degradation of the extracellular matrix (ecm) by matrix metalloproteinase (MMP)-2, constitutively produced by endothelial cells (ECs), is essential for concrete angiogenic steps such as EC proliferation, differentiation, and motility, processes important for physiological as well as pathological vascularization (10, 20). However, MMP-2 is also linked with EC apoptotic death because of its participation in the loss of EC adhesion to the ECM, and thus the loss of survival signals (4). Although the requirement of EC limited apoptosis in the angiogenic process is established (29), the role of MMP-2 and the mechanisms associated with the activity of the enzyme in these seemingly opposing effects are not yet known.

The zymogen form of MMP-2 (72 kDa), which is considered to be devoid of proteolytic activity, undergoes stepwise activation (18). The main molecule implicated in the first step of MMP-2 activation is membrane type (MT)1-MMP, which on its own is also capable of ECM degrading activity. MT1-MMP leads to the cleavage of MMP-2, generating an active intermediate form (64–68 kDa). Autocatalysis of the intermediate form, in the presence of integrins or other MMPs, results in the fully activated form of MMP-2 (∼62 kDa). Although a clear distinction between the different forms exists, the intermediate form is rarely mentioned in the literature when referring to MMP-2 activity, and the majority of research relates to the fully active enzyme.

During the process of MMP-2 activation, MT1-MMP itself undergoes degradation, shedding, and endocytosis. However, both membranal as well as soluble fragments containing the catalytic site are still capable of MMP-2 activation (23).

Paradoxically, the presence of a low level of the main tissue inhibitor of MMP, tissue inhibitor of metalloproteinase-2 (TIMP-2), participates in the MT1-MMP-dependent activation of MMP-2. High levels of TIMP-2 block this activation of MMP-2 as well as the enzymatic activity of the fully active form of the enzyme (18).

Caspases and p38 MAP kinase (p38) are two important signaling pathways associated with EC apoptosis (5, 6). The interrelationship between caspases and p38 is unclear as several authors demonstrated that p38 is upstream (9, 31), while others claim that it is downstream (19) or does not interact (21) with caspases. Both the caspases and p38 pathways have been linked to MMP-2 activity. Levkau et al. (13) demonstrated that caspases upregulated MMP-2 activation, resulting in apoptosis. In contrast, Preaux et al. (27) showed that apoptosis led to enhanced MMP-2 expression, while we and others demonstrated that inhibition of MMP-2 downregulated apoptosis (1, 30), supporting interactions between MMP-2 and apoptotic pathways, without addressing specific molecules involved. The relationship between p38 and MMP-2 is also controversial, as p38 was reported to increase (17, 24), decrease (15), or not influence (2, 8) its expression.

In this study, we wished to explore the perturbation of constitutive, homeostatic expression of MMP-2 in ECs when directed toward one main activity, “survival.” We rationalized that inhibition of EC main death pathways, the caspases and p38, would accentuate MMP-2 survival-associated activities and allow us to better understand MMP-2 regulation and activity. To this end, specific inhibitors of these two apoptotic signaling pathways in ECs were used. To further elucidate the role of MMP-2 in determining the fate of ECs, its reciprocal effects on caspase and p38 activation, as well as on specific angiogenic processes, were studied. In addition, due to the divergent reported results concerning p38/caspases relationships, their mutual influences were measured in our assay conditions.

MATERIALS AND METHODS

Cell cultures.

Human umbilical vein ECs (HUVEC) were isolated from umbilical cords and cultured as previously described (1). The study was approved by the Carmel Medical Center Review Committee, and signed informed consent was obtained from all donors. For experiments, ECs from different donors (passages 2-5) were pooled and cultured in serum-free medium, Bio-MPM1 (Biological Industries, Bet Haemek, Israel), containing 0.1% BSA.

Noncytotoxic concentrations of all reagents [evaluated by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays as described below] were chosen in preliminary dose-response experiments. The concentration chosen for the experiments of the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (100 μmol/l; Z-VAD, R&D Systems, Minneapolis, MN) and of the p38 inhibitor, SB203580 (50 μmol/l; Calbiochem), showed gradual enhanced potency to prevent natural apoptosis as well as gradual changes in MMP-2 expression and its activation. The concentration of the commercial human MMP-2 (R&D Systems) added to cultures was 49 ng/ml and 8 ng/ml. To obtain fully active MMP-2, the commercial product was first incubated with the chemical activator, p-aminophenylmercuric acetate (APMA 0.5 mM, Sigma, St. Louis, MO) for 2 h. This activated MMP-2 was added to cultures at the concentration of 8 ng/ml and 2 ng/ml. The same concentration of APMA alone was not cytotoxic and had no influence on the ECs in the different tests performed.

