Research
Abstract
Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2 to 3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over one year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR-ISR axis could be a potential therapeutic strategy for PVOD.
Authors
Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata
Abstract
Spinal muscular atrophy (SMA) is a recessive, developmental disorder caused by the genetic loss or mutation of the gene SMN1 (Survival of Motor Neuron 1). SMA is characterized by neuromuscular symptoms and muscle weakness. Several years ago, SMA treatment underwent a radical transformation, with the approval of three different SMN-dependent disease modifying therapies. This includes two SMN2 splicing therapies - Risdiplam and Nusinersen. One main challenge for Type II SMA patients treated with these drugs is ongoing muscle fatigue, limited mobility, and other skeletal problems. To date, few molecular studies have been conducted on SMA-patient derived tissues after treatment, limiting our understanding of what targets remain after the principal spinal cord targeted therapies are applied. Therefore, we collected paravertebral muscle from eight Type II patients undergoing spinal surgery for scoliosis and seven controls. We used RNA-sequencing to characterize their transcriptional profiles and correlate these with muscle histology. Despite the limited cohort size and heterogeneity, we observed a consistent loss of oxidative phosphorylation machinery of the mitochondria, a decrease in mitochondrial DNA copy number, and a correlation between signals of cellular stress, denervation and increased fibrosis. This work provides new putative targets for combination therapies for Type II SMA.
Authors
Fiorella Grandi, Stéphanie Astord, Sonia Pezet, Elèna Gidaja, Sabrina Mazzucchi, Maud Chapart, Stéphane Vasseur, Kamel Mamchaoui, Piera Smeriglio
Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many Mendelian disease patients. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here we describe Targeted Long-read Sequencing of Mendelian Disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 subjects with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in two IRD cases with inconclusive testing, which uncovered non-coding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
Abstract
Epstein Barr virus (EBV) contributes to around 2% of all tumors worldwide. Simultaneously, more than 90% of healthy human adults persistently carry EBV without clinical symptoms. In most EBV carriers it is thought that virus-induced tumorigenesis is prevented by cell-mediated immunity. Specifically, memory CD8+ T cells recognize EBV-infected cells during latent and lytic infection. Using a symptomatic primary infection model, similar to infectious mononucleosis (IM), we found EBV-induced CD8+ tissue-resident memory T cells (TRMs) in mice with a humanized immune system. These human TRMs were preferentially established after intranasal EBV infection in nasal-associated lymphoid tissues (NALT), equivalent to tonsils, the primary site of EBV infection in humans. They expressed canonical TRM markers, including CD69, CD103, and BLIMP-1, as well as Granzyme B, CD107a and CCL5. Despite cytotoxic activity and cytokine production ex vivo, these TRMs demonstrated reduced CD27 expression and proliferation and failed to control EBV viral loads in the NALT during infection although effector memory T cells (TEMs) controlled viral titers in spleen and blood. Overall, TRMs are established in mucosal lymphoid tissues by EBV infection, but primarily systemic CD8+ T cell expansion seems to control viral loads in the context of IM-like infection.
Authors
Daniel Kirchmeier, Yun Deng, Lisa Rieble, Michelle Böni, Fabienne Läderach, Patrick Schuhmachers, Alma Delia Valencia-Camargo, Anita Murer, Nicole Caduff, Bithi Chatterjee, Obinna Chijioke, Kyra Zens, Christian Münz
Abstract
Immune cell mediated inflammation is important in normal tissue regeneration but can be pathologic in diabetic wounds. Limited literature exists on the role of CD4+T cells in normal or diabetic wound repair, however, the imbalance of CD4+TH17/Treg cells has been found to promote inflammation in other diabetic tissues. Here, using human tissue and murine transgenic models, we identified that the histone methyltransferase MLL1 directly regulates the TH17 transcription factor RORγ via an H3K4me3 mechanism and increases expression of Notch receptors and downstream Notch signaling. Further, we found that Notch receptor signaling regulates CD4+TH cell differentiation and is critical for normal wound repair, and loss of upstream Notch pathway mediators or receptors in CD4+T cells resulted in the loss of CD4+TH cell differentiation in wounds. In diabetes, MLL1 and Notch-receptor signaling were upregulated in wound CD4+TH cells, driving CD4+ T cells towards the TH17 cell phenotype. Treatment of diabetic wound CD4T cells with a small molecule inhibitor of MLL1 (MI-2) yielded a significant reduction in CD4+TH17 cells and IL17A. This is the first study to identify the MLL1-mediated mechanisms responsible for regulating the TH17/Treg balance in normal and diabetic wounds and define the complex role of Notch signaling in CD4+T cells in wounds, where increased or decreased Notch signaling both result in pathologic wound repair. Therapeutic targeting of MLL1 in diabetic CD4+TH cells may decrease pathologic inflammation through regulation of CD4+T cell differentiation.
