mucus in human cells - Wiley Online Library

Angiogenesis enhanced by Phyllocaulis boraceiensis mucus in human cells Ana R. de Toledo-Piza and Durvanei A. Maria Laboratory of Biochemistry and Biophysics, Butantan Institute, S~ ao Paulo, Brazil

Keywords angiogenesis; CD34; molluscs; Phyllocaulis boraceiensis mucus; proliferation; vascular endothelial growth factor (VEGF) Correspondence Ana Rita de Toledo-Piza, Laboratory of Biochemistry and Biophysics, Butantan Institute, Av. Vital Brazil, 1500, 05503-900 S~ ao Paulo, Brazil Fax: +55 11 37917989 Tel: +55 11 26279777 E-mail: [email protected] (Received 27 June 2013, revised 2 August 2013, accepted 12 August 2013) doi:10.1111/febs.12487

Phyllocaulis boraceiensis mucus is known to be a compound capable of inducing cell proliferation and enhancing the wound healing process. The process of angiogenesis is a chain of mechanisms responsible for the formation of new vessels, which are are involved in cell proliferation, and factors that will act in the healing process. Our aim was to demonstrate that the angiogenesis process is enhanced in cultures of endothelial cells and fibroblasts treated with P. boraceiensis mucus. Experiments were carried out with 105 cellsmL 1 of endothelial cells and fibroblasts treated with P. boraceiensis mucus in concentrations that have significant effects in proliferation assays, i.e. 0.012 lglL 1 and 0.18 lglL 1, both of which cause extreme responses. Aliquots of 100 lL of cell suspensions were incubated for 1 h at 4 °C with 1 lL of antibodies specific for the cell markers vascular endothelial growth factor receptor 1 and cluster of differentiation 34, and negative isotype controls. Reading and expression analysis of cell markers was performed on a FACSCalibur flow cytometer. Expression levels of vascular endothelial growth factor receptor 1 and cluster of differentiation 34 expression were significantly increased in endothelial cells cultivated with 0.012 lglL 1 P. boraceiensis mucus, suggesting that this compound is capable of enhancing angiogenesis.

Introduction Natural products constitute the single most productive source of leads for the development of drugs. Over 100 new products are in clinical development, particularly as anticancer and anti-infective agents [1]. More than 60% of commercial drugs are derived from natural sources. In the last 20 years, there has been increasing interest in natural products, because of the urgent need for new drugs [2]. Peptide therapeutics are increasingly being used clinically [3]. Some of them are isolated from natural products, and others are chemically synthesized [4]. Human dermal tissue represents a complex that promotes mechanical support and maintenance of the avascular epidermis, promoting nutrient distribution and thermal regulation of epidermal cells. This is possible

because of an elaborate microvasculature in the papillary dermis [5]. This microvasculature is physically supported by elements of connective tissue synthesized by local fibroblasts, which play an important role in restoring the vascular plexus in wound healing repair through the production of angiogenic factors [6]. Wound healing has been described as an ongoing process of coagulation, inflammation, and repair, which involves a series of growth factors. Also, this process regulates inflammatory infiltration and cell proliferation [7]; all of these processes promote coagulation, fibrinolysis, angiogenesis and bone formation [8]. Cluster of differentiation 34 (CD34) is a glycophosphoprotein expressed in primitive hematopoietic stem cells and early in cell differentiation. It is also present

Abbreviations CD34, cluster of differentiation 34; ECM, extracellular matrix; NS, non-significant; VEGF, vascular endothelial growth factor; VEGF-R1, vascular endothelial growth factor receptor 1.

