DTD, an anti-inflammatory ditriazine, inhibits angiogenesis in vitro and in vivo

The ditriazine derivative DTD (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3,2-d]-1,2,3-ditriazine) has been previously reported to reduce the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The aim of this study was to test whether DTD affects angiogenesis. Our results show that DTD inhibits in vivo angiogenesis in the chorioallantoic membrane (CAM) assay at doses equal or lower than 0.3 nmol/egg. Different in vitro assays were used to study the potential effects of this compound on key steps of angiogenesis, namely, a colorimetric assay of cell proliferation/viability, a morphogenesis on Matrigel assay, zymographic assays for gelatinases and nuclear morphology and cell cycle analysis for apoptosis induction. Our data indicate that DTD inhibits proliferation but does not induce apoptosis in endothelial cells in vitro. DTD suppresses the endothelial capillary-like chord formation at concentrations lower than those required to inhibit proliferation. DTD treatment inhibits the matrix metalloproteinase-2 production in endothelial and fibrosarcoma cells, but does not affect the cyclooxygenase-2 expression in endothelial cells, as assessed by western blot analysis. Taken together, results here presented indicate that DTD exhibits an anti-angiogenic activity that is independent of inflammatory processes and make it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.


4,10-dichloropyrido
thieno [3,2-d':3,2-d]-1,2,3ditriazine (DTD) (Fig.1), is a ditriazine derivative that modulates acute inflammation in murine models by inhibition of leukocyte functions and expression of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) [10]. Oral administration of DTD reduced the degree of granulomatous inflammation and vascular density in a murine air pouch granuloma model. The inhibition of the endogenous production of angiogenic cytokines and COX-2 expression in the granuloma, was then suggested to participate in the inhibition of vascularization by DTD in this model of inflammation [11].
Results presented here show for the first time that the antiangiogenic effect of DTD is not only due to a putative modulation of the production of angiogenic cytokines in inflammation, but it is exerted directly on endothelial cells. DTD inhibits angiogenesis in vivo in a widely employed angiogenesis assay (chorioallantoic membrane assay). Furthermore, in vitro assays show that DTD interferes several functions of the activated endothelial cells, namely proliferation, proteases production and differentiation. Taken together, our data suggest the possibility of utilising this compound for the treatment of angiogenesis-related diseases.  (Roskilde, Denmark). DTD was prepared by diazotation of 3,6-diamino-2,5-dicyanothieno [2,3-b]pyridines according to modified procedures from the literature [12], dissolved in dimethylsulfoxide (DMSO) at a concentration of 2 mg/ml and stored at -20ЊC until use. COX-2 monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA), the peroxidase-conjugated antimouse IgG was purchased from Amersham Biosciences (Buckinghamshire, UK), and the ␤-actin, monoclonal antibody was from Sigma. Fertilised chick eggs were obtained from Granja Santa Isabel (Córdoba, Spain [13] and maintained in Medium 199 containing HEPES (10 mM), L-glutamine (2 mM), heparine (10 mg/mL), penicillin (50 IU/ml), streptomycin (50 g/ml) and amphotericin (1.25 g/ml), supplemented with 3 mg/l endothelial cell growth supplement (ECGS, Sigma) and 20 % FBS in 5% CO2 and 37°C.

Endothelial cell differentiation assay: chord formation on Matrigel
Wells of a 96-well plate were coated with 50 L of Matrigel (10.5 mg/ml) at 4ЊC and allowed to polymerise at 37ЊC for a minimum of 30 min [14]. 5 ϫ 10 4

