Angiogenesis is a highly regulated multistep process that involves endothelial cell chemotactic migration, invasion, proliferation, differentiation into tubular capillaries, and the production of a basement membrane around the vessels (Folkman, 1995; Kesisis et al., 2007). In this study, CBD exhibited potent anti-angiogenic properties, inhibiting HUVEC growth, migration and invasion in vitro as well as angiogenesis in the Matrigel sponge assay in vivo. Molecular studies in vitro demonstrated that CBD exerts its effects through the down-regulation of several angiogenic mechanisms. Taken together, these data suggest that CBD has great potential as a new anti-angiogenic drug.
Our results demonstrated that CBD was effective in inhibiting endothelial cell proliferation without inducing endothelial cell apoptosis or necrosis, suggesting a cytostatic action. Several chemotherapeutic drugs have anti-angiogenic properties only at near or fully cytotoxic concentrations; therefore, their clinical relevance is controversial. Interestingly, CBD did not induce HUVEC apoptosis or necrosis even at the highest dose tested (12 µM). Several anti-angiogenic molecules inhibit endothelial cell proliferation, without exerting any cytotoxic action and can even inhibit apoptosis in endothelial cells (Fassina et al., 2004; Lorusso et al., 2009; Noonan et al., 2011b). This seems to be in contrast to their effects on tumour cells, where often these compounds are cytotoxic, at least at high doses. However, this may depend on several characteristics of tumour cells, including chronic stress related to high-level production of oxygen and other radicals, metabolic alterations and oncogene dependence (Ferrari et al., 2010). Further cellular stress in tumour cells pushes the cell over the threshold and into apoptosis, whereas in normal cells, this may act as a form of ‘preconditioning’ stimulus that renders the cells more resistant to subsequent insults (Ferrari et al., 2010).
CBD’s lack of cytotoxicity towards endothelial cells is quite different from that reported in previous studies with other cannabinoids (Blázquez et al., 2003). In these studies, endothelial cell cytotoxicity was considered a potential mechanism of action. Our data suggest that, unlike other cannabinoids, the effects of CBD are not due to endothelial cell toxicity but rather to modulation of intracellular pathways leading to a decrease in several pro-angiogenic factors.
CBD showed potent inhibition of endothelial cell migration, both in the scratch wound-healing assay and, with an even stronger effect, in the Boyden chamber assay. The different efficacy of CBD in these tests could be ascribed to the different sensitivity of the assays, since the wound-healing assay is generally less sensitive than the Boyden chamber. Moreover, the use of primary fresh HUVECs in the Boyden chamber in comparison with the commercially available cells employed in the wound-healing assay could account for the different potency observed. However, in our hands, CBD elicited significant effects on migration at concentrations lower than those causing 50% inhibition of proliferation (9 µM and 1 µM, respectively, in wound healing and Boyden chamber assays versus IC50 value of 10 µM in the MTT assay run in parallel to the migration tests). We previously demonstrated the same order of potency of CBD on U87-MG glioma cell proliferation versus invasion (Vaccani et al., 2005). In agreement with these results, Marcu et al. (2010) showed that CBD was more potent at inhibiting U251 invasiveness compared to their proliferation. Thus, similar to glioma, CBD is highly potent at inhibiting endothelial cell migration compared to proliferation, suggesting that factors influencing cell migration and invasion may represent its primary targets.
In line with this, our molecular investigation showed that CBD affected the expression of several prominent factors involved in primary vascular endothelial cell functions; in particular compounds that induce invasion and migration, which included MMP2 and MMP9, TIMP1, SerpinE1/PAI1, uPA, CXCL16, IL-8, ET-1 and PDGF-AA.
CBD inhibited MMP2 and MMP9, two fundamental proteases that, through the remodelling of the extracellular matrix and basement membrane, are involved in distinct vascular events, and whose levels are increased in numerous malignancies, including glioma (Cantelmo et al., 2010; Pisanti et al., 2011; Noonan et al., 2011b).
CBD down-regulated the expression of TIMP1, a stromal factor with multiple functions. TIMPs are commonly described as negative regulators of MMPs. Nasser et al. (2006) showed that TIMP1 is an inhibitor of high-grade glioma invasion. In line with this, Ramer et al. (2010a) recently reported a CBD-driven increase in TIMP1 in lung cancer cells that correlated with diminished invasiveness. Nevertheless, there is increasing evidence to suggest that TIMPs are multifunctional proteins, possessing a dual role in regulating cell proliferation and angiogenesis. In vitro, TIMP1 promotes growth of human keratinocytes and several other cell types (Bertaux et al., 1991; Hayakawa et al., 1992), inhibits apoptosis (Alexander et al., 1996; Guedez et al., 1998; Li et al., 1999) and regulates angiogenesis (Yoshiji et al., 1998; Lafleur et al., 2002). Moreover, increased expression of TIMP1 protein has been observed in multiple tumour types, including breast, colon, gastric and lung cancers, as well as in lymphoma and carcinomas of unknown primary origin (Zeng et al., 1995; Mimori et al., 1997; Ree et al., 1997; Guedez et al., 2001; Schrohl et al., 2004; Gouyer et al., 2005; Karavasilis et al., 2005).