Viability, proliferation, and apoptosis assays.

The following commercial assays were employed, according to the manufacturers’ instructions. To determine cell viability, XTT assay (Biological Industries) was used, and cell proliferation was assessed using a bromodeoxyuridine incorporation assay (Chemicon International, Temecula, CA). To determine death of HUVEC, we used a commercial ELISA (Roche Applied Science, Mannheim, Germany), which detects histone-associated DNA fragments, both cytosolic (apoptosis) and released into supernatant (necrosis). Triplicate samples were evaluated in each experiment.

Zymography.

Volumes of supernatants from cultured cells, proportional to the number of viable ECs, were loaded onto gelatin-containing gels, and zymography, followed by computerized densitometry, was performed to assess levels of MMP-2, as previously described (1). Samples from the same experiment were run in the same gel.

mRNA expression.

Total cellular RNA was extracted from ECs and cDNA was prepared as previously described (1). Quantitative real-time PCR (Corbett) was performed, which included parallel amplification of MMP-2, MT1-MMP, or TIMP-2 with ubiquitin. To ensure that amplification was within the linear phase of the PCR reaction, serial dilutions (determined empirically in preliminary experiments) were included in each assay. The expression of nontreated ECs was fixed as 100%, and relative expression of specific gene/ubiquitin was calculated for treated samples. The primers and probe (TibMolbiol, Berlin, Germany) used were as follows: MMP-2: sense-TgCTggAgACAAATTCTggAgATAC, antisense-gCACCCTTgAAgAAgTAgCTgT, probe-FAM-ATCCTggCTTCCCCAAgCTCATC-BHQ1; TIMP-2: sense-ACAggCgTTTgCAATgCA, antisense-TCggCCTTTCCTgCAATgA, probe-FAM-AAgCggTCAgTgAgAAggAAgTggACT-BHQ1; MT1-MMP: sense-ggCAgCgATgAAgTCTTCAC, antisense-CTCAATgATgATCACCTCCgTCT, probe-FAM-ACCAgAAgCTgAAggTAgAACCgggCT-BHQ1; and ubiquitin: sense-TgTgATCgTCACTTgACAATgCA, antisense-CCTTATCTTggATCTTTgCCTTgA, probe-YAK-ATggTgTCACTgggCTCAACCTCgA-BHQ1.

ELISA.

The levels of the following proteins were evaluated using commercial assays according to the manufacturers’ instructions: TIMP-2 in culture supernatants (after 24-h incubation) and intracellular active caspase 3, using Quantikine Kits (R&D Systems); MMP-2 in culture supernatants and MT1-MMP, cellular extracts, and supernatants (after 24 h), using Biotrak activity assay system (Amersham Bioscience).

Western blot analysis.

Extracts of cellular protein were separated through 8–10% SDS-PAGE and transferred onto nitrocellulose. Membranes were exposed to blocking solution followed by specific antibodies to p38 MAPK (Cell Signaling Technology, Beverly MA), followed by secondary horseradish-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). End products were visualized using an ECL detection kit (Biological Industries).

Flow cytometric analysis.

Phosphorylated p38 [anti-phospho-p38 MAPK (Thr180/Tyr182)] (Cell Signaling Technology) and phosphorylated heat shock protein 27 [HSP27; anti-HSP27 (Ser82), Upstate, Lake Placid, NY] were assessed by indirect intracellular staining, and fluorescence levels of cells were evaluated by flow cytometry (Epics XL/MCL; Beckman Coulter, Miami, FL).

In vitro wound migration and tube formation assays.

Migration and tubelike formation assays were performed as previously described (1).

Statistical evaluation.

All experiments were repeated three to five times with good agreement between the results. The data shown are the relative mean ± SE relative to nontreated cells. Statistical significance of results was determined by analysis of variance to determine differences within a treated group. Further comparisons within a group were determined by Tukey-Kramer analysis. Values of P < 0.05 were considered significant.

RESULTS

Inhibitors of caspases and p38 reduce EC apoptotic death.

