Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis

Endothelial Colony Forming Cells (ECFCs) were isolated from > 50 ml human umbilical cord blood (UCB) of healthy newborns, as described previously [14, 15], after maternal informed consent and in compliance with Italian legislation, and analyzed for the expression of surface antigens (CD45, CD34, CD31, CD105, ULEX, vWF, KDR, uPAR) by flow-cytometry [14]. ECFCs were grown in EGM-2 culture medium (Lonza), supplemented with 10% FBS (Euroclone) onto gelatin coated dishes. Human microvascular endothelial cells (HMVEC) were purchased from Lonza and were grown in the same conditions of ECFCs.

In vitro capillary morphogenesis was performed as described [14, 15] in tissue culture wells coated with Matrigel (BD Biosciences). ECFCs or HMVECs were plated (18 × 10³ /well) in EGM-2 medium, supplemented with 2% FCS and incubated at 37 °C-5% CO2. Results were quantified at 6 h by measuring the branching points of capillary projections. Six to nine photographic fields from three plates were scanned for each point. Results were expressed as % increase/decrease of branching points/field ± SD with respect to control fixed at 100%.

Speed of capillary structures formation was measured by time-lapse capillary morphogenesis assay (Additional file 1).

Invasion was studied in Boyden chambers in which the upper and lower wells were separated by 8 μm–pore size polycarbonate filters coated with Matrigel (BD Biosciences), as previously described [13]. For details, see (Additional file 1).

Protease-indipendent angiogenic properties and invasion were evaluated by in vitro capillary morphogenesis and 3D-Boyden chamber assays, as described above, with Matrigel coating in the presence of a physiological protease inhibitor cocktail, consisting of α2-antiplasmin (plasmin inhibitor; 5 μg/ml), Cystatin (cysteine protease inhibitor; 5 μM), PAI 1 (plasminogen activator inhibitor; 10 ng/ml), TIMP1, TIMP2 and TIMP3 (metallo-protease inhibitors; 0,5 μg/ml each one) purchased by Abcam. A completely artificial protease inhibitor cocktail (composed by Ilomastat, leupeptin, pepstatin A, E-64 and aprotinin; Sigma Aldrich) used in a previous study [13] was also used. Concentrations used were selected according to the manufacturer’s instructions and literature [16, 17]. Protease inhibitor cocktails were added to un-polymerized Matrigel solution on the upper surface of the porous filter. To induce the amoeboid phenotype, cells were treated overnight with the protease inhibitor cocktails at the same concentrations used in the invasion assay. Cell viability upon protease inhibitors treatment was evaluated by Trypan blue dye (Sigma) exclusion assay.

ECFC and HMVEC cell suspensions were co-polymerized with Matrigel containing 2% FITC-labeled collagen monomers (Molecular Probes). Digestion was allowed for 40 h at 37 °C and solid-phase Matrigel containing the cells was pelleted, whereas FITC released into the supernatant was analyzed by spectrofluorometry. One hundred percent values were obtained by complete collagenase digestion of cell-free Matrigel lattices. Background fluorescence was obtained by pelleting non-digested cell-free FITC-collagen-enriched Matrigel layers.

Cells from different experimental conditions (control, physiological protease inhibitor cocktail) were lysed in radio-immunoprecipitation assay buffer, the lysates were clarified by centrifugation, and RhoA GTP or Rac1 GTP was quantified. Briefly, lysates were incubated with 10 μg rhotekin–glutathione S-transferase (GST) fusion protein (Millipore) or p21-activated kinase-GST fusion protein, both absorbed on glutathione–Sepharose beads for 1 h at 4 °C. Ratios between activated (GTP-bound) RhoA and Rac1 adsorbed to the beads were quantified by Western blot densitometry.

Specific electrophoretic conditions and the source of used antibodies are reported Additional file 1.

Total RNA preparation and reverse transcription were performed as previously reported. The levels of messenger RNA for the integrin chains were determined by an internal-based semiquantitative RT-PCR, using procedures and primers previously described [18].

