The function of tyrosine phosphatases in the regulation of endothelial activation and inflammation
As a first line of defence in inflammatory conditions, cellular components of the innate immunity release pro-inflammatory cytokines. The endothelium, lining the vessels and constituting the barrier between blood and tissue, becomes activated by these inflammatory factors, resulting in for instance the upregulation of adhesion molecules, generation of reactive oxygen species (ROS) and increased vascular permeability. The activation of the endothelium results in the recruitment of inflammatory cells and increased vascular leakage. These responses contribute to tissue oedema and organ failure in systemic inflammatory conditions, such as sepsis or SIRS (systemic inflammatory response syndrome). In chronic inflammatory settings, this promotes endothelial dysfunction and subsequent cardiovascular disease. We have demonstrated the tyrosine phosphatase SHP-2 to be inactivated in endothelial cells under inflammatory conditions, resulting in increased expression of ICAM-1 and VCAM-1 and subsequently in enhanced endothelial leukocyte recruitment and transmigration in vitro and in vivo. Importantly, re-installing SHP-2 activity prevented these inflammatory changes. Moreover, we found inflammatory vascular permeability to be enhanced in mice treated with an SHP-2 inhibitor, pointing towards a protective role of SHP-2 in this context. These data demonstrate that an inactivation of SHP-2 may indeed drive endothelial dysfunction and promote vascular events. Ongoing projects aim to further characterize the underlying mechanisms and direct targets of SHP-2 within this context as well as investigating the function of SHP-2 in endothelial activation and dysfunction in cardiovascular disease under insulin resistant conditions mimicking type 2 diabetes.
Role of SHP-2 in IL-β dependent endothelial cell signalling and vascular inflammation
Upon inflammatory activation by IL-1β, the adaptor protein MyD88 is phosphorylated on Y257 enabling binding of SHP-2. The binding of SHP-2 results in dephosphorylation of other tyrosine residues and inhibition of downstream signalling. However, inflammation-induced generation of ROS inactivate SHP-2 and consequently cause recruitment of p85/PI3-K to MyD88 and subsequent activation of inflammatory downstream signalling. ICAM-1 and VCAM-1 surface expression is upregulated leading to leukocyte recruitment and vascular leakage is enhanced. In experiments, the forced expression of an inactive SHP-2 protein (CS) or SHP-2 inhibition by PTPI IV enhances the inflammatory response, whereas expression of a constitutively active SHP-2 (EA) prevents this. Figures and illustration are adapted from our publication Heun et al., eBioMedicine, 2019 used under CC-BY-NC-ND 4.0
Publications within this subject:
Heun Yvonn, Pircher Joachim, Czermak Thomas, Bluem Philipp, Hupel Georg, Bohmer Monica, Kraemer Bjoern F., Pogoda Kristin, Pfeifer Alexander, Woernle Markus, Ribeiro Andrea, Hübner Max, Kreth Simone, Claus Ralf A., Weis Sebastian, Ungelenk Luisa, Krötz Florian, Pohl Ulrich, Mannell Hanna.
Inactivation of the tyrosine phosphatase SHP-2 drives vascular dysfunction in sepsis.
https://doi.org/10.1016/j.ebiom.2019.03.034
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Grundler Groterhorst Katharina, Mannell Hanna, Pircher Joachim, Kraemer Bjoern F..
Platelet proteasome activity and metabolism is upregulated during bacterial sepsis.
https://doi.org/10.3390/ijms20235961
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Koch Elisabeth, Pircher Joachim, Czermak Thomas, Gaitzsch Erik, Alig Stefan, Mannell Hanna, Niemeyer Markus, Krötz Florian, Wörnle Markus.
The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular haemostasis in TNFα-induced inflammation in vivo.