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A. R. de Toledo-Piza and D. A. Maria

in progenitor cells, microvascular endothelial cells and fibroblasts that functions as a cell adhesion factor and acts mediating binding of hematopoietic stem cells to extracellular matrix (ECM) or stromal cells [9]. Vascular endothelial growth factor receptor 1 (VEGF-R1) is a molecule that evidences growth factor or vascular endothelial angiogenesis. Besides being a potent mitogen of endothelial cells, vascular endothelial growth factor (VEGF) increases vascular permeability by inhibiting endothelial cell apoptosis and promoting the migration of endothelial cell precursors. VEGF is not the only molecule whose expression is increased in pathological angiogenesis. Basic fibroblast growth factor angiopoietins, pigment epitheliumderived factor and factors related to adhesion to the ECM also play an important role in the balance between proangiogenic and antiangiogenic factors [10]. In a study using pure mucus of Achatina fulica applied on injured rabbit skin, clinical and histological analysis demonstrated faster healing processes and abundant collagen fibers [11]. When breeding the snail Helix aspersa M€ uller in Chile, the animal handlers claimed that mucus released by these animals acted to heal wounds on their hands [12]. To verify this healing property, experiments were performed to compare the cicatricial activity of two creams made of mucus from this snail, Elicina and Novobase, and indicated that some creams that contain mucus of snails are effective in the treatment of some burns, and also speed up the apoptotic process in burned tissue and the epithelialization of deep partial burns [13]. Phyllocaulis boraceiensis is a Brazilian slug that releases mucus composed mainly of proteins [14]. This natural compound is being studied as an inducer of proliferation. It was demonstrated that fibroblasts treated with 0.012 lglL 1 P. boraceiensis mucus presented high rates of proliferation and dose-dependent effect. Also, the production and secretion of ECM components and collagen type I fibers was enhanced after 24 h of treatment, revealing a biphasic dose response, a proliferative low dose, and a toxic high dose. These results demonstrate that the treatment with P. boraceiensis mucus produces pronounced changes in fibroblast cell number and morphology and in the amount of well-ordered collagen deposition [15]. This same product was used in essays of cicatrization, and it was shown that mice subjected to a dorsal incision and treated daily with a ointment composed of P. boraceiensis mucus demonstrated a more efficient healing process and wound closure after application of 0.012 lglL 1 P. boraceiensis mucus [16]. According to these results and advances in scientific research based on the mucus of molluscs as a FEBS Journal 280 (2013) 5118–5127 ª 2013 FEBS

Angiogenesis enhanced by mollusc mucus

compound to be used in healing, our aim was demonstrate that the angiogenesis process is enhanced in cultures of endothelial cells and fibroblasts treated with P. boraceiensis mucus.

Results Evaluation and characterization of normal human dermal fibroblast primary cultures by cytochemistry Fibroblasts obtained after the third passage of culture showed up as adherent cells, spindle core and monolayers formed over time. The results of flow cytometry showed 97.7% 2.8% of ER-TR7 marker specific for fibroblast human nonspecific low markup, obtained by isotype control IgG2a–phycoerythrin, and immunohistochemical analysis with antibody against collagen revealed human collagen type I synthesis and storage of fibrillar protein in all cells observed in the culture under a polarized microscope. Photomicrographs of type I collagen fibers are shown in Fig. 1. Expression analysis of cell markers involved in pathways of angiogenesis by flow cytometry For further comparison, Fig. 2 shows representative histograms of negative control fibroblasts and endothelial cells. CD34 expression in human fibroblasts after treatment with 0.18 lglL 1 P. boraceiensis mucus showed a significant decrease as compared with the control group and the group treated with

Fig. 1. Immunostaining for collagen fibers produced by normal human fibroblasts maintained in culture. Note cytoplasmic expression of newly synthesized collagen fibers. Magnification: 9 400.

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Endothelial cells


Fibroblast cells Control negative Mean 9.6 ± 1.8

FSC

Control negative Mean 6.4 ± 1.3

Fig. 2. Representative histograms of negative control of fibroblasts and endothelial cells stained with antibody against human CD3. FSC, forward scatter.

