Impact of delivery mode of hyaluronan oligomers on elastogenic responses of adult vascular smooth muscle cells

Abstract

Our prior studies demonstrated that exogenous supplements of pure hyaluronan (HA) tetramers (HA4) dramatically upregulate elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). Some studies suggest that exogenous HA likely only transiently contacts and signals cells, and may elicit different cell responses when presented on a substrate (e.g., scaffold surface). To clarify such differences, we used a carbodiimide-based chemistry to tether HA4 onto glass, and compared elastin matrix synthesis by SMCs cultured on these substrates, with those cultured with equivalent amounts of exogenous HA4. Tethered HA4-layers were first characterized for homogeneity, topography, and hydrolytic stability using SEM, XPS, AFM, and FACE. In general, mode of HA4 presentation did not influence its impact on SMC proliferation, or cell synthesis of tropoelastin and matrix elastin, relative to non-HA controls; however, surface-tethered HA4 stimulated SMCs to generate significantly greater amounts of elastin-stabilizing desmosine crosslinks, which partially accounts for the greater resistance to enzymatic breakdown of elastin derived from these cultures. Elastin derived from both sets of cultures contained peptide masses that correspond to the predominant peptides present in rat aortic elastin. SEM and TEM showed that HA4-stimulated fibrillin-mediated elastin matrix deposition, and organization into fibrils. Surface-immobilized HA4 was particularly conducive to organization of elastin into aggregating fibrils, and their networking to form closely woven sheets of elastin fibers, as seen in cardiovascular tissues. The results suggest that incorporation of elastogenic HA4 mers onto cell culture substrates or scaffolds is a better approach than exogenous supplementation for in vitro or in vivo regeneration of architecturally and compositionally faithful-, and more stable mimics of native vascular elastin matrices.

Figure 1

Scanning electron micrographs of untreated glass, aminosilane-treated glass, and HA4mer-tethered glass. Glass surfaces appear mostly smooth with occasional spots that are owed to commercial plasma coating (A). Glass surfaces treated with the aminosilane show the presence of APTMS molecules (B). Panel (C) shows aminated surfaces further reacted with enzymatic digests of long-chain HA, containing primarily HA4 (75% w/w). Panels D-F show atomic force micrographs and corresponding peak heights of uncoated glass (D), APTMS (E), and HA4-tethered (F) surfaces. As seen, the HA4mers reacted uniformly across the surface. Magnification: 1500× (A and B) and 900× (C).

Figure 2

Comparison of enzymatic degradation profiles of insoluble rat aortal elastin, and that isolated from HA4-treated and untreated cell layers. In each case, elastin pellets were digested with 1 ml of elastase (20 U/ml) over 8 hours at 37 °C (n = 9/ case/ time point). In panel A, values shown represent mean ± SD of fractions of the respective pre-digestion elastin pellet amounts retained after the designated period of enzymatic breakdown. Note that the error bars are too small to be visible on this plot. Silver staining of soluble elastin peptides within the digestate solutions in each case show greater intensity, signifying greater amounts of elastin degradation products generated in the case of elastin from aortae and HA-free cultures than upon elastase treatment of elastin pellets from HA4-exposed cell layers.

Figure 3

Representative mass spectroscopy spectra for peptides derived by elastase digestion of elastin matrices isolated from native rat aortae (A), and smooth muscle cell layers exposed to exogenous HA4 supplements (B) and cultured on HA4-tethered substrates. Irrespective of source, the spectra contain almost identical peaks, primarily contained within a mass range of 100−922 Da, that reflect similar relative abundance of content of peptides of designated mass or mass/charge ratios contained in a rat protein database accessed through Bioworks 3.2 software for protein identification. A total of n = 9 samples/case were analyzed and good repeatability of outcomes was observed.

Figure 4

Scanning electron micrographs comparing ultrastructures of alkali-insoluble matrix elastin isolated from rat aortae, and cell layers cultured for 3 weeks. Aortal elastin was mostly organized into continuous (A) and fenestrated sheets (B) with a few visible areas exhibiting elastin fibers closely networked into sheets. Elastin isolated from HA-free control cell layers were however mostly clumpy and formless with no fibers or distinct organization visible (C). Cell layers that received exogenous HA4 supplements or were cultured atop HA4-tethered glass appeared mostly similar, organized into continuous and fenestrated sheets (D). However, in addition, the latter cultures contained sheets of tightly intertwined/ woven elastin fibers (E), with individual fibers visible at the sheet periphery (F); only individual elastin fibers, not woven sheets were seen cell layers that received exogenous HA4.

Figure 5

Representative transmission electron micrographs showing elastin within 21 day-old cell layers cultured HA-free (Panel A) in the presence of exogenous HA4 (4 μg/ well; panel B), and on HA4-tethered substrates (2 μg/cm², 8 cm² culture area; panel C-E). In each case, representative images were selected from nearly 20 captured micrographs. A qualitative comparison of elastin matrix within the control and test cell layers reveals differences in the amount and nature of elastin deposited. HA free cultures contained only small clumps of amorphous elastin and no fibers. Cultures that received HA4 exogenously however contained larger clumps of amorphous elastin and more elastin fibers than controls, while still greater amounts of elastin were seen within cell layers cultured on HA4-tethered surfaces, which were predominantly assembled into aggregating fiber bundles. Immunogold labeling confirmed deposition of fibrillin microtubules (deep staining spots), scaffolds that precede elastin deposition and fibril organization (see arrows in panel E).