Oxidative-nitrosative stress and poly(ADP-ribose) polymerase in cardiovascular pathophysiology, ischemia/reperfusion injury and diabetic complications: cellular and molecular mechanisms.

Oxidative-nitrosative stress and consequent poly(ADP-ribose) polymerase (PARP) activation are key events in the development of endothelial and myocardial dysfunction in various models of cardiovascular injury and heart failure (ischemic, drug-induced and aging-associated; Fig. 1). Importantly, novel drug candidates targeting this pathway are entering or being evaluated in Phase II trials for a variety of critical care diseases associated with reperfusion injury and inflammation. These include, but are not limited to, ischemic stroke, acute respiratory distress syndrome, thoraco-abdominal aortic aneurism, repair surgery and the complications associated with cardiopulmonary bypass, and myocardial infarction as well as primary percutaneous coronary intervention.
We have demonstrated that peroxynitrite, a highly reactive oxidant formed from the reaction of nitric oxide and superoxide anion, is a key mediator of Doxorubicin (a widely-used chemotherapeutic drug)-induced cell death in cardiomyocytes and endothelial cells.

Diabetic vascular dysfunction is a major clinical problem which can lead to retinopathy, nephropathy, neuropathy and increased risk of stroke, hypertension and myocardial infarction. We have demonstrated that the aldose reductase inhibitor, fidarestat, counteracts diabetes- associated cataract formation, retinal oxidative-nitrosative stress, glial activation, and apoptosis. We have also described that the novel inosine analogue, INO-2002, protected against development of diabetes in mice. We have also shown that AdenosineA2A receptor activation inhibited T helper 1 and T helper 2 cell development and effector functions and protected against the development of diabetes.

Our impeding studies will also be directed towards the investigation of the role of oxidative/nitrosative stress related pathways in the development of complex hemodynamic alterations associated with diabetic cardiomyopathy, and the identification of novel therapeutic targets to counteract these pathological processes.

There is accumulating evidence indicating that endocannabinoids and synthetic cannabinergic ligands exert potent antioxidant, cytoprotective and antiinflammatory effects. Our recent studies showed that the non-psychoactive cannabinoid, cnnabidiol, attenuated high glucose-induced endothelial cell activation and barrier disruption, which are crucial early events underlying the development of various diabetic complications and atherosclerosis.

In collaboration with Professor David Kass we have created, for the benefit of the cardiovascular research community, a comprehensive online resource tool for complex hemodynamic measurements in mice using a sophisticated pressure-volume (P-V) system. We have also used the P-V system in various collaborations to characterize complex cardiac function in interesting knockout mouse models of cardiovascular disorders (e.g., in collaboration with Drs. Usdin and Kunos). In the future we will develop novel multisegment catheters, which are expected to improve volume recordings in situations where the heart axis is abnormal (e.g.. in various models of heart failure and myocardial hypertrophy).

Publications 2007-2008:

Csiszar A, Labinskyy N, Perez V, Recchia FA, Podlutsky A, Mukhopadhyay P, Losonczy G, Pacher P, Austad SN, Bartke A, Ungvari Z. Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice.

Am J Physiol Heart Circ Physiol 295: H1882-94, 2008.

Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, Zhang C, Mukhopadhyay P, Pacher P, Hu F, de Cabo R, Ballabh P, Ungvari Z. Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations.

Am J Physiol Heart Circ Physiol 294: H272 1-35, 2008.

Csoka B, Himer L, Selmeczy Z, Vizi ES, Pacher P, Ledent C, Deitch EA, Spolarics Z, Németh ZH, Haskó G. Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function.

FASEB J 22: 3491-9, 2008.

Csoka B, Németh ZH, Selmeczy Z, Koscsó B, Pacher P, Vizi ES, Deitch EA, Haskó G. Role of A(2A) adenosine receptors in regulation of opsonized E. coli-induced macrophage function.

Purinergic Signal 3: 447-52, 2007.

Csoka B, Németh ZH, Virág L, Gergely P, Leibovich SJ, Pacher P, Sun CX, Blackburn MR, Vizi ES, Deitch EA, Haskó G. A2A adenosine receptors and C/EBPbeta are crucially required for IL-10 production by macrophages exposed to Escherichia coli.
 
Blood 110: 2685-95, 2007.

Drel VR, Pacher P, Ali TK, Shin J, Julius U, El-Remessy AB, Obrosova IG. Aldose reductase inhibitor fidarestat counteracts diabetes-associated cataract formation, retinal oxidative-nitrosative stress, glial activation, and apoptosis.

Int J Mol Med 21: 667-76, 2008.

