Supplementary Material

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

Schuchardt et al. J Mol Med. 2011

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Materials

All cell culture nutrients were obtained from Biochrom (Berlin, Germany). Up4A was synthesized and purified as published previously [5]. All other nucleotides were purchased from Jena BioScience (Jena, Germany). PD98059, SB202190, U0126 and NSC23766 were purchased from Tocris (Bristol, UK). Apocynin was obtained from Calbiochem (Merck, Nottigham, UK). All other chemicals were provided from Sigma-Aldrich (Munich, Germany) unless otherwise indicated.

Methods

General methods

RNA was isolated with the RNeasy Mini kit from Qiagen (Chatsworth, CA) according to the manufacturer´s instructions. For determination of protein content the BCA™ assay kit (Pierce, Rockford, USA) were used. Fluorescence measurement was done in a multi-mode reader (Mithras LB940, Berthold Technologies, Bad Wildbad, Germany).

Detection of P2X subtypes

Total RNA was extracted from rat VSMCs using RNeasy Mini kit. RNA from Aorta was isolated using Trizol® (Invitrogen, Karlsbad, Germany). RT-PCR was performed using iScript™ cDNA Synthesis Kit (Biorad, Munich, Germany) in a thermal cycler (Mastercycler EP gradient, Eppendorf, Germany). Oligonucleotide sequences are summarized in Table 1. The resulting PCR products were separated on a 2% agarose gel containing ethidium bromide (EtBr) and observed under ultraviolet illumination. Negative controls were performed using no cDNA and always appeared blank. The thermal cycling protocol was: 94°C for 2 min, then 35 cycles of 95°C for 45 s, 60°C for 30 s and 72°C for 1 min and finally 10 min 72°C.


Proliferation assay

Proliferation of VSMCs was determined by bromodeoxyuridine (BrdU) incorporation using the cell proliferation ELISA kit from Roche (Mannheim, Germany). VSMCs were plated in 96-well plates, serum-starved for 24 h and afterwards stimulated for 24 h. BrdU was added for the last 4 h of stimulation. Incorporation of BrdU was detected by chemiluminescence measurement in a multimode reader.

Measurement of MCP-1 secretion

Aorta was isolated from Wistar Kyoto rats. Adventitia was carefully removed. Aorta was cut in rings of 2 mm and stimulated with indicated agonists/antagonists for 48 h in serum-free medium. Afterwards media were collected for MCP-1 detection using Luminex™ technology. The dry weight of the aorta rings of each probe were used for normalization.

Results

P2X expression in aorta and cultured VSMCs

Figure 2A shows the expression profile of P2Y subtypes in cultured VSMCs. Supplementary Figure IA shows additional expression profiles of P2Y in freshly cultured VSMCs and after one passage of the cells, with comparable expression of P2Y2/4/6 and no expression of P2Y1 (suppl. Figure IA). Besides P2Y, the dinucleoside polyphosphate Up4A, with its purine and pyrimidine moiety, activates P2X [1, 2]. Here we could show that P2X is lost in cultured VSMCs, exept P2X4. Aorta homogenates showed an expression of P2X1/4, whereas all other P2X subtypes are not expressed (suppl. Figure IB). As positive control for P2X2/3/6 brain homogenates were used.

P2X1-mediated effect on MCP-1 secretion from aorta

To verify possible P2X1-mediated effects of Up4A on MCP-1 production, we investigated the influence of α,β-methylene ATP (α,β-meth-ATP) as P2X1 activator. We used cultured VSMCs in our study so that we cannot exclude a P2X1 effect in vivo. MCP-1 secretion is not induced in VSMCs after stimulation of the cells with α,β-meth ATP for 24 h (suppl. Figure IC). Furthermore, we stimulated aortic rings from rats with thrombin, Up4A ± suramin, ATPγS ± suramin, or α,β-meth-ATP. As shown in figure ID, Up4A and ATPγS induced MCP-1 secretion, whereas, α,β-meth-ATP did not induce MCP-1 formation ex vivo (suppl. Figure ID). These data indicate that a P2X1-mediated effect on MCP-1 formation seems not obvious.

