Abstract T P207: Matrix Metalloproteinase 3 (MMP3) Exacerbates the Hemorrhagic Transformation in Hyperglycemic Stroke

Acute hyperglycemia (HG) worsens stroke outcomes and increases the risk of cerebral hemorrhage especially with the co-administration of tissue plasminogen activator (tPA). MMP3 mediates tPA-induced hemorrhagic transformation (HT) after stroke. However, the role of MMP3 in hyperglycemic stroke is unknown. The working hypothesis of the current study is that HG upregulates MMP3 activity and worsens vascular injury after stroke and this response is independent of the method of reperfusion. Methods: Control and mildly HG rats (160-200 mg/dl, achieved by 30% glucose injection (i.p.) 15 min prior to surgery, n=7-9/group) were subjected to either 90 min middle cerebral artery (MCA) suture occlusion and 22.5 h reperfusion, or to humanized thromboembolic stroke. At 24 h, neurological deficit, infarct size, edema, HT occurrence rate (HT index) and tissue hemoglobin (Hb) were measured. MMP3 activity in isolated cerebral microvasculature and/or brain homogenates was quantified by FRET assay. In addition, MMP-3 expression was assessed in brain microvascular endothelial cells (BVEC) subjected to 90 min hypoxia followed by 22.5 h reoxygenation. Results: While HG did not increase infarct size when compared to control animals, this mild elevation in blood glucose (BG) significantly increased vascular injury indicated by HT index, edema and Hb content in ischemic hemispheres . This was associated with a significant increase in MMP3 activity in both cerebral micro-vasculature and brain homogenates (Table, *p<0.05 vs control). In vitro, the combination of hypoxia and HG has increased MMP3 expression more than each alone (*p<0.05 vs control). Conclusion: Even mild elevations in BG increased MMP3 activity and augmented vascular injury following ischemic stroke. Our findings suggest that MMP3 might be playing an important role in worsening the outcomes in hyperglycemic stroke and MMP3 inhibition may be a potential therapeutic target.

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Abstract W MP96: Low Dose Tissue Plasminogen Activator Amplifies Vascular Injury and Worsens Functional Outcomes in Acute Hyperglycemic Stroke Independent of the Model of Reperfusion

Stroke ◽

10.1161/str.46.suppl_1.wmp96 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Sherif Hafez ◽

Md Nasrul Hoda ◽

Xinyue Guo ◽

Susan Fagan ◽

Adviye Ergul

Keyword(s):

Infarct Size ◽

Vascular Injury ◽

Functional Outcomes ◽

Low Dose ◽

Hemorrhagic Transformation ◽

Occurrence Rate ◽

Translational Study ◽

Flow Monitoring ◽

Tissue Plasminogen ◽

Tissue Hemoglobin

Clinically, hyperglycemia (HG) exacerbates reperfusion injury and aggravates tPA-induced hemorrhagic transformation (HT). Yet, most of the experimental hyperglycemic stroke studies exclusively employed suture occlusion model. Only few studies involved tPA in hyperglycemic setting and employed a 10-fold higher dose of tPA than that is used in patients. Thus, in this translational study using suture and thromboembolic occlusion of middle cerebral artery (MCA), with and without human tPA dose, we tested the hypothesis that even acute mild HG worsens the neurovascular injury and functional outcomes irrespective of the method of reperfusion, and that the use of low dose tPA amplifies this injury. Methods: Control and mildly HG rats (140-200 mg/dl, achieved by 30% glucose ip injection 15 min prior to surgery, n=7-9/group) were subjected to MCA occlusion by either suture (90 min) or humanized clot and 24 h reperfusion. tPA (1mg/kg) was injected IV 2 h after induction of ischemia. At 24 h, neurological deficit, infarct size, edema, HT occurrence rate (HT index) and tissue hemoglobin (Hb) were measured. Results: Cerebral blood flow monitoring indicated that % drop at occlusion were similar across the groups and that low dose tPA effectively resolved the clot in the embolic model. tPA did not increase the infarct size in either control or hyperglycemic animals when compared to no tPA groups (Table). HG increased vascular injury (HT index and Hb content) in both suture and embolic occlusion models. The combination of HG and tPA exacerbated the vascular injury and worsened functional outcomes more than each alone. Conclusion: The interaction between HG and even low dose tPA has a significant deleterious effect on cerebrovasculature and functional outcomes independent of the method of reperfusion.