First, we verified that in our assay conditions the chosen caspases (7) and p38 (3) inhibitors indeed impede the low level of apoptotic death in quiescent cultured ECs and that we were capable of detecting this low level of apoptosis. The ECs were incubated with a nontoxic concentration of the pan-caspase inhibitor, Z-VAD, and/or the specific p38 inhibitor, SB203580, according to preliminary dose-response assays (data not shown), and the levels of their death (necrotic and apoptotic), viability, and proliferation (Fig. 1) were measured. Only apoptotic and not necrotic death (data not shown) was observed in cultures. The degree of inhibition of EC apoptosis was higher in the presence of Z-VAD compared with SB203580, 92% and 40%, respectively (P < 0.001), while incubation of cells with both inhibitors together reduced apoptosis by 90% (Fig. 1A). Each of the inhibitors significantly increased the number of viable cells by ∼20% (P < 0.001), whereas Z-VAD reduced their proliferation by 19% (P < 0.001) and SB203580 enhanced it by 10% (P < 0.05; Fig. 1, B and C). The results point to differences in the effects of the two apoptotic pathways on EC fate: both caspases and p38 induce apoptosis; however, p38 also enhances EC proliferation. Not surprising, the effects of both pathways on viability and proliferation of the nontriggered cells were minor, reflecting the nontriggered in vivo quiescent conditions.

Inhibitors of caspases and p38 decrease the accumulation of MMP-2 mRNA, pro- and active enzyme, but differentially modulate the intermediate form.

To evaluate whether the caspases and p38 apoptotic pathways influence MMP-2, in addition to their effects on EC survival, inhibitors were added to cultures, and MMP-2 mRNA and protein activation forms were determined (Fig. 2). Z-VAD and SB203580 reduced MMP-2 mRNA by 30% (P < 0.01) and 62% (P < 0.001), respectively, while incubation of cells with both inhibitors downregulated its level by 70% (P < 0.001; Fig. 2B). The zymography assay showed that Z-VAD and SB203580 reduced the level of pro-MMP-2 by 14% (P < 0.01) and 30% (P < 0.001), respectively, and the combination of both inhibitors by 30% (P < 0.001). A decrease in the fully active enzyme was also observed (57%, P < 0.001, and 27%, P < 0.01, respectively; Fig. 2, A, C, and E). However, the pan-caspase inhibitor increased (20%, P < 0.001) and the p38 inhibitor decreased (40%, P < 0.01) the level of the MMP-2 intermediate form (Fig. 2, A and D). The results obtained with both inhibitors together were similar to those obtained with SB203580 alone. A commercial ELISA, which enables the evaluation of fully active MMP-2, verified the zymography results of total and fully active MMP-2 (data not shown). These results confirm that caspases and p38 modulate MMP-2: both elevate its mRNA accumulation, protein expression, and full activation. However, caspases reduce and p38 enhances its partial activation.

Inhibitors of caspases and p38 oppositely modulate MT1-MMP mRNA and protein.

To further explore the contradictory effects of the caspases and p38 on the partial activation of MMP-2, their influence on its main activator, MT1-MMP, and its main inhibitor, TIMP-2, were examined. The mRNA level of MT1-MMP was enhanced by Z-VAD (37%, P < 0.001), while SB203580 reduced it by 22% (P < 0.05; Fig. 3). Similar results were obtained using ELISA and Western blot assays that measured the catalytically active MT1-MMP forms in whole cell lysates and supernatants (only ELISA results shown). No significant effects of the inhibitors on TIMP-2 protein expression were observed (data not shown).

Inhibitors of caspases and p38 differently affect morphology and migration of ECs while similarly interfering with tubelike formation.

To evaluate the effects of the apoptotic pathways on specific angiogenic steps, the inhibitors were added to cultures and cellular morphology, migration, and tube formation were examined. Confluent ECs in culture exhibit cobblestone morphology (Fig. 4A1), while cells incubated with Z-VAD acquired an elongated phenotype with cytoplasmic protrusions and were loosely attached to plates (as observed by their fast detachment following trypsin addition), concordant with their mobility capacity (Fig. 4A2). ECs cultured with the p38 inhibitor alone, or both inhibitors, were flat and elongated, with no protrusions, and strongly attached to the plate, concordant with their stationary capacity (Fig. 4, A3 and A4). In the wound assay (designed to evaluate EC migration), the caspase inhibitor enhanced, while the p38 inhibitor reduced the number of migrating cells in comparison with untreated ECs (P < 0.05; Fig. 4, B and C). The differences in the number of migrating cells were abolished in the presence of both inhibitors together.