Chorioallantoic membrane (CAM) assay
CAM assay was performed as described [16]. Fertilised chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. DTD was added to a 0.7% solution of methylcellulose in water, and 10 l drops of this solution were allowed to dry on a Teflon-coated surface in a laminar flow hood. After implanting the methylcellulose discs on the CAM, the eggs were sealed with adhesive tape and returned to the incubator for 48 hrs. Negative controls were made with DMSO mixed with the methylcellulose. After re-incubation, the CAM was examined under a stereomicroscope by two different observers. The assay was scored as positive when both of them reported a significant reduction of vessels in the treated area. The gelatinolytic activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) delivered to the conditioned media were detected as previously described [17]. Aliquots of conditioned media normalized for equal cell numbers were subjected to non-reducing SDS-PAGE with gelatin (1mg/ml) added to the 10% resolving gel. After electrophoresis, gels were washed twice with 2% Triton X-100 in 50 mM Tris/HCl, pH 7.4, and twice with 50 mM Tris/HCl, pH 7.4. Each wash was for 10 min and with continuous shaking. After incubation (overnight at 37°C) in a substrate buffer (50 mM Tris/HCl, pH 7.4, 1% Triton X-100, 5 mM CaCl2, and 0.02% Na3N), gels were stained with Coomassie blue R-250 and the bands of gelatinase activity were detected as non-stained bands in a dark, stained background.

DTD inhibits in vivo angiogenesis in the chick chorioallantoic membrane assay
The CAM assay is frequently used to determine the ability of test compounds to inhibit in vivo angiogenesis. In controls, blood vessels form a dense and spatially oriented branching network composed by vascular structures of progressively smaller diameter as they branch ( Fig. 2A). Table 1 summarises the evaluation of the in vivo inhibition of angiogenesis in the CAM assay by DTD, showing that the anti-angiogenic activity of this compound is maintained as low as 0.3 nmol per CAM, where 80% of the eggs scored positive. DTD anti-angiogenic effect was observed as an inhibition of the ingrowth of new vessels in the area covered by the methylcellulose discs. The peripheral vessels (relative to the position of the disc) grew centrifugally, avoiding the treated area, where a decrease in the vascular density could be observed ( Fig. 2B and C). Signs of inflammation, such as a whitening of the CAM, were not observed.

DTD inhibits the growth of endothelial and tumour cells
Angiogenesis involves local proliferation of endothelial cells. We investigated the ability of DTD to inhibit the growth of endothelial cells. Survival curves obtained with the MTT assay showed that DTD inhibited, in a concentration-dependent manner, the growth of cultured endothelial cells (Fig. 3A). IC50 value of this anti-proliferative effect was 21 (Fig. 3A).

As a first approach to determine whether DTD could induce apoptosis in endothelial cells, nuclear morphology and flow cytometric analysis of the cell sub-population distribution were investigated in BAE cells after 14 hrs treatment with 3 or 15 M of DTD. Our data
show that neither significant changes on the nuclear morphology (Fig. 3B) nor significant increases in sub-G1 population (Fig. 3C (Fig. 3C). This pattern is consistent with blocks to progression at the S and G2/M phases of the cell cycle and may underlie the growth inhibitory activity observed for this compound.

DTD inhibits the capillary chord formation by endothelial cells
The final event during angiogenesis is the organisation of endothelial cells in a three-dimensional network of tubes. In vitro, endothelial cells plated on Matrigel align themselves forming tubelike structures, already evident a few hours after plating ( Fig. 4A and C

Effect of DTD on endothelial and tumour cells gelatinases secretion
Angiogenesis involves the acquisition by endothelial cells of the capability to degrade the basement membrane and, in general, to remodel the extracellular matrix. As shown in Fig. 5

, in our hands HUVE cells express the 72 kD pro-form of MMP-2. However, no MMP-9 secretion by HUVEC could be detected. Gelatin zymography of conditioned media of HUVE cells untreated and treated for 24 hrs with 3 or 15 M of DTD (Fig. 5), shows that 15 M of this compound produced complete inhibition of MMP-2 secretion by endothelial cells. The effect on MMP-2 production does not seem to be endothelial-specific, since a decrease in MMP-2 secretion by HT1080 tumour cells was also observed after treatment with 15 M DTD.
No effect on MMP-9 levels was observed after HT1080 treatment with 15 M DTD (Fig. 5).

