Dynamic Hyaluronan drives liver endothelial cells towards angiogenesis

Abstract

Background: Angiogenesis, the formation of new blood vessels from pre-existing vasculature is essential in a number of physiological processes such as embryonic development, wound healing as well as pathological conditions like, tumor growth and metastasis. Hyaluronic acid (HA), a high molecular weight polysaccharide, major component of extracellular matrix is known to associate with malignant phenotypes in melanomas and various other carcinomas. Hyaluronic acid binding protein 1 (HABP1) has been previously reported to trigger enhanced cellular proliferation in human liver cancer cells upon its over-expression. In the present study, we have identified the HA mediated cellular behaviour of liver endothelial cells during angiogenesis.

Methods: Endothelial cells have been isolated from perfused liver of mice. Cell proliferation was studied using microwell plates with tetrazole dye. Cell migration was evaluated by measuring endothelial monolayer wound repair as well as through transwell migration assay. Alterations in proteins and mRNA expression were estimated by immunobloting and quantitative real time PCR using Applied Biosystems. The paraformaldehyde fixed endothelial cells were used for immuno- florescence staining and F-actin detection with conjugated antibodies. The images were captured by using Olympus florescence microscope (IX71).

Results: We observed that administration of HA enhanced cell proliferation, adhesion, tubular sprout formation as well as migration of liver endothelial cells (ECs). The effect of HA in the rearrangement of the actins confirmed HA -mediated cytoskeleton re-organization and cell migration. Further, we confirmed enhanced expression of angiogenic factors like VEGF-A and VEGFR1 in endothelial cells upon HA treatment. HA supplementation led to elevated expression of HABP1 in murine endothelial cells. It was interesting to note that, although protein levels of β- catenin remained unaltered, but translocation of this protein from membrane to nucleus was observed upon HA treatment, suggesting its role not only in vessel formation but also its involvement in angiogenesis signalling.

Conclusions: The elucidation of molecular mechanism (s) responsible for HA mediated regulation of endothelial cells and angiogenesis contributes not only to our understanding the mechanism of disease progression but also offer new avenues for therapeutic intervention.

Keywords: Angiogenesis; Hyaluronic acid or Hyaluronan; Liver endothelial cells.

Fig. 1

HA Influence the proliferation, wound healing, migration and adhesion of endothelial cells. a Cell survivability assay (MTT) was performed for untreated and HA-treated (10μg/ml) ECs to examine the effect of HA on proliferation rate. Three wells for each condition were taken for different time points. Same number of cells were seeded in triplicates for each time point along with a set of untreated cells as controls b Images of scratch assay showing the influence of HA on ECs migration to recover the wound. Scale bar, 100 μm. c Percentage of scratch covered after HA treatment were quantified after 24 and 48 h d Graph showing the number of migrated cells through the porous transwell membrane in untreated control and HA treated (10μg/ml for 48 h) ECs. e Graph showing change of adhesion property of cells with HA treatment (10μg/ml for 48 h) after 2 and 4 h in comparison to equal number of untreated control ECs. *p < 0.05; **p < 0.01, ***p < 0.001

Fig. 2

Effect of HA treatment on tubule formation of ECs. The influence of HA on vascular tubule formation of ECs are shown with DIC images of Matrigel assay. Murine ECs with HA treatment (right panel) were found with more growing vascular sprouts in comparison to untreated ECs (left panel) after 18 h (a, b) and 48 h (c, d) respectively. e The quantification of tubules (indicated with arrows) were measured per microscopic field after indicated time points. *p < 0.05; **p < 0.01

Fig. 3

HA treatment up-regulates pro angiogenic factors within ECs. a Reverse transcriptase PCR with VEGFR1, VEGFA and β- catenin primers confirmed increased mRNA level in murine ECs upon HA treatment at mentioned time points. b Immunoblot with anti-HABP1, anti- VEGFR1, anti- VEGFA and anti-β-catenin antibody showed increased VEGFR1, VEGFA and β- catenin protein levels after HA treatment of 24 and 48 h. Equal protein loading is confirmed by probing the blot with β-actin antibody

Fig. 4

Alteration in mRNA expression of angiogenic factors in ECs upon HA treatment. Real-time PCR showing the relative fold change in mRNA level of several pro-angiogenic factors like a VEGF (Vascular endothelial growth factor) and VEGFR1 (Vascular endothelial growth factor receptor 1), b HABP1 (Hyaluronic acid binding protein1) and RHAMM (Hyaluronan mediated motility receptor) c β-catenin (Catenin β1) and VECAM (Vascular cell adhesion molecule) d NF-κB1 (p50 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells) and p65 (nuclear factor NF-kappa-B p65 subunit)

Fig. 5

HA induces morphological and cytoskeletal alterations in ECs. a, d Cells were stained with Phalloidin to detect F-actin, (b, e) stained with Hoechst and (c, f) showing merged picture of phalloidin and Hoechst of untreated ECs (upper panel) vs HA treated (10μg/ml) ECs for 48 h (lower panel). The higher fluorescence intensity of phalloidin in ECs were indicated with arrows. Scale bar, 20 μm

Fig. 6

HA treatment influences localization of HABP1 and β-catenin in ECs. a Immunofluorescence staining showed cellular localization of endogenous HABP1 in mouse liver ECs, without HA treatment and with HA treatment. (i, ii) showing localization of HABP1, (iii, iv) staining with hoechst for detecting nucleus and (v, vi) merged picture of HABP1 and hoechst. b Cellular localization of endogenous β-catenin in mouse liver ECs, without HA treatment and with HA treatment. (vii, viii) showing localization of β-catenin, (ix, x) staining with hoechst for detecting nucleus and (xi, xii) merged picture of β-catenin and hoechst

Fig. 7

Schematic diagram showing influence of HA on angiogenesis in liver ECs. In the liver microenvironment the sinusoidal pericyte, hepatic stellate cells (HSCs) synthesize HA and this compound degraded by the sinusoidal ECs via the process of endocytosis [16]. Elevated HA level or administration of HA are co-ordinate with expression of HABP1 and internalization of β- catenin in ECs. HA treatment enhanced the tubular sprout formation, rearrangement of the actin cytoskeleton, cell migration and adhesion along with overexpression of angiogenic factors VEGFA and VEGFR1 in liver ECs