In the present study, we evaluated the contribution of three host MMPs (MMP-2, MMP-3, and MMP-9) to the invasion and vascularization of malignant keratinocyte tumors transplanted into syngeneic MMP-deficient mice. The angiogenic and invasive phenotype of malignant cells was not affected by the single deficiency of host MMP-2, MMP-3, or MMP-9, or the combined deficiency of MMP-3 and MMP-9. Thus, although MMP-3 has a skin wound healing phenotype (32
) and can activate MMP-9, acting upstream in the cascade of MMP activation (2
), it has no significant contribution in this tumor model. However, we provide evidence that both MMP-2 and MMP-9 contribute to tumor invasion and vascularization. Tumor invasion and angiogenesis were impaired by the combined deficiency in both gelatinases.
In other models, the single deficiency of one of the gelatinase gene demonstrated a partial role of either MMP-9 or MMP-2 in the angiogenic switch (16
). We have recently reported the synergistic contribution of MMP-2 and MMP-9 to choroidal neoangiogenesis induced by a laser burn (34
). While both incidence and severity of choroidal angiogenesis were partially affected in single MMP-deficient mice, they were strongly attenuated in double-deficient mice.
A direct role for MMP-9 in the regulation of angiogenesis, at least via a modulation of vascular endothelial growth factor (VEGF) availability, has been reported in previous studies (6
). The important contribution of MMP-9 in cancer progression has been further supported by the correlation of a low level of active MMP-9 and a suppression of spontaneous tumor growth in thrombospondin-deficient mice (35
). However, MMP-9 also suppresses tumor angiogenesis through production of angiostatic fragments of basement membrane collagens (36
We detected MMP-9 promoter activity in vivo in the host stroma, when we transplanted MMP-9/LacZ mice with malignant keratinocytes, as demonstrated by β-galactosidase staining. However double immunostaining showed that neutrophils are the main cellular source of MMP-9 in our model. This is consistent with previous studies of inflammatory processes such as asthma (38
). The inflammatory cell origin of MMP-9 in experimental tumor models has already been reported. Indeed, MMP-9 has been shown to be expressed by mast cells and neutrophils in a mouse model of skin carcinogenesis (16
) and by macrophages in breast and ovarian tumors (17
). Our finding supports the importance of neutrophils as the inflammatory cell origin of MMP-9 in an experimental cancer model.
Although MMP-9 promotor activity was detected in microvessels outgrowth from aortic rings issued from MMP-9/LacZ transgenic mice in vitro (data not shown), the single deficiency of MMP-9 did not affect microvessel sprouting. This observation highlights the importance of MMP-9 produced by inflammatory host cells.
Similarly to MMP-9 deficiency, the lack of MMP-2 did not affect tumor progression. Therefore, genetic ablation might underestimate the respective importance of MMP-2 or MMP-9 because of some compensatory response by the other gelatinase or other protease(s). In situ gelatin zymography revealed a partial and a strong reduction of gelatinolytic activity in MMP-9 or MMP-2 deficient mice, respectively, suggesting a lack of compensation of one gelatinase by the other. Although MMP-2 and MMP-9 are endowed with similar enzymatic activities in vitro, these MMPs may have distinct activities in vivo against nonmatrix substrates. For instance, MMP-9 is unable to cleave the MMP-2 cleavage site of monocyte chemoattractant protein-3 (8
) and fibroblast growth factor receptor 1 (9
), whereas MMP-2 is much poorer at cleaving type IV collagen α3 chain to generate tumstatin than MMP-9 (36
The requirement of both MMP-2 and MMP-9 for successful tumor invasion and vascularization might reflect the necessity of specific interactions between stromal cells producing MMP-2 and inflammatory cells secreting MMP-9. The intact outgrowth of microvessels from aortic rings of mice with a combined gelatinase deficiency demonstrates that endothelial cell migration in a pure collagen matrix can occur in the absence of gelatinases. The normal in vitro microvessel outgrowth in combined deficient mice could be due to the lack of involvement of a gelatinase substrate in this in vitro model. In sharp contrast, in vivo, endothelial cell activation and migration might require gelatinases produced by other host cells to generate angiogenic or chemoattractant factors. Such a contribution of the two gelatinases produced by different cell types has been recently reported in a model of aneurysm (42
). For aneurysm formation, both the local mesenchymal cell expression and the macrophage expression of gelatinases were required. Furthermore, we have previously shown that choroidal neoangiogenesis was fully prevented in double MMP-2−/−:MMP-9−/− mice, whereas it was only partly impaired in each single deficient mice (34
). In our experimental cancer model, although both enzymes are separately provided by different host cell types, the enzymatic activity of a single gelatinase is sufficient to circumvent the absence of the other one. As assessed by in situ zymography, in both single MMP-2- and MMP-9-deficient mice, a gelatinolytic activity was detected in the host tissue, while it was completely absent in double-deficient mice. Therefore, a low level of gelatinolytic activity in the host tissue is likely to be sufficient to remodel the extracellular matrix or to activate growth factors or cytokines/chemokines leading to an appropriate microenvironment both for vessel sprouting and tumor invasion. Altogether, these studies underline the complexity of the dialogues occurring between inflammatory cells and mesenchymal cells which are likely to differ from a pathological condition to another.
In mice with combined deficiency of both MMP-2 and MMP-9 genes, pathological angiogenesis such as tumor angiogenesis and choroidal neovascularization (34
) is significantly impaired. In sharp contrast, these mice develop normally suggesting that physiological angiogenesis was unaffected. This is the case for the effects of the MMP-9 generated antiangiogenic fragment, tumstatin, which only suppresses pathological angiogenesis, not physiological angiogenesis in wound healing or development (36
). These observations highlight the difference between physiological and pathological angiogenic processes and are consistent with the differential effect of plasminogen activator inhibitor (PAI-1) deficiency in physiological and pathological angiogenesis (27
MMP-9-deficient mice exhibit an abnormal pattern of skeletal growth plate vascularization and ossification 3 weeks after birth. However, a skeletal of normal appearance is found in adult animals, suggesting that compensating mechanisms occur. This transient phenotype evokes a possible temporal regulation of MMP-9 and/or compensating mechanisms in adult life. In support of this hypothesis, autoimmune experimental models have shown a phenotype in MMP-9-deficient mice only in the early stage of life (47
Our data emphasize the fact that lack of requirement is not synonymous with lack of involvement in a specific process. Therefore, it is necessary to be cautious with conclusions as to function from single gene ablation of an MMP. When two enzymes have overlapping activities an apparently normal gross phenotype may be manifested, even though there may be significant molecular differences. Because of putative redundancy, it is crucial to determine when and which of the MMPs are the most important for tumor progression and metastasis dissemination. Indeed, first therapeutic approaches using broad spectrum MMP inhibitors have led to disappointing results (2
). It is likely that broad spectrum inhibitors might repress some beneficial effects such as, for instance, the generation of anti-angiogenic factors (e.g., angiostatin, endostatin, and tumstatin) (1
) or the inactivation or activation of chemokines (49
). Our study suggests that the specific and combined inhibition of MMP-2 and MMP-9 may be a promising strategy for anticancer treatment.