Melatonin promotes angiogenesis during protection ...

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J. Pineal Res. 2010; 49:130–140

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/j.1600-079X.2010.00776.x

 2010 Indian Institute of Chemical Biology, CSIR, India Journal compilation  2010 John Wiley & Sons A/S

Journal of Pineal Research

Melatonin promotes angiogenesis during protection and healing of indomethacin-induced gastric ulcer: role of matrix metaloproteinase-2 Abstract: Matrix metalloproteinase (MMP)-2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N-acetyl-5-methoxy tryptamine), an antioxidant and anti-inflammatory agent, prevents indomethacin-induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP-2mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin-induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP-2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over-expression of MMP-2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP-2. In addition, upregulation of MMP-2 was parallel to MMP-14 and inverse to tissue inhibitor of metalloprotease (TIMP)-2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP-2 and VEGF over-expression during protection and healing of gastric ulcers. This study highlights for the first time a phase-associated regulation of MMP-2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin-induced gastropathy.

Krishnendu Ganguly1*, Anamika Vivek Sharma1*, Russel J. Reiter2 and Snehasikta Swarnakar1 1 Department of Physiology, Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Kolkata, India; 2Department of Cellular and Structural Biology, University of Texas Health Science Center 7703 Floyd Curl Drive, San Antonio, TX, USA

Key words: angiogenesis, extracellular matrix, indomethacin, matrix metalloproteinase, melatonin, tissue inhibitor of metalloproteinase, vascular endothelial growth factor Address reprint requests to Dr. Snehasikta Swarnakar, Ph. D., Head, Department of Physiology, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata-700032, India. E-mail: [email protected] *Both authors contributed equally. Received February 4, 2010; accepted April 1, 2010.

Introduction Repair of gastric damage is a natural wound-healing process of regenerating submucosal and mucosal tissues. A set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage. These events overlap in time and may be categorized into separate steps, i.e., inflammatory, proliferative, maturation and remodeling phases [1]. The proliferative phase is characterized by angiogenesis, collagen deposition and formation of granulation tissue, epithelialization, and ulcer wound contraction [2]. Angiogenesis, the growth of new blood capillaries from the existing vessels, is the key physiologic process and is controlled by signals from pro- and anti-angiogenic molecules in the gastric mucosa. Numerous mediators, including growth factors, transcription factor and signaling molecule have been reported to play a major role in inflammation-associated angiogenesis [3]. However, limited studies have reported the efficacy of angiogenic molecules to promote gastric ulcer healing [4–8]. Vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis, while nitric oxide (NO) 130

is a downstream effector molecule for increasing the number of capillaries [8, 9]. VEGF increases NO levels in endothelial cells (ECs) by elevating endothelial nitric oxide synthase (eNOS) [8, 10, 11]. Thereafter, EC proliferates to form microvessels and a capillary network [8, 10]. Recently, the role of eNOS in EC migration was demonstrated both in in vivo and in vitro studies [9, 12], but the precise mechanism by which VEGF regulate angiogenesis during gastric ulcer healing is unknown. Matrix metalloproteinases (MMP)s are extracellular matrix (ECM)-degrading endopeptidases that can influence physiological and pathological situations, such as tissue development, embryogenesis, wound healing, atherosclerosis, osteoarthritis and cancer [13, 14]. Gastric ulcer healing is a complex process involving enzymatic activities that converge toward damage repair. The process is tightly regulated by matrix turnover, in which MMPs play a pivotal role [13–15]. Of late, research has focused on degradation of ECM in ulcerated margins of gastric tissues [8, 16, 17]. MMPs are involved in angiogenesis, cell proliferation, apoptosis and metastasis. The activity of MMPs is tightly regulated in a complex fashion that

Melatonin accelerates angiogenesis in gastric tissues includes pro-enzyme activation and the action of specific tissue inhibitor of metalloproteinases (TIMPs) [18]. Dysregulation of MMPs has been demonstrated in impairment of angiogenesis during pathological processes such as arthritis, tumor invasion and gastric ulceration [13, 14]. Some MMPs regulate wound healing by removal of damaged tissues and thus may facilitate migration of different cell types during neovascularization and collagenization [13]. MMPs also release ECM-bound proangiogenic factors, viz. VEGF, transforming growth factor b and basic fibroblast growth factor [18]. Lower expression or absence of these growth factors or receptors may limit angiogenesis and thereby decelerate the healing process of gastric ulcer. Herein, the preliminary studies on angiogenesis using rat corneal micropocket assay revealed that melatonin exerted significant proangiogenic activities through induced expression and activities of MMP-2 which occur in parallel with increased protein and gene expression of VEGF. Moreover, during prevention of indomethacin-induced gastric ulcer, melatonin accelerated new blood vessel formation and modulated ECM turnover through secretion and activation of MMP-2. The study documented for the first time a mechanistic basis of angiogenesis via MMP-2 expression and activity during protection and healing of indomethacin-induced acute gastric ulcer by melatonin. It is plausible that melatonin may be used therapeutically for the treatment of gastric ulcer.

Materials and methods Chemicals Gelatin from porcine skin, indomethacin, melatonin, Triton X-100 (TX), protease inhibitors cocktail, gelatin fused with 4% beaded agarose and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium solution were obtained from Sigma (St. Louis, MO, USA). MMP-2 for standard was purchased from Chemicon (Hampshire, UK). Monoclonal and polyclonal anti-antibodies and florescent-conjugated and nonconjugated secondary antibody were purchased from Santa Cruz (CA, USA). 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium solution was obtained from Sigma. TRIzol reagent, Superscript II Reverse Transcriptase and oligo (dT)15 primer were purchased from Invitrogen (Carlsbad, CA, USA). Real-time assay kits were purchased from Applied Biosystems (Carlsbad, CA, USA). Collagenase, Type III and IV collagens were purchased from Calbiochem (San Diego, CA, USA) and Sigma.

