In this study, the expression of gal-1, gal-3, pSTAT3 and pSTAT5 along with the MVD in bone marrow cells was immunohistochemically measured in ET, PV, PMF and control bone marrows.
Gal-1 is known to be involved in tumour angiogenesis [7
]. The higher expression of gal-1 and MVD in the total group of MPN patients in our study together with a significant correlation between gal-1 and MVD, suggests a role of gal-1 in the increased angiogenesis in MPN patients. These results assign a possible target for the angiogenesis inhibitor anginex, as gal-1 was identified as receptor for anginex. Anginex blocks the adhesion and migration of angiogenically activated endothelial cells, leading to apoptosis and inhibition of angiogenesis [22
]. In gal-1-null mice treatment with anginex did not inhibit tumour growth in contrast to the wild type mice where tumour growth and vessel density was significantly inhibited with anginex treatment [7
Increased expression of gal-3 has been associated with liver fibrosis secondary to diverse types of injury [11
]. However, in the mf 0/1 group we saw a higher gal-3 expression compared to the mf 2/3 group. Also we saw no significant correlation between gal-3 and MVD. These findings contradict the relation between increasing fibrosis, MVD and gal-3 expression in MPN trephines. On the other hand we were able to show higher gal-3 expression in PV patients. Recently, it was also demonstrated that gal-3 is predominantly expressed in Chronic Myeloid Leukemia (CML) cells, where gal-3 expression support the molecular signalling pathways for maintaining CML in the bone marrow and resistance to therapy [23
]. Therefore there are indications that gal-3 might play a role in MPN pathogenesis.
Constitutive activation of STAT proteins is present in a variety of haematological disorders [25
]. STAT3 activation has been reported in PV and ET and low pSTAT3 levels in PMF patients [17
]. However, our study does not confirm these results, possibly due to a relative high amount of PMF patients and lower amounts of PV and ET patients.
Activated STAT3 has an important role in the regulation of megakaryopoiesis and thrombopoiesis in vivo
, via activation of Bcl-xL inhibiting apoptosis of megakaryocytes [31
]. The bone marrow of PMF patients is characterized by a proliferation of the megakaryocytic cell line. The megakaryocytes often demonstrate dense clustering with cloud like nucleus [20
]. The increased megakaryocytes with deviated forms in the bone marrow of PMF patients might be due to the decreased megakaryocyte apoptosis as result of increased STAT3 activation in PMF patients. The higher pSTAT3 expression in JAK2V617F
positive patients indicates an increased STAT3 activation generated by the presence of the JAK2V617F
mutation. In diverse cancer types it was shown that constitutive activation of STAT3 induces vascular endothelial growth factor (VEGF) expression [19
]. In our study we show a correlation between pSTAT3 and MVD, indicating that the increased MVD seen in MPN patients, especially in PMF patients, might be induced by the constitutive activation of STAT3 resulting in increased expression of VEGF.
Our finding of higher pSTAT5 expression in PV and JAK2V617F
positive patients is in line with earlier published data [14
]. This indicates that the presence of the JAK2V617F
mutation generates increased levels of pSTAT5. However, in our study the pSTAT5 expression did not reach statistical significant difference but only showed a trend between patients carrying the JAK2V617F
mutation and patients without the mutation as well as in PV patients compared to ET and PMF patients. This might be due to the high number of patients with an unknown JAK2 status and also to the small PV patient population. The correlation between pSTAT5 and MVD might suggest other pathways involved in the increased MVD seen in MPN patients. pSTAT5 can interact with p85, a regulatory subunit of PI3K/Akt pathway, and might increase VEGF via the PI3K/Akt and mammalian target of rapamycine (mTor) pathway as was already shown in chronic myeloid leukaemia (CML) [34
In line with other studies[37
], we found the bone marrow MVD in the total MPN group and in PV and PMF patients to be significantly higher compared to the control group. The increased MVD reflects increased angiogenic activity which might be induced by hypoxia, via hypoxia-inducible factor (Hif) and VEGF, or by normoxia, directly via VEGF.
Regarding the MVD and fibrosis in MPN patients, Boveri et al. [39
] found a higher MVD along with a higher grading of fibrosis, which is line with our study. Other studies showed higher MVD in PMF, post-ET myelofibrosis and post-PV myelofibrosis patients compared to ET and PV patients indicating that angiogenesis is primarily involved in later stages of the disease [38
In conclusion, the characteristic megakaryopoietic abnormalities and also the higher MVD expression in PMF trephines can be explained by a higher pSTAT3 expression in PMF patients. Also gal-1 expression is correlated with the MVD with anginex as potential new therapy for MPN patients. pSTAT5 expression showed a trend of higher expression in PV and JAK2V617F positive patients, possible induced by the JAK2V617F mutation and also gal-3 expression seems correlated with PV. Further, the increased MVD expression in MPN patients with higher myelofibrosis grading suggests the important role of angiogenesis in the development of myelofibrosis.
Based upon these data we support the concept that the microenvironment plays an important role in haematological malignancies [42
]. Interactions between stroma and haematopoietic cells in MPNs constitute possible targets for therapy.