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Role of Activated Platelets in the Homing, Maturation, and Differentiation of Endotheleal Progenitor Cells (EPCs) to Vascular Subendotheleal Matrix.

Blood ◽

10.1182/blood.v108.11.3938.3938 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3938-3938

Author(s):

Eli I. Lev ◽

Jing-fei Dong ◽

Marcin Bujak ◽

Khatira Aboulfatova ◽

Neal S. Kleiman ◽

...

Keyword(s):

Progenitor Cells ◽

Vascular Injury ◽

Human Platelets ◽

In Vitro Experiments ◽

Cell Markers ◽

Femoral Arteries ◽

In Vivo Experiments

Abstract We and others have found that platelets play an important role in the recruitment of endothelial progenitor cells to sights of vascular injury. However, it is not clear whether the EPCs mature and differentiate to endothelial cells following recruitment to the vascular injury sites. In addition, there is limited in vivo data to support the role of EPCs in re-endothlialization following vascular injury. We conducted in vitro experiments to investigate the maturation of EPCs on platelet based-media and in vivo experiments to evaluate the recruitment of EPCs following vascular injury. In in vitro experiments human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as cell markers. Isolated viable EPCs (CD133+, VEGFR-2+ cells) were plated on human fibronectin or a monolayer of washed human platelets. Cell colonies were counted 7 days after plating and stained for the endothelial cell markers CD31 (PECAM-1) and CD144 (VE-cadherin). The mean number of colony-forming cells was 35±2.6 colonies/106 cells on platelets, which was significantly higher than 18±4.2 colonies/106 cells on fibronectin (n = 4, P<0.01). Apart from the difference in colony numbers, the EPC colonies grew faster on the platelet substrate, were larger, and had more spindle-shaped cells (Figure 1 - staining of EPC colonies for CD31 and CD144). In the in vivo experiments a model of transluminal injury to mouse femoral arteries was used. Femoral artery denudation was performed by 0.25-mm-diameter angioplasty guidewire. Injured femoral arteries were compared to the contra-lateral controls (uninjured), and were harvested 1.5 hours following the injury and immunostaining performed with an anti-VEGFR-2 antibody. Four experiments showed a markedly higher number of VEGFR-2+ cells in the artery that has undergone denudation. These experiments indicate that a media composed of platelets promotes the maturation and differentiation of EPCs. Furthermore, in vivo, EPCs are recruited early following vascular injury. Thus, homing, maturation, and differentiation of EPCs are mediated by platelets.

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Vascular Protection in Diabetic Stroke: Role of Matrix Metalloprotease-Dependent Vascular Remodeling

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2010.120 ◽

2010 ◽

Vol 30 (12) ◽

pp. 1928-1938 ◽

Cited By ~ 52

Author(s):

Mostafa M Elgebaly ◽

Roshini Prakash ◽

Weiguo Li ◽

Safia Ogbi ◽

Maribeth H Johnson ◽

...

