Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia.

BACKGROUND: Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. OBJECTIVES: To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. METHODS: Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. RESULTS: Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. CONCLUSION: These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

Platelet CD40 ligand and bleeding during P2Y12 inhibitor treatment in acute coronary syndrome.

Antiplatelet therapy through inhibition of the adenosine diphosphate (ADP)/P2Y12 pathway is commonly used in the treatment of acute coronary syndrome (ACS). Although efficient in preventing platelet activation and thrombus formation, it increases the risk of bleeding complications. In patients with ACS receiving platelet aggregation inhibitors, that is, P2Y12 blockers (n = 923), we investigated the relationship between plasma and platelet-associated CD40L levels and bleeding events (n = 71). Treatment with P2Y12 inhibitors in patients with ACS did not affect plasma-soluble CD40L levels, but decreased platelet CD40L surface expression (pCD40L) and platelet-released CD40L (rCD40L) levels in response to stimulation as compared to healthy controls. In vitro inhibition of the ADP pathway in healthy control platelets reduced both pCD40L and rCD40L levels. In a multivariable analysis, the reduced pCD40L level observed in ACS patients was significantly associated with the risk of bleeding occurrence (adjusted odds ratio = 0.15; 95% confidence interval = 0.034-0.67). P2Y12 inhibitor-treated (ticagrelor) mice exhibited a 2.5-fold increase in tail bleeding duration compared with controls. A significant reduction in bleeding duration was observed on CD40L+/+ but not CD40L-/- platelet infusion. In addition, CD40L blockade in P2Y12 inhibitor-treated blood samples from a healthy human reduced thrombus growth over immobilized collagen under arterial flow. In conclusion, measurement of pCD40L may offer a novel approach to assessing bleeding risk in patients with ACS who are being treated with P2Y12 inhibitors.

Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis.

Increased platelet activity occurs in type 2 diabetes mellitus (T2DM) and such platelet dysregulation likely originates from altered megakaryopoiesis. We initiated identification of dysregulated pathways in megakaryocytes in the setting of T2DM. We evaluated through transcriptomic analysis, differential gene expressions in megakaryocytes from leptin receptor-deficient mice (db/db), exhibiting features of human T2DM, and control mice (db/+). Functional gene analysis revealed an upregulation of transcripts related to calcium signaling, coagulation cascade and platelet receptors in diabetic mouse megakaryocytes. We also evidenced an upregulation (7- to 9.7-fold) of genes encoding stefin A (StfA), the human ortholog of Cystatin A (CSTA), inhibitor of cathepsin B, H and L. StfA/CSTA was present in megakaryocytes and platelets and its expression increased during obesity and diabetes in rats and humans. StfA/CSTA was primarily localized at platelet membranes and granules and was released upon agonist stimulation and clot formation through a metalloprotease-dependent mechanism. StfA/CSTA did not affect platelet aggregation, but reduced platelet accumulation on immobilized collagen from flowing whole blood (1200 s-1). In-vivo, upon laser-induced vascular injury, platelet recruitment and thrombus formation were markedly reduced in StfA1-overexpressing mice without affecting bleeding time. The presence of CA-074Me, a cathepsin B specific inhibitor significantly reduced thrombus formation in-vitro and in-vivo in human and mouse, respectively. Our study identifies StfA/CSTA as a key contributor of platelet-dependent thrombus formation in both rodents and humans.

An evaluation of the effects of Lactobacillus ingluviei on body weight, the intestinal microbiome and metabolism in mice.

BACKGROUND: Food can modify the intestinal flora, and Lactobacillus ingluviei has been shown to cause weight gain in chicks and ducks but not in mammals. METHODOLOGY: Female BALB/c mice were divided into a control and two experimental groups and were inoculated either once or twice with L. ingluviei or with PBS. Faecal samples were collected and tested using qPCR in order to detect and quantify Lactobacillus spp., Bacteroidetes spp. and Firmicutes spp. Gene expression was examined in liver and adipose tissue by microarray and qPCR. Metabolic indicators in the plasma were also measured. RESULTS: Mice that were inoculated with 4 × 10(10) L. ingluviei presented a significant increase in weight gain and liver weight and significant increases in Lactobacillus spp. and Firmicutes DNA copy numbers in their faeces. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumour necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1), 3-phosphoinositide-dependent protein kinase-1 (Pdpk1), acyl-Coenzyme A dehydrogenase 11 (Acad11), ATP-binding cassette sub family member G (ABCG2) and DEAD box polypeptide 25 (Ddx25) were significantly elevated in the liver tissues of animals in the experimental group. In gonadal adipose tissue, the expression levels of leptin, peroxisome proliferator-activated receptor γ (Pparg) and Srebp1c were significantly higher in animals from the experimental group, whereas the expression of adiponectin was significantly lower in these animals. CONCLUSIONS: The inoculation of L. ingluviei in mice resulted in alterations in the intestinal flora, increased weight gain and liver enlargement, accelerated metabolism and increased inflammation.

