A Naturally Derived, Growth Factor-Binding Polysaccharide for

Letter pubs.acs.org/macroletters

A Naturally Derived, Growth Factor-Binding Polysaccharide for Therapeutic Angiogenesis Qiu Li,† Guangxing Guo,‡ Fancheng Meng,† Helena H. Wang,† Yiming Niu,† Qingwen Zhang,† Junfeng Zhang,‡ Yitao Wang,† Lei Dong,*,‡ and Chunming Wang*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, China ‡ State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China S Supporting Information *

ABSTRACT: We herein report the discovery of a naturally derived carbohydrate with binding affinities for two proangiogenic growth factors−fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB). This galacturonic acid-containing polysaccharide (EUP3) sequestered endogenous FGF-2 and PDGF-BB in vivo and promoted in situ formation and maturation of new blood vessels. Our findings suggest EUP3 as the first nonglycosaminoglycan, nonanimal-originated carbohydrate molecule that binds two pro-angiogenic growth factors to stimulate angiogenesis. Further investigations into this carbohydrate may lead to the development of new tools for therapeutic angiogenesis.

T

polysaccharides of these origins are often immunogenic (such as carrageenan)29,30 or anticoagulant (like heparin).25,26 In addition, as a possible biomimicry of heparin/HS, it could be a polysaccharide containing acidic groups to interact with basic amino acids (AAs) in GFs; such molecules exist in herbs and often contain uronic acids.31 Based upon the above-mentioned criteria and hypothesis, we started with extensive literature search and raw material screening (Supporting Information), before we eventually targeted a medicinal herb Eucommia ulmoides (EU) for its polysaccharide components. We report here the discovery of a galacturonic acid (GalA)presenting polysaccharide that possesses the desirable features in sequestering pro-angiogenic GFs in situ. The found acidic heteroglycan was the third of the three polysaccharide fractions purified from this herb, hence, named EUP3. It exhibited not only an affinity for FGF-2, a primary mediator of angiogenesis,32 but also a high binding capacity for platelet-derived growth factor-BB (PDGF-BB), a key player in promoting blood vessel maturation.33,34 Its affinity for PDGF-BB is unfound in the other sugar fractions of EU and is also rare, if any, in natural carbohydrates. This newly identified polysaccharide is the first nonheparin/HS, nonanimal-derived carbohydrate with binding affinity for two major pro-angiogenic GFs which, respectively, regulate new capillary formation and blood vessel maturation. EUP3 demonstrated promising potential to orchestrate blood vessel formation both in vitro and in vivo.

he restoration of blood supply to damaged tissue is crucial for both the treatment of ischemic diseases and regeneration of complex tissue.1−5 Physiologically, this process is mediated by various growth factors (GFs), suggesting that administration of these GFs to stimulate angiogenesis may be a therapeutic mode.6−10 However, clinical practices to deliver exogenous GFs have achieved very limited success, because they rapidly degrade in vivo,11 become carcinogenic,12,13 or induce immature vessel formation.14−17 Meanwhile, endogenous angiogenic GFs are usually overexpressed in abundance at the site of injury of ischemia.18 Therefore, instead of adding excessive exogenous proteins into the body, delivering a nonGF molecule to locally sequester, and harness the functions of, endogenous GFs has been proposed as a more effective strategy for therapeutic angiogenesis.19 The search for the GF-sequestering molecule was inspired by the physiological role of heparin/heparan sulfate (HS).20−23 The heparin/HS polysaccharides bind and enrich angiogenic GFs in the extracellular matrix, stabilizing and prolonging the interactions between GFs and corresponding receptors.24 Their action is required for these GFs but, in the absence of GFs, they do not trigger angiogenesis on their own accord. Although heparin molecules are precluded for use in therapeutic angiogenesis, due to their unwanted side effects such as anticoagulation25,26 and immunogenicity commonly found with animal-derived products,27,28 their unique “bioresponsive” activity sets rational criteria for this molecule. Specifically, it is expected to (i) bind at least one endogenous angiogenic GF; (ii) contain no sulfate groups; and (iii) originate from sources other than animal or ocean, considering that sulfated © XXXX American Chemical Society

