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The aim of this study was to assess the power of the polygenic risk score (PRS) in estimating the overall genetic risk of women carrying germline BRCA1 pathogenic variants (PVs) c.4035del or c.5266dup to develop breast (BC) or ovarian cancer (OC) due to additional genetic variations. In this study, PRSs previously developed from two joint models using summary statistics of age-at-onset (BayesW model) and case–control data (BayesRR-RC model) from a genome-wide association analysis (GWAS) were applied to 406 germline BRCA1 PV (c.4035del or c.5266dup) carriers affected by BC or OC, compared with unaffected individuals. A binomial logistic regression model was used to assess the association of PRS with BC or OC development risk. We observed that the best-fitting BayesW PRS model effectively predicted the individual’s BC risk (OR = 1.37; 95% CI = 1.03–1.81, p = 0.02905 with AUC = 0.759). However, none of the applied PRS models was a good predictor of OC risk. The best-fitted PRS model (BayesW) contributed to assessing the risk of developing BC for germline BRCA1 PV (c.4035del or c.5266dup) carriers and may facilitate more precise and timely patient stratification and decision-making to improve the current BC treatment or even prevention strategies.

Contains fulltext : 292339.pdf (Publisher’s version ) (Open Access) OBJECTIVE: Netherton syndrome (NS) (OMIM:256500) is a very rare autosomal recessive multisystem disorder mostly affecting ectodermal derivatives (skin and hair) and immune system. It is caused by biallelic loss-of-function variants in the SPINK5 gene, encoding the protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI). MATERIAL, METHODS AND RESULTS: Here, we describe NS clinical and genetic features of homogenous patient group: 9 individuals from 7 families with similar ethnic background and who have the same SPINK5 variant (NM_006846.4: c.1048C > T, p.(Arg350*)) in homozygous or compound heterozygous states, suggesting that it is a common founder variant in Latvian population. Indeed, we were able to show that the variant is common in general Latvian population, and it shares the same haplotype among the NS individual. It is estimated that the variant arose >1000 years ago. Clinically, all nine patients exhibited typical NS skin changes (scaly erythroderma, ichthyosis linearis circumflexa, itchy skin), except for one patient who has a different skin manifestation-epidermodysplasia. Additionally, we show that developmental delay, previously underrecognized in NS, is a common feature among these patients. CONCLUSIONS: This study shows that the phenotype of NS individuals with the same genotype is highly homogeneous. 01 april 2023

According to the FAIR principles, data produced by scientific research should be findable, accessible, interoperable, and reusable—for instance, to be used in machine learning algorithms. However, to date, there is no estimate of the quantity or quality of dental research data evaluated via the FAIR principles. We aimed to determine the availability of open data in dental research and to assess compliance with the FAIR principles (or FAIRness) of shared dental research data. We downloaded all available articles published in PubMed-indexed dental journals from 2016 to 2021 as open access from Europe PubMed Central. In addition, we took a random sample of 500 dental articles that were not open access through Europe PubMed Central. We assessed data sharing in the articles and compliance of shared data to the FAIR principles programmatically. Results showed that of 7,509 investigated articles, 112 (1.5%) shared data. The average (SD) level of compliance with the FAIR metrics was 32.6% (31.9%). The average for each metric was as follows: findability, 3.4 (2.7) of 7; accessibility, 1.0 (1.0) of 3; interoperability, 1.1 (1.2) of 4; and reusability, 2.4 (2.6) of 10. No considerable changes in data sharing or quality of shared data occurred over the years. Our findings indicated that dental researchers rarely shared data, and when they did share, the FAIR quality was suboptimal. Machine learning algorithms could understand 1% of available dental research data. These undermine the reproducibility of dental research and hinder gaining the knowledge that can be gleaned from machine learning algorithms and applications.

