Research
Abstract
Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic low-density lipoprotein receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL cholesterol (LDL-C) level and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3’UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3’UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p level. In mice, hepatic miR-483 overexpression increased LDLR level by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3’UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3’UTR (deltaBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.
Authors
Jianjie Dong, Ming He, Jie Li, Ariane R. Pessentheiner, Chen Wang, Jin Zhang, Yameng Sun, Wei-Ting Wang, Yuqing Zhang, Junhui Liu, Shen-Chih Wang, Po-Hsun Huang, Philip L.S.M. Gordts, Zu-Yi Yuan, Sotirios Tsimikas, John Y-J Shyy
Abstract
Resident vascular adventitial SCA1(+) progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling are significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM-specific expression of Gli1, we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell-specific Klf4 depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a pro-fibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis, but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable anti-fibrotic approach.
Authors
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
Abstract
Atrial fibrillation (AF) commonly occurs after surgery and is associated with atrial remodeling. TRPV4 is functionally expressed in the heart, and its activation affects cardiac structure and functions. We hypothesized that TRPV4 blockade alleviates atrial remodeling and reduces AF induction in sterile pericarditis (SP) rats. TRPV4 antagonist GSK2193874 or vehicle was orally administered 1 d before pericardiotomy. AF susceptibility and atrial function were assessed using in vivo electrophysiology, ex vivo optical mapping, patch-clamp, and molecular biology on day 3 after surgery. TRPV4 expression increased in the atria of SP rats and patients with AF. GSK2193874 significantly reduced AF vulnerability in vivo and the frequency of atrial ectopy and AF with a reentrant pattern ex vivo. Mechanistically, GSK2193874 reversed the abnormal action potential duration (APD) prolongation in atrial myocytes through the regulation of voltage-gated K+ currents (IK), reduced the activation of atrial fibroblasts by inhibiting P38, AKT, and STAT3 pathways, and alleviated the infiltration of immune cells. Our results reveal that TRPV4 blockade prevents abnormal changes in atrial myocyte electrophysiology and ameliorated atrial fibrosis and inflammation in SP rats, and, therefore, might be a promising strategy to treat AF, particularly post-operative AF.
Authors
Jie Liao, Qiongfeng Wu, Cheng Qian, Ning Zhao, Zhaoyang Zhao, Kai Lu, Shaoshao Zhang, Qian Dong, Lei Chen, Qince Li, Yimei Du
Abstract
A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus (P). papatasi, the vector of zoonotic cutaneous leishmaniasis has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. We here show by Far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a molecular weight of about 170kDa. Co-immunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg (1 and 3) was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice a week exhibited significantly increased levels of anti-Dsg1 and anti-Dsg3 antibodies from day 75 to day 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of crossreactivity to PpSP32. Herein, we demonstrated for the first time a specific binding between the PpSP32 and the dsg1 and 3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.
Authors
Soumaya Marzouki, Ines Zaraa, Maha Abdeladhim, Chaouki Ben Abdessalem, Fabiano Oliveira, Shaden Kamhawi, Mourad Mokni, Hechmi Louzir, Jesus Valenzuela, Mélika Ben Ahmed
Abstract
Cardiac ischemia is associated with arrhythmias, but effective therapies are limited. The cardiac voltage gated sodium channel α-subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a microRNA (miR), miR-448. The expression of miR-448 is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3’-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF1α and NF-κB were major transcriptional regulators for MIR448. Hypoxia also relieved MIR448 transcriptional suppression by RE1 silencing transcription factor (REST). Inhibition of miR-448 reduced arrhythmic risk after myocardial infarction. These results indicated that ischemia drove miR-448 expression, reduced Nav1.5 current and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy.
Authors
Gyeoung-Jin Kang, An Xie, Hong Liu, Samuel C. Dudley, Jr.
Abstract
Pituitary developmental defects lead to partial or complete hormone deficiency and significant health problems. The majority of cases are sporadic and of unknown cause. We screened 28 patients with pituitary stalk interruption syndrome (PSIS) for mutations in the FAT/DCHS family of protocadherins that have high functional redundancy. We identified seven variants, four of which putatively damaging, in FAT2 and DCHS2 in six patients with pituitary developmental defects recruited through a cohort of patients with mostly ectopic posterior pituitary gland and/or pituitary stalk interruption. All patients had growth hormone deficiency and two presented with multiple hormone deficiencies and small glands. FAT2 and DCHS2 were strongly expressed in the mesenchyme surrounding the normal developing human pituitary. We analyzed Dchs2-/- mouse mutants and identified anterior pituitary hypoplasia and partially penetrant infundibular defects. Overlapping infundibular abnormalities and distinct anterior pituitary morphogenesis defects were observed in Fat4-/- and Dchs1-/- mouse mutants but all animal models displayed normal commitment to the anterior pituitary cell type. Together our data implicate FAT/DCHS protocadherins in normal hypothalamic-pituitary development and identify FAT2 and DCHS2 as candidates underlying pituitary gland developmental defects such as ectopic pituitary gland and/or pituitary stalk interruption.