Metabolic endotoxemia initiates obesity and insulin resistance.

Diabetes and obesity are two metabolic diseases characterized by insulin resistance and a low-grade inflammation. Seeking an inflammatory factor causative of the onset of insulin resistance, obesity, and diabetes, we have identified bacterial lipopolysaccharide (LPS) as a triggering factor. We found that normal endotoxemia increased or decreased during the fed or fasted state, respectively, on a nutritional basis and that a 4-week high-fat diet chronically increased plasma LPS concentration two to three times, a threshold that we have defined as metabolic endotoxemia. Importantly, a high-fat diet increased the proportion of an LPS-containing microbiota in the gut. When metabolic endotoxemia was induced for 4 weeks in mice through continuous subcutaneous infusion of LPS, fasted glycemia and insulinemia and whole-body, liver, and adipose tissue weight gain were increased to a similar extent as in high-fat-fed mice. In addition, adipose tissue F4/80-positive cells and markers of inflammation, and liver triglyceride content, were increased. Furthermore, liver, but not whole-body, insulin resistance was detected in LPS-infused mice. CD14 mutant mice resisted most of the LPS and high-fat diet-induced features of metabolic diseases. This new finding demonstrates that metabolic endotoxemia dysregulates the inflammatory tone and triggers body weight gain and diabetes. We conclude that the LPS/CD14 system sets the tone of insulin sensitivity and the onset of diabetes and obesity. Lowering plasma LPS concentration could be a potent strategy for the control of metabolic diseases.

Polymorphism A36G of the tumor necrosis factor receptor 1 gene is associated with PAI-1 levels in obese women.

The tumor necrosis factor (TNF) pathway may be implicated in etiopathogenesis of PAI-1 overexpression during obesity. The aim of this study was to investigate the influence of polymorphism A36G of the TNF receptor 1 (TNFRSF1A +36A/G) on plasma concentrations of PAI-1 in 163 obese (31 with the metabolic syndrome, MetS) and 150 lean, healthy women. Genotypic and allele frequencies did not significantly differ between obese and lean subjects. TNFRSF1A genotypes were significantly associated with sTNFR1 plasma levels in obese women only (p < 0.01); TNFRSF1A +36G/G obese carriers exhibited higher sTNFR1 and PAI-1 levels than A carriers (p < 0.01 and p < 0.05, respectively). In obese women, the presence of the MetS significantly potentiated the elevation of sTNFR1 and PAI-1 levels observed in the TNFRSF1A + 36G/G carriers. Our results suggest that association between TNFRSF1A +36G/G genotype and the MetS renders obese women more prone to activation of the TNF pathway reflected by high circulating sTNFR1 and PAI-1 levels.

Plasma PAI-1 levels are more strongly related to liver steatosis than to adipose tissue accumulation.

OBJECTIVE: Because obesity and insulin resistance (IR) are strongly associated with liver steatosis (LS), we investigated the relation between the degree of LS and plasminogen activator inhibitor-1 (PAI-1) in ob/ob mice, in C57/BL6 mice with alcoholic LS, and in severely obese humans. METHODS AND RESULTS: In both mouse models, plasma PAI-1 levels were associated with PAI-1 expression in the liver and with the degree of LS. Liver PAI-1 antigen was associated with the tumor necrosis factor receptor-II (TNFRII) antigen, whereas association with TNF antigen content was found in ob/ob mice only. No significant correlation between plasma PAI-1 and PAI-1 expression in adipose tissue of ob/ob mice was observed. Furthermore, the relation between plasma PAI-1 levels and body weight was positive in ob/ob mice but negative in C57/BL6 mice (both P<0.001). In humans, PAI-1 levels were correlated with the degree of LS, and 26% of plasma PAI-1 activity was independently explained by LS and serum insulin levels. CONCLUSIONS: Plasma PAI-1 levels are more closely related to fat accumulation and PAI-1 expression in the liver than in adipose tissue. In steatotic liver, PAI-1 antigen content is associated with those of TNF and TNFRII. Therefore, we suggest that TNF pathway dysregulation in LS could be involved in increased plasma PAI-1 in obesity with IR.