0.012 lglL 1 P. boraceiensis mucus. The decrease in the group treated with 0.18 lglL 1 P. boraceiensis was time-dependent (Fig. 3). Moreover, expression of receptor to formation of new vessels (VEGF-R1) (Fig. 4) demonstrates significant decrease on marking population of positive cells after 48 h and 72 h of treatment, with both concentrations of mucus. Endothelial cells treated with P. boraceiensis mucus, presents CD34 expression in an average of 18.7 3.7 and 58.3 8.2 VEGF-R1 expression. Treatment with mucus at concentrations of 0.18 lglL 1 and 0.012 lglL 1 presented an increased CD34 expression within 24 h on the contrary, experiments carried out during periods of 48 h and 72 h showed significant reduction on its expression (Fig. 5). VEGF-R1 expression in endothelial cells treated with 0.18 lglL 1 P. boraceiensis mucus showed significant difference in all periods analyzed greater than those observed in cells treated with 0.012 lglL 1 (Fig. 6).

Discussion The results reported here concern the action of P. boraceiensis mucus as an angiogenic inducer in endothelial cells and fibroblasts. However, there is no scientific literature in this area to allow comparison of results. The activity of P. boraceiensis mucus on endothelial cells and fibroblasts was studied with flow cytometry to compare results obtained in cells treated with mucus and those not treated with mucus. Blood vessel formation plays a key role in both physiological and pathological tissue growth and healing [17]. Therefore, angiogenesis, i.e. the formation of blood vessels from pre-existing vessels that occurs in physiological and pathological conditions, is a complex phenomenon involving numerous molecules that stim5120

ulate and inhibit the formation of new vessels. Neovascularization occurs in pathological situations such as the repair of tissue damage through the formation of local neovessels [10]. Vasculogenesis involves endothelial cell precursors called angioblasts, which arise in the mesoderm of the yolk sac. These cells are organized in clusters, differentiating into a network in which primordial vascular endothelial channels have a relatively uniform size. Subsequently, during angiogenesis, vascular remodeling occurs before new capillaries emerge from the primary vessel, forming a vascular network and stable complex with blood vessels of different sizes. Vascular remodeling involves both growth and regression of vessels, especially in major physiological events in childhood, during the growth of tissues and organs of different systems. Vascular remodeling is also present in the adult, e.g. in hair growth, in repair of damaged tissue (healing), and in the female reproductive cycle, with regard to the vasculature in the ovary, genital tracts, mammary gland, and placenta [18]. In the adult organism, vascularization, which is normally stable, can be reactivated by various angiogenic factors that trigger the formation of blood vessels (neovascularization). Disturbances in the delicate balance between growth and regression of existing vessels in the adult organism may contribute to the development of various pathological processes. The growth of tumors, for example, depends on neovascularization, directly or indirectly induced by tumor cells themselves during the transition between hyperplasia to neoplasia, such as in hemangioma [19]. Fibroblasts play a central role in wound healing, because of their ability to migrate to the site of injury, to produce and remodel ECM components such as collagen, and to exert paracrine stimulation in wound healing process, including angiogenesis. Fibrocytes are inactive fibroblasts whose cellular activity is fibrosis; FEBS Journal 280 (2013) 5118–5127 ª 2013 FEBS



A 48 h

72 h

0.18 μg/μl 0.012 μg/μl

FSC

Control

24 h

FL-1 H B

Fig. 3. (A) Representative histograms of control fibroblasts and fibroblasts treated with 0.18 and 0.012 µgµL 1 P. boraceiensis mucus and stained with CD34 for cell quantification. (B) Detection of CD34 by flow cytometry in fibroblasts treated with 0.18 and 0.012 lglL 1 P. boraceiensis mucus. The data are presented as NS or significant at *P < 0.05, **P < 0.005, or ***P < 0.0001. FSC, forward scatter.


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A 48 h

72 h

0.18 µg/µl 0.012 µg/µl

FSC

Control

24 h

FL-1 H B

Fig. 4. (A) Representative histograms of control fibroblasts and fibroblasts treated with 0.18 and 0.012 µgµL 1 P. boraceiensis mucus and stained with VEGF-R1 for cell quantification. (B) Detection of VEGF-R1 by flow cytometry in fibroblasts treated with 0.18 and 0.012 lglL 1 P. boraceiensis mucus. The data are presented as NS or significant at *P < 0.05, **P < 0.005, or ***P < 0.0001. FSC, forward scatter.