BackgroundLarge-scale genome-wide association studies have identified hundreds of single-nucleotide variants (SNVs) significantly associated with coronary artery disease (CAD). However, collectively, these explain HypothesisHere, we hypothesize that mitochondrial (MT) SNVs might present one potential source of this “missing heritability”.MethodsWe analyzed 265 MT-SNVs in ∼500,000 UK Biobank individuals, exploring two different CAD definitions: a more stringent (myocardial infarction and/or revascularization; HARD=20,405), and a more inclusive (also angina and chronic ischemic heart disease; SOFT=34,782).ResultsIn HARD cases, the most significant (PT (control region) and m.12612A>G (ND5), found more frequently in cases (OR=1.05), potentially related to reduced cardiorespiratory fitness in response to exercise, as well as for m.12372G>A (ND5) and m.11467A>G (ND4), present more frequently in controls (OR=0.97), previously associated with lower ROS production rate. In SOFT cases, four MT-SNVs survived multiple testing correction (at FDRG (ND4) and m.15452C>A (CYB) have previously shown significant associations with body height. In line with this, we observed that CAD cases were slightly less physically active and their average body height was ∼2.00 cm lower compared to controls, both traits known to be related to an increased CAD risk. Gene-based tests identified CO2 associated with HARD/SOFT CAD, whereas ND3 and CYB associated with SOFT cases (PConclusionsWe found only spurious associations between MT genome variation and HARD/SOFT CAD and conclude that more MT-SNV data in even larger study cohorts may be needed to conclusively determine the role of MT-DNA in CAD.

Introduction: Large-scale genome-wide association studies have identified >200 single-nucleotide variants (SNVs) significantly associated with coronary artery disease (CAD), collectively explaining Hypothesis: Mitochondrial (MT) genetic variants presents an underexplored one potential source of the “missing heritability”. Methods: We analyzed 265 MT-SNVs in ∼500,000 UK Biobank individuals, exploring two different CAD definitions: including (SOFT=34,782) vs. excluding (HARD=20,405) angina, using the array, sex, birth year and first five principal components as covariates. Results: In SOFT cases, four MT-SNVs survived multiple testing correction (at FDRT (rs28358278, OR=1.28, MAF=0.02, T114T in MT-ND3), m.15301G>A (rs527236045, OR=1.03, MAF=0.04, L185L in MT-CYB), m.11251A>G (rs869096886, OR=1.03, MAF=0.2, L164L in MT-ND4) and m.15452C>A (rs193302994, OR=1.03, MAF=0.2, L236I in MT-CYB), the latter two previously associated with height in the UK Biobank. In HARD cases, no MT-SNVs survived multiple testing correction, the most significant nominal (P T (rs41528348, OR=1.05, MAF=0.1, in the D-LOOP) and m.12612A>G (rs28359172, OR=1.05, MAF=0.04, V92V in MT-ND5) could potentially increase CAD risk, whereas m.12372G>A (rs2853499, OR=0.97, MAF=0.22, L12L in MT-ND5), m.11467A>G (rs2853493, OR=0.97, MAF=0.22, L236L in MT-ND4) and m.15301G>A (rs193302991, OR=0.97, MAF=0.04, L185L in MT-CYB) may confer protective effects. Previously, several of these variants have been associated with a number of blood cell traits in the UK Biobank, including the mean corpuscular hemoglobin levels (m.295C>T, m.12612A>G, m.12372G>A) and volume (m.295C>T and m.12612A>G). In addition, m.12372G>A was significantly association with five other blood cell traits, including the mean platelet volume and white/red blood cell count, as well as with several renal biomarkers, and m.12612A>G was associated with height. Conclusions: Our results indicated only spurious associations of the MT-SNV with the occurrence of CAD. More data in larger study cohorts may be needed to conclusively determine the role of MT-DNA in CAD.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Non-coding RNAs have already been linked to CVD development and progression. While microRNAs (miRs) have been well studied in blood samples, there is little data on tissue-specific miRs in cardiovascular relevant tissues and their relation to cardiovascular risk factors. Tissue-specific miRs derived from Arteria mammaria interna (IMA) from 192 coronary artery disease (CAD) patients undergoing coronary artery bypass grafting (CABG) were analyzed. The aims of the study were 1) to establish a reference atlas which can be utilized for identification of novel diagnostic biomarkers and potential therapeutic targets, and 2) to relate these miRs to cardiovascular risk factors. Overall, 393 individual miRs showed sufficient expression levels and passed quality control for further analysis. We identified 17 miRs–miR-10b-3p, miR-10-5p, miR-17-3p, miR-21-5p, miR-151a-5p, miR-181a-5p, miR-185-5p, miR-194-5p, miR-199a-3p, miR-199b-3p, miR-212-3p, miR-363-3p, miR-548d-5p, miR-744-5p, miR-3117-3p, miR-5683 and miR-5701–significantly correlated with cardiovascular risk factors (correlation coefficient &gt, 0.2 in both directions, p-value (p &lt, 0.006, false discovery rate (FDR) &lt, 0.05). Of particular interest, miR-5701 was positively correlated with hypertension, hypercholesterolemia, and diabetes. In addition, we found that miR-629-5p and miR-98-5p were significantly correlated with acute myocardial infarction. We provide a first atlas of miR profiles in IMA samples from CAD patients. In perspective, these miRs might play an important role in improved risk assessment, mechanistic disease understanding and local therapy of CAD.

