Mediator subunits: Gene expression pattern, a novel transcript identification and nuclear localization in human endothelial progenitor cells

Abstract Abstract 997 Growth Factor Independence 1 (GFI1) is a transcriptional repressor, which plays an important role in myeloid differentiation. We have recently described a Single Nucleotide Polymorphism (SNP) in the GFI1 gene that is found in 15% of all AML patients and increases risk of development of acute myeloid leukemia (AML) in homozygous or heterozygous carriers of this variant. The SNP generates a GFI1 variant that carries an Asparagine residue instead of a Serine at amino acid position 36 (GFI136N). To better understand how this variant might cause leukmemia, we replaced the murine Gfi1 locus with the common form of human GFI1 (denominated GFI36S) or the variant form (GFI136N) using a previously described knockin strategy. Replacement of murine Gfi1 by human GFI136S did not affect lymphoid or myeloid differentiation demonstrating that “knocked-in” human GFI1 (Gfi1hu36S/hu36S) can fully substitute the endogenous murine gene. However, mice homozygous for the variant GFI136N allele (Gfi1hu36N/hu36N) featured a 3-fold increase in the number of both Common myeloid progenitors (CMPs) and Granulocytic monocytic progenitors (GMPs) compared with wt or Gfi1hu36S/hu36S mice (p’0.001). In addition, in the GMP fraction from Gfi1hu36N/hu36N mice more dividing cells (20 %) were found compared to the GMP fraction originating from wt or Gfi1hu36S/hu36S mice (p≤0.01). Moreover, we observed a 3-fold increased expression of the proleukemogenic transcription factors Hoxa9 and Pbx1 in GMPs from Gfi1hu36N/hu36N compared to wt or Gfi1hu36S/hu36S animals (p≤0.01). This increased level of Hoxa9 would be consistent with recent publications showing that Gfi1 is required for the repression of Hoxa9 expression in GMP/CMP subsets. Interestingly, AML patients heterozygous for the GFI136N variant showed a tendency toward a 2-fold higher expression of the Hoxa9 gene when compared with GFI136S homozygous patients. To explain how the presence of the variant might predispose to leukemia, we performed a gene expression array of GMPs from Gfi1hu36N/hu36N, wt mice, Gfi1ko/ko or Gfi1hu36S/hu36S mice. Whereas wt and Gfi1hu36S/hu36S mice showed a similar gene expression pattern, the presence of the GFI136N variant led to a reprogramming of the GMP population with dysregulation of genes known to be involved in AML such as Idh1, Tet2 and Trib1. The gene expression pattern was also distinct from the pattern of sorted Gfi1ko/ko GMPs, pointing to a specific role of GFI136N in reprogramming the progenitor fraction and thus predisposing to AML. Interestingly, some of the genes differentially regulated between Gfi1hu36N/hu36N GMPs and the other GMPs were also differentially expressed between GFI36S patients and GFI136N heterozygous AML patients. We examined the biochemical features of the two Gfi1 variants in order to explain how the variant might reprogramm the GMPs and predispose to AML. Both GFI136N and GFI136S are still able to bind to previously published GFI1interaction partners such as PU.1 or CEBPe. We observed that GFI136N exhibited a different intranuclear localization than GFI136S. Whereas murine Gfi1 and human GFI136S localize in dots within the nucleus, GFI136N is mainly found along the nuclear border suggesting that nuclear localization is important for Gfi1 function. This was in accordance with a previously reported AML patient homozygous for GFI136N who also exhibited a similar aberrant nuclear localization. Finally to study a potential impact of GFI136N on myeloid leukemogenesis, we used an established mouse model in which expression of a mutated form of KRAS (K12D) can be induced causing a 100% penetrance of an AML like disease. GFI136N significantly accelerated the onset of KRAS driven leukemia in these mice which was associated with a higher number of blast cells in the bone marrow, blood and spleen (p’0.05 for blast number and latency). These data suggest that the GFI136N variant predisposes to the development of AML since it is inefficient in repressing Hoxa9 owing to an aberrant subnuclear localization. To our knowledge this is the first knock-in of a human protein variant predisposing to AML and this mouse model could help developing a targeted therapy for these patients. Disclosures: No relevant conflicts of interest to declare.