Drel VR, Pacher P, Vareniuk I, Pavlov I, Ilnytska O, Lyzogubov VV, Tibrewala J, Groves JT, Obrosova IG. A peroxynitrite decomposition catalyst counteracts sensory neuropathy in streptozotocin-diabetic mice.

Eur J Pharmacol 569: 48-58, 2007.

Drel VR, Pacher P, Vareniuk I, Pavlov IA, Ilnytska O, Lyzogubov VV, Bell SR, Groves JT, Obrosova IG. Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes.

Int J Mol Med 20: 783-92, 2007.

Fegley DB, Holmes A, Riordan T, Faber CA, Weiss JR, Ma S, Batkai S, Pacher P, Dobolyi A, Murphy A, Sleeman MW, Usdin TB. Increased fear- and stress-related anxiety-like behavior in mice lacking tuberoinfundibular peptide of 39 residues.

Genes Brain Behav 7:933-42, 2008.

Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases.

Nat Rev Drug Discov 7: 759-70, 2008.

Hasko G, Pacher P. A2A receptors in inflammation and injury: lessons learned from transgenic animals.

J Leukoc Biol 83: 447-55, 2008.

Hasko G, Pacher P, Deitch EA, Vizi ES. Shaping of monocyte and macrophage function by adenosine receptors.

Pharmacol Ther 113: 264-75, 2007.

Lengyel C, Virág L, Kovács PP, Kristóf A, Pacher P, Kocsis E, Koltay ZM, Nánási PP, Tóth M, Kecskeméti V, Papp JG, Varró A, Jost N. Role of slow delayed rectifier K+-current in QT prolongation in the alloxan-induced diabetic rabbit heart.

Acta Physiol (Oxf) 192: 359-68, 2008.

Levrand S, Pacher P, Pesse B, Rolli J, Feihl F, Waeber B, Liaudet L. Homocysteine induces cell death in H9C2 cardiomyocytes through the generation of peroxynitrite.

Biochem Biophys Res Commun 359: 445-50, 2007.

Mabley JG, Pacher P, Murthy KG, Williams W, Southan GJ, Salzman AL, Szabo C. The novel inosine analogue, INO-2002, protects against diabetes development in multiple low-dose streptozotocin and non-obese diabetic mouse models of type I diabetes.

J Endocrinol 198: 58 1-9, 2008.

Mabley JG, Wallace R, Pacher P, Murphy K, Szabo C. Inhibition of poly(adenosine diphosphate-ribose) polymerase by the active form of vitamin D.

Int J Mol Med 19:947-52, 2007.

Selmeczy Z, Csoka B, Pacher P, Vizi ES, Hasko G. The adenosine A2A receptor agonist CGS 21680 fails to ameliorate the course of dextran sulphate-induced colitis in mice.

Inflamm Res 56:204-9, 2007.

Selmeczy Z, Vizi ES, Csoka B, Pacher P, Hasko G. Role of nonsynaptic communication in regulating the immune response.

Neurochem Int 52:52-9, 2008.

Moon KH, Hood BL, Mukhopadhyay P, Rajesh M, Abdelmegeed MA, Kwon YI, Conrads TP, Veenstra TD, Song BJ, Pacher P. Oxidative inactivation of key mitochondrial proteins leads to dysfunction and injury in hepatic ischemia reperfusion.

Gastroenterology 135:1344-57, 2008.

Nemeth, Z.H. Bleich D, Csóka B, Pacher P, Mabley JG, Himer L, Vizi ES, Deitch EA, Szabó C, Cronstein BN, Haskó G. Adenosine receptor activation ameliorates type 1 diabetes.

FASEB J 21: 2379-88, 2007.

Pacher P, Szabo C. Role of poly(ADP-ribose) polymerase 1 (PARP-1) in cardiovascular diseases: the therapeutic potential of PARP inhibitors.

Cardiovasc Drug Rev 25:235-60, 2007.

Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease.

Physiol Rev 87: 315-424, 2007.

Pacher P. Poly(ADP-ribose) polymerase inhibition as a novel therapeutic approach against intraepidermal nerve fiber loss and neuropathic pain associated with advanced diabetic neuropathy: a commentary on "PARP Inhibition or gene deficiency counteracts intraepidermal nerve fiber loss and neuropathic pain in advanced diabetic neuropathy".

Free Radic Biol Med 44:969-71, 2008.

Pacher P, Sharma K, Csordas G, Zhu Y, Hajnoczky G. Uncoupling of ER-Mitochondrial Calcium Communication by Transforming Growth factor-J3.

