Innate Inflammation as the Common Pathway of Risk Factors Leading to Transient Ischemic Attacks and Stroke

Agenda

* Presentation times are subject to change

DAY 2: MAY 10, 2010

Specific Pathways for the Down-Regulation of Innate Inflammation 

Robert A. Campbell, Guy A. Zimmerman, and Andrew S. Weyrich, PhD, University of Utah

There is a growing appreciation that secondary stroke and related ischemic events are manifestations of progressive inflammation. Inflammatory events are controlled by several key cells that include platelets, leukocytes and endothelial cells. Interactions among platelets and leukocytes at the vascular wall induce an array of thrombotic and inflammatory processes that, in some cases, leads to the initiation and development of deleterious blood clots. Previous work from our group has demonstrated that interactions between activated human platelets and monocytes induce inflammatory gene expression in target monocytes. Monocyte chemotactic protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) are among a group of genes that are generated by platelet-monocyte aggregates. Dipyridamole, which is used clinically in combination with low-dose aspirin to treat patients with prior ischemic stroke, significantly decreases MCP-1 and MMP-9 synthesis by platelet-monocyte aggregates. This indicates that dipyridamole and aspirin combination therapy has anti-platelet and anti-inflammatory properties. Recently we developed more complex model systems to examine the effects of dipyridamole and aspirin on global gene expression patterns in endothelial cells and monocytes. In the first model, platelet-rich clots were formed adjacent to endothelial cells and after two hours, mRNA was collected for illumina-based RNA deep sequence analyses. In the second model, clots that spontaneously lysed were formed in human whole blood and RNA was subsequently extracted from platelet-monocyte aggregates. In both model systems, cells were pretreated with dipyridamole and aspirin or its vehicle. Our results indicate that thousands of genes were down or upregulated in both model systems. Many of these genes were differentially expressed in the presence of dipyridamole and aspirin providing further evidence that this combination therapy regulates the expression of select transcripts.

Endothelial and Blood-Brain Barrier Function: Inflammation, Ischemia, and Hemorrhage

Mark Fisher, MD, Departments of Neurology, Anatomy & Neurobiology, and Pathology & Laboratory Medicine, UC Irvine

Traditional concepts of stroke focus on presence of clinically-evident neurological deterioration, affecting a large but still limited population of patients. Insights from brain imaging studies suggest that this is the proverbial tip of the iceberg. Specifically, cerebral white matter disease, usually viewed as a variant of cerebrovascular disease, is known to affect more than 95% of the population over the age of 65 years. Cerebral white matter disease is substantial in approximately one-third of cases, and presence of multiple vascular risk factors amplifies the extent of white matter disease. Moreover, cerebral microbleeds as seen on MRI are known to affect nearly 20% of the population over age 60 and nearly 40% of the population over the age of 80. Recent pathological analysis suggests that prevalence of cerebral microbleeds in the elderly may be higher. Imaging studies indicate a direct relationship between presence of cerebral white matter disease and presence of cerebral microbleeds. Limited pathology studies suggest cerebral white matter disease abnormalities occurring at level of blood-brain barrier, while capillary involvement appears to underly some cerebral microbleeds. Microbleeds occurring in the microcirculation are associated with inflammatory component, implying inflammatory reaction occurring as a consequence of blood-brain barrier alteration rather than simply the cause of barrier dysfunction. These varied observations suggest ubiquity of cerebrovascular disease in the aging population and imply some common underlying mechanisms at the level of the blood-brain barrier for both cerebral white matter disease and cerebral microbleeds.

Connexins and Gap Junctions in Ischemic Conditions and Inflammation: Is Gap Junction Coupling in Vascular Cells A Pharmacological Target?

Daniela Begandt, Willem Bintig, Karina Oberheide, Natalie Eisert, Thilo Reeck, Anaclet Ngezahayo, PhD, Institute of Biophysics, Leibniz University of Hannover, Hannover, Germany

Because of its involvement in regulation of vascular functions, gap junction coupling in the vascular cells has been proposed as a possible pharmacological target in management of cardiovascular pathologies. We applied the scrape loading technique to analyse the effects dipyridamole on gap junction coupling of GM-7373 cell line, a model of aortic endothelial cell. We found that pharmacological relevant concentrations of dipyridamole (1-75 µM) induced an increase of gap junction coupling. With respect to the involved mechanism, we found that the activator of adenylyl cyclase forskolin as well as the cAMP analogue 8-Br-cAMP mimicked the effect of dipyridamole on gap junction coupling of endothelial cells. Moreover the PKA inhibitors H-89 or Rp-cAMPs antagonised the dipyridamole induced increase of gap junction coupling. Considering the time course at which dipyridamole affected the gap junction coupling, we observed that it required a drug presence on the cells for at least 6 h to induce a significant increase of gap junction coupling, indicating that dipyridamole probable affected newly formed gap junction channels. All results observed in endothelial cells could be reproduced in the rat A-10 cells, a model of aortic smooth muscle cells. It is concluded that dipyridamole increases gap junction coupling by a cAMP/PKA dependent mechanism. This action could be involved in the beneficial effect of dipyridamole treatment of the cardiovascular impairments. Furthermore the results indicate that gap junction coupling of the vascular cells is a suitable target for dipyridamole.