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Effect of Interval Training Intensity on Gene Expression of Endothelial Progenitor Cells and Cardiac Stem Cells in Aged Rats

journal of ilam university of medical sciences ◽

10.29252/sjimu.26.3.27 ◽

2018 ◽

Vol 26 (3) ◽

pp. 27-37

Author(s):

Sharif Rezaei ◽

Hassan Matinhomaee ◽

Mohammad ali Azarbayjani ◽

Parvin Farzanegi ◽

◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Interval Training ◽

Cardiac Stem Cells ◽

Aged Rats ◽

Training Intensity ◽

Endothelial Progenitor

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Angiogenesis‐Related Genes in Endothelial Progenitor Cells May Be Involved in Sickle Cell Stroke

Journal of the American Heart Association ◽

10.1161/jaha.119.014143 ◽

2020 ◽

Vol 9 (3) ◽

Author(s):

Mirta T. Ito ◽

Sueli M. da Silva Costa ◽

Letícia C. Baptista ◽

Gabriela Q. Carvalho‐Siqueira ◽

Dulcinéia M. Albuquerque ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cell ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Sickle Cell ◽

Cell Biology ◽

Vascular Diseases ◽

Clinical Aspects ◽

Endothelial Progenitor ◽

Matrix Metalloproteinase 1

Background The clinical aspects of sickle cell anemia ( SCA ) are heterogeneous, and different patients may present significantly different clinical evolutions. Almost all organs can be affected, particularly the central nervous system. Transient ischemic events, infarcts, and cerebral hemorrhage can be observed and affect ≈25% of the patients with SCA . Differences in the expression of molecules produced by endothelial cells may be associated with the clinical heterogeneity of patients affected by vascular diseases. In this study, we investigated the differential expression of genes involved in endothelial cell biology in SCA patients with and without stroke. Methods and Results Endothelial progenitor cells from 4 SCA patients with stroke and 6 SCA patients without stroke were evaluated through the polymerase chain reaction array technique. The analysis of gene expression profiling identified 29 differentially expressed genes. Eleven of these genes were upregulated, and most were associated with angiogenesis (55%), inflammatory response (18%), and coagulation (18%) pathways. Downregulated expression was observed in 18 genes, with the majority associated with angiogenesis (28%), apoptosis (28%), and cell adhesion (22%) pathways. Remarkable overexpression of the MMP 1 (matrix metalloproteinase 1) gene in the endothelial progenitor cells of all SCA patients with stroke (fold change: 204.64; P =0.0004) was observed. Conclusions Our results strongly suggest that angiogenesis is an important process in sickle cell stroke, and differences in the gene expression profile of endothelial cell biology, especially MMP 1 , may be related to stroke in SCA patients.

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Endothelial progenitor cells promote osteogenic differentiation in co-cultured with mesenchymal stem cells via the MAPK-dependent pathway

10.21203/rs.3.rs-66113/v2 ◽

2020 ◽

Author(s):

chu xu ◽

haijie liu ◽

yuanjia he ◽

yuanqing li ◽

xiaoning he

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Western Blotting ◽

Nodule Formation ◽

Endothelial Progenitor ◽

Alp Activity

Abstract Background: The role of bone tissue engineering is to regenerate tissue using biomaterials and stem cell based approaches. Combination of two or more cell types is one of the strategies to promote bone formation. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of mesenchymal stem cells (MSCs) and promote bone healing, this study aimed to investigate the possible mechanisms of EPCs on promoting osteogenic differentiation of MSCs.Methods: MSCs and EPCs were isolated and co-cultured in Transwell chambers, the effects of EPCs on the regulation of MSC biological properties was investigated. Real-time PCR array and western blotting were performed to explore possible signaling pathways involved in osteogenesis. The expression of osteogenesis markers and calcium nodule formation was quantified by qRT-PCR, western blotting and Alizarin Red staining. Results: Results showed that MSCs exhibited greater alkaline phosphatase (ALP) activity and increased calcium mineral deposition significantly when co-cultured with EPCs. The mitogen-activated protein kinase (MAPK) signaling pathway was involved in this process. p38 gene expression and p38 protein phosphorylation levels showed significant up-regulation in co-cultured MSCs. Silencing expression of p38 in co-cultured MSCs reduced osteogenic gene expression, protein synthesis, ALP activity and calcium nodule formation. Conclusions: These data suggest paracrine signaling from EPCs influence the biological function and promote MSCs osteogenic differentiation. Activation of the p38MAPK pathway may be the key to enhancing MSCs osteogenic differentiation via indirect interactions with EPCs.

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Role of Canagliflozin on function of CD34+ endothelial progenitor cells (EPC) in patients with type 2 diabetes

10.21203/rs.3.rs-109671/v1 ◽

2020 ◽

Author(s):

Seshagiri Rao Nandula ◽

Nabanita Kundu ◽

Hassan Awal ◽

Beda Brichacek ◽

Mona Fakhri ◽

...

