Hemodynamics and Axial Strain Additively Increase Matrix Remodeling and MMP-9, But Not MMP-2, Expression in Arteries Engineered by Directed Remodeling

Abstract

We previously demonstrated the ability to create engineered arteries by carefully controlling the mechanical environment of intact arteries perfused ex vivo, yielding engineered arteries with native appearance and vasoactive response. Increased axial strain was sufficient to increase length up to 20% in 9 days through a growth and remodeling response. The amount of the achievable length increase, however, was highly dependent on the hemodynamic conditions acting through unknown mechanisms. Because matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activity is increased, and often required, in mechanically induced remodeling in vivo, MMP-2 and MMP-9 expression was investigated to elucidate the hemodynamic mediation of artery length. Carotid arteries from 30 kg pigs were perfused for 9 days ex vivo at either in situ axial strain or with a gradual 50% increase in axial strain, under either arterial or reduced hemodynamics (*10% of arterial hemodynamics). MMP-2 protein expression increased roughly twofold, while MMP-9 expression increased threefold under either reduced hemodynamics or increased axial strain ( p < 0.05). The combination of reduced hemodynamics with increased axial strain demonstrated an additive increase in MMP-9 protein ( p < 0.05) with no further change in MMP-2 expression. To investigate the mechanism by which axial strain and hemodynamics could additively increase MMP-9 expression, the expression of nuclear factor kappa B (NF-kB) subunits p50 and p65 was evaluated. Axial strain stimulated p65 expression and localization, while hemodynamics increased p50 expression, with both molecules being expressed only when both mechanical stimuli were applied. These data suggest that MMP-9 expression can be simultaneously stimulated by separate mechanical stimuli mediated by p50 and p65 expression, and that by using conditions that maximize MMP-9 expression, we can create an optimal remodeling environment to better direct the growth of engineered arteries and other tissues.

Arterial pO2 stimulates intimal hyperplasia and serum stimulates inward eutrophic remodeling in porcine saphenous veins cultured ex vivo

“…The ex-vivo perfusion system has been previously described (Clerin et al 2003;Gusic et al 2005a,b;Nichol et al 2005Nichol et al , 2009Lawrence and Gooch 2009a,b) and is similar to those used by others (Porter et al 1996b;Han et al 2003Han et al , 2006Davis et al 2005;Gleasonm et al 2007;Wayman et al 2008;Yao et al 2009). PSV isolated from 25-35 kg pigs were cultured in low-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, CA), 100 μg/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 25 mM HEPES (all from Invitrogen Corp., Rockville, MD).…”

“…Harvesting veins and pressure testing them for leaks briefly exposes veins to an altered mechanical environment, which may result in subsequent remodeling during culture. For example, both axial and circumferential stretch has been reported to up-regulate molecular mediator of vascular remodeling including matrix metalloproteinase (MMP),mmP-2 and MMP-9 (Chesler et al 1999;Nichol et al 2009). Axial stretch of HSV ex vivo for less than 10 s results in elevated MMP levels that peaks 3 days later (Meng et al 1999 during vessel harvest or preparation might manifest itself as remodeling days later.…”

“…A primary benefit of these ex-vivo models is that they afford much better control and monitoring of the mechanical and chemical environments than possible with vessels in vivo while allowing the study of whole-vessel behavior not captured in cell culture. Capitalizing on these strengths of the exvivo culture systems, investigators have used them to study the independent contribution of mechanical factors (e.g., flow, pressure, and pulsatility) (Clerin et al 2003;Han et al 2003;Davis et al 2005;Gusic et al 2005a,b;Han et al 2006;Gleasonm et al 2007;Wayman et al 2008;Yao et al 2009) on different aspects of vascular remodeling and to understand how multiple mechanical factors (e.g., pressure and axial loading) interactively regulate vascular remodeling (Nichol et al 2005(Nichol et al , 2009Lawrence and Gooch 2009a,b). Many aspects of mechanically induced remodeling of blood vessels originally observed in vivo have also been observed in vessels cultured ex vivo including pressure-induced hypertrophy (Gusic et al 2005a,b), axial loading-induced growth/remodeling (Clerin et al 2003;Nichol et al 2005Nichol et al , 2009Gleasonm et al 2007;Lawrence and Gooch 2009a,b), and flow-induced changes in luminal area (Nichol et al 2005(Nichol et al , 2009.…”

“…For example, vessels cultured ex vivo with mechanical environments similar to their native in vivo environments exhibit hypertrophy (Clerin et al 2003;Gusic et al 2005a,b), lengthen (Nichol et al 2005(Nichol et al , 2009Gleasonm et al 2007;Lawrence and Gooch 2009a,b), eutrophically remodel (Gusic et al 2005a,b), develop intimal hyperplasia (Gusic et al 2005a,b), as well as alter their extracellular matrix content (Gusic et al 2005a,b), matrix metalloproteinase activity (Nichol et al 2009) and biomechanical properties (Clerin et al 2003;Gusic et al 2005a,b;Lawrence and Gooch 2009a,b). There are at least three general explanations for these changes.…”

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

“…In particular content and organization of collagen fibers in the extracellular matrix contribute to the load-bearing properties and dominate overall tissue strength 1 . In tissue engineering mechanical conditioning of the construct is used -typically consisting of (cyclic) straining regimens -to enhance tissue organization and mechanical properties 2,3 . Full understanding of strain-induced collagen organization in complex tissue geometries to create tissues with predefined collagen architecture has not yet been achieved.…”