TFAP2C is a key regulator of intrauterine trophoblast cell invasion and deep hemochorial placentation
Dominguez et al. report that TFAP2C is critical for trophoblast cell specialization, including development of the invasive trophoblast cell lineage and trophoblast cell–guided remodeling of the uterine vasculature. The cover image shows a rat placentation site at late gestation, with Tfap2c (magenta) transcript localized in the placenta as well as in invasive trophoblast cells in the uterus of the mother. In contrast, the Prl7b1 (cyan) transcript is primarily localized in the invasive trophoblast cells.
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Research Letter
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Abstract
Authors
Huafeng Fu, Qinbo Cai, Zhijun Zhou, Yulong He, Min Li, Dongjie Yang
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Research Articles
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Abstract
Regulatory T (Treg) cells are essential for maternal immune tolerance of the fetus and placenta. In preeclampsia, aberrant Treg cell tolerance is implicated, but how Treg cells affect the uterine vascular dysfunction thought to precede placental impairment and maternal vasculopathy is unclear. We used Foxp3-diphtheria toxin receptor mice to test the hypothesis that Treg cells are essential regulators of decidual spiral artery adaptation to pregnancy. Transient Treg cell depletion during early placental morphogenesis caused impaired remodeling of decidual spiral arteries, altered uterine artery function, and fewer Dolichos biflorus agglutinin+ uterine natural killer (uNK) cells, resulting in late-gestation fetal loss and fetal growth restriction. Replacing the Treg cells by transfer from wild-type donors mitigated the impact on uNK cells, vascular remodeling, and fetal loss. RNA sequencing of decidua revealed genes associated with NK cell function and placental extravillous trophoblasts were dysregulated after Treg cell depletion and normalized by Treg cell replacement. These data implicate Treg cells as essential upstream drivers of uterine vascular adaptation to pregnancy, through a mechanism likely involving phenotypic regulation of uNK cells and trophoblast invasion. The findings provide insight into mechanisms linking impaired adaptive immune tolerance and altered spiral artery remodeling, 2 hallmark features of preeclampsia.
Authors
Shanna L. Hosking, Lachlan M. Moldenhauer, Ha M. Tran, Hon Y. Chan, Holly M. Groome, Evangeline A.K. Lovell, Ella S. Green, Stephanie E. O’Hara, Claire T. Roberts, Kerrie L. Foyle, Sandra T. Davidge, Sarah A. Robertson, Alison S. Care
×Abstract
Autoimmune uveitis (AU) is a sight-threatening ocular autoimmune disorder that often manifests as retinal vasculitis. Increased neutrophil infiltration around retinal vessels has been reported during the progression of AU, while how they function is not fully recognized. Neutrophil extracellular traps (NETs), produced by activated neutrophils, have been suggested to be detrimental in autoimmune diseases. Here, we found that NETs were elevated in patients with active AU, and this was verified in an experimental AU (EAU) mouse model. Depletion of neutrophils or degradation of NETs with deoxyribonuclease-I (DNase I) could decrease CD4+ effector T cell (Teff) infiltration in retina and spleen to alleviate EAU. Moreover, we found that the expression of adhesion molecules, selectin, and antigen-presenting molecules was elevated in EAU retina and in retinal microvascular endothelial cells (RMECs) cocultured with NETs. The stimulated RMECs further facilitated CD4+ T cell adhesion, activation, and differentiation into Teffs. Mechanistically, NETs trigger RMEC activation by hastening cell senescence through the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Slowing down senescence or inhibiting the cGAS/STING pathway in RMECs reduces the activation and differentiation of CD4+ T cells. These results suggest a deleterious role of NETs in AU. Targeting NETs would offer an effective therapeutic method.
