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1 The role of proteins in the angiogenic activity of Hancornia speciosa latex
2
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3ABSTRACT
4
5Previous research has been demonstrated that H. speciosa latex present angiogenic, osteogenic, anti-
6inflammatory and antioxidant activities. All those properties can assist in the wound healing process.
7Particularly, the angiogenesis is the initial and crucial step of tissue healing process. In this work we
8try understanding the role of proteins in angiogenesis process, which it was previous determined in
9tissues treated with H. speciosa serum fraction latex. To test the hypothesis that the proteins present
10in the latex serum are the main responsible for angiogenic activity, this work inactivated the latex
11proteins using proteinase K. After the enzymatic inactivation, the angiogenic activity was evaluated
12with the use of chick embryo chorioallantoic membrane (CAM) assay. CAM assays are among the
13most common in vivo models used to study different aspects of angiogenesis. After the CAM
14experiments, biochemical analyzes were performed to quantify the total proteins and the activity of
15the enzymes present in the SE fraction of the latex. As a result, it was observed that the proteins
16present in the SE fraction of H. speciosa have angiogenic activity and can be beneficial in the
17development of therapeutic materials for wound healing and tissue regeneration. Despite the low
18protein content, it was possible to identify enzymatic activity of at least 3 enzymes in the SE fraction
19of latex, they are: β-1,3 glucanase, β glucosidase and proteases. In general, these enzymes seem to
20influence the healing process, aiding in debridement, aiding in the remodeling of the extracellular
21matrix and in the collagen deposit, decreasing the chances of contamination by microorganisms. In
22conclusion, the wound healing process depends not only on cells and polypeptides available in the
23tissue, but also on enzymes from the microenvironment of the extracellular matrix.
24Keywords: chick embryo chorioallantoic membrane; wound healing; enzymes, proteinase K.
25
26RESUMO
27
28Pesquisas anteriores demonstraram que o látex de H. speciosa apresenta atividades angiogênicas,
29osteogênicas, antiinflamatórias e antioxidantes. Todas essas propriedades podem auxiliar no processo
30de cicatrização de feridas. De particular interesse nesse estudo, a angiogênese é a etapa inicial e
31crucial do processo de cicatrização do tecido. Neste trabalho buscamos compreender o papel das
32proteínas no processo de angiogênese, previamente determinado em tecidos tratados com látex da
33fração sérica de H. speciosa. Para testar a hipótese de que as proteínas presentes no soro do látex são
34as principais responsáveis pela atividade angiogênica, este trabalho inativou as proteínas do látex
35utilizando a proteinase K. Após a inativação enzimática, a atividade angiogênica foi avaliada com o
36uso do ensaio da membrana corioalantóide de embrião de galinha (CAM ). Os ensaios CAM estão
37entre os modelos in vivo mais comumente empregados para estudar diferentes aspectos da
38angiogênese. Após os experimentos do CAM, análises bioquímicas foram realizadas para quantificar
39a proteínas totais e a atividade das enzimas presentes na fração SE do látex. Como resultados,
40observou-se que as proteínas presentes na fração SE de H. speciosa possuem atividade angiogênica e
41podem ser benéficas no desenvolvimento de materiais terapêuticos para cicatrização de feridas e
42regeneração de tecidos. Apesar do baixo conteúdo de proteínas, foi possível identificar atividade
43enzimática de pelo menos 3 enzimas na fração SE do látex, são elas: β-1,3 glucanase, β glucosidase e
44proteases. De maneira geral, essas enzimas parecem influenciar o processo de cicatrização,
45auxiliando no desbridamento, auxiliando na remodelação da matriz extracelular e no depósito de
46colágeno, diminuindo as chances de contaminação por microrganismos. Em conclusão, o processo de
47cicatrização de feridas depende não apenas de células e polipeptídeos disponíveis no tecido, mas
48também de enzimas do microambiente da matriz extracelular.