Authors
William J. Melvin, Tyler M. Bauer, Kevin D. Magnum, Christopher O. Audu, James Shadiow, Emily Barrett, Amrita Joshi, Jadie Y. Moon, Rachel Bogel, Purba Mazumder, Sonya J. Wolf, Steven Kunkel, Johann E. Gudjonsson, Frank M. Davis, Katherine A. Gallagher
Abstract
Ataxia telangiectasia and Rad3-related protein (ATR) is a key DNA damage response protein that facilitates DNA damage repair and regulates cell cycle progression. As such, ATR is an important component of the cellular response to radiation, particularly in cancer cells which show altered DNA damage response and aberrant cell cycle checkpoints. Therefore, ATR’s pharmacological inhibition could be an effective radiosensitization strategy to improve radiotherapy. We assessed the ability of an ATR inhibitor, AZD6738, to sensitize cancer cell lines of various histologic types to photon and proton radiotherapy. We found that radiosensitization took place through persistent DNA damage and abrogated G2 cell cycle arrest. We also found that AZD6738 increased the number of micronuclei after exposure to radiotherapy. We found that combining radiation with AZD6738 led to tumor growth delay and prolonged survival relative to radiation alone in a breast cancer model. Combining AZD6738 with photons or protons also led to increased macrophage infiltration at the tumor microenvironment. These results provide a rationale for further investigation of ATR inhibition in combination with radiotherapy and with other agents such as immune checkpoint blockade.
Authors
Scott J. Bright, Mandira Manandhar, David B. Flint, Rishab Kolachina, Mariam Ben Kacem, David K.J. Martinus, Broderick X. Turner, Ilsa Qureshi, Conor H. McFadden, Poliana Camila Marinello, Simona F. Shaitelman, Gabriel O. Sawakuchi
Abstract
Post-transplantation, T helper 1 (Th1)-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ dendritic cells (DC2) and CD103+ dendritic cells (DC1). This interaction necessitates further investigation in context of transplant immunity. We use a well-established pre-clinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+DC1 demonstrate a tolerogenic phenotype that modulate the immunogenic capacity of CD11b+DC2 primarily mediated by IL-10, suppressing alloreactive CD4+Th1 cells via the PD-L1/PD-1 pathway, and enhancing Treg-mediated tolerance via αvβ8 integrin-activated TGFβ1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+Th1 cells induces migratory CD103+DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers including αvβ8 integrin and IL-10 and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex-vivo induced tolerogenic CD103+DC1(iDC1) effectively inhibits Th1 polarization and preserves the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+DC1 in modulating host alloimmune responses.
Authors
Tomas Blanco, Hayate Nakagawa, Aytan Musayeva, Mark Krauthammer, Rohan Bir Singh, Akitomo Narimatsu, Hongyan Ge, Sara I. Shoushtari, Reza Dana
Abstract
Our objective was to interrogate infant mesenchymal stem cell (MSC) lipid metabolism and gestational exposures that may contribute to child obesity risk. MSCs were cultured from term infants of mothers with obesity (n=16) or normal-weight (n=15). In MSCs undergoing myogenesis in vitro, we used lipidomics to distinguish phenotypes by unbiased cluster analysis and lipid challenge (24h excess fatty acid, 24hFA). We measured MSC AMP-activated protein kinase (AMPK) activity and fatty acid oxidation (FAO), and a composite index of maternal glucose, insulin, triglycerides, free fatty acids, tumor necrosis factor-α, high density lipoprotein- and total- cholesterol in fasting blood from mid- and late-gestation (~17, ~27wks). We measured child adiposity at birth (n=29), 4-6m (n=29), and 4-6y (n=13). Three MSC clusters were distinguished by triacylglycerol (TAG) stores, with greatest TAGs in Cluster-2. All Clusters increased acylcarnitines and TAGs with 24hFA, though Cluster-2 was more pronounced and corresponded to AMPK activation and FAO. Maternal metabolic markers predicted MSC Clusters and child adiposity at 4-6y (both highest in Cluster-3). Our data supports that MSC phenotypes are predicted by comprehensive maternal metabolic milieu exposures, independent of maternal BMI, and suggest utility as an at-birth predictor for child adiposity, though validation with larger longitudinal samples is warranted.