Authors
Zuoyi Li, Zhuang Li, Yunwei Hu, Yanyan Xie, Yuxun Shi, Guanyu Chen, Jun Huang, Zhiqiang Xiao, Wenjie Zhu, Haixiang Huang, Minzhen Wang, Jianping Chen, Xiaoqing Chen, Dan Liang
×Abstract
African American (AA) women are disproportionately affected by obesity and hyperlipidemia, particularly in the setting of adverse social determinants of health (aSDoH) that contribute to health disparities. Obesity, hyperlipidemia, and aSDoH appear to impair NK cells. As potential common underlying mechanisms are largely unknown, we sought to investigate common signaling pathways involved in NK cell dysfunction related to obesity and hyperlipidemia in AA women from underresourced neighborhoods. We determined in freshly isolated NK cells that obesity and measures of aSDoH were associated with a shift in NK cell subsets away from CD56dim/CD16+ cytotoxic NK cells. Using ex vivo data, we identified LDL as a marker related to NK cell function in an AA population from underresourced neighborhoods. Additionally, NK cells from AA women with obesity and LDL-treated NK cells displayed a loss in NK cell function. Comparative unbiased RNA-sequencing analysis revealed DUSP1 as a common factor. Subsequently, chemical inhibition of Dusp1 and Dusp1 overexpression in NK cells highlighted its significance in NK cell function and lysosome biogenesis in a mTOR/TFEB-related fashion. Our data demonstrate a pathway by which obesity and hyperlipidemia in the setting of aSDoH may relate to NK cell dysfunction, making DUSP1 an important target for further investigation of health disparities.
Authors
Yvonne Baumer, Komudi Singh, Abhinav Saurabh, Andrew S. Baez, Cristhian A. Gutierrez-Huerta, Long Chen, Muna Igboko, Briana S. Turner, Josette A. Yeboah, Robert N. Reger, Lola R. Ortiz-Whittingham, Sahil Joshi, Marcus R. Andrews, Elizabeth M. Aquino Peterson, Christopher K.E. Bleck, Laurel G. Mendelsohn, Valerie M. Mitchell, Billy S. Collins, Neelam R. Redekar, Skyler A. Kuhn, Christian A. Combs, Mehdi Pirooznia, Pradeep K. Dagur, David S.J. Allan, Daniella M. Schwartz, Richard W. Childs, Tiffany M. Powell-Wiley
×Abstract
Exposure to loud noise is a common cause of acquired hearing loss. Disruption of subcellular calcium homeostasis and downstream stress pathways in the endoplasmic reticulum and mitochondria, including the unfolded protein response (UPR), have been implicated in the pathophysiology of noise-induced hearing loss. However, studies on the association between calcium homeostasis and stress pathways have been limited due to limited ability to measure calcium dynamics in mature-hearing, noise-exposed mice. We used a genetically encoded calcium indicator mouse model in which GCaMP6f is expressed specifically in hair cells or supporting cells under control of Myo15Cre or Sox2Cre, respectively. We performed live calcium imaging and UPR gene expression analysis in 8-week-old mice exposed to levels of noise that cause cochlear synaptopathy (98 db sound pressure level [SPL]) or permanent hearing loss (106 dB SPL). UPR activation occurred immediately after noise exposure, and the pattern of UPR activation was dependent on noise level, with the proapoptotic pathway upregulated only after 106 dB noise exposure. Spontaneous calcium transients in hair cells and intercellular calcium waves in supporting cells, which are present in neonatal cochleae, were quiescent in mature-hearing cochleae but reactivated upon noise exposure. Noise exposure of 106 dB was associated with more persistent and expansive intercellular Ca2+ signaling wave activity. These findings demonstrate a strong and dose-dependent association between noise exposure, UPR activation, and changes in calcium homeostasis in hair cells and supporting cells, suggesting that targeting these pathways may be effective to develop treatments for noise-induced hearing loss.
Authors
Yesai Park, Jiang Li, Noura Ismail Mohamad, Ian R. Matthews, Peu Santra, Elliott H. Sherr, Dylan K. Chan
×Abstract
Biliary obstruction and cholangiocyte hyperproliferation are important features of cholangiopathies affecting the large extrahepatic bile duct (EHBD). The mechanisms underlying obstruction-induced cholangiocyte proliferation in the EHBD remain poorly understood. Developmental pathways, including WNT signaling, are implicated in regulating injury responses in many tissues, including the liver. To investigate the contribution of WNT signaling to obstruction-induced cholangiocyte proliferation in the EHBD, we used complementary in vivo and in vitro models with pharmacologic interventions and transcriptomic analyses. To model obstruction, we used bile duct ligation (BDL) in mice. Human and mouse biliary organoids and mouse biliary explants were used to investigate the effects of WNT activation and inhibition in vitro. We observed an upregulation of WNT ligand expression associated with increased biliary proliferation following obstruction. Cholangiocytes were identified as both WNT ligand–expressing and WNT-responsive cells. Inhibition of WNT signaling decreased cholangiocyte proliferation in vivo and in vitro, while activation increased proliferation. WNT effects on cholangiocyte proliferation were β-catenin dependent, and we showed a direct effect of WNT7B on cholangiocyte growth. Our studies suggested that cholangiocyte-derived WNT ligands can activate WNT signaling to induce proliferation after obstructive injury. These findings implicate the WNT pathway in injury-induced cholangiocyte proliferation within the EHBD.
