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expression. Consistent with this decrease, there was a correlation between advanced CLL (Binet stages B and
C) and low TSP-1 plasma levels, in agreement with the studies mentioned above. Additionally, MMP-9 was
also dramatically upregulated (25.9-fold change) upon CLL cell contact with the fibroblastic cells, compared
[32]
[85]
with freshly isolated CLL cells , a fact also confirmed by Schulz et al. . In line with this upregulation, we
showed that culturing CLL cells on primary stromal cells (derived from a CLL patient) for 48 h increases the
[81]
amount of MMP-9 bound to the CLL cell surface by two-fold . Moreover, CLL cells isolated from the bone
marrow or lymph nodes of patients consistently showed higher levels of surface-bound MMP-9 than their
peripheral blood counterparts, confirming the increased production of MMP-9 in a pathophysiological
context . This MMP-9, which is produced by the CLL cells as well as by stromal cells, likely contributes to
[81]
the increased angiogenic status observed in CLL tissues (see below).

Endothelial cells are also an important stromal component of lymphoid tissues [Figure 1]. Maffei et al.
[33]
showed that physical interaction of CLL cells with human umbilical vein endothelial cells protected CLL
cells from spontaneous and drug-induced apoptosis. This interaction was mediated by β1 and β2 integrins,
and it also resulted in the modulation of 1944 genes, 1217 upregulated and 727 downregulated, determined
[33]
by gene microarray analyses . Many of the upregulated genes were related to angiogenesis and included
the Ang-2 receptors Tie-1 and Tie-2, VEGFC, TSP-1, MMP-2, and MMP-14, while VEGFR3 was
downregulated. The C-C motif chemokine-2 (CCL2), which is known to regulate angiogenesis and recruit
monocytes/macrophages to tissues , was also significantly upregulated in CLL cells upon coculture with
[86]
[33]
endothelial cells . It can be concluded that, in lymphoid tissues, particularly in the bone marrow, these
CLL-produced factors disturb the angiogenic balance and contribute to disease progression.
Autocrine and inter-regulation among angiogenic factors
The fact that CLL cells express angiogenic factors and their receptors supports the existence of autocrine
and crosstalk regulations of the expression and function of these factors. These regulations may take place
on circulating CLL cells as well as in CLL cells located in niches. On isolated CLL cells, Bauvois et al.
[40]
showed that antibodies to VEGF decreased MMP-9 expression, suggesting a link between both proteins. We
showed that binding of VEGF to its VEGFR2 receptor in CLL cells downregulates the expression of MMP-9
as well as the migration of these cells through Matrigel or endothelial cells . Regulation of MMP-9 by the
[87]
VEGF/VEGFR2 axis was at the transcriptional level and was mediated by the phosphorylation and
[87]
translocation to the nucleus of the signal transducer and activator of transcription 1 (STAT1) , a factor
[88]
known to suppress MMP-9 gene transcription in response to interferons in several cell systems .
Using immunofluorescence analyses, our group also demonstrated that MMP-9 is present in the bone
marrow and lymph nodes of CLL patients, in partial association with the macrophages in these tissues .
[79]
We studied whether, as a component of the CLL microenvironment, MMP-9 affected the CLL cell
angiogenic pattern. Culturing CLL cells on immobilized MMP-9 for 24 h increased the expression of
MMP-9 and VEGF and reduced the expression of the angiostatic molecules TSP-1 and Ang-2, all at the
gene and protein levels , establishing that isolated MMP-9 is able to induce a proangiogenic profile in CLL
[79]
cells. The fact that MMP-9 interaction with its receptors in CLL cells increases its own production may
[20]
represent an autocrine positive feedback loop, similar to that described for VEGF in these cells .

Further mechanistic studies demonstrated that downregulation of TSP-1 by MMP-9 involved α4β1 integrin
(MMP-9 receptor), Src kinase family activity, and the STAT3 transcription factor . The fact that STAT3 is
[79]
also a transcription factor for VEGF, regulates the expression and degradation of HIF-1α, and is a key factor
in angiogenesis [89-92] strongly suggests that upregulation of VEGF by MMP-9 is also mediated by STAT3
activation.