doi: 10.1161/CIRCRESAHA.107.170548
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Editorials |
Another TRP to Endothelial Dysfunction
TRPM2 and Endothelial Permeability
From the Institute of Pharmacology and Toxicology, Philipps-University of Marburg, Germany.
Correspondence to Alexander Dietrich, PhD, Institute of Pharmacology and Toxicology, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, 35043 Marburg, Germany. E-mail dietrica{at}staff.uni-marburg.de
See related article, pages 347–355
Key Words: TRPM2 • H2O2-induced Ca2+ influx • reactive oxygen species • oxidative stress • endothelial permeability
The vascular endothelium acts not only as a passive barrier between plasma and extracellular fluid but is intimately involved in various physiological processes including the regulation of systemic and regional vascular tone, blood coagulation, cell–cell adhesion, wound healing, cellular proliferation, and angiogenesis. The implications of endothelial dysfunction in many pathological states have rendered modulation of endothelial functions as a promising therapeutic strategy for cardiovascular and cerebrovascular disease. Increasing endothelial permeability by oxidative stress through the production of oxygen metabolites is an important trigger for endothelial dysfunction.
Until now, the general belief was that the resulting reactive oxygen species would directly damage the endothelium.1 The study by Hecquet et al in this issue of Circulation Research identified transient receptor potential melastatin (TRPM)2 as a nonselective cation channel inducing increases in the cytosolic Ca2+ concentration ([Ca 2+]i) in primary cultured human pulmonary artery endothelial cells in response to reactive oxygen species (Figure).2 Endothelial cells are generally viewed as electrically nonexcitable, lacking functional voltage-gated Ca2+ channels. A major mode of Ca2+ entry in these cells in response to both chemical and mechanical stimuli is the so-called nonselective cation entry through TRP channels.3 Several members of the TRP superfamily have been identified and characterized in the endothelium to date: the classic TRP channels TRPC1, -3, -4, and -6; TRPP1 and -2 of the P family; TRPV1, -2, and -4 representing the V family; as well as the melastatin-related TRPM2, -4, and -7. Whereas TRPC1, -C4, -C6, and -M7 have been linked to endothelial barrier dysfunction and perturbed angiogenic processes, TRPC3, -C4, -M2, and -M7 have been suggested to be responsible for oxidative damage and cell death.4
|
) and activating (
) agents used in the report are indicated. P indicates putative channel pore. See the text for details.
TRPM2 is an oxidant-activated channel belonging to the melastatin family of TRP cation channels, named after the first identified member of the family, the tumor suppressor melastatin, now TRPM1. This subfamily shares a TRPM homology region of 
700 aa in the N terminus. The general structure, with 6-membrane-spanning 
helices, a pore between S5 and S6, and 2 cytosolic tails is common to the TRP superfamily.5 The predominant feature of TRPM2, however, is the so called Nudix box, a consensus region for pyrophosphatases, which other members of the TRPM family have lost during evolution.6 The Nudix box, localized in the cytoplasmic C-terminal tail of the channel protein, confers a unique activation mechanism, gating by adenosine 5'-diphosphoribose (ADPR), on TRPM2.5 However, other activators, like cyclic ADPR and NAD+, as well as inhibitors have been reported recently.7
Oxidative stress, for which application of H2O2 is an experimental paradigm, induces TRPM2 currents and increases in [Ca 2+]i in various cell types transfected with TRPM2,8 as well as in pancreatic β-cells,9 neutrophils,10 U937 monocytes,11 and Jurkat T cells.12 The mode of action of H2O2, however, is a matter of debate.13 H2O2 activates the mitochondrial production of ADPR and may also result in the activation of poly-ADPR polymerases (PARPs). PARP enzymes catalyze the breakdown of NAD into nicotinamide and ADPR.13 Subsequently, ADPR can activate TRPM2 by binding to the C-terminal Nudix domain, inducing large cation currents in monocytic U937 cells.11 Direct agonist action of H2O2 on TRPM2, however, also has been described in myeloid cells.14,15
In the report by Hecquet et al, the authors now show H2O2-induced, TRPM2-like cation currents in human pulmonary artery endothelial cells that were increased by transfection of a TRPM2 cDNA and by the application of 3-deaza-cADP ribose but inhibited by a specific TRPM2 small interfering RNA, by a TRPM2-specific antibody, and by a PARP inhibitor.2 These data clearly favor an indirect activation of TRPM2 channels by H2O2 via the formation of ADPR. Using a recalcification protocol, the authors were also able to demonstrate H2O2-induced Ca2+ entry from the extracellular medium through TRPM2 channels, whereas emptying of internal Ca2+ stores was detectable only after adding high concentrations of H2O2 (500 µmol/L), most probably reflecting an unspecific cellular reaction. Moreover, the authors stress the importance of TRPM2 for the H2O2-induced reduction of transendothelial resistance, an effect that could be prevented partly by application of 2 PARP inhibitors, TRPM2 small interfering RNA and a TRPM2-specific antiserum. Most intriguingly, the short variant of TRPM2 (TRPM2s), which lacks the pore domain and acts as a dominant-negative form by inhibiting the formation of functional homotetrameric channels, was also able to significantly diminish the decrease in transendothelial resistance (Figure).2 Because both forms of TRPM2 are expressed in human pulmonary artery endothelial cells, the control of the relative expression levels is an enticing potential regulatory mechanism of TRPM2 activity in these cells.