Keyword(s):

Gene Expression ◽

Type 2 Diabetes ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Functional Improvement ◽

P Value ◽

Risk Indicator ◽

Cvd Risk ◽

Endothelial Progenitor

Abstract Background: Endothelial Progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Effect of sodium glucose channel inhibitors (SGLT2 inhibitors) such as Canagliflozin (CG) on a cellular CVD risk indicator such as CD34+ve cells, in patients with T2DM with established CKD has not been explored,Methods: 29 subjects taking metformin and/or Insulin were enrolled in a 16 weeks, double blind, randomized placebo matched trial, with a low dose 100 mg CG compared to placebo. Type 2 diabetes subjects (30–70 years old), HbA1c of 7–10%,. CD34+ cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, biochemistry, resting energy expenditure and body composition were measured. Data were collected at week 0, 8 and 16. A mixed model regression analysis was done with p value less than 0.05 were considered significant.Results: Gene expression analysis of CD34+ve cells showed increase in antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX). A significant increase in gene expression of endothelial markers PECAM1 (p=0.002), VEGF-A(p= 0.04) and CXCR4 receptor (p= 0.06) followed by an increase in migratory function of CD34+ve cells is observed in Canagliflozin treated group as compared to placebo group. A significant reduction in glucose (p=0.01) levels was observed along with improved systolic and diastolic blood pressure in the CG group. Significant increase in adiponectin (p=0.02) was also noted in treatment group. Urinary exosomal protein examining podocyte health (podocalyxin, Wilm’s tumor and nephrin) showed improvement.Conclusion: We conclude that Canagliflozin on addition to metformin and/or Insulin, in subjects with type 2 diabetes demonstrates a functional improvement of CD34+ Endothelial Progenitor Cell function and gene expression and improves endothelial function

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Abstract 359: Disordered Chemokine Expression in Diabetes Following Experimental Myocardial Infarction: Potential Role in Impaired Progenitor Cell Recruitment

Circulation ◽

10.1161/circ.118.suppl_18.s_290 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Jean-Francois Desjardins ◽

Kim A Connelly ◽

Kerri Thai ◽

Cory A Johnson ◽

Thomas G Parker ◽

...

Keyword(s):

Gene Expression ◽

Myocardial Infarction ◽

Infarct Size ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Diabetic Rats ◽

Endothelial Progenitor ◽

Vessel Formation ◽

Chemokine Expression ◽

Time Point

BACKGROUND: Impaired collateral vessel formation is thought to contribute to the excessive morbidity in diabetic subjects with ischaemic heart disease where following myocardial infarction (MI) patients with diabetes are twice more likely to develop heart failure than non-diabetic subjects. Since the trafficking of bone marrow derived endothelial progenitor cells is thought to contribute to new vessel formation in ischemia, we postulated that altered chemokine expression may contribute to the impaired angiogenic response in diabetes. METHODS: Six week old F344 rats were randomized to vehicle or streptozotocin (35mg/kg i.p.) in order to induce diabetes. Once diabetes was confirmed (plasma glucose >15 mM), a total of 40 animals then underwent sham procedure or LAD ligation (MI) at age 10 weeks, and sacrificed at day 0 (sham) and days 1, 2, 4 and 7 post-MI (4 per group). Infarct size was determined by echocardiography 24 hours after ligation. RT q PCR was used to assess mRNA expression levels of SDF-1 and its receptor CXCR4 at each time point, from tissue obtained from the peri-MI zone. RESULTS: There was no difference in mortality or infarct size between the groups. In control rats an increase in SDF-1 gene expression was apparent at Day 2 post-MI, persisting through Day 7 (p<0.05). In contrast, diabetic rats showed high basal expression (6-fold vs. control, p<0.01) that fell abruptly after MI and remained persistently low throughout the 7-day time course. Diabetes was also associated with increased basal gene expression of CXCR4 (2-fold vs. control, p<0.05). Post-MI, CXCR4 gene expression increased in control rats at each time point, with fold differences up to maximally 3 times at Day 2 (p<0.05 vs. Day 0). In diabetic rats, the level of expression remained high at Day 1, significantly decreased at Day 2 (3-fold vs. non-diabetic, p<0.01) and was comparable to that of non-diabetic rats at days 4 and 7. CONCLUSIONS: Diabetes was associated with paradoxical alterations in gene expression of both SDF-1 and its receptor CXCR4 with high baseline expression and diminution following an ischemic insult. These findings suggest that disordered chemokine expression may reduce the diabetic heart’s ability to recruit endothelial progenitor cells to sites of ischemic injury.

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Exposure to Fine Particulate Matter Air Pollution Alters mRNA and miRNA Expression in Bone Marrow-Derived Endothelial Progenitor Cells from Mice

Genes ◽

10.3390/genes12071058 ◽

2021 ◽

Vol 12 (7) ◽

pp. 1058

Author(s):

Xiaohong Li ◽

Petra Haberzettl ◽

Daniel J. Conklin ◽

Aruni Bhatnagar ◽

Eric C. Rouchka ◽

...