Pharmacological Modulation Of Vascular Inflammation In Atherothrombosis

R. De Caterina, MD, PhD, M. Massaro*, PhD, M.A. Carluccio*, DBiol, and E. Scoditti*, PhD
Institute of Cardiology, “G. d’Annunzio” University – Chieti , and CNR Institute of Clinical Physiology, Lecce – Italy

Vascular inflammation has been shown to play a pivotal role in the inception, progression and clinical complications of atherosclerosis. Animal models of atherosclerosis have shown a causal role of several inflammatory mediators, such as vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), CD-40/CD-40 ligand. Gene knock-out or pharmacological inhibition strategies against these targets have demonstrated to impact atherosclerosis. The common denominators for the activation of inflammatory genes appear however to be a small subset of transcription factors (among which nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and GATA), functioning as the central hub of vascular inflammation. Strategies directed to inhibit both secondary mediators and the primary triggers (atherosclerosis risk factors) appear both viable to inhibit atherosclerosis. Attempts have been now made also to address the central hub of vascular inflammation. “Old” drugs, such as dipyridamole, can also be now revisited for properties related to inhibition of vascular inflammation, probably by acting on the same common targets. In in vivo models of endothelial activation on cultured endothelial cells, dipyridamole exerts inhibitory effects on phorboly myristate acetate- and tumor necrosis factor (TNF)-α-induced expression of cyclooxygenase-2 (COX-2), in turn linked to reduced expression of matrix metalloproteinase-9, through a mechanism involving the reduced intracellular production of reactive oxygen species. This is linked to inhibition of endothelial cell migration and angiogenesis, potentially leading to a stabilization of atherosclerotic plaques and explaining some of the protection found with this old drug on the recurrence of stroke and ischemic cerebro-vascular events.

Role of NF-κB and Innate Immune Response in Ischemic Stroke

Hyung-Hwan Kim, PhD¹, Olivier A. Harari, MD², Daniela Ahl, PhD¹, Emiri Tejima, MD⁴, Hongwei Wang, MD, PhD¹, Ruth Schmidt-Ullrich, PhD³, Eng H. Lo, PhD⁴, Michael A. Moskowitz, MD⁴, James K. Liao, MD¹
1. Brigham & Women’s Hospital, Boston, MA, USA
2. Hammersmith Hospital, Imperial College London, UK
3. Max-Delbrück-Center of Molecular Medicine, Berlin, Germany
4. Massachusetts General Hospital, Boston, MA, USA

Cerebral ischemia induces an innate immune response, which is mediated in part by the pro-inflammatory transcription factor, NF-κB. However, the contributory role of NF-κB in vascular wall cells and circulating leukocytes to ischemia-induced cerebral injury is not known. Mice with endothelial-specific inhibition of NF-κB (ecNFκB-/-) were established by crossing mice with loxP-flanked IκBαsr (inhibitor of NF-κB) alleles with transgenic mice expressing a Cre-fusion protein under control of the endothelial-specific Tie2 promoter. Transient cerebral ischemia was produced by left middle cerebral artery occlusion (MCAO) for 2 hrs followed by reperfusion. After 24 hrs, cerebral infarct areas were quantified using TTC. Changes in blood-brain barrier integrity were assessed using Evans-blue staining. Because Tie2 is also expressed in hematopoietic stem cells, bone marrow transplantation (BMT) of WT BM (donor) into irradiated recipient mice (WT or ecNFκB-/- mice) was performed to distinguish the effect of NF-κB in endothelial cells versus leukocytes. Compared to controls, ecNFκB-/- mice exhibited increased infarct size and higher neurological deficit score following MCAO. However, vascular leakage was smaller in ecNFκB-/- mice compared to controls. After BMT, infarct size tended to be larger in ecNFκB-/- mice compared to that of controls. In contrast, vascular leakage in recipient ecNFκB-/- mice receiving WT BMT was similar compared to that of controls receiving WT BMT. These findings indicate that endothelial NF-κB may protect against cerebrovascular injury while leukocyte NF-κB worsens vascular leakage. These results suggest that NF-κB may have protective and detrimental effects depending upon the cell type in which NF-κB is being modulated.

Role of Socs Proteins in Influencing Glial Cell Inflammatory Responses

Etty (Tika) Benveniste, PhD, Hongwei Qin, Ph.D., Xiangyu Ma, MD, University of Alabama at Birmingham, AL

The Suppressor Of Cytokine Signaling (SOCS) family contains eight members: CIS and SOCS1-SOCS3, best known for their negative regulation of the JAK/STAT signaling pathway. SOCS1 and SOCS3 are expressed by immune cells and cells of the Central Nervous System (CNS), and have the potential to impact immune processes within the CNS, including cytokine and chemokine production; activation of microglia, macrophages and astrocytes; immune cell infiltration; and autoimmunity. We describe the role of SOCS1 and SOCS3 in regulating astrocyte function with respect to cytokine and chemokine production, and how this impacts immune cell migration. IFN-β treatment of astrocytes induces robust expression of chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL10. However, this expression is self-regulated by concurrent IFN-β-induced expression of SOCS1 and SOCS3, as shown by increased production of these chemokines when SOCS1 or SOCS3 is experimentally depleted. Functionally, this increase in chemokine expression correlates with enhanced migration of macrophages and T-cells. Thus, SOCS1 and SOCS3 expression in astrocytes may limit inflammatory cell migration into the CNS. In addition, SOCS3 is able to influence the NF-κB and MAPK signaling pathways in astrocytes, thereby affecting cytokine/chemokine production. SOCS proteins have both detrimental and beneficial effects in neuroinflammatory diseases, depending on the neurological insult and cell type(s) expressing SOCS proteins.