The data presented in the article by Hecquet et al2 add another promising piece to the signal transduction puzzle underlying increased endothelial permeability. The same group has identified TRPC4, TRPC1, and TRPC6 as important TRP channels for the thrombin-induced decrease in transendothelial resistance.16–19 Do agonists like thrombin and different activators like reactive oxygen species signal to different TRP channel families to increase endothelial permeability? The answer is not completely clear, because earlier TRPC3, a member of the TRPC family, was also characterized as a channel activated by reactive oxygen species,20,21 and, recently, we have been able to show that TRPC6 is essential for acute hypoxic vasoconstriction induced by a mechanism relying on changes in the amount of reactive oxygen species.22 Moreover, cation influx through another member of the TRPM family, TRPM7, in response to reactive oxygen species results in anoxic neuronal cell death.23 The analysis of gene-deficient mouse models will be enlightening in this regard. Along these lines, TRPC4-deficient mice showed reduced agonist-induced Ca2+ influx in pulmonary endothelial cells associated with a lack of thrombin-induced stress fiber formation and a reduced endothelial cell retraction response. Most remarkably, in TRPC4–/– mice, the increase in the microvessel filtration coefficient (Kfc) of isolated perfused mouse lungs, an in vivo measure of vascular permeability in response to proteinase-activated receptor 1 agonist peptide, was reduced by 50% when compared to wild-type mice.16 These data clearly demonstrate that, at least for agonist-induced increases in vascular permeability, more than 1 TRP channel appears to be responsible. Unfortunately, a TRPM2-deficient mouse model was not yet available to investigate whether TRPM2 has an essential role in the oxidant-activated reduction in transendothelial permeability in isolated perfused lungs.
Nevertheless, Hecquet et al have not only identified TRPM2 as an interesting pharmacological target to inhibit increases in endothelial permeability, but they also present a possible means to interfere with TRPM2 activity in vivo.2 Increased endothelial permeability resulting in lung endothelial injury is a crucial event in the pathophysiological scenario accompanying sepsis and ischemia/reperfusion of lungs intended for transplantation. In both cases, invasion of granulocytes and T lymphocytes, which also express TRPM2,5 is the driving force for lung injury. Therefore, inhibition of TRPM2 by TRPM2 small interfering RNAs or overexpression of the dominant-negative TRPM2s isoform represents a potential pharmacological intervention to reduce lung endothelial injury. The proof of principle for such an approach has been demonstrated recently in HEK293T cells heterologously expressing TRPM2. In these cells, cell death can be induced by H2O2 but prevented by heterologous expression of the dominant-negative TRPM2s.14 The identification of TRPM2 as a key component of the endothelial Ca2+ entry pathway in response to reactive oxygen species sheds new light on the physiology and pathophysiology of the endothelium. In the future, manipulating TRPM2 function in the endothelium may be highly useful for experimental therapies of endothelial dysfunction.
 |
Acknowledgments |
|---|
Sources of Funding
Work performed in the laboratory of the authors has been supported by a grant from the Deutsche Forschungsgemeinschaft.
Disclosures
None.
|
Footnotes |
|---|
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
|
References |
|---|
 Top References |
|---|
1. Lum H, Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol. 2001; 280: C719–C741.
2. Hecquet CM, Ahmmed GU, Vogel SM, Malik AB. Role of TRPM2 channel in mediating H2O2-induced Ca2+ entry and endothelial hyperpermeability. Circ Res. 2008; 102: 347–355.
3. Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem. 2007; 76: 387–417.[CrossRef][Medline] [Order article via Infotrieve]
4. Kwan HY, Huang Y, Yao X. TRP channels in endothelial function and dysfunction. Biochim Biophys Acta. 2007; 1772: 907–914.[Medline] [Order article via Infotrieve]
5. Eisfeld J, Luckhoff A. TRPM2. Handb Exp Pharmacol. 2007; (179): 237–252.
6. Mederos Y Schnitzler M, Wäring J, Gudermann T, Chubanov V. Evolutionary determinants of divergent calcium selectivity of TRPM channels. FASEB J. 2007 Dec 18; Epub ahead of print.
7. Naziroglu M. New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochem Res. 2007; 32: 1990–2001.[CrossRef][Medline] [Order article via Infotrieve]
8. Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, Yamada H, Shimizu S, Mori E, Kudoh J, Shimizu N, Kurose H, Okada Y, Imoto K, Mori Y. LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell. 2002; 9: 163–173.[CrossRef][Medline] [Order article via Infotrieve]
9. Inamura K, Sano Y, Mochizuki S, Yokoi H, Miyake A, Nozawa K, Kitada C, Matsushime H, Furuichi K. Response to ADP-ribose by activation of TRPM2 in the CRI-G1 insulinoma cell line. J Membr Biol. 2003; 191: 201–207.[CrossRef][Medline] [Order article via Infotrieve]
10. Heiner I, Radukina N, Eisfeld J, Kuhn F, Luckhoff A. Regulation of TRPM2 channels in neutrophil granulocytes by ADP-ribose: a promising pharmacological target. Naunyn Schmiedebergs Arch Pharmacol. 2005; 371: 325–333.[CrossRef][Medline] [Order article via Infotrieve]
11. Perraud AL, Fleig A, Dunn CA, Bagley LA, Launay P, Schmitz C, Stokes AJ, Zhu Q, Bessman MJ, Penner R, Kinet JP, Scharenberg AM. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature. 2001; 411: 595–599.[CrossRef][Medline] [Order article via Infotrieve]
12. Beck A, Kolisek M, Bagley LA, Fleig A, Penner R. Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes. FASEB J. 2006; 20: 962–964.
13. Miller BA. The role of TRP channels in oxidative stress-induced cell death. J Membr Biol. 2006; 209: 31–41.[CrossRef][Medline] [Order article via Infotrieve]
14. Zhang W, Chu X, Tong Q, Cheung JY, Conrad K, Masker K, Miller BA. A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death. J Biol Chem. 2003; 278: 16222–16229.
15. Zhang W, Hirschler-Laszkiewicz I, Tong Q, Conrad K, Sun SC, Penn L, Barber DL, Stahl R, Carey DJ, Cheung JY, Miller BA. TRPM2 is an ion channel that modulates hematopoietic cell death through activation of caspases and PARP cleavage. Am J Physiol Cell Physiol. 2006; 290: C1146–C1159.
16. Tiruppathi C, Freichel M, Vogel SM, Paria BC, Mehta D, Flockerzi V, Malik AB. Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability. Circ Res. 2002; 91: 70–76.
17. Ahmmed GU, Mehta D, Vogel S, Holinstat M, Paria BC, Tiruppathi C, Malik AB. Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+ entry in endothelial cells. J Biol Chem. 2004; 279: 20941–20949.
18. Paria BC, Vogel SM, Ahmmed GU, Alamgir S, Shroff J, Malik AB, Tiruppathi C. Tumor necrosis factor-alpha-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability. Am J Physiol Lung Cell Mol Physiol. 2004; 287: L1303–L1313.
19. Singh I, Knezevic N, Ahmmed GU, Kini V, Malik AB, Mehta D. Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem. 2007; 282: 7833–7843.
20. Balzer M, Lintschinger B, Groschner K. Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells. Cardiovasc Res. 1999; 42: 543–549.
21. Groschner K, Rosker C, Lukas M. Role of TRP channels in oxidative stress. Novartis Found Symp. 2004; 258: 222–230.[Medline] [Order article via Infotrieve]
22. Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U, Mederos y Schnitzler M, Ghofrani HA, Schermuly RT, Pinkenburg O, Seeger W, Grimminger F, Gudermann T. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci U S A. 2006; 103: 19093–19098.
23. Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M. A key role for TRPM7 channels in anoxic neuronal death. Cell. 2003; 115: 863–877.[CrossRef][Medline] [Order article via Infotrieve]
Related Article:
Circ. Res. 2008 102: 347-355.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||