Pieter-Jan Guns

Knockout Mice Reveal a Role for P2Y6 Receptor in Macrophages, Endothelial Cells, and Vascular Smooth Muscle Cells

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y6 receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y6-null mice by gene targeting. The P2Y6 knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y6 is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-α, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y6 KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y6 mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y6-null mice. In conclusion, we generated P2Y6-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta.

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′‐[γ‐thio] triphosphate (ATPγS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4‐deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53±0.07) was compatible with literature, but the pKb for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non‐P2Y2 receptor subtype(s). Further, pyridoxal‐phosphate‐6‐azophenyl‐2′‐4′‐disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2′‐deoxy‐N6‐methyladenosine3′,5′‐bisphosphate (MRS2179) inhibited ADP‐evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP‐evoked vasodilatation.

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium‐dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2‐deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2‐deficient and wild‐type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2‐deficient mice showed impaired ATP‐ and adenosine 5′[γ‐thio] triphosphate (ATPγS)‐evoked relaxation, suggesting that in WT mice, ATP and ATPγS activate predominantly the P2Y2 subtype. The ATP/ATPγS‐evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2′‐deoxy‐N6‐methyladenosine 3′,5′‐bisphosphate (MRS2179), a P2Y1‐selective antagonist. In contrast to ATP, the UTP‐evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype. In conclusion, we demonstrated that ATP‐evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP‐evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.

P2Y receptors and atherosclerosis in apolipoprotein E‐deficient mice

Background and purpose:  P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Diffusion kurtosis imaging to detect amyloidosis in an APP/PS1 mouse model for Alzheimer's disease

Amyloid deposition in the brain is considered an initial event in the progression of Alzheimers disease. We hypothesized that the presence of amyloid plaques in the brain of APP/presenilin 1 mice leads to higher diffusion kurtosis measures due to increased microstructural complexity. As such, our purpose was to provide an in vivo proof of principle for detection of amyloidosis by diffusion kurtosis imaging (DKI).

Transcription Profiles of Aortic Smooth Muscle Cells from Atherosclerosis-Prone and -Resistant Regions in Young Apolipoprotein E-Deficient Mice before Plaque Development

Background/Aims: Site-specific atherosclerosis is generally attributed to differential gene expression in endothelial cells. We investigated whether the transcriptome of smooth muscle cells is different between atherosclerosis-prone and atherosclerosis-resistant regions in apolipoprotein E-deficient (apoE–/–) mice before plaque development, and in C57Bl/6 mice. Methods: De-endothelialized aortas (both strains: 3 males, 3 females, age 4 months) were divided into atherosclerosis-prone (AA: ascending aorta, aortic arch and proximal 2 mm of thoracic aorta) and -resistant (CTA: central thoracic aorta, i.e. 6 mm distal from the proximal 2 mm) regions. The transcriptome of these two regions was compared using whole-genome mouse microarrays. Results: Microarray analysis revealed differential expression (>2-fold difference) of 70 and 244 genes in C57Bl/6 and apoE–/– mice. This was confirmed for 6 genes using the real-time quantitative polymerase chain reaction. Up- or downregulation in the AA was observed for 33 and 37 genes in C57Bl/6, and for 186 and 58 genes in apoE–/– mice, respectively. The 201 genes that showed exclusively differential expression in apoE–/– mice were related to atherosclerotic processes, such as cell adhesion, proliferation, differentiation, motility, cell death, lipid metabolism and immune responses. Conclusion: Our findings indicate that smooth muscle cells display an altered transcriptome at atherosclerosis-prone locations before actual lesion development.

Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein

OBJECTIVE Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. METHODS AND RESULTS Apolipoprotein E-deficient (apoE(-/-)) mice were crossbred with animals lacking APP (APP(-/-)). After 16 weeks on a Western-type diet, apoE(-/-) and APP(-/-)/apoE(-/-) mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP(-/-)/apoE(-/-) mice as compared to apoE(-/-). Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP(-/-)/apoE(-/-) mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. CONCLUSION Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability.