Modulation of Cell Surface by Lipophylic Compounds and Its Distribution into Different Blood Cell Types over Time

Victor Serebruany, MD, PhD,HeartDrug™ Research Laboratories, Johns Hopkins University, Baltimore, MD

Background and Purpose -Earlier studies have shown that a lipophylic compound with strong antioxidative properties, such as dipyridamole (D)in vitro reduces the number of putative binding sites for the prothrombinase complex on platelets in patients indentified as resistant to antiplatelet treatment. This phenomenon seems to indicate that under yet unknown conditions the membrane of platelets exhibit a higher number of negatively charged phospholipids on the outer leaflet with yet unknown potential immunogenicity. To further elucidate the role of a potential approach to reduce membrane disturbance, as an example for such lipophyllic radical scavenger ex vivo was treated for its differential distribution amongst different blood. We hypothesized that since D is known to be highly lipophylic, the drug may be present not only in plasma, but also accumulated in platelets, leukocytes, and erythrocytes.
Methods - Fifteen patients after documented ischemic stroke were treated with Aggrenox BID for 30 days, and 12 of them completed the study. Extended released (ERD) concentrations in blood cells and platelet poor plasma were measured by spectrofluorimetry at Baseline, Day 14, and Day 30 after the initiation of therapy. Results - The background level of spectrofluorometry readings differs slightly among the blood components (132-211 ng/mL) due to the differences in the preparation of samples and cell isolation techniques. As expected, 2 weeks of D therapy produced steady state plasma concentration of D already at Day 14 (1680±542ng/mL), followed by a slight not significant decrease at one month (1619±408). Two weeks of therapy was sufficient to achieve a consistent D accumulation in erythrocytes (361±43), but not in platelets (244±78), or leukocytes (275±49). In fact, white blood cells continued membrane D intake beyond 14 days period, and this increase (398±66) was significant (p=0.02) at thirty days. Conclusion - Treatment with Aggrenox in post-stroke patients resulted not only in the achieving of therapeutic plasma D concentrations, but also deposition of the drug in erythrocytes and leukocytes, but not in platelets. If confirmed, these data will affect our better understanding of D pleiotropy, and may explain long term benefit of ERD formulation.

The Role of miRNA/Gene Expression and Reactive Oxygen Species in Innate Inflammation

Jane E. Freedman, MD, Boston University School of Medicine, Boston, MA

Objective: Diabetes is associated with an increased inflammatory state characterized by enhanced oxidative stress. Diabetes is also known to increase the risk for cerebrovascular disease and stroke. Antithrombotic therapy is effective in preventing stroke, however, platelet inhibition, particularly in the setting of fibrinolytic treatment may increase the risk of hemorrhagic events. Using an embolic stroke model, the present study was designed to examine the interaction between increased inflammatory state and stroke as well as identify novel markers of clinical outcome. An embolic stroke/thrombolytic model was studied as was the effect of the redox modulating agent dipyridamole. Distinct patterns of stroke, inflammatory biomarkers, and miRNA were associated with treatment and outcome.

The Neurovascular Unit, Matrix Proteases, and Innate Inflammation

Gregory J. del Zoppo, MD^1,2, Yu-Huan Gu, PhD¹, Harold Frankowski, PhD¹, Takashi Osada, MD¹, Richard Milner, MD²
1. University of Washington, School of Medicine, Department of Medicine (Hematology) and Department of Neurology, Seattle, WA
2. The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA

Cerebral microvessels and their recipient neurons, as well as other components of the “neurovascular unit,” suffer a sequence of consistent and complex changes following focal cerebral ischemia. The neurovascular unit is both a structural and conceptual framework that recognizes functional interactions among the cells and their environment. Microglial cells interact with the microvessel endothelium-matrix-astrocyte complex, which communicates with the neuron. While focal cerebral ischemia initiates rapid expression of surface adhesion receptors on the endothelium to initiate cellular inflammation, events within the neuropil promote microglial activation. During focal ischemia, hemorrhagic transformation is associated with the appearance of pro-MMP-9 in the non-human primate. Studies using primary murine endothelial cells, like astrocytes and microglia, suggest that this protease (and other matrix proteases) is generated when microglial cells interact with matrix substrates found only in the plasma. Exposure to specific matrix substrates found predominantly in the plasma stimulates pro-MMP-9, and not pro-MMP-2, generation by activated microglial cells under conditions of experimental ischemia. This suggests that the microglial compartment is poised to initiate matrix protease release under conditions of plasma exposure, and focal ischemia. These observations support a number of implications about how stroke acutely injures both nonvascular and vascular tissues within the brain, how components of the neurovascular unit might communicate under conditions of injury, and the roles of a matrix environment in the function of cells within the neurovascular unit.

Biphasic Inflammatory Responses in Injury and Repair after Stroke

Eng H. Lo, PhD, Departments of Radiology and Neurology, Massachusetts General Hospital

Inflammation may be an important contributor to the pathophysiology of brain injury after stroke. During the acute phase, various neurovascular mediators may augment endothelial activation and infiltration of inflammatory cells into damaged brain. These early events may serve to increase blood-brain barrier leakage, edema, hemorrhage and cell death. During the delayed phase however, continued inflammatory signals may conversely serve to mediate endogenous substrates of remodeling. These mechanisms of neurovascular plasticity and repair may underlie angiogenesis and neurogenesis as stroke patients recover over time. In this presentation, two representative examples of this overall theme wil be discussed: matrix metalloproteinases and the alarmin protein HMGB1. During the initial stages of cerebral ischemia, dysregulated matrix metalloproteinases may degrade blood-brain barrier integrity and amplify neurovascular injury. But during delayed stages post-ischemia, matrix metalloproteinases may contribute to neuroblast migration and peri-infarct cortical remodeling. During the early stages of cerebral ischemia, releae of HMGB1 from dying cells in the core may serve to expand neuronal necrosis and inflammation. But during the later stages of stroke, upregulation of HMGB1 in reactive astrocytes may contribute to dendritic plasticity and angiogenesis. Taken together, these findings suggest that biphasic responses in neurovascular inflammation may blur the boundaries between injury and repair after stroke.

