Anti-Angiogenic Property of Free Human Oligosaccharides

Angiogenesis, a fundamental process in human physiology and pathology, has attracted considerable attention owing to its potential as a therapeutic strategy. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are deemed major mediators of angiogenesis. To date, inhibition of the VEGF-A/VEGFR-2 axis has been an effective strategy employed in the development of anticancer drugs. However, some limitations, such as low efficacy and side effects, need to be addressed. Several drug candidates have been discovered, including small molecule compounds, recombinant proteins, and oligosaccharides. In this review, we focus on human oligosaccharides as modulators of angiogenesis. In particular, sialylated human milk oligosaccharides (HMOs) play a significant role in the inhibition of VEGFR-2-mediated angiogenesis. We discuss the structural features concerning the interaction between sialylated HMOs and VEGFR-2 as a molecular mechanism of anti-angiogenesis modulation and its effectiveness in vivo experiments. In the current state, extensive clinical trials are required to develop a novel VEGFR-2 inhibitor from sialylated HMOs.


Introduction
Milk oligosaccharides are milk components with diverse biological functions [1]. In particular, human milk has been utilized as a medicinal food due to its nutritional composition and non-nutritive bioactive factors [2]. Breast milk, which is ideal for infants, contains numerous complex ingredients, including proteins, lipids, carbohydrates, minerals, and other minor nutrients [3]. Among human milk components in colostrum, oligosaccharides are the third most abundant, present at concentrations of up to 20-25 g/L [4]. However, newborn babies lack enzymes that digest complex milk oligosaccharides. Thus, the precise physiological role of milk oligosaccharides remained elusive until the 1960s [4,5]. However, pioneering studies in the early 20th century have discovered that the carbohydrate fraction of human milk contains growth-promoting factors for Lactobacillus bifidus [4]. Currently, accumulated data suggest that human milk oligosaccharides (HMOs) are prebiotics, as well as modulators of the intestinal mucosal and systemic immune response [4,6].
All HMOs contain five different monosaccharides, including D-glucose, D-galactose, L-fucose, N-acetylglucosamine and N-acetylneuraminic acid [3]. Approximately 150 different types of oligosaccharides have been identified in human milk, with all possessing a lactose unit at the reducing end [3,5]. Typically, HMOs are classified as neutral and acidic oligosaccharides based on the respective presence or absence of negatively charged N-acetylneuraminic acid, that is, sialic acid [6]. In several aspects, the biological activities of acidic HMOs tend to differ from those of neutral HMOs [7,8]. Naturally occurring free oligosaccharides harboring sialic acid have been found in both plasma and urine in healthy men and women, especially in pregnant and lactating women [9][10][11]. Free sialylated oligosaccharides frequently present in human milk inhibit the adhesion of immune cells, cholera toxin, and influenza virus with endothelial or epithelial cells [12][13][14]. Moreover, the sialic acid-containing portion of HMOs is essential for early neurodevelopment and cognition [15,16].
In the present review, we focused on the modulation of angiogenesis via humanderived oligosaccharides, especially free sialylated oligosaccharides, to ameliorate diseases associated with excessive angiogenesis.

Physiological and Pathological Angiogenesis
In vertebrates, the vascular system plays a crucial role in organ homeostasis by transporting oxygen and nutrients [17]. Closed blood vessel systems, like those in vertebrates, first appeared in their common ancestor over 500 million years ago to optimize flow dynamics and barrier function [18]. Reportedly, metabolic requirements for oxygen and nutrients induce new blood vessel formation from the existing ones, a process termed angiogenesis [19]. Angiogenesis was initially considered as a physiological process for maintaining metabolic homeostasis in the field of developmental biology [20]. Inevitably, new blood vessel formation plays a critical role in early development, tissue growth and wound healing [19]. Furthermore, female reproductive physiology, including oocytogenesis, embryo implantation and the menstruation cycle, are regulated by angiogenesis ( Figure 1) [21]. Conversely, angiogenesis could be employed as a therapeutic target for treating pathological conditions characterized by either insufficient vascularization or excessive vasculature [22,23]. Tumor-derived factors to promote neovascularization were first postulated in the late 1930s [23]. In 1971, Folkman [24] suggested that inhibition of angiogenesis might have potential therapeutic implications in cancer therapy. These pioneering studies have highlighted the concept that angiogenesis is an important biological process and therapeutic target in diverse diseases, including cancer [25]. The excessive growth of new vessels can aggravate diverse disorders, ranging from cancer and obesity to retinopathy, such as age-related macular degeneration [22,26]. Psoriasis, arthritis, inflammatory bowel disease, benign prostate hyperplasia, endometriosis, ovarian cysts, and uterine bleeding also have been reported to have a mutual correlation with excessive angiogenesis [22,27,28]. In contrast, insufficient angiogenesis can contribute to various diseases, such as stroke, myocardial infarction, diabetic ulcers, atherosclerosis, coronary artery disease, systematic lupus erythematosus, preeclampsia, Alzheimer's disease, and Crohn's disease [22,26,29]. Thus, balanced regulation of the angiogenic process might be a key factor for maintaining human health and preventing or treating numerous diseases.