Authors
Ashley N. Calder, Mirabelle Q. Peter, John W. Tobias, Nureen H. Mohamad Zaki, Theresa M. Keeley, Timothy L. Frankel, Linda C. Samuelson, Nataliya Razumilava
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T cells targeting a KRAS mutation can induce durable tumor regression in some patients with metastatic epithelial cancer. It is unknown whether T cells targeting mutant KRAS that are capable of killing tumor cells can be identified from peripheral blood of patients with pancreatic cancer. We developed an in vitro stimulation approach and identified HLA-A*11:01–restricted KRAS G12V–reactive CD8+ T cells and HLA-DRB1*15:01–restricted KRAS G12V–reactive CD4+ T cells from peripheral blood of 2 out of 6 HLA-A*11:01–positive patients with pancreatic cancer whose tumors expressed KRAS G12V. The HLA-A*11:01–restricted KRAS G12V–reactive T cell receptor (TCR) was isolated and validated to specifically recognize the KRAS G12V8–16 neoepitope. While T cells engineered to express this TCR specifically recognized all 5 tested human HLA-A*11:01+ and KRAS G12V+ pancreatic cancer organoids, the recognition was often modest, and tumor cell killing was observed in only 2 out of 5 organoids. IFN-γ priming of the organoids enhanced the recognition and killing by the TCR-engineered T cells. The TCR-engineered T cells could significantly slow the growth of an established organoid-derived xenograft in immunodeficient mice. Our data suggest that this TCR has potential for use in TCR-gene therapy, but additional strategies that enhance tumor recognition by the TCR-engineered T cells likely will be required to increase clinical activity.
Authors
Xiongfei Xu, Shiwei Guo, Haihui Gu, Zhanshan Cha, Xiaohan Shi, Xiaoyi Yin, Huan Wang, Suizhi Gao, Bo Li, Lingyu Zhu, Wei Jing, Kailian Zheng, Zhuo Shao, Peng Cheng, Chunhong Zheng, Yi-Ping Shih, Yunguang Li, Baohua Qian, Dong Gao, Eric Tran, Gang Jin
×Abstract
We characterized the longitudinal serum protein signatures of women 6 and 10 years after having gestational diabetes mellitus (GDM) to identify factors associated with the development of type 2 diabetes mellitus (T2D) and prediabetes in this at-risk post-GDM population, aiming to discover potential biomarkers for early diagnosis and prevention of T2D. Our study identified 75 T2D-associated serum proteins and 23 prediabetes-associated proteins, some of which were validated in an independent T2D cohort. Machine learning (ML) performed on the longitudinal proteomics highlighted protein signatures associated with progression to post-GDM diabetes. We also proposed prognostic biomarker candidates that were differentially regulated in healthy participants at 6 years postpartum who later progressed to having T2D. Our longitudinal study revealed T2D risk factors for post-GDM populations who are relatively young and healthy, providing insights for clinical decisions and early lifestyle interventions.
