Transforming growth factor beta 1 and hyaluronan oligomers synergistically enhance elastin matrix regeneration by vascular smooth muscle cells

Abstract

Elastin is a vital structural and regulatory matrix protein that plays an important role in conferring elasticity to blood vessel wall. Previous tissue engineering approaches to regenerate elastin in situ or within tissue engineering constructs are curtailed by innate poor elastin synthesis potential by adult vascular smooth muscle cells (SMCs). Currently, we seek to develop cellular cues to enhance tropoelastin synthesis and improve elastin matrix yield, stability, and ultrastructure. Our earlier studies attest to the elastogenic utility of hyaluronan (HA)-based cellular cues, though their effects are fragment size dependent and dose dependent, with HA oligomers deemed most elastogenic. We presently show transforming growth factor beta 1 (TGF-beta1) and HA oligomers, when provided concurrently, to synergistically and dramatically improve elastin matrix regeneration by adult vascular SMCs. Together, these cues suppress SMC proliferation, enhance synthesis of tropoelastin (8-fold) and matrix elastin protein (5.5-fold), and also improve matrix elastin yield (45% of total elastin vs. 10% for nonadditive controls), possibly by more efficient recruitment of tropoelastin for crosslinking. The density of desmosine crosslinks within the elastin matrix was itself attenuated, although the cues together modestly increased production and activity of the elastin crosslinking enzyme, lysyl oxidase. TGF-beta1 and HA oligomers together induced much greater assembly of mature elastin fibers than they did separately, and did not induce matrix calcification. The present outcomes might be great utility to therapeutic regeneration of elastin matrix networks in situ within elastin-compromised vessels, and within tissue-engineered vascular graft replacements.

FIG. 1.

Effect of HA fragments and TGF-β1 on RASMCs. Proliferation ratios represent DNA content at 21 days relative to day 1 of culture. Values shown represent mean ± SD of nine repeat samples/case. Differences versus controls were significant (*) for p < 0.05.

FIG. 2.

Impact of HA fragments and TGF-β1 on (A) collagen matrix protein synthesis and (B) total collagen output (pooled medium and matrix fractions). Cells were cultured for 21 days. Data shown represent mean ± SD of nine repeats/case and are shown normalized to controls. Differences versus controls were significant (*) for p < 0.05.

FIG. 3.

Effect of exogenous HA fragments and TGF-β1 alone and together on synthesis of tropoelastin (A: Fastin assay; B: SDS-PAGE), alkali-soluble (C), and -insoluble elastin matrix proteins (D). Case-wise trends for DNA-normalized amounts of desmosine mirrored that in insoluble matrix elastin protein (E). DNA-normalized data represent mean ± SD of nine repeats/case, and are shown as fold change compared to controls. Differences versus controls were significant (*) for p < 0.05.

FIG. 3.

Effect of exogenous HA fragments and TGF-β1 alone and together on synthesis of tropoelastin (A: Fastin assay; B: SDS-PAGE), alkali-soluble (C), and -insoluble elastin matrix proteins (D). Case-wise trends for DNA-normalized amounts of desmosine mirrored that in insoluble matrix elastin protein (E). DNA-normalized data represent mean ± SD of nine repeats/case, and are shown as fold change compared to controls. Differences versus controls were significant (*) for p < 0.05.

FIG. 4.

(A) LOX enzyme activities in test cultures at 21-day culture periods. (B) SDS-PAGE/Western blots showed that TGF-β1 alone or together with HA oligomers/fragments enhanced LOX protein synthesis. All the data are shown normalized to LOX activities in controls. Data shown represent mean ± SD of nine repeats/case and are shown normalized to controls. Differences versus controls were significant (*) for p < 0.05.

FIG. 5.

(A) Phase contrast images of 21-day cultures that did not receive additives (controls), or received TGF-β, or TGF-β and HA oligomers show that confluence was attained at the time of immunolabeling, although cell density in the last-said culture was much lower. (B) TGF-β1 alone or together with HA oligomers enhanced elastin, fibrillin, and LOX (red) production relative to nonadditive control cultures. (C) Negative immunolabeling controls did not exhibit any red fluorescence, confirming lack of nonspecific binding of the fluorophore-conjugated secondary probe(s). Although distribution of the labeled proteins was mostly homogenous (E), occasional clumps of high cell density were seen (D), which colocalized with a higher density of elastin matrix. DAPI-stained cell nuclei fluoresce blue. Immunofluorescence images are typical of ≥3 cultures/case. All cultures were imaged under identical conditions. Magnification: 20× (panels A, D, and E) and 40× (B, C). Color images available online at www.liebertonline.com/ten.

FIG. 6.

TGF-β1 and HA oligomers alone, or together, promote elastin fiber formation, as seen in transmission electron micrographs. Control cultures contained only amorphous clumps. Elastin was assembled as fibrils within a peripheral fibrillin scaffold (see black dots) and aggregated to form mature fibers ∼400 nm in diameter, typical of fully formed fibers.