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. 2007 Jun;170(6):1942-53.
doi: 10.2353/ajpath.2007.060887.

Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells

Erica Novo  1 , Stefania Cannito, Elena Zamara, Lorenzo Valfrè di Bonzo, Alessandra Caligiuri, Carlo Cravanzola, Alessandra Compagnone, Sebastiano Colombatto, Fabio Marra, Massimo Pinzani, Maurizio Parola
Affiliations

Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells

Erica Novo et al. Am J Pathol. 2007 Jun.

Abstract

Pathological angiogenesis is associated with the fibrogenic progression of chronic liver diseases. Experimental data suggest that hypoxia and vascular endothelial growth factor (VEGF) may stimulate proliferation and synthesis of type I collagen in activated, myofibroblast-like rat hepatic stellate cells (HSC/MFs). In this study, we investigated whether hypoxia, recombinant VEGF, or angiopoietin 1 (Ang-1) may affect other crucial profibrogenic features. In human HSC/MFs, which constitutively express VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2) and the Ang-1 receptor Tie-2, exposure to hypoxia, VEGF, or Ang-1 resulted in a Ras/Erk-dependent stimulation of chemokinesis and chemotaxis. Migration of human HSC/MFs under hypoxic conditions involved up-regulation of VEGF-A, Ang-1, and related receptors and was mainly dependent on VEGFR-2 (Flk-1). In specimens from either cirrhotic rat livers or from patients with hepatitis C virus-related cirrhosis, HSC/MFs expressed proangiogenic factors and related receptors in areas of active fibrogenesis (ie, at the leading or lateral edge of developing incomplete fibrotic septa). Data presented herein suggest that VEGF and Ang-1 may contribute to fibrogenesis by acting as hypoxia-inducible, autocrine, and paracrine factors able to recruit myofibroblast-like cells. Moreover, HSC/MFs, in addition to their established profibrogenic role, may also contribute to neoangiogenesis during chronic hepatic wound healing.

