Search

Background: Developmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished., Methods: We applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal-to-adult transition. A bioinformatics pipeline was developed to identify stable, neonatal-restricted platelet biomarkers for clinical application., Results: Cord blood (CB) platelets retained the capacity for linear agonist-receptor coupling linked to phosphatidylserine (PS) exposure and α-granule release, although a restricted block in cross-agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c-Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome-protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB- or adult-derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5-fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at-risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age-dependent expression., Conclusions: These studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal-to-adult megakaryocytopoiesis in vivo., (© 2022 International Society on Thrombosis and Haemostasis.)

Inflammatory stimuli have divergent effects on peripheral platelet counts, although the mechanisms of thrombocytopenic and thrombocytotic responses remain poorly understood. A candidate gene approach targeting 326 polymorphic genes enriched in thrombopoietic and cytokine signaling pathways was applied to identify single nucleotide variants (SNVs) implicated in enhanced platelet responses in cohorts with reactive thrombocytosis (RT) or essential (myeloproliferative neoplasm [MPN]) thrombocytosis (ET). Cytokine profiles incorporating a 15-member subset, pathway topology, and functional interactive networks were distinct between ET and RT, consistent with distinct regulatory pathways of exaggerated thrombopoiesis. Genetic studies using aggregate (ET + RT) or ET-restricted cohorts identified associations with 2 IFNA16 (interferon-α16) SNVs, and the ET associations were validated in a second independent cohort (P = .0002). Odds ratio of the combined ET cohort (n = 105) was 4.92, restricted to the JAK2V617F-negative subset (odds ratio, 5.01). ET substratification analysis by variant IFNA16 exhibited a statistically significant increase in IFN-α16 levels (P = .002) among 16 quantifiable cytokines. Recombinantly expressed variant IFN-α16 encompassing 3 linked non-synonymous SNVs (E65H95P133) retained comparable antiviral and pSTAT signaling profiles as native IFN-α16 (V65D95A133) or IFN-α2, although both native and variant IFN-α16 showed stage-restricted differences (compared with IFN-α2) of IFN-regulated genes in CD34+-stimulated megakaryocytes. These data implicate IFNA16 (IFN-α16 gene product) as a putative susceptibility locus (driver) within the broader disrupted cytokine network evident in MPNs, and they provide a framework for dissecting functional interactive networks regulating stress or MPN thrombopoiesis., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Genetic pathways regulating hematopoietic lineage commitment at critical stages of development remain incompletely characterized.  To better delineate genetic sources of variability regulating cellular speciation during steady-state hematopoiesis, we applied a factorial single-cell latent variable model (f-scLVM) to decompose single-cell transcriptome heterogeneity into interpretable biological factors (refined pathway annotations or gene sets without annotation) dynamically regulating cell fate.  Hematopoietic single cell transcriptomic raw sequencing data extracted from 1,920 hematopoietic stem and progenitor cells (HSPCs) derived from 12-week-old female mice were used for data analysis and model development. These single cell RNA sequencing data were subsequently analyzed using the factorial single-cell latent variable model (f-scLVM), with their heterogeneity decomposed into interpretable biological factors. The top biological factors underlying the basal hematopoiesis were subsequently identified for the aggregate, and lineage-restricted (myeloid, megakaryocyte, erythroid) progenitor cells. For a subset of factors, data were independently verified experimentally in a companion research paper [1]. These data facilitate the identification of novel subpopulations and adjust gene sets to discover new marker genes and hidden confounding factors driving basal hematopoiesis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article., (© 2021 The Author(s). Published by Elsevier Inc.)

Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/β dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXβ tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts., (© 2016 by The American Society of Hematology.)

IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer.