49Palavras-chave: membrana corioalantóide de ovo de galinha; cicatrização de feridas, enzimas,
50proteinase K.
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52INTRODUCTION
53 Angiogenesis is the growth of blood vessels from the existing vasculature (Adair; Montani,
542010). This is a vital process in human growth and development, as well as in wound healing and in
55the formation of granulation tissue (Folkman, 2003). For decades, researchers are looking for
56products or strategies to stimulate the angiogenesis and consequently accelerate the wound healing
57process (Singer and Clark, 1999, Scheneider et al., 2019). It is known that plants produce a variety of
58angiogenic compounds that can be important for the development of wound healing drugs
59(Wittenauer et al. 2015, Danciu et al. 2015, Karim et al. 2014). Among the plant exudates, latex has
60been shown to have high angiogenic potential (de Almeida et al., 2015). The latex is an organic
61milky fluid that flows out of some plants after a tissue injury. The general functions of this exudate
62in plants are associated with the excretion of waste metabolites, coverage of damaged tissue, and
63defense against pathogens (Konno 2011). Among the latex with angiogenic activities, our research
64group has been working with the latex bioprospecting of Hancornia speciosa Gomes.
65 H. speciosa, popularly called by mangabeira, is native species from Brazil that belongs to the
66family Apocynaceae. It is typically found in the Amazon Rainforest, Caatinga and Cerrado
67vegetations. Previous ethnobotanical studies have been showed the use of H. speciosa latex in
68traditional medicine for treating of: acne and others skin diseases, fungal diseases, tuberculosis,
69gastric ulcers, and bone fractures (Pott and Pott 1994, Neto and Guarim-Morais 2003, Macedo and
70Ferreira 2004, Sampaio and Nogueira 2006, Santos et al. 2007). In addition of this ethnobotanical
71knowledge, our group and other researchers have been observed that the H. speciosa latex possess
72high angiogenic (D'Abadia et al., 2020; Almeida et al., 2014) and osteogenic potentials (Floriano et
73al., 2016; Dos Santos Neves et al., 2016), as well as antioxidant (D’Abadia et al., 2020) and anti-
74inflammatory (Marinho et al., 2011) activities. In relation to the toxicity potential, it was observed
75that H. speciosa latex no present cytogenotoxicity effects against human cells (Costa, 2019), animals
76(Almeida et al., 2014) and vegetables (Ribeiro et al., 2016).
77 To continue our bioprospecting work, it is important identifying which substances present in
78the latex have angiogenic activity. The previous study determined that the compounds responsible
79for the angiogenic activity are present in the serum (SE) fraction of H. speciosa latex. The SE latex
80fraction containing large variety of proteins, secondary metabolites and luteoids compounds. Among
81the different components present in serum latex fraction, the present study aims to evaluate the
82influence of proteins on the angiogenic activity of H. speciosa latex. To test the hypothesis that the
83proteins present in the latex serum are the main responsible for angiogenic activity, this work intends
84to inactivate the latex proteins using proteinase K. After the enzymatic inactivation, the angiogenic
85activity was evaluated with the use of chick embryo chorioallantoic membrane (CAM) assay. This is
86one of the most employed methods to study angiogenesis and has the main advantages of to be fast,
87has low cost, be simple, has good reproducibility, easy dynamic observation, and no major ethical
88concerns involved (Nowak-Sliwinska et al., 2014; Aleksandrowicz; Herr, 2015). After the CAM
89experiments detected that the proteins are responsible for angiogenic activity, biochemical analyzes
90were performed to quantify the total amount of proteins and the main classes of enzymes present in
91the SE fraction latex. The enzymes can give us clue of biological activity of SE fraction in the wound
92healing.
93
94MATERIAL AND METHODS
95
96Latex collection
97 H. speciosa latex was obtained from trees of the collection belonging to Universidade
98Estadual de Goiás, in the city of Ipameri, state of Goiás, Brazil (17°43’19’’S, 48°9’35’’W, 773 m).