Effect of DTD on endothelial COX-2 expression
Inhibition of either endothelial COX-2 expression or of its enzymatic activity has recently been considered a new strategy to inhibit Fig. 3  angiogenesis. Taking into account the previously described downregulation of COX-2 expression levels by DTD in inflammatory cells [10,11], the effect of DTD on endothelial COX-2 expression was investigated. As shown in Fig. 6, 15 M DTD did not affect the expression level of COX-2 in HUVE cells.

Discussion
The new ditriazine derivative DTD exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 (PGE2) production, which could be due to a decreased expression of inducible NO synthase and COX-2 in activated macrophages [10].

DTD inhibits the vascularisation in an inflammatory model, what has been suggested to be related to inhibition of cytokine and PGE2 production by interfering with NF-B activation [11]. The objective of our work has been to determine if DTD could inhibit angiogenesis in vivo in a model where inflammation was not directly involved, and to elucidate if DTD might directly interfere any of the following key steps mediated by endothelial cells: degradation of the basement membrane, proliferation of endothelial cells, and the formation of capillary-like chords.
The evaluation of the potential anti-angiogenic activity of test compounds in the in vivo CAM assay is one of the most widely used in vivo angiogenesis assays [18]. Our results show that DTD inhibits the neovascularisation of the chick chorioallantoic membrane at concentrations that are several orders of magnitude lower than those required for other inhibitors of angiogenesis [19][20][21], demonstrating that this compound exhibits a potent anti-angiogenic activity, independently of a putative  modulation of the production of angiogenic cytokines in an inflammatory environment.
Capillary endothelial cells proliferate in response to an angiogenic stimulus during neovascularisation. Our data show that DTD inhibits the growth of endothelial cells by slowing down proliferation. DTD caused accumulation of endothelial cells in the G1 phase of the cell cycle, with concomitant depletion of S and G2/M phases of the cell cycle, while inducing little or no apoptosis. IC50 values obtained with DTD in BAE and HUVE cells are higher than those reported for compounds to be considered to exert their anti-angiogenic activity by inhibition of endothelial cell proliferation [22,23]. DTD is not a specific inhibitor or endothelial cell growth. This lack of specificity is also exhibited by other anti-angiogenic compounds that are considered to act mainly through inhibition of endothelial cell proliferation [23]. Although in our hands the anti-proliferative activity has not been the most prominent effect of DTD on endothelial cells, its contribution to the anti-angiogenic potential role of DTD can not be ruled out.
Our data indicate that DTD inhibits capillary-like chord formation by endothelial cells at concentrations that are lower than or in the same range as those required for other previously described inhibitors of angiogenesis [19-21, 24, 25]. The concentrations required for a complete abrogation of tubulogenesis were lower than those required to inhibit cell proliferation, and do not affect human neutrophils or murine macrophages viability [10,11]. Therefore, although a role of the anti-proliferative activity of DTD could not be discarded, our results suggest that DTD anti-angiogenic activity could be dependent on prevention of capillary-like chord formation rather than endothelial cell proliferation. Recently Miller et al. [26] have suggested criteria by which a chemotherapeutic agent might reasonably be considered to have meaningful anti-angiogenic activity. Taking into account that DTD interferes with endothelial cell differentiation at concentrations that do not cause cell death, this compound could be considered an anti-angiogenic compound, assessment that is reinforced by the angiogenesis inhibitory activity of DTD in vivo. It should be pointed out that the previously reported effects of DTD on the functions of inflammatory cells were also exerted at concentrations that did no affect their viability [3,4].
