Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Osei-Tutu, K.
RNA expression analysis of selected components of sphingolipid metabolism in primary porcine microvascular brain endothelial cells
Humanmedizin; [ Diplomarbeit ] Medical University Graz; 2021. pp. 48
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Bernhart Eva Maria
- Sattler Wolfgang
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- Abstract:
- Aims of the study: This study aimed to identify selected gene products of sphingolipid (SL) metabolism in primary porcine brain microvascular endothelial cells (pBMVEC) – the morphological basis of the blood-brain barrier (BBB). SLs maintain BBB function by structural and signaling input. In a first step, I tested primers for enzymes involved in SL turnover in subsequent conventional RT-PCR and RT-qPCR reactions. I also tested whether 2-chlorohexadecanal (2-ClHDA) – a myeloperoxidase-derived chlorinated fatty aldehyde – regulates the expression of these genes. Background: The neurovascular unit (NVU) separates the brain from peripheral circulation and regulates the neuronal microenvironment. The cerebral microvessels are sensitive to oxidative stress (i.e., aberrant redox control). Oxidative processes can alter the integrity of the NVU and are implicated in the pathophysiological sequelae of neurodegenerative diseases. The phagocyte heme peroxidase, myeloperoxidase (MPO) converts H2O2 into the strong oxidant hypochlorous acid (HOCl) in the presence of chloride ions (Cl-). HOCl can attack and modify macromolecules generating a series of chlorinated products that can inflict tissue/organ damage on the host. Methods: RNA isolated from pBMVEC was reverse transcribed into complementary DNA of enzymes involved in SL metabolism. The cDNA was used in PCR and real time quantitative PCR (qPCR) analyses and in a first set of experiments primer efficiency was analyzed. I then went on to assess whether 2-ClHDA regulates the transcription of these genes. Cell cultures of pBMVEC were incubated in the absence or presence of 2-ClHDA and changes in gene expression were analyzed. Results: PCR conditions for the following set of genes involved in SL synthesis/degradation were established: serine palmitoyltransferase 1, serine palmitoyltransferase 2, ceramide synthase 5, ceramide synthase 6, sphingomyelin synthase 2, sphingosine kinase 2, alkaline ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3, neutral ceramidase2, sphingomyelin phosphodiesterase, sphingosine-1-phosphate phosphatase 1, and sphingosine-1-phosphate lyase 1. In response to 2-ClHDA, expression of CerS1, CerS6, SPHK2, and SGPL1 were upregulated. Conclusion: The results obtained during the present pilot study demonstrate the expression of genes known to be involved in SL homeostasis. In addition, my data suggest that MPO-derived 2-ClHDA has the potential to interfere with SL biosynthetic pathways in pBMVEC.