It is broadly accepted that genetically engineered animal models do not always recapitulate human pathobiology. Therefore identifying best-fit mouse models of human cancers that truly reflect the corresponding human disease is of vital importance in elucidating molecular mechanisms of tumorigenesis and developing preventive and therapeutic approaches. A new hepatocellular carcinoma (HCC) mouse model lacking a novel putative tumor suppressor IQGAP2 has been generated by our laboratory. The aim of this study was to obtain the molecular signature of Iqgap2(-/-) HCC tumors and establish the relevance of this model to human disease. Here we report a comprehensive transcriptome analysis of Iqgap2(-/-) livers and a cross-species comparison of human and Iqgap2(-/-) HCC tumors using Significance Analysis of Microarray (SAM) and unsupervised hierarchical clustering analysis. We identified the Wnt/β-catenin signaling pathway as the top canonical pathway dysregulated in Iqgap2(-/-) livers. We also demonstrated that Iqgap2(-/-) hepatic tumors shared genetic signatures with HCC tumors from patients with advanced disease as evidenced by a 78% mouse-to-human microarray data set concordance rate with 117 out of 151 identified ortholog genes having similar expression profiles across the two species. Collectively, these results indicate that the Iqgap2 knockout mouse model closely recapitulates human HCC at the molecular level and supports its further application for the study of this disease.

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of (5')AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.

Adeno-associated virus (AAV) is a single-stranded parvovirus retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3' cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints.

Background: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC., Methods: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA., Results: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6)., Conclusions: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V(617)F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V(617)F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the importance of parallel transcriptomic studies to optimally dissect platelet function. Applications of these global profiling approaches to investigate platelet genetic diseases and platelet-related disorders are also addressed.

Objective: Two major protease-activated receptors (PARs), PAR1 and PAR4, are involved in the activation of human platelets by thrombin. A third, PAR3, is preferentially expressed by tissues of hematopoietic origin and megakaryocytes. Although PAR3 is also a thrombin substrate, its low-level expression on human platelets suggests a function distinct from that of PAR1, the major receptor involved in thrombin-mediated platelet activation. We studied the expression of PARs during megakaryocyte differentiation of human erythroleukemia (HEL) cells in order to determine the role of PAR3 in megakaryocytopoiesis., Methods: HEL cells exposed to phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation were examined by light microscopy and flow cytometry (DNA ploidy, surface expression of PAR1, PAR3, GPIIb-IIIa). Northern blot, RT-PCR, and quantitative RT-PCR were used to evaluate the expression of PARs 1, 3, and 4 mRNA. HEL cells were also exposed to thrombin and thrombopoietin (TPO)., Results: In baseline studies, unstimulated HEL cells were found to express comparable levels of PAR1 and PAR3 by Northern blot. Minimal expression of PAR4 was detected by RT-PCR, but not by Northern analysis. Exposure to PMA, but not thrombin or TPO, resulted in megakaryocytic differentiation as evident by increased cell size and nuclear complexity, increased ploidy, and enhanced expression of GPIIb-IIIa, a specific marker of megakaryocytes/platelets. PMA-stimulated HEL cells showed enhanced PAR3 cell-surface expression (approximately threefold increase by day 2) by flow cytometry. In contrast, there was no change in cell-surface PAR1 expression. Northern blot analysis (approximately 10-fold) and quantitative RT-PCR (approximately threefold) confirmed the upregulation of PAR3 mRNA expression (by 24 hours) in cells exposed to PMA. This did not occur with exposure to TPO., Conclusion: These data demonstrate increased expression of PAR3 mRNA and protein in HEL cells undergoing megakaryocytic maturation following PMA exposure, suggesting a developmental role for PAR3. Furthermore, regulation of PAR3 expression appears to be specifically coupled to the protein kinase C system, but independent of the Ras/Raf/MAP kinase pathway.

Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HG-U95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating 21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags represented mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. Quantitative reverse transcription-polymerase chain reaction (PCR) confirmed the highest frequency of mt-derived transcripts, along with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and the complement lysis inhibitor clusterin among the top 5 most abundant transcripts. For confirmatory characterization, immunoblots and flow cytometric analyses were performed, establishing abundant cell-surface expression of clusterin and intracellular expression of NGN. These observations demonstrate a strong correlation between high transcript abundance and protein expression, and they establish the validity of transcript analysis as a tool for identifying novel platelet proteins that may regulate normal and pathologic platelet (and/or megakaryocyte) functions.

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78- or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.