Research Article
Abstract
When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 μM, individuals are at increased risk of developing large vessel atherothrombosis and small vessel dysfunction. The annexin A2 complex (termed “A2”) is the cell surface coreceptor for plasminogen and TPA and accelerates the catalytic activation of plasmin, the major fibrinolytic agent in mammals. We previously showed that HC prevents A2-mediated, TPA-dependent activation of plasminogen in vitro by disulfide derivatization of the “tail” domain of A2. We also demonstrated that fibrinolysis and angiogenesis are severely impaired in A2-deficient mice. We now report here that, although hyperhomocysteinemic mice had a normal coagulation profile and normal platelet function, fibrin accumulated in their tissues due to reduced perivascular fibrinolytic activity and angiogenesis was impaired. A2 isolated from hyperhomocysteinemic mice failed to fully support TPA-dependent plasmin activation. However, infusion of hyperhomocysteinemic mice with fresh recombinant A2, which localized to neoangiogenic endothelial cells, resulted in normalization of angiogenesis and disappearance of peri- and intravascular fibrin. We therefore conclude that hyperhomocysteinemia impairs postnatal angiogenesis by derivatizing A2, preventing perivascular fibrinolysis, and inhibiting directed endothelial cell migration. These findings provide a mechanistic explanation for microvascular dysfunction and macrovascular occlusion in individuals with hyperhomocysteinemia.
Authors
Andrew T. Jacovina, Arunkumar B. Deora, Qi Ling, M. Johan Broekman, Dena Almeida, Caroline B. Greenberg, Aaron J. Marcus, Jonathan D. Smith, Katherine A. Hajjar
Abstract
Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4–/– mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene–knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.
Authors
Sophia Bornstein, Ruth White, Stephen Malkoski, Masako Oka, Gangwen Han, Timothy Cleaver, Douglas Reh, Peter Andersen, Neil Gross, Susan Olson, Chuxia Deng, Shi-Long Lu, Xiao-Jing Wang
Abstract
Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain–binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.
Authors
Qi Shen, Shawn C. Little, Meiqi Xu, Julia Haupt, Cindy Ast, Takenobu Katagiri, Stefan Mundlos, Petra Seemann, Frederick S. Kaplan, Mary C. Mullins, Eileen M. Shore
Abstract
The heterogeneous cellular composition of the mammalian renal collecting duct enables regulation of fluid, electrolytes, and acid-base homeostasis, but the molecular mechanism of its development has yet to be elucidated. The Notch signaling pathway is involved in cell fate determination and has been implicated in proximal-distal patterning in the mammalian kidney. To investigate the role of Notch signaling in renal collecting duct development, we generated mice in which Mind bomb-1 (Mib1), an E3 ubiquitin ligase required for the initiation of Notch signaling, was specifically inactivated in the ureteric bud of the developing kidney. Mice lacking Mib1 in the renal collecting duct displayed increased urinary production, decreased urinary osmolality, progressive hydronephrosis, sodium wasting, and a severe urinary concentrating defect manifested as nephrogenic diabetes insipidus. Histological analysis revealed a diminished number of principal cells and corresponding increase in the number of intercalated cells. Transgenic overexpression of Notch intracellular domain reversed the altered cellular composition of mutant renal collecting duct, with principal cells occupying the entire region. Our data demonstrate that Notch signaling is required for the development of the mammalian renal collecting duct and principal cell differentiation and indicate that pathway dysregulation may contribute to distal renal tubular disorders.
Authors
Hyun-Woo Jeong, Un Sil Jeon, Bon-Kyoung Koo, Wan-Young Kim, Sun-Kyoung Im, Juhee Shin, Yunje Cho, Jin Kim, Young-Yun Kong
Abstract
Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1+/–;Chd7+/– double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.
Authors
Victoria Randall, Karen McCue, Catherine Roberts, Vanessa Kyriakopoulou, Sarah Beddow, Angela N. Barrett, Francesca Vitelli, Katrina Prescott, Charles Shaw-Smith, Koen Devriendt, Erika Bosman, Georg Steffes, Karen P. Steel, Subreena Simrick, M. Albert Basson, Elizabeth Illingworth, Peter J. Scambler
Abstract
Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes. Given that dysregulation of intracellular calcium and reactive species has been described in patients with atrial fibrillation, we investigated the role of DCT in this process. Here, we characterize a unique DCT-expressing cell population within murine and human hearts that populated the pulmonary veins, atria, and atrioventricular canal. Expression profiling demonstrated that this population expressed adrenergic and muscarinic receptors and displayed transcriptional profiles distinct from dermal melanocytes. Adult mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias. Cultured primary cardiac melanocyte-like cells were excitable, and those lacking DCT displayed prolonged repolarization with early afterdepolarizations. Furthermore, mice with mutations in the tyrosine kinase receptor Kit lacked cardiac melanocyte-like cells and did not develop atrial arrhythmias in the absence of DCT. These data suggest that dysfunction of melanocyte-like cells in the atrium and pulmonary veins may contribute to atrial arrhythmias.
