Utility of Hyaluronan Oligomers and Transforming Growth Factor-Beta1 Factors for Elastic Matrix Regeneration by Aneurysmal Rat Aortic Smooth Muscle Cells

Abstract

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 mg=mL of hyaluronan oligomers and 1 ng=mL of transforming growth factor-b1 (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 *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 ( p < 0.01), transforming growth factorb1 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.

Elastogenic Inductability of Smooth Muscle Cells from a Rat Model of Late Stage Abdominal Aortic Aneurysms

“…In EaRASMC cultures, unlike in healthy RASMC cultures, low dose combinations of HA-o ( < 20 mg/mL) and TGF-b ( < 10 ng/mL) had no effect on tropoelastin production; this agrees well with our prior findings that diseased SMCs require higher doses of these factors for elastogenic stimulation. 16 Also, as seen in our trend curves (Fig. 5C), the dependence of tropoelastin synthesis on HA-o dose is enhanced at higher doses of concurrently delivered TGF-b.…”

“…28 More recently, we demonstrated that SMCs isolated from rat aortal expansions created by peri-adventitial caustic injury, which resemble early stage AAAs, (a) continue to exhibit an abnormal, activated phenotype when passaged in vitro culture, (b) are much more elastogenically challenged relative to healthy, passagematched RASMCs, and can (c) be elastogenically stimulated in vitro with HA-o and TGF-b factors. 16 The relevance of the present study lies in the fact that AAAs are often only diagnosed at advanced stages of development, when matrix deterioration is severe. Since cell behavior is influenced by their microenvironment, there is a need for evidence that SMCs from such severely afflicted tissues can also respond favorably to elastogenic stimulation, and if so, if the effective doses of elastogenic factors differ significantly from that we ascertained useful with healthy RASMCs.…”

“…We subsequently investigated the dose-specific effects of the same factors (0-20 mg/mL HA-o and 0-10 ng/mL TGF-b) on elastogenesis by SMCs derived from CaCl 2 induced rat aortal expansions (aRASMCs), 16 which simulated very early stage inflammatory human AAAs; we showed the aRASMCs isolated from these expansions to exhibit an activated phenotype in culture and to be amenable to elastogenic stimulation. However, AAAs are frequently diagnosed at advanced stages of development when matrix deterioration is severe.…”

Induced Elastic Matrix Deposition Within Three-Dimensional Collagen Scaffolds

“…6 The present results suggest that (a) interaction with the collagenous ECM alters SMC behavior compared to 2D cultures, and (b) such collagen-SMC interaction de-sensitizes the cells to TGFb1 and HA-o, attested to by a significant decrease in cell number only at the highest provided doses. It is also apparent from our results that similar to our recently published observations in 2D cultures, TGF-b1 effects dominate with a biphasic dose-dependent proliferative response to TGF-b1 at both HA-o doses.…”

“…5 Of potential benefit to overcoming the poor elastogenicity of adult vascular cells, our laboratory previously determined the synergistic benefits of hyaluronan oligomer (HA-o) and transforming growth factor-beta1 (TGF-b1) to elastin precursor synthesis, elastic matrix deposition, and fiber formation by cultured adult rat and human SMCs. 6,7 We also showed that TGF-b1 increases expression levels of mRNA for tropoelastin, and that of the matrix crosslinking enzyme, lysyl oxidase (LOX), while suppressing the activity of matrix degrading matrix metalloproteinases (MMPs). 7,8 Negatively charged HA is also thought to coacervate tropoelastin molecules for more efficient localized crosslinking and organization of these precursors into mature fibers, much like the role of glycosaminoglycans (GAGs) in a developing aorta.…”