The caspase inhibitor interrupted tubelike formation by 30% (P < 0.001), and tubes were often open-ended (arrowheads, Fig. 4, E2 and E4). The p38 inhibitor or both inhibitors together also led to a reduction in tubelike formation, and many nondifferentiated cells were observed (long arrows, Fig. 4, E3 and E4). Therefore caspases reduce but p38 elevates EC migration, accompanied by concordant changes in morphology. Both pathways appear to elevate tube formation.

Fully active but not pro-/intermediately active MMP-2 forms cause EC death.

To determine the direct effects of MMP-2 on EC fate, we first established the culture conditions required to obtain fractions that were enriched with “MMP-2 proenzyme,” “MMP-2 intermediate form,” or the “MMP-2 fully active form.” To this end, two concentrations of commercial MMP-2, without or following APMA activation (22), were added to cultures. The concentrations chosen were based on data from ELISA measuring the level of pro- and active MMP-2 in EC culture supernatants. The proenzyme and the active form, but not the intermediate form, were found in the commercial MMP-2 without addition of APMA, whereas mainly the active form was found following incubation of the commercial enzyme with APMA (Fig. 5A). The forms of MMP-2 in supernatants following 24-h incubation with non- and APMA-activated MMP-2 were then compared with those present in supernatants from untreated ECs (Fig. 5, B and C). Zymography of EC supernatants after 24-h incubation revealed the appearance of the intermediate form in cultures of untreated ECs as well as in those to which nonactivated MMP-2 was added. In supernatants to which the higher concentration of APMA-activated MMP-2 was added, almost no pro-MMP-2 was found, while a slightly larger amount of pro-MMP-2 was found in cultures where the lower concentration of activated MMP-2 had been added. These results suggest that the active synthesis of the proenzyme and its gradual activation occur in cultures containing live cells.

To directly address the supposition that active MMP-2 kills ECs, the influence of the same concentrations of nonactivated and APMA-activated MMP-2 on apoptosis of ECs was examined, following incubation of 2 h, 8 h, and 24 h (Fig. 5D). The higher concentration of APMA-activated MMP-2 led to early (2 h) apoptosis, whereas the lower concentration of the activated form led to cell death at a later time (8–24 h), confirming the ability of fully active MMP-2 to cause EC apoptotic death. Of note, in the presence of the high concentration of commercial nonactivated MMP-2, which contained proenzyme as well as a large fraction of fully active MMP-2, the level of apoptosis after 24-h incubation was similar to that of untreated cells. In agreement with the induced death of ECs by APMA-activated MMP-2, this fully activated form prevented EC proliferation (data not shown).

Pro- but not active-MMP-2 enhances migration of ECs, while active form decreases tube formation.

To determine the effects of MMP-2 forms on specific angiogenic parameters, pro-rich and active-rich forms of MMP-2 were added to cultures and their influence on migration and tube formation was followed. Since only a few live cells remained after the addition of the higher concentration of APMA-activated MMP-2, only the lower concentration (2 ng/ml) was used in the following assays. The wound assay demonstrated that the lower concentration of nonactivated MMP-2, containing the intermediate form (with almost no active form) enhanced EC migration (60%, P < 0.001; Fig. 6A). The higher concentration of nonactivated MMP-2, containing a large amount of the fully active form, did not enhance migration of cells. The active MMP-2 reduced migration of ECs by eliminating the cells. The nonactivated enzyme did not affect tube formation, while the lower concentration of the APMA-treated, fully activated MMP-2 led to a decrease (>50%, P < 0.001) in the number of tubelike structures (Fig. 7B), probably via the elimination of cells.

Active MMP-2 enhances phosphorylation of p38 but does not influence active caspase 3.

To determine the reciprocal effects of MMP-2 on the two apoptotic pathways, nonactivated and the lower concentration of APMA-activated MMP-2 were added to ECs and their influence on p38 and caspase 3 was measured. After 24-h incubation, the higher concentration of nonactivated and the APMA-activated MMP-2 enhanced the level of p38 MAPK protein (up to 1.5-fold and 2.7-fold, respectively; P < 0.001) and to a lesser extent its phosphorylation (Fig. 7, A and B). In contrast, neither the pro- nor the active-MMP-2 forms significantly influenced the level of active caspase 3 (Fig. 7C), suggesting that MMP-2 is localized downstream to caspases.