Inhibition of uPAR-integrin interaction was obtained with the M25 peptide, previously identified in a phage display library [19], able to uncouple uPAR interaction with integrin α-chain. The peptide was produced in collaboration with PRIMM srl, Milan, Italy. In the β-propeller model of α-chain folding, the sequence of this peptide (STYHHLSLGYMYTLN) spans an exposed loop on the ligand-binding surface of α-chain, thus impairing integrin α chain-uPAR interaction. In cell culture both M25 and scramble-M25 (sM25) were used at 50 μM for 15 h at 37 °C.

For co-immunoprecipitation, ECFCs and HMVECs were plated at 500 × 10³ cells/100 mm dish in complete medium. One of two dishes for each cell line was treated with M25 peptide at 50 μM for 15 h. After two washes in ice-cold PBS, cells were lysed on ice with Ripa buffer, centrifuged (15,000 rpm, 15 min) and the supernatant was used for co-immunoprecipitation. For details, see Additional file 1.

Immunofluorescence and confocal microscopy were performed as previously described [18]. For details, see Additional file 1.

See details under Additional file 1.

All procedures involving animals were performed in accordance with the ethical standards and according to the Declaration of Helsinki and to national guidelines approved by the ethical committee of Animal Welfare Office of Italian Health Ministry and conformed to the legal mandates and Italian guidelines for the care and maintenance of laboratory animals.

Two groups of 8 and 10 four-week-old male SCID beige mice (two for each experimental condition) were purchased from Charles River. The Matrigel plug assay was used to study a possible role of the amoeboid movement in vivo as previously described [14]. VEGFA (10 ng/ml), was added to unpolymerized Matrigel at 4 °C at a final volume of 0.6 ml. Protease inhibitors were added to unpolymerized Matrigel at the same concentrations used for in vitro assays. Heparin (50 U/ml) was added to each solution. To study the role of mice vessels, the Matrigel suspension was carefully injected subcutaneously into both flanks of mice using a cold syringe. As the Matrigel warms to body temperature, it polymerizes to a solid gel, which then becomes vascularized within five days in response to the angiogenic substance. The extent of vascularization was quantified by measuring the hemoglobin content of the recovered plugs. Groups of four pellets were injected for each treatment. The eight animals were subdivided as follows: two controls (Matrigel alone); two animals injected with a Matrigel plug containing VEGF-A; two injected with Matrigel plus the physiologic protease-inhibitor cocktail; and two with Matrigel, the physiologic protease-inhibitor cocktail plus VEGFA. The reagents for all treatments were added to the Matrigel solution prior to injection. Five days after injection, the pellets were removed, minced and diluted in water to measure the hemoglobin content with a Drabkin reagent kit (Sigma). Vascularization was evaluated by sight taking a representative photograph of individual Matrigel plugs recovered at autopsy for the corresponding condition. Samples were also fixed in formalin, embedded in paraffin for histological analysis and stained with hematoxylin-eosin.

The second group of 10 mice was used to verify the role of uPAR in amoeboid angiogenesis in vivo. Before implantation we added to the plugs, composed of Matrigel plus physiologic protease-inhibitor cocktail, murine uPAR-aODN (ISIS Pharmaceuticals, Carlsbad Research Center, California) or M25 peptide used in vitro. At the third day, another dose of ODNs and M25 was injected subcutaneously into the plugs. Treatments consisted in plug administration of liposome-encapsulated vehicle alone (DOTAP; Roche, Germany), DOTAP + scramble ODN, DOTAP + uPAR-aODN, scramble M25 peptide and M25 peptide. Upon sacrifice (fifth day), isolated plugs were stained with hematoxylin-eosin.