Based on these considerations, it is noteworthy that inhibition of proteins such as MMP2 and MMP9 and TIMP1 further confirms the wide spectrum of CBD action on MMP and TIMP molecules, key factors in cell motility, invasion and proliferation, and suggests a complex picture through which CBD can impair cell growth and invasion.
In addition to the MMP/TIMP system, CBD also down-regulated the uPA and the plasminogen activator inhibitor SerpinE1/PAI-1, two important factors in extracellular matrix remodelling and consequent angiogenesis. The uPA plays a pivotal role in the degradation of extracellular matrix, and suppression of uPA and uPAR by shRNA attenuates angiogenin-mediated angiogenesis in endothelial and glioblastoma cell lines (Raghu et al., 2010). Thus, CBD shares similarities with other therapeutic approaches that, by inhibiting the uPA/uPAR functions, have been shown to possess anti-angiogenic and anti-tumour effects (for review, see Ulisse et al., 2009).
Since SerpinE1/PAI-1 inhibits uPA, low levels of this protein would be expected to favour cell growth. However, recent data have revealed a two-faced role in the modulation of apoptosis in tumour cells in comparison with non-tumour cells. At present, the reason for these discrepant effects is still unclear and some recent reports point to other multifunctional roles of this protein in angiogenesis, invasiveness and cell adhesion (Ulisse et al., 2009).
CBD also significantly inhibited two potent angiogenic factors: the chemokines CXCL16 and IL-8 (Rabquer et al., 2011). Stimulation of HUVECs with CXCL16 leads to increases in cell proliferation, chemotactic motility and network formation (Zhuge et al., 2005), whereas IL-8 can induce angiogenesis through both direct and indirect mechanisms (Benelli et al., 2002; 2003; Lai et al., 2011). The decreased level of both chemokines following CBD treatment would be consistent with its in vitro and in vivo anti-angiogenic effects.
Two growth factors were also down-regulated by CBD: ET-1 and PDGF-AA. ETs modulate various stages of neovascularization. Increased levels of ET-1 and its cognate receptor are significantly associated with microvessel density and VEGF expression in tumour cells, whereas its down-regulation correlates well with diminished endothelial cell growth and migration (Bagnato et al., 2008).
PDGF-AA is a member of the well-known PDGF family that exerts its angiogenic effect in endothelial cells by binding to a specific protein tyrosine kinase receptor, which in its turn engages several signalling molecules involved in multiple cellular and developmental responses.
Taken together, these observations suggest a broad effect of CBD on vascular endothelial cell biology. This wide spectrum modulation of angiogenesis-related factors leads to a decreased ability of endothelial cells to properly form new vessels in vitro and, to an even more prominent extent, impaired angiogenesis in in vivoplugs.
However, the molecular mechanism through which CBD exerts these effects still remains unknown. Vascular endothelial cells express various functional receptors for cannabinoids, including the CB1 receptor (Liu et al., 2000), the CB2 receptor (Blázquez et al., 2003), the tentative abnormal CBD receptor (Járai et al., 1999), the TRP receptors (Golech et al., 2004; Curry and Glass, 2006; Kwan et al., 2007) and the PPARγ (O’Sullivan et al., 2009; Yokoyama et al., 2011): each of these could, at least in part, be involved in CBD anti-angiogenic effects. These receptors control important cell functions such as migration (Blázquez et al., 2003; Mo et al., 2004), survival (Blázquez et al., 2003), vascular tone (Wagner et al., 1997; Bátkai et al., 2001) and tumour-derived endothelial cell migration (Fiorio Pla et al., 2012). Recently, blockade of the CB1 receptor has been closely linked to inhibition of angiogenesis (Pisanti et al., 2011). Our present data do not allow us to indicate a receptor-dependent versus -independent mechanism of CBD in HUVECs. Since both cannabinoid-dependent (McKallip et al., 2002; 2006; Ligresti et al., 2006; Ramer et al., 2010a,b; Aviello et al., 2012) and -independent (Massi et al., 2004; Vaccani et al., 2005; Shrivastava et al., 2011) mechanisms were previously shown for CBD anti-tumour effects, it is also possible that its anti-angiogenic activity may be due to a receptor-independent mechanism, involving different primary cellular targets. In line with this, recent studies (McAllister et al., 2007; 2011) have demonstrated that the anti-invasive and anti-proliferative effects of CBD in breast cancer are closely associated with inhibition of Id-1, an inhibitor of basic helix-loop-helix transcription factors that is over-expressed in tumour cells. Id proteins play a vital role in regulating angiogenesis during embryonic development and tumourigenesis and ectopic Id-1 expression in HUVECs leads to increased migration of the cells, while suppression of its endogenous expression results in reduced migration (Qiu et al., 2011). Thus, Id-1 could represent a key signalling pathway for CBD in HUVECs.
In conclusion, our results indicate that CBD exerts a potent anti-angiogenic effect by widely affecting several pathways involved in this process. Its dual effect on both tumour and endothelial cells further suggests that CBD could represent a potential effective agent in cancer therapy.
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