https://doi.org/10.1155/2013/279781
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Functional role of endothelial tyrosine phosphatases in angiogenesis in vitro and in vivo
The formation of new blood vessels (angiogenesis) plays an essential role not only in physiological processes such as development (embryogenesis) and wound healing, but also in pathophysiological processes such as tumour growth and diabetic retinopathy. A condition, which is a strong inducer of angiogenesis is hypoxia. Hypoxia induces the accumulation and activation of the α subunit of the transcription factor hypoxia inducible factor 1 (HIF-1), which is otherwise rapidly degraded by the proteasome under normoxic conditions. HIF-1 induces expression of several angiogenic genes, thus being the target for therapeutic strategies. We found that downregulation of the tyrosine phosphatase SHP-1 enhanced HIF-1α accumulation and increased hypoxia induced VEGF expression as well as endothelial cell (EC) proliferation. Interestingly, we previously showed that the SHP-1 homologue SHP-2 is additionally important for angiogenesis. In contrast to SHP-1, down regulation or inhibition of SHP-2 impairs growth factor mediated EC proliferation, migration and vessel sprouting in vitro and ex vivo. In follow-up projects, we observed that overexpression of an inactive SHP-2 mutant in EC under hypoxia led to reduced HIF-1α accumulation and activity, demonstrating that SHP-2 activity is essential for HIF-1α activity. Moreover, we were able to modulate hypoxic wound healing angiogenesis in vivo by site-directed gene transfer using magnetic lentiviruses expressing different SHP-2 constructs. Whereas introduction of an inactive SHP-2 mutant impaired wound healing angiogenesis, expression of a constitutively active SHP-2 mutant accelerated this. We found that SHP-2 promotes hypoxic angiogenesis by preventing HIF-1α degradation via a Src dependent mechanism. In a further study, we detected that SHP-2 influences 26S proteasomal activity under hypoxia in vitro and in vivo. We are now interested in studying the mechanism of SHP-2 dependent regulation of proteasomal activity under hypoxia in more detail as well as a possible role for SHP-2 in hypoxia dependent vascular permeability and the identification of the underlying mechanisms. Additionally, ongoing projects investigate the role of SHP-2 for angiogenesis and HIF-1α activity under insulin resistant conditions.
SHP-2 activity promotes hypoxic angiogenesis via HIF-1α activation
Publications within this subject:
Mannell Hanna, Kameritsch Petra, Beck Heike, Pfeifer Alexander, Pohl Ulrich, Pogoda Kristin.
Cx43 promotes endothelial cell migration and angiogenesis via the tyrosine phosphatase SHP-2.
https://doi.org/10.3390/ijms23010294
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Heun Yvonn, Grundler Groterhorst Katharina, Pogoda Kristin, Kraemer Bjoern F., Pfeifer Alexander, Pohl Ulrich, Mannell Hanna.
The phosphatase SHP-2 activates HIF-1α in wounds in vivo by inhibition of 26S proteasome activity.
https://doi.org/10.3390/ijms20184404
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Rieck Sarah, Heun Yvonn, Heidsieck Alexandra, Mykhaylyk Olga, Pfeifer Alexander, Gleich Bernhard, Mannell Hanna, Wenzel Daniela.
Local anti-angiogenic therapy by magnet-assisted downregulation of SHP2 phosphatase.
https://doi.org/10.1016/j.jconrel.2019.05.031
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Heun Yvonn, Pogoda Kristin, Anton Martina, Pircher Joachim, Pfeifer Alexander, Woernle Markus, Ribeiro Andrea, Kameritsch Petra, Mykhaylyk Olga, Plank Christian, Kroetz Florian, Pohl Ulrich, Mannell Hanna.
HIF-1α dependent wound healing angiogenesis in vivo can be controlled by site-specific lentiviral magnetic targeting of SHP-2.
https://doi.org/10.1016/j.ymthe.2017.04.007
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Alig Stefan K., Stampnik Yvonn, Pircher Joachim, Rotter Raffaela, Gaitzsch Erik, Ribeiro Andrea, Wörnle Markus, Krötz Florian, Mannell Hanna.
The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.
https://doi.org/10.1371/journal.pone.0121113
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Mannell Hanna, Hellwig Nicole, Gloe Torsten, Plank Christian, Sohn Hae-Young, Groesser Leopold, Walzog Barbara, Pohl Ulrich, Krötz Florian.
Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo.
https://doi.org/10.1159/000110081
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Mannell Hanna, Hammitzsch Ariane, Mettler Ramona, Pohl Ulrich, Krötz Florian.
Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt.
https://doi.org/10.1016/j.cellsig.2009.09.015
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Mannell Hanna, Krotz Florian.
SHP-2 regulates growth factor dependent vascular signalling and function.
https://doi.org/10.2174/1389557514999140506094738
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Mannell Hanna, Krotz Florian.