Keyword(s):

Gene Expression ◽

Air Pollution ◽

Particulate Matter ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Mirna Expression ◽

Fine Particulate Matter ◽

Cell Movement ◽

Endothelial Progenitor ◽

Fine Particulate

Exposure to fine particulate matter (PM2.5) air pollution is associated with quantitative deficits of circulating endothelial progenitor cells (EPCs) in humans. Related exposures of mice to concentrated ambient PM2.5 (CAP) likewise reduces levels of circulating EPCs and induces defects in their proliferation and angiogenic potential as well. These changes in EPC number or function are predictive of larger cardiovascular dysfunction. To identify global, PM2.5-dependent mRNA and miRNA expression changes that may contribute to these defects, we performed a transcriptomic analysis of cells isolated from exposed mice. Compared with control samples, we identified 122 upregulated genes and 44 downregulated genes in EPCs derived from CAP-exposed animals. Functions most impacted by these gene expression changes included regulation of cell movement, cell and tissue development, and cellular assembly and organization. With respect to miRNA changes, we found that 55 were upregulated while 53 were downregulated in EPCs from CAP-exposed mice. The top functions impacted by these miRNA changes included cell movement, cell death and survival, cellular development, and cell growth and proliferation. A subset of these mRNA and miRNA changes were confirmed by qRT-PCR, including some reciprocal relationships. These results suggest that PM2.5-induced changes in gene expression may contribute to EPC dysfunction and that such changes may contribute to the adverse cardiovascular outcomes of air pollution exposure.

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Gene expression pattern in hepatic stem/progenitor cells during rat fetal development using complementary DNA microarrays

Hepatology ◽

10.1002/hep.20088 ◽

2004 ◽

Vol 39 (3) ◽

pp. 617-627 ◽

Cited By ~ 47

Author(s):

Petko M. Petkov ◽

Jiri Zavadil ◽

David Goetz ◽

Tearina Chu ◽

Robert Carver ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Expression Pattern ◽

Fetal Development ◽

Dna Microarrays ◽

Gene Expression Pattern ◽

Complementary Dna

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Mortalin-2 (mthsp70/PBP74/Grp75) Mediates the Erythropoietin Signal To Stimulate Growth of Purified Human Erythroid Progenitor Cells.

Blood ◽

10.1182/blood.v108.11.4165.4165 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4165-4165

Author(s):

Rie Ohtsuka ◽

Yasunobu Abe ◽

Tomomi Fujii ◽

Junji Nishimura ◽

Koichiro Muta

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Progenitor Cells ◽

Expression Pattern ◽

Gene Expression Pattern ◽

Janus Kinase ◽

Expression Vectors ◽

Brdu Incorporation ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

Abstract Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal; however, the entire process of EPO stimulation remains undetermined. Previously, we used siRNA to show that mortalin-2, which is a member of the heat shock protein 70 family of chaperones, mediates EPO signaling to stimulate the growth of human erythroid colony forming cells (ECFCs). In the present study, we examined the relationship between cell growth and mortalin-2 overexpressed in ECFCs, and analyzed the gene expression pattern of ECFCs treated with mortalin-2 siRNA using a DNA microarray, to further clarify the intracellular mechanism by which EPO and mortalin-2 interact. In the presence of different concentrations of EPO (0–1.0 U/ml), the effect of mortalin-2 overexpression on ECFC growth was determined by MTT assay. There was tendency to better viability of ECFCs treated with mortalin-2 expression vectors than control cells, especially in the EPO 0 U/ml group (p=0.08). BrdU ELISA, used to investigate the effects of mortalin-2 on the DNA synthesis of ECFCs, revealed that when ECFCs were treated with mortalin-2 expression vectors, the cells showed an increase in the amount of BrdU incorporation into DNA without EPO. Next we analyzed the gene expression pattern using mRNA obtained from ECFCs cultured with or without EPO after treatment with mortalin-2 siRNA or control siRNA. When ECFCs were cultured with EPO after treatment with mortalin-2 siRNA, the expression of 19 genes was suppressed to less than 0.6 fold, and these genes included those involved in cell growth, apoptosis or transport, such as interleukin 6 receptor, ATP-binding cassette, Mdm2, BCL2 interacting protein and interleukin 10 receptor alpha. Furthermore, the expression of 8 genes was upregulated to over 1.5 fold, and these genes included transcription or signal transmission related genes, such as ubiquitin A-52 residue ribosomal protein fusion product 1 and serum/glucocorticoid regulated kinase 2. There were also some genes whose expressions overlapped with genes obtained from ECFCs cultured without EPO after treatment with control siRNA. Our data suggests that mortalin-2 expression depends on various pathways, and that one of these pathways mediates EPO signaling to stimulate mortalin-2 expression, which is related to the growth of erythroid progenitor cells.

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