Fibronectin EIIIA splice variant promotes liver sinusoid repair following hepatectomy

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA) and increase expression of this isoform in response to liver injury, although its function is not well understood. Here, I examined the role of EIIIA in sinusoid repair and liver regeneration by carrying out partial hepatectomies in mice lacking EIIIA or their wild type littermates. In vitro, I studied LSEC adhesion on decellularized, EIIIA-containing matrices and investigated the role of cellular fibronectins in LSEC tubulogenesis. I found that liver regeneration was significantly delayed and that sinusoidal repair was impaired in EIIIA-/- mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, I observed that primary LSECs were more adhesive to cell-deposited matrices containing EIIIA and that cellular fibronectin enhanced LSEC tubulogenesis and vascular cord formation. The integrin α9β1 , which specifically binds EIIIA, promoted adhesion of LSECs to EIIIA and tubulogenesis. My graduate project’s findings identify a role for EIIIA in liver regeneration. The study suggests that sinusoidal repair and the ability to deliver lipids mobilized from peripheral stores as an energy source for regeneration is enhanced by increased LSEC adhesion, which is mediated by EIIIA

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy

<div><p>Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. <i>In vitro</i>, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α₉β₁, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.</p></div

EIIIA promotes expression of the vasculature marker VE-cadherin following PHx.

<p>Frozen liver sections taken at day 2 after PHx were stained for VE-cadherin (white). Livers from female and male wild type mice showed increased staining for VE-cadherin <b>(A, C)</b> compared to livers from EIIIA-cFN null mice <b>(B, D)</b>. Scale bar, 50 μm. <b>(E, F)</b> Quantification = minimum to maximum % VE cadherin positive area measurements with line at mean, EIIIA^+/+ (n = 10; 5 female, 5 male); EIIIA^-/- (n = 10; 5 female, 5 male). * p<0.05.</p

Expression of EIIIA-cFN is upregulated early after PHx.

<p><b>(A, B)</b> Wild type mice were euthanized at days 1, 2 and 5 following PHx. mRNA transcript levels for <b>(A)</b> EIIIA-cFN and <b>(B)</b> total fibronectin were measured by qRT-PCR normalized to the expression of <i>tbp</i>. n = 3–4 mice per time point, error bars are mean +/- SEM, *p < 0.05. <b>(C, D)</b> Liver tissue from wild type <b>(C)</b> control mice or <b>(D)</b> mice at day 2 after PHx was stained for EIIIA-cFN (magenta) and with DAPI (blue). Scale bar, 20 μm.</p

Expression of EIIIA-cFN is upregulated early after PHx.

<p><b>(A, B)</b> Wild type mice were euthanized at days 1, 2 and 5 following PHx. mRNA transcript levels for <b>(A)</b> EIIIA-cFN and <b>(B)</b> total fibronectin were measured by qRT-PCR normalized to the expression of <i>tbp</i>. n = 3–4 mice per time point, error bars are mean +/- SEM, *p < 0.05. <b>(C, D)</b> Liver tissue from wild type <b>(C)</b> control mice or <b>(D)</b> mice at day 2 after PHx was stained for EIIIA-cFN (magenta) and with DAPI (blue). Scale bar, 20 μm.</p

Female EIIIA-cFN null mice have decreased accumulation of intracellular lipids at day 2 after PHx.

<p>Frozen liver sections from animals at day 2 following PHx were stained with Oil Red O; lipid droplets (red), hematoxylin (blue). Oil Red O staining was decreased in female EIIIA-cFN null mice <b>(B)</b> in comparison to wild type littermates <b>(A)</b>. Staining for males was comparable between EIIIA-cFN null <b>(D)</b> and wild type littermates <b>(C). (E, F)</b> Quantification of percent Oil Red O covered area, mean +/- SD, * p<0.05. Scale bar, 50 μm. (EIIIA^+/+ = 10; 5 female, 5 male; EIIIA^-/- = 10; 4 female, 6 male).</p

Liver endothelial cells are more adhesive to cell-deposited matrices containing EIIIA.

<p><b>(A)</b> Mouse liver endothelial cells were allowed to bind to cell-deposited matrices with or without EIIIA-cFN, and with or without blocking antibodies to the integrin α₉ subunit. Graph shows number of cells adhering to either EIIIA⁺ or EIIIA^- matrix, normalized to cells adhering to EIIIA⁺ matrix. Images shown represent 3 independent experiments, mean +/- SEM. <b>(B)</b> Integrin profiling of primary liver endothelial cells. RNA was collected from primary liver endothelial cells at day 8 after isolation. qRT-PCR shows expression of EIIIA-cFN and total FN binding integrins α₄, α₅, α₉, and α_V relative to the housekeeping gene ribosomal protein S12.</p

Cellular fibronectin enhances tubulogenesis and vascular cord formation in vitro.

<p><b>(A)</b> In a tubulogenesis assay, TSECs were premixed with cellular fibronectin (cFN), plasma fibronectin (pFN), or no fibronectin (control) and cultured on Geltrex for 18 h. TSECs incubated with cellular fibronectin had enhanced tubulogenesis and aligned to form cords that were absent in the plasma fibronectin and control conditions. <b>(B)</b> Quantification shows mean tube number +/- SD, *p<0.05. Data from three independent experiments, each with three technical replicates. <b>(C)</b> TSECs were incubated with blocking antibodies to α₅, α₉, or α_V or with control IgG for 1 h, premixed with cellular fibronectin and cultured for 18 h on Geltrex. <b>(D)</b> Quantification shows mean tube number +/- SD, *p<0.05. Data from three independent experiments, each with three technical replicates. <b>(E)</b> A model for the role of EIIIA-cFN in sinusoidal repair and liver regeneration. We propose that 1) the presence of EIIIA-cFN promotes LSEC adhesion, which leads to 2) enhanced sinusoid integrity and hepatocyte lipid accumulation, which in turn results in 3) timely regeneration following PHx.</p