Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-alpha-inhibitor (HC*HA) purified from extracts of human amniotic membrane

Abstract

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of approximately 3000 kDa) was predominantly extracted in isotonic Extract A (70.1 +/- 6.0%) and PBS (37.7 +/- 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-alpha-inhibitor (IalphaI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-alpha stimulated gene-6 protein (TSG-6). This HC.HA complex (nHC*HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC*HA) by mixing HMW HA, serum IalphaI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-beta1 promoter activation in corneal fibroblasts and induced mac ro phage apoptosis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC*HA or rcHC*HA. These data collectively suggest that the HC*HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.

FIGURE 1.

Presence of HMW HA in AM extracts. Extracts A, B, and C with or without HAase digestion (HAase) or NaOH treatment (NaOH) were resolved by 0.5% agarose gel electrophoresis and stained with Stains-all dye. The M_r of the Select-HA HiLadder (M, lane 1) is marked at the left of the gel. Five μg of HMW HA, with an average molecular mass of ∼1000 kDa (Sigma), was loaded as a comparison.

FIGURE 2.

Covalent linkage between HA and HCs of IαI in AM extracts. Extract A was treated (in duplicate) with a series of NaOH concentrations (0, 0.02, 0.05, 0.10, and 0.2 n) before Western blot with an anti-IαI antibody to determine the optimal NaOH concentration for cleaving the linkage between HA and HCs (A: M, protein ladder markers, and IαI, purified from the human plasma). Extracts A–C with or without HAase digestion or 0.05 n NaOH treatment were analyzed by Western blot with anti-IαI antibody (B).

FIGURE 3.

TSG-6 was present in AM extracts. TSG-6 was found to be present in Extract A using three different antibodies that recognized the control TSG-6Q (25 ng) as a ∼32 kDa protein (A, bands of ∼35 and ∼50 kDa were seen in Extract A).

FIGURE 4.

Suppression of TGF-β1 promoter activation by Extract PBS. A dose-dependent relationship was noted in the suppression of the TGF-β1 promoter activity by a series of concentrations of Extract PBS (A). In contrast, HMW HA (Healon), with an average molecular mass of HA ∼4000 kDa, showed some suppression of TGF-β1 promoter activity but not statistically significant (B). The suppressive effect of the TGF-β1 promoter activity was lost when HMW HA (125 μg/ml) or Extract PBS (125 μg/ml proteins) was digested with hyaluronidase (C). Heat treatment (95 °C for 10 min) also eliminated the suppressive activity by Extract PBS but this was not significant for HMW HA (D). Data are represented as the mean ± S.D. of four independent experiments (n = 4) with a sample size of 4 for each condition. In A–D, an asterisk indicated p < 0.05.

FIGURE 5.

Purification of HC·HA (nHC·HA) complex from Extract PBS by ultracentrifugation. Fractions 8–15 from the first CsCl, 4 m guanidine HCl ultracentrifugation (1st) started at the initial density of 1.35 g/ml (A) and fractions 3–15 from the second ultracentrifugation (2nd) started at the initial density of 1.40 g/ml (B) were pooled according to the presence of HA but the absence of proteins. The latter fraction was dialyzed in distill water to remove CsCl and guanidine. Extract PBS, 1st and 2nd pooled fractions were then treated with or without 0.05 n NaOH at 25 °C for 1 h (2nd pooled fractions was also digested with 20 units/ml HAase at 37 °C for 2 h) and analyzed on 0.5% agarose gel before being stained with Stains-all dye (C), stained with the Coomassie Blue dye (D), or on Western blot using an anti-IαI antibody (E). The results confirmed the nHC·HA complex was formed by HMW HA and HC of IαI via a NaOH-sensitive bond. Please note the pooled fractions from the second ultracentrifugation (labeled as 2nd) in D were concentrated ∼20-fold by lyophilization before loading to enhance the detection by the Coomassie Blue dye. AME, amniotic membrane extract.

FIGURE 6.

In vitro reconstitution of HC·HA (rcHC·HA) complex. The HA binding capacity (%) on HABP cross-linked wells was determined to be maximal at 25 μg/ml HMW HA (Healon) by addition of both human IαI and recombinant human TSG-6 (A, ●) when compared with HMW HA alone (A, ▴) or HMW HA with IαI (A, ■). Four independent experiments (n = 4) with a sample size of 4 for each condition were performed and data were represented as the mean ± S.D. IαI directly bound to HABP, which was covalently cross-linked to the 96-well surface, as shown by Western blot with anti-IαI antibody (B). Lane 1, protein marker; lane 2, purified IαI; lane 3, HABP only; lane 4, HABP + IαI; lane 5, HABP + HA; lane 6, HABP + IαI + HA. Western blot using an anti-IαI antibody (C) revealed that the bound HMW HA on HABP cross-linked wells formed the HC·HA complex when added with both IαI and TSG-6 (HA + IαI + TSG-6, lanes 6, 10, and 14) when compared with HMW HA alone (lanes 3, 7, and 11), with IαI alone (HA + IαI, lanes 4, 8, and 12) or TSG-6 alone (HA + TSG-6, lanes 5, 9, and 13) either without (lanes 3-6) or with HAase digestion (lanes 7–10) or NaOH treatment (lanes 11–14).

FIGURE 7.

Suppression of TGF-β1 promoter activity and promotion of macrophage death by rcHC·HA and nHC·HA complexes. As compared with the control (A, Ctrl, PBS only), rcHC·HA complex or nHC·HA significantly suppressed TGF-β1 promoter activity, i.e. as measured by the TGF-β1 promoter assay (A, p = 0.004 and 0.005, respectively), and promoted macrophage death as measured by the MTT assay (B, p = 0.0003 and 0.0007, respectively). In contrast, Healon HA alone (HA) or with additional IαI (HA + IαI) or TSG-6 (HA + TSG-6) did not show a statistically significant effect (all p > 0.05). Four independent experiments (n = 4) with a sample size of 4 for each condition were performed and data were represented as the mean ± S.D.