Bone Marrow Stromal Cell-Derived Vascular Endothelial Growth Factor (VEGF) Rather Than Chronic Lymphocytic Leukemia (CLL) Cell-Derived VEGF Is Essential for the Apoptotic Resistance of Cultured CLL Cells

Abstract

Chronic lymphocytic leukemia (CLL) cells feature a pronounced apoptotic resistance. The vascular endothelial growth factor (VEGF) possesses a role in this apoptotic block, although underlying functional mechanisms and the involvement of the microenvironment are unclear. In this study, the VEGF status in CLL was assessed by enzyme-linked immunosorbent assay and immunofluorescence. VEGF receptor 2 (VEGFR2) phosphorylation was determined flow cytometrically and by immunofluorescence. For coculture, CLL cells were cultivated on a monolayer of the bone marrow-derived stromal cell (BMSC) line HS5. Secreted VEGF was neutralized using the monoclonal antibody mAb293 (R&D Systems, Minneapolis, MN, USA). To block protein secretion, we used Brefeldin A. VEGF was downregulated in BMSCs by small interfering RNA (siRNA), and we assessed survival by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. CLL cells express and secrete VEGF and possess phosphorylated VEGFR2. This positive VEGF status is not sufficient to prevent spontaneous apoptosis in vitro. Coculture with BMSCs, which secrete vast amounts of VEGF, maintains in vitro CLL cell survival. Blockage of secreted VEGF using the monoclonal antibody mAb293 significantly reduced the survival support for cocultured CLL cells. Both general blockage of protein secretion by Brefeldin A in BMSCs, but not in CLL cells, and siRNA-mediated downregulation of VEGF in BMSCs, significantly reduced the coculture-mediated survival support for CLL cells. It can be concluded that BMSC-derived proteins and VEGF, in particular, but not CLL cell-derived VEGF, is essentially involved in the coculture-mediated survival support for CLL cells. Hence, therapeutic targeting of VEGF signaling might be a promising approach to overcome the apoptotic resistance CLL cells feature within their natural microenvironment.

Angiogenic factors in chronic lymphocytic leukemia

“…A recent in vitro study showed that bone marrow stromal cell (BMSC)-derived VEGF was essentially involved in the stromal cell-mediated survival support for CLL cells. Researchers found only the blocking of protein secretion of BMSCs, rather than CLL cells, could cause a significant reduction of CLL cell survival in coculture [25].…”

Vascular Endothelial Growth Factor A (VEGFA) Gene Polymorphisms Have an Impact on Survival in a Subgroup of Indolent Patients with Chronic Lymphocytic Leukemia

“…Several reports have shown an enhanced microvessel density in the bone marrow and lymph nodes in patients with CLL, as a result of a VEGF-dependent angiogenesis associated with an advanced stage of disease [1]–[3]. Furthermore, it has also been reported that the resistance to apoptosis of leukemic cells in CLL is mediated by VEGF-dependent autocrine and paracrine mechanisms of cell survival [4]–[7]. In addition to the angiogenic and antiapoptotic effects on CLL cells, VEGF regulates CLL cell motility [8], [9] and the microenvironment-tumor interactions [10], [11].…”

Vascular Endothelial Growth Factor Levels in Childhood Acute Lymphoblastic and Myeloblastic Leukemia

“…They found that these cell lines express many cytokines, growth factors, and negative regulators (VEGF, FGF, IL1-6-7-8, TGF, and PDGF). Also in a recent study it is found that leukemia cell derived VEGF is not effective as bone morrow stromal cell derived ones on the VEGF receptor for apoptotic resistance of the leukemic cells [27]. VEGF expression is affected from other factors as RUNX1 negatively in the AML patients although VEGF is found to be independent prognostic importance in this patient group [28].…”