Authors
Heaseung Sophia Chung, Lawrence Middleton, Manik Garg, Ventzislava A. Hristova, Rick B. Vega, David Baker, Benjamin G. Challis, Dimitrios Vitsios, Sonja Hess, Kristina Wallenius, Agneta Holmäng, Ulrika Andersson-Hall
×CD34hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis
Abstract
Osteoarthritis (OA) shows various clinical manifestations depending on the status of its joint components. We aimed to identify the synovial cell subsets responsible for OA pathophysiology by comprehensive analyses of human synovium samples in single-cell resolution. Two distinct OA synovial tissue groups were classified by gene expression profiles in RNA-Seq: inflammatory and fibrotic. The inflammatory group exhibited high expression of inflammatory cytokines, histologically inflammatory infiltrate, and a more severe pain score. The fibrotic group showed higher expression of fibroblast growth factor (FGFs) and bone morphogenetic proteins (BMPs), showed histologically perivascular fibrosis, and showed a lower pain score. In single-cell RNA-Seq (scRNA-Seq) of synovial cells, MERTKloCD206lo macrophages and CD34hi fibroblasts were associated with the inflammatory and fibrotic groups, respectively. Among the 3 fibroblast subsets, CD34loTHY1lo and CD34loTHY1hi fibroblasts were influenced by synovial immune cells, whereas CD34hi fibroblasts were influenced by mural and endothelial cells. Particularly, in CD34hi fibroblast subsets, CD34hiCD70hi fibroblasts promoted proliferation of Tregs, potentially suppressing synovitis and protecting articular cartilage. Elucidation of the mechanisms underlying the regulation of these synovial cell subsets may lead to novel strategies for OA therapeutics.
Authors
Junya Miyahara, Yasunori Omata, Ryota Chijimatsu, Hiroyuki Okada, Hisatoshi Ishikura, Junya Higuchi, Naohiro Tachibana, Kosei Nagata, Shoichiro Tani, Kenichi Kono, Kohei Kawaguchi, Ryota Yamagami, Hiroshi Inui, Shuji Taketomi, Yasuhide Iwanaga, Asuka Terashima, Fumiko Yano, Masahide Seki, Yutaka Suzuki, Roland Baron, Sakae Tanaka, Taku Saito
×Abstract
Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II–induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.
Authors
Brennan Callow, Xiaobing He, Nicholas Juriga, Kevin D. Mangum, Amrita Joshi, Xianying Xing, Andrea Obi, Abhijnan Chattopadhyay, Dianna M. Milewicz, Mary X. O’Riordan, Johann Gudjonsson, Katherine Gallagher, Frank M. Davis
×Abstract
Aniridia is a rare congenital condition of abnormal eye development arising principally from heterozygous mutation of the PAX6 gene. Among the multiple complications arising in the eye, aniridia-associated keratopathy (AAK) is a severe vision-impairing condition of the cornea associated with a progressive limbal stem cell deficiency that lacks suitable treatment options. Current mouse models of aniridia do not accurately represent the onset and progression dynamics of human AAK, hindering therapy development. Here, we performed deep phenotyping of a haploinsufficient Pax6+/– small-eye (Sey) mouse model on the 129S1/SvImJ background, which exhibits key features of mild presentation at birth and progressive AAK with aging, mimicking human disease. The model exhibits a slowly progressing AAK phenotype and provides insights into the disease, including disturbed basal epithelial cell organization, function, and marker expression; persistent postnatal lymphangiogenesis; disrupted corneal innervation patterns; and persisting yet altered limbal stem cell marker expression with age. The model recapitulates many of the known features of human disease, enabling investigation of underlying disease mechanisms and, importantly, access to a well-defined temporal window for evaluating future therapeutics.
Authors
Dina Javidjam, Petros Moustardas, Mojdeh Abbasi, Ava Dashti, Yedizza Rautavaara, Neil Lagali
×Abstract
Mechanisms underpinning signals from genome-wide association studies remain poorly understood, particularly for noncoding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where noncoding single nucleotide variants (SNVs) are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPR interference (CRISPRi) screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro; CRISPR excision of SNV-containing noncoding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes; and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes compared with homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of noncoding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.
Authors
Yang Zhang, Natalie L. David, Tristan Pesaresi, Rosemary E. Andrews, G.V. Naveen Kumar, Hongyin Chen, Wanning Qiao, Jinzhao Yang, Kareena Patel, Tania Amorim, Ankit X. Sharma, Silvia Liu, Matthew L. Steinhauser
×Abstract
Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 (MCL1) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.
