Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Angiogenic Potential of HMECs Is Driven by HIF-2 Overcoming the Effects of HIF-1.
Hahne2, Martin, Luetkecosmann1, Steffi, Tran1, Cam Loan, Lohanatha1, Ferenz, Jakstadt6, Manuela, Kasper4, Grit, Duda4, Georg
Berlin-Brandenburg Center for Regenerative Medicine
Berlin-Brandenburg School for Regenerative Medicine, Germany
Charite - University Medicine, Berlin, Germany
Charite - University Medicine
Charite University Med-Berlin, Berlin, Germany
German Rheumatism Research Center
Hypoxia and angiogenesis are features of inflamed tissues. For the development of the rheumatoid pannus, subsequent synovial inflammation and joint destruction, the growth of new vessels is necessary. Inhibition of angiogenesis is one potential therapeutic approach to manage inflammatory diseases like rheumatoid arthritis (RA). In this regard the transcription factors Hypoxia inducible factor (HIF)-1a and (HIF)-2a control cellular response to decreased oxygen tension thereby promoting angiogenesis. Although HIF-1a and HIF-2a share structural similarities, they are supposed to have distinct transcriptional targets with implications on the pathogenesis of RA.
We focused on the effects of HIF-2a in the process of angiogenesis. Therefore, we developed a stable human microvascular endothelial cells (HMEC) lentiviral based knockdown system for HIF-2a. This model allowed us to analyze the ability of HMECs to perform angiogenesis under hypoxia in the absence of HIF-2a.
Specific knockdown of HIF-2a was achieved using lentiviral-based shRNA technology. Absence of HIF-2a and presence of HIF-1a was proved on transcriptional level by realtime RT-PCR as well as on translational level via Western blot. Angiogenic potential of HMECs was studied using an angiogenesis assay by investigating both tubuli and node formation under hypoxia (<1% O2). Furthermore, realtime RT-PCR was carried out for expression analysis of hypoxia driven genes HIF1A, HIF2A, GAPDH, PGK, GLUT1, LDHA and angiogenesis related genes VEGFA and IL8.
Evidencing the successful knockdown of HIF-2a, the gene expression levels of HIF2A were reduced by 69% under normoxia (scr vs. HIF2shRNA; p=0.0045) with the same reductive effect of 69% under hypoxia. Moreover, strongly reduced HIF-2a protein levels were detected by Western blot. HIF-1a levels were not affected by HIF-2a knockdown. Targeting HIF-2a led to a significantly decreased node formation by factor 2 under normoxia (Nox) and factor 4.5 under hypoxic (Hox) incubation (Nox scr vs. Nox HIF2shRNA, p=0.0255 and Hox scr vs. Hox HIF2shRNA, p=0.0002). Similar effects were observed at tubuli formation with a reduction by 70% in HIF-2a targeted cells (Nox scr vs. Nox HIF2shRNA, p=0.0128 and Hox scr vs. Hox HIF2shRNA, p=0.0053). Furthermore, targeting of HIF-2a gave rise to reduced levels of VEGFA and IL8 expression, whereas the expression of metabolic relevant genes GAPDH, PGK, GLUT1, LDHA were not affected by HIF-2a knockdown under hypoxia.
Our findings show for the first time in a functional assay HIF-2a (i) to be responsible for the angiogenic potential of endothelial cells and (ii) to overcome HIF-1a's impact on angiogenesis. These findings provide new insights into basic principles of angiogenesis in inflamed tissues and therefore could be of clinical importance.
To cite this abstract, please use the following information:
Hahne, Martin, Luetkecosmann, Steffi, Tran, Cam Loan, Lohanatha, Ferenz, Jakstadt, Manuela, Kasper, Grit, et al; Angiogenic Potential of HMECs Is Driven by HIF-2 Overcoming the Effects of HIF-1. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1418