Received: March 3, 2016 Accepted: May 2, 2016

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DOI: 10.1021/acsmacrolett.6b00182 ACS Macro Lett. 2016, 5, 617−621

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ACS Macro Letters We first extracted the crude polysaccharide from EU, following an optimized procedure (Supporting Information). Since in the case of heparin/HS, acidity is one of the key factors underpinning polysaccharide-GF binding,35 we separated different fractions of the crude EU polysaccharides according to their acidic degrees, by using an anion-exchange chromatography (DEAE-52) eluted with gradient saline buffer (NaCl; 0.1, 0.2, 0.35, and 0.5 M). Further purification with gel-permeation chromatography (Sephadex G-100) generated polysaccharides EUP1, EUP2, and EUP3 (Figure 1a). Monosaccharide analyses revealed that the galA content in the three EUPs ranked in an order of EUP3 > EUP2 > EUP1 (Figure S1b).

Table S1a), 1H and 13C NMR, COSY, HSQC, and HMBC NMR (Supporting Information). Complete hydrolysis of EUP3, followed by gas chromatography analysis, revealed its monosaccharide composition as mannose, galactose, glucose, arabinose, rhamnose, and galA. Further partial acid hydrolysis plus GC-MS analysis suggested that the backbone of EUP3 comprises mannose, glucose, galactose, and galA, while the side chain comprises rhamnose, galactose, and glucose. The carbon signals at 174 ppm (13C NMR) confirmed the carboxyl group of Galp, in which the carboxyl group is not esterified, indicating that the −COOH of GalA could freely interact with other cationic groups. The comprehensive analysis to dissect the composition and structure of EUP3 is detailed in Supporting Information, with the full NMR spectra and assignments (Figure S4a−e; Table S1b). All the above data allowed us to propose the repeated unit of EUP3 (Figure 1c). We postulated that the electrostatic interaction between GalA and the basic AAs of the PDGF-BB underpinned the sugar-GF binding (Figure S5a). To study whether the basic AAs are required for this binding, we synthesized (i) the segment of PDGF-BB (152−169) that contained two pairs and one triplet of adjacent basic AAs (P0) and (ii) three other peptides (P1−P3) with basic AAs mutated to serine (S), a neutral polar amino acid (Figure S5b). No significant difference was found between the binding affinities of EUP3 for P0 and for PDGF-BB; however, mutation of the basic AAs of either 160 RKK162 or 166KK167 substantially weakened the affinity of EUP3 for the peptides, and replacement of the both basic AA segments completed abolished the binding (Figure 2a). This

Figure 1. (a) Three EU polysaccharide fractions (EUP1−3) were acquired based upon their acidity; (b) Binding kinetics of EUP3 against PDGF-BB as assessed by SPR; (c) Proposed structure of EUP3.

We assessed the binding affinity of EUP1, 2, and 3 for proangiogenic growth factors36 VEGF-A, FGF-2, and PDGF-BB (isoelectric point: 8.5, 9.6, and 9.8, respectively), by using two assays based on different principles, namely, surface plasmon resonance (SPR) and GAG-binding ELISA (Iduron, U.K.). The SPR outcomes suggested that both the unfractioned EUP and EUP3 bound to FGF-2 but none of the tested sugars was affinitive for VEGF-A (Figure S2 and Table 1). Notably, EUP3 Table 1. Binding Affinity of Total and Various Fractions of EU-Derived Polysaccharides for Growth Factors KD (μM)

EUP

EUP1

EUP2

EUP3

VEGF-A FGF-2 PDGF-BB

0.0653 0.997

0.0344 1.55

0.0188 0.219

1.334 0.0121 0.0135

Figure 2. (a) Binding capacity of EUP3 for the original (P0) and three modified (P1−3) segment of PDGF-BB; (b) binding capacity of EUP3 with or without GalA for PDGF-BB and FGF-2; (c) estimation of the specificity and portion of binding between EUP3 and PDGF-BB; (d) binding capacity of degraded EUP3 in different sizes for PDGF-BB and FGF-2.

