Publications by authors named "Madhu M Ouseph"

18 Publications

  • Page 1 of 1

EGCG, a Green Tea Catechin, as a Potential Therapeutic Agent for Symptomatic and Asymptomatic SARS-CoV-2 Infection.

Molecules 2021 Feb 24;26(5). Epub 2021 Feb 24.

Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, WA 99202, USA.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules26051200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956763PMC
February 2021

Guanylate Binding Protein 5 (GBP5) regulates synovial fibroblast mediated inflammation and tissue destruction in rheumatoid arthritis.

Arthritis Rheumatol 2020 Dec 5. Epub 2020 Dec 5.

Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, Washington, United States.

Objective: Rheumatoid arthritis synovial fibroblasts (RASFs) are crucial mediators of synovial inflammation and joint destruction. However, their intrinsic immunoregulatory mechanisms under chronic inflammation remain unclear. Thus, present study was aimed to understand the role of newly identified GTPase, guanylate-binding protein 5 (GBP5) in RA pathogenesis.

Methods: The expression of GBP1-GBP7 transcripts was evaluated using qRT-PCR in RA synovial tissues (RASTs) or non-diseased STs (NLSTs). Transient siRNA knockdown and lentiviral overexpression studies in human RASFs examined the regulatory role of GBP5 on proinflammatory cytokine signaling pathways. Unbiased whole transcriptome RNA-sequencing analysis examined the impact of GBP5 on RASF molecular functions. These findings were confirmed using in vivo rat model of adjuvant-induced arthritis (AIA).

Results: Among different GBPs evaluated, GBP5 was selectively upregulated in RASTs (p<0.05; n=4) and the joints of AIA rats (p<0.05; n=6), and was significantly induced in human RASFs by IL-1β, TNF-α and/or IFN-γ (p<0.05; n=3). Bioinformatics analysis of RNA-sequencing data identified cytokine-cytokine receptor signaling as a major function altered by GBP5, with IL-6 signaling as a primary target. Knockdown of GBP5 amplified IL-1β-induced IL-6, IL-8, ENA-78/ CXCL5 by 44%, 54%, 45%, respectively, and MMP-1 production by several-folds, effects which reversed with exogenously delivered GBP5. Lack of GBP5 increased IFN-γ-induced proliferation and migration in human RASFs. In vivo GBP5 knockdown using intra-articular siRNA exacerbated disease onset, severity, synovitis, and bone destruction in AIA.

Conclusion: Expressed by RASFs in response to cytokine stimulation, GBP5 has potential to restore cellular homeostasis and blunt inflammation and tissue destruction in RA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/art.41611DOI Listing
December 2020

Single cell RNA-seq reveals developmental plasticity with coexisting oncogenic and immune evasion programs in ETP-ALL.

Blood 2020 Nov 23. Epub 2020 Nov 23.

Boston Children's Hospital, United States.

Lineage plasticity and stemness have been invoked as the cause of therapy resistance in cancer, as these flexible states allow cancer cells to de-differentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed / refractory early T-cell progenitor acute lymphoblastic leukemia carrying activating NOTCH1 mutations, by full-length single cell RNA sequencing of malignant and microenvironmental cells. We identified two highly distinct stem-like states that critically differ in their cell-cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrate Notch activation and are effectively eliminated in patients by Notch inhibition, while slow cycling stem-like cells are Notch-independent but rather rely on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we find that both stem-like states can differentiate into a more mature leukemia state with prominent immune-modulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promote an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that express HAVCR2, the cognate receptor for LGALS9. Our study identifies complex interactions between signaling programs, cellular plasticity and immune programs that characterize T-ALL and illustrates the multi-dimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem appear most promising to successfully eliminate tumor cells that escape treatment through co-existing transcriptional programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019004547DOI Listing
November 2020

Everolimus in the treatment of metastatic thymic epithelial tumors.

