Page 183 - 2019_10 resto del Mondo_web

BCL2L2 promotes MK numbers and PPF
in MKpoiesis.12,13,20 However, BCL2L2 (encoding Bcl-w) has not been studied in Mkpoiesis and became the focus of further investigation. The increase in expression of BCL2L2 observed by RNA sequencing was verified by qPCR on RNA isolated from CD61-purified MK (Figure 4B). This led to the hypothesis that those MK with increasing levels of anti-apoptotic BCL2L2 would be the LLG, whereas those MK with stable or decreasing levels of anti-apoptotic BCL2L2 would be the SHG. LLG MK and SHG MK were purified by cell sorting and analyzed for changes in BCL2L2 expression over time in culture. There was no difference between LLG and SHG cells for BCL2L2 expression at days 6 and 9, but a significant difference was observed by day 13 due to a fall in BCL2L2 in SHG cells and concomitant rise in LLG cells (Figure 4C).
We next considered the effects of BCL2L2 on cultured MK apoptosis and number. Initially, we used the general Bcl-2 family inhibitor, ABT-263, to screen for an effect on CD41a+ LLG MK numbers, and observed a significant reduction (Online Supplementary Figure S5). Since ABT-263 inhibits all Bcl-2 family members, we specifically tested the effects of BCL2L2 on the cultured MK. CB-derived MK cultures were transduced with lentiviral vectors con- taining BCL2L2, and both mRNA and protein increased by day 13 (Figure 5A-C). BCL2L2 overexpression significant- ly reduced the percentage of annexin V+ CD41a+ MK (Figure 5D) and increased the number of CD41a+ LLG MK by 19% (1.36x105 to 1.61x105; P=0.049).
A
BC
Figure 4. Expression of the anti-apoptotic gene BCL2L2 increases functional megakaryocytes (MK). CD61+ MK RNA was isolated from day 6 and day 13 cultures for analyses in (A) and (B). (A) CD61-positive (+) MK expression of 24 apoptosis-related genes was assessed by RNA sequencing (n=3). Average fold changes in expres- sion for day 13 versus day 6 are shown. (B) CD61+ MK BCL2L2 quantification by quantitative polymerase chain reaction (qPCR) (n=4). (C) Larger size, lower granu- larity (LLG) and smaller size, higher granularity (SHG) MK were flow cytometrically sorted and BCL2L2 expression was quantified by qPCR. Fold changes were deter- mined for LLG and SHG MK compared to their respective day 6 expression (n=4 for day 6 and day 13; n=6 for day 9).
BCL2L2 regulates megakaryocyte proplatelet formation Megakaryocyte PPF is believed to be a critical process in thrombopoiesis. By day 13 we observed MK PPF primari- ly in LLG MK (Figure 3C), and asked whether Bcl-w might affect PPF. Importantly, when we scored MK PPF blinded to lentiviral transduction treatment group, we found that BCL2L2 overexpression induced a significant 58%
increase in PPF MK (Figures 5E and F).
Relationship between BCL2L2 expression and platelet number
Megakaryocyte PPF is tightly linked to PB platelet count,10,21,22 raising the possibility that BCL2L2 may regu- late platelet number as well as MKpoiesis. Because we had previously performed genome-wide platelet gene expression profiling in 154 healthy individuals,14 we were able to query this dataset for such an association. Platelet BCL2L2 mRNA levels were positively correlated with platelet count in the PRAX1-1 study (Figure 6A).
As is typical of MK culture systems, the day 13 cultures also contained a population of small, hypogranular parti- cles that overlapped the forward and side scatter proper- ties of normal human platelets (Figure 6B). These particles are typically referred to as platelet-like particles (PLP), and we assessed the effect of MK BCL2L2 overexpression on PLP number and function. Compared to control lentiviral transduction, BCL2L2 overexpression induced a modest but significant increase in CD41a+ PLP from approximate-
haematologica | 2019; 104(10)
2079