Overexpression of tumstatin in genetically modified megakaryocytes changes the proangiogenic effect of platelets

Thrombocytopenia is a common side effect of tumor chemotherapy, the main management approach to which is based on platelet (PLT) transfusion. However, PLTs, containing angiogenesis regulators, play a major role in boosting tumor growth and metastasis. The purpose of the study was to determine whether PLTs have the capacity to overexpress tumstatin by modified megakaryocyte (MK) and PLT precursors using lentivirus-mediated gene transfer, which might lead to alteration in proangiogenic effect of PLTs.

CD34+ hematopoietic stem cells (HSCs) were transduced with recombinant lentivirus carrying tumstatin and induced to produce MKs and PLTs in the culture medium containing a cytokine cocktail. Flow cytometry and aggregation test were used to detect the generation and function of MKs and PLTs. Western blot analysis and confocal microscopy were applied to examine the expression and distribution of tumstatin in transgenic MKs and PLTs. Capillary tube formation of human umbilical vein endothelial cells (HUVECs) was used to evaluate the inhibitory effect of transgenic PLTs.

CD34+ HSCs can be efficiently transduced with lentivirus vectors and successfully differentiated into MKs and PLTs. Large amounts of functional MKs and PLTs could be generated and had correct biologic characteristics. The tests demonstrated the feasibility of tumstatin expression in MKs and PLTs under control of the cytomegalovirus promoter, that thus tumstatin was stored in the α-granules of PLTs, and that the releasate of thrombin or A543 cell-stimulated transgenic PLTs obviously inhibited the growth of capillary tube network structures of HUVECs.

Gene-modified CD34+ HSCs not only successfully differentiated into MKs and PLTs but also expressed tumstatin protein. Release of tumstatin in transgenic PLT granules led to antiangiogenic effect of PLTs.