Authors
Emily J. Lodge, Paraskevi Xekouki, Tatiane S. Silva, Cristiane Kochi, Carlos A. Longui, Fabio R. Faucz, Alice Santambrogio, James L. Mills, Nathan Pankratz, John Lane, Dominika Sosnowska, Tina Hodgson, Amanda L. Patist, Philippa Francis-West, Francoise Helmbacher, Constantine Stratakis, Cynthia L. Andoniadou
Abstract
Epigenetic dysregulation is implicated in the pathogenesis of lupus. We performed a longitudinal analysis to assess changes in DNA methylation in lupus neutrophils over 4 years of follow up and across disease activity levels using 229 patient samples. We demonstrate that DNA methylation profiles in lupus are partly determined by ancestry-associated genetic variations and are highly stable over time. DNA methylation levels in two CpG sites correlated significantly with changes in lupus disease activity. Progressive demethylation in SNX18 was observed with increasing disease activity in African-American patients. Importantly, demethylation of a CpG site located within GALNT18 was associated with the development of active lupus nephritis. Differentially methylated genes between African-American and European-American lupus patients include type I interferon-response genes such as IRF7 and IFI44, and genes related to the NFkB pathway. TREML4, which plays a vital role in toll-like receptor signaling, was hypomethylated in African-American patients and demonstrated a strong cis-meQTL effect among 8855 cis-meQTL associations identified in our study.
Authors
Patrick Coit, Lourdes Ortiz-Fernandez, Emily E. Lewis, W. Joseph McCune, Kathleen Maksimowicz-McKinnon, Amr H. Sawalha
Abstract
Caspase 8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCC), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, in combination with pan-caspase inhibitors zVAD FMK or Emricasan and radiation. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels render many HNSCC cell lines resistant to necroptosis, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.
Authors
Burak Uzunparmak, Meng Gao, Antje Lindemann, Kelly Erikson, Li Wang, Eric Lin, Steven J. Frank, Frederico O. Gleber-Netto, Mei Zhao, Heath D. Skinner, Jared M. Newton, Andrew G. Sikora, Jeffrey N. Myers, Curtis R. Pickering
Abstract
We remain largely without effective prophylactic/therapeutic interventions for COVID-19. Although many human COVID-19 clinical trials are ongoing, there remains a deficiency of supportive preclinical drug efficacy studies to help guide decisions. Here we assessed the prophylactic/therapeutic efficacy of hydroxychloroquine (HCQ), a drug of interest for COVID-19 management, in two animal disease models. The standard human malaria HCQ prophylaxis (6.5 mg/kg given weekly) and treatment (6.5 mg/kg) did not significantly benefit clinical outcome nor reduce SARS-CoV-2 replication/shedding in the upper and lower respiratory tract in the rhesus macaque disease model. Similarly, when used for prophylaxis or treatment neither the standard human malaria dose (6.5 mg/kg) nor a high dose (50 mg/kg) of HCQ had any beneficial effect on clinical disease or SARS-CoV-2 kinetics (replication/shedding) in the Syrian hamster disease model. Results from these two preclinical animal models may prove helpful in guiding clinical use of HCQ for prophylaxis/treatment of COVID-19.
Authors
Kyle Rosenke, Michael A. Jarvis, Friederike Feldmann, Benjamin Schwarz, Atsushi Okumura, Jamie Lovaglio, Greg Saturday, Patrick W. Hanley, Kimberly Meade-White, Brandi N. Williamson, Frederick A. Hansen, Lizette Pérez-Pérez, Shanna Leventhal, Tsing-Lee Tang-Huau, Julie Callison, Elaine Haddock, Kaitlin A. Stromberg, Graham Sewell, Dana Scott, Catharine M. Bosio, David W. Hawman, Emmie de Wit, Heinz Feldmann
Abstract
In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Authors
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
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