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24 h

48 h

72 h

0.18 µg/µl 0.012 µg/µl

FSC

Control

A

FL-1 H B

Fig. 5. (A) Representative histograms of control endothelial cells and endothelial cells treated with 0.18 and 0.012 µgµL 1 P. boraceiensis mucus and stained with CD34 for cell quantification. (B) Detection of CD34 by flow cytometry in endothelial cells treated with 0.18 and 0.012 lglL 1 P. boraceiensis mucus. The data are presented as NS or significant at *P < 0.05, **P < 0.005, or ***P < 0.0001. FSC, forward scatter.


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A

48 h

72 h

0.18 µg/µl 0.012 µg/µl

FSC

Control

24 h


FL-1 H B

Fig. 6. (A) Representative histograms of control endothelial cells and endothelial cells treated with 0.18 and 0.012 µgµL 1 P. boraceiensis mucus and stained with VEGF-R1 for cell quantification. (B) Detection of VEGF-R1 by flow cytometry in endothelial cells treated with 0.18 and 0.012 lglL 1 P. boraceiensis mucus. The data are presented as NS or significant at *P < 0.05, **P < 0.005, or ***P < 0.0001. FSC, forward scatter.

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they help in the healing process in granuloma formation; they have antigenic activity; they act as collagen producers or, as in the ECM, are involved in remodeling; in inflammation, they are a rich source of cytokines [20,21]; as growth factor producers and angiogenic factor producers, they contribute to the formation of new blood vessels and in some fibrotic disorders [21,22]. In this study, fibroblasts from blepharoplasty have been shown to be a well-stabilized and proliferative culture that can be used in experiments on angiogenesis, and can be treated with different concentrations of P. boraceiensis mucus. These same aspects have been observed in cultured endothelial cells. During human development, nutrients and oxygen are consumed continuously. As there is cell division in embryonic life, the vascular system undergoes expansion to ensure the supply of these substances throughout the process of body formation in a process called angiogenesis [23,24]. Mature endothelial cells that constitute the vessel wall originate from the splanchnic mesoderm of the embryo, and subsequently differentiate into vascular structures in different tissues and organs [25]. Neovascularization is essential not only during fetal development, but also in the female reproductive cycle, and has a role in tissue repair [26]. Proliferation of endothelial cells and fibroblasts was observed from 48 h after treatment with P. boraceiensis mucus, showing that this compound acts as an inducer of proliferation, suggesting that, once it has induced an increase in cell proliferation, all correlated factors will increase, as shown by the results reported here. The authors also present images of endothelial cell cultures treated with P. boraceiensis mucus forming blood vessels, which is clear evidence of angiogenesis [15]. VEGF was initially called vascular permeability factor, because of its ability to promote increased permeability and proliferation of endothelial cells. Currently, it is considered to be major factor in vessel formation, in periods of both vasculogenesis and angiogenesis [27]. One of the aspects evaluated in the present article is that endothelial cells treated with P. boraceiensis mucus undergo significant changes in the expression of these receptors for vascular growth, which is possibly one of the reasons for the efficacy of treatment in surgical wounds. P. boraceiensis mucus was shown to be an efficient compound to accelerate the wound healing process in mice treated with 0.012 lglL 1 P. boraceiensis mucus, which is the same concentration as used in our experiments [16]. CD34 and VEGF-R1 were used in this study to investigate the number of endothelial cells and fibroFEBS Journal 280 (2013) 5118–5127 ª 2013 FEBS


blasts treated with P. boraceiensis mucus that were susceptible to these markers, and their relationship after treatment with different concentrations and periods of culture. Fibroblasts treated with 0.012 lglL 1 P. boraceiensis mucus showed a subtle increase in CD34 expression. In contrast, experiments with endothelial cells with the same concentration showed significant increases in CD34 and VEGF-R1 expression levels, indicating that this compound acts as an inducer of angiogenesis. P. boraceiensis mucus is a natural compound released by a slug found in Brazil. This substance is composed of a pool of proteins, is easily collected by mechanical stimulation, and is simply applied, making it cheap and abundant [14]. Our results have shown that VEGF and CD34 expression levels are increased significantly in endothelial cells as compared with controls. These data indicate that P. boraceiensis mucus is an inducer of the angiogenic process, although these results must be extended with other techniques for the identification of the angiogenesis process.