Aims Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. Methods and results Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. Conclusion Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability., Graphical Abstract This study provides novel mechanistic insights into how acute mental stress fuels vascular inflammation and promotes plaque rupture. EC, endothelial cells; HPA, hypothalamic-pituitary-adrenal axis; MACS, macrophages; SAM, sympathetic-adrenal-medullary axis.

As clinicians, we understand the development of atherosclerosis as a consequence of cholesterol deposition and inflammation in the arterial wall, both being triggered by traditional risk factors such as hypertension, hyperlipidaemia or diabetes mellitus. Another risk factor is genetic predisposition, as indicated by the predictive value of a positive family history. However, we had to wait until recently to appreciate the abundant contribution of genetic variation to the manifestation of atherosclerosis. Indeed, by now 164 chromosomal loci have been identified by genome-wide association studies (GWAS) to affect the risk of coronary artery disease. By design, practically all risk variants discovered by GWAS are frequently found in our population, resulting in the fact that principally every Western European individual carries between 130 and 190 risk alleles at the known, genome-wide significant loci (there are 0, 1, or 2 risk alleles per locus). One can assume that it is this widespread disposition that makes mankind susceptible to the detrimental effects of lifestyle factors, which likewise increase the risk of atherosclerosis. In this review, we summarize the recent genetic discoveries and attempt to group the multiple genetic risk variants in functional groups that may become actionable from a preventive or therapeutic perspective.

The phenolic compound profiles of 393 raw mono- and polyfloral Latvian honey samples were investigated using a targeted ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method. This strategy allowed simultaneous determination of the content of 11 phenolic acids, 18 flavonoids, 2 plant hormones, and 3 water-soluble vitamins in honey samples of botanically diverse origin, including buckwheat (Fagopyrum esculentum), heather (Calluna vulgaris), linden (Tilia cordata), and rapeseed (Brassica napus). Honey samples were collected directly from the beekeepers and were analyzed in their natural, raw form. A selective high-throughput analysis was implemented using sugaring-out assisted liquid-liquid extraction (SULLE) combined with the pentafluorophenyl (PFP) stationary phase. The robustness of the method was evaluated by using the design of experiments (DoE) approach and validated. Pooled quality control (QC) samples allowed monitoring of the method performance across the analysis sequences and principal components analysis (PCA) of QC samples resulted in a small amount of total variance in the total projection, indicating that the data were of good quality. The acquired occurrence data were compiled and compared with other studies. To the best of our knowledge, this study is the first of this scale and purpose, providing unique insights into the phenolic compound composition of fresh, regional European honey.

Genome-wide association studies (GWAS) have identified over two hundred chromosomal loci that modulate risk of coronary artery disease (CAD). The genes affected by variants at these loci are largely unknown and an untapped resource to improve our understanding of CAD pathophysiology and identify potential therapeutic targets. Here, we prioritized 68 genes as the most likely causal genes at genome-wide significant loci identified by GWAS of CAD and examined their regulatory roles in 286 metabolic and vascular tissue gene-protein sub-networks (“modules”). The modules and genes within were scored for CAD druggability potential. The scoring enriched for targets of cardiometabolic drugs currently in clinical use and in-depth analysis of the top-scoring modules validated established and revealed novel target tissues, biological processes, and druggable targets. This study provides an unprecedented resource of tissue-defined gene–protein interactions directly affected by genetic variance in CAD risk loci.

Summary Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage− SCA-1+ KIT+ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34− CD48− CD150+ LSK (CD34− SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34− SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27Kip1 expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity., Graphical Abstract, Highlights • LSK cells induce the expression of CTGF in stromal cells • Stromal CTGF drives cell-cycle progression and proliferation of CD34− LSK cells • Decreased stromal CTGF activates TGFB signaling to decrease LSK cell cycle • Stromal CTGF improves maintenance of HSCs with long-term repopulating activity, Oostendorp and colleagues show that hematopoietic stem cells (HSCs) induce expression of connective tissue growth factor (CTGF) in stromal cells. A decrease in stromal CTGF impairs the maintenance of HSC activity in co-culture by rapid modulation of HSC signaling and cell-cycle entry. Thus, HSCs regulate their own cell cycle by modulating gene expression in stromal cells.