Modulation of Blood Brain Barrier Leakage in Amyloid Angiopathy in Rodents

David H. Cribbs, PhD^1,2, Giselle Passos, PhD², Daniel Quiring², Vitaly Vasilevko, PhD²
1. Department of Neurology, University of California, Irvine, Irvine, CA
2. Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, Irvine, CA

Cerebral amyloid angiopathy (CAA) and hypertension are the most common causes of intracerebral hemorrhage, and preexisting vascular pathology in the elderly increases CAA and microhemorrhages (AN1792) and vasogenic edema (bapineuzumab AAB-001) in response to anti-amyloid-beta-immunotherapy (IT). Old amyloid-precursor-protein-transgenic (APP-Tg) mice develop CAA and microhemorrhages, and IT further exacerbates the CAA and microhemorrhages. Therefore we have used APP-Tg2576 mice to investigate the effect of dipyridamole and minocycline on the safety and efficacy of IT. Old APP-Tg2576 mice with extensive CAA were passively immunized (PI), dosed weekly (i.p.) at 10mg/kg per mouse, with anti-Aß40 C-terminal specific monoclonal antibody. Minocycline diet (normal chow) and dipyridamole diet (high-fat chow) were given to mice ad libitum. The high-fat-diet complicated the interpretation of the data in the dipyridamole study. Dipyridamole attenuated the increase in the CNS amyloid caused by the high-fat-diet, and no adverse events were detected in PI mice receiving the dipyridamole diet. Thus our data suggest that dipyridamole may not present a significant risk factor in elderly patients with CAA. Passive immunization of APP-Tg2576 mice reduced amyloid plaque load and microglial activation. Minocycline treatment further attenuated the amyloid-induced microglial response, however minocycline also attenuated plaque clearance. Both PI groups had extensive vascular CAA and antibody-induced microhemorrhages, however chronic anti-inflammatory therapy reduced microhemorrhage progression in APP-Tg2576 mice. Our data suggest that microglia are involved in antibody-mediated plaque clearance in older APP-Tg mice. Ultimately, the safety of immunotherapy may be dependent on the health of the blood-brain-barrier, which unfortunately is significantly compromised in many Alzheimer’s patients.

Examining Cerebral Hemodynamics, Inflammation and Outcome in Rabbit Model of Middle Cerebral Artery Embolic Occlusion: A Pilot Study

C.D. d’Esterre^1,2,3 BMSc, K.M. Tichauer^1,2 PhD , R.I. Aviv⁴ MD , F. Sun⁵ PhD, B. Garcia⁵ PhD, W. Eisert⁶ MD, T-Y Lee^1,2,3 PhD
1 Medical Biophysics, TheUniversity of Western Ontario, London, Ontario, Canada
2 Lawson Health Research Institute, London, Ontario, Canada
3 Robarts Research Institute, London, Ontario, Canada
4 Medical Imaging, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada
5 Department of Pathology, The University of Western Ontario, London, Ontario, Canada
6 University of Hannover, Hannover, Germany

Background and Purpose – Dipyridamole (DP) is becoming a routine prescription for reducing stroke in high-risk patients. Clinical observations have indicated that DP may also ameliorate the effects of stroke through anti-platelet and anti-inflammatory mechanisms. The purpose of this study was to explore the potential of DP treatment to improve outcome in a controlled animal model of middle cerebral artery occlusion. Methods – Twenty male New Zealand white rabbits were randomly selected for intravenous treatment with DP (n=10) or saline (n=10) for 7 days prior to a middle cerebral artery occlusion by an autologous blood clot. Multiple Computed Tomography Perfusion (CTP) scans were acquired out to 28 days post-stroke to map cerebrohemodynamics, in conjunction with neurological assessments and histology. Results – The DP group fared better than the saline group on several accounts: 66% of them survived to 28 days, while saline animals all had to be euthanized by day 7 due to severe neurological deficits. Extent of hemorrhage, apoptosis, and peri-infarct inflammation were significantly lessened in the DP group (p<0.05). They presented with significantly more viable tissue in the ischemic hemisphere (69 ± 7% vs. 34 ± 10%, p<0.05), as well as fewer neurological deficits on days 4 and 7 (p<0.05). Furthermore, DP-treated animals exhibited improved cerebrohemodynamics by 72 h (p<0.05). Conclusions – We used an animal stroke model to demonstrate that continuous pre-morbid treatment with therapeutic concentrations of DP can be neuroprotective post ischemic insult.

