Artificial and natural sialic acid precursors influence the angiogenic capacity of human umbilical vein endothelial cells

Abstract

N-acetylneuraminic acid (Neu5Ac) represents the most common terminal carbohydrate residue in many mammalian glycoconjugates and is directly involved in a number of different physiological as well as pathological cellular processes. Endogenous sialic acids derive from the biosynthetic precursor molecule N-acetyl-D-mannosamine (ManNAc). Interestingly, N-acyl-analogues of D-mannosamine (ManN) can also be incorporated and converted into corresponding artificial sialic acids by eukaryotic cells. Within this study, we optimized a protocol for the chemical synthesis of various peracetylated ManN derivatives resulting in yields of approximately 100%. Correct molecular structures of the obtained products ManNAc, N-propanoyl-ManN (ManNProp) and N-butyl-ManN (ManNBut) were verified by GC-, ESI-MS- and NMR-analyses. By applying these substances to human umbilical vein endothelial cells (HUVECs), we could show that each derivative was metabolized to the corresponding N-acylneuraminic acid variant and subsequently incorporated into nascent glycoproteins. To investigate whether natural and/or artificial sialic acid precursors are able to modulate the angiogenic capacity of HUVECs, a spheroid assay was performed. By this means, an increase in total capillary length has been observed when cells incorporated N-butylneuraminic acid (Neu5But) into their glycoconjugates. In contrast, the natural precursor ManNAc inhibited the growth of capillaries. Thus, sialic acid precursors may represent useful agents to modulate blood vessel formation.

Figure 1

Biosynthetic pathway of sialic acids and feasible implementations of artificial precursors. R, acyl chains; GNE/MNK, uridinediphospho-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase; NANS, N-acetylneuraminic acid synthase; NANP, N-acylneuraminate-9-phosphatase.

Figure 2

Chemical synthesis of peracetylated N-acyl-ManN derivatives. (A) Reaction sequence including peracylation, cleavage of O-acyl-residues and peracetylation. GC analyses were performed to confirm the synthesized structures to be peracetylated (B) ManNAc, (C) ManNProp, and (D) ManNBut by comparing respective retention times to those of (E) standard substances, which were confirmed by NMR analysis. Further structural verification was performed by ESI-MS analyses of peracetylated (F) ManNAc, (G) ManNProp, and (H) ManNBut registered as sodium adducts ([M+Na]⁺) as well as sodium adducts after the loss of acetic acid. Hydrogen adducts ([M+H]⁺) formed after the loss of acetic acid are marked by trianglels (▼) and unidentified signals are labeled by asterisks (*). Retention times as well as monoisotopic masses are assigned with the first decimal place.

Figure 3

Biocompatibility of peracetylated ManNProp, ManNBut, and ManNAc. HUVECs were incubated with 50 µM of per-O-acetylated ManNProp, ManNBut, and ManNAc for 48 h and supernatants were checked for LDH activity. 100% was set for Triton-X 100 (1% v/v) treated HUVECs. All values ± S.D. in this figure are means of 3 independent experiments.

Figure 4

Analysis of sialic acids present in whole cell lysate from HUVECs treated with peracetylated ManNProp, ManNBut, and ManNAc. (A) DMB-labeled sialic acid residues were separated by RP-HPLC. For control, HUVECs were not incubated with any sialic acid precursors. (B) EIC of sodium adducts ([M+Na]⁺) of Neu5Ac (m/z 448) and Neu5Prop (m/z 462) as well as Neu5Ac (m/z 448) and Neu5But (m/z 476) present in HUVECs incubated with peracetylated ManNProp and ManNBut, respectively. Peaks related to DMB reagent are labeled with an asterisk (*) and those related to further unidentified impurities are marked with a triangle (▼). (C) ESI-MS/MS analyses of Neu5Ac (upper profile), Neu5Prop (central profile) and Neu5But (lower profile) are registered as proton adducts ([M+H]⁺). For fragmentation analyses, respective parent ions generated after loss of water were selected. Annotation of fragment ions was performed according to the fragmentation pathway of DMB-labeled sialic acids proposed by Manzi and co-workers [12] with monoisotopic masses of respective fragment ions printed in bold. Corresponding structures of generated fragments are given exemplarily in the upper profile. For the structure of Neu5Ac (m/z 426.1) prior to loss of water see inset.

Figure 5

Analysis of sialic acid residues of glycoproteins isolated from HUVECs after incubation with per-O-acetylated ManNProp, ManNBut, and ManNAc. (A) Sialic acid residues obtained after DMB-labeling were separated by RP-HPLC. For control, HUVECs were not incubated with any sialic acid precursors. Peaks related to DMB reagent are labeled with an asterisk (*) and those related to unknown impurities are marked with a triangle (▼). (B) EIC of sodium adducts ([M+Na]⁺) of Neu5Prop (m/z 462) and Neu5But (m/z 476) present in glycoproteins from HUVECs incubated with ManNProp and ManNBut, respectively. (C) ESI-MS/MS analyses of Neu5Prop (upper profile) and Neu5But (lower profile) registered as proton adducts ([M+H]⁺). Annotation of fragment ions was carried out as defined in Figure 4.

Figure 6

Influence of per-O-acetylated ManNR derivatives on capillary sprouting of endothelial cell spheroids induced by bFGF. (A) Scheme of the applied three dimensional angiogenesis assay. HUVECs spheroids were treated with bFGF to induce capillary sprouting in absence (control) or presence of indicated sialic acid precursor molecules (R, acyl chains) [13,14]. (B) Representative spheroids after bFGF stimulation. (C) The lengths of arising capillaries were measured and obtained values were summed up using NIS Elements AR 3.0 Software (Nikon, Düsseldorf, Germany). Results are shown as relative increase in total capillary length after stimulation with bFGF. To this end, total capillary lengths of spheroids were measured after bFGF stimulation and compared with the unstimulated control. All values ± S.D. in this figure are means of 3 independent experiments using at least 12 spheroids per set. The statistical evaluation was performed by Student´s t test (unequal variances, two tailed). Significance levels are indicated by n.s. (not significant), p > 5%; *, p < 5%; **, p < 1%; ***, p < 0.1%.