99After botanical identification, a voucher specimen was deposited at the Universidade Estadual de
100Goiás Herbarium (Anápolis, Goiás, Brazil) under voucher number 4875. Latex was extracted in a
101sterile tube by drilling in the tree trunk according Arruda et al. (2016) methodology. To collect latex,
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102a knife was used to make a cut of approximately 5 cm length and 0.5 cm depth in the bark. The latex
103was centrifuged for 5min at 3,000rpm to remove the debris derived from the collection process, and
104the supernatant was transferred to a new collection tube.
105
106Latex fractionation
107 H. speciosa latex was centrifuged at 4 °C for 1 h at 22.000 g (Heraeus Megafuge 16R,
108Thermo Scientific). After initial centrifugation, the rubber was carefully separated, and a second
109centrifugation was performed. Two different fractions were obtained after this process: the top white
110zone with the rubber particles, and the serum layer (SE). The same fractionation method was
111employed in Hevea brasilensis latex to obtain the sample control for the total protein quantification
112analysis.
113
114Protein inactivation using Proteinase K
115Proteinase K, also known as protease K or endopeptidase K, is commonly used in molecular biology
116to digest protein. The H. speciosa SE fraction latex was treated with proteinase K according
117manufacturer's instructions.
118
119Angiogenic potential/CAM assay
120This study was approved by the Ethics Committee on the use of animals at the Universidade Estadual
121de Goiás, Brazil (Protocol no007/2018). A CAM assay with few modifications was performed as
122described by Almeida et al (2014). Four groups of 12 fertilized chicken eggs (Gallus domesticus)
123were incubated at 37 °C in a humidified environment (around 70% of humidity). After five days of
124incubation, the CAM was accessed through a window cut in the eggshell, and the eggs were returned
125to the incubator. On day 13 of incubation, the CAM was exposed to the different treatments. Four
126these treatments, filter paper disks were soaked in 3 μl of the following solutions: (1) serum fraction
127(SE); (2) protein inactivated serum fraction (IPSE); (3) water (WA, negative control); (4)
128dexamethasone (DE, angiogenesis inhibitor). After exposure of CAM to different treatments, the
129eggs were returned to the incubator for 72 h, following which CAM were fixed in formaldehyde
130(3.7%) for 5 min. A curved blunt scissors was used to remove the CAM from egg and the CAM was
131maintained in Petri dishes with formaldehyde solution. The new-formed vascular net was analysed
132and quantificated through captured images using the two different software. Gimp (version 2.0.5)
133was used to normalize saturation, light, and contrast of the CAM images; and the ImageJ (NIH,
134version 1.28) was used to quantify the number of pixels in each image. The angiogenic activity of SE
135latex fraction was evaluated by comparing the treated and control groups using one way analysis of
136variance (ANOVA), followed by Tukey’s post-hoc test to compare treatment means. A p value <0.05
137was used to indicate statistical significance. After image analysis, the CAM were embedded in
138paraffin for histological analysis, stained with hematoxylin and eosin, and examined by optical
139microscopy (40×). The parameters evaluated were conjunctive cells, inflammation, and
140neovascularization. The results were classified based on their intensity, and the data were
141transformed into quantitative variables by assigning the following scores: absent (0), discrete (1-
14225%), moderate (26-50%), and accentuated (over 51%). The results were analyzed using a Kruskal-
143Wallis test with a significance level of p<0.05 followed by Bonferroni test to compare treatment
144means.
145
146Total protein quantification
147 The total proteins present in SE fraction was determined using Bradford method (Bradford,
1481976). For this test, the standard curve was first prepared using the albumin (1 mg/mL BSA) and its
149serial dilutions in H2O (0.5 mg/mL; 0.25 mg/mL and 0.125 mg/mL). SE fraction latex were prepared
150using 2.37mL of water, 30 µL SE fraction latex and 600 µL of Bradford. The mixture was incubated
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151for 5 min in the dark and was measured at 595 nm (SpectraMax Paradigm, Molecular Devices). The
152same protocol was used for measuring the total protein in H. brasiliensis serum latex.