Am J Physiol Renal Physiol 293: R1 303-12, 2008.

Pacher P, Szabo C. Role of the peroxynitrite-poly(ADP-ribose) polymerase pathway in human disease.

Am J Pathol 173: 2-13, 2008.

Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass DA. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.

Nat Protoc 3: 1422-34, 2008.

Role of the endocannabinoid system in tissue injury and inflammation.

During the last two years the main focus of our laboratory has been to understand the interplay of oxidative/nitrosative stress and inflammation with the endocannabinoid system (ES; an emerging very promising therapeutic target against various inflammatory and other diseases) in tissue injury associated with ischemia/reperfusion (I/R), doxorubicin-induced heart failure, and various in vitro and in vivo models of cardiovascular inflammation, as well as other pathologies associated with inflammation and tissue injury. These studies have demonstrated that oxidative/nitrosative stress is involved in the activation of the ES, and the stimulation of peripheral CB2 cannabinoid receptors protected against I/R-induced tissue injury by decreasing endothelial cell activation and inflammatory response and interrelated oxidative/nitrosative stress  

Since the endothelial and smooth muscle cell activation and inflammatory response play a pivotal role in the development of atherosclerosis, we have also explored the effects of selective CB2 receptor agonists (JWH1 33 and HU308) on these critical events using primary cultures of human coronary artery endothelial and smooth muscle cells (HCAEs and HCASMCs) and isolated vessels. We found that CB2 agonists dose-dependently attenuated the TNF-α induced NF-κB and RhoA activation, up-regulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of MCP-1, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Furthermore, the endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas, and the adhesion of monocytes to aortic vascular endothelium were also decreased by CB2 agonists. TNF-α triggered proliferation and migration of HCASMCs and activation of various interrelated signaling pathways (Ras, p38 MAPK, ERK 1/2, SAPK/JNK and Akt), which could be dose-dependently attenuated by CB2 agonists.

Our studies have also demonstrated marked protection of CB1 antagonists against Doxorubicin-induced cell death in cardiomyocytes and associated heart failure.

Our recent collaborative studies with Dr. Rohini Kuner have established an important role for the endocannabinoid system in acute and chronic pancreatitis and pain.

The above mentioned studies may identify new pharmacological targets in various forms of tissue injury and cardiovascular dysfunction associated with increased inflammation and tissue injury.

Publications 2007-2008:

Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, Michalski CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer MJ, Reeh P, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R. Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors.

Nat Neurosci 10: 870-9, 2007.

Batkai S, Mukhopadhyay P, Harvey-White J, Kechrid R, Pacher P, Kunos G. Endocannabinoids acting at CB1 receptors mediate the cardiac contractile dysfunction in vivo in cirrhotic rats.

Am J Physiol Heart Circ Physiol 293:H1689-95, 2007.

Batkai S, Osei-Hyiaman D, Pan H, El-Assal O, Rajesh M, Mukhopadhyay P, Hong F, Harvey- White J, Jafri A, Haskó G, Huffman JW, Gao B, Kunos G, Pacher P. Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury.

FASEB J 21 :1788-1800, 2007.

Batkai S, Rajesh M, Mukhopadhyay P, Haskó G, Liaudet L, Cravatt BF, Csiszár A, Ungvári Z, Pacher P. Decreased age-related cardiac dysfunction, myocardial nitrative stress, inflammatory gene expression, and apoptosis in mice lacking fatty acid amide hydrolase.

Am J Physiol Heart Circ Physiol 293: H909-18, 2007.

Engeli S, Heusser K, Janke J, Gorzelniak K, Bátkai S, Pacher P, Harvey-White J, Luft FC, Jordan J. Peripheral endocannabinoid system activity in patients treated with sibutramine. Obesity (Silver Spring) 16:1135-7, 2008.
Michalski CW, Laukert T, Sauliunaite D, Pacher P, Bergmann F, Agarwal N, Su Y, Giese T, Giese NA, Bátkai S, Friess H, Kuner R. Cannabinoids ameliorate pain and reduce disease pathology in cerulein-induced acute pancreatitis.

Gastroenterology 132: 1968-78, 2007.

Michalski CW, Maier M, Erkan M, Sauliunaite D, Bergmann F, Pacher P, Batkai S, Giese NA, Giese T, Friess H, Kleeff J. Cannabinoids reduce markers of inflammation and fibrosis in pancreatic stellate cells.

PLoS ONE 3:e1701, 2008.

Michalski CW, Oti FE, Erkan M, Sauliunaite D, Bergmann F, Pacher P, Batkai S, Müller MW, Giese NA, Friess H, Kleeff J. Cannabinoids in pancreatic cancer: correlation with survival and pain.

