J. Prüfer

Pharmacological relevance and potential of sphingosine 1-phosphate in the vascular system.

Sphingosine‐1‐phosphate (S1P) was identified as a crucial molecule for regulating immune responses, inflammatory processes as well as influencing the cardiovascular system. S1P mediates differentiation, proliferation and migration during vascular development and homoeostasis. S1P is a naturally occurring lipid metabolite and is present in human blood in nanomolar concentrations. S1P is not only involved in physiological but also in pathophysiological processes. Therefore, this complex signalling system is potentially interesting for pharmacological intervention. Modulation of the system might influence inflammatory, angiogenic or vasoregulatory processes. S1P activates G‐protein coupled receptors, namely S1P1–5, whereas only S1P1–3 is present in vascular cells. S1P can also act as an intracellular signalling molecule. This review highlights the pharmacological potential of S1P signalling in the vascular system by giving an overview of S1P‐mediated processes in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). After a short summary of S1P metabolism and signalling pathways, the role of S1P in EC and VSMC proliferation and migration, the cause of relaxation and constriction of arterial blood vessels, the protective functions on endothelial apoptosis, as well as the regulatory function in leukocyte adhesion and inflammatory responses are summarized. This is followed by a detailed description of currently known pharmacological agonists and antagonists as new tools for mediating S1P signalling in the vasculature. The variety of effects influenced by S1P provides plenty of therapeutic targets currently under investigation for potential pharmacological intervention.

Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification.

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up(4)A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up(4)A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up(4)A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up(4)A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up(4)A-mediated process indicated that P2Y(2/6) receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up(4)A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.

The endothelium-derived contracting factor uridine adenosine tetraphosphate induces P2Y2-mediated pro-inflammatory signaling by monocyte chemoattractant protein-1 formation

It is very well established that purinergic signaling plays a relevant role in vascular physiology and pathophysiology. Recently, a new purinoceptor agonist uridine adenosine tetraphosphate (Up4A) has been identified as a highly potent endothelial-derived contracting factor (EDCF). The purpose of the study was to investigate Up4As influence on pro-inflammatory mechanisms. An early component of the inflammatory response in atherogenesis is the oxidative stress-induced formation of monocyte chemoattractant protein-1 (MCP-1). Here, we investigated the influence of Up4A on MCP-1 formation and characterized the underlying signaling transduction mechanisms in rat vascular smooth muscle cells (VSMCs). Up4A induced MCP-1 expression and secretion in VSMCs via the activation of P2Y2 in a concentration-dependent manner. MCP-1 formation depends on generation of reactive oxygen species (ROS). To determine whether the predominant source of ROS in the vasculature, the NAD(P)H oxidase (Nox), is involved, we used different approaches. The ROS scavenger, tiron, the Nox inhibitor, apocynin and diphenyl-iodonium, as well as Nox1 knockdown, diminished the Up4A-induced MCP-1 formation. Rac1 activation and p47phox translocation from cytosol to the plasma membrane—both required for assembling and activation of Nox, were stimulated by Up4A. ERK1/2 and p38 activation is essential for the intracellular signal transduction. In summary, Up4A induced Nox1-dependent ROS generation, which further stimulated MCP-1 formation via MAPK phosphorylation in VSMCs. This process requires the activation of the G-protein coupled receptor P2Y2. Therefore, Up4A is not only a potent EDCF but also a potent inductor of pro-inflammatory response in the vascular wall.

Harmful effects of the azathioprine metabolite 6-mercaptopurine in vascular cells: induction of mineralization.

Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells in vitro. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in in vitro and ex vivo models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.

MYCOPHENOLIC ACID DIMINISH THE DEXAMETHASONE-INDUCED TRANSFORMATION OF VASCULAR SMOOTH MUSCLE CELLS IN OSTEOBLAST-LIKE CELLS: 3A.07

Objective: Arteriosclerosis is a major problem in advanced chronic kidney disease (CKD) as well as in kidney transplanted patients. Vascular calcification has long been considered to be a passive process; however recent evidence indicates actively inducing changes in vascular smooth muscle cell behavior toward an osteoblast-like phenotype. Mycophenolic acid (MPA) is a non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) that exerts cytostatic effects on proliferating T-lymphocytes as well as other immunmodulating effects. In this study the non-immunmodulating effects of MPA should be investigated by proving anti-arteriosclerotic properties of this substance. Methods: In vitro calcification in rat vascular smooth muscle cells (VSMC) were induced with calcification medium (DMEM containing 4.5 g/L glucose supplemented with 15% FCS, 10 mmol/L sodium pyruvat, 50 μg/mL Vitamin C, and 10 mmol/L ß-glycero phosphate) and dexamethasone [100 nmol/L]. Calcium deposition was monitored by Alizarin staining and quantified by O-cresolphthalein complexone method. Cbfa1 gene expression was measured by real-time PCR. Results: Calcification medium (CM) with dexamethasone [100 nmol/L] induced mineralization of VSMC visualized by Alizarin Red staining quantified by measuring the extracellular calcium content. In the presence of MPA [10 μmol/L] this mineralization process could be significantly diminished (28 days: 0.86 ± 0.2 (mg/dL)/μg protein vs. 0.22 ± 0.1 (mg/dL)/μg protein). For the transformation of VSMC to a osteoblast-like phenotype which is essential for mineralization, the transcription factor core binding factor alpha 1 (cbfa1) is crucial. Therefore, cbfa1 expression was measured. The dexamethasone [100 nmol/L]-induced expression of the cbfa1 is significantly and dose-dependently decreased in co-stimulation with MPA [1–100 μmol/L] with a maximum decrease at 50 μmmol/L (23 ± 8% decrease). Conclusions: Here, we could show that the immunosuppressant agent MPA has other, non-immunomodulating properties by diminishing transformation of VSMCs to osteoblast-like cells. Therefore, MPA might have influence on arteriosclerotic alterations in the vascular wall which might have benefit for cardiovascular outcome after renal transplantation.