionine residues within the regulatory domain. We previously showed that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model, in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice post-IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.
Simon Lebek, Xurde M. Caravia, Leon G. Straub, Damir Alzhanov, Wei Tan, Hui Li, John R. McAnally, Kenian Chen, Lin Xu, Philipp E. Scherer, Ning Liu, Rhonda Bassel-Duby, Eric N. Olson
A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20∆CNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL as adhesion molecule expressed by A20-deficient CNS-ECs. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20∆CNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in wildtype mice. In addition, blocking of ICOSL on primary mouse brain microvascular endothelial cells (pMBMECs) impaired the adhesion of T cells in vitro. Taken together, we here propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.
Lisa Johann, Sasha Soldati, Kristin Müller, Josephine Lampe, Federico Marini, Matthias Klein, Eva Schramm, Nathalie Ries, Carsten Schelmbauer, Ilaria Palagi, Khalad Karram, Julian C. Assmann, Mahtab A. Khan, Jan Wenzel, Mirko H.H. Schmidt, Jakob Körbelin, Dirk Schlüter, Geert van Loo, Tobias Bopp, Britta Engelhardt, Markus Schwaninger, Ari Waisman
The G protein-coupled receptor 84 (GPR84), a medium-chain fatty acid receptor, has garnered attention because of its potential involvement in a range of metabolic conditions. However, the precise mechanisms underlying this effect remain elusive. Our study has shed light on the pivotal role of GPR84, revealing its robust expression and functional significance within the brown adipose tissue (BAT). Mice lacking GPR84 exhibited increased lipid accumulation in BAT, rendering them more susceptible to cold exposure, and displaying reduced BAT activity compared to their wild-type counterparts. Our in vitro experiments with primary brown adipocytes from GPR84 knockout mice revealed diminished expression of thermogenic genes and reduced O2 consumption. Furthermore, the application of the GPR84 agonist 6-OAU counteracted these effects, effectively reinstating the brown adipocyte activity. These compelling in vivo and in vitro findings converge to highlight mitochondrial dysfunction as the primary cause of BAT anomalies in GPR84 knockout mice. The activation of GPR84 induced an increase in intracellular Ca2+ levels, which intricately influences mitochondrial respiration. By modulating mitochondrial Ca2+ levels and respiration, GPR84 has emerged as a potent molecule involved in BAT activity. These findings suggested that GPR84 is a potential therapeutic target for invigorating BAT and ameliorating metabolic disorders.
Xuenan Sun, Yu A. An, Vivian A. Paschoal, Camila O. De Souza, May-yun Wang, Lavanya Vishvanath, Lorena M.A. Bueno, Ayanna S. Cobb, Joseph A. Nieto Carrion, Madison E. Ibe, Chao Li, Harrison A. Kidd, Shiuhwei Chen, Wenhong Li, Rana K. Gupta, Da Young Oh
A paucity of chemotherapeutic options for metastatic brain cancer limits patient survival and portends poor clinical outcomes. Using a central nervous system (CNS) small-molecule inhibitor library of 320 agents known to be blood-brain barrier permeable and approved by the U.S. Food and Drug Administration, breast cancer brain metastases vulnerabilities were interrogated to identify an effective agent. Metixene, an antiparkinsonian drug, was identified as a top therapeutic agent that was capable of decreasing cellular viability and inducing cell death across different metastatic breast cancer subtypes. This agent significantly reduced mammary tumor size in orthotopic xenograft assays and improved survival in an intracardiac model of multiorgan site metastases. Metixene further extended survival in mice bearing intracranial xenografts and in an intracarotid model of multiple brain metastases. Functional analysis revealed that metixene induced incomplete autophagy through N-Myc Downstream Regulated 1 (NDRG1) phosphorylation thereby leading to caspase-mediated apoptosis in both primary and brain metastatic cells, regardless of cancer subtype or origin. CRISPR Cas9 knockout of NDRG1 led to autophagy completion and reversal of the metixene apoptotic effect. Metixene is a promising therapeutic agent against metastatic brain cancer, with minimal reported side effects in humans, which merits consideration for clinical translation.