Lung Cancer 2020 11 17;149:97-102. Epub 2020 Sep 17.

Stanford Cancer Institute/Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Introduction: There is emerging evidence to support the use of mTOR inhibitor everolimus in patients with advanced, relapsed-refractory thymic epithelial tumors (TETs). However, patient selection and identifying predictive biomarkers of response remains a challenge. Here, we describe a single-center experience with everolimus in patients with TETs and provide detailed molecular analysis of their thymic tumors.

Materials And Methods: Data on all patients with advanced TETs who were prescribed everolimus at Stanford University were retrospectively assessed. Time to treatment failure (TTF) and overall survival (OS) were calculated. STAMP, a 130-gene targeted next generation sequencing (NGS) panel, was performed on each tumor sample.

Results: Twelve patients with thymoma (T) and three with thymic carcinoma (TC) treated with everolimus were included. Patients had been heavily pre-treated with an average of three prior lines of therapy. Three patients discontinued treatment due to adverse events. The average TTF was 14.7 months in T and 2.6 months in TC with median OS of 27.6 months in the entire cohort (NR T and 5.3 months TC). Two patients with paraneoplastic autoimmune diseases had improvement in autoimmunity on everolimus. Pathogenic mutations were observed in 4/15 (27 %) of patients and includedTP53, KEAP1 and CDKN2A. Several variants of unknown significance in key genes responsible for modulating tumor response to mTOR inhibition were also found.

Conclusion: As previously reported in a prospective trial, patients with previously treated advanced TETs appear to benefit from everolimus in this single institution cohort. Moreover, there was a manageable toxicity profile and no cases of everolimus-induced pneumonitis. A targeted NGS panel revealed several pathogenic mutations but there was no association between detectable tumor mutations and time to treatment failure in this cohort.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lungcan.2020.09.006DOI Listing
November 2020

Genomic alterations in patients with somatic loss of the Y chromosome as the sole cytogenetic finding in bone marrow cells.

Haematologica 2021 Feb 1;106(2):555-564. Epub 2021 Feb 1.

Department of Pathology, Boston Children Hospital, Boston, USA.

Loss of the Y chromosome (LOY) is one of the most common somatic genomic alterations in hematopoietic cells in men. However, due to the high prevalence of LOY as the sole cytogenetic finding in the healthy older population, differentiating isolated LOY associated with clonal hematologic processes from aging-associated mosaicism can be difficult in the absence of definitive morphological features of disease. In the past, various investigators have proposed that a high percentage of metaphases with LOY is more likely to represent expansion of a clonal myeloid disease-associated population. It is unknown whether the proportion of metaphases with LOY is associated with the incidence of myeloid neoplasia-associated genomic alterations. To address this question, we identified marrow samples with LOY as isolated cytogenetic finding and used targeted next generation sequencing-based molecular analysis to identify common myeloid neoplasia-associated somatic mutations. Among 73 patients with median age of 75 years (range 29-90), the percentage of metaphases with LOY was <25% in 23 patients, 25-49% in 10, 50-74% in 8 and ≥75% in 32. A threshold of ≥75% LOY was significantly associated with morphologic diagnosis of myeloid neoplasm (p = 0.004). Further, ≥75% LOY was associated with a higher lifetime incidence of diagnosis of myelodysplastic syndromes (MDS; p < 0.0001), and in multivariate analysis ≥75% LOY was a statistically significant independent predictor of myeloid neoplasia [OR 6.17; 95% CI = 2.15-17.68; p = 0.0007]. Higher LOY percentage (≥75%) was associated with greater likelihood of having somatic mutations (p = 0.0009) and a higher number of these mutations (p = 0.0002). Our findings indicate that ≥75% LOY in marrow is associated with increased likelihood of molecular alterations in genes commonly seen in myeloid neoplasia and with morphologic features of MDS. These observations suggest that ≥75% LOY in bone marrow should be considered an MDS-associated cytogenetic aberration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.240689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849577PMC
February 2021

TKI-resistant -rearranged lung adenocarcinoma with secondary CTNNB1 p.S45V and tertiary ALK p.I1171N mutations.