153
154Enzyme activity quantification
155The protease activity was determined using casein as a substrate, according to the methodology
156Sarath et al. (2001). The enzymatic activity was expressed in U / mL, defined as the amount of
157enzyme needed to promote the release of 1 µmol of tyr / min under the test conditions. b-1,3-
158glucanase activity was determined using laminarin as a substrate (Noronha and Ulhoa, 2000). The
159amount of reducing sugar was determined at 540 nm. A unit of enzymatic activity (U) was defined as
160the amount of enzyme needed to release 1 µmol of reducing sugar per minute. N-
161acetylglycosaminidase (NAGase) activity was determined with the use of Nacetyl-β-D-
162diacetylglicosaminide (Ulhoa and Perberdy, 1992). One unit (U) of N-acetyl-β-D-glycosaminidase
163was defined as the amount of enzyme needed to hydrolyze one µmol of p-nitrophenol per minute.
164Acid phosphatase was determined using substrate derived from ƿ-nitrophenyl-phosphate (2.5 mM)
165according Monteiro et al (2015) methodology. One unit of phosphatase was defined as the amount of
166enzyme required to produce 1 μMol of ρ-nitrophenol per minute. Beta-glucosidase activity was
167determined using substrate derived from p-nitrophenyl-β-D-glucopyranoside (2.5 mM). An enzyme
168unit of N-acetyl-β-D-glucosity was defined as the amount of enzyme required to produce 1 μMol of
169ρ-nitrophenol per minute. Chitian activity was determined using substrate derived from ƿ-
170nitrophenyl-N-N-diacetylchitobiose (2.5 mM) according Qualhato et al. (2013) methodology. A
171chitinase enzyme unit was defined as the amount of enzyme required to produce 1 μMol of ρ-
172nitrophenol per minute. Lipase activity was performed using the synthetic substrate p-nitrophenyl
173palmitate, following the release of the p-nitrophenyl palmitate ion (410nm). The unit (U) was defined
174as the amount of enzyme needed to hydrolyze a micromol of the substrate in one minute (μmols /
175min), under the test conditions.
176
177
178RESULTS
179
180 To evaluate the rule of H. speciosa SE fraction latex proteins in the angiogenesis, the
181percentages of CAM vascular area in the treatments groups and controls were calculated.
182Representative images of the vascular nets of each condition are shown (Figure 1). The amount of
183blood vessels was significant decreased in inactivated protein serum fraction latex (IPSE). The mean
184percentage of vascularization obtained for each group was: WA 14.03 ± 1.4; DE 9.30 ± 1.3; SE
18526.55 ± 1.5; IPSE 11.7 ± 2.4. The SE fraction, how it was expected, showed an increase in the
186percentage vascularization compared with water (negative control), which confirm the angiogenic
187activity of H. speciosa SE fraction. However, the serum fraction treated with proteinase K and
188without actives protein do not show the angiogenic activity. The vascularization in IPSE group
189showed no significant differences in the percentage of vascularization with inhibitor control
190(dexamethasone).
191
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192
193Figure 1. Angiogenic potential of Hancornia speciosa latex on chorioallantoic membrane (CAM)
194model. Representative images of different CAM treatments: WA (water, neutral control); DE
195(dexamethasone, angiogenesis inhibitor); SE (H. speciosa serum fraction); IPSE (inactivated proteins
196serum latex of H. speciosa using Proteinase K)
197
198
199 After CAM morphology analysis, the histological analyses were performed (Figure 2). Three
200different parameters were observed. For inflammation, SE fraction group is significant different from
201dexamethasone (Table 1). The conjunctive cells from SE group also are significant different from
202dexamethasone and water (Table 1). In addition, SE group has significant increase in angiogenesis
203parameter showing a significant difference with all other groups (WA, DE, IPSE) (Table 1). Thus,
204the histological results agreed with the findings based on CAM morphology, indicating that H.