The role of SHP-2 in cell signalling and human disease.
https://doi.org/10.2174/157340807782330264
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Function of cytohesins in the vascular system
The family of cytohesins, which are small guanine nucleotide exchange factors (GEFs), consisting of cytohesin-1, cytohesin-2 (ARNO), cytohesin-3 (Grp-1) and cytohesin-4, has been shown to contribute to cellular adhesion and transmigration of leukocytes as well as adhesion and migration of other cell types. They also seem to play an important role in insulin signalling. In earlier studies, we demonstrated that inhibition of cytohesin-2 (ARNO) reduced vascular leakage in vivo. We discovered that ARNO directly affects VEGFR-2 signalling in endothelial cells by controlling internalization and degradation of VEGFR-2. Furthermore, we discovered that ARNO is important for the phenotypic switch of vascular smooth muscle cells (VSMC). We found that an inhibition or inactivation of ARNO affected the morphology, reduced the synthetic activity and migration of VSMC induced by the adipokine resistin. Furthermore, ARNO was shown to be important for resistin dependent MMP-2 production as well as migration through activation of the JNK/AP-1 pathway and p38 MAPK. An ongoing project aims to characterize the function of the cytohesins 1, -2, -3 and -4 in hypoxia induced permeability and angiogenesis in endothelial cells.
Role of ARNO (cytohesin-2) in endothelial cells and vascular smooth muscle cells
In endothelial cells (EC) ARNO stabilizes surface VEGFR-2 thereby promoting VEGFR downstream signalling, leading to proliferation and EC permeability. In vascular smooth muscle cells (VSMC), ARNO promotes the production of MMP-2, the phenotypic switch and migration of these cells via activation of p38 MAPK and JNK, therefore potentially contributing to the formation of atherosclerotic plaques.
Publications within this subject:
Heun Yvonn, Gräff Pascal, Lagara Aikaterini, Schelhorn Romina, Mettler Ramona, Pohl Ulrich, Mannell Hanna.
The GEF cytohesin-2/ARNO mediates resistin induced phenotypic switching in vascular smooth muscle cells.
https://doi.org/10.1038/s41598-020-60446-z
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Mannell Hanna, Pircher Joachim, Chaudhry Daniel I., Alig Stefan K.C., Koch Elisabeth G., Mettler Ramona, Pohl Ulrich, Krötz Florian.
ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.
https://doi.org/10.1093/cvr/cvr265
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Mechanisms of endothelial activation induced by cardiopulmonary bypass
Cardiopulmonary bypass (CPB) is routinely used in cardiac surgery because it allows temporary suppression of cardiac activity while keeping systemic circulation maintained by redirecting the blood from the heart and lungs and instead pump it through an extra-corporal circuit. Several components of the CPB process, such as the shear forces generated by the pumps, hemodilution, exposure of blood to the artificial surfaces of the circuit and the aortic cross clamping may contribute to the induction of a systemic inflammatory response. Moreover, the CPB may influence the pharmacodynamics of administered drugs. The inotropic drug levosimendan is often used as therapeutic approach perioperatively in cardiac surgery patients with CPB to improve hemodynamic stabilization and to reduce the risk of post-surgical low cardiac output syndrome. In cooperation with the Department of Pharmacy, LMU, we recently established a LS-ESI-MS/MS protocol for therapeutic drug monitoring (TDM) of levosimendan and its metabolites OR-1855 and OR-1896, enabling rapid and simultaneous detection of these compounds in serum. Subsequently, in cooperation with the Department of Anaesthesiology at the LMU University Hospital we detected low total serum concentrations as well as unbound fractions of these compounds in cardiac surgery patients with CPB. Moreover, we investigated the effects of levosimendan and its metabolites on inflammatory endothelial activation and found levosimendan and both metabolites to reduce reactive oxygen species, while only levosimendan impaired endothelial adhesion molecule upregulation. An ongoing study investigates if the low concentrations of levosimendan and metabolites, which were measured in cardiac surgery patients, still have protective effects on endothelial activation and vascular permeability. We are furthermore interested in studying the underlying cellular mechanisms for the enhanced endothelial barrier disruption and endothelial activation and dysfunction in patients with CPB.
Publications within this subject:
Kipka Hannah, Liebchen Uwe, Hübner Max, Höfner Georg, Frey Otto, Wanner Klaus T., Kilger Erich, Hagl Christian, Tomasi Roland, Mannell Hanna.
Serum concentrations of levosimendan and its metabolites OR-1855 and OR-1896 in cardiac surgery patients with cardiopulmonary bypass.
https://doi.org/10.3389/fcvm.2024.1406338
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Kipka Hannah, Schaflinger Rebecca, Tomasi Roland, Pogoda Kristin, Mannell Hanna.
The effects of the levosimendan metabolites OR-1855 and OR-1896 on endothelial pro-inflammatory responses.
https://doi.org/10.3390/biomedicines11030918
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Kipka Hannah, Tomasi Roland, Hübner Max, Liebchen Uwe, Hagl Christian, Wanner Klaus T., Mannell Hanna, Höfner Georg.