Experimental procedures Culture of human fibroblasts and endothelial cells Normal human fibroblasts were isolated from eyelid blepharoplasty performed at the University Hospital of University of S~ao Paulo (research project number 921/06). Skin fragments were transferred to a 35-mm Petri dish containing RPMI-1640 with antibiotics, and washed to remove the excess of blood and red blood cells. Fat tissue was removed with the aid of forceps and scissors. The fragments were cut into 51 fragments and distributed into three Petri dishes ( 15 fragments each) with culture medium. Cultures of human umbilical vein endothelial cells were isolated from human umbilical cord vein from a commercial strain (ATCC CRL-1730) [28]. Both types of cells in the exponential growth phase were cultured in bottles (75 cm2) with RPMI-1640 supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine, and antibiotics. They were incubated at 37 °C in an atmosphere of 5% CO2, and examined under an inverted microscope three times a week. Culture medium was exchanged three times a week. When cells reached subconfluence, they were trypsinized (0.005% trypsin) to allow detachment from the culture bottles. They were centrifuged at 324 000 g twice and rehydrated in culture medium, and the cell concentration was adjusted to 5 9 105 cellsmL 1. After cells had been transferred to 96-well plates, they were allowed 24 h to adhere to the plate and reach confluence, and treated with P. boraceiensis mucus at extreme concentrations that showed significant responses in proliferation assays: these

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concentrations were 0.012 lglL 1 (proliferative dose) and 0.18 lglL 1 (cytotoxic dose) [15]. These concentrations were evaluated for induction of the healing process and hindrance of it, respectively, when applied in mice subjected to a 1-cm2 dorsal incision and treated with a ointment composed of papain cream enriched with mucus [16].

Expression analysis of cell markers involved in pathways of angiogenesis by flow cytometry In order to study the angiogenesis process, we used two cell markers, CD34 and VEGF-R1. After detachment of fibroblasts and endothelial cells treated with P. boraceiensis mucus, samples were centrifuged for 10 min at 324 000 g the supernatant was discarded, the pellet was solubilized in NaCl/Pi, and the sample was frozen at 80 °C; this condition was maintained until the time of sample preparation for analysis. Aliquots of 100 lL of cell suspension were incubated for 1 h at 4 °C with 1 lL of antibodies specific for VEGF-R1 and CD34. In order to permeabilize the cell membrane, we added 10 lL of 0.1% Triton X-100. Cells were centrifuged for 10 min at 225 000 g and washed with cold NaCl/Pi. The supernatant was discarded, and the pellet was solubilized in NaCl/Pi containing 0.1% paraformaldehyde. Experiments were designed after expansion of cells. Analysis of expression of specific markers of the surface cell membrane of fibroblasts was performed on a Flow Cytometer (FACSCalibur; Becton Dickinson), and the following markers were used: anti-human fibroblast Ig serum [fibroblast marker (ER-TR7): sc-73355] labeled with phycoerythrin (Santa Cruz), mouse monoclonal IgG2a serum control (Santa Cruz), and anti-collagen type I Ig (CSI 008-01; Genese). As an isotype negative control, nonspecific antibody against human CD3 labeled with fluorescein isothiocyanate was used. Results were analyzed with WINMDI 2.8 software.

Statistical analysis Statistical analysis was performed with GRAPHPAD PRISM 5 software (GraphPad, USA), and the statistical significance of differences between groups was determined with unpaired one-way ANOVA. Means were compared by use of Tukey’s multiple comparison tests. Regression analysis was used to examine the data obtained and infer relationships between dependent and independent variables. Comparisons were considered to be either non-significant (NS) or significant at *P < 0.05, **P < 0.005, or ***P < 0.0001.

Acknowledgements Contract grant sponsor: CNPq. Contract grant number: 142935/2010-7.

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