Background/Purpose: Hypoxia is a feature of RA synovitis. In the present study, we focused on the two transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2. These factors are known to regulate the cellular and metabolic responses to pathophysiologically reduced oxygen tension thereby promoting both angiogenesis and metabolic adaptation of endothelial cells. However, functional differences and similarities between these factors in these regards have not been investigated so far. Our aim was to knockdown either HIF-1α or HIF-2α in human microvascular endothelial cells (HMEC), respectively, in order to understand the differential 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 was studied by performing gene expression studies (Agilent Human Whole Genome 60K Microarrays) of (un-)transduced HMECs incubated under normoxia (18% O2) vs. hypoxia (1% O2) for 20h. Obtained data were analyzed by the classification of significantly regulated genes (≥2-fold change, p<0.01) into angiogenic and metabolic processes using Panther database.

Results:

In untransduced HMECs, we identified 73 angiogenesis related genes in 11 different pathways and 17 cellular metabolism related genes in 9 different pathways, respectively, which are differentially expressed under hypoxia vs. normoxia. Interestingly, 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 (VEGF pathway, HIF activation, EGFR  pathway)  showed that  74%  of  the  differentially  expressed  genes  are  controlled  by  both  HIF-1  and  HIF-2. Another 14% of the regulated genes depend on the presence of HIF-1, among them the genes GRB2, PDGFRB, PLD, WNT5A and MMP3. The remaining 12% of regulated genes are under the control of HIF-2, among them the genes DLL3, HSP27b2, 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 FOXJ1, FOXQ1 and Cyt C.

Conclusion: Both HIF-1α and HIF-2α are key regulators driving 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. In contrast, HIF-2α does influence angiogenesis more 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-hif-1-and-hif-2-during-angiogenesis-and-metabolic-adaptation-of-human-microvascular-endothelial-cells-towards-hypoxia/