Unless otherwise stated, all the experiments were performed five times in duplicate for a reliable application of statistics. Statistical analysis was performed with GraphPad Prism5 software. Results are expressed as means ± SD. Multiple comparisons were performed by Anova and paired Student T test. Statistical significances were accepted at p < 0.05. (*p < 0,005, **p < 0,001, ***p < 0,0001).

Selected families of membrane-associated proteases acting at specific steps of angiogenesis and vasculogenesis are required to perform a suitable angiogenic program [3, 20]. In order to investigate if the presence of protease inhibitors cocktail produced a protease-independent invasion in vitro, we first performed a Boyden chamber invasion assay. Notably, we added protease inhibitor cocktail to ECFC and HMVEC cell suspension and to Matrigel solution before polymerization. We used two different kinds of protease inhibitors cockatils: the chemical cocktail (Ch-C) [13, 21], and the physiological cocktail (Ph-C or MIX), composed as reported in M&M. Under the effect of the Ph-C both ECFCs and HMVECs showed a greatly enhanced Matrigel invasion, as opposed to the very poor movement and high toxicity observed with the Ch-C (Fig. 1a). Indeed, while the Ch-C proved to be very toxic for both ECFCs and HMVECs, the percent of cell death induced by the Ph-C was similar to that of untreated cells (Fig. 1b). Therefore, in the next experiments we always used the physiological cocktail that will be indicated as MIX. To be sure that the invasion capacity of endothelial cells in amoeboid conditions was independent from the compactness of the Matrigel, we tested the ECFC and HMVEC invasion capacity in a Matrigel layer five times more concentrated (250 μg) than the usually used (50 μg), observing that the ratio between the percentage of migrated cells in mesenchymal or amoeboid conditions was independent of the Matrigel density (Fig. 1c). Moreover, single inhibitors of the MIX produced no or scarce decrease of cell invasion as compared to the intense invasion-promoting activity of the full-range cocktail, demonstrating that the effect of the mix was due to the synergistic effect of all inhibitors mixed together and not to any biological activity of a single one at the used concentrations (Fig. 1d).

Capillary morphogenesis is considered a reliable in vitro analog of in vivo angiogenesis and has been recognized to be mainly dependent on MT1-MMP, MMP2, MMP9 and uPAR-bound uPA [22, 23]. We observed that both ECFCs and HMVECs produced similar tubular-like structures under mesenchymal or amoeboid conditions (Fig. 1e) and evaluated the speed of capillary structures formation by time-lapse capillary morphogenesis assay. It’s known that the low-adhesion attachment to the substrate enables cells that adopt an amoeboid movement to translocate at relatively high velocities [1]. We have observed that also the speed of tube formation for is higher in amoeboid conditions than in the presence of proteases (videos): at 25th second of the recorded video, corresponding to about 312 min, cells treated with the MIX (video: https://​vimeo.​com/​246963233) formed a complex capillary network compared to the control (video: https://​vimeo.​com/​246963182), with a ratio of about 2:1 evaluated counting the branching points formed.

As previously reported for melanoma and prostate cancer cells [13], the residual collagenolytic activity shown by endothelial cells under amoeboid conditions did not justify the number of invasive cells (Fig. 2a).

Cell morphology and its association with actin cytoskeleton assembly is a characteristic of the movement style. Mesenchymal motility is connoted by elongated, fibroblast-like cell morphology with established cell polarity, dependent on the small GTPase Rac which, in turn, organizes actin polymerization to form stress fibers, filopodia and lamellipodia [24], giving origin to actin-rich protrusions. These features were exhibited by both ECFCs and HMVECs under control mesenchymal conditions (Fig. 2b-c). Under protease inhibition, both cell lines acquired a round morphology with sub-membranous cortical actin localization, a feature connoting the amoeboid phenotype (Fig. 2c). Nevertheless, immune-phenotyping by FACS analysis (Table 1) revealed that both ECFCs and HMVECs, even after actin cytoskeletal reorganization, maintain endothelial characteristics.

