Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation ihas been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired re-epithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, whilst its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif knockout mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.
Ritu Bansal, Monica Torres, Matthew Hunt, Nuoqi Wang, Margarita Chatzopoulou, Mansi Manchanda, Evan P. Taddeo, Cynthia Shu, Orian S. Shirihai, Etty Bachar-Wikstrom, Jakob D. Wikstrom
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD-cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin null (mdx:utrn–/–) mice in sarcolipin (SLN) heterozygous knockout (sln+/–) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn–/– mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes show that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiac myocytes. These findings indicate that SLN upregulation plays a significant role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD-cardiomyopathy.
Satvik Mareedu, Nadezhda Fefelova, Cristi L. Galindo, Goutham Prakash, Risa Mukai, Junichi Sadoshima, Lai-Hua Xie, Gopal J. Babu
People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts, however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4, CD8 T-cells, nadir CD4 count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197-PWH with median age of 42-years, using a 56-gene panel. Seventy-nine percent had a CD4 nadir < 200, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8 T-cells and nadir CD4 counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of ≥ 35-years-old patients. Inflammatory biomarkers were higher in CH carriers compared to non-carriers supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4 and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.
Joseph M. Rocco, Yifan Zhou, Nicholas S. Liu, Elizabeth Laidlaw, Frances Galindo, Megan V. Anderson, Adam Rupert, Silvia Lucena Lage, Ana M. Ortega-Villa, Shiqin Yu, Andrea Lisco, Maura Manion, George S. Vassiliou, Cynthia E. Dunbar, Irini Sereti
Fibroblast Growth Factor 23 (FGF23) production has recently been shown to increase downstream of G⍺q/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding G⍺11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the impact of G⍺11 deficiency on FGF23 production in mice with heterozygous (Gna11+/–) or homozygous (Gna11–/–) ablation of Gna11. Both Gna11+/– and Gna11–/– mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11–/– mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and evidence of systemic inflammation with elevated serum IL1β levels. Furin gene expression was significantly increased in the Gna11–/– liver, suggesting enhanced FGF23 cleavage despite the observed rise in intact FGF23 levels. Gna11–/– mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, G⍺11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that FHH patients, at least those with GNA11 mutations, may be at risk for these complications.
Birol Ay, Sajin Marcus Cyr, Kaitlin Klovdahl, Wen Zhou, Christina M. Tognoni, Yorihiro Iwasaki, Eugene P. Rhee, Alpaslan Dedeoglu, Petra Simic, Murat Bastepe
Skeletal muscle wasting results from numerous pathological conditions impacting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss of function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 results in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fibre integrity. We further implicate a disruption in atrogin-1 mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We reveal that BiP is not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorates pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicates atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy, and highlights atrogin-1’s essential role in maintaining muscle homeostasis.
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
Spine metastases can result in severe neurologic compromise and decreased overall survival. Despite treatment advances, local disease progression is frequent, highlighting the need for novel therapies. Tumor treating fields (TTFields) impair tumor cell replication and are influenced by properties of surrounding tissue. We hypothesize bone’s dielectric properties will enhance TTFields mediated suppression of tumor growth in spine metastasis models. Computational modeling of TTFields intensity was performed following surgical resection of a spinal metastasis and demonstrated enhanced TTFields intensity within the resected vertebral body. Additionally, luciferase-tagged human KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized bone grafts and exposed to TTFields. Following TTFields exposure, BLI signal decreased 10-80% of baseline while control cultures displayed 4.48-9.36 fold increase in signal. Lastly, TTFields were applied in an orthotopic murine model of spinal metastasis. After 21 days of treatment, control mice demonstrated a 5-fold increase in BLI signal compared to TTFields treated mice. TTFields similarly prevented tumor invasion into the spinal canal and development of neurologic symptoms. Our data suggest that TTFields can be leveraged as a local therapy within minimally-conductive bone of spine metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spine metastases.
Daniel Ledbetter, Romulo de Almeida, Xizi Wu, Ariel Naveh, Chirag B. Patel, Queena Gonzalez, Thomas H. Beckham, Robert North, Laurence Rhines, Jing Li, Amol Ghia, David Aten, Claudio Tatsui, Christopher Alvarez-Breckenridge
HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that Caspase 6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) leads to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment can restrain p65 NF-κB transcriptional activity by cytoplasmic sequestration NF-κB signaling component, p65 NF-κB, and attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidney of mice with unilateral ureteral obstruction and lipopolysaccharide-induced acute kidney injury. Collectively, our findings indicate that the C-terminal region of HIPK2 is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential anti-inflammatory agents to treat kidney disease.
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.
Lisa Duvick, W. Michael Southern, Kellie A. Benzow, Zoe N. Burch, Hillary P. Handler, Jason S. Mitchell, Hannah Kuivinen, Udaya Gadiparthi, Praseuth Yang, Alyssa Soles, Carrie A. Sheeler, Orion Rainwater, Shannah Serres, Erin B. Lind, Tessa Nichols-Meade, Brennon O'Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement of cholesterol for activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and non-responsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells detects T cell activation and has potential to assess the success of ICI therapy.
Nicholas G. Ciavattone, Jenny Nan Guan, Alex Farfel, Jenelle Stauff, Timothy J. Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
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