exhibited a distinct, high affinity for PDGF-BB (Figure 1b and Table 1). Consistent results were obtained from the ELISA tests (Figure S3). The results from the two assays suggested that EUP3 bound to both PDGF-BB and FGF-2. Particularly, its affinity for PDGF-BB was unfound in other EU fractions. This is a unique feature rarely found in other natural carbohydrates and may suggest its potential to promote blood vessel maturation.37 To better investigate its biological functions, we sought to thoroughly characterize the structure of EUP3 through a procedure of partial and complete hydrolysis (Figure S1a,b;

result confirmed basic AAs were crucial for PDGF-BB to bind EUP3. On the other hand, to validate the importance of GalA for the GF-binding capacity of EUP3, we reduced EUP3 with NaBH4 to remove the carboxyl groups (de-GalA), and found that the de-GalA-EUP3, in sharp contrast to EUP3, completely lost its ability to bind PDGF-BB and FGF-2 (Figure 2b). This finding emphasized the essential role of GalA for EUP3 to bind both GFs. 618

DOI: 10.1021/acsmacrolett.6b00182 ACS Macro Lett. 2016, 5, 617−621

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ACS Macro Letters

PDGF receptor in mesenchymal stem cells (MSCs), the progenitor of pericytes,38,39 as well as that of FGFR1 and VEGF receptor 2 (VEGFR2) in human umbilical vein endothelial cells (HUVECs).40,41 In agreement with the IP result, EUP3, but not unfractioned EUP, enhanced PDGF signaling by increasing the levels of phosphorylated PDGFR (pPDGFR)34 and phosphorylated Erk1/2 (pErk1/2), a key downstream signaling regulating cell growth.42 Relatively weaker, but still significant, enhancement was found with FGF2-FGFR1, but not with VEGF signaling at all (Figures 3b and S6). As hypothesized, EUP3 did not activate PDGFR on its own accord; it exerted its effect only via enhancing PDGF-BB’s activity. This important finding confirms the “bioresponsiveness” of EUP3, emphasizing its safety for potential therapeutic uses. To evaluate the performances of EUP3 in sequestering GFs and stimulating angiogenesis in vivo, we loaded this carbohydrate into agarose-gelatin gels (AgaGel) and implanted them subcutaneously in mice. AgaGels loaded with unfractioned EUP or saline were set as controls. The samples were collected after 15 days post implantation. By gross view we could see much denser blood vessels formed in the EUP3-laden gels than in other groups (Figure 4a). Determination of various

We performed a mathematical analysis of the above data, in order to understand the contribution of each specific interaction to the total binding between EUP3 and PDGF-BB (Supporting Information). Nonspecific interactions constitute 23% of the total binding capacity, while the contribution from nonelectrostatic interactions was about 10%. Electrostatic interactions were perhaps the major kind of interaction and required the presence of BAAs: 154KK155, 160RKK162, and 166 KK167. We estimated the contribution from each BAAs group was about 14, 22, and 20% of the total binding capacity, respectively. Another 11% of total binding might be due to the copresence of all three basic-AAs group (Table S2 and S3, Figure 2c). In addition to the presence of GalA, we asked whether molecular size was also important for EUP3 to bind the GFs. We hydrolyzed EUP3 with trifluoroacetic acid (TFA) in different concentrations to generate EUP3 fragments of various lengths. Decreasing EUP3’s size significantly weakened its binding affinity (Figure 2d). A 10% decrease in size led to a 45−70% loss of affinity to PDGF-BB; while 50% and higher percentages of size reduction resulted in almost completely loss of the affinity to either GF. We set out to evaluate the biological functions of EUP3, particularly its ability to enhance the GFs’ activities both in vitro and in vivo. First, we evaluated whether it could sequester GFs and aid them in binding their corresponding receptors. Results from the immunoprecipitation (IP) assay indicated that, in the presence of EUP3 (10 or 100 g mL−1), significantly more FGF-2 (2−3-fold) and PDGF-BB (5−6-fold) bound to FGFR1 and PDGFR, respectively (Figure 3a). Interestingly, total EUP,

Figure 4. (a) Gross view of (b) the levels of various GFs in and (c) immunohistological analyses of AgaGel samples preloaded with saline, unfractioned EUP, or EUP3, after 15 days post-subcutaneous implantation.