Reciprocal downregulation of caspase 3 and p38 activity by Z-VAD and SB203580.

Lastly, the interrelationships between the two apoptotic pathways, which profoundly differed in their influences on MMP-2 activity, were examined. Incubation with Z-VAD temporarily reduced the level of phosphorylated p38 and its downstream substrate, HSP27, by 25% and 50% respectively (P < 0.001), following 0.25 and 2-h incubation, returning to basal levels at 24 h (Fig. 8, A and B). Incubation of ECs with the p38 inhibitor also led to changes in active caspase: a temporary reduction of 25% (P < 0.001) was observed at 15 min followed by a twofold increase at 2 h and a return to pretreatment level at 24 h (Fig. 8C). Thus each pathway seems to rapidly modulate the other's activity in our experimental conditions.

DISCUSSION

The main question addressed in this study is the supposedly contradictory effects of MMP-2 on survival of ECs. We rationalized that the constitutive production and activity of MMP-2 in quiescent ECs is concordant with balanced activity of death-inducing pathways, resulting in basal apoptosis, required for the maintenance of angiogenic activity (4, 10, 20, 29). We hypothesized that there is a link between MMP-2, death-inducing pathways, and angiogenic parameters, which will be accentuated when blocking this low level of apoptosis and directing the function of MMP-2 toward prosurvival behavior.

First, we verified that spontaneous apoptosis could be detected in our culture conditions (Fig. 1A) and chose inhibitors to block apoptosis. The pan-caspases and p38 MAPK inhibitors were chosen for this study because of their specificity (6, 26), ascertained by dose-response curves and the inability of inhibitors of additional candidate pathways (such as ERK kinase, NF-κB, JNK, and Rho-associated kinase) to exert dose-response effects and influence the MMP-2 activation forms (data not shown). The two chosen inhibitors differed in their effects on EC apoptosis, proliferation, and migration, though both inhibitors impeded tubelike formation (Fig. 1, B and C, and Fig. 4). Two main conclusions can be drawn from these differences. First, the p38 pathway may possess simultaneous pro- and antiapoptotic effects (14) unlike the absolute basal proapoptotic effects of the caspases. Second, apoptosis is not essential for migration but is required for tube formation, supporting divergent effects of apoptosis on concrete angiogenic steps (25, 29).

The interactions of the death pathways with MMP-2 and its main regulators MT1-MMP and TIMP-2 were directly examined. Consistent with the death-inducing activity of MMP-2 (1, 11, 30), both inhibitors downregulated the expression of its mRNA, pro and active-enzyme forms (Fig. 2). Since pro-MMP-2 is considered to be devoid of activity, these results suggest that the active enzyme is responsible for EC death. However, a dichotomy was observed between the enhancing effects of the caspase inhibitor, as opposed to the suppressive effects of the p38 inhibitor, on MMP-2 intermediate form, suggesting a prosurvival effect of this form. The modulatory influences of the caspase inhibitor on the intermediate and active forms of MMP-2 are consistent with a previous report (13), though the bulk of literature concerning MMP-2 does not relate to the activities of its separate forms. In addition, data concerning the influences of p38 on MMP-2 are partial and conflicting depending on the type of cells and the trigger studied (8, 15, 19).

Because the inhibitors did not significantly alter the levels of TIMP-2 protein (data not shown), we suspect that this molecule does not play a major role in the activation of the different forms of MMP-2 in the present culture conditions.

Active MT1-MMP cleaves pro-MMP-2 to its intermediate form, which then matures to the fully active form (18). Therefore our results implying a reduction of active MT1-MMP by caspases and its elevation by p38 (Fig. 3) are compatible with changes observed in the MMP-2 intermediate form and suggest that the two apoptotic pathways regulate the level of the partially activated form via MT1-MMP. The elevation of MT1-MMP, intermediate, and fully active MMP-2 by the p38 pathway is in line with the classical stepwise mode of MMP-2 activation requiring the formation of the ternary complex, MT1-MMP/TIMP-2/MMP-2. However, the caspase-induced reduction of MT1-MMP and partially active MMP-2, while enhancing the fully active MMP-2, is not in accordance with the classical activation dogma suggesting the participation of additional molecules, not addressed in this study, in the caspase-dependent activation of MMP-2 (16, 27). Further support of the participation of other molecules as well as interactions between the two death pathways is implied by the simultaneous elevation of MT1-MMP and reduction of intermediate-MMP-2 observed in cultures containing both inhibitors (Figs. 2 and 3). In summary, these results imply that, in physiological conditions, p38 upregulates MT1-MMP synthesis and activity, leading to enhancement of the antiapoptotic, promigratory intermediate form of MMP-2, while caspases carry out the opposite effect. Nonetheless, both pathways upregulate proapoptotic, fully active MMP-2.

