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Inflammatory processes play a critical role in the development of atherosclerosis and its fatal outcomes, such as myocardial infarction and ischemic stroke [ 1 , 2 ]. The roles of inflammatory cytokines and ROS in cardiovascular diseases are inseparably linked, and elevated endothelial-derived ROS activate proatherogenic signalling pathways and stimulate vascular smooth muscle cell proliferation [ 10 , 11 ], scavenge endothelium-derived nitric oxide, and, thus, aggravate endothelial dysfunction [ 10 , 12 ].
In addition, platelets represent an important link between inflammation, atherogenesis, and thrombosis [ 13 — 16 ]. It is not surprising that patients with chronic inflammatory disorders are at increased risk for cardiovascular morbidity and atherothrombotic events [ 19 ], which could not be explained by traditional cardiovascular risk factors [ 20 — 22 ]. The SH2-domain containing protein tyrosine phosphatase 1 SHP-1 is known to be a negative regulator of immune receptor signalling in lymphocytes, macrophages, and platelets, in which it is typically coactivated upon cellular stimulation to exert an autoinhibitory function [ 25 — 27 ].
We could previously show that SHP-1 is expressed in vascular endothelial cells and negatively regulates NAD P H-oxidase-dependent endothelial superoxide production by inhibition of PI3 K activity and subsequent inactivation of the small GTPase Rac1; SHP-1 mRNA knockdown as well as inhibition with the pharmacological inhibitor sodium stibogluconate leads to oxidative stress in endothelial cells [ 29 ].
Sodium stibogluconate and all other chemicals were from Sigma-Aldrich Germany. All experiments were conducted in accordance with the German animal protection law and approved by the district government of Upper Bavaria approval reference number AZ The dorsal skinfold chamber microcirculatory model was used in mice as described previously [ 24 ].
Animals with an intact microcirculation underwent carotid artery catheterization for application of drugs or injection of isolated platelets, respectively. Intravital fluorescence microscopy was performed using a modified microscope Zeiss Axiotech Vario, Germany. Whole blood was drawn from anesthetized mice by cardiac puncture.
The ability of the isolated and stained platelets to aggregate was tested by platelet aggregometry. For intravital studies of platelet interaction with the intact vessel wall isolated and fluorescent-stained murine platelets from a donor animal were injected via a carotid artery catheter and observed in the dorsal skinfold chamber model.
Movie sequences of 30 s in 4—6 vessel segments in each animal were recorded and analyzed using AxioVision Software Carl Zeiss, Germany. Vessels with abnormal flow were excluded from analysis.
From the resulting length of the platelet trace in single images, velocities of single platelets were calculated using the exposure time of each single picture. Platelet vessel wall interaction PVWI was expressed in frequency histograms consisting of all platelet velocities analyzed.
Histograms were normalized to the maximum platelet speed within a vessel to exclude biasing influences of altered blood flow velocities between different arterioles. As a consequence a rightward shift in platelet velocity distribution within a histogram expresses less PVWI, whereas a leftwards shift signalises increased PVWI at the arteriolar wall. For induction of intra-arteriolar thrombosis, the ferric chloride superfusion method was used as described previously [ 24 ].
Before the experiments blood vessel flow was digitally recorded and regular blood flow was confirmed for all analyzed arterioles.
Platelet aggregation in human platelet-rich plasma PRP was measured using the turbidimetric method described by Born [ 31 ]. Human PRP was obtained by centrifugation of whole citrated blood, drawn from human cubital veins at g. Written consent was obtained from platelet donors.
For mice studies blood was collected from the inferior vena cava in anesthetized mice with a syringe containing heparin. Whole blood aggregation was performed by impedance aggregometry with the Multiplate multiple platelet function analyzer assay Dynabyte, Munich, Germany according to the manufacturer's protocol.
Changes in impedance expressed as aggregation amplitudes were recorded over 6 minutes in duplicates and results were expressed as mean arbitrary aggregation units AU. The procedure was approved by a university ethic review board and the investigation conforms with the principles outlined in the Declaration of Helsinki. Lysates were subjected to Western blot analysis as previously described [ 33 ] and SHP-1 was detected using a polyclonal rabbit anti-SHP-1 antibody C To measure SHP-1 activity phosphorylation of SHP-1 on Y was detected using a rabbit anti-phoshpho-YSHP-1 antibody Abcam , as phosphorylation at this site has been described to correlate with the phosphatase activity [ 34 ].
GAPDH was used as loading control. SigmaStat Software was used to calculate statistical differences. We assessed transient interaction of injected labelled platelets to the vessel wall in vivo by intravital microscopy of vessels in the dorsal skinfold chamber. The maximum platelet velocities in the analyzed vessels as an approximate measure of flow velocity were not significantly different between the treatment groups.