GF contents revealed that PDGF-BB was preferentially enriched in EUP3-laden gels, FGF-2 accumulated in both EUP and EUP3 gels, while the VEGF-A level was similar in all groups (Figure 4b). These outcomes were in agreement with the binding affinity results, suggesting that EUP3 has particular affinity for PDGF-BB and FGF-2 in situ. Histological analyses of the gel samples further highlighted the desired potential of EUP3 in promoting revascularization (Figures 4c and S7). No acute inflammation or mass apoptosis were found with any groups after the 15-day implantation, as evidenced by H&E staining. The number of CD31 (an endothelial marker)-positive cells was higher in EUP3-laden gels than in the crude EUP group, with hardly any signal being found in blank control. Also, in agreement with the ELISA result, the density of PDGF-BB was found significantly higher in the EUP3 group than any other groups. Notably, the signal of SMA-alpha, marking the maturation of newly formed blood vessels, was clearly higher in the EUP3 group than in the other groups, with a visible shape of vascular cross sections. All these

Figure 3. (a) Amount of GFs bound to their corresponding receptors in the presence or absence of unfractioned EUP or EUP3; (b) The activation of PDGFR signaling pathway in MSCs by PDGF-BB (left) and that of FGFR1 and VEGFR2 signaling by FGF-2 and VEGF-A, respectively (right), in the presence or absence of EUP3.

which was also found to recruit FGF2, showed little effect in increasing FGF2-FGF receptor 1 (FGFR1) binding. On the other hand, total EUP moderately increased the amount of PDGF-BB in the pull-down sample, which should partly come from the effect of its EUP3 fraction. Next, we assessed whether the binding between EUP3 and GFs could empower cell signaling in the living system, by testing the phosphorylation of 619

DOI: 10.1021/acsmacrolett.6b00182 ACS Macro Lett. 2016, 5, 617−621

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ACS Macro Letters findings suggest that EUP3 can effectively sequester endogenous PDGF-BB and FGF-2 and, without extra addition of exogenous growth factors, orchestrate formation of blood vessels with evident signs of maturation. The above findings demonstrate that the unique affinity of EUP3 for FGF-2 and PDGF-BB can translate into in situ neovascularization in the living system. Successful sequestering and enhancement of these two GFs is particularly encouraging for two reasons: first, this highlights that enrichment of endogenous GFs is sufficient to initiate revascularization, without the need to supplement exogenous GFs; second, these two GFs are representative key mediators of blood vessel formation and maturation, respectively. By using a bioresponsive macromolecule to simultaneously harness their functions, especially those of PDGF-BB, we provide a new approach to regenerate blood vessels that are both structurally integrate and functionally comprehensive, which is highly desirable for various regenerative and therapeutic applications. In summary, we report in this study the discovery of EUP3, the first non-tissue-derived, nonsulfated polysaccharide, with demonstrated affinity to sequester pro-angiogenic factors of FGF-2 and PDGF-BB in situ and in vivo, thereby promoting angiogenesis at the local site. Further development of EUP3 may provide a safer and more efficient agent for the unmet medical challenge in therapeutic angiogenesis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00182. Experimental procedures and supporting data (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by The Science and Technology Development Fund, Macau SAR (048/2013/A2), and the University of Macau Research Committee (MRG006/WCM/ 2014/ICMS, MYRG2014-00069-ICMS-QRCM, and MYRG2015-00160-ICMS-QRCM).

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DOI: 10.1021/acsmacrolett.6b00182 ACS Macro Lett. 2016, 5, 617−621