205speciosa SE fraction presented angiogenic activity, but this property is lost after the enzymatic
206treatment of SE fraction. The result indicated that proteins have angiogenic activity in H. speciosa
207latex.
208
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209
210Figure 2. Histological analysis of chorioallantoic membranes CAMs treated with Hancornia
211speciosa SE fractions. Paraffin sections stained with hematoxylin-eosin for different groups: WA
212(water, neutral control); DE (dexamethasone, angiogenesis inhibitor); SE (H. speciosa serum
213fraction); IPSE (inactivated proteins serum latex of H. speciosa using Proteinase K)
214
215Table1. Histological parameters of the chorioallantoic membranes treated with the control solutions
216(W: water and DE: dexamethasone) and H. speciosa serum fractions (SE: serum latex; IPSE:
217inactivated proteins serum latex using Proteinase K).
218
Samples Inflammation Conjunctive cells Angiogenesis
Median Bonferroni Median Bonferroni Median Bonferroni
test test test
W 1 A 0 A 0 A
DE 0 AB 0 AC 0 A
SE 2 AC 3 BD 2,5 B
IPSE 1 A 1 A 0 A
P=0,005 P=0,003 P=0,003
219* The data were submitted to non-parametric Kruskal-Wallis analysis.
220Same letters represent no significant difference by the Bonferroni t-test.
221
222 For the evaluation of protein content in SE fraction we quantified the total protein using
223Bradfold and compare the results with the latex of H. brasiliensis, which is well known in literature.
224H. speciosa serum latex obtained was 0.17 mg/mL, which is smaller than the total protein found in
225H. brasiliensis serum latex (1.1 mg/mL). In addition, Table 1 show the enzymatic activities of SE
226fraction. As result no enzymatic activity was observed for NAGase, Phosphatase, Chitian, and very
227small activity for Lipase. A remarkable activity of β-1,3-glucanase was detected in H. speciosa SE
228fraction.
229
230
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231
232Table 2: Enzymatic activity of Hancornia speciosa SE fraction and in H. brasiliensis SE fraction.
233The enzymatic activity was expressed in U/mL.
234
Samples Protease β-1,3 NAGase Phosphatas β- Chitian Lipase
glucanase e glucosidas
e
H. speciosa SE fraction 1.95 92.05 0 0 2.45 0 0.2
H. brasiliensis SE fraction 8.37 5.35 5.92 8.54 7.02 4.73 2.52
235
236
237DISCUSSION
238 The repair process is extremely complex and begins immediately after the injury. It is initially
239characterized by vascular and cellular events, such as: the increase in vascular permeability and
240angiogenesis. Wound healing cannot occur without the formation of new blood vessels
241(angiogenesis). Then, in this work we try understanding the role of proteins in angiogenesis process
242previous detected in tissues treated with H. speciosa latex (Almeida et al., 2014; D’Abadia et al.,
2432020; Bonete et al., 2020; Pegorin et al. 2020). The obtained results can be a clue to identify the
244agents that control angiogenesis, and which are beneficial to the development of therapeutics for
245treatment of wound healing and tissue regeneration.
246 The CAM assay results confirm the angiogenic activity of SE fraction from H. speciosa latex
247(Figure 1, 2). In addition, the lack of angiogenesis in IPSE group, where there are no proteins,
248indicated that the stimulation of angiogenesis process in CAM membrane are associated with
249proteins present in SE fraction. Then, if the proteins have important role in angiogenesis, we decide
250investigated the total protein present in SE fractions, as well as the enzymatic activities identified in
251this latex fraction.
252 Regards to total amount of proteins, it important to mention that different from others, H.
253speciosa latex showed low concentration of proteins. This could be an advantage in the use of this
254species for development new medicinal products with hipoallergenic response. This because it well
255known that the main responsible for latex skin sensitization are proteins (Kelly and Sussman, 2017).