To further define the effects of partially and fully active MMP-2, we used a commercial MMP-2 that is mainly composed of the pro-form but contains also the fully active enzyme (Fig. 5A). Though a clear distinction of the two forms was not possible in this product, the addition of APMA, which cleaves MMP-2 to its fully active form (22), enabled us to compare between the effects of pro-rich to those of active-rich enzyme on different parameters of ECs. Following 24-h culture with the nontreated commercial enzyme, we detected the three MMP-2 forms in EC supernatants, supporting ongoing MMP-2 synthesis and activation. In cultures where active-rich enzyme was added, the active form was mainly observed, suggesting the decline or absence of ongoing MMP-2 synthesis and activation. Indeed, the high and low concentration of active-rich MMP-2 dose-dependently induced early and later apoptosis, respectively, reducing and ablating EC proliferation, migration, and tube formation (Figs. 5 and 7). Interestingly, the higher concentration of nontreated MMP-2 containing a prominent prosurvival/intermediate fraction and death-inducing active MMP-2 did not induce either apoptosis or migration. At the same time, the lower concentration of nontreated MMP-2 containing a lower quantity of the active form promoted EC migration and did not induce apoptosis. Therefore, although active MMP-2 induces EC apoptosis and the intermediate form enhances migration, it may be surmised that the ratio between the different forms determines the fate of ECs.

Tubulogenesis was differently influenced by the MMP-2 forms: in nontreated ECs, with unperturbed caspases and p38, and therefore physiologically produced active MMP-2, tubes were rapidly formed, while in ECs treated with the inhibitors, with less active-MMP-2, tubes were not formed (Fig. 4). We suggest that a basal degree of apoptosis, dependent on a critical concentration of local active-MMP-2, is required for tube formation. Nonetheless, higher concentrations of active MMP-2, as added in these experiments and may be found in inflammatory environments, are detrimental for EC survival and thus tube formation. The pro/intermediate fractions of MMP-2 did not influence tubulogenesis, in line with a previous study (12) that proposed MT1-MMP and not MMP-2 responsible for this angiogenic process.

In addition to examining the effects of the death pathways on MMP-2, we also evaluated possible feedback effects of MMP-2 on caspases and p38. Both pro-rich and active MMP-2 did not influence active caspase 3, suggesting that MMP-2 induced by this pathway is an end point executioner product in ECs. In contrast, elevation of p38 and its phosphorylation were observed following the addition of active MMP-2 (Fig. 7). Thus active MMP-2 generated via p38, as found in an inflammatory environment, could further elevate p38 activity (via a positive regulatory loop) and affect EC survival.

As to caspases and p38 interactions, the multiple effects of the inhibitors did not allow us to resolve whether caspases are upstream or downstream to p38 activation. In fact, opposing influences, dominance of one, the mean, or the sum of the effects of the inhibitors were all observed. Therefore caspases and p38 may share the same signaling pathway and also participate in independent signaling pathways.

Cumulatively, the results of this study demonstrate that diverse apoptotic pathways specifically influence the constitutive production and activation of MMP-2 in ECs. Alterations in these pathways affect EC life and death decisions as well as concrete angiogenic steps via changes in local concentrations of the three different MMP-2 forms. The full significance of the intermediate MMP-2 form cannot be fully determined unless this species is purified and tested alone. Moreover, deeper knowledge of the signaling loops controlling MMP-2 activation in ECs will require the use of additional methods to block or enhance these apoptotic pathways. Such knowledge may be of therapeutic importance for improving physiological neovascularization in injured tissues as well as inhibiting pathological angiogenesis in tumors.

GRANTS

This study was supported by the Chief Scientist, Ministry of Health, Jerusalem, Israel (Grant 3-1659).

DISCLOSURES

No conflicts of interest are declared by the author(s).