Keyword(s):

Infarct Size ◽

Vascular Remodeling ◽

Hemorrhagic Transformation ◽

Clinical Implications ◽

Matrix Metalloprotease ◽

Vascular Protection ◽

Gk Rats ◽

Vascular Tortuosity ◽

Stimulation Of

Temporary focal ischemia causes greater hemorrhagic transformation (HT) in diabetic Goto-Kakizaki (GK) rats, a model with increased cerebrovascular matrix metalloprotease (MMP) activity and tortuosity. The objective of the current study was to test the hypotheses that (1) diabetes-induced cerebrovascular remodeling is MMP dependent and (2) prevention of vascular remodeling by glucose control or MMP inhibition reduces HT in diabetic stroke. Control and GK rats were treated with vehicle, metformin, or minocycline for 4 weeks, and indices of remodeling including vascular tortuosity index, lumen diameter, number of collaterals, and middle cerebral artery (MCA) MMP activity were measured. Additional animals were subjected to 3 hours MCA occlusion/21 hours reperfusion, and infarct size and HT were evaluated as indices of neurovascular injury. All remodeling markers including MMP-9 activity were increased in diabetes. Infarct size was smaller in minocycline-treated animals. Both metformin and minocycline reduced vascular remodeling and severity of HT in diabetes. These results provide evidence that diabetes-mediated stimulation of MMP-9 activity promotes cerebrovascular remodeling, which contributes to greater HT in diabetes. Metformin and minocycline offer vascular protection, which has important clinical implications for diabetes patients who are at a fourfold to sixfold higher risk for stroke.

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Abstract P781: Ischemic Postconditioning Protects Against Hemorrhagic Transformation Induced by Hyperglycemia in Ischemic Stroke

Stroke ◽

10.1161/str.52.suppl_1.p781 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Hansen Chen ◽

Michelle Y Cheng ◽

Tonya Bliss ◽

Heng Zhao ◽

Gary Steinberg

Keyword(s):

Ischemic Stroke ◽

Mortality Rate ◽

Infarct Size ◽

Brain Edema ◽

Neurological Deficit ◽

Hemorrhagic Transformation ◽

Artery Occlusion ◽

Ischemic Postconditioning ◽

Brain Infarct ◽

Acute Hyperglycemia

Background: Hyperglycemia occurs in over 40% of ischemic stroke patients, which induces hemorrhagic transformation (HT) and worsens stroke outcomes. The management of hyperglycemia with insulin did not show favorable outcomes. Thus, strategies for managing hyperglycemia-exacerbated stroke injury are urgently needed. We previously demonstrated that ischemic postconditioning (IPostC) (repeated transient interruption of cerebral blood flow during reperfusion) can reduce brain infarct size and improve neurological outcomes. In this study, we hypothesized that IPostC can reduce HT in ischemic stroke with acute hyperglycemia. Method: Male mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour, followed by reperfusion to mimic ischemic stroke. Glucose was injected before MCAO to induce hyperglycemia. IPostC was initiated upon reperfusion with 3 cycles of 30-second reperfusion followed by 10 seconds of MCA occlusion. Brain infarct was visualized by TTC staining and quantitated using Image J. Hemorrhagic transformation was evaluated by hemorrhagic scores. Result: Acute hyperglycemia significantly increased the brain infarct size (by 25%, p<0.01), brain edema (p<0.001) and hemorrhagic transformation (HT) (average HT scores: 0.75 in MCAO group vs 15.6 in MCAO plus hyperglycemia group, p<0.001), Mice with hyperglycemia also exhibited more severe neurological deficit and higher mortality rate at 24 hours after MCAO. 2) IPostC treatment significantly reduced brain infarct size (p<0.01), brain edema (p<0.05) and attenuated HT (p<0.001). Neurological deficit and mortality rate was reduced with IPostC treatment. Conclusion: Our findings suggest that IPostC can counteract the effects of acute hyperglycemia and reduce brain injury, edema and HT after stroke. Grant/Other Support: NIH Grant R01NS064136C

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The role of connexin43 in hemorrhagic transformation after thrombolysis in vivo and in vitro

Neuroscience ◽

10.1016/j.neuroscience.2016.04.040 ◽

2016 ◽

Vol 329 ◽

pp. 54-65 ◽

Cited By ~ 10

Author(s):

Xiaobo Yang ◽

Heling Chu ◽

Yuping Tang ◽

Qiang Dong

Keyword(s):

Hemorrhagic Transformation

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Exosomes Secreted from CXCR4 Overexpressing Mesenchymal Stem Cells Promote Cardioprotection via Akt Signaling Pathway following Myocardial Infarction

Stem Cells International ◽

10.1155/2015/659890 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 87

Author(s):

Kai Kang ◽

Ruilian Ma ◽

Wenfeng Cai ◽

Wei Huang ◽

Christian Paul ◽

...