Vascular Endothelial Cell Growth Factors (VEGFs) and Their Receptors (VEGFRs) as Therapeutic Targets for Pathological Angiogenesis
Angiogenesis involves the formation and maintenance of new blood vessels via the cooperation of multiple cells in vascular networks, including vascular endothelial cells, their progenitor cells and pericytes [30,31]. Based on the dynamic interplay between these cells, new blood vessel-like structures are formed via multistep processes, such as sprouting, tip cell migration and tube formation [22,32]. These processes are regulated by signaling between endothelial cells and the perivascular cell layer by secreting growth factors, direct cell-cell interaction, and extracellular matrix production [31][32][33]. Among the secretory growth factors that regulate angiogenesis, VEGFs are the most important, as they play key roles in multiple steps of neovascular formation ( Figure 2) [31,34]. Thus, the axis of VEGFs and their receptors (VEGFRs) has been considered a therapeutic target for modulating angiogenesis since the beginning of anti-angiogenic studies [24,35].
In mammals, the VEGF family consists of five members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PLGF). These ligands bind to their respective receptors (VEGFRs), which belong to the type IV receptor tyrosine kinase (RTK) family and are composed of three members, VEGFR-1, VEGFR-2, and VEGFR-3 [36,37]. Once the ligand binds to the receptor, homo-or hetero-dimeric interactions of VEGFRs initiate the autophosphorylation of intracellular tyrosine residues, as well as downstream signaling pathways responsible for the proliferation, migration and remodeling of the vascular endothelial cells [34,36]. Among them, VEGFR-1 and VEGFR-2 play critical roles in physiological and pathological angiogenesis. In blood vascular endothelial cells, angiogenesis is predominantly mediated via VEGFR-2 activation [38]. VEGF-A, VEGF-B, and PLGF are high-affinity ligands of VEGFR-1, but the kinase activity of VEGFR-1 is relatively weak for the progression of the angiogenesis [39]. In some cancers, VEGF-C sustains VEGFR-2 activation by binding to VEGFR-2 even when inhibiting VEGF-A [40,41]. VEGF-C and VEGF-D stimulate VEGFR-3 activation, which plays an indispensable role in both angiogenesis and lymphangiogenesis [42]. Furthermore, the cooperative signaling between VEGFR-2 and -3 is involved in forming new lymphatic vessels ( Figure 3) [43]. In response to stimulators such as VEGFs, vascular endothelial cells sprout from the basement membrane and migrate to the site of new vessel formation. The formation of tip and stalk cells is regulated by the VEGF-DLL4/NOTCH signaling pathway. The other stimulating molecules, such as PDGF, EGF, FGF, BMPs, and TGF-β, also cooperate to regulate pericytes and tip cells. ALK1/5, actin receptor-like kinase 1/5; BMP, bone morphogenic protein (yellow dots); DLL4, delta-like protein 4; EGF, epidermal growth factor (black dot); EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor (gray dot); FGFR, fibroblast growth factor receptor; NICD, intracellular domain of the notch protein; PDGF, platelet-derived growth factor (green dot); PDGFR, platelet-derived growth factor receptor; TGF-β, transforming growth factor-β (dark yellow dots); VEGF-A, vascular endothelial cell growth factor A (orange dots); VEGF-C, vascular endothelial cell growth factor C (red dots); VEGFR, vascular endothelial cell growth factor receptor. Therefore, considerable efforts to inhibit VEGFR-2 activation have been made to suppress angiogenesis as pathogenic angiogenesis is predominantly mediated by the VEGF-A/VEGFR-2 axis [44]. Several strategies for suppressing excessive angiogenesis have been exploited, including neutralizing monoclonal antibodies, VEGF-trapping recombinant proteins and small molecule tyrosine kinase inhibitors [34,35,45]. Among them, neutralizing VEGF-A using the anti-VEGF-A monoclonal antibody, bevacizumab (brand name Avastin), has been inhibiting angiogenesis most successfully in clinical settings. It has been approved as a conventional treatment for several cancers, such as colorectal cancer, lung cancer, glioblastoma, renal cell carcinoma, and age-related macular degeneration [46][47][48]. However, the use of bevacizumab has revealed several limitations, such as high cost, no effect on overall survival in a few cancer cases, and adverse effects on coronary and peripheral artery disease [49,50]. Accordingly, several researchers have focused on developing an effective and safe anti-angiogenic agent from small molecule compounds [34,35]. The majority of small molecule anti-angiogenic agents target the tyrosine kinase activity of VEGFRs [51,52]. However, these chemical inhibitors for tyrosine kinases have been unsuccessful owing to their low specificity and mutation-induced drug resistance [51,53]. As the extracellular ligand-binding region of RTKs is markedly diverse in terms of the protein structure, they are considered more suitable for developing specific inhibitors [54,55]. Accordingly, several investigations reported that interactions between VEGF-A and its receptor could be intercepted by employing recombinant peptides and peptidomimetic chemicals [56][57][58].

