Format

Send to

Choose Destination
See comment in PubMed Commons below
Mol Vis. 2012;18:1684-95. Epub 2012 Jun 20.

Upregulation of heparanase in high-glucose-treated endothelial cells promotes endothelial cell migration and proliferation and correlates with Akt and extracellular-signal-regulated kinase phosphorylation.

Author information

  • 1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic center of Sun Yat-sen University, Guangzhou, China.

Abstract

PURPOSE:

The objectives of this study were to determine whether high-glucose-induced upregulation of heparanase (HPSE) expression and differential heparanase expression in human retinal vascular endothelial cells (HRECs) can alter HREC migration and proliferation. We also aimed to determine whether HREC migration and proliferation correlate with the levels of protein kinase B (Akt) and extracellular-signal-regulated kinase (ERK) phosphorylation and activation.

METHODS:

HRECs were treated with either 5 mM glucose (Glu5) or high (30 mM) glucose (Glu30) for 48 h. Untransfected HRECs were grown in human endothelial serum-free medium (HE-SFM) in the presence of 5 mM glucose and supplemented with 30 mM mannitol for 48 h as an osmotic control (mannitol). HRECs were also infected with a heparanase small interfering RNA recombinant lentiviral vector (HPSE-LV) or a control vector (Con-LV) at a multiplicity of infection (MOI) of 60 for three days. Then the con-LV and HPSE-LV-infected cells were treated with 30 mM glucose for 48 h (Con-LV-Glu30 and HSPE-LV-Glu30, respectively). The expression levels of heparanase mRNA and protein and HREC proliferation and migration were examined using quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, 3-(4,5-dimethylthiahiazol-2-y1)-2,5-diphenyltetrazolium bromide assay, bromodeoxyuridine histochemical staining, and the Boyden chamber assay. The expression level of paxillin was examined using immunofluorescent staining. Akt and ERK phosphorylation was evaluated using western blot analysis.

RESULTS:

We successfully transfected the HPSE RNAi lentiviral vector into HRECs and demonstrated that it can suppress the expression of the heparanase gene in these cells. Western blot and qRT-PCR analyses showed that HRECs treated with a high concentration of glucose exhibited increased heparanase protein and mRNA levels, while the levels were decreased in HRECs that had been infected with HPSE-LV before treatment with high glucose (HPSE-LV-Glu30; p<0.05). The observed increase or decrease in the levels of heparanase correlated with increased or decreased HREC migration and proliferation, respectively (p<0.05). HREC proliferation and migration were found to correlate with Akt and ERK phosphorylation levels (p<0.5).

CONCLUSIONS:

Our results indicate that heparanase plays a significant role in mediating retinal vascular endothelial cell proliferation and migration after the HRECs are exposed to high levels of glucose. Signaling inducing heparanase-stimulated HREC proliferation and migration appears to be related to the activation of Akt and ERK via their phosphorylation.

PMID:
22773906
PMCID:
PMC3388982
[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for PubMed Central
    Loading ...
    Write to the Help Desk