Potential Interactions between Anti-Platelet Drugs and Statins on Protection against Ischemia-Reperfusion Injury

Yochai Birnbaum, MD^1,2, Yumei Ye, MD²
1. The Section of Cardiology, Baylor College of Medicine, Houston, TX
2. The Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX

Background: Statins protect against ischemia-reperfusion injury and limit myocardial infarct size (IS). This effect is dependent on increased generation of adenosine by ecto-5’ nucleotidase and downstream activation of cyclooxygenase-2 (COX2). Dipyridamole (DIP) augments the IS-limiting effects of statins by blocking the cellular reuptake of adenosine; whereas aspirin (ASA) attenuates the effect by inhibiting COX2. A combination of DIP with low-dose ASA is as effective as clopidogrel in secondary stroke prevention; however, the role of DIP+ASA in acute myocardial infarction is unclear. We studied the effect of acute administration of DIP, ASA and their combination on the IS-limiting effect of simvastatin (SIM). Methods: Rats received oral SIM (10mg/kg/d) or vehicle for 3 days. Rats underwent 30min of coronary artery occlusion and 4h reperfusion. At 5min of ischemia rats received i.v. DIP (5mg/kg), ASA (20mg/kg or 2mg/kg) or DIP+ASA (2mg/kg) or vehicle alone. Ischemia area at risk (AR) was assessed by blue dye and IS by TTC. Results: SIM limited IS. ASA 2 and 20 alone had no effect on IS. DIP alone or with ASA 2 significantly reduced IS. ASA 2 did not attenuate the SIM effect, whereas ASA 20 completely blocked the effect. The combination of DIP+ASA2+SIM resulted in the smallest IS. Both SIM and DIP+ASA2 augmented Akt phosphorylation and their effect was additive. Both SIM and DIP+ASA2 augmented ERK 1/2 and cyclic-AMP-response-element-binding-protein (CREB) phosphorylation. Conclusions: Administration of DIP alone or with low-dose ASA during acute myocardial ischemia limited IS and did not attenuate the IS-limiting effect of SIM, as high-dose ASA.

Reperfusion of Chronic Tissue Ischemia: Redox Regulation of Innate Immune Responses

Christopher G Kevil, PhD and Christopher B. Pattillo, PhD, Department of Pathology, LSU Health Sciences Center Shreveport, LA

Chronic and intermittent ischemic vascular disorders represent a burgeoning clinical challenge. Previous studies have focused on the idea that functional revascularization of affected tissue through therapeutic angiogenesis strategies would alleviate the disorder. However, it is becoming increasingly appreciated that ischemic vascular dysfunction is not simply limited to defective vascular cell wall responses but also influenced by chronic inflammatory responses which simultaneously occur. Our laboratory has discovered that pharmacological treatment of experimental permanent tissue ischemia with dipyridamole significantly augments ischemic tissue reperfusion, endothelial cell growth, angiogenesis, and arteriogenesis over time. We have found that the beneficial effects of dipyridamole therapy are due to its ability to activate the recently described nitrite/nitric oxide endocrine system which invokes redox tissue reduction of nitrite anion to bonafide NO only under permissive conditions. Importantly, we have recently discovered that nitrite anion dependent reperfusion of chronically ischemic tissue results in selective down regulation of innate immune response genes such as chemokine ligands, toll like receptors, and leukotriene and formyl peptide receptors, while increasing expression of vascular growth and repair genes such as VE cadherin, thrombospondin 2, and serotonin receptor 2B. These data demonstrate that dipyridamole induced nitrite effects work to simultaneously decrease inflammatory responses while increasing vascular remodeling of ischemic tissues. Novel approaches targeting redox regulation of innate immune responses may represent useful clinical strategies in which to treat ischemic vascular disease.

Effect of Intravenous Administration of Dipyridamole in a Rat Model of Chronic Cerebral Ischemia

Alessia Melani, Sara Cipriani, Francesca Corti, and Felicita Pedata, Department of Pharmacology, University of Florence, Florence, Italy

Purpose of this study, performed in the animals, is to verify whether Dipyridamole improves cognitive functions in a model of chronic cerebral ischemia in the rat obtained by occlusion of the two carotids (2VO). Such animal model mimics, although with some limitation, a situation of chronic hypoperfusion in humans that may be present in aging. Dipyridamole (Boehringer Ingelheim, 5mg/ml) or vehicle were administered (10 µl/h per 7 days) into the jugular vein by a mini-osmotic pump. Fifteen rats for each group (sham-operated, vehicle-treated and Dypiridamole-treated) were tested. The body weight of Dipyridamole-treated rats was not different from that of sham-operated rats while it decreased in vehicle-treated rats. In vehicle- and Dipyridamole-treated rats, the neurological deficit score decreased at 60 and 90 days after 2VO. Sixty and ninty days after 2VO, vehicle-treated rats showed alternation impairment in the Y maze test that evaluates spatial working memory. Dipyridamole-treated rats showed alternation impairment at 60 days after 2VO but anymore at 90 days. Non spatial working memory evaluated by object recognition test was not different among the three animal groups. Histological assessment by cresyl violet staining showed that, 90 days after operation, there was no cell damage in the CA1 area and gyrus dentate of the hippocampus of hypoperfused rats. Immunohistochemical evaluation by myelin associated glycoprotein (MAG) staining, showed that myelin distribution was not disordered in the hippocampus. In conclusion, 90 days after 2VO, dipyridamole, significantly restores working memory although it does not significantly ameliorates neurological deficit.

Impact of Innate Inflammation in Population Studies

Mitchell S. V. Elkind, MD, MS, FAAN, Columbia University, New York, NY

Acute phase proteins have been the most extensively studied inflammation-related biomarkers in relation to risk of stroke. High sensitivity C-reactive protein (hsCRP), in particular, has many features that recommend it as a molecular marker of the risk of stroke associated with inflammation. CRP is part of the innate immune response, and it is capable of several immunological actions that may contribute to the chronic inflammatory process that constitutes atherosclerosis. Assays for hsCRP, moreover, have several advantages. Disadvantages to the use of acute phase proteins as measures of vascular risk, however, are that they are non-specific, they are associated with a number of other risk factors, and acute increases in levels may occur in the setting of acute infection or other illness. The mechanisms by which hsCRP may be associated with stroke remain uncertain, moreover. The relationship of hsCRP to incident stroke, therefore, is controversial and probably depends on study design and population. Nonetheless, several studies and meta-analyses support the potential for measurements of hsCRP to aid prediction of future vascular risk, including stroke, and patients with elevated levels of inflammatory markers might be candidates for anti-inflammatory therapies, including statins. The role of measurement of hsCRP and other acute phase proteins after stroke is also uncertain. Other biomarkers related to inflammation, such as cytokines, cellular adhesion molecules, and lipoprotein-associated phospholipase A2, have also been associated with vascular risk and stroke recurrence, and further studies are needed to confirm their utility in different populations.