In order to feature the motility shift we also evaluated the activation of the small GTPases RhoA and Rac1, two accepted regulators of the cytoskeleton. It’s described that mesenchymal motility is associated with the inhibition of Rho GTPases and activation of Rac, which drives motility organizing actin polymerization and lamellipodium formation, whereas amoeboid motility is characterized by an opposite phenotype [25]. Western blotting and relative quantification for the activated forms of RhoA and Rac1, compared to the total amount of RhoA and Rac1, revealed an increase of activated RhoA and a decrease of activated Rac1, in both ECFCs and HMVECs after the induction of amoeboid motility in presence of the MIX (Fig. 2c).

To investigate the role of uPAR in amoeboid angiogenesis, after validating the silencing activity of pooled small interfering RNAs targeting PLAUR mRNA (siPLAUR), that produced an evident reduction of uPAR expression in terms of mRNA and protein in both ECFCs and HMVECs (Fig. 3a), we have studied the effect of uPAR knockdown on the small Rho-GTPases activation. In mesenchymal conditions, siPLAUR treatment of ECFCs (Fig. 3b) and HMVECs (data not shown) resulted into increase of RhoA and Rac1 activation, whereas in the presence of inhibitor cocktail the same cells showed a reduced RhoA activation paralleled by an increased Rac1 activation. Therefore, even though after uPAR knockdown ECFCs and HMVECs still activate amoeboid/mesenchymal-related transductions, they are unable to invade 3D matrices (Fig. 3c) and to form tubular structures in vitro (Fig. 3d), indicating that both movement styles demand the presence of uPAR. The residual movement observed in the absence and in the presence of protease inhibitors, in cells treated with si-PLAUR, may possibly be ascribed to proteases and protease receptors of different families [26].

ECFCs and HMVECs showed an integrin pattern in line with previous studies [18] (Fig. 4a). Immunoprecipitation experiments with lysates of ECFCs and HMVECs, demonstrated the activity of M25 peptide in uncoupling uPAR-integrin αvβ3 interaction (Fig. 4b). Figure 4c shows the confocal immune-fluorescence analysis of integrin αvβ3 and uPAR in both cell lines. These data showed that uPAR-integrin interactions persist under both mesenchymal and amoeboid conditions but, after cell treatment with 50 μM M25 peptide, we assist to an uncoupling between uPAR and αvβ3 integrin in the absence and in the presence of the inhibitor- IX, resulting in abolition of mesenchymal and amoeboid invasion and angiogenesis as shown by invasion assay and capillary morphogenesis (Fig. 4d), Taken together, these results indicate a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis.

Endothelial cells respond to the most potent of the proangiogenic regulators, the vascular endothelial growth factor (VEGF), that binds VEGF receptors on the cell surface stimulating endothelial cell migration and proliferation, new blood vessels formation and sprouting, and triggers the caveolar-raft recruitment of proteins able to maintain a proper angiogenic function of endothelial cells and preserve the integrity of the actin cytoskeleton [27].

Here we examined the role in amoeboid conditions of VEGF and VEGFR2/KDR/Flk-1, considered the major mediator of proangiogenic signaling. In endothelial cells VEGF controls the delivery of KDR from the endosomal storage pool to the plasma membrane [28].

We first examined the cellular distribution of endogenous KDR in ECFCs in mesenchymal conditions before and after VEGF stimulation. Under control conditions, KDR showed mainly a clustered staining pattern in the cytoplasm. VEGF stimulation leaded to a redistribution of KDR, which lost the clustered pattern and spread within the cytoplasm to reach the plasma membrane, with no substantial differences between mesenchymal and amoeboid conditions (Fig. 5a).

