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The progression of aortic aneurysms (AAs) is typically associated with an activated smooth muscle cell (SMC) phenotype, diminished density of mature medial elastic fibers, and an elevated presence of matrix-degrading enzymes, which ultimately leads to vessel rupture. Currently, no surgical or nonsurgical methods are available to regress aneurysms via regeneration of new elastic matrices, particularly because of inherently poor elastin synthesis by adult vascular cells and absence of tools to stimulate the same. We seek to address this void in this study. We recently showed 0.2 microg/mL of hyaluronan oligomers and 1 ng/mL of transforming growth factor-beta1 (termed elastogenic factors) to dramatically enhance elastin synthesis and matrix formation by healthy aortic SMCs. In this study, the effect of these factors, alone or together, on suppressing procalcific and elastolytic activities of aneurysmal vascular cells, and improving their elastin matrix synthesis and assembly is examined. Periadventitial injury with calcium chloride was used to induce AAs in rats, and approximately 45% increase in aortic diameter was observed after 4 weeks. Aneurysmal SMCs isolated from these AA segments produced higher levels of inflammatory markers matrix metalloproteinases-2 and 9 elastase activity and calcific deposits, while synthesizing significantly less collagen, tropoelastin, and matrix elastin proteins over a 3-week culture period, relative to healthy SMCs. While hyaluronan oligomers alone significantly suppressed aneurysmal cell proliferation and promoted 20-50% increases in collagen and elastin synthesis (p0.01), transforming growth factor-beta1 alone had no effect on cellular proliferation and elastin synthesis. However, provision of factors together resulted in significantly higher amounts of collagen/elastin protein synthesis and crosslinking, by upregulating lysyl oxidase and desmosine. Compared to their individual contributions, the factors together were highly effective in minimizing the release of inflammatory enzymes, and encouraging elastic fiber formation. Since elastic matrix amounts were one order of magnitude lower than that observed with healthy cells, even upon elastogenic stimulation at doses optimized previously for healthy cells, increased doses are likely required and must be reoptimized for diseased cells. Despite this, the results point to the potential utility of these elastogenic factors in regenerating elastic matrices within AAs.

Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo- and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu(2+)) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu(2+) ions slowed cell proliferation (5.6 +/- 2.3-fold increase over 21 days vs 22.9 +/- 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 +/- 0.4-fold), tropoelastin (4.1 +/- 0.05-fold) and cross-linked matrix elastin (4.2 +/- 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu(2+) ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu(2+) ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu(2+) ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers ( approximately 1 mum diameter). However, 0.01 M of Cu(2+) ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu(2+) ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.