Innate Inflammation in Myocardial Perfusion and Its Applications in Heart Failure

Eugenio Picano, MD, PhD, Rosa Sicari, MD; Maria Aurora Morales, MD, CNR, Institute of Clinical Physiology, Pisa, Italy

Cytokines are involved in the pathophysiology of heart failure, which is characterized by a chronic inflammatory status. Cells of the immune system express adenosine receptors and are responsive to the modulating effects of adenosine in an inflammatory environment. In particular, of all adenosine receptors, A2A receptors are abundantly expressed in the heart, and cardiac myocytes, fibroblasts and endothelial cells as well as infiltrating hemopoietic cells are the major cell types in which A2A receptor expression was been detected but the relevance of adenosine-mediated anti-inflammatory effects in heart failure remains unclear. The inflammatory status may mediate, at least in part, the impairment in coronary microvascular function which has been implicated in the pathogenesis and prognosis of ischemic and non-ischemic dilated cardiomyopathy. Improvements in microvascular function can be obtained with cellular (bone marrow derived progenitor cells) or drug (adenosine-modulating agents) therapy, with some preliminary clinical evidence supporting the improvement in clinical status and myocardial perfusion in both ischemic and non-ischemic cardiomyopathy. Pilot clinical observational studies showed a striking beneficial effect of endogenous adenosine accumulation (achieved with different drugs) on symptoms, exercise capacity and left ventricular function in chronic heart failure. In addition, a combination of adenosine, heparin and exercise (inducing myocardial ischemia or increase in shear stress) may promote formation of coronary collaterals and improvement of function in ischemic cardiomyopathy. In summary, coronary microcirculatory dysfunction, and underlying inflammation, can be a trigger of disease, and a potential target for therapeutic interventions in heart failure patients.

Inflammatory Biomarkers Time Course after Ischemic Stroke in German Stroke Victims

Worthmann, Hans, MD¹, Tryc, Anita Blanka, MD¹, Deb, Milani, MD¹, Goldbecker, Annemarie, MD¹, Ma, YueTao, MD^1,3, Tountopoulou, Argyro, MD¹, Lichtinghagen, Ralf, PhD² and Weissenborn, Karin, MD¹.
1. Department of Neurology, Medical School of Hannover, Germany
2. Department of Clinical Chemistry, Medical School of Hannover, Germany
3. Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, China

Success in stroke treatment strategies is limited due to the low tolerance of brain tissue to ischemia and the small number of drugs that could efficiently interrupt the fatal cascade of ischemia and subsequent inflammatory damage. After occlusion of cerebral arteries and rapid depletion of substrates one of the predominant mechanisms of secondary progression of brain injury is represented by inflammation. In animal models a great number of studies investigated this cellular and noncellular inflammatory response in the lesioned tissue. Since a direct approach is unfeasible in ischemic stroke patients surrogate markers based on imaging and biochemistry were used to reflect the inflammatory process in humans. To illuminate the importance of discrete biochemical markers that have been shown to take part in the complex inflammatory response after stroke, and to gain further insights into their interplay in this condition our group and some others have measured the time courses of a selection of plasma markers in acute ischemic stroke patients. Here we highlight the complex inflammatory response in acute ischemic stroke patients by focussing on own data in german stroke victims. Thereby we show characteristic time courses for selected markers of inflammation and brain damage depending on stroke outcome and concomitant infectious diseases. Thus sequential dynamics of the early inflammation must be considered for upcoming mechanism-targeting anti-inflammatory strategies in ischemic brain damage.

Time Course of Inflammatory Markers after Acute Ischemic Stroke and Were Likely To Correlate With Stroke Severity and Outcome

Liping Liu¹ ,Yuetao Ma¹, Xingquan Zhao¹, H Worthmann², Yilong Wang¹, Yongjun Wang¹
1. Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
2. Departments of Neurology, Hannover Medical School, Germany.

Background: A non-specific systemic inflammatory response occurs after ischemic stroke, higher levels of these markers are associated with worse outcome and can even improves prediction. But the delay between blood taking and stroke did leave time for the development of complications in some of the more severely stroke. Purpose: To investigate the time course of selected inflammatory markers such as high sensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), S-100, monocyte chemoattractant protein-1 (MCP-1), Tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-9 (MMP-9) from 3 hrs after acute ischemic stroke , and to seek its potential clinical implications. Methods: We prospectively recruited 48 consenting super acute ischemic stroke patients and 30 age and sex matched healthy controls. Demographic and baseline characteristics were collected for all controls and the patients. Blood samples were withdrawn at serial time points (3or 6h, 12h, 24h, 48h, 3d and 7d) after symptom onset for stroke patients and only once at admission for control group. All the inflammatory markers were tested by commercially available assays. Clinical severity and 3 months outcome were measured by NIHSS score and modified Rankin Scale (mRS) respectively. Results: Hs-CRP and IL-6 level at each time point in stroke patients was significantly higher than those in controls (all p≤0.01 for hs-CRP and IL-6). Both elevated from 3h after symptom onset, peak at 3 days, and started to fall down again at 7 days, but keep the high levels continued at higher than healthy controls. IL-6 showed persistent higher levels in both hypertensive and diabetic patients compared to without respectively. While hs-CRP and MCP-1 showed higher levels in diabetic patients compared with non diabetic patients. At each time point, Hs-CRP, IL-6 as well as MMP-9 showed higher levels in smoking patients than those do not. Adjustment for age, vascular risk factors, sex and prior independence led to only minor changes, there were strong positive associations between hs-CRP, IL-6, s-100 and MMP-9 time course and worse clinical outcome(mRS≥2) or stroke severity (NIHSS≥ 8) . Conclusion: Hs-CRP and IL-6 increased at early stage (3-6h) after acute ischemic stroke, peak at 3 days. Combined with s-100 and MMP-9, they were likely to correlate with stroke severity and poor clinical outcome. Patients with Diabetes were more likely to develop more significant inflammatory reactions after stroke.