Figure 5b-c show an “indifference” of ECFCs to VEGF stimulation under amoeboid conditions in terms of invasion and capillary morphogenesis. While Matrigel invasion under mesenchymal conditions was dependent on the chemotactic activity of VEGF, amoeboid invasion was VEGF-indifferent (Fig. 5b). Similar results were obtained in capillary morphogenesis assay (Fig. 5b), possibly due to the endothelial cells capacity to migrate and differentiate into tubular structures at the maximum levels under amoeboid conditions. We performed the same experiments of migration and capillary morphogenesis after uPAR silencing. uPAR knockdown greatly impaired ECFC invasion and tubular structure formation in the presence of VEGF with or without MIX in vitro, indicating that both movement styles, also under VEGF stimulation, demand the presence of uPAR (Fig. 5c).

We then investigated the effect of VEGF on the intracellular signaling molecules like the small GTPases RhoA and Rac1, pKDR, pMLC2 and WAVE2 (Fig. 5d). Our results reveal that VEGF seems to induce an amoeboid signaling pattern in both mesenchymal and amoeboid conditions, as we can see from the results of Rho and Rac activity assay. We found obviously an increment of pKDR after VEGF stimulation in both conditions. As we anticipated above, in the presence of inhibitor-MIX, even though VEGF signaling is active, the cells can’t use that stimulus because they already invade and differentiate to the maximum levels. Because amoeboid movement is associated with elevated levels of Myosin Light Chain 2 (MLC2) phosphorylation, we found an increment in the presence of protease inhibitor-MIX and, under VEGF stimulation, elevated levels in mesenchymal conditions compared to the control and a reduction in amoeboid conditions. WAVE2 is responsible for downregulation of amoeboid motility and therefore of actomyosin contractility and membrane blebbing. We indeed found a downregulation in the presence of inhibitor MIX, before and after VEGF treatment, compared to the control in mesenchymal conditions. After VEGF treatment in mesenchymal conditions, WAVE2 expression is similar to the control. Each result shown in Fig. 5 reveals representative data obtained with ECFCs. HMVECs gave similar results (not shown).

We performed the Matrigel plug assay in SCID mice to study a possible role of the amoeboid movement in vivo. In the first in vivo experiment, we used 8 SCID mice to examine murine angiogenesis in control conditions and after addition of inhibitor-MIX to unpolymerized Matrigel, both in the presence and in the absence of VEGF. Individual Matrigel plugs were recovered at autopsy 5 days after implants. Vascularization was evaluated by sight taking a representative photograph, of individual Matrigel plugs recovered at autopsy for the corresponding condition and after histological analysis (Fig. 6).

The plugs recovered from mice showed that whereas control conditions displayed trace of vessels, the inhibitor-MIX stimulated angiogenesis, as shown also by increases in hemoglobin content. This effect was the result of protease inhibitor activity and was particularly relevant when VEGF was added to the MIX (Fig. 6a). Hematoxylin/eosin staining revealed that plugs containing the protease inhibitor MIX showed a strong angiogenic response with an increased number of vascular structures with lumens and red blood cells as compared to control plugs.

In the second experiment we performed another Matrigel plug assay using 10 SCID mice to show uPAR dependence in amoeboid angiogenesis in vivo. We evaluated uPAR silencing by murine uPAR-aODN and uPAR-integrin uncoupling by the M25 peptide, already reported to efficiently inhibit mesenchymal angiogenesis [13] and invasion of tumor cells [14]. We added both the relevant molecules (uPAR-aODN and M25 peptide) and the respective negative controls to unpolymerized Matrigel, containing inhibitor-MIX, before implantation. At the third day, other treatments were performed subcutaneously into the plugs: such treatments consisted in plug administration of liposome-encapsulated vehicle alone (DOTAP), DOTAP + scramble ODN, DOTAP + uPAR-aODN, scramble M25 peptide and M25 peptide. As shown in Fig. 6b, uPAR-integrin uncoupling by the M25 peptide as well as uPAR silencing produced an evident reduction of amoeboid angiogenesis, as shown also by decreases in hemoglobin content, indicating a role of uPAR and uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis also in vivo. These results were confirmed in histological analysis after staining with hematoxylin-eosin that revealed the presence of murine vessels.