Abstract
BackgroundMacrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.MethodsDifferentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 μg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.ResultsHIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.ConclusionsExpression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1α protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.
Highlights
Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue
Macrophages are known to play an important role in inflammatory diseases such as rheumatoid arthritis (RA), as the rheumatoid synovium is intensively infiltrated by macrophages and their numbers correlate well with articular destruction [1] and clinical scores [2]
In this study we investigated expression of HIF-1a in macrophages with subsequent activation both in an inflammatory and hypoxic environment, and evaluated whether this activation leads to production of proangiogenic factors
Summary
Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages. It has long been recognized that synovial fluids from RA patients are hypoxic, acidotic and have low glucose and high lactate levels [3]. This is indicative of an anaerobe situation, which has been confirmed by measuring (GLUT-1), promoting angiogenesis, erythropoiesis, cell growth and migration, and a switch to a glytolytic cell metabolism [6]. Frede et al [11] reported involvement of the ERK (p44/42) MAPK pathway in differentiation of the human monocytic cell line THP-1 along with increased HIF-1 activity, while increased expression of HIF-1a correlated to differentiation was reported by others [12]
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