Int J Cancer 122:742-50, 2008.

Mukhopadhyay P, Bátkai S, Rajesh M, Czifra N, Harvey-White J, Haskó G, Zsengeller Z, Gerard NP, Liaudet L, Kunos G, Pacher P. Pharmacological inhibition of CB 1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity.

J Am Coll Cardiol 50:528-36, 2007.

Pacher P, Gao B. Endocannabinoids and liver disease. III. Endocannabinoid effects on immune cells: implications for inflammatory liver diseases.

Am J Physiol Gastrointest Liver Physiol 294:G850-4, 2008.
 
Pacher P, Hasko G. Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning.

Br J Pharmacol 153:252-62, 2008.

Pacher P, Mukhopadhyay P, Mohanraj R, Godlewski G, Bátkai S, Kunos G. Modulation of the endocannabinoid system in cardiovascular disease: therapeutic potential and limitations.

Hypertension 52:601-7, 2008.

Pacher P, Ungvari Z. Pleiotropic effects of the CB2 cannabinoid receptor activation on human monocyte migration: implications for atherosclerosis and inflammatory diseases.

Am J Physiol Heart Circ Physiol 294:H1133-4, 2008.

Pacher, P. Endocannabinoids, cannabinoid receptors and inflammatory stress: an interview with Dr. Pal Pacher. Interviewed by Helene F. Rosenberg.

J Leukoc Biol 82:1390-2, 2007.

Rajesh M, Mukhopadhyay P, Bátkai S, Haskó G, Liaudet L, Drel VR, Obrosova IG, Pacher P. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption.

Am J Physiol Heart Circ Physiol 293:H610-9, 2007.

Rajesh M, Mukhopadhyay P, Bátkai S, Haskó G, Liaudet L, Huffman JW, Csiszar A, Ungvari Z, Mackie K, Chatterjee S, Pacher P. CB2-receptor stimulation attenuates TNF-alpha-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte-endothelial adhesion.

Am J Physiol Heart Circ Physiol 293:H2210-8, 2007.

Rajesh M, Pan H, Mukhopadhyay P, Bátkai S, Osei-Hyiaman D, Haskó G, Liaudet L, Gao B, Pacher P. Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis.

J Leukoc Biol 82:1382-9, 2007.

Rajesh M, Mukhopadhyay P, Haskó G, Huffman JW, Mackie K, Pacher P. CB2 cannabinoid receptor agonists attenuate TNF-alpha-induced human vascular smooth muscle cell proliferation and migration.

Br J Pharmacol 153: 347-57, 2008.

Role of oxidative-nitrosative stress in ethanol-induced tissue-damage.

Moderate and heavy drinking may significantly influence cardiovascular function and aging in different ways. During the course of the last decade, several research groups have reported that, in animal models of myocardial ischemia/reperfusion, ethanol and non-ethanol components of wine may have a specific protective effect on the myocardium, independent of the classical risk factors implicated in vascular atherosclerosis and thrombosis. Apoptosis is a mechanism of cell death implicated in the pathogenesis of alcohol-induced organ damage. Experimental studies have suggested alcohol-mediated apoptosis in cardiac muscle, and there is also evidence of skeletal muscle apoptosis in long-term high-dose alcohol drinkers. Apoptosis is present to a similar degree in the heart muscle of high-dose alcohol drinkers and long-standing hypertensive subjects and is related to structural damage.

We have recently developed an assay allowing simultaneous quantitative detection of oxidative stress and apoptosis in virtually any live cells, which we utilized in our studies and in various collaborations. Our future studies will be focused on the understanding of the mechanisms of ethanol-induced oxidative/nitrosative stress and apoptosis in the cardiovascular system and also in other organ systems.
Publications 2007-2008:

Horiguchi N, Wang L, Mukhopadhyay P, Park O, Jeong WI, Lafdil F, Osei-Hyiaman D, Moh A, Fu XY, Pacher P, Kunos G, Gao B. Cell type-dependent pro- and anti-inflammatory role of signal transducer and activator of transcription 3 in alcoholic liver injury.

Gastroenterology 134: 1148-58, 2008.

Mukhopadhyay P, Rajesh M, Haskó G, Hawkins BJ, Madesh M, Pacher P. Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy.

Nat Protoc 2:2295-301, 2007.

Mukhopadhyay P, Rajesh M, Yoshihiro K, Hasko G, Pacher P. Simple quantitative detection of mitochondrial superoxide production in live cells.

Biochem Biophys Res Commun 358:203-8, 2007.