Acute modulation of the cholinergic system in the mouse brain detected by pharmacological resting-state functional MRI

INTRODUCTION The cholinergic system is involved in learning and memory and is affected in neurodegenerative disorders such as Alzheimers disease. The possibility of non-invasively detecting alterations of neurotransmitter systems in the mouse brain would greatly improve early diagnosis and treatment strategies. The hypothesis of this study is that acute modulation of the cholinergic system might be reflected as altered functional connectivity (FC) and can be measured using pharmacological resting-state functional MRI (rsfMRI). MATERIAL AND METHODS Pharmacological rsfMRI was performed on a 9.4T MRI scanner (Bruker BioSpec, Germany) using a gradient echo EPI sequence. All mice were sedated with medetomidine. C57BL/6 mice (N = 15/group) were injected with either saline, the cholinergic antagonist scopolamine, or methyl-scopolamine, after which rsfMRI was acquired. For an additional group (N = 8), rsfMRI scans of the same mouse were acquired first at baseline, then after the administration of scopolamine and finally after the additional injection of the cholinergic agonist milameline. Contextual memory was evaluated with the same setup as the pharmacological rsfMRI using the passive avoidance behavior test. RESULTS Scopolamine induced a dose-dependent decrease of FC between brain regions involved in memory. Scopolamine-induced FC deficits could be recovered completely by milameline for FC between the hippocampus-thalamus, cingulate-retrosplenial, and visual-retrosplenial cortex. FC between the cingulate-rhinal, cingulate-visual and visual-rhinal cortex could not be completely recovered by milameline. This is consistent with the behavioral outcome, where milameline only partially recovered scopolamine-induced contextual memory deficits. Methyl-scopolamine administered at the same dose as scopolamine did not affect FC in the brain. CONCLUSION The results of the current study are important for future studies in mouse models of neurodegenerative disorders, where pharmacological rsfMRI may possibly be used as a non-invasive read-out tool to detect alterations of neurotransmitter systems induced by pathology or treatment.

Neuregulin-1 attenuates stress-induced vascular senescence

Aims Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence. Methods and results Senescence was induced in cultured rat aortic endothelial cells (ECs) and aortic smooth muscle cells (SMCs) by 2 h exposure to 30 µM hydrogen peroxide (H2O2). Cellular senescence was confirmed after 72 h using senescence-associated-β-galactosidase staining (SA-β-gal), cell surface area, and western blot analyses of SA pathways (acetyl-p53, p21). Recombinant human NRG-1 (rhNRG-1, 20 ng/mL) significantly reduced H2O2-induced senescence, as shown by a lower number of SA-β-gal positive cells, smaller surface area and lower expression of acetyl-p53. In C57BL/6 male mice rendered diabetic with streptozotocin (STZ), rhNRG-1 attenuated cellular senescence in aortic ECs and SMCs. Next, we created mice with SMC-specific knockdown of the NRG-1 receptor ErbB4. Aortic SMCs isolated from SMC-specific ErbB4 deficient mice (ErbB4f/+ SM22α-Cre+) showed earlier cellular senescence in vitro compared with wild-type (ErbB4+/+ SM22α-Cre+) SMCs. Furthermore, when rendered diabetic with STZ, ErbB4f/+ SM22α-Cre+ male mice showed significantly more vascular senescence than their diabetic wild-type littermates and had increased mortality. Conclusions This study is the first to explore the role of NRG-1 in vascular senescence. Our data demonstrate that NRG-1 markedly inhibits stress-induced premature senescence in vascular cells in vitro and in the aorta of diabetic mice in vivo. Consistently, deficiency in the NRG-1 receptor ErbB4 provokes cellular senescence in vitro as well as in vivo.

Magnetization transfer contrast imaging detects early white matter changes in the APP/PS1 amyloidosis mouse model.

•MTC visualizes cortical Aβ-induced changes at very old ages.•MTC visualizes longitudinal Aβ-induced white matter changes in young APP/PS1 mice.•Changes in white matter MT-ratios correlate to Aβ plaque load, astro- and microgliosis.