Metalloproteinase and Stroke Infarct Size: Role for Anti-Inflammatory Treatment? 

Joan Montaner, MD, PhD; Lidia García-Bonilla, PhD; Anna Rosell, PhD.
Neurovascular Research Laboratory & Neurology Department, Institut de Recerca, Hospital Vall d’Hebron, Barcelona, Spain

Matrix metalloproteinases (MMPs), the largest class of human proteases, have been implicated in both animal and human studies of brain damage. MMP-9 is elevated in humans after stroke both in plasma and in brain infarction. The exact cellular source of MMP-9 remains unknown, and although brain endothelium, astrocytes and neurons may release MMP-9, also invading inflammatory activated cells, such as neutrophils contain several MMPs. In the context of the neurovascular unit, t-PA, the only approved drug for stroke treatment, may degrade extracellular matrix integrity and increase risks of neurovascular cell death, blood–brain barrier leakage, edema and hemorrhage. Although thrombolytic therapy in acute stroke is effective since it accelerates clot lyses, up to 6-15% of treated patients suffer hemorrhagic transformations with high death rates. We have shown that t-PA treatment increases and activates MMP-9 in human stroke and might be used as a biomarker to select patients at higher risk of hemorrhagic transformation. Our data suggest that neutrophils are good candidates to be the main source of MMP-9 following t-PA stroke treatment and, in consequence, partially responsible of brain bleedings. Moreover, high levels of MMP-9 and MMP-13 are involved in DWI lesion growth in spite of thrombolytic therapy suggesting its ultra-early role in brain injury. MMP-13 is mainly produced by neurons, and its activation and nuclear translocation is an early consequence of the ischemic stimulus. Several drugs used for stroke secondary prevention (statins and antiplatelet agents) might decrease MMPs and have also a role in the acute treatment of human brain ischemia.

Innate Inflammation - A Challenge for Drug Absorption and Pharmacokinetics

Hartmut Derendorf, PhD¹
1. Dept. of Pharmaceutics, University of Florida, Gainesville, FL

For any drug oral drug treatment to be pharmacologically active it has to be assured that the active compound will reach its site of action in a sufficiently high concentration. However, there are many potential hurdles that can be in the way. For example, the drug needs to have sufficient solubility and chemical stability in the gastrointestinal fluids. The permeability has to allow crossing of the membranes in the gastrointestinal wall. Active efflux transporter systems such as P-glycoprotein can further block access to the systemic circulation, and enzymatic first-pass metabolism can inactivate the compound both in the gastrointestinal wall or during the first-pass across the liver. Only the fraction of the intact drug that reaches the systemic circulation is bioavailable and can contribute to the desired activity. One compound under investigation for the treatment of innate inflammation is dipyridamole (DP) in combination with low-dose aspirin. DP is shows decreased oral bioavailability with higher gastric pH that occurs with commonly prescribed antacids and in elderly patients. An extended-release formulation of DP (ER-DP) that employs tartaric acid to improve bioavailability of DP in the presence of elevated gastric pH was developed as a combination product with immediate-release aspirin. DP bioavailability from this ER-DP formulation combined with low-dose aspirin is not diminished with high gastric pH. Since the use of antacids is widespread among elderly patients, improved absorption with the ER-DP formulation may insure a more reliable bioavailability in this patient population.

Acute Anti-Inflammatory Approaches to Ischemic Stroke

Gregory J. del Zoppo, MD, University of Washington, School of Medicine, Department of Medicine (Hematology) and Department of Neurology, Seattle, WA

A series of prospective blinded controlled clinical trials have examined the thesis that humoral or cellular elements of inflammation contribute significantly to extension of the early ischemic cerebral injury into the irreversible injury of infarction. Based upon experimental studies, both cellular and humoral inflammation have been shown to contribute to the extent of ischemic injury. Inhibition of endothelial cell-leukocyte adhesion, PMN leukocyte activation, the respiratory burst, and other aspects of inflammatory cell activation reduce infarction volume in small animal models. Flow disturbances caused by leukocyte activation in microvessels are significantly reduced. However, when translated to clinical trials, the study outcomes have been uniformly disappointing. Among the clinical studies, the Enlimomab acute stroke trial of inhibition of endothelial cell ICAM-1-mediated adhesion of PMN leukocytes, using a humanized murine anti-ICAM-1 antibody, demonstrated increased disability and mortality compared to placebo. The HALT stoke study using the anti-leukocyte 2-integrin CD18 antibody Hu23F2G was discontinued when a “futility analysis” suggested that no benefit of the treatment would occur” if the study continued. The recombinant neutrophil inhibitory factor (rNIF) study, a phase 2 dose-finding study using a Bayesian strategy, found no dose-rate of effect. Hence, no phase 3 study was undertaken. A trial of the interleuken-1 receptor antagonist IL-1ra has now been completed. The agent was found to be safe, but efficacy data in the acute setting of ischemic stroke is still wanting. These experiences demonstrate a number of limitations to clinical trial success that are remediable. Nonetheless, there is substantial evidence that focal ischemia of the cerebral tissues initiates reactive inflammatory responses. It may be concluded that the fundamental hypothesis has not yet been adequately or appropriately tested. Therefore, it is critical that both well-designed experimental and clinical studies of anti-inflammatory approaches to ischemic stroke proceed.