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Figures

Figure 1
Figure 1
Expression of VEGF and Ang-1 receptors in human HSC/MFs under normoxic or hypoxic conditions. A: Constitutive expression of Flt-1, Flk-1, and Tie-2 as shown by indirect immunofluorescence staining. B: Western blot analysis of kinetics of expression of the three receptors under normoxia (C) or under hypoxic conditions (HYP). Data from a typical experiment (of three performed) are expressed in arbitrary units as densitometric analysis of bands observed at the different time points as compared with respective bands observed in control (normoxic) cell lysates at time 0. Insets provide a representative image of blots comparing bands for the three antigens obtained in control or hypoxic condition at 24 hours (Flt-1), 6 hours (Flk-1), or 16 hours (Tie-2). Molecular masses of receptors are provided in the insets. Sample loading was evaluated by reblotting the same membranes with antibodies raised against β-actin. Original magnifications, ×1000.
Figure 2
Figure 2
VEGF and Ang-1 stimulate nonoriented migration and chemotaxis of human HSC/MFs. Nonoriented migration was assessed by means of WHA (A, B), whereas chemotaxis was assessed by means of Boyden’s chamber (C). WHA was performed on cells seeded on 24-well plates coated with collagen type I, grown to confluence in complete medium, and then incubated for an additional 24 hours in serum-free medium. An artificial lesion was generated in the cell layer to remove a linear area of cells, and then cultured cells were allowed 18 hours to migrate in the absence (control) or in the presence of increasing concentrations of human recombinant VEGF or Ang-1 or in the presence of human recombinant PDGF-BB (10 ng/ml) used as a positive control. Chemotaxis assay (C) was performed in trypsinized 24-hour-starved cells placed in Boyden’s chambers and then exposed to VEGF or Ang-1 (both used at 100 ng/ml) or to PDGF-BB (10 ng/ml) used as positive control. Data in bar graphs (A, C) represent mean ± SEM (n = 5, in triplicate) and are expressed as number of cells migrated in the artificial lesion (WHA, A) or in the filter (C), respectively. *P < 0.05 and **P < 0.01 versus control values. Representative images of nonoriented migration of human HSC/MFs under the different experimental conditions indicated are provided in B. Original magnifications, ×100.
Figure 3
Figure 3
VEGF- and Ang-1-dependent migration of human HSC/MFs involves activation of Ras/Erk signaling and, for VEGF action, Flk-1 receptor. For evaluation of Ras/Erk signaling (A), confluent and 24-hour-starved HSC/MFs were incubated for 15 minutes in the presence of increasing concentrations of human recombinant VEGF or Ang-1 (range, 1 to 100 ng/ml) or in the presence of human recombinant PDGF-BB (10 ng/ml) used as positive control. Cell lysates, processed as described in Materials and Methods, were used in Western blot analysis to detect the state of phosphorylation of extracellular regulated kinase (Erk1/2, p42, and p44; A) or c-Akt (C) by using specific antibodies directed against the phosphorylated form of Erk. Sample loading was evaluated by reblotting the same membranes with antibodies raised against Erk or c-Akt. Representative blots are shown of three independent experiments. To characterize nonoriented migration, WHA (B, D) was performed as described in the legend of Figure 2. Basal conditions were cells not exposed (C) or exposed to human recombinant VEGF (100 ng/ml), Ang-1 (100 ng/ml), PlGF (100 ng/ml), or PDGF-BB (10 ng/ml). When required, cells to be treated with VEGF or Ang-1 were pretreated for 30 minutes with the following agents: 30 μmol/L PD98095 (PD); 2 μmol/L of the TyrK inhibitor SU1498; 0.045 mg/ml (final dilution) of monoclonal neutralizing antibodies against Flk-1 that, although originally raised against mouse epitope were found to also cross-react with human Flk-1, as confirmed by the block of Erk1/2 phosphorylation in human HSC/MFs treated with VEGF (data not shown). Data in bar graphs (B, D) represent mean ± SEM (n = 4, in triplicate) and are expressed as number of cells migrated in the artificial lesion. *P < 0.05 and **P < 0.01 versus control values. #P < 0.01 versus values in cells stimulated with VEGF or Ang-1.
Figure 4
Figure 4
VEGF- and Ang-1-dependent chemotaxis of human HSC/MFs is prevented by inhibitors of TyrKs and of Ras/Erk signaling and, for VEGF action, involves Flk-1 receptor. Chemotaxis experiments were performed as described in the legend of Figure 2. Basal and experimental conditions were identical to those described in the legend of Figure 3. Data in bar graphs (A, B) represent mean ± SEM (n = 4, in triplicate) and are expressed as number of cells migrated in the artificial lesion. **P < 0.01 versus control values. #P < 0.01 versus values in cells stimulated with VEGF or Ang-1.
Figure 5
Figure 5