Rat corneal micropocket assay Rats were anesthetized by an intraperitoneal administration of a ketamine hydrochloride solution (12 mg/kg bw). Eyes were irrigated with ringers lactate, proptosed with a dental dam, and topically anesthetized with a 0.5% propacaine hydrochloride solution. A 0.3-mm transverse incision was made centrally, penetrating about halfway through the corneal stroma with a cataract knife (E-63; Storz, St Louis, MO, USA). The corneal micropocket was formed with a dulled keratome blade (No. OP-600, Muller, Chikage.I.II) and extended to 1 mm from the cornoscleral limbus. The micropocket was enlarged to a final size of 0.3 by 1.5 mm by insertion of a fine forceps. Vehilcle control (20% MeOH), melatonin (20 and 40 lg), indomethacin (5 lg) and melatonin plus indomethacin were impregnated in carboxymethyl cellulose (CMC) disks and were implanted at the base of each pocket with fine forceps. The disks had previously been placed in sterile ringers lactate solution. The micropocket was closed by sliding the forceps over the corneal epithelium overlaying the pocket with the side of the forceps. The eyes were irrigated with antibiotics (Neosporin ophthalmic solution; Burroughs Wellcome Co, Triangle Park, NC, USA). All procedures were performed aseptically [19]. Macroscopic images were captured through a high resolution Canon Camera associated with Camedia software (E- 20P 5.0 Megapixel) and processed under Adobe Photoshop version 7.0. The quantitative measurement of angiogenic index was calculated after taking the printed photographs in graph paper manually. Indomethacin-induced acute gastric lesions in mice Indomethacin at a dose of 80 mg/kg bw dissolved in alkaline water was administered orally to different groups of BALB/c mice and was kept at room temperature for 4 hr to generate acute gastric ulcers. The 80 mg/kg bw was considered as an effective dose for generation of acute gastric lesions. The control group received vehicle orally. Animals were anesthetized by ketamine (12 mg/kg bw) followed by cervical dislocation for killing. After 4 hr, the animals were killed, and gastric lesions in the fundic stomach were scored and expressed as ulcer index as follows: 0 = no pathology; 1 = a small pinhead ulcer spot; and 2–5 = a band-like lesion of 2–5 mm length. The sum of the total scores divided by the number of animals was expressed as the mean ulcer index [17, 20].

Experimental model

Preventive and healing studies of indomethacininduced gastric lesions in mice

Animal experiments were carried out in male SpragueDawley rats (180–220 g) and BALB/c mice (20–25 g) bred in-house and maintained with free access of food and water following the approval and guidelines of animal ethics committee of the Institute. Experiments were designed to minimize animal suffering and to use the minimum number associated with valid statistical evaluation. Before commencement of experiments, animals of both control and experimental groups were kept separately under standard controlled condition and were fasted for 12 hr with free access to water.

To evaluate the preventive and healing effect of melatonin, indomethacin at a dose of 80 mg/kg bw was used in all experiments. Melatonin at a dose of 80 mg/kg bw was administered ip 30 min prior to indomethacin treatment to test its preventive effect. To study the healing effect, one group of animals served as auto healing where indomethacin at a dose of 80 mg/kg bw was administered orally for 4 hr, and the other group was melatonin treated (60 mg/kg bw) following indomethacin treatment. All animals were then kept for 24 hr with vehicle treatment and were killed at different time intervals [17, 20]. 131

Ganguly et al. Tissue extraction and partial purification The fundic portion of the gastric mucosa was suspended in phosphate-buffered saline containing protease inhibitors, minced and incubated for 10 min at 4C. After centrifugation at 12,000 · g for 15 min, the supernatant was collected in new a tube marked as phosphate buffered saline (PBS) samples. The pellet was then extracted in lysis buffer (10 mm Tris-HCl pH 8.0, 150 mm NaCl, 1% Triton X-100 and protease inhibitors) and centrifuged at 12,000 g for 15 min to obtain extracts. Both PBS and tissue extracts were preserved at )70C. PBS extracts from all tissue types were used for purification of MMP-2. Briefly, an extract was mixed with gelatin–agarose bead, incubated at 4C for 1 hr followed by centrifugation at 1500 · g for 5 min. The pellet was washed twice with PBS through centrifugation at 1500 · g for 5 min, and MMP-2 was eluted by incubating the pellet with Lammeli sample loading buffer for 10 min at room temperature [21]. SDS-PAGE, Gelatin zymography and Western Blotting Proteins separated by SDS-PAGE were detected in the gels by either Coomassie Blue R-250 or silver staining. Gelatin Zymography was performed for detection of MMP-2 activity. Briefly, partial purified PBS extracts of gastric tissues were electrophoresed in 8% SDS-polyacrylamide gel containing either gelatin (0.5 mg/mL), under nonreducing conditions. Gel was washed twice in 2.5% Triton X-100 and incubated in CAB (40 mm Tris-HCl, pH 7.4, 0.2 m NaCl, 10 mm CaCl2) at 37C for 18 hr. Gels were stained with 0.1% coomassie blue followed by destaining [19–21]. The zones of gelatinolytic activities came as negative staining. Quantification of zymographic bands was carried out using densitometry linked to proper software (Lab Image). For Western blotting, SDS-PAGE-separated proteins were transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST), the membranes were incubated overnight at 4C with 1 lg/mL primary polyclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA), washed four times with PBST, incubated for 1 or 2 hr at room temperature with horseradish peroxidaseconjugated goat anti-rabbit IgG (Pierce) diluted at 1:2, 500 in PBST containing 5% milk. The bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazonium substrate solution. Gelatin (0.28 mg/mL) zymography were performed on 8% SDS-PAGE gels as described [21, 22]. Following electrophoresis, gels were washed twice in 2.5% Triton X-100, incubated overnight at 37C in calcium assay buffer, and stained with Coomassie Blue R-250.