Authors
Shazia Adjumain, Paul Daniel, Claire Xin Sun, Gabrielle Bradshaw, Nicole J. Chew, Vanessa Tsui, Hanbyeol Lee, Melissa Loi, Nataliya Zhukova, Dilru Habarakada, Abigail Yoel, Vijesh G. Vaghjiani, Shaye Game, Louise E. Ludlow, Naama Neeman, E. Alejandro Sweet-Cordero, David D. Eisenstat, Jason E. Cain, Ron Firestein
×Abstract
Despite effective treatment, human immunodeficiency virus (HIV) persists in optimally treated people as a transcriptionally silent provirus. Latently infected cells evade the immune system and the harmful effects of the virus, thereby creating a long-lasting reservoir of HIV. To gain a deeper insight into the molecular mechanisms of HIV latency establishment, we constructed a series of HIV-1 fluorescent reporter viruses that distinguish active versus latent infection. We unexpectedly observed that the proportion of active to latent infection depended on a limiting viral factor, which created a bottleneck that could be overcome by superinfection of the cell, T cell activation, or overexpression of HIV-1 transactivator of transcription (Tat). In addition, we found that tat and regulator of expression of virion proteins (Rev) expression levels varied among HIV molecular clones and that tat levels were an important variable in latency establishment. Lower rev levels limited viral protein expression whereas lower Tat levels or mutation of the Tat binding element promoted latent infection that was resistant to reactivation even in fully activated primary T cells. Nevertheless, we found that combinations of latency reversal agents targeting both cellular activation and histone acetylation pathways overcame deficiencies in the Tat/TAR axis of transcription regulation. These results provide additional insight into the mechanisms of latency establishment and inform Tat-centered approaches to cure HIV.
Authors
Francisco Gomez-Rivera, Valeri H. Terry, Cuie Chen, Mark M. Painter, Maria C. Virgilio, Marianne E. Yaple-Maresh, Kathleen L. Collins
×Abstract
Peptidoglycans (PGNs) are structural polymers of the bacterial cell wall and a common microbial molecular pattern encountered by the immune system daily. Low levels of PGNs are constitutively present in the systemic circulation in humans and rise during inflammatory pathologies. Since all known PGN sensors are intracellular, PGN internalization is a prerequisite for the initiation of cellular immune responses. Here, we report the mechanisms controlling the recognition and uptake of polymeric PGNs by circulating human mononuclear phagocytes. We found that complement C3 and C4 opsonins govern PGN recognition and internalization, but no single opsonin is indispensable because of multiple uptake redundancies. We observed a bimodal internalization of polymeric PGNs with distinct requirements for complement C4. At low PGN concentrations, C3 mediated PGN recognition by surface receptors while the efficient internalization of PGN polymers critically required C4. Supraphysiologic PGN concentrations triggered a secondary uptake modality that was insensitive to C4 and mediated instead by C3 engagement of complement receptors 1 and 3. To our knowledge, this is the first description of nonoverlapping C3 and C4 opsonophagocytoses working in parallel. Controlling these uptake mechanisms has the potential to modulate PGN clearance or the dysregulated immune responses during bacterial infections.
Authors
Narcis I. Popescu, Jędrzej Kluza, Megan A. Reidy, Elizabeth Duggan, John D. Lambris, Linda F. Thompson, K. Mark Coggeshall
×Abstract
Transcription factor AP-2 gamma (TFAP2C) has been identified as a key regulator of the trophoblast cell lineage and hemochorial placentation. The rat possesses deep placentation characterized by extensive intrauterine trophoblast cell invasion, which resembles human placentation. Tfap2c is expressed in multiple trophoblast cell lineages, including invasive trophoblast cells situated within the uterine-placental interface of the rat placentation site. Global genome editing was used to explore the biology of Tfap2c in rat placenta development. Homozygous global disruption of Tfap2c resulted in prenatal lethality. Heterozygous global disruption of Tfap2c was associated with diminished invasive trophoblast cell infiltration into the uterus. The role of TFAP2C in the invasive trophoblast cell lineage was explored using Cre-lox conditional mutagenesis. Invasive trophoblast cell–specific disruption of Tfap2c resulted in inhibition of intrauterine trophoblast cell invasion and intrauterine and postnatal growth restriction. The invasive trophoblast cell lineage was not impaired following conditional monoallelic disruption of Tfap2c. In summary, TFAP2C contributes to the progression of distinct stages of placental development. TFAP2C is a driver of early events in trophoblast cell development and reappears later in gestation as an essential regulator of the invasive trophoblast cell lineage. A subset of TFAP2C actions on trophoblast cells are dependent on gene dosage.