Keyword(s):

Myocardial Infarction ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Infarct Size ◽

Functional Restoration ◽

Akt Inhibitor ◽

Cardiac Functions

Background and Objective.Exosomes secreted from mesenchymal stem cells (MSC) have demonstrated cardioprotective effects. This study examined the role of exosomes derived from MSC overexpressing CXCR4 for recovery of cardiac functions after myocardial infarction (MI).Methods. In vitro, exosomes from MSC transduced with lentiviral CXCR4 (ExoCR4) encoding a silencing sequence or null vector were isolated and characterized by transmission electron microscopy and dynamic light scattering. Gene expression was then analyzed by qPCR and Western blotting. Cytoprotective effects on cardiomyocytes were evaluated and effects of exosomes on angiogenesis analyzed.In vivo, an exosome-pretreated MSC-sheet was implanted into a region of scarred myocardium in a rat MI model. Angiogenesis, infarct size, and cardiac functions were then analyzed.Results. In vitro, ExoCR4significantly upregulatedIGF-1αand pAkt levels and downregulated active caspase 3 levelin cardiomyocytes. ExoCR4also enhanced VEGF expression and vessel formation. However, effects of ExoCR4were abolished by an Akt inhibitor or CXCR4 knockdown.In vivo, ExoCR4treated MSC-sheet implantation promoted cardiac functional restoration by increasing angiogenesis, reducing infarct size, and improving cardiac remodeling.Conclusions.This study reveals a novel role of exosomes derived from MSCCR4and highlights a new mechanism of intercellular mediation of stem cells for MI treatment.

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Abstract 176: Platelet Cyclophilin D Mediates Neutrophil Recruitment and Ischemic Stroke Outcomes in Mice

Stroke ◽

10.1161/str.51.suppl_1.176 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Frederik Denorme ◽

Bhanukanth Manne ◽

Yasuhiro Kosaka ◽

Jennifer Majersik ◽

Benjamin Kile ◽

...

Keyword(s):

Ischemic Stroke ◽

Infarct Size ◽

Infarct Volume ◽

Human Platelets ◽

Artery Occlusion ◽

Cyclophilin D ◽

Stroke Outcomes ◽

Fold Reduction ◽

Cerebral Artery Occlusion

Rationale: Besides their role in thrombosis, platelets also mediate inflammation through platelet-neutrophil aggregates (PNA). Recently, cyclophilin D (CypD)-mediated platelet necrosis emerged as a potential mediator of detrimental PNA during thrombosis. However, the role of platelet CypD in ischemic stroke has never been examined. Objective: Here, we investigate the contribution of platelet CypD following ischemic stroke. Methods: We generated mice lacking CypD specifically in platelets (KO). Both wild-type (WT) and KO mice were subjected to a 1h transient middle cerebral artery occlusion (tMCAO) stroke model. Twenty-four hours after occlusion, neurological and motor function, and stroke infarct size were determined. We also examined if the CypD pathway was altered in human platelets after ischemic stroke. Results: Loss of platelet CypD significantly improved neurological (p<0.001) and motor (p<0.005) functions compared to WT mice after tMCAO. Furthermore, platelet CypD deficient significantly reduced ischemic stroke infarct volume (39.1±15.7mm 3 vs. 78.6±27.7mm 3 , n=15; p<0.0001). Smaller infarcts in KO mice was not due to difference in blood flow during the ischemia stage. Twenty-four hours after stroke, a greater than 2-fold reduction in neutrophils was observed in the brains from KO mice (p<0.0001). In addition, we observed significantly fewer circulating and cerebral PNA (p<0.01). Depletion of neutrophils significantly (p<0.05) reduced infarct size and neurological damage following ischemic stroke in WT mice, however, no additional protective effect was observed in KO mice, suggesting necrotic PNAs are critical during ischemic stroke. RNA-sequencing on platelets isolated from ischemic stroke patients (n=8) and healthy aged, gender matched controls (n=7) revealed significant increases in several targets involved in CypD-mediated necrosis, including MCUR1, TMEM16F and calpain2 (p<0.005). Conclusion: Our results demonstrate necrotic platelets interact with neutrophils to exacerbate brain injury following ischemic stroke. As inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage after ischemic stroke.