Jawad Fares, Edgar Petrosyan, Deepak Kanojia, Crismita Dmello, Alex Cordero, Joseph T. Duffy, Ragini Yeeravalli, Mayurbhai H. Sahani, Peng Zhang, Aida Rashidi, Victor A. Arrieta, Ilya Ulasov, Atique U. Ahmed, Jason Miska, Irina V. Balyasnikova, C. David James, Adam M. Sonabend, Amy B. Heimberger, Maciej S. Lesniak
The progression of proteinuric kidney diseases is associated with podocyte loss but the mechanisms underlying this process remain unclear. Podocytes re-enter the cell cycle to repair double-stranded DNA (dsDNA) breaks. However, the unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes results in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle re-entry was identified using FUCCI2aR mice and observed altered expression of cell-cycle associated proteins such as p21, p53, Cyclin B1, and Cyclin D1. Podocyte-specific translating ribosome affinity purification (TRAP) and RNAseq revealed the downregulation of Ribosomal RNA-processing protein 8 (Rrp8). Over-expression of Rrp8 in Pfn1 KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.
Xuefei Tian, Christopher E. Pedigo, Ke Li, Xiaotao Ma, Patricia Bunda, John Pell, Angela Lek, Jianlei Gu, Yan Zhang, Paulina X. Medina Rangel, Wei Li, Eike Schwartze, Soichiro Nagata, Gabriel Lerner, Sudhir Perincheri, Anupama Priyadarshini, Hongyu Zhao, Monkol Lek, Madhav C. Menon, Rongguo Fu, Shuta Ishibe
We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have extremely poor prognosis, and most of these TP53 mutations are missense mutations. Here, we report that in contrast to mixed AML and T-cell malignancy developed in NrasG12D/+; p53-/- (NP-/-) mice, NrasG12D/+; p53R172H/+ (NPmut) mice rapidly developed an inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and largely normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergize to establish an NPmut-AML transcriptome distinct from that of NP-/- cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated expression of inflammatory genes, including those linked to NFκB signaling. NFκB was also upregulated in human NRAS;TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NFκB inhibition in vitro. The proteasome inhibitor bortezomib stabilized NFκB inhibitory protein IκBα reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergizes with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.
Adhithi Rajagopalan, Yubin Feng, Meher B. Gayatri, Erik A. Ranheim, Taylor Klungness, Daniel R. Matson, Moon Hee Lee, Mabel Minji Jung, Yun Zhou, Xin Gao, Kalyan V.G. Nadiminti, David T. Yang, Vu L. Tran, Eric Padron, Shigeki Miyamoto, Emery H. Bresnick, Jing Zhang
Tumor burden, considered a common chronic stressor, can cause widespread anxiety. Evidence suggests that cancer-induced anxiety can promote tumor progression, but the underlying neural mechanism remains unclear. Here, we used neuroscience and cancer tools to investigate how the brain contributes to tumor progression via nerve-tumor crosstalk in mice model of breast cancer. We showed that the tumor-bearing mice exhibited significant anxiety-like behaviors and that corticotropin-releasing hormone (CRH) neurons in the central medial amygdala (CeM) were activated. Moreover, newly formed sympathetic nerves were detected in tumors, which established a polysynaptically connected with the brain. Pharmacogenetic or optogenetic inhibition of CeMCRH neurons and CeMCRH→LPGi circuit significantly alleviated anxiety-like behaviors and slowed tumor growth. Conversely, artificial activation of CeMCRH neurons and CeMCRH→LPGi circuit increased anxiety and tumor growth. Importantly, alprazolam, an anti-anxiety drug, was found to be a promising intervention for slowing tumor progression. Furthermore, we showed that manipulation of CeMCRH→LPGi circuit directly regulates the activity of the intratumoral sympathetic nerves and peripheral nerve-derived norepinephrine, then affecting tumor progression by modulating the antitumor immunity. Together, these findings reveal a brain–tumor neural circuit contributing to breast cancer progression and provide new therapeutic insights for breast cancer.