Hypoxia as well as hypoxic-conditioned medium stimulate nonoriented migration and chemotaxis of human HSC/MFs. WHA (A) was performed as reported in the legends of Figures 2 and 4 in both normoxic (control and PDGF, 24 hours) and hypoxic conditions (16 hours or 24 hours, HYP16 and HYP24). Human HSC/MFs in normoxic conditions behaved as usual: a low number of control cells was found to invade the artificial lesion after a 24-hour incubation, whereas a massive invasion was detected after exposure to PDGF-BB. Western blot analysis of activation of Ras/Erk signaling (B) was performed as described in the legend of Figure 2 using cell lysates obtained after 16 or 24 hours from either cells incubated in normoxic (control) or hypoxic conditions. Wound healing (C) and chemotaxis (D) assays were also performed using normoxic cells exposed to hypoxic-conditioned medium of human HSC/MFs exposed to hypoxia for 16 or 24 hours as well as to PDGF-BB (10 ng/ml) used as positive control. Data in bar graphs (A, C, and D) represent mean ± SEM (n = 4, in triplicate) and are expressed as number of cells migrated in the artificial lesion or in the filter of Boyden’s chambers. *P < 0.05 and **P < 0.01 versus control values.
Figure 6
Figure 6
Migration in hypoxic conditions or stimulated by hypoxic conditioned medium involves Flk-1 receptor. Wound healing (A) and chemotaxis (B) assays were performed as described in the legend of Figure 2. When required, cells designed to be exposed at experimental time 0 to hypoxic medium collected after 16 hours were pretreated for 30 minutes with the following agents: 30 μmol/L PD98095 (PD); 2 μmol/L of the TyrK inhibitor SU1498; 0.045 mg/ml (final dilution) of monoclonal neutralizing antibodies against Flk-1. Data in bar graphs represent mean ± SEM (n = 4, in triplicate) and are expressed as number of cells migrated in the artificial lesion or found in the filter of Boyden’s chambers. *P < 0.05 and **P < 0.01 versus control values; #P < 0.05 and ##P < 0.01 versus values obtained in the presence of hypoxia-conditioned medium.
Figure 7
Figure 7
In vivo localization of HSC, proangiogenic cytokines, and related receptors in normal liver (A–D). Immunofluorescence was performed on liver cryostat sections from the liver of control (ie, untreated) rats at 9 weeks in the CCl4-dependent chronic protocol for fibrosis induction. All panels include the following: 1) a larger image on the left side offering electronic merging of images acquired for single fluorescence (blue fluorescence, DAPI staining; green fluorescence, desmin staining; red fluorescence, growth factor or receptor under analysis: A: Flk-1; B: Tie-2; C: VEGF; D: Ang-1); and 2) a higher digital magnification (right) of the boxed area. Hn, hepatocyte nucleus; EC, endothelial cell. Original magnifications, ×400.
Figure 8
Figure 8
In vivo localization of HSC/MFs, proangiogenic cytokines, and related receptors in fibrotic rat livers. Immunofluorescence was performed on liver cryostat sections from the liver of chronically injured rats obtained at 9 weeks in the CCl4-dependent chronic protocol for fibrosis induction. A: Reported images (green fluorescence) of immunostaining for VEGF (a1 to a4) or α-SMA (a5) in the presence or absence of concomitant nuclear stain (blue fluorescence, DAPI staining). In B, C, E, F, and G are reported the following: 1) tiny images representing image acquisition of single fluorescence always identifying in the same section nuclei (1, blue fluorescence, DAPI staining), α-SMA (2, green fluorescence), or the growth factor or receptor under analysis (3, red fluorescence; B: Flk-1; C: Ang-1; E: VEGF; F: Flk-1; G: Tie-2); 2) a larger image offering combined immunofluorescence (4, electronic merging of images); and 3) a higher digital magnification of the boxed area (5, when present). In D are reported the following: tiny images representing (see before) in the same section nuclei (1), CD-31 (2, green fluorescence), and Flk-1 (3, red fluorescence); and a larger image (4, electronic merging of images) offering combined immunofluorescence. Areas of co-localization between α-SMA and the antigen under analysis (yellow/orange fluorescence) are indicated by arrows. Hn, hepatocyte nucleus; EC, endothelial cell. Original magnifications: ×200 (Aa1–Aa3); ×400 (Aa4, Aa5, B, C, F); ×1000 (D, E, G).
Figure 9
Figure 9
In vivo localization of HSC/MFs, proangiogenic cytokines and related receptors in cirrhotic human livers from chronic HCV patients (A–F). Immunofluorescence was performed on cryostat sections from the liver of HCV patients scored METAVIR F4. In A–F are reported the following: tiny images representing image acquisition of single fluorescence always identifying in the same section nuclei (1, blue fluorescence, DAPI staining), α-SMA (2, green fluorescence), or the growth factor or receptor under analysis (3, red fluorescence; proangiogenic factors identified: A, D: Tie-2; B: Flk-1; C, E: Ang-1; F: VEGF); and a larger image offering combined immunofluorescence (4, electronic merging of images). Areas of co-localization between α-SMA and the antigen under analysis (yellow/orange fluorescence) are indicated by arrows. HSC/MFs, activated hepatic stellate cells; Hn, hepatocyte nucleus; EC, endothelial cell. Original magnifications: ×400 (C, F); ×1000 (A, B, D, E).
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