Proliferation

Proliferation and migration are important processes in atherosclerosis. As shown in figure II, Up4A and ATPγS induced a significant and dose-dependent increase in VSMC proliferation. PDGF-BB was used as positive control. Co-stimulation of PDGF-BB and Up4A further increase the proliferation rate of VSMCs (suppl. Figure II).

References of supplementary material

1. Jankowski V, Tolle M, Vanholder R, Schonfelder G, van der Giet M, Henning L, Schluter H, Paul M, Zidek W, Jankowski J (2005) Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor. Nat Med 11: 223-227

2. Jankowski V, Meyer AA, Schlattmann P, Gui Y, Zheng XL, Stamcou I, Radtke K, Tran TN, van der Giet M, Tolle M, Zidek W, Jankowski J (2007) Increased uridine adenosine tetraphosphate concentrations in plasma of juvenile hypertensives. Arterioscler Thromb Vasc Biol 27: 1776-1781

3. Hayes IM, Jordan NJ, Towers S, Smith G, Paterson JR, Earnshaw JJ, Roach AG, Westwick J, Williams RJ (1998) Human vascular smooth muscle cells express receptors for CC chemokines. Arterioscler Thromb Vasc Biol 18: 397-403

4. San Martin A, Foncea R, Laurindo FR, Ebensperger R, Griendling KK, Leighton F (2007) Nox1-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products. Free Radic Biol Med 42: 1671-1679

5. Johansson PA, Burnstock G, Dziegielewska KM, Guida E, McIntyre P, Saunders NR (2007) Expression and localization of P2 nucleotide receptor subtypes during development of the lateral ventricular choroid plexus of the rat. Eur J Neurosci 25: 3319-3331

6. Erlinge D, Hou M, Webb TE, Barnard EA, Moller S (1998) Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR. Biochem Biophys Res Commun 248: 864-870

7. Hou M, Harden TK, Kuhn CM, Baldetorp B, Lazarowski E, Pendergast W, Moller S, Edvinsson L, Erlinge D (2002) UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y(6) receptors. Am J Physiol Heart Circ Physiol 282: H784-792

8. Greenwood D, Jagger DJ, Huang LC, Hoya N, Thorne PR, Wildman SS, King BF, Pak K, Ryan AF, Housley GD (2007) P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea. Development (Cambridge, England) 134: 1407-1417

9. Syed NI, Tengah A, Paul A, Kennedy C (2010) Characterisation of P2X receptors expressed in rat pulmonary arteries. Eur J Pharmacol: 649:342-348.


Supplementary Figure I

Suppl. Figure I Detection of P2X and P2Y involvement in MCP-1 production. (A) P2Y and (B) P2X expression in VSMCs and aorta. Spleen and brain were used as positive control. PCR products were separated over an agarose gel and stained with EtBr. (C) MCP-1 secretion of VSMCs and (D) aortic rings after stimulation as indicated for 48 h (thrombin: 8 IU/mL; Up4A/ATPγS/α,β-methATP: 10 µmol/L). MCP-1 concentration was normalized to protein content of VMSCs or dry weight of aortic rings. *p<0.05 vs. control, #p<0.05 vs. Up4A/ATPγS.


Supplementary Figure II

Suppl. Figure II Up4A-induced proliferation of VSMCs. Proliferation was measured via BrdU incorperation and detected by chemiluminescence-based ELISA. The cells were stimulated for 24 h with PDGF-BB and indicated Up4A/ATPγS concentrations. *p<0.05 vs. control.

Table 1 Oligonucleotide sequences

All oligonucleotides were synthesized from TibMolBiol (Berlin, Germany).