(calcium assay buffer). Individual MMPs or organomecurial p-aminophenylmercuric acetate (APMA) were added to collagen mixtures at the concentrations indicated in the text. Reactions were performed at 23C to maintain the native triple helical collagen status and then terminated by the addition of 10· reducing SDS sample buffer. The samples were analyzed for specific collagen cleavage by SDS-PAGE [22]. Real-time PCR Total cellular RNA was extracted with TRIzol reagent (Invitrogen, Foster city, CA, USA) according to the manufacturerÕs protocol and quantified by measuring the absorbance at 260 nm. Complementary DNA was synthesized using 1 lg of total RNA in a 20 lL reaction buffer using Superscript II Reverse Transcriptase with an oligo(dT)15 primer (Invitrogen). The real-time polymerase chain reaction (PCR) was used for quantitative detection of VEGF and MMP-2 gene in control, indomethacin and melatonin-pretreated gastric tissues. The reaction was carried out in a 20 lL final volume with the following conditions containing 50 ng of cDNA, 10 pmol of primer and SYBR Premix Ex Taq (Takara, Japan). Polymerase activation was carried out at 95C for 3 min followed by 40 cycles at 94C for 30 s, 58C for 30 s and 72C for 60 s using 10 pmol of the following primers: for MMP-2, sense, 5¢-ACCAGAACACCATCGAGACC-3¢ and antisense 5¢TACTTTTAAGGCCCGAGCAA-3¢ (expected product 182 bp); for VEGF, sense, 5¢-TTGAGACCCTGGTGGACATC-3¢ and antisense, 5¢-CTCCTATGTGCTGGCTTTGG-3¢ (expected product 192 bp) and for GAPDH, sense, 5¢-CCACCCAGAAGACTGTGGAT-3¢, and antisense, 5¢CACATTGGGGGTAGGAACAC-3¢, (expected product 173 bp). A quantitative measure of VEGF and MMP-2 was obtained through amplification of GAPDH and VEGF and MMP-2 cDNA in each sample using the same PCR conditions. The amount of VEGF and MMP-2 expression relative to the total amount of cDNA was calculated as DCt = Ct GAPDH – Ct VEGF/MMP-2 where Ct VEGF, Ct MMP-2 and Ct GAPDH were fractional cycle number at which fluorescence generated by reporter dye exceeds fixed level above baseline for VEGF and MMP-2 and GAPDH cDNA, respectively. The changes of VEGF/ MMP-2 expressions in respective samples compared to control were expressed as DDCt = DCt control – DCt respective samples. Relative expressions in VEGF/MMP-2 genes in respective samples were calculated as 2- DDCt. The real-time PCR products were analyzed by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. PCR product sizes were estimated by 100-bp marker (Invitrogen) in each case [23]. Immunofluorescence analyses

Collagenase assay Assays for the cleavage of native, acid-soluble collagens by specific MMPs were performed essentially as described. Briefly, collagens were solubilized in 10 mm acetic acid and diluted to the indicated concentrations in 40 mm Tris-HCl, pH 7.5, 200 mm NaCl, 10 mm CaCl2, 0.05% Brij-35 buffer 132

For tissue histology control, indomethacin and melatoninpretreated indomethacin-treated mouse stomachs were fixed in 4% parformaldehyde solution in PBS (40 mm phosphate buffer, 150 mm NaCl, pH 7.4), paraffin-embedded and cut into sectioned into (2–3) mm2 pieces. Sections of 5 lm thick were deparaffinized and processed for

Melatonin accelerates angiogenesis in gastric tissues immunohistochemical analyses. For the immunoflorescence study, paraformaldehyde-fixed tissues were washed in PBS (40 mm phosphate buffer, 150 mm NaCl, pH 7.4) and processed through ascending alcohol series for dehydration at 4C. After dehydration, the tissue sections were embedded in paraffin and sectioned at 5 lm thickness using a microtome. The sections were passed through the dehydration procedure by dipping them in descending alcohol series. Thereafter, the slides were dipped in the antigen retrieval buffer system, and the sections were encircled by using pap-pen (Sigma), dipped in the blocking solution for 2 hr at room temperature (10% BSA in PBST) followed by the incubation over night at 4C in primary antibody solution (1:200 dilutions in PBST 5% BSA) in a humid chamber. The tissue sections were washed four times with PBST followed by incubation with fluorescence-conjugated secondary antibody solution (1:400 dilutions in PBST containing 5% BSA) for 2 hr at room temperature under shaking conditions. After washing in 5–10 mL of PBST, the images were observed in an Olympus microscope. Images at different magnification were captured using Camedia software (E- 20P 5.0 Megapixel) and processed under Adobe Photoshop version 7.0.

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Statistical analysis The activity bands of zymogram, protein bands of western blots and DNA bands of real-time PCR were quantified using LabImage (version 2.7.1, Kapelan GmbH)-based densitometry software. Data are presented as the mean ± S.E.M. Statistical analysis was performed using StudentÕs t-test and Student–Newman–Keuls test (ANOVA) through GraphPad InStat3 (version 3.06, San Diego, CA, USA) software [21].