Background and aims Mitochondrial damage and augmented production of reactive oxygen species (ROS) may represent an intermediate step by which hypercholesterolemia exacerbates atherosclerotic lesion formation. Methods To test this hypothesis, in mice with severe but genetically reversible hypercholesterolemia (i.e. the so called Reversa mouse model), we performed time-resolved analyses of mitochondrial transcriptome in the aortic arch employing a systems-level network approach. Results During hypercholesterolemia, we observed a massive down-regulation (>28%) of mitochondrial genes, specifically at the time of rapid atherosclerotic lesion expansion and foam cell formation, i.e. between 30 and 40 weeks of age. Both phenomena - down-regulation of mitochondrial genes and lesion expansion - were largely reversible by genetically lowering plasma cholesterol (by >80%, from 427 to 54 ± 31 mg/L) at 30 weeks. Co-expression network analysis revealed that both mitochondrial signature genes were highly connected in two modules, negatively correlating with lesion size and supported as causal for coronary artery disease (CAD) in humans, as expression-associated single nucleotide polymorphisms (eSNPs) representing their genes overlapped markedly with established disease risk loci. Within these modules, we identified the transcription factor estrogen related receptor (ERR)-α and its co-factors PGC1-α and -β, i.e. two members of the peroxisome proliferator-activated receptor γ co-activator 1 family of transcription regulators, as key regulatory genes. Together, these factors are known as major orchestrators of mitochondrial biogenesis and antioxidant responses. Conclusions Using a network approach, we demonstrate how hypercholesterolemia could hamper mitochondrial activity during atherosclerosis progression and pinpoint potential therapeutic targets to counteract these processes.

Background: A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3 , which encodes the α 1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. Methods: Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses. Results: Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo–studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. Conclusions: Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.

Cardiovascular diseases are leading causes for death worldwide. Genetic disposition jointly with traditional risk factors precipitates their manifestation. Whereas the implications of a positive family history for individual risk have been known for a long time, only in the past few years have genome‐wide association studies (GWAS) shed light on the underlying genetic variations. Here, we review these studies designed to increase our understanding of the pathophysiology of cardiovascular diseases, particularly coronary artery disease and myocardial infarction. We focus on the newly established pathways to exemplify the translation from the identification of risk‐related genetic variants to new preventive and therapeutic strategies for cardiovascular disease.

Domain Interaction MAp (DIMA, available at http://webclu.bio.wzw.tum.de/dima) is a database of predicted and known interactions between protein domains. It integrates 5807 structurally known interactions imported from the iPfam and 3did databases and 46,900 domain interactions predicted by four computational methods: domain phylogenetic profiling, domain pair exclusion algorithm correlated mutations and domain interaction prediction in a discriminative way. Additionally predictions are filtered to exclude those domain pairs that are reported as non-interacting by the Negatome database. The DIMA Web site allows to calculate domain interaction networks either for a domain of interest or for entire organisms, and to explore them interactively using the Flash-based Cytoscape Web software.

Objective— Genome-wide association studies have to date identified 159 significant and suggestive loci for coronary artery disease (CAD). We now report comprehensive bioinformatics analyses of sequence variation in these loci to predict candidate causal genes. Approach and Results— All annotated genes in the loci were evaluated with respect to protein-coding single-nucleotide polymorphism and gene expression parameters. The latter included expression quantitative trait loci, tissue specificity, and miRNA binding. High priority candidate genes were further identified based on literature searches and our experimental data. We conclude that the great majority of causal variations affecting CAD risk occur in noncoding regions, with 41% affecting gene expression robustly versus 6% leading to amino acid changes. Many of these genes differed from the traditionally annotated genes, which was usually based on proximity to the lead single-nucleotide polymorphism. Indeed, we obtained evidence that genetic variants at CAD loci affect 98 genes which had not been linked to CAD previously. Conclusions— Our results substantially revise the list of likely candidates for CAD and suggest that genome-wide association studies efforts in other diseases may benefit from similar bioinformatics analyses.