Simultaneous LC-ESI-MS/MS quantification of levosimendan and its metabolites for therapeutic drug monitoring of cardiac surgery patients.
https://doi.org/10.3390/pharmaceutics14071454
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Establishment and implementation of local vascular gene transfer by magnetic targeting
Gene delivery to cells constitutes an efficient way to specifically modulate signalling pathways and is a promising approach to induce physiological responses (gene therapy) in both research and medical therapy. Regarding the vasculature, local gene therapy shows great potential in improving ischemic cardiovascular disease and wound healing, but also to inhibit tumor growth and diabetic retinopathy, to mention a few. Upon intravascular application, gene vectors are distributed systemically and apart from reaching the organ of interest, they are also delivered to other sites, causing undesired side-effects. Other hurdles with this technique yet to overcome are poor gene delivery and expression efficiency at the desired site. We have developed an innovative effective gene delivery approach for in vivo use, which combines high efficiency and site-specificity following intravascular application. By using lipid microbubbles coated with superparamagnetic nanoparticles (magnetic microbubbles, MMB) which are associated to lentiviruses, DNA vectors, or AS-ODN we were able to target these to vessels at a specific site in vivo by applying an external magnetic field and subsequent ultrasound mediated disruption of the MMB ("Ultrasound-Targeted Microbubble Destruction"; UTMD) to enable the delivery of their cargo. This resulted in high transgene expression at the targeted site, whereas the non-specific delivery at other sites was strongly reduced. We further established a technique to efficiently deliver genes to wounds in the dorsal skin of mice to study hypoxic wound healing angiogenesis by using magnetic nanoparticle associated lentiviruses and external magnetic fields. This technique now enables the investigation of cellular mechanisms during vascular regeneration and vascular remodelling in the intact organ.
Local vascular gene transfer by magnetic targeting
Publications within this subject:
Heun Yvonn, Pogoda Kristin, Anton Martina, Pircher Joachim, Pfeifer Alexander, Woernle Markus, Ribeiro Andrea, Kameritsch Petra, Mykhaylyk Olga, Plank Christian, Kroetz Florian, Pohl Ulrich, Mannell Hanna.
HIF-1α dependent wound healing angiogenesis in vivo can be controlled by site-specific lentiviral magnetic targeting of SHP-2.
https://doi.org/10.1016/j.ymthe.2017.04.007
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Heun Yvonn, Hildebrand Staffan, Heidsieck Alexandra, Gleich Bernhard, Anton Martina, Pircher Joachim, Ribeiro Andrea, Mykhaylyk Olga, Eberbeck Dietmar, Wenzel Daniela, Pfeifer Alexander, Woernle Markus, Krötz Florian, Pohl Ulrich, Mannell Hanna.
Targeting of magnetic nanoparticle-coated microbubbles to the vascular wall empowers site-specific lentiviral gene delivery in vivo.
https://doi.org/10.7150/thno.16192
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Mannell Hanna, Pircher Joachim, Fochler Franziska, Stampnik Yvonn, Räthel Thomas, Gleich Bernhard, Plank Christian, Mykhaylyk Olga, Dahmani Chiheb, Wörnle Markus, Ribeiro Andrea, Pohl Ulrich, Krötz Florian.
Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles.
https://doi.org/10.1016/j.nano.2012.03.007
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Mannell Hanna, Pircher Joachim, Räthel Thomas, Schilberg Katharina, Zimmermann Katrin, Pfeifer Alexander, Mykhaylyk Olga, Gleich Bernhard, Pohl Ulrich, Krötz Florian.
Targeted endothelial gene delivery by ultrasonic destruction of magnetic microbubbles carrying lentiviral vectors.
https://doi.org/10.1007/s11095-012-0678-8
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Rieck Sarah, Heun Yvonn, Heidsieck Alexandra, Mykhaylyk Olga, Pfeifer Alexander, Gleich Bernhard, Mannell Hanna, Wenzel Daniela.
Local anti-angiogenic therapy by magnet-assisted downregulation of SHP2 phosphatase.
https://doi.org/10.1016/j.jconrel.2019.05.031
BibTeX | RIS | DOI |
Räthel T., Mannell Hanna, Pircher Joachim, Gleich B., Pohl Ulrich, Krötz F..
Magnetic stents retain nanoparticle-bound antirestenotic drugs transported by lipid microbubbles.
https://doi.org/10.1007/s11095-011-0643-y
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Gleich Bernhard, Hellwig Nicole, Bridell Hanna, Jurgons Roland, Seliger Christian, Alexiou Christoph, Wolf Bernhard, Weyh Thomas.
Design and evaluation of magnetic fields for nanoparticle drug targeting in cancer.
https://doi.org/10.1109/tnano.2007.891829
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