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

Abstract

Although abdominal aortic aneurysms (AAA) can be potentially stabilized by inhibiting inflammatory cell recruitment and their release of proteolytic enzymes, active AAA regression is not possible without regeneration of new elastic matrix structures. Unfortunately, postneonatal vascular smooth muscle cells (SMCs), healthy, and likely more so, diseased cells, poorly synthesize or remodel elastic fibers, impeding any effort directed at regenerative AAA treatment. Previously, we determined the eleastogenic benefits of oligomers (HA-o; 4-6 mers) of the glycosaminoglycan, hyaluronan (HA) and transforming growth factor-b1 (TGF-b1) to healthy SMCs. Since AAAs are often diagnosed only late in development when matrix disruption is severe, we now determine if elastogenic upregulation of SMCs from late-stage AAAs ( > 100% diameter increase) is possible. AAAs were induced by perfusion of rat infrarenal aortae with porcine pancreatic elastase. Elastic matrix degradation, vessel expansion (*120%), inflammatory cell infiltration, and enhanced activity of matrix-metalloproteases (MMPs) 2 and 9 resulted, paralleling human AAAs. Aneurysmal SMCs (EaRASMCs) maintained a diseased phenotype in 2D cell culture and exhibited patterns of gene expression different from healthy rat aortic SMCs (RASMCs). Relative to passage-matched healthy RASMCs, unstimulated EaRASMCs produced far less tropoelastin and matrix elastin. Exogenous TGF-b and HA-o (termed ''factors'') significantly decreased EaRASMC proliferation and enhanced tropoelastin synthesis, though only at the highest provided dose combination (20 mg/mL of HA-o, 10 ng/mL of TGF-b); despite such enhancement, tropoelastin amounts were only *40% of amounts synthesized by healthy RASMC cultures. Differently, elastic matrix synthesis was enhanced beyond amounts synthesized by healthy RASMCs (112%), even at lower doses of factors (2 mg/mL of HA-o and 5 ng/mL of TGF-b). The factors also enhanced elastic fiber deposition over untreated EaRASMC cultures and restored several genes whose expression was altered in EaRASMC cultures back to levels expressed by healthy RASMCs. However, the activity of MMPs 2 and 9 generated by EaRASMC cultures was unaffected by the factors/factor dose. The study confirms that SMCs from advanced AAAs can be elastogenically induced, although much higher doses of elastogenic factors are required for induction relative to healthy SMCs. Also, the factors do not appear to inhibit MMP activity, vital to preserve existing elastic matrix structures that serve as nucleation sites for new elastic fiber deposition. Thus, to enhance net accumulation of newly regenerated elastic matrix, toward possibly regressing AAAs, codelivery of MMP inhibitors may be necessitated.

Induced Elastic Matrix Deposition Within Three-Dimensional Collagen Scaffolds

“…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. 32 However, from the standpoint of a tissue engineering application, wherein growth in tissue mass is contingent on increased availability of ECM-generating cells, the HA-o/TGF-b1 dose combination that provides greater impetus to SMC proliferation (i.e., 0.1 ng/mL of TGF-b1 and 0.2 or 2 mg/mL of HA-o) is most desirable.…”

Magnetically Responsive Bone Marrow Mesenchymal Stem Cell-Derived Smooth Muscle Cells Maintain Their Benefits to Augmenting Elastic Matrix Neoassembly

“…Unlike collagen fibers, which undergo natural regenerative repair postdisruption, elastic fibers are poorly restored, due to poor elastogenesis, and impaired elastic fiber assembly by postneonatal and diseased vascular SMCs. 6,7,29,30 Thus, to achieve AAA growth arrest, elastin regeneration must significantly exceed its breakdown. 31 Previously, 10 we showed that BM-SMCs exhibit high elastogenesis and generate paracrine secretions that stimulate elastogenesis by EaRASMCs, findings which support their use for matrix regenerative AAA therapy, provided they can be efficiently delivered to the AAA wall.…”

“…6 Similarly, control healthy rat aortic SMCs (RASMCs) were isolated from healthy adult rats (n = 3). 6 The primary cells were cultured for 2 weeks in Dulbecco's modified Eagle's medium-F12 (Invitrogen) supplemented with 10% v/v fetal bovine serum (FBS; Invitrogen) and 1% v/v penicillin-streptomycin (Penstrep; Thermo Fisher) and passaged before use.…”

“…As we previously published, 6,7 21-day cultures within Permanox chamber slides were fixed with 2% w/v cacodylate glutaraldehyde (12 h), postfixed in 1% w/v osmium tetroxide (1 h), dehydrated in a graded ethanol series (50-100% v/v), and embedded in Epon 812 resin. The sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and visualized on a Hitachi TEM (Model H7600T).…”

“…Although inhibiting MMPs may slow AAA growth, 4 AAA growth arrest and restoration of vessel recoil properties are challenged by poor regenerative repair of elastic fibers by adult vascular smooth muscle cells (SMCs). [5][6][7] A proelastogenesis stimulus is thus needed that may be effective in the proteolytic AAA tissue milieu.…”