Si-Yi Xiong, Hui-Zhong Wen, Li-Meng Dai, Yun-Xiao Lou, Zhao-Qun Wang, Yi-Lun Yi, Xiao-Jing Yan, Ya-Ran Wu, Wei Sun, Peng-Hui Chen, Si-Zhe Yang, Xiao-Wei Qi, Yi Zhang, Guang-Yan Wu
Three sisters, born from consanguineous parents, manifested a unique Mullerian anomaly characterized by uterine hypoplasia with thin estrogen-unresponsive endometrium, primary amenorrhea, but spontaneous tubal pregnancies. Through whole-exome sequencing followed by comprehensive genetic analysis, a missense variant was identified in the OSR1 gene. We therefore investigated OSR1/OSR1 expression in postpubertal human uteri, and the prenatal and postnatal expression pattern of Osr1/Osr1 in murine developing Mullerian ducts (MDs) and endometrium, respectively. We then investigated whether Osr1 deletion would affect MD development, using wild-type and genetically engineered mice. Human uterine OSR1/OSR1 expression was found primarily in the endometrium. Mouse Osr1 was expressed prenatally in MDs and Wolffian ducts (WDs), from rostral to caudal segments, in E13.5 embryos. MDs and WDs were absent on the left side and MDs were rostrally truncated on the right side of E13.5 Osr1-/- embryos. Postnatally, Osr1 was expressed in mouse uteri throughout lifespan, peaking at postnatal days 14 and 28. Osr1 protein was present primarily in uterine luminal and glandular epithelial cells and in the epithelial cells of mouse oviducts. Through this translational approach, we demonstrated that OSR1/Osr1 is important for MD development and endometrial receptivity and may be implicated in uterine factor infertility.
Adriana Lofrano-Porto, Sidney Alcântara Pereira, Andrew Dauber, Jordana C.B. Bloom, Audrey N. Fontes, Naomi Asimow, Olívia Laquis de Moraes, Petra Ariadne T. Araujo, Ana Paula Abreu, Michael H. Guo, Silviene F. De Oliveira, Han Liu, Charles Lee, Wendy Kuohung, Michella S. Coelho, Rona S. Carroll, Rulang Jiang, Ursula B. Kaiser
Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we suspected that the underlying cause was reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5–/– mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific monoclonal antibodies, the binding of LPL antibodies to heart and BAT capillaries (relative to CD31 or GPIHBP1 antibodies) was reduced in Apoa5–/– mice. LPL levels in the postheparin plasma were also lower in Apoa5–/– mice. We suspected that a recent biochemical observation—that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity—could be related to the low intracapillary LPL levels in Apoa5–/– mice. We showed that an ANGPTL3/8-specific monoclonal antibody (IBA490) and APOA5 normalize plasma triglyceride levels and intracapillary LPL levels in Apoa5–/– mice. We also showed that ANGPTL3/8 detaches LPL from HSPGs and GPIHBP1 on the surface of cells and that the LPL detachment is blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5–/– mice and further illuminate the molecular mechanisms that regulate plasma triglyceride metabolism.
Ye Yang, Anne P. Beigneux, Wenxin Song, Le Phuong Nguyen, Hyesoo Jung, Yiping Tu, Thomas A. Weston, Caitlyn M. Tran, Katherine Xie, Rachel G. Yu, Anh P. Tran, Kazuya Miyashita, Katsuyuki Nakajima, Masami Murakami, Yan Q. Chen, Eugene Y. Zhen, Joonyoung R. Kim, Paul H. Kim, Gabriel Birrane, Peter Tontonoz, Michael Ploug, Robert J. Konrad, Loren G. Fong, Stephen G. Young
The metabolic syndrome, today affecting more than 20% of the US population, is a group of five conditions that often co-exist and that strongly predispose to cardiovascular disease. How these conditions are linked mechanistically remains unclear, especially two of these: obesity and elevated blood pressure. Here we show that high fat consumption in mice leads to the accumulation of lipid droplets in endothelial cells throughout the organism, and that lipid droplet accumulation in endothelium suppresses endothelial nitric oxide synthase (eNOS), reduces NO production, elevates blood pressure, and accelerates atherosclerosis. Mechanistically, the accumulation of lipid droplets destabilizes eNOS mRNA and activates an endothelial inflammatory signaling cascade that suppresses eNOS and NO production. Pharmacological prevention of lipid droplet formation reverses the suppression of NO production in cell culture and in vivo, and blunts blood pressure elevation in response to high fat diet. These results highlight lipid droplets as a critical and unappreciated component of endothelial cell biology, explain how lipids increase blood pressure acutely, and provide a mechanistic account for the epidemiological link between obesity and elevated blood pressure.
Boa Kim, Wencao Zhao, Soon Yew Tang, Michael G. Levin, Ayon Ibrahim, Yifan Yang, Emilia M. Roberts, Ling Lai, Jian Li, Richard K. Assoian, Garret A. FitzGerald, Zoltan Arany
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