Adipose tissue and atherothrombosis.

Obesity is associated with increased cardiovascular mortality and morbidity mainly through insulin resistance. Dysregulation of protein secretion by adipose tissue is involved in obesity-related diseases. Adipose tissue contributes to create a subinflammatory status which could explain the disturbances in the haemostatic and fibrinolytic systems observed in obesity. Elevated plasma levels of PAI-1 demonstrated the strongest association with the degree of insulin resistance and could be an underlying mechanism for the thrombotic tendency and the progression of atherothrombosis during obesity. The effect of PAI-1 was examined on adipose tissue growth in several mouse models as well as on adipocyte differentiation in vitro. Most of the data indicate that PAI-1 can effectively modulate weight gain and may be a potential therapeutic target for controlling cardiovascular morbidity in obese subjects.

Relationships between fibrinolytic and inflammatory parameters in human adipose tissue: strong contribution of TNFalpha receptors to PAI-1 levels.

Plasminogen activator inhibitor type 1 (PAI-1), a risk marker of atherosclerosis, is highly expressed in adipose tissue from obese subjects. PAI-1 is also considered as an acute phase protein. Recently, adipose tissue has been described as a source of inflammatory cytokines. Therefore, our aim was to study the relationships between PAI-1, and IL-6, TNF, TNF receptors (TNFRSF1s) and TGFbeta1, in plasma and adipose tissue from obese (n = 60) and lean (n = 28) subjects. Study has been extended to plasminogen activators (t-PA and u-PA). Compared to lean subjects, obese subjects exhibited higher plasma levels of all the studied parameters (except for TGFbeta1) whereas in adipose tissue only PAI-1, t-PA and TGFbeta antigen levels differed. In the obese population, plasma PAI-1 levels were weakly associated with circulating TNF, and this relationship disappeared after adjustment for plasma t-PA. Adipose tissue PAI-1 levels were positively associated with TNFRSF1s and TGFbeta, the strongest relationship being observed with TNFRSF1A, which explained 82% of PAI-1 variability. TNF and IL-6 were the main contributors to t-PA variability in plasma and in adipose tissue, respectively. Our results argue on the relevance of TNFRSF1s in the regulation of PAI-1 expression by adipose tissue. Association between t-PA, which is mainly produced by endothelial cells, and IL-6 or TNF suggest that inflammation might be involved in angiogenesis in adipose tissue.

Stromal cells are the main plasminogen activator inhibitor-1-producing cells in human fat: evidence of differences between visceral and subcutaneous deposits.

Elevated plasma plasminogen activator inhibitor (PAI)-1 observed during insulin resistance has been connected with an excessive PAI-1 adipose tissue secretion mainly by visceral fat. Our aim was to compare the localization of PAI-1 in human visceral and subcutaneous fats. PAI-1 secretion was also investigated in vitro during human adipocyte differentiation. PAI-1 antigen and mRNA were localized in the stromal area of the tissue and were also present in a few CD14-positive monocytes, in direct contact with adipocytes. In addition, in subcutaneous tissue, PAI-1 mRNA contents, determined by using real-time polymerase chain reaction, were higher in the stromal fraction than in the adipocyte fraction. PAI-1 mRNA-positive cells were 5-fold more frequent in the visceral area than in the subcutaneous stromal area (P=0.004). Such a difference was also observed for PAI-1 mRNA content between both whole adipose tissues. In contrast to leptin, during adipocyte differentiation, PAI-1 secretion did not follow adipocyte maturation. In situ hybridization in culture did not reveal PAI-1 mRNA in lipid-filled cells. Our results demonstrate that PAI-1 production is mainly due to stromal cells, which were more numerous in the visceral than in the subcutaneous depot. These results could explain the strong relationship observed between circulating PAI-1 levels and the accumulation of visceral fat.