Results Melatonin is known to possess gastroprotective action because of its antioxidant and anti-inflammatory effects. Herein, we addressed whether melatonin exerts any effect in angiogenic processes during gastroprotection? Therefore, we investigated melatoninÕs angiogenic potential in rat corneal micropocket assay. The results shown in Fig. 1 and Table 1 prove that melatonin possesses significant angiogenic potential. We found that melatonin dose dependently ameliorated the parameters of angiogenic index i.e. total length and branch points of capillaries by 1.8–3.6-fold and 2.2–4.1-fold, respectively, when compared with vehicletreated control in both assay systems (Fig. 1A–C, Table 1). However, only indomethacin disrupted the existing blood vessels demonstrating its anti-angiogenic potential (Fig. 1D). Herein, indomethacin caused 2–4-fold suppression of each parameters of angiogenic index (Table 1). To address whether melatonin protected against the antiangiogenic property of indomethacin in vivo, we applied melatonin plus indomethacin-soaked CMC disks in rat cornea assay. Surprisingly, we found that melatonin dose dependently suppressed the indomethacin-induced attenuation of angiogenic indices (Fig. 1E,F). Herein, melatonin at its highest doses, elevated the angiogenic index significantly by increasing the length of blood vessels at 1.5–3-

Mela (40 µg)

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Fig. 1. Angiogenic effect of melatonin on rat corneal micropocket assay (CMA): The experiments were conducted by implanting various molecules impregnated in carboxymethyl cellulose disk containing in rat cornea as mentioned in ÔMethodsÕ. Photograph of new blood vessels in rat eye treated with vehicle (A), 20 lg/mm2 melatonin (B), 40 lg/mm2 melatonin (C), 5 lg/mm2 indomethacin (D), indomethacin plus 20 lg/mm2 melatonin (E) and indomethacin plus 40 lg/mm2 melatonin (F).

fold and, branch points at 1.5–3.5-fold, respectively, while arresting the anti-angiogenic action of indomethacin (Table 1). To understand the mechanism of melatonin-mediated angiogenesis in rat CMA, we examined the regulation of MMP-2 in melatonin assisted angiogenesis. Among MMPs, MMP-2 (gelatinase A, 72 kDa enzyme) is considered as one of the major ECM modifiers during angiogenic process. Therefore, regulation of MMP-2 in melatonin assisted angiogenesis in rat CMA was investigated. Our study proves that increased angiogenesis was tightly regulated by the MMP-2 induction at both activity and expression levels (Fig. 2). Herein, melatonin alone or when applied with indomethacin caused auxiliary amelioration of MMP-2 activity and expression in a dose and time-dependent fashion and significantly triggered angiogenesis in rat cornea (Fig. 2A–C). However, anti-angiogenic property of indomethacin was associated with downregulation of MMP-2 activity and expression (Fig. 2A–C). Figure 2A shows that indomethacin caused 2-fold reduction, 133

Ganguly et al. Table 1. Angiogenic index in rat corneal micropocket assay

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Blood vessel Treatment (lg/mm2) None 20 lg melatonin 40 lg melatonin 5 lg indomethacin 5 lg indomethacin + 20 lg melatonin 5 lg indomethacin + 40 lg melatonin

Total length 100 180 ± 360 ± 25 ± 150 ±

5.2* 8.5* 3.5* 4.3*

Branch point 100 225 ± 415 ± 45 ± 210 ±

6.4* 9.8* 4.5* 3.5*

(B) 295 ± 6.5*

335 ± 5.5*

The experiments were conducted by implanting carboxymethyl cellulose disk containing various molecules in cornea as mentioned in the ÔMaterials and MethodÕ. The snaps were taken, and photographs were printed in a graduated graph paper that is divided into 1-in2 boxes by Adobe PhotoShop. Total length and branch points of newly formed blood vessel were measured from three independent experiments of each group. Results are reported as the means ± S.E.M. *P < 0.01 versus none (control), using t test to compare means.

whereas only melatonin dose dependently caused 1.2–1.4fold induction in MMP-2 activity. In contrary, melatonin plus indomethacin caused almost 3-fold increase of MMP-2 activity when compared with only indomethacin treatment (Fig. 2A). Because growth factors like VEGF are considered as potential angiogenic modulators, we analyzed their expression profile and examined the effect of melatonin thereon in rat CMA model by Western blot analysis (Fig. 2C). Herein, melatonin exerted significant proangiogenic activity by 2fold increase in VEGF expression. However, indomethacin caused inhibition of proangiogenic stimulus by moderate reduction of VEGF, whereas melatonin plus indomethacin caused supplementary augmentation of both molecules almost up to control values (Fig. 2C). More interestingly, we found that induced expression of MMP-2 followed almost a parallel expression profile with that of VEGF. Herein, indomethacin caused a moderate reduction in MMP-2 expression while only melatonin or melatonin plus indomethacin treatment caused little changes (Fig. 2C). As the activation of MMP-2 is critically dependent upon the ratio of MMP-14 and TIMP-2, we examined their expression as well. We found an analogous expression profile of MMP-14 and inverse expression profile of TIMP-2 when compared with the expression profile of MMP-2 (Fig. 2C). MMP-14 expression was reduced to 2-fold during indomethacin treatment whereas melatonin or melatonin plus indomethacin treatment caused an elevation. In contrary, indomethacin treatment caused an increase in TIMP-2 expression while melatonin or melatonin plus indomethacin treatment caused a reduction in its expression even lower that of control value (Fig. 2C). Western blot for GAPDH was performed to confirm equal protein loading in all the blots. Rat cornea micropocket assay revealed the mechanistic basis of melatonin‘s angiogenic and indomethacin‘s antiangiogenic potential. This result prompted us to investigate whether melatonin hastened the ulcer healing process by promoting angiogenesis during prevention of indomethacin-induced gastric lesions. To elucidate whether the 134