ß-actin / fwd / 5'-TCGCTGACAGGATGCAGAAG-3'
rev / 5'-CTCAGGAGGAGCAATGATCTTGAT- 3'
probe (FAM-TAMRA) / 5'-AGATTACTGCCCTGGCTCCTAGCACCA- 3'
MCP-1 / fwd / 5'-CTGTCTCAGCCAGATGCAGTTAAT-3'
rev / 5'-TTCTCCAGCCGACTCATTGG-3'
probe (FAM-TAMRA) / 5'-CACCTGCTGCTACTCATTCACTGGCAAGA-3'
Nox1 / fwd / 5'-CTGCTCTCCTTCCTGAGGGGCACCTGCT-3' / [4]
rev / 5'-GACAATCCCCCCCAGGCCATGGATCCCTA-3'
Nox4 / fwd / 5'-TGCCCACTTGGTGAACGCCC-3'
rev / 5'-CCTGTCAGGCCCGGAACAGTTG-3'
P2Y1 / fwd / 5'-CCTGCGAAGTTATTTCATCTA-3' / [5]
rev / 5'-GTTGAGACTTGCTAGACCTCT-3'
P2Y2 / fwd / 5'-CTGCCAGGCACCCGTGCTCTACTT-3' / [5]
rev / 5'-CTGAGGTCAAGTGATCGGAAGGAG-3'
P2Y4 / fwd / 5`-TGGGTGTTTGGTTGGTAGTA-3' / [6]
rev / 5'-GTCCCCCGTGAAGAGATAG-3'
P2Y6 / fwd / 5'-GTTATGGAGCGGGACAATGG-3' / [7]
rev / 5'- AGGATGCTGCCGTGTAGGTT -3'
P2X1 / fwd / 5'- CAGAAAGGAAAGCCCAAGGTATTC -3' / [8]
rev / 5'- TGACGACGGTTTGTCCCATTC -3'
P2X2 / fwd / 5'- CTGGGACTACGAGACGCCTAAGG -3' / [8]
rev / 5'- TTGATGATGACTCCAATGACACCG -3'
P2X3 / fwd / 5'- GGACATAAAGAGGTGCCGCTTC -3' / [8]
rev / 5'- AACACTGGGTTGGTTGACGCAG -3'
P2X4 / fwd / 5'- GGAACATCCTCCCCAACATCAC -3' / [9]
rev / 5'- TTCATCTCCCCCGAAAGACC -3'
P2X5 / fwd / 5'- TGTCACGCTGGGGAGTCTGTTGTAG -3' / [9]
rev / 5'- TTGCTATTCTGCTTCCTGCCAC -3'
P2X6 / fwd / 5'- CAACTTCCTTGTGACACCAGCTCA -3' / [8]
rev / 5'- GGAGAGTGAATCGTAGAGACAGTA -3'
P2X7 / fwd / 5'- AATGAGTCCCTGTTCCCTGGCTAC -3' / [8]
rev / 5'- CAGTTCCAAGAAGTCCGTCTGG -3'

Table 2 P2X and P2Y expression in rat

Receptor subtypes detected via RT-PCR and PCR products were separated via agarose gel electrophoresis. Representative pictures are in figure 1 and supplementary figure I.

P2X
P2X1 / P2X2 / P2X3 / P2X4 / P2X5 / P2X6 / P2X7
VSMC p0 / - / - / - / + / + / - / +
VSMC p1 / - / - / - / + / + / - / +
VSMC p3 / - / + / + / ++ / + / - / +
VSMC p6 / - / - / - / + / - / - / -
Aorta / ++ / - / - / + / + / - / +
Brain / - / + / + / - / - / ++ / -
P2Y
P2Y1 / P2Y2 / P2Y4 / P2Y6
VSMC p0 / - / ++ / ++ / ++
VSMC p1 / - / +++ / ++ / ++
VSMC p4 / - / +++ / + / ++
Aorta / - / +++ / + / ++
Spleen / +++ / +++ / ++ / +++