Authors
Esteban M. Dominguez, Ayelen Moreno-Irusta, Regan L. Scott, Khursheed Iqbal, Michael J. Soares
×Abstract
Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor–dependent (VEGF-dependent) angiogenesis remain unclear. In our study, the molecular underpinnings of endothelial dysfunction in diabetes are investigated, focusing on the roles of disabled-2 (Dab2) and Forkhead box M1 (FOXM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high-glucose-treated primary mouse skin endothelial cells. In diabetic mice with endothelial deficiency of Dab2, in vivo and in vitro angiogenesis and wound healing were reduced when compared with wild-type diabetic mice. Restoration of Dab2 expression by injected mRNA-containing, LyP-1–conjugated lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. Furthermore, FOXM1 was downregulated in skin endothelial cells under high-glucose conditions as determined by RNA-sequencing analysis. FOXM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FOXM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. Our study provides evidence of the crucial roles of Dab2 and FOXM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.
Authors
Sudarshan Bhattacharjee, Jianing Gao, Yao Wei Lu, Shahram Eisa-Beygi, Hao Wu, Kathryn Li, Amy E. Birsner, Scott Wong, Yudong Song, John Y-J. Shyy, Douglas B. Cowan, Wendong Huang, Wenyi Wei, Masanori Aikawa, Jinjun Shi, Hong Chen
×Abstract
Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms of and have minimal treatment options for liver fibrosis. Therefore, we engineered a live-cell imaging system that assessed fibrosis in a human multilineage hepatic organoid in a microwell (i.e., microHOs). Transcriptomic analysis revealed that TGFB converted mesenchymal cells in microHOs into myofibroblast-like cells resembling those in fibrotic human liver tissue. When pro-fibrotic intracellular signaling pathways were examined, the antifibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates their antifibrotic efficacy would be limited to fibrotic diseases solely mediated by that growth factor. Based upon transcriptomic and transcription factor activation analyses in microHOs, glycogen synthase kinase 3β and p38 MAPK inhibitors were identified as potential new broad-spectrum therapies for liver fibrosis. Other new therapies could subsequently be identified using the microHO system.
Authors
Yuan Guan, Zhuoqing Fang, Angelina Hu, Sarah Roberts, Meiyue Wang, Wenlong Ren, Patrik K. Johansson, Sarah C. Heilshorn, Annika Enejder, Gary Peltz
×Abstract
Diabetes mellitus (DM) is acknowledged as an independent risk factor for acute kidney injury. Ras guanine nucleotide-releasing protein-4 (RasGRP4) exerts a notable role in modulating immune-inflammatory responses and kidney disease progression in diabetes. Herein, we delved into the specific role and mechanism of RasGRP4 in diabetic renal ischemia-reperfusion injury. Diabetes was induced by a high-fat diet and streptozocin (STZ) injections, followed by creating an ischemia-reperfusion kidney injury via renal pedicle clamping and reperfusion. In vitro, a high glucose and hypoxia-reoxygenation modeled cellular inflammatory injury. We found RasGRP4-KO mice, compared with C57BL/6J (WT) mice, showed markedly less renal dysfunction and fibrosis in diabetic ischemia-reperfusion injury. There was a significant decrease in the renal infiltration of M1 macrophages and Th17 cells, along with downregulated IL-17 pathway proteins and effectors. In vitro, RasGRP4 deletion restrained M1 macrophage polarization and Th17 cell differentiation, inhibiting the IL-17 signaling pathway in HK-2 cells. Hyperglycemia intensified renal inflammation state. Together, RasGRP4, through the regulation of interactions among M1 macrophages, CD4+ T cells, and HK-2 cells, formed a cascade that intensified the inflammatory storm activity, ultimately exacerbating the inflammatory injury of diabetic ischemia-reperfusion kidneys. DM intensified this inflammatory injury mechanism, worsening the injury from renal ischemia-reperfusion.
Authors
Li Zhang, Zhanglong Wang, Yunqi Wu, Binshan Zhang, Zhongli Wang, Sisi Chen, Xuying Meng, Pei Yu, Saijun Zhou