Authors
Lauren E. Gyllenhammer, Vincent Zaegel, Allison M. Duensing, Manoel Lixandrao, Dana Dabelea, Bryan C. Bergman, Kristen E. Boyle
Abstract
The prevalence of chronic kidney diseases (CKD) varies by race due to genetic and environmental factors. The Glu504Lys polymorphism in aldehyde dehydrogenase 2 (ALDH2), commonly observed among East Asians, alters the enzyme's function in detoxifying alcohol-derived aldehydes, impacting kidney function. This study investigated the association between variations in ALDH2 levels within the kidney and the progression of kidney fibrosis. Our clinical data indicates that diminished ALDH2 levels are linked to worse CKD outcomes, with correlations between ALDH2 expression, estimated glomerular filtration rate, urinary levels of acrolein, an aldehyde metabolized by ALDH2, and fibrosis severity. In mouse models of unilateral ureteral obstruction and folic acid nephropathy, reduced ALDH2 levels and elevated acrolein were observed in kidneys, especially in ALDH2 Glu504Lys knock-in mice. Mechanistically, acrolein modifies pyruvate kinase M2, leading to its nuclear translocation and co-activation of HIF-1α, shifting cellular metabolism to glycolysis, disrupting mitochondrial function, contributing to tubular damage and the progression of kidney fibrosis. Enhancing ALDH2 expression through adeno-associated virus vectors reduces acrolein and mitigates fibrosis in both wild-type and Glu504Lys knock-in mice. These findings underscore the potential therapeutic significance of targeting the dynamic interaction between ALDH2 and acrolein in CKD.
Authors
Szu-yuan Li, Ming-Tsun Tsai, Yu-Ming Kuo, Hui-Min Yang, Zhen-Jie Tong, Hsiao-Wei Cheng, Chih-Ching Lin, Hsiang-Tsui Wang
Abstract
Dual endothelin-1 (ET-1) and angiotensin II (AngII) receptor antagonism with sparsentan has strong antiproteinuric actions via multiple potential mechanisms that are more pronounced, or additive compared to current standard of care using angiotensin receptor blockers (ARB). Considering the many actions of ET-1 and AngII on multiple cell types, this study aimed to determine glomeruloprotective mechanisms of sparsentan compared to the ARB losartan by direct visualization of its effects in the intact kidney in focal segmental glomerulosclerosis (FSGS) using intravital multiphoton microscopy. In both healthy and FSGS models, sparsentan treatment increased afferent/efferent arteriole diameters, increased or preserved blood flow and single nephron glomerular filtration rate, attenuated acute ET-1+AngII-induced increases in podocyte calcium, reduced proteinuria, preserved podocyte number, increased both endothelial and renin lineage cells and clones in vasculature, glomeruli and tubules, restored glomerular endothelial glycocalyx, attenuated mitochondrial stress and immune cell homing. These effects were either not observed or of smaller magnitude with losartan. The pleiotropic nephroprotective effects of sparsentan included improved hemodynamics, podocyte and endothelial cell functions, and tissue repair. Compared to losartan, sparsentan was more effective in the sustained preservation of kidney structure and function, which underscores the importance of the ET-1 component in FSGS pathogenesis and therapy.
Authors
Georgina Gyarmati, Urvi Nikhil Shroff, Audrey K. Izuhara, Sachin Deepak, Radko Komers, Patricia W. Bedard, Janos Peti-Peterdi
No posts were found with this tag.
