Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury

Abstract

Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins by myofibroblasts derived from hepatic stellate cells and portal fibroblasts. Activation of these precursors to myofibroblasts requires matrix stiffness, which results in part from increased collagen cross-linking mediated by lysyl oxidase (LOX) family proteins. The aims of this study were to characterize the mechanical changes of early fibrosis, to identify the cells responsible for LOX production in early injury, and to determine which cells in normal liver produce collagens and elastins, which serve as substrates for LOXs early after injury. Hepatocytes and liver nonparenchymal cells were isolated from normal and early-injured liver and examined immediately for expression of LOXs and matrix proteins. We found that stellate cells and portal fibroblasts were the major cellular sources of fibrillar collagens and LOXs in normal liver and early after injury (1 day after bile duct ligation and 2 and 7 days after CCl(4) injury). Activity assays using stellate cells and portal fibroblasts in culture demonstrated significant increases in LOX family enzymatic activity as cells became myofibroblastic. LOX family-mediated deoxypyridinoline and pyridinoline cross-links increased after CCl(4)-mediated injury. There was a significant association between liver stiffness (as quantified by the shear storage modulus G') and deoxypyridinoline levels; increased deoxypyridinoline levels were also coincident with significantly increased elastic resistance to large strain deformations, consistent with increased cross-linking of the extracellular matrix. These data suggest a model in which the liver is primed to respond quickly to injury, activating potential mechanical feed-forward mechanisms.

Fig. 1.

Mechanical properties of the liver change in early fibrosis. Livers from normal and CCl₄-intoxicated rats at days 7, 10, and 14 were analyzed by shear rheometry, with shear storage and loss moduli G′ and G″, respectively, determined over a range of strains. A: liver stiffness (G′) was increased by day 7. Livers demonstrated marked softening with increased strain. For G′ at minimal strain, all values are significantly different from normal: P < 0.01 at day 7, P < 0.001 at day 10, and P < 0.001 at day 14. B: data in A normalized to 1 at low (1%) strain to demonstrate different shapes of strain curves. For G′ at maximum strain, P < 0.01 for normal vs. day 7 and normal vs. day 14. C: increased elasticity of livers after CCl₄, as manifested by differences in curves of G″/G′ vs. strain (normalized to 1 at starting strain); n = 3–6 for each time point. *P < 0.05; **P < 0.01; ***P < 0.001 vs. normal.

Fig. 2.

Mechanical properties of edematous liver. Livers were removed from rats 1 or 2 days after CCl₄ injection, when edema is pronounced. A: liver stiffness (G′) is increased compared with normal, although strain softening is unchanged. By 1-way ANOVA there is no difference between the 3 curves. At low strain, days 1 and 2 are significantly different from normal (P < 0.05). B: G″/G′ vs. strain demonstrates that, despite increased stiffness, edematous livers can be distinguished from livers with early fibrosis (day 14). Normalized curves are shown; n = 3–5 for each time point. Similar results were obtained with day 1 livers (not shown). For G″/G′ at 40% strain, normal vs. day 2, no significant difference; normal vs. day 14, significant at strains >30%. *P < 0.05; **P < 0.01.

Fig. 3.

Lysyl oxidase (LOX)-mediated cross-links increase early after CCl₄-mediated injury. A and B: mature LOX-mediated collagen cross-links [pyridinoline and deoxypyridinoline (D-pyridinoline)] in livers from rats with CCl₄-induced fibrosis and normal, noninjected controls. Deoxypyridinoline cross-links were increased as early as 10 days. Values are means ± SD; n = 3–6 per time point. *P < 0.05; **P < 0.01. C and D: correlation between whole liver shear modulus (G′) and pyridinoline and deoxypyridinoline.

Fig. 4.

Liver stiffness and LOX family expression increase rapidly after bile duct ligation (BDL). Rats underwent BDL or sham surgery, and livers were removed at days 0–14. A: G′ was measured by rheometry. B: α-smooth muscle actin (α-SMA) expression was assessed by immunostaining. *P < 0.05; **P < 0.01; ***P < 0.005 vs. sham. C and D: expression of LOXs and collagens in liver lysates determined by real-time PCR; n = 5–6 for all data points. For each individual experiment, values were normalized to 18S expression; values shown in graphs were normalized to those for samples from sham-operated animals from the same time point. *P < 0.05; ***P < 0.005 vs. 6 h after surgery.

Fig. 5.

LOX is significantly upregulated in hepatic stellate cells immediately after injury. Primary hepatocytes (Hep), Kupffer cells (KC), hepatic stellate cells (HSC), portal fibroblasts (PF), and sinusoidal endothelial cells (SEC) were isolated from livers of healthy rats, 2 days after CCl₄ injection, and 1 day after BDL. Cells were lysed for mRNA isolation immediately, and real-time PCR was carried out to determine expression of all 5 LOX family members: LOX, LOXL1, LOXL2, LOXL3, and LOXL4. All are expressed; however, hepatic stellate cells and, to a lesser extent, portal fibroblasts are the major producers of LOXs in normal liver and shortly after injury. *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 6.

mRNA expression of LOX, but not other family LOX members, is upregulated in hepatic stellate cells and portal fibroblasts after injury. Cells were isolated from rat livers 7 days after initiation of CCl₄ treatment (after a total of 2 injections, on days 1 and 4) and analyzed by real-time PCR for LOX family expression. Only LOX was significantly increased at this time point. *P < 0.05; **P < 0.01.

Fig. 7.

Hepatic stellate cells and portal fibroblasts increase secretion of active LOX family members during myofibroblastic differentiation. Primary cells were placed in culture for up to 7 days. Conditioned media (CCM) were examined by a LOX family activity assay at 1 and 7 days after isolation. *P < 0.05.

Fig. 8.

Hepatic stellate cells and portal fibroblasts produce collagens and elastin in normal and acutely injured liver. mRNA isolated as described in Fig. 5 legend was analyzed by real-time PCR to determine expression of collagen chains (A–C) or elastin (D). Data represent cells from ≥3 independent isolations per cell type, with 3 technical repeats per isolation. **P < 0.01; ***P < 0.005.

Fig. 9.

Hepatic stellate cells and portal fibroblasts from normal liver express α-SMA. mRNA was prepared as described in Fig. 5 legend and analyzed by real-time PCR for expression of α-SMA. *P < 0.05.