Oligosaccharides and Angiogenesis
Several different types of oligosaccharide chains exist in mammals as bound to proteins, lipids, or repeating sugar units [59,60]. Oligosaccharide-linked proteins or lipids, that is, glycoproteins or glycolipids, respectively, are mainly located on the cell surface and have distinct biological functions, including mediation of viral/bacterial infection, immune response, cell-cell interaction and cancer progression [61]. Additionally, numerous ligand-receptor interactions might be regulated by the glycosylation status, especially those involving G protein-coupled receptors and growth factor receptors [61][62][63]. It is well known that ligand binding and trafficking of epidermal growth factor receptor (EGFR), fibroblast growth factor-1 and VEGFR-2, the most crucial RTKs in cancer-associated angiogenesis, are affected by site-specific N-glycosylation [64][65][66]. Conversely, in the present review, we focused on the pro-or anti-angiogenic roles of unbound oligosaccharides to determine their potential as therapeutic modulators of angiogenesis.

Roles of Sialylated HMOs in Pathologic Angiogenesis
Several cell-surface proteins, such as mucins, ion channels, receptors, and adhesion molecules, are highly glycosylated with terminal sialic acid residues [61]. Numerous studies have revealed that cell surface glycosylated molecules bound to growth factor receptors regulate their proangiogenic function [99][100][101]. N-glycosylation, especially that of terminal sialic acid residues, regulates ligand-dependent activation of VEGFR-2 [102]. Not only proteins, a sialylated glycosphingolipid, GM3, reportedly exhibits an anti-angiogenic effect by inhibiting VEGFR2 activation [103,104]. Although two review papers have discussed the role of glycosylation as a novel therapeutic target for diseases associated with excessive angiogenesis [105,106], none of them described the exact functions of sialylated HMOs in angiogenesis. In 2004, Rudloff et al. [107] demonstrated that sialylated HMOs have an anti-angiogenic effect on bovine vascular endothelial cells by employing an in vitro tube formation assay. However, the study failed to identify the precise components of acidic HMOs and the mechanism underlying their anti-angiogenic effects.
As described above, antibody-and recombinant protein-based drugs are superior to small molecules, such as oligosaccharides, in terms of target specificity. However, their clinical application might be limited due to the high cost, the risk of immunogenicity following long-term treatment, and limited accessibility to target pathological foci owing to their large size [110,112]. Natural sialylated HMOs reportedly possess several valuable properties, including low molecular weight, low immunogenicity, and high accessibility to therapeutic targets. Moreover, the safety of 3 -and 6 -sialyllactoses has previously been confirmed for use in infants as well as in the general population, based on rodent and porcine models [113][114][115]. Therefore, these in vivo efficacy and safety assessment studies potentiate sialylated oligosaccharides as an anti-angiogenic agent via suppressing the VEGF-A/VEGFR-2 axis. However, to develop sialylated HMOs as clinically available VEGFR-2 inhibitors, further extensive preclinical studies using animal models of pathological angiogenesis, as well as clinical trials, should be warranted. Moreover, identifying superior oligosaccharide analogs with higher binding affinities might help guarantee improved anti-angiogenic effects.

Conclusions
In the present review, we highlighted the emerging role of sialic acid-containing HMOs in the suppression of VEGFR-2-mediated angiogenesis. Disparately from other glycans contained in HMOs, sialyllactose, and sialylgalactose could inhibit the activation of VEGFR-2 by binding to its immunoglobulin-like domain 2. Although the anti-angiogenic effects of sialylated HMOs have been evaluated in limited in vivo models, such as several cancers, premature retinopathy, and benign prostate hyperplasia, their anti-angiogenic efficacy still has the potential to cure other pathological conditions associated with excessive angiogenesis. Moreover, extensive clinical trials using sialylated oligosaccharides would lead us to new and novel strategies to develop clinically available VEGFR-2 inhibitors from sialylated HMOs.

Data Availability Statement:
The data presented in this study are openly available.