Enteral Protease Inhibition Preserves Aortic Ultrastructure and Mechanics in Experimental Trauma/Hemorrhagic Shock

Abstract

Introduction: Vascular compliance of large arteries is often overlooked in acute trauma/hemorrhagic shock (T/HS), as standard interventions, such as vasopressors, typically target systemic vascular resistance (SVR) regulated by small arterioles. However, patients may become refractory to vasopressors, and efforts to raise SVR may not restore mean arterial blood pressure (MAP), which is crucial for organ perfusion. We have previously reported compromised vascular compliance after T/HS in vivo with increased pulse wave velocity and ex vivo with diminished circumferential distensibility, that were both partially mitigated by enteral serine protease inhibition. To further elucidate the mechanisms behind these adverse changes, our aim was to directly assess the material stiffness, extracellular matrix (ECM) morphology, and matrix metalloproteinase (MMP) activity within the aortic wall tissue after T/HS, as well as to evaluate the effectiveness of enteral protease inhibition in this context.Methods: Anesthetized Wistar rats (350 - 400g) underwent laparotomy (trauma) and exsanguination to maintain a MAP of 35-40 mmHg for 90 minutes (shock). Animals were randomly assigned to receive either enteral gabexate mesilate protease inhibitor treatment (GM-treated) or vehicle-only (Untreated) via an orally-inserted catheter; investigators were blinded to the treatment assignation. At the conclusion of the shock period, animals were resuscitated with intravenous Lactated Ringer’s solution (LR) for two hours to achieve and maintain a MAP of at least 60 mmHg. A sham control group was also included for comparison. In all groups, the thoracic aorta was bisected, with one half sectioned for atomic force microscopy and histology and the other half homogenized for gelatin zymography. Liquid-phase force mapping was conducted on random areas (1600 μm2) of the medial layer in triplicate to determine the average Young’s modulus of the tissue. Collagen fibers were stained with picrosirius red, and elastin fibers were stained with Verhoeff Van Gieson. Results: After being subjected to T/HS and LR reperfusion, the aortic media of the Untreated group had a significantly higher average Young’s modulus than that of healthy sham vessels (862.7 kPa vs. 556.5 kPa, p < 0.05). The GM-treated group, however, better preserved its material elasticity (~650 kPa). These stiffness values negatively correlate with corresponding animals’ final MAP values measured at the end of the reperfusion period (r2 = 0.5031), supporting an association between vascular compliance and hemodynamic stability. The area of collagen within the tunica media was found to be significantly decreased in the Untreated compared to the sham group (4.93% vs. 10.26%, p < 0.05), but GM treatment was able to prevent collagen degradation (8.48%). Disruptions in elastin fibers were also observed at a higher frequency in the Untreated T/HS aorta compared to sham controls (3.3 vs. 1.7, p < 0.05) or GM-treated aortas (3.3 vs. 1.6, p < 0.05). Gelatin zymography revealed that enteral GM treatment also significantly attenuated elevated plasma MMP-9 (0.85 vs. 1.97 A.U., p < 0.01) and tissue MMP-2 activity (0.86 vs. 1.80 A.U., p < 0.05) seen following T/HS. Conclusions: Fluid-resuscitated traumatic hemorrhagic shock resulted in ultrastructural and biomechanical alterations in the aorta, indicating a loss in compliance. Enteral gabexate mesilate treatment effectively mitigated these pathological changes to the vasculature, suggesting the potential involvement of serine proteases in aortic ECM remodeling and large artery dysfunction in T/HS. Interventions against central artery impairment may be a promising strategy for improving blood pressure and survival in circulatory shock. Department of Defense Award W81XWH-17-2-0047. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.