Angiopoietins in Human Breast Milk

Abstract

Introduction:

Angiogenesis is an important process after birth for neonates. Vascular endothelial growth factor is a potent proangiogenic protein, which stimulates endothelial cell proliferation, survival, and migration. For vessel maturation, all components have to move together. Angiopoietins (ANG) are very important signal proteins for pericytes and have not yet been determined in human breast milk. The aim of this study was to show whether there were ANG in human breast milk.

Methods:

Human breast milk samples were collected from 9 mothers of preterm (≤33 weeks) and 17 mothers of term and late preterm (>33 weeks). Milk samples were collected on 3rd, 7th, and 28th days from delivery. We analyzed ANG-1 and ANG-2 levels in human milk and compared these levels considering the gestational age and day of lactation from delivery.

Results:

There was a significant difference between the ANG-1 levels of the two groups of gestational age (p = 0.008), while ANG-2 levels were not significantly different (p = 0.821), without considering the days of lactation from delivery. ANG-1 and 2 levels of milk samples of 3rd, 7th, and 28th days from lactation showed no significant difference in both gestational age groups.

Conclusion:

The presence of ANG in human breast milk was proved with this study and ANG-1 levels were found to be lower in preterm group. These results may be important for neonates in terms of angiogenesis and also lymphangiogenesis.

Introduction

Angiogenesis is formation of new blood vessels from preexisting vasculature.¹ During prenatal and postnatal period, one of the most important steps in development is angiogenesis. Endothelial cells are the basic and constant components that proliferate in response to vascular endothelial growth factor (VEGF) signaling. When the new vessels are formed, endothelial cells secrete some mediators to recruit pericytes that promote vessel maturation. One of these important mediators is the angiopoietin (ANG) family. ANG are a family of growth factors affecting Tie-1 and Tie-2 receptors and secreted by endothelial and perivascular cells.^2,3 The ANG–Tie system has functions about endothelial cell survival, proliferation, periendothelial cell recruitment, vascular stability, and remodeling.^4,5 ANG are also involved in vascular permeability and signaling the smooth muscle cells around vessels for vasoconstriction and vasodilation.⁶ Angiopoietin-1 (ANG-1) is essential for endothelial cell migration, adhesion, vessel maturation, and survival; while angiopoietin-2 (ANG-2) promotes endothelial cell death and breaks vascularization. ANG-2 behaves as an antagonist of ANG-1 and causes vessel regression in the abscence of VEGF.⁷ Deregulation of ANG and Tie receptor pathway results in some pathologies related to angiogenesis.

The purpose of this study was to show whether ANG were present in human breast milk. In addition, we compared the concentrations of ANG in human breast milk collected from mothers of preterm and term infants, as well as on the day of lactation from delivery.

Methods

Twenty-six healthy volunteer mothers were enrolled to this study, who gave birth in the Department of Obstetrics, Gazi University Faculty of Medicine, and divided into two groups: (a) preterm delivery (≤33 weeks): 9 mothers and (b) term and late preterm delivery (>33 weeks): 17 mothers

This was a descriptive study and performed between April 2015 and July 2015. Five milliliters of human breast milk was collected from each mother on 3rd, 7th, and 28th day from delivery. Volunteered mothers were included to this study. Infants with congenital malformations were excluded. All the samples were stored at −80°C till the day of analysis. After thawing the frozen samples, they were centrifuged at 1000 g for 10 minutes. The aqueous part was carefully seperated without the contamination of fat layer on the surface.⁸ ANG-1 and ANG-2 analyses were performed by ELISA method (Wuhan EIAAB Science Co., Ltd.). Absorbance values were read on an ELISA plate reader (model ELx 800 bioelisa microplate reader; Biokit). The statistical analysis was performed using SPSS software for Windows, Version 15.00 (SPSS, Inc.). Between-group comparisons were made using Mann–Whitney U test, within-group comparisons were made using Friedman and Wilcoxon test. All participants gave informed consent and this study was approved by Gazi University Faculty of Medicine Local Ethics Committee.

Results

Characteristics of healthy volunteer mothers have been presented in Table 1. The difference was not statistically significant between the two groups, considering the maternal age, mode of delivery, and sex of infants. The indicators of intrauterine growth (birth weight, length, and head circumference) and gestational age were significantly different, as expected.

Table 1.