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Fig. 2. Regulation in Matrix metalloproteinase (MMP)-2 activity and expression in rat cornea following melatonin treatment: Dose and time-dependent studies were carried out in rat cornea by implantation of disk containing either vehicle or melatonin or indomethacin and melatonin plus indomethacin as described in ÔMethodsÕ. To monitor the secreted MMP-2 activities, phosphate buffered saline (PBS) extracts (80 lg) of rat cornea treated with different melatonin doses (A) and time periods (B) were subjected to gelatin zymography. The fold changes of individual bands were given below the respective gels. PBS extracts (120 lg) from dosedependent studies in rat cornea (C) were subjected to Western blot and probed separately with different polyclonal anti-vascular endothelial growth factor, anti-MMP-2, anti-MMP-14, anti-TIMP2 and anti-GAPDH antibodies. Fold changes of individual bands were given below the respective Western blot pictures. Fold changes in individual bands were measured by Lab Image-based designed densitometry program. Indo, M, +M, D3 and D7 are represented for Indomethacin, melatonin, indomethacin plus melatonin, day 3 and day 7, respectively.

angiogenic potential of melatonin was directly associated with MMP-2-mediated ECM turnover or not, mouse gastric tissues from control, ulcerated and melatonintreated group were subjected to immunofluorescence staining with MMP-2-specific primary antibodies. We found that MMP-2-specific proteins were localized throughout the

Melatonin accelerates angiogenesis in gastric tissues mucosal ECM (lamina propria) encircling the pits and glandular region in control tissues and hence maintains matrix turnover in normal gastric ECM (Fig. 3Aa,d and 3Ba,d). However, the localization of MMP-2 was significantly reduced in almost diffused pattern throughout the ulcerated margins along with the inside of pits and glandular cells (Fig. 3Ab, e and 3Bb, e). Interestingly, melatonin significantly reversed the appearance of MMP-2 signal in both mucosal and submucosal areas which were decreased during ulcerated situation (Fig. 3Ac, f and 3Bc, f). Altogether, it is confirmed that during acute gastric lesions, the structure of gastric ECM became completely disorganized whereas melatonin restored the proper orientation of ECM structure by increased localization of MMP2 during gastroprotection (Fig. 3A,B). Previous experiments revealed that indomethacin exhibited anti-angiogenic property while melatonin controlled angiogenesis through regulation of MMP-2. We examined the status of MMP-2 activity during ulceration and healing by gelatin zymography and Western blot. Figure 4A shows that melatonin alone increased total MMP-2 activity by 30% while indomethacin ulceration decreased the activity by 80% when compared with control. Interestingly, melatonin pretreatment augmented MMP-2 activity by 8-fold when compared with ulcerated tissues (Fig. 4A). In addition, ulceration caused reduction in VEGF expressions by 50% in each cases and melatonin treatment augmented VEGF expressions to control value (Fig. 4B). As MMP-2 activity is dependent upon the MMP-14 and TIMP-2, hence

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we determined their expression. Melatonin in each case caused an upregulation of MMP-14 expression by  2.5fold while ulceration caused a reduction by 5-fold when compared with control (Fig. 4B). On contrary, TIMP-2 expression was increased in ulcerated tissues by 2-fold and melatonin reversed it back to control (Fig. 4B). Western blot for GAPDH was performed to confirm equal protein loading in all the blots. Using real-time PCR, direct quantitative expression of MMP-2 and VEGF mRNA expression was measured in melatonin-treated and ulcerated tissue samples. The relative expressions of MMP-2 were reduced to 0.33 in ulcerated tissues and melatonin-augmented MMP-2 expression to 0.66 in comparison with 1.0 in control tissues (Fig. 4C,D). Similarly, Fig. 4E show that the relative mRNA expressions of VEGF were reduced to 0.2 in ulcerated tissues whereas melatonin pretreatment induced VEGF expression to 0.7 values when compared with 1 value of control. GAPDH gene was used as an internal control for VEGF and MMP-2 mRNA expression. To elucidate the therapeutic potential of melatonin in MMP-2-mediated angiogenesis, we investigated MMP-2 status and its mechanistic aspects in melatonin-mediated and indomethacin-mediated auto healing in mice (Fig. 5A). First, we determined the time course regulation of secreted MMP-2 activities in vivo in the ulcerated and healed tissues. We found that during both auto- and melatonin-mediated healing, activity of secreted MMP-2 gradually reached a saturation phase after 12 hr of healing process (Fig. 5A).

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Phase @ 10X Fig. 3. Angiogenic effect of melatonin in mouse gastric tissues and localization of Matrix metalloproteinase (MMP)-2 in gastric mucosa during prevention of acute gastric lesions: Gastric lesions were induced in mice by oral administration of indomethacin (80 mg/kg bw) and, melatonin (60 mg/kg bw) treatment was carried out prior to indomethacin. Control mice received vehicle only. After 4 hr, mice were killed, and stomachs were processed; immunofluorescence slides were prepared as described in ÔMethodsÕ. The phase contrast picture of immunofluorescence slides at 10· and 40· magnifications of vehicle (Aa and Ba), indomethacin treated (Ab and Bb) and melatonin-pretreated indomethacin-treated (Ac and Bc) gastric tissues, respectively. The FITC immunofluorescence MMP-2 proteins at 10· and 40· magnifications of vehicle (Ad and Bd), indomethacin treated (Ae and Be) and melatonin-pretreated indomethacin-treated (Af and Bf) gastric tissues, respectively. Extracellular matrix of gastric mucosa (lamina propria) is indicated by blue arrows.

