Background Chronically inflamed tissues as well as injured tissues are characterized by local hypoxia and – as a consequence – enhanced angiogenesis. The two transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2 regulate the cellular and metabolic responses to pathophysiologically reduced oxygen tension thereby promoting both angiogenesis and metabolic adaptation of endothelial cells with implications on the pathogenesis of rheumatoid arthritis (RA).
Objectives To knockdown either HIF-1α or HIF-2α in human microvascular endothelial cells (HMEC), respectively, in order to investigate the impact of HIF-1 and HIF-2 on the angiogenic and metabolic transcriptome under hypoxic versus normoxic conditions.
Methods Specific knockdown of either HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. Angiogenic and metabolic transcriptome of transduced HMECs (and untransduced control cells) was studied by performing gene expression studies (Agilent Human Whole Genome 60K Microarrays) under normoxic (18% O2) vs. hypoxic (1% O2) conditions. Obtained data were analyzed in silico by the classification of significantly? regulated genes (≥2-fold change, p<0.01) into angiogenic and metabolic processes using Panther database.
Results In comparison to normoxia the incubation of untransduced HMECs at 1% O2 resulted in 73 regulated angiogenesis related genes in 11 different pathways and 17 regulated cellular metabolism related genes in 9 different pathways, respectively.
In both HIF-1α and HIF-2α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but the effect was much less pronounced if compared with cells without knockdown.
Analysis of effects on angiogenesis related processes showed (such as i.e. angiogenesis, VEGF pathway, HIF activation, EGF receptor pathway) that 74% of the differentially expressed genes are controlled by both HIF-1 and HIF-2, respectively. Another 14% of the regulated genes are dependent on the presence of HIF-1, among them i.e. the genes GRB2, PDGFRB, PLD, WNT5A and MMP3. The remaining 12% of regulated genes are under control of HIF-2, among them i.e. the genes DLL3, HSP27beta2, NOTCH4, PKC and MMP1.
The differentially regulated genes encoding proteins/enzymes involved in the cellular metabolism (i.e. glycolysis, ATP synthesis, TCA cycle) were found to be to 80% controlled by both HIF-1 and HIF-2, respectively. The remaining 20% are dependent on the presence of HIF-1, among them the genes GRB2, FOXJ1, FOXQ1 and Cyt C.
Conclusions HIF-1α and HIF-2α are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. However, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. This leads us to conclude that HIF-1α affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. HIF-2α does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown).
These findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by HIF-1 and HIF-2 and are, therefore, considered to be of clinical relevance in RA.
Disclosure of Interest None Declared
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