Lung Cancer (Auckl) 2019 15;10:81-86. Epub 2019 Aug 15.

Department of Pathology, Rhode Island Hospital and Alpert Medical School at Brown University, Providence, RI 02903, USA.

Anaplastic lymphoma kinase ()-rearranged non-small cell lung cancer (NSCLC) is an important molecular subgroup of tumors that are typically sensitive to tyrosine kinase inhibitors (TKIs). Although a substantial portion of patients benefit from TKIs, this approach is complicated by intrinsic and acquired resistance. We report a patient with -rearranged NSCLC who showed an initial response to targeted therapy, but developed resistance to multiple TKIs. Serial comprehensive genomic profiling (CGP) was performed at four independent points during the clinical course. We review the pathology and clonal progression of the tumor, with CGP identifying a secondary CTNNB1 p.S45V mutation after the initiation of targeted therapy, followed by tertiary ALK p.I1171N. The presence of an alteration in a second oncogenic driver gene suggests a possible mechanism for resistance, and a secondary therapeutic target. Due to the involvement of Wnt signaling in the pathogenesis of many tumors and its association with immune evasion, a variety of therapeutic strategies are being developed to target this pathway. This case exemplifies the challenges of targeted therapeutics in the face of tumor progression, as well as the increasing role of genomics in understanding tumor biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2147/LCTT.S212406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699522PMC
August 2019

Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-β-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system.

Cell Mol Immunol 2021 Jan 11;18(1):162-170. Epub 2019 Sep 11.

Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, WA, USA.

Monosodium urate (MSU) crystals activate inflammatory pathways that overlap with interleukin-1β (IL-1β) signaling. However, the post-translational mechanisms involved and the role of signaling proteins in this activation are unknown. In the present study, we investigated the intracellular signaling mechanisms involved in MSU-induced activation of THP-1 macrophages and human nondiseased synovial fibroblasts (NLSFs) and the in vivo efficacy of an inhibitor of tumor growth factor-β (TGF-β)-activated kinase 1 (TAK1), 5Z-7-oxozeaenol, in MSU-induced paw inflammation in C57BL/6 mice. THP-1 macrophage activation with MSU crystals (25-200 µg/ml) resulted in the rapid and sustained phosphorylation of interleukin-1 receptor-activated kinase 1 (IRAK1 Thr) and TAK1 (Thr) and their association with the E3 ubiquitin ligase TRAF6. At the cellular level, MSU inhibited the deubiquitinases A20 and UCHL2 and increased 20s proteasomal activity, leading to a global decrease in K-linked ubiquitination and increase in K-linked ubiquitination in THP-1 macrophages. While MSU did not stimulate cytokine production in NLSFs, it significantly amplified IL-1β-induced IL-6, IL-8, and ENA-78/CXCL5 production. Docking studies and MD simulations followed by TAK1 in vitro kinase assays revealed that uric acid molecules are capable of arresting TAK1 in an active-state conformation, resulting in sustained TAK1 kinase activation. Importantly, MSU-induced proinflammatory cytokine production was completely inhibited by 5Z-7-oxozeaenol but not IRAK1/4 or TRAF6 inhibitors. Administration of 5Z-7-oxozeaenol (5 or 15 mg/kg; orally) significantly inhibited MSU-induced paw inflammation in C57BL/6 mice. Our study identifies a novel post-translational mechanism of TAK1 activation by MSU and suggests the therapeutic potential of TAK1 in regulating MSU-induced inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41423-019-0284-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853128PMC
January 2021

- An Uncommon but Noteworthy Cause of Intrauterine Fetal Demise and Acute Necrotizing Funisitis.