A positive proteolytic balance is required for capillary sprouting and lumen formation during angiogenesis. Matrix metalloproteinases, particularly the gelatinases MMP-2 and MMP-9, play a central role during angiogenesis [27]. Endothelial cells constitutively secrete MMP-2, which is required for the tumour to trigger the angiogenic response [28]. MMP-2 is secreted as an 72 kD inactive pro-form; when it is converted to a 62 kD active form it can degrade collagen types IV and V, laminin and elastin. In intact cells, MMP-2 is activated at the cell surface by a process involving interaction of the C-terminal component of MMP-2 with a plasma membrane activation mechanism [27]. Our data show that incubation with DTD inhibits MMP-2 proform secretion by HUVE cells. Similar decreases of pro-MMP-2 levels in the conditioned media of endothelial cells have been described for halofuginone [29] and aeroplysinin-1 [24], and they have been suggested to lead to the inhibition of the endothelial cell tube formation in vitro. This is in agreement with previously reported data showing that when endothelial cells are cultured on Matrigel, the formation of tubular networks is increased by the addition of recombinant MMP-2 and decreased when a neutralizing antibody is added [30]. The inhibition of MMP-2 production by DTD does not seem to be endothelial-specific, since a similar effect was observed in HT-1080 fibrosarcoma cells. However MMP-9 secretion by HT1080 cells is not affected by DTD incubation. MMP-9 is not constitutively expressed by endothelial cells, but it may be induced in response to several factors, including the tumour promoter chemical phorbol myristate acetate (PMA), cytokines or stress [27,31,32]. Our data show that incubation with DTD does not induce the 92 KD pro-MMP-9 secretion by HUVE cells.
The zinc-finger transcription factor Ets-1 seems to play a key role in activation of the proteolytic system by transactivation of the promoters of many proteases, including the MMPs. Expression of Ets-1 is mediated by the mitogen activated protein kinase (MAPK, ERK1 and ERK2) pathway. The effect of DTD on MMP-2 expression could suggest a possible intervention of this pathway, what could also explain DTD inhibition of the endothelial cell morphogenesis and proliferation, also mediated by the MAPK/ERK pathway. On the other hand, a role of integrin-mediated pathways in the mechanism of action of DTD can not been discarded, since they are involved in the transduction of the signals leading to the proliferation, differentiation and extracellular matrix degradation by endothelial cells [8].
Cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumour cells, stromal cells and angiogenic endothelial cells during tumour progression. The contributions of COX-2 in tumour angiogenesis include: (a) the increased expression of the proangiogenic growth factors; (b) the production of the eicosanoid products thromboxaneA2, PGE2 and PGI2 that can directly stimulate endothelial cell migration and growth factor-induced angiogenesis; and potentially, (c) the inhibition of endothelial cell apoptosis by stimulation of Bcl-2 or Akt activation [33]. Therefore, the targeting of endothelial COX-2, either by inhibiting its enzymatic activity or by blocking its transcription, might be useful in combating angiogenesis-dependent diseases [34]. Previous results showing that DTD inhibited the COX-2 expression by lipopolysaccharide-stimulated murine peritoneal macrophages in vitro [10] and that a decreased COX-2 expression was observed in murine air pouch granuloma after oral administration of DTD [11], suggested the possibility that the anti-angiogenic activity of DTD could be also due to a direct modulation of the endothelial COX-2 expression, what could induce endothelial apoptosis cells by inhibition of the Akt signalling axis [35]. Our results show that incubation with DTD neither affect the expression of COX-2 in HUVE cells, nor induces detectable apoptosis in endothelial cells, suggesting that different signalling pathways are modulated by DTD in endothelial and inflammatory cells.
In conclusion, we have shown for the first time that DTD is able to inhibit endothelial cell proliferation, differentiation and MMP-2 secretion in vitro, and it exhibits a potent inhibition of in vivo angiogenesis in the chick chorioallantoic mem-brane. Although additional studies will be needed to elucidate the molecular mechanisms underlying the anti-angiogenic activity of DTD, data presented here suggest its potential in therapeutic applications for the treatment of angiogenesis related diseases.