Authors
Mark D. Levin, Min Min Lu, Nataliya B. Petrenko, Brian J. Hawkins, Tara H. Gupta, Deborah Lang, Peter T. Buckley, Jeanine Jochems, Fang Liu, Christopher F. Spurney, Li J. Yuan, Jason T. Jacobson, Christopher B. Brown, Li Huang, Friedrich Beermann, Kenneth B. Margulies, Madesh Muniswamy, James H. Eberwine, Jonathan A. Epstein, Vickas V. Patel
Abstract
Mutations in the neuronal protein α-synuclein cause familial Parkinson disease. Phosphorylation of α-synuclein at serine 129 is prominent in Parkinson disease and influences α-synuclein neurotoxicity. Here we report that α-synuclein is also phosphorylated at tyrosine 125 in transgenic Drosophila expressing wild-type human α-synuclein and that this tyrosine phosphorylation protects from α-synuclein neurotoxicity in a Drosophila model of Parkinson disease. Western blot analysis of fly brain homogenates showed that levels of soluble oligomeric species of α-synuclein were increased by phosphorylation at serine 129 and decreased by tyrosine 125 phosphorylation. Tyrosine 125 phosphorylation diminished during the normal aging process in both humans and flies. Notably, cortical tissue from patients with the Parkinson disease–related synucleinopathy dementia with Lewy bodies showed less phosphorylation at tyrosine 125. Our findings suggest that α-synuclein neurotoxicity in Parkinson disease and related synucleinopathies may result from an imbalance between the detrimental, oligomer-promoting effect of serine 129 phosphorylation and a neuroprotective action of tyrosine 125 phosphorylation that inhibits toxic oligomer formation.
Authors
Li Chen, Magali Periquet, Xu Wang, Alessandro Negro, Pamela J. McLean, Bradley T. Hyman, Mel B. Feany
Abstract
The relative balance between the quantity of white and brown adipose tissue can profoundly affect lipid storage and whole-body energy homeostasis. However, the mechanisms regulating the formation, expansion, and interconversion of these 2 distinct types of fat remain unknown. Recently, the lysosomal degradative pathway of macroautophagy has been identified as a regulator of cellular differentiation, suggesting that autophagy may modulate this process in adipocytes. The function of autophagy in adipose differentiation was therefore examined in the current study by genetic inhibition of the critical macroautophagy gene autophagy-related 7 (Atg7). Knockdown of Atg7 in 3T3-L1 preadipocytes inhibited lipid accumulation and decreased protein levels of adipocyte differentiation factors. Knockdown of Atg5 or pharmacological inhibition of autophagy or lysosome function also had similar effects. An adipocyte-specific mouse knockout of Atg7 generated lean mice with decreased white adipose mass and enhanced insulin sensitivity. White adipose tissue in knockout mice had increased features of brown adipocytes, which, along with an increase in normal brown adipose tissue, led to an elevated rate of fatty acid, β-oxidation, and a lean body mass. Autophagy therefore functions to regulate body lipid accumulation by controlling adipocyte differentiation and determining the balance between white and brown fat.
Authors
Rajat Singh, Youqing Xiang, Yongjun Wang, Kiran Baikati, Ana Maria Cuervo, Yen K. Luu, Yan Tang, Jeffrey E. Pessin, Gary J. Schwartz, Mark J. Czaja
Abstract
West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.
Authors
Marion C. Lanteri, Katie M. O’Brien, Whitney E. Purtha, Mark J. Cameron, Jennifer M. Lund, Rachel E. Owen, John W. Heitman, Brian Custer, Dale F. Hirschkorn, Leslie H. Tobler, Nancy Kiely, Harry E. Prince, Lishomwa C. Ndhlovu, Douglas F. Nixon, Hany T. Kamel, David J. Kelvin, Michael P. Busch, Alexander Y. Rudensky, Michael S. Diamond, Philip J. Norris
Abstract
Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8– fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-α. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease.
Authors
Michael K. Connolly, Andrea S. Bedrosian, Jon Mallen-St. Clair, Aaron P. Mitchell, Junaid Ibrahim, Andrea Stroud, H. Leon Pachter, Dafna Bar-Sagi, Alan B. Frey, George Miller
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ISSN: 0021-9738 (print), 1558-8238 (online)