Characteristics of the Participants

	Preterm (n = 9)	Late preterm & term (n = 17)	p
Primiparous	8	11
Maternal age at delivery, years	28.5 ± 4.8	30 ± 3.9	0.386
Gestational age, weeks	30.67 ± 2.45	37.06 ± 1.95	<0.001
Mode of delivery (cesarean/vaginal)	7/2	12/5	1.000
Sex of infants (male/female)	6/3	8/9	0.429
Birth weight of child, g	1576.1 ± 451.2	3090.8 ± 603.8	<0.001
Birth length of child, cm	40.4 ± 4.1	48.4 ± 3	<0.001
Head circumference, cm	29.2 ± 3.1	33.7 ± 1.9	<0.001

Values were given as mean ± standard deviation.

ANG were detected in human breast milk. Comparison of ANG levels of human breast milk samples are presented in Table 2. While ANG-1 levels were ranging from 1.12 to 29.59 pg/mL and ANG-2 levels were ranging from 0.4 to 0.8 ng/mL for mothers of preterm group breast milk, in the other group, the ANG-1 range was from 3.12 to 36.64 pg/mL and ANG-2 range was from 0.4 to 1.58 ng/mL. ANG-1 levels were significantly different (p = 0.008) when the total 27 milk samples from mothers of preterm infants were compared to the total 51 milk samples from mothers of the other group, without dividing the groups according to the day of lactation. With these pooled data, we aimed to investigate this lactation process not for a single day, but approximately for a month. ANG-1 levels of mothers of preterm infants were significantly lower than those of the other group, whereas ANG-2 levels were not significantly different (p = 0.821). When the comparisons of ANG levels were made between the two groups, for each lactation day (3rd, 7th, and 28th days), the difference was not significant for both ANG-1 and 2 levels.

Table 2.

Comparison of Angiopoietin-1 and -2 Levels in Breast Milk of the Participants

					Within-group comparisons (p^**)
	3rd day	7th day	28th day	All	3–7–28 days	3–7 days	3–28 days	7–28 days
Angiopoietin-1 (pg/mL)
≤33 weeks (n = 9)	9.3 (4.6–10.9)	7.4 (2.9–10.2)	8.9 (5.3–13.4)	8.2 (4.7–11.8)	0.690	0.314	0.484	0.441
>33 weeks (n = 17)	14.2 (5.9–20.6)	11.4 (6–18.4)	8.2 (6.8–16)	11.8 (6.3–18.7)	0.731	0.653	0.298	0.836
p^*	0.225	0.112	0.426	0.008
Angiopoietin-2 (ng/mL)
≤33 weeks (n = 9)	0.57 (0.50–0.64)	0.53 (0.44–0.65)	0.54 (0.48–0.59)	0.56 (0.47–0.59)	0.895	0.594	0.678	0.953
>33 weeks (n = 17)	0.54 (0.46–0.58)	0.49 (0.44–0.60)	0.52 (0.46–0.62)	0.52 (0.46–0.59)	0.589	0.687	0.906	0.554
p^*	0.345	0.746	0.935	0.821

Angiopoietin levels were given as median values with 25th–75th percentiles in parentheses.

Between-group comparisons: Mann–Whitney U test.

Within-group comparisons: Friedman and Wilcoxon test.

In each delivery group, correlation of ANG-1 and 2 levels were tested on day 3, 7, and 28. The strongest correlation was found on day 28 for each group (r = 0.424 for preterm group, r = 0.478 for the other group), while there was no significant correlation on day 3 and 7.

Discussion

Human breast milk is essential for growth and maturation in neonates, which includes many trophic factors and proteins.⁹ Human milk components and their mission are still under investigation. After birth, the neonate develops very fast, considering the size and maturation in a limited time. Siafakas et al. showed VEGF in human milk for the first time.¹⁰ The presence of many potent angiogenic factors in human breast milk was proved in time.⁹ In our previous study, we showed that some important angiogenic factors, VEGF, basic fibroblast growth factor, and insulin-like growth factor-1, had been significantly different in milk of mothers of preterm and term groups.⁸ A study showed that VEGF and its receptors might have a role in the development of newborn intestine.¹¹ However, there have been no reports of ANG in human milk. Our results proved the presence of ANG in human breast milk for the first time, and these results are compatible with findings of other studies about VEGF in human breast milk. High concentrations in early milk in both preterm and term groups with a decrease in sample collected on day 28 can be determined and distinguished. While the mean concentrations during the first month seem to be lower in the preterm group, on day 28, the two groups seem to be similar. So, VEGF and ANG-1 can be considered to be in relation during the first month of lactation. Correlation analysis of ANG-1 and 2 for each group on days 3, 7, and 28 showed that in early periods of lactation, there was no significant correlation, while a balance between ANG-1 and 2 was set later, on day 28.