Fetal Pediatr Pathol 2019 Aug 3;38(4):352-358. Epub 2019 Apr 3.

b Warren Alpert Medical School of Brown University , Providence , RI , USA.

: (. ) is an uncommon cause of amniotic fluid infection and intrauterine fetal demise. : A 39-year-old G8P2052 presented with preterm premature rupture of membrane at 22 weeks gestation and had a spontaneous vaginal delivery of a neonate who soon expired. Placental examination revealed retroplacental hematoma, acute necrotizing chorioamnionitis, acute three-vessel vasculitis and necrotizing funisitis of the umbilical cord. Postmortem examination demonstrated features of amniotic fluid infection syndrome with blood culture growing Antenatal screening does not typically quantify . infection, but small series have found vaginal colonization in fewer than 1% of women. Intrauterine or peritoneal infection derives primarily from ascending infection although other routes are hypothetically possible. Intra-amniotic and neonatal infections by are associated with high morbidity and mortality. : should be considered in perinatal death of immature fetus with severe amniotic fluid infection syndrome and acute necrotizing funisitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15513815.2019.1587121DOI Listing
August 2019

Autophagy in Platelets.

Methods Mol Biol 2019 ;1880:511-528

Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY, USA.

Anucleate platelets are produced by fragmentation of megakaryocytes. Platelets circulate in the bloodstream for a finite period: upon vessel injury, they are activated to participate in hemostasis; upon senescence, unused platelets are cleared. Platelet hypofunction leads to bleeding. Conversely, pathogenic platelet activation leads to occlusive events that precipitate strokes and heart attacks. Recently, we and others have shown that autophagy occurs in platelets and is important for platelet production and normal functions including hemostasis and thrombosis. Due to the unique properties of platelets, such as their lack of nuclei and their propensity for activation, methods for studying platelet autophagy must be specifically tailored. Here, we describe useful methods for examining autophagy in both human and mouse platelets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8873-0_32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039316PMC
June 2019

Endoreduplication of the mouse genome in the absence of ORC1.

Genes Dev 2018 07;32(13-14):978-990

Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.311910.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075035PMC
July 2018

Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation.

Oncogene 2018 08 3;37(32):4428-4442. Epub 2018 May 3.

Department of Pathology, Ohio State University, Columbus, OH, 43210, USA.

The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-018-0263-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063845PMC
August 2018

Fatal Sepsis in a Patient With Celiac Disease-Associated Hyposplenism.

ACG Case Rep J 2016 Aug 12;3(4):e140. Epub 2016 Oct 12.

Department of Pathology and Laboratory Medicine, Alpert Medical School of Brown University and Rhode Island Hospital, Providence, RI.

We present a 59-year-old male with poorly controlled celiac disease (CD) and fatal sepsis, describe the morphologic findings, and stress the need for monitoring splenic function and pneumococcal vaccination in these patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14309/crj.2016.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064423PMC
August 2016

Systemic Amyloidosis Masquerading as Intractable Cardiomyopathy.

R I Med J (2013) 2016 Oct 4;99(10):54-56. Epub 2016 Oct 4.

Associate Professor of Diagnostic Imaging and Associate Professor of Medicine, Department of Diagnostic Imaging, Alpert Medical School of Brown University, Providence, RI.

View Article and Find Full Text PDF

Download full-text PDF

Source
October 2016

Can Sentinel Lymph Node Biopsy Be Spared in Papillary Carcinoma of the Breast?

Clin Breast Cancer 2017 04 8;17(2):127-133. Epub 2016 Sep 8.

Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Lifespan Medical Center, The Warren Alpert Medical School of Brown University, Providence, RI.

Background: Papillary carcinoma (PC) of the breast represents ∼0.5% of all newly diagnosed cases of breast cancer and usually has an indolent course. The current data suggest lack of a consensus in the surgical management of this disease. Because patients can occasionally develop metastatic disease, sentinel lymph node (SLN) biopsy is often performed during surgery.