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Interestingly, melatonin facilitated the healing process by production of more active forms of MMP-2 at last phases (24 hr) of healing (Fig. 5A,B). As VEGF modulate the physiological angiogenesis, we determined the expression levels in this therapeutic model (Fig. 5B). We found that indomethacin gradually reduced the expression of VEGF almost 90% at 12 hr of healing process. However, in melatonin-mediated healing, VEGF expression remained almost unaltered up to 12 hr and was diminished by 60% at 24 hr (Fig. 5B). Most interesting fact of our therapeutic experiment is that expression of MMP-2 followed an inverse pattern when compared with other angiogenic modulators like VEGF. Overall, MMP-2 activities were time dependently increased during auto as well as melatonin-mediated healing process (Fig. 5B). Herein, MMP-2 expression was increased by 15-fold in melatonin-mediated healing when compared with only 2-fold for auto healing (Fig. 5B). As MMP-14 and TIMP-2 expression ratio are vital for MMP-2 activity in vivo, we investigated their expressions from time-dependent experiment. MMP14 expression followed the parallel while TIMP-2 expres136

Fig. 4. Effect of melatonin on activity, expression and transcription of Matrix metalloproteinase (MMP)-2 and vascular endothelial growth factor (VEGF) during prevention of acute gastric lesions: phosphate buffered saline extracts were prepared from gastric tissues of control, ulcerated and melatonin-treated mice. Equal amount of extract was subjected to gelatin zymography (A) and Western blot (B). The later was probed separately with anti-VEGF, anti-MMP-2, anti-MMP-14, anti-TIMP-2 and anti-GAPDH antibodies. Fold changes were measured by using Lab Image-based densitometry program, and the values were given below each representative gel pictures or Western blot. Complementary (c)DNA were prepared by using reverse transcriptase (RT)PCR from gastric tissues of control, ulcerated and melatonin-treated mice as mentioned in ÔMethodsÕ. Equal concentrations (0.6 lg) of cDNA were used for real-time PCR analysis with specific primers of MMP-2, VEGF and GAPDH mRNA. Graphical representation of linear amplification plot of MMP-2 (C), VEGF (D) and GAPDH mRNA-specific products of control, ulcerated and melatonin-pretreated gastric tissues where DRn values were plotted against cycle number. The agarose gels in inset of C and D showing the MMP-2, VEGF and GAPDH mRNA-specific products from real-time PCR analysis in control, ulcerated and melatonin-treated tissues. Histographic representation of relative expression of MMP-2 (E) and VEGF (F)-specific transcript in acute ulcerated and melatonintreated tissues as measured by real-time RT-PCR.

sion followed an inverse pattern with MMP-2 expression (Fig. 5B). Herein, melatonin allowed more MMP-14 production and less TIMP-2 production at late phases (12– 24 hr) of healing process; therefore, MMP-2 production enhanced rigorously during melatonin-mediated healing process (Fig. 5B). We postulate that during both melatonin-mediated and nonmediated healing process, the active forms of MMPs can cleave collagen types III and IV which are principal constituents of blood vessel and basement membrane and hence facilitate the angiogenesis in gastric tissues. Therefore, we have activated the proMMP-2 into active forms by treatment with APMA and allowed reaction in in vitro condition with mouse-specific collagens III and IV separately as substrates. The PBS extract of control mouse gastric tissues were passed through gelatin–agarose matrix beads, and MMP-2 was partially purified. The purified fractions consisted of 75% proMMP-2 and 25% active MMP-2, as judged by gelatin zymography (data not shown). To activate the pro-MMP-2 (72 kDa), partially purified MMP-2 was treated with different concentrations

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(D)

(B)

Fig. 5. Effect of melatonin on regulatory molecules during healing of indomethacin-induced gastric lesions: Dose and timedependent gastric lesions and healing studies were carried out in mice as described in ÔMethodsÕ. To monitor the secreted Matrix metalloproteinase (MMP)-2 activities, phosphate buffered saline (PBS) extracts (80 lg) of gastric tissues from different group of mice (A) were subjected to gelatin zymography. The fold changes of individual bands were measured by using Lab Image-based densitometry program, and the values were given below each representative gel pictures. PBS extracts (120 lg) from of mouse gastric tissues under therapeutic studies (B) were subjected to Western blot and probed separately with polyclonal anti-vascular endothelial growth factor, anti-MMP-2, anti-MMP-14, antiTIMP-2 and anti-GAPDH antibodies. Fold changes in individual protein bands were measured by Lab Image-based designed densitometry program, and the values were given below respective Western blots.

of APMA for 15 min at 23C followed by gelatin zymography. Figure 6A reveals that 1 mm APMA was optimal for activation of proMMP-2. Keeping APMA concentration fixed at 1 mm, the reactions were run for various time points and analyzed in gelatin zymography for conversion of 62-kDa active forms (Fig. 6B). Conversion to active MMP-2 (62 kDa) was detectable within 5–10 min, and nearly 50% conversion occurred at 30 min. To assess the interstitial collagenase-like activity of MMP-2, the APMAgenerated active form of the enzyme was incubated at 23C with purified mouse collagen types III (Fig. 6C) and IV (Fig. 6D). True collagenase-like activity of MMP-2 was measured by comparing the cleavage site at distinct 3/4 and 1/4 fragments by comparing mouse-specific collagenase activity. The activity of mouse MMP-2 was more pronounced with type III collagen (Fig. 6C) compared to that of collagen type IV (Fig. 6D) suggesting the function of active MMP-2 toward blood vessels formation than ECM remodeling thereby elucidating its strong angiogenic potential.