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Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions

Molecules ◽

10.3390/molecules26216365 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6365

Author(s):

David Couret ◽

Cynthia Planesse ◽

Jessica Patche ◽

Nicolas Diotel ◽

Brice Nativel ◽

...

Keyword(s):

Cerebral Ischemia ◽

Infarct Size ◽

Neuroprotective Effect ◽

Hemorrhagic Transformation ◽

High Density ◽

Cerebral Infarct ◽

High Density Lipoproteins ◽

Protective Effects ◽

Neuroprotective Effects ◽

Acute Hyperglycemia

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.

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Abstract 237: The Role of Lymphocyte Specific Protein-1 in the Phenotypic Switch of Smooth Muscle Cells After Arterial Injury

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.237 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Mirnal A Chaudhary ◽

Go Urabe ◽

Alex Hayden ◽

Sarah Franco ◽

Xudong Shi ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Western Blotting ◽

Vascular Injury ◽

Muscle Cells ◽

Specific Protein ◽

Phenotypic Switch

Background: After vascular injury, vascular smooth muscle cells (SMCs) switch from a differentiated contractile state to synthetic de-differentiated phenotype which contributes to the pathophysiology of restenosis. Experimental data generated by our lab indicate that TGF-β downregulates contractile proteins and stimulates migration. To understand how TGF-β promotes SMC phenotypic switch in injured arteries, we performed an Affymetrix Array analysis and identified Lymphocyte Specific Protein-1 (LSP1) among other upregulated genes. LSP1 is known to play a role in neutrophil extravasation, however the role of LSP1 within SMCs is unknown. We hypothesize that LSP1 contributes to SMC pathophysiological behavior through changes in cell architecture and migration in-vivo and in-vitro. Methods and Results: After carotid artery angioplasty, male Sprague-Dawley rats were sacrificed at 3, 7, and 14 days after injury for immunohistochemistry. Immunofluorescence staining revealed a unique upregulation of LSP1 within the neointima, media, and adventitia at 7 and 14 days, but not at 3 days after injury. Confocal images revealed that the LSP1 positive cells minimally express α-SMA (Pierson’s Coefficient, r=.017). Additional characterization experiments using immune cell markers CD3 and CD45 show no co-localization with LSP1 positive cells. To mimic the in-vivo neointimal cells and vascular injury induced de-differentiation in-vitro , rat A10 cells were treated with solvent or PDGF-bb (10 ng/mL). Quantitative RT-PCR demonstrated an upregulation of LSP1 mRNA after 24 hrs of PDGF-BB stimulation. Using Western Blotting, we confirm an upregulation of LSP1 protein after 48 hrs of PDGF-BB stimulation. Lastly, we performed nuclear and cytoplasmic fractionation followed by Western Blotting which demonstrated that LSP1 is remained within cytoplasmic fraction of the A10 cell after treatment with PDGF-BB. Conclusion: These results demonstrate that LSP1 is increased in-vivo after balloon injury, and in-vitro after PDGF-BB stimulation. Experiments to characterize the identity of these LSP1 cells in-vivo are in process, with future in-vitro experiments to focus on the role of LSP1 phosphorylation as a part of cytoskeletal remodeling and cellular migration.

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