256According to the International Allergen Nomenclature Committee of the IUIS (International Union
257of Immunological Societies), 15 allergenic proteins were identified and characterized in the rubber
258latex, which are called Hev 1 to Hev 15 and each protein has a molecular weight which allows
259differentiate between them (http://www.allergen.org). Our results are in accordance with Malmonge
260et al. (2009), who also identified a low concentration of proteins in H. speciosa crude latex (1,900 
26132 g/g dw) and suggested the hypoallergenic potential.
262 Although low concentration of proteins comparing to other lactiferous, it was possible
263detected enzymatic activity in H. speciosa SE fraction. Three enzymes showed considerable activity:
264β-1,3 glucanase, β glucosidase and proteases (Table 2). We tried to understand how these enzymes
265could help in the wound healing process. The 1,3-β-D-glucanases are fibrolytic enzymes, playing
266irreplaceable role in hydrolysis of carbohydrates for energy production in fungi and bacteria
267(Usoltseva et al., 2020). Because of the fibrolytic activity, this enzyme could be help in debridement
268and physiological remodeling during the healing process. In those steps, the fibroblast produces the
269fundamental substance amorphous (mucopolysaccharides) involved with the production, size, and
270orientation of collagen fibers (Junqueira and Carneiro, 1993). Besides help in the collagen fibers
271orientation, it was suggested that 1,3-β-D-glucanases could promote cell proliferation (Jose et al.,
2722014). In addition, no cytotoxic effect was observed when animal cells were exposed to 1,3-β-D-
273glucanases, even in high concentrations (Usoltseva et al., 2020). Other possible effect of 1,3-β-D-
274glucanases in the wound it is antifungal activity (Usoltseva et al., 2020; Hong and Meng., 2003),
275which could reduce the chance of wound contamination.
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276 Regards to β glucosidase, some reports have been shown that glycohydrolases play an
277important role in the destruction of connective tissue matrix during the inflammatory process
278(Chithra et al., 1998). β glucosidase act alternatively on the blinkage of glucuronic acid in
279glycosaminoglycan to release monosaccharides and make the hydrolyze the terminal non-reducing b-
280glucosyl residues of glycolipids residues of glycosaminoglycan. A species with wound healing
281activity that present β glucosidase in its extract is Aloe vera (Chithra et al., 1998)
282 Regards to proteases activity, some studies have been demonstrated that these enzymes could
283act as clot-inducing and as clot-hydrolyzing properties (Urs et al., 2017). Clot formation is vital for
284hemostasis, the initial phase of wound healing, where proteases present procoagulant and thrombin
285like activities. Whereas clot hydrolysis is a prerequisite for the events of regenerative phase, where
286fibrionolytic, gelatinolytic, and collagenolytic activities of proteases can help in wound debridement
287(Urs et al., 2017). Overall, the proteases give excellent conditions for physiological healing by
288complementing the endogenous proteases in hemostasis, microbial attenuation, wound debridement,
289angiogenesis, and cell proliferation (Urs et al., 2017). Some examples of proteases used in wound
290healing process are papain (Udod and Storozhuk, 1979), bromelin (Maurer, 2001), curcain (Nath and
291Dutta, 1991), and ficins (Devaraj et al., 2008; Shiksharthi and Mittal, 2011).
292
293CONCLUSION
294 The proteins present in H. speciosa SE fraction possess angiogenic activity and can be
295beneficial to the development of therapeutics materials to wound healing and tissue regeneration.
296The protein content of SE fraction is low; however, 3 enzymes show activity in this material: β-1,3
297glucanase, β glucosidase and proteases. Overall, these enzymes seem to influence the healing
298process, helping in debridement, assistance remodeling of the extracellular matrix and collagen
299deposit, decreasing the chances of contamination by microorganisms. In this way, healing depends
300not only on available cells and polypeptides, but also on enzymes from the extracellular matrix
301microenvironment.
302
303
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