Fig. 6. Collagenolytic activity of mouse Matrix metalloproteinase (MMP)-2 in the presence of APMA: ProMMP-2 (62-kDa) from control BALB/c mice was partially purified by affinity chromatography and was incubated with different doses of APMA (A) and for different time periods with fixed 1 mm APMA concentration (B) at 23C for 2 hr. Nonreducing SDS-PAGE of mouse collagen IV (C) and collagen III (D) prior incubation with proMMP-2 at 23C in the presence (+) or absence ()) of APMA for 16 and 32 hr. Positions of molecular weight markers and, collagen chains and its 1/4 and 3/4 cleavage products were indicated on the left and right, respectively.

Discussion Gastric ulcer healing is a complex process including cell proliferation, angiogenesis, blood circulation and matrix remodeling [2, 11]. Angiogenesis is the most crucial event for ulcer healing which is regulated by several growth factors including VEGF and eNOS etc. [2, 8, 11]. VEGF induces angiogenesis by stimulating EC proliferation and migration [24, 25]. It has been demonstrated by Ma et al., [26] that platelets help in gastric ulcer healing by secreting VEGF in serum. Daily application of melatonin (20 mg/ kg bw) was found to be accelerated the ulcer healing process by affecting cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis, expression of hypoxia inducible factor and activation of cNOS-nitric oxide (NO) system thereby restoration of mucous secretion and microcirculation in the ulcer bed [27]. We, herein, investigated the action of melatonin on VEGF expression to explore their influence on angiogenesis, particularly, in relation to the healing process of non steroidal antiinflammatory drug (NSAID)-induced gastric ulcers. The angiogenic potential of melatonin was tested in rat cornea assay as well as in gastric ulcer model separately. We found that when melatonin was applied alone or prior to indomethacin treatment, it significantly induced angiogenesis via upregulation of VEGF. This observation for the first time indicates that melatonin not only alters the pathologic condition of indomethacin-induced gastric lesions by regulation of angiogenesis but also maintains the capillary homeostasis of gastric mucosa in normal 137

Ganguly et al. condition, i.e., melatonin regulates both physiological and pathological conditions as a proangiogenic accelerator in gastric mucosa. Previous studies indicated that indomethacin blocked VEGF-mediated angiogenesis through mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase signaling cascades [8, 28]. Studies have also demonstrated that NSAIDs such as aspirin or indomethacin inhibits the healing process of gastric wounds by blocking the angiogenesis in granulation tissues [8, 28, 29]. It has been reported that circadian variation increases the occurrence of stress-induced gastric lesions, depending upon the interaction of COX-PG and free radicals, probably mediated by the changes in local melatonin concentration in gastric tissues [30]. The in vivo result of our present study has shown that attenuation pattern of VEGF expressions in indomethacin-induced ulcerated tissues while melatonin blocked their suppression during gastroprotection. Thus, two different wound-healing models of this report universally followed a common pathway regarding proangiogenic stimulus i.e. via VEGF overexpression. It seems that anti-angiogenesis was governed by indomethacin via blocking of VEGF expression whereas melatonin promotes angiogenesis via increased expression of VEGF. A contrary observation was reported by Blask et al. [31]; in this report during neoplastic growth in patients with cancer, melatonin blocked angiogenesis by attenuation of VEGF secretion, which suggest melatonin‘s anti-angiogenic property during prevention of cancer proliferation. This tumor growth inhibitory property of melatonin is mediated by the suppression of epidermal growth factor receptor (EGFR)/ MAPK-mediated signaling mechanism [32]. During the natural wound-healing process, melatonin accelerates angiogenesis via increased secretion of proangiogenic growth factors. On contrary, during cancer prevention, it suppresses the upregulation of proangiogenic growth factors. To understand whether melatonin acts therapeutically in regression of physiological wounds, we conducted healing experiments in indomethacin-induced gastric ulcer. We found that expression of VEGF was enhanced time dependently during natural gastric ulcer healing process (auto healing), whereas melatonin accelerated healing process suggesting its strong angiogenic potential. To address the question about how proangiogenic stimulus like VEGF expression facilitated the angiogenesis process, we especially focused on the regulation of a downstream effector molecule i.e. on MMP-2 expression, activity and localization in vivo, which is found to be absolutely critical for angiogenesis. Studies have also demonstrated that enhanced expression of VEGF is associated with upregulation of MMP-2 and downregulation of TIMP1 and 2 in human umbilical vein ECs [18]. In this study, we have found that the downregulation of MMP-2 and MMP14 and, upregulation of TIMP-2 expression in ulcerated tissues. In support of this, the reports of Stetler-Stevenson and Jones et al. [33, 34], documented the inhibitory effect of indomethacin on angiogenesis via suppression of MMP-2. Herein, melatonin pretreatment reversed the indomethacininduced changes of MMP-2, MMP-14 and TIMP-2 expressions, respectively, and thus promoted the healing process in those wound-healing models. Moreover, TIMP-2 was downregulated in melatonin-pretreated healed tissues sug138