Combating Innate Inflammation - New Paradigm for Acute Treatment of Stroke?

Guido Stoll, MD¹, Christoph Kleinschnitz, MD¹, Bernhard Nieswandt, PhD¹
1. University of Würzburg, Würzburg, Germany

Interference with early steps of platelet adhesion/activation by inhibition of the von Willebrand factor (vWF) receptor glycoprotein (GP) Ib, its ligand vWF, or the collagen receptor GPVI profoundly limits infarction while not increasing the risk of intracranial bleeding in the mouse stroke model of transient middle cerebral artery occlusion (tMCAO) (Circulation 115, 2323-30, 2007; Blood 113, 3600-3603, 2009). A similar pathogenic role was revealed for coagulation factor XII (FXII) (J Exp Med 203, 513-8, 2006). Although these data strongly suggest that microvascular thrombus formation is the leading pathophysiological event in ischemic lesion development, recent studies have shown that these molecules also have the capacity to guide inflammatory processes, thereby providing an intriguing alternative mechanistic explanation for their pathogenic function in stroke. This alternate view is further fuelled by unexpected observations: (i) Mice lacking T cells are profoundly protected from cerebral ischemia, but show no primary defect in thrombus formation. These T-cell effects are antigen-independent and do not require formation of an immunological synapse (Kleinschnitz et al. Blood 2010, epub March 9). (ii) Inhibition of platelet aggregation by GPIIb/IIIa had no beneficial effect on infarct growth (Stroke 39, 87-99, 2008; Circulation 115, 2323-30, 2007). In conclusion, reperfusion injury in acute stroke encompasses a significant innate immune component, but it is unclear at present how this is mechanistically linked to platelets. Thus, acute ischemic stroke can be defined as a thrombo-inflammatory disorder, and multifunctional signaling molecules such as GPIb, GPVI, and FXII may provide new therapeutic targets linking inflammation and thrombus formation.

Inflammation and Cognition

Philip B. Gorelick, MD MPH, Department of Neurology and Rehabilitation, University of Illinois College of Medicine, Chicago, Illinois

Alzheimer’s disease (AD) was once considered a traditional “neurodegenerative” disease characterized by neuropathological changes of neuritic plaques, neurofibrillay tangles, and neuronal loss. In recent years it has been shown that Inflammation may be an important mechanism for cognitive decline and AD dementia. The most common cause of irreversible cognitive impairment of later life, AD has been linked to both systemic and focal brain inflammation. Experimental and brain necropsy study suggest that inflammation is a component of the pathologic cascade leading to amyloid neuritic plaque formation. Brain microglia which mediate the brain’s immune response are thought to promote neuritic plaque formation via an inflammatory cascade. Furthermore, in AD patients chemokines, cytokines and complement are found in cerebrospinal fluid and amyloid neuritic plaques, and systemic inflammatory markers such as hs-CRP, IL-6, IL-10, and TNF-alpha are elevated.

The linkage between AD and inflammation has led to the study of treatment strategies to reduce inflammation in the hopes of preventing cognitive decline and AD dementia. Some observational epidemiological studies have shown a relationship between non-steroidal anti-inflammatory drug (NSAID) use and lower risk of AD. Various NSAIDs that are COX1- and/or COX2-inhibitors have been evaluated in clinical trials to assess risk of AD or cognitive decline. In this discussion we review the inflammatory hypothesis of AD and cognitive impairment, the results of observational epidemiological studies, treatment strategies related to NSAIDs, intravenous immune globulin, and vaccination, and the angiogenesis hypothesis of AD and vascular risk factors which may contribute to AD and inflammation.

Long Term Outcome after Stroke and Tia - Impact of Inflammation on Repair? 

JP Mohr, Neurological Institute, Columbia University Medical Center, New York, NY

Many large clinical trials of TIA, and prevention of first and recurrent stroke have had limited documentation of inflammatory markers as part of the original dataset. These deficiencies reflect the initial ignorance, not willful neglect, of the original investigators. Well-known inflammatory vasculopathies such as giant cell arteritis were assumed unsuitable for inclusion, thought too infrequent, and subject to competing therapies, so were usually excluded from such trials. Recent studies in cohorts suffering a wide variety of medical illnesses, some focused on stroke – as cited by speakers in the symposium - have demonstrated the importance infection (clinically obvious or occult) and also of biomarkers of inflammation on stroke risk and subtype. Simultaneously evidence is emerging that the effects of antithrombotic therapies are not static, some subject to decline over time. New antithrombotic agents are also blunting the importance of prior studies. The process of post-stroke improvement is also now known not just to be subsidence of edema or ‘recovery’ but also includes functional reorganization. Such challenges to prior clinical trial results should be welcomed as proof of progress. They also serve as reminders that the revolutionary implications of the latest studies are subject to similar future eclipse, the time-honored notion of ‘Sic fugit gloria’. Efforts to clarify these effects through ‘outcomes research’ are likely to founder on complexity and inadequate documentation. Advances would be best achieved by more focused new trials

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