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Enhanced tissue factor expression by blood eosinophils from patients with hypereosinophilia: a possible link with thrombosis.

Cugno M, Marzano AV, Lorini M, Carbonelli V, Tedeschi A - PLoS ONE (2014)

Bottom Line: Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis.TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05).The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milano, Italy; Medicina Interna, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

ABSTRACT
Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis. In particular, eosinophils have been described as source of tissue factor (TF), the main initiator of blood coagulation; however, this aspect is still controversial. This study was aimed to evaluate whether TF expression varies in eosinophils isolated from normal subjects and patients with different hypereosinophilic conditions. Eosinophils were immunologically purified from peripheral blood samples of 9 patients with different hypereosinophilic conditions and 9 normal subjects. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were performed to test eosinophil TF expression. For comparison, TF expression was evaluated in monocytes from blood donors and in human endothelial (ECV304) and fibroblast (IMR90) cell lines. Western blot analysis revealed a major band of 47,000 corresponding to native TF in homogenates of purified eosinophils with a higher intensity in the 9 patients than in the 9 controls (p<0.0001). According to RT-PCR cycle threshold (Ct), TF gene expression was higher in eosinophils from patients than in those from controls, median (range) 35.10 (19.45-36.50) vs 37.17 (35.33-37.87) (p = 0.002), and was particularly abundant in one patient with idiopathic hypereosinophilic syndrome and ischemic heart attacks (Ct: 19.45). TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05). Our results indicate that human blood eosinophils contain variable amounts of TF. The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

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Panel A shows real-time polymerase chain reaction (RT-PCR) analysis of tissue factor (TF) in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines).Panel B shows RT-PCR analysis of beta-actin in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines). Panel C shows RT-PCR analysis of TF in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines). Panel D shows RT-PCR analysis of beta-actin in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines).
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pone-0111862-g004: Panel A shows real-time polymerase chain reaction (RT-PCR) analysis of tissue factor (TF) in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines).Panel B shows RT-PCR analysis of beta-actin in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines). Panel C shows RT-PCR analysis of TF in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines). Panel D shows RT-PCR analysis of beta-actin in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines).

Mentions: Real-time polymerase chain reaction (RT-PCR) analysis revealed different amplifications in 9 patients with hypereosinophilia using TF specific sets of primers and probes (Figure 4). As shown in table 2 and in figure 4, TF cycle threshold was significantly lower in patients with hypereosinophilia than in healthy subjects, median (range) 35.10 (19.45–36.50) vs 37.17 (35.33–37.87) (p = 0.002), indicating that TF gene expression was higher in hypereosinophilic disorders. Interestingly, the two patients with idiopathic hypereosinophilic syndrome and ischemic heart attacks, showed the lowest TF cycle threshold (19.45 and 33.68) indicating an enhanced TF gene expression. The cycle thresholds of the housekeeping gene beta actin in patients and controls ranged between 27.79 and 36.31, without any significant differences between the two groups (Table 2). Considering the beta-actin controls, the relative quantification of PCR data confirmed a significantly higher expression of TF mRNA in patients with hypereosinophilia than in normal subjects (Table 2).


Enhanced tissue factor expression by blood eosinophils from patients with hypereosinophilia: a possible link with thrombosis.

Cugno M, Marzano AV, Lorini M, Carbonelli V, Tedeschi A - PLoS ONE (2014)

Panel A shows real-time polymerase chain reaction (RT-PCR) analysis of tissue factor (TF) in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines).Panel B shows RT-PCR analysis of beta-actin in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines). Panel C shows RT-PCR analysis of TF in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines). Panel D shows RT-PCR analysis of beta-actin in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222944&req=5

pone-0111862-g004: Panel A shows real-time polymerase chain reaction (RT-PCR) analysis of tissue factor (TF) in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines).Panel B shows RT-PCR analysis of beta-actin in purified eosinophils from 9 patients with hyperesinophilia (HE, red lines) and 9 normal controls (N, blue lines). Panel C shows RT-PCR analysis of TF in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines). Panel D shows RT-PCR analysis of beta-actin in purified monocytes from 4 normal controls (green lines), in 4 samples of endothelial cell line ECV304 (blue lines) and in 8 samples of fibroblast cell line IMR90 (red lines).
Mentions: Real-time polymerase chain reaction (RT-PCR) analysis revealed different amplifications in 9 patients with hypereosinophilia using TF specific sets of primers and probes (Figure 4). As shown in table 2 and in figure 4, TF cycle threshold was significantly lower in patients with hypereosinophilia than in healthy subjects, median (range) 35.10 (19.45–36.50) vs 37.17 (35.33–37.87) (p = 0.002), indicating that TF gene expression was higher in hypereosinophilic disorders. Interestingly, the two patients with idiopathic hypereosinophilic syndrome and ischemic heart attacks, showed the lowest TF cycle threshold (19.45 and 33.68) indicating an enhanced TF gene expression. The cycle thresholds of the housekeeping gene beta actin in patients and controls ranged between 27.79 and 36.31, without any significant differences between the two groups (Table 2). Considering the beta-actin controls, the relative quantification of PCR data confirmed a significantly higher expression of TF mRNA in patients with hypereosinophilia than in normal subjects (Table 2).

Bottom Line: Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis.TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05).The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milano, Italy; Medicina Interna, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

ABSTRACT
Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis. In particular, eosinophils have been described as source of tissue factor (TF), the main initiator of blood coagulation; however, this aspect is still controversial. This study was aimed to evaluate whether TF expression varies in eosinophils isolated from normal subjects and patients with different hypereosinophilic conditions. Eosinophils were immunologically purified from peripheral blood samples of 9 patients with different hypereosinophilic conditions and 9 normal subjects. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were performed to test eosinophil TF expression. For comparison, TF expression was evaluated in monocytes from blood donors and in human endothelial (ECV304) and fibroblast (IMR90) cell lines. Western blot analysis revealed a major band of 47,000 corresponding to native TF in homogenates of purified eosinophils with a higher intensity in the 9 patients than in the 9 controls (p<0.0001). According to RT-PCR cycle threshold (Ct), TF gene expression was higher in eosinophils from patients than in those from controls, median (range) 35.10 (19.45-36.50) vs 37.17 (35.33-37.87) (p = 0.002), and was particularly abundant in one patient with idiopathic hypereosinophilic syndrome and ischemic heart attacks (Ct: 19.45). TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05). Our results indicate that human blood eosinophils contain variable amounts of TF. The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

Show MeSH
Related in: MedlinePlus