Leukotrienes and leukotriene receptors : potential roles in cardiovascular diseases

Leukotrienes (LTs) are a family of lipid mediators that are involved in host defense and inflammatory responses. They are synthesized from arachidonic acid through the 5-lipoxygenase (5-LO) pathway, and are divided into two classes, the classical chemoattractant leukotriene B4 (LTB4), and spasmogenic cysteinyl leukotrienes (cys-Us) including LTC4, LTD4 and LTE4. Us exert their actions through stimulation of specific G-protein coupled receptors, and so far two receptors for LTB4, namely BLT1, and BLT2, and two receptors for cys-LTs, namely CysLT1 and CysLT2, have been identified. The present thesis is aimed at investigating the expression and regulation profiles of LT biosynthesis enzymes and receptors in the vascular system in order to define their potential roles in the pathogenesis of cardiovascular diseases.

The LT receptor expression was studied in human umbilical vein endothelial cells (HUVECs). We found that HUVECs abundantly express CysLT2 receptor mRNA in vast excess (>4000-fold) of CysLT1 mRNA. Challenge of HUVECs with BAY u9773, a specific CysLT2 agonist, triggered diagnostic Ca2+ transients. LTC4 and LTD4 are equipment agonists, and their actions can be blocked by the dual-receptor antagonist BAY u9773, but not by the CysLT1 - selective antagonist MK571. These data identify CysLT2 as the predominant functional receptor for cys-LTs in endothelial cells. In addition, Lipopolysaccaride (LPS), turner necrosis factor-á (TNF-á), or interleukin-1â (IL-1â) caused a rapid and partially reversible suppression of CysLT2 mRNA. With regard to BLT receptors, we found that both BLT1 and BLT2 are expressed at low levels in an apparently silent state in HUVECs. However, treatment with LPS leads to a Fivefold increase in the levels of BLT mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT protein. TNF-á increases the expression of BLT2 mRNA approximately six times above basal levels with only a modest increase in BLT, mRNA. IL-1â causes variable and parallel increases of both BLT, and BLT2 mRNA The natural ligand LTB4 also increases BLT1, but not BLT2 mRNA and protein expression. Along with the induction of BLT, and/or BLT2, HUVECs acquire the capacity to respond to LTB4 with increased levels of intracellular calcium, and isotype selective BLT antagonists, CP-105696 and LY-255283, can block these signals. In addition, treatment of HUVECs with LTB4 causes increased release of nitrite, presumably reflecting nitric oxide, and monocyte chemoattractant protein-1. These data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines and ligand, which in rum may have functional consequences during the early vascular responses to inflammation.

The expression of LT pathway proteins was assessed in human carotid atherosclerotic plaques. We found that mRNA levels for the three key proteins in LTB4 biosynthesis, namely 5-LO, 5-LO activating protein (FLAP), and LTA4 hydrolase (LTA4H), are significantly increased in human atherosclerotic plaques compared with healthy controls. Immunohistochemical staining revealed abundant expression of 5-LO, FLAP, and LTA4H protein colocalizing in macrophages of intimal lesions. Human lesion tissue converts arachidonic acid into significant amounts of LTB4, and a selective, tight-binding LTA4H inhibitor can block this activity. Furthermore, expression of 5-LO and LTA4H is increased in patients with recent or ongoing symptoms of plaque instability. The abundant expression of LTA4H and correlation with plaque instability identify LTA4H as a potential target for pharmacological intervention in treatment of human atherosclerosis.

Human cytomegalovirus (HCMV) has been detected in the tissues of cardiovascular diseases such as atherosclerosis and re-stenosis. However, a cause-effect relationship has yet to be determined. We found in our study that in human vascular smooth muscle cells (VSMCs), HCMV infection induces an up to 170 fold upregulation of 5-LO mRNA in a dose- and time- dependent manner. Immunohistochemistry studies demonstrate profound expression of 5-LO protein in HCMV infected cells, which renders these cells the capacity to synthesize LTB4. Moreover, we found that double positive cells for HCMV and 5-LO can readily be detected in VSMCs in tissue samples obtained from patients with inflammatory bowel disease or AIDS suffering from acute HCMV infection. These data indicate that in HCMV infected tissues, pathogen-induced LT production may play an important role in local inflammation. These observations uncover a novel, viral-induced, process that can trigger LT biosynthesis in non-myeloid cells and offers a molecular mechanism underlying HCMV-induced pathogenesis in inflammatory diseases including atherosclerosis.

Put together, the findings in the present thesis have revealed the potential involvement of LTs in pathophysiological conditions in the vascular system, and might have profound implications for the development of new therapeutic strategies that could reduce the devastating impact of cardiovascular diseases.

List of scientific papers

I. Sjostrom M, Johansson AS, Schroder O, Qiu H, Palmblad J, Haeggstrom JZ. (2003). "Dominant expression of the CysLT2 receptor accounts for calcium signaling by cysteinyl leukotrienes in human umbilical vein endothelial cells." Arterioscler Thromb Vasc Biol 23(8): e37-41
https://pubmed.ncbi.nlm.nih.gov/12816881

II. Qiu H, Johansson AS, Sjostrom M, Wan M, Schroder O, Palmblad J, Haeggstrom JZ. (2006). "Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4." Proc Natl Acad Sci U S A 103(18): 6913-8
https://pubmed.ncbi.nlm.nih.gov/16624877

III. Qiu H, Gabrielsen A, Agardh HE, Wan M, Wetterholm A, Wong CH, Hedin U, Swedenborg J, Hansson GK, Samuelsson B, Paulsson-Berne G, Haeggstrom JZ. (1970). "Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability." Proc Natl Acad Sci U S A 103(21): 8161-6
https://pubmed.ncbi.nlm.nih.gov/16698924

IV. Qiu H, Strååt K, Rahbar A, Wan M, Söderberg-Nauclér C, Haeggström JZ. (2006). "Human cytomegalovirus infection induces 5-lipoxygenase expression and leukotriene B4 production in vascular smooth muscle cells." (Manuscript)