Pleiotrophin (Ptn) is strongly expressed by stromal cells which maintain HSCs. However, in vivo, Ptn deficiency does not alter steady-state hematopoiesis. However, knockdown of Ptn (PtnKD) in stromal cells increases production of hematopoietic progenitors as well as HSC activity in cocultures, suggesting that Ptn may have a role in HSC activation. Indeed, transplantations of wild-type (Ptn+/+) HSCs into Ptn−/− mice show increased donor cell production in serial transplantations and dominant myeloid regeneration caused by Ptn-dependent regulation of HSC repopulation behavior. This regulation of Lin−Kit+Sca1+ function is associated with increased proliferation and, on a molecular level, with up-regulated expression of cyclin D1 (Ccnd1) and C/EBPα (Cepba), but reduced of PPARγ. The known HSC regulator β-catenin is, however, not altered in the absence of Ptn. In conclusion, our results point to different Ptn-mediated regulatory mechanisms in normal hemostasis and in hematopoietic regeneration and in maintaining the balance of myeloid and lymphoid regeneration. Moreover, our results support the idea that microenvironmental Ptn regulates hematopoietic regeneration through β-catenin–independent regulation of Ccnd1 and Cebpa.

Recently, mutations of additional sex comb-like 1 (ASXL1) have been identified as an independent poor prognostic factor in MDS patients. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced MDS-like disease in mice by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27. Intriguingly, the patients with ASXL1 mutation have significantly higher incidences of concurrent SET binding protein 1 (SETBP1) mutation than those with wild-type ASXL1, which prompted us to investigate whether SETBP1 mutation plays a critical role in leukemic transformation of MDS. Most SETBP1 mutations, such as D868N, have been shown to occur in the PEST domain of ski homology region, resulting in the prevention from proteasomal degradation. In in vitro experiment, the expression of both SETBP1-D868N and ASXL1-MT enhanced myeloid colony output, reinforced ASXL1-MT-induced differentiation block and inhibited apoptosis. Of note, SETBP1-D868N collaborated with ASXL1-MT in inducing MDS/AML in mice BMT model compared to control mice (0.0001). Using GSEA, we demonstrated that SETBP1-MT enriched hematopoietic stem cells-related genes and deregulated p53 and Myc pathway. In addition, SETBP1-MT inhibited PP2a leading to the activation of the Akt pathway and exhibited characteristic histone modification pattern. In conclusion, our data provide evidence for the role of SETBP1-MT in leukemic transformation and suggest the deregulated pathways as a potential therapeutic target in MDS to prevent disease progression.