gesting a balance between MMPs and TIMPs in the physiological setting during healing. It is evidenced that only active forms of MMPs cleave collagen types III and IV which are principal constituents of blood vessel and basement membrane [22]. Our in vitro results revealed that APMA facilitated the activation of MMP-2 molecule in a dose and time-dependent manner hence may cleave the collagen type III and IV efficiently. The immunofluorescence and in vitro collagenase studies revealed that during gastroprotection, melatonin induced the availability of active MMP-2 molecules in gastric ECM (lamina propia) which assisted in alteration of the basement membrane of blood vessels resulting in proper homeostasis in angiogenic process. Furthermore, we also found that upregulation of MMP-2 was parallel to MMP-14 and inverse to TIMP-2 expression. We postulate that MMP-2 gets activated by MMP-14 and TIMP-2-dependent mechanism in vivo [21] and modulate ECM homeostasis and angiogenesis process during melatonin-mediated healing process. To substantiate the preventive as well as therapeutic role of melatonin and to elucidate the proper regulation of MMP-2 thereon, we conducted the preventive and healing experiment. Our preventive and healing studies conjugately corroborate the molecular mechanism i.e. the availability of MMP-2 is imperative at ulcer milieu for guidance of physiological angiogenesis. The present study for the first time revealed that the presence of melatonin in the system prior or after the ulcer development may arrest microcirculatory damage or initiate the neovessel formation by upregulation of MMP-2, thereby escalate the angiogenic process. Thus, melatonin acts as preventive and therapeutic modulator of angiogenesis in physiological wound-healing process by ameliorating MMP-2 expression and activity. In recent years, it has become clear that MMPs do more than just degrade structural ECM proteins to promote invasion and tissue remodeling [18]. Are the opposing functions of MMPs involved in disease progression or healing? To effectively target MMPs, the challenge is to identify a protein circuits as part of complete regulatory cascades and signal-transduction pathways that operate on a system-wide basis for MMP production in disease outcome and progression. Although complex, these multiple mechanisms of MMP regulation render new opportunities for therapeutic intervention of NSAIDinduced gastric lesions. A new appreciation has emerged recently regarding the role of growth factor‘s regulation of basement membrane of blood vessels which is an important functional regulator of angiogenesis and ulcer healing [33]. Consistent with the report of Aimes and Quigley [22], our results document that MMP-2 has significant collagenolytic activities in vitro. We hypothesize that increased MMP-2 activity in melatonin-pretreated stomach may in part be because of proper remodeling of gastric ECM especially in relation to removal of cellular debris and excess angiogenesis. Because, collagen is the primary component of gastric ECM, ulceration appears because of differences in the balance between deposition in collagen content and degradation of collagen in gastric tissues. This multiple effect of the melatonin on angiogenesis could be explained by the different environments of tissues on which it is exerting its effect or differences in

Melatonin accelerates angiogenesis in gastric tissues the organization of the ECM of responding cells. So far, no body has given a clear cut idea that how the disease can be initiated, what molecular machineries are really involved in the onset, stabilization and healing processes? Furthermore, literature on ECM remodeling of gastric mucosa and the role of MMPs thereon are absolutely necessary to reveal the severity of gastric lesions and subsequent healing process. NSAIDs inhibit nonspecifically the two rate-limiting isoenzymes COX-1 or -2 and suppress PG synthesis thereby causing gastric ulcers [34, 35]. A few studies deal with the direct involvement of NSAIDs on COX-2-mediated inhibition of tumor growth and angiogenesis by downregulation of PGE2 and VEGF which is parallel to MMP-2 suppression in many types of human cancer [36, 37]. Herein, we also observed that indomethacin exhibits same mechanism of anti-angiogenesis i.e. by downregulation of VEGF and MMP-2 expression, which leads to gastric lesions. Our results also elucidate that the reduction in blood vessel numbers as well as their disruption during ulceration were reported that during ulceration collagen synthesis were decreased in different gastric ulcer wounds. In contrary, melatonin exerts positive effect on monocyte, cytokine and fibroblast proliferations, which influence angiogenesis [38]. This multiple effect of the melatonin on angiogenesis could be explained by the different environments of tissues on which it is exerting its effect or differences in the organization of the ECM of responding cells. Herein, melatonin participated in releasing ECM-bound growth factors and MMP-2 in ulcer milieu via VEGF-mediated signaling while vascularization of basement membrane in repairing ulcerated areas. Over the last century, researchers have whittled away at the mystery of what causes NSAID-induced gastric lesions, and we have gained a considerable amount of knowledge restored by new blood vessel formation to normalcy because of melatonin treatment during healing. Considerable knowledge about the gastric ulcer disease has blossomed into an array of drugs that not only effectively heal gastric lesions but also are likely to prevent or forestall its development. This study showed that melatonin exerted a beneficial role as protective and therapeutic agent of NSAID-induced gastric lesions through a double edged sword i.e. by accelerating angiogenesis via induction of VEGF in one hand and ECM remodeling on the other via upregulation of MMP-2. The present study provides a glimpse of a novel report that how pathophysiology of a disease like gastric ulcer can be explained in the context of ECM remodeling and angiogenesis. Also, one may speculate that a novel molecule like melatonin or its metabolites, e.g., kynuramines [39, 40] may be useful in therapeutic interventions to annihilate gastrointestinal disorders. The fact that induction of proangiogenic factors by melatonin may be therapeutically exploited in gastric ulcer risk individuals by co-administering melatonin along with anti-ulcer drugs. It is to be hoped that the new knowledge that has been derived from recent study of MMPs will lead to the introduction of MMPinhibition strategies as an essential component of the new generation of molecularly targeted therapies for other diseases.

Acknowledgements KG and AVS were recipients of Senior Research Fellowships and Research Associateship, respectively, from Council of Scientific and Industrial Research (New Delhi, India). The work was supported by grant IAP001 of CSIR, CLP261 of NTRF and NBA07 of DBT, India. Authors are grateful to Prof. Siddhartha Roy, Director, Indian Institute of Chemical Biology, Kolkata for his constant encouragement and support.

Author contributions KG and AVS contributed to the experimental design, data acquisition, analysis and interpretation, drafting of the manuscript and revision of the manuscript. RJR contributed to the critical revision of the manuscript. SS contributed to the research designing, data interpretation, drafting of the manuscript and critical revision of the manuscript. All authors approved for the publication of the article.

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