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Page 8 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103

Other levels of regulation - epigenetic and gene polymorphism
Angiogenic molecules in CLL cells can also be regulated through other molecular mechanisms, including
gene polymorphisms and epigenetic alterations. The genomic polymorphism in VEGF-A of CLL cells has
been extensively studied. According to Lozano-Santos et al. , a specific VEGF haplotype correlates with
[93]
shorter survival in CLL patients. Furthermore, patients who carry this haplotype in combination with other
[93]
clinical features, such as negative CD38 expression or early age at diagnosis, show a poor survival ratio .
Other studies have also indicated that VEGF polymorphism might be relevant as a genetic risk and adverse
survival marker in CLL [94,95] . On the other hand, epigenetic profiles might serve to stratify patients and
identify different molecular pathways involved in CLL progression. For example, the expression of the
[96]
Ang-2 gene promoter in CLL cells is highly dependent on the DNA methylation status . This methylation
is regulated by VEGF, which promotes Ang-2 expression and neovascularization . In agreement with the
[96]
notion that DNA methylation regulates critical steps during CLL angiogenesis, it has been reported that
Ang-2 expression is also regulated by the tumor suppressor gene microcephalin . Furthermore, other
[97]
[98]
angiogenic molecules such as endothelin-1 are also regulated by DNA methylation in CLL cells .
CONTRIBUTION OF CLL CELLS TO THE ANGIOGENIC STATUS OF LYMPHOID TISSUES
Functional effects on endothelial cells
The above-mentioned studies have clearly established that the molecular and cellular components of the
microenvironment in CLL tissues modulate the gene expression pattern of CLL cells, inducing a pro-
survival and proangiogenic phenotype in the malignant cells. It is also known that modulation of angiogenic
[38]
molecules in CLL has functional consequences in the CLL niches [Figure 1]. Chen et al. showed that the
conditioned medium (CM) of CLL cells cultured for 24 h contained VEGF and induced proliferation of
endothelial cells. Moreover, this CM also induced moderate in vitro angiogenesis, and this effect was
[38]
prevented by a neutralizing anti-VEGF antibody . It was later demonstrated that the CM of cultured CLL
cells also contained Ang-2, and the levels of this protein correlated with the degree of bone marrow
vascularization of the patients studied . These authors also showed that both Ang-2 and VEGF were
[84]
responsible for the CM-induced tube formation by endothelial cells on Matrigel matrices, as neutralizing
antibodies to either factor diminished the angiogenic effect . In both studies, a hypoxic stimulus further
[83]
increased the functional effects of the CLL cell CM on endothelial cells. Additionally, we showed that the
CM of CLL cells that had been incubated with MMP-9 for 24 h had a significantly higher effect on
[79]
endothelial cell proliferation compared to the CM of CLL cells incubated on 0.5% BSA . This is likely due
to the MMP-9-induced production of proangiogenic factors (MMP-9 and VEGF) by CLL cells mentioned
above.

Functional effects on mesenchymal stromal cells
Besides affecting endothelial cell behavior, CLL cells also influence the function of other cells present in the
microenvironment of CLL niches . An active crosstalk between CLL and mesenchymal stromal cells
[29]
(MSC), resulting in malignant cell survival and changes in the pattern of cytokine production by MSC, has
been reported . Moreover, the CM of CLL cells was shown to drive migration and proliferation of MSC .
[99]
[52]
This effect was mediated by the interaction of the PDGF present in the CM of CLL cells with its PDGFR in
mesenchymal cells, which resulted in activation of the receptor and induction of Akt phosphorylation.
Binding of the CLL-derived PDGF to PDFGR also induced the production of VEGF (but not bFGF or
TSP-1) by mesenchymal cells through a PI3-K-dependent mechanism . This is a clear example of how
[52]
CLL cells actively modulate their microenvironment, inducing an angiogenic switch that is permissive for
CLL progression.