Hematopoietic stem cells (HSC) are regulated by an interplay of intrinsic and extrinsic signals, the latter of which are mostly transmitted by the niche. The processes involved and their interactions are largely unknown. We studied the dynamic interaction of HSC and niche stromal cells, using co-cultures of HSC (lineage-negative Sca-1+ c-Kit+: LSK) cells and HSC-maintaining UG26-1B6 stromal cells. Microarray analyses from cells prior to co-culture and cells sorted separately from the cultures revealed that most changes in gene expression take place in the first 24 hours of co-culture. Analyses using STEM clustering, LIMMA, and ToppGene databases showed early activation of cell cycle progression In LSK cells and extensive remodeling of chomatin structure and transcriptional activation in both LSK and stromal cells. Interestingly, connective tissue growth factor (Ctgf/Ccn2), which is involved in TGFb, BMP and Wnt signaling, was strongly upregulated in both stromal and LSK cells. To study the role of Ctgf as a stromal mediator, LSK cells were co-cultured with siCTGF knockdown stromal cells. We showed that although short-term HSC activity was unchanged, siCtgf-stromal cells were unable to sustain long-term repopulating ability. To study underlying mechanisms, a Boolean model simulating possible signaling mechanisms leading to cell cycle activation was extracted from the data. We validated this model by co-cultures of LSK cells with control and Ctgf-knockdown stroma, separating LSK and stromal cells and assessing protein levels and phosphorylation using immunocytofluorescence in LSK cells. We found that in the absence of extrinsic Ctgf, expression of Pten was increased in LSK cells. However, phosphorylation of Akt (both p308, and p473) and Erk was unchanged. In contrast, both canonical Wnt (LRP6, Gsk3b, b-catenin) and Tgfb (Smad2/3) signaling were significantly affected in that Wnt signaling was turned off, whereas Tgf signaling was turned on, by the lack of extrinsic Ctgf. This resulted in a downregulation of G1 transition, as was exemplified by downregulation of Cyclin D1, upregulation of p27Kip1 and modulations in the phosphoryalation of both Rb and p53. Equally interestingly, we could show that extrinsic Ctgf deficiency also downregulates induction of Ctgf in HSC, suggesting the existence of intrinsic-extrinsic feedback signaling. In summary, co-culture of LSK cells with stromal cells results in cellular activation of both stromal cells and LSK cells, involving Tgf and canonical Wnt signaling pathways. Furthermore, reduced expression of extrinsic Ctgf, regulates mediators of G1 cell cycle progression in LSK cells an a biochemical level and functionally results in an increased production of myeloid progenitors and decreased long-term repopulating ability. Our studies show the dynamics of reciprocal signaling between HSC and niche stromal cells and give insights how the niche regulates early regenerative responses in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 2353 Hematopoiesis is maintained throughout life by the constant production of mature blood cells from hematopoietic stem cells (HSC). One mechanism by which the number of HSC is maintained is self-renewal, a cell division in which at least one of the daughter cells is a cell with the same functional potential as the mother cell. The mechanisms of this process are largely unknown. We have described cell lines that maintain self-renewal in culture. To study possible mechanisms and mediators involved in self-renewal, we performed co-cultures of HSC model cells: Lineage-negative Sca-1+ c-Kit+ (LSK) cells and HSC maintaining UG26–1B6 stromal cells. Microarray analyses were performed on cells prior to co-culture and cells sorted from the cultures. STEM clustering analysis of the data revealed that most changes in gene expression were due to early cell activation. Functional enrichment analysis revealed dynamic changes in focal adhesion and mTOR signaling, as well as changes in epigenetic regulators, such as HDAC in stromal cells. In LSK cells, genes whose products are involved in inflammation, Oxygen homeostasis and metabolism were differentially expressed after the co-culture. In addition, genes involved in the regulaton of H3K27 methylation were also affected. Interestingly, connective tissue growth factor (CTGF), which is involved in TGF-b, BMP and Wnt signaling, was upregulated in both stromal and LSK cells in the first day of co-culture. To study a possible extrinsic role of CTGF as a stromal mediator, we co-cultured siCTGF knockdown stromal cells with wild-type LSK cells. Since self-renewal requires cell division, we focused on cell cycle regulation of LSK cells. We found that knockdown of CTGF in stromal cells downregulates CTGF in LSK cells. In addition, knockdown of stromal CTGF downregulated Ccnd1, Cdk2, Cdkn1a (p21), Ep300 and Fos. On the other hand, decreased CTGF in stromal cells upregulates Cdkn1b (p27) and phosphorylation of Smad2/3. These results show that stromal CTGF regulates the cell cycle of LSK cells. On a functional level, we found that decreased stromal CTGF results in an increased production of MPP and myeloid colony-forming cells in 1-week co-cultures. We will present data showing whether and how a decrease in CTGF in stromal cells affects the maintenance of transplantable HSC. In summary, our current results indicate that reduced expression of CTGF in stromal cells regulates mediators of cell cycle and Smad2/3-mediated signaling in LSK cells, resulting in an increased production of myeloid progenitors. Disclosures: No relevant conflicts of interest to declare.

Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Cancer pathogenesis involves tumor-intrinsic genomic aberrations and tumor-cell extrinsic mechanisms such as failure of immunosurveillance and structural and functional changes in the microenvironment. Using Myc as a model oncogene we established a conditional mouse bone marrow transduction/transplantation model where the conditional activation of the oncoprotein Myc expressed in the hematopoietic system could be assessed for influencing the host microenvironment. Constitutive ectopic expression of Myc resulted in rapid onset of a lethal myeloproliferative disorder with a median survival of 21 days. In contrast, brief 4-day Myc activation by means of the estrogen receptor (ER) agonist tamoxifen did not result in gross changes in the percentage/frequency of hematopoietic lineages or hematopoietic stem/ progenitor cell (HSPC) subsets, nor did Myc activation significantly change the composition of the non-hematopoietic microenvironment defined by phenotyping for CD31, ALCAM, and Sca-1 expression. Transcriptome analysis of endothelial CD45- Ter119- cells from tamoxifen-treated MycER bone marrow graft recipients revealed a gene expression signature characterized by specific changes in the Rho subfamily pathway members, in the transcription-translation-machinery and in angiogenesis. In conclusion, intra-hematopoietic Myc activation results in significant transcriptome alterations that can be attributed to oncogene-induced signals from hematopoietic cells towards the microenvironment, e. g. endothelial cells, supporting the idea that even pre-leukemic HSPC highjack components of the niche which then could protect and support the cancer-initiating population.