Previous studies have shown that there are many proteins related to angiogenesis. There are both angiogenic⁹ and antiangiogenic factors¹² in human breast milk. Endothelial cells are essential for neovascularization. However, without pericytes, it seems impossible to maintain the stabilization of vessels. Considering the previous data, we thought that ANG, which are very important for pericytes, might be present in human breast milk. Furthermore, we investigated whether ANG levels changed according to the gestational age (preterm, term). ANG-1 levels were lower in preterm group than those in the term group, and results were significantly different (p = 0.008), while they were similar for the two groups on day 28. Considering these results, we affirm that ANG-1 levels in milk are high at the beginning of first 28 days for a term delivery, while it decreases through this time. However, these high levels at the beginning could not be seen for the preterm group, maybe due to the unprepared mammalian glands for early delivery. This difference at the beginning of the first 28 days may be important for some processes related with lymphangiogenesis. The difference between the groups of gestational age was not significant, considering the ANG-2 levels (p = 0.821). We thought that these results might be significant in the angiogenic process.

ANG-1 in human breast milk may have multiple functions. First, this may help control of angiogenic process and stabilization of the vasculature in mammary tissue preparing for lactation. Also, transport of the angiogenic process from mother to the infant may cause secondary effects on digestive system of the infant, as we stated in our previous study.⁸ Intestinal villi have lymphatic capillaries named as lacteals. During the digestion process in small intestine, some nutrients especially lipids and triglycerides are absorbed through these lacteals and transported in lymphatic vessels. If there is a defect in growth and differentiation of these lacteals, this results in problems in lymphatic circulation from intestines and other pathologies such as lipid absorption defects and even change in microbiota of the gastrointestinal tract. So, a defect in mediators of lymphangiogenesis may result in problems with lacteals and some digestive pathologies.

It was reported that ANG-1 also stimulated lymphangiogenesis and ANG-2 maintained the organization of lymphatic vessels through its effect on periendothelial cells.^13–15 It was shown that ANG-1-deficient mice had dilated vessels, simpler vasculature, and weak perivascular support.¹⁶ In another study, ANG-2-deficient mice were shown to develop lymphatic vessels, but there were defects and disorganizations such as the missing of lymphatic central lacteals in the intestinal villi, plasma leakage, and edema.¹⁷ In our results, we can see that ANG-1 levels in breast milk of mothers of preterm group are lower, while ANG-2 levels seem similar. Considering the aforementioned effects of lymphangiogenic mediators on intestinal lymphatics of the infants, low levels of ANG-1 levels in preterm infants may even be one of the etiological factors for necrotizing enterocolitis (NE), which is mostly seen in premature infants with very low birth weight. ANG-1 levels in cord plasma were reported as a possible predictor of development of bronchopulmonary dysplasia in premature infants.¹⁸ ANG-1 levels may also be a possible marker for NE development in preterm infants. Additional studies designed with infants having NE have to be performed about this possible relation.

In addition, platelet-derived growth factor (PDGF) levels might be evaluated in milk samples because not only ANG but also PDGF has important effects on pericytes. In our previous study, we had evaluated the PDGF levels in human breast milk and found no significant difference between the mothers of preterm and term groups.⁸

The limitation of this study was the number of participants. Collection of samples on different days provided larger sample size for between-group comparisons, while the sample size for within-group comparisons was small. Also, we had not enough opportunity to show the Tie receptors in mammalian tissue of mothers and digestive system of the infants to affirm the clear effect of ANG on both mothers' milk-secreting glands and intestine of the infants. Serum ANG levels of infants and mothers might also increase the scientific value of this study; however, the difficulty of blood sampling from newborns and mothers not giving consent for this process prevented us doing this.

Conclusions

As known, preterm infants commonly have intestinal disorders and malabsorption problems resulting in growth retardation. Low levels of ANG-1 levels in breast milk may also be a predisposing factor during growth and development, besides the prematurity of the infant.

As a result, we proved that ANG can be quantified in human breast milk and found ANG-1 levels lower in the preterm group. Detailed studies are needed to introduce the targets of ANG in human breast milk. Complex roles and targets of ANG may affect both mother and infant.

Footnotes

Acknowledgment

The authors would like to thank Hakan Ozturk, who assisted with the organization of specimens.

Disclosure Statement

All authors declare that there is no relationship that may pose a conflict of interest.

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