Materials And Methods: In the present study, we retrospectively evaluated the histologic characteristics of 99 cases of PC with or without associated frank invasive carcinoma, including 43 encapsulated or intracystic PCs, 24 solid PCs, and 32 intraductal PCs, and correlated these with the incidence of nodal metastasis.

Results: Of the 99 cases, 64 were tumor stage Tis (noninvasive), 5 were T1 microinvasive, 17 T1a, 5 T1b, 5 T1c, and 3 were T2. A total of 37 patients (37%) underwent axillary staging, including 31 SLN biopsies and 6 axillary dissections. Only 1 patient (2.7%) with noninvasive solid PC had evidence of nodal metastasis. Follow-up information was available for 81 patients, with a mean follow-up period of 4.9 years (range, 1-13 years). Two local recurrences, no distant metastases, and no disease-related deaths were recorded.

Conclusion: PC rarely involves the lymph nodes even in tumors with an associated frank invasive component, and the overall prognosis and long-term survival is excellent. We propose that evaluation of the SLN should not be routinely indicated for patients with PC treated by local control lumpectomy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.clbc.2016.08.009DOI Listing
April 2017

Vision loss in juvenile neuronal ceroid lipofuscinosis (CLN3 disease).

Ann N Y Acad Sci 2016 05 8;1371(1):55-67. Epub 2016 Jan 8.

Department of Molecular and Cellular Biochemistry.

Juvenile neuronal ceroid lipofuscinosis (JNCL; also known as CLN3 disease) is a devastating neurodegenerative lysosomal storage disorder and the most common form of Batten disease. Progressive visual and neurological symptoms lead to mortality in patients by the third decade. Although ceroid-lipofuscinosis, neuronal 3 (CLN3) has been identified as the sole disease gene, the biochemical and cellular bases of JNCL and the functions of CLN3 are yet to be fully understood. As severe ocular pathologies manifest early in disease progression, the retina is an ideal tissue to study in the efforts to unravel disease etiology and design therapeutics. There are significant discrepancies in the ocular phenotypes between human JNCL and existing murine models, impeding investigations on the sequence of events occurring during the progression of vision impairment. This review focuses on current understanding of vision loss in JNCL and discusses future research directions toward molecular dissection of the pathogenesis of the disease and associated vision problems in order to ultimately improve the quality of patient life and cure the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nyas.12990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025599PMC
May 2016

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis.

Blood 2015 Sep 24;126(10):1224-33. Epub 2015 Jul 24.

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY;

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2014-09-598722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559933PMC
September 2015

Canonical and atypical E2Fs regulate the mammalian endocycle.

Nat Cell Biol 2012 Nov 14;14(11):1192-202. Epub 2012 Oct 14.

Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Department of Molecular Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.

The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb2595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616487PMC
November 2012

Atypical E2F repressors and activators coordinate placental development.

Dev Cell 2012 Apr;22(4):849-62

Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA.

The evolutionarily ancient arm of the E2f family of transcription factors consisting of the two atypical members E2f7 and E2f8 is essential for murine embryonic development. However, the critical tissues, cellular processes, and molecular pathways regulated by these two factors remain unknown. Using a series of fetal and placental lineage-specific cre mice, we show that E2F7/E2F8 functions in extraembryonic trophoblast lineages are both necessary and sufficient to carry fetuses to term. Expression profiling and biochemical approaches exposed the canonical E2F3a activator as a key family member that antagonizes E2F7/E2F8 functions. Remarkably, the concomitant loss of E2f3a normalized placental gene expression programs, corrected placental defects, and fostered the survival of E2f7/E2f8-deficient embryos to birth. In summary, we identified a placental transcriptional network tightly coordinated by activation and repression through two distinct arms of the E2F family that is essential for extraembryonic cell proliferation, placental development, and fetal viability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2012.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483796PMC
April 2012