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. 2021 Nov 2;78(18):1782-1795.
doi: 10.1016/j.jacc.2021.08.047.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support

Amrut V Ambardekar  1 , Matthew S Stratton  2 , Evgenia Dobrinskikh  3 , Kendall S Hunter  4 , Philip D Tatman  5 , Madeleine E Lemieux  6 , Joseph C Cleveland  7 , Rubin M Tuder  3 , Mary C M Weiser-Evans  8 , Karen S Moulton  2 , Timothy A McKinsey  2
Affiliations

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support

Amrut V Ambardekar et al. J Am Coll Cardiol. .

Abstract

Background: The effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood.

Objectives: The authors sought to quantify indexes of fibrosis and determine the molecular signature of post-CF-LVAD vascular remodeling.

Methods: Paired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD-mediated changes in aortic mRNA and protein expression.

Results: The mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 μm pre-LVAD to 410 ± 209 μm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix-degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted.

Conclusions: There is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix-degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.

Keywords: aorta; congestive heart failure; fibrosis; left ventricular assist device; mechanical circulatory support; vascular remodeling.

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Conflict of interest statement

Funding Support and Author Disclosures REDCap was provided by National Institutes of Health/NCATS Colorado CTSA Grant Number UL1 TR002535. Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core supported in part by National Institutes of Health/NCATS Colorado CTSI Grant Number UL1 TR002535. Dr Ambardekar was supported by a Scientist Development Grant from the American Heart Association and by the Boettcher Foundation’s Webb-Waring Biomedical Research Program. Dr Stratton was supported by National Institutes of Health grants HL126354 and AG056848. Dr Weiser-Evans was supported by the National Heart, Lung, and Blood Institute National Institutes of Health Grant Numbers R01 HL121877 and R01 HL123616. Dr McKinsey was supported by the National Institute of Health (grants HL116848, HL147558, DK119594, HL127240, and HL150225) and by the American Heart Association (16SFRN31400013); has received support from the Colorado Office of Economic Development and International Trade (CTGGI 19-3579) through the University of Colorado SPARK Program; is on the scientific advisory boards of Artemes Bio, Inc., and Eikonizo Therapeutics; has received funding from Italfarmaco for an unrelated project, and has a subcontract from Eikonizo Therapeutics related to an SBIR grant from the National Institutes of Health (HL154959). The contents are the authors’ sole responsibility and do not necessarily represent official National Institutes of Health views. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

Figure 1.
Figure 1.. Human aorta is remodeled by continuous-flow LVAD support.
A, Movat’s Pentachrome-stained aorta sections are shown from the same individual before and after LVAD exposure. Scale bars 600 microns. Right panel is higher power image of expanded adventitia layer post-LVAD. Scale bar 200 microns. B through D, Data points represent the mean thickness (microns) of adventitia (B), media (C) and intima (D) layers in the pre-LVAD aorta and post-LVAD aorta collected from N=20 individuals. Values represent means +SEM. *Indicates the paired t-test, two-tailed P-value =0.0018 for the adventitia thickness pre-LVAD compared to post-LVAD aorta.
Figure 2.
Figure 2.. CF-LVADs increase collagen and reduce elastin in a time-dependent manner.
A and B, Second harmonic generation (SHG) microscopy of human aorta sections revealing fibrillar collagen (red) and elastin (green) (scale bar = 50 μm). The boundaries of the adventitia (A) and media (M) are indicated. Quantification of collagen (C) and elastin-to-collagen ratios (D). N indicates the total number of images analyzed from 13 (2–6 months), 5 (6–12 months) and 3 (>13 months) aortas. Values represent means +SEM. P < 0.0005 by one-way ANOVA with Sidak’s multiple comparisons test.
Figure 3.
Figure 3.. Stress-strain testing reveals increased stiffness with longer duration of CF-LVAD.
A, Post-LVAD aortic tissue strips were attached to a motor and force transducer. Uniaxial extension in the circumferential direction was applied and stress-strain curves were generated. The high strain elastic modulus was calculated from these stress-strain curves. B, Stiffening of post-LVAD aortas greater with longer versus shorter duration of CV-LVAD support.
Figure 4.
Figure 4.. Aorta transcriptome remodeling in response to CF-LVAD implantation.
A, Row normalized heat map for 235 protein coding transcripts that were differentially expressed Pre- versus Post-LVAD. B, Principal component analysis (PCA) of gene expression clearly segregated Pre- versus Post-LVAD, with 61% of variance accounted for by principal component 1 (PC1, x-axis) (color indicates patient and shape indicates LVAD status). C, Ingenuity Pathway Analysis (IPA), with P-values plotted for representative associated diseases and functions, as well as predicted effect of LVAD on the activity of identified upstream regulators. D, IPA identified suppression of a network that could explain ECM accumulation Post-LVAD. Downregulation of several inflammation associated signals (TNFα, IL-1β, IL-32) was predicted to also reduce expression of specific metalloproteases (ADAMTS4, ADFAMTS9, MMP19) and lead to increased ECM protein half-life and accumulation. E, Heat map of 75 long noncoding RNAs (lncRNAs) that were differentially expressed. F, Pre- and Post-LVAD samples were separable via PCA of lncRNA differential expression. G, Expression (LogFC values) for the 10 most activated and 10 most inhibited lncRNAs.
Figure 5.
Figure 5.. MMP19 and ADAMTS4 abundance is reduced after CF-LVAD implantation.
A, Representative immunofluorescence microscopy images of MMP19 (red) and alpha smooth muscle cell actin (α-SMA, green) abundance in the aorta media Pre-LVAD (top row) and Post-LVAD (lower row). B, The abundance of MMP19 protein is reported as the mean area of positive MMP19 immunoreactivity per media area. Data points indicate the abundance of MMP19 in Pre-LVAD (black circle) and Post-LVAD (red circle) aorta. C, Representative immunofluorescence microscopy images of ADAMST4 (red) and α-SMA (green) protein abundance in the aorta media Pre- and Post-LVAD. D, The abundance of ADAMTS4 protein is shown as the mean area of ADAMTS4 immunoreactivity per media area. Each data point is the mean value from 4–5 non-overlapping high power (400x) images of aorta media per sample. Dashed lines link pairs of pre- and post-LVAD data from the same individual. Scale bar is 50 microns.
Central Illustration.
Central Illustration.. Continuous-flow LVAD-induced aortic fibrosis and stiffening.
The non-physiologic flow after continuous-flow LVAD placement leads to downregulation of extracellular matrix degrading enzyme expression in the aorta, with resulting accumulation of extracellular matrix and deposition of fibrillar collagen. This aortic fibrosis results in vascular stiffening. We posit that these changes contribute to vascular complications in continuous-flow LVAD patients. Therapeutic strategies based on restoring physiologic blood flow and/or enhancing extracellular matrix turnover are warranted to improve patient outcomes.
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