The histograms display all platelet velocities from 5 different animals per group. SG: sodium stibogluconate. To analyze the effect of SHP-1 inhibtion on thrombus formation in vivo , we assessed the time to thrombotic arteriolar vessel occlusion following injury by ferric chloride superfusion to the vascular wall. Inhibition of SHP-1 leads to accelerated arteriolar thrombus formation in vivo.
Inhibtion SHP-1 does not affect platelet aggregation in vitro and ex vivo. SG: sodium stibogluconate, AU: arbitrary unite. To test effects of systemic SHP-1 inhibition on platelets aggregation was measured in whole blood of mice after oral treatment with sodium stibogluconate, which did not change ADP-induced ADP 6.
In this study we show that inhibition of the tyrosine phosphatase SHP-1 results in increased platelet-endothelium interaction and accelerated arteriolar thrombus formation in vivo , possibly by upregulation of adhesion molecules on endothelial cells.
In our in vivo studies inhibition of treatment with the SHP-1 inhibitor sodium stibogluconate led to a slightly greater amount of rolling platelets as compared to control treated animals under physiological conditions. When the endothelium is activated, similar to leukocytes, platelets can roll on the endothelium [ 17 ]. We have previously identified SHP-1 as a negative regulator of endothelial NADPH-oxidase dependent superoxides already under basal conditions [ 29 ], which highly suggests a role for ROS to be involved in mediating these effects.
Moreover inhibition of SHP-1 already under basal condition i. Elevated expression of vWF on endothelial cells has been shown to contribute to microvascular thrombosis in vivo [ 36 ] and oxidative stress is well described to induce prothrombotic changes [ 12 ]. It should be mentioned, though, that from the thrombus formation experiment direct effects of SHP-1 inhibition in platelets cannot be excluded.
To elucidate whether the observed prothrombotic effects in vivo could be due to direct effects of SHP-1 inhibition by sodium stibogluconate in platelets we performed in vitro human PRP and ex vivo mouse blood aggregation studies. Therefore we conclude that the effects we observed in the in vivo experiments are due to endothelial mechanisms rather than direct effects on platelets.
Autoinhibitory functions for SHP-1 have been described in other cell types, such as lymphocytes, macrophages, and platelets, where the phosphatase is coactivated and serves as a negative regulator of immune receptor signaling [ 25 — 27 ]. In this sense we could previously show that inhibition of SHP-1 in endothelial cells leads to increased VEGF-dependent superoxide formation [ 29 ]. Accordingly, Sugano et al. Several proteins are tyrosine phosphorylated downstream of TNF-receptor activation, involving src- and jak-family kinases [ 40 ], which in part have been shown to be regulated by SHP-1 [ 41 ].
Considering the crucial role of inflammatory cytokines and oxidative stress in the pathophysiology of cardiovascular diseases these factors are interesting targets for pharmacological approaches. Numerous studies targeting inflammatory cytokines by antibodies or ROS by antioxidants have been performed, which, however, led to conflicting results regarding a clinical benefit and have not become established as standard therapy in cardiovascular diseases [ 42 — 44 ].
The understanding of the pathophysiological processes leading to cardiovascular diseases is therefore fundamental to develop new therapeutic strategies. SHP-1 has already been tested as a therapeutic target in ischemic conditions such as stroke or myocardial infarction, where inhibition of its activity resulted in a reduction of infarct sizes [ 45 — 47 ].
Inhibition of another tyrosine phosphatase, namely PTP1B, resulted in improvement of peripheral endothelial dysfunction in heart failure [ 48 ]. The cellular effects and functions of the numerous different cytosolic and membrane-bound tyrosine phosphatases are multiple and many times antagonistic.
Thus, preservation of tyrosine phosphorylation as a therapeutic principle for vascular medicine, especially using tyrosine phosphatase inhibitors, must be viewed in a very differentiated manner and evaluated with caution. Eventually decreased SHP-1 activity in an inflammatory setting increases platelet-endothelium interaction and accelerates arteriolar thrombus formation in vivo. Koch designed and performed experiments and interpreted the data.
Pircher interpreted the data and wrote the paper. Czermak, E. Gaitzsh, S. Alig, H. Mannell, and M. Niemeyer performed experiments.
Koch and J. Pircher contributed equally to this paper. All authors read and approved the final paper. Read article at publisher's site DOI : Free to read. Thromb Haemost , 6 , 31 Jul Cited by 40 articles PMID: Pain , 4 , 01 Apr Cited by 18 articles PMID: PLoS One , 10 3 :e, 23 Mar J Mol Med Berl , 92 9 , 27 May Cited by 1 article PMID: This data has been provided by curated databases and other sources that have cited the article.
To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Arthritis Res Ther , 14 5 :R, 18 Oct J Thromb Haemost , 7 10 , 19 Aug Cited by 11 articles PMID: J Am Coll Cardiol , 45 10 , 01 May Cited by 32 articles PMID: J Thromb Haemost , 4 11 , 08 Aug Cited by 6 articles PMID: Immunol Res , 47 , 01 Jul Review Free to read.
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