Mouse Cremaster Muscle Research Articles

Hemorrhagic and procoagulant P-III snake venom metalloproteinases differ in their binding to the microvasculature of mouse cremaster muscle

Binding of two P-III snake venom metalloproteinase (SVMPs), one procoagulant and one hemorrhagic, to microvessels was compared in an ex vivo model. The procoagulant SVMP did not bind to the microvasculature, in contrast to the clear localization on microvessels of the hemorrhagic SVMP. Deglycosylation of the procoagulant enzyme did not enable this toxin to bind to microvessels, suggesting that glycosylation is not interfering with binding. These observations suggest that procoagulant SVMPs lack exosites for interaction with microvessels components.

Using Intravital Microscopy to Study the Role of MIF in Leukocyte Trafficking In Vivo.

In vivo visualization of the microvasculature of the mouse cremaster muscle has been fruitful in the evaluation of the role of macrophage migration inhibitory factor in promotion of leukocyte trafficking. Here we explain how to undertake this preparation, including details on mouse anesthesia, securing intravenous access, and cremaster muscle exteriorization. We also provide information on the various microscopy modalities now available for imaging microvascular preparations of this nature.

Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.

Interleukin-8 Secreted by Glioblastoma Cells Induces Microvascular Hyperpermeability Through NO Signaling Involving S-Nitrosylation of VE-Cadherin and p120 in Endothelial Cells.

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Blood flow guides sequential support of neutrophil arrest and diapedesis by PILR-β1 and PILR-α.

Arrest of rapidly flowing neutrophils in venules relies on capturing through selectins and chemokine-induced integrin activation. Despite a long-established concept, we show here that gene inactivation of activating paired immunoglobulin-like receptor (PILR)-β1 nearly halved the efficiency of neutrophil arrest in venules of the mouse cremaster muscle. We found that this receptor binds to CD99, an interaction which relies on flow-induced shear forces and boosts chemokine-induced β2-integrin-activation, leading to neutrophil attachment to endothelium. Upon arrest, binding of PILR-β1 to CD99 ceases, shifting the signaling balance towards inhibitory PILR-α. This enables integrin deactivation and supports cell migration. Thus, flow-driven shear forces guide sequential signaling of first activating PILR-β1 followed by inhibitory PILR-α to prompt neutrophil arrest and then transmigration. This doubles the efficiency of selectin-chemokine driven neutrophil arrest by PILR-β1 and then supports transition to migration by PILR-α.

Dystrophin deficiency promotes leukocyte recruitment in mdx mice.

A growing body of evidence defines inflammation as a hallmark feature of disease pathogenesis of Duchenne muscular dystrophy. To tailor potential immune modulatory interventions, a better understanding of immune dysregulation in Duchenne muscular dystrophy is needed. We now asked whether dystrophin deficiency affects the cascade of leukocyte recruitment. We performed intravital microscopy on the cremaster muscle of wild-type and dystrophin-deficient mdx mice. Recruitment was triggered by preparation alone (traumatic inflammation) or in combination with scrotal TNFα injections. Neutrophilic infiltration of the cremaster muscle was assessed on tissue sections. Integrin expression on circulating neutrophils and serum levels of pro-inflammatory cytokines were measured by flow cytometry. Mdx mice show increased rolling and adhesion at baseline (traumatic inflammation) and a more profound response upon TNFα injection compared with wild-type animals. In both models, neutrophilic infiltration of the cremaster muscle is increased. Upregulation of the integrins LFA-1 and Mac-1 on circulating leukocytes and pro-inflammatory cytokines IL-6 and CCL2 in the serum points toward systemically altered immune regulation in mdx mice. We are the first to show exaggerated activation of the leukocyte recruitment cascade in a dystrophin-deficient organism in vivo.

Activated protein C inhibits lipopolysaccharide‐mediated acetylation and secretion of high‐mobility group box 1 in endothelial cells

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated proteinC (APC) inhibits high-mobility group box1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial proteinC receptor (EPCR)-dependent activation of protease-activated receptor1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of proteinC/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein1 in LPS-treated endothelial cells. The invivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

The anti-inflammatory effects of the Chromogranin A hormone cleavage product catestatin on human monocytes and macrophages

Abstract Objective High levels of the prohormone chromogranin A (CgA) have been detected in the plasma of patients with various cancers and inflammatory diseases such as rheumatic arthritis, inflammatory bowel disease as well as type 1 and 2 diabetes mellitus (T2DM). In contrast, T2DM and hypertensive patients show reduced levels of the CgA-derived peptide catestatin (CST). Here we show the anti-inflammatory effects of CST on human macrophages and phagocyte migration. Method Mouse phagocyte (monocytes and granulocytes) migration was imaged in the cremaster muscle of mice superperfused with buffer containing CST or the chemokine MIP-2. Human blood monocytes were used for in vitro migration to CCL2 and/or CST and macrophage differentiation assays. Macrophages were treated with CST and polarized towards pro-inflammatory or anti-inflammatory phenotypes. Afterwards, cytokine production was measured by ELISA (Il-10, TNF-a, IL-6) and cells were phenotyped by EM and RNA sequencing. Results In vivo and in vitro imaging shows that CST works as a chemo-attractant for monocytes. Interestingly, the combination of CST with the well-known chemoattractant CCL2 results in an anti-inflammatory effect. Moreover, CST treatment of inflammatory macrophages inhibited TNF-α and IL-6 production. Additionally, several proinflammatory gene levels in CST treated macrophages were decreased. Conclusions CST influences monocyte migration and promotes M1-M2 polarization by decreasing expression of pro-inflammatory cytokines and increasing anti-inflammatory cytokine expression. These factors are important for tissue homeostasis, which makes CST an interesting therapeutic target for treatment and/or diagnosis of various metabolic and immune diseases.

Platelets determine the onset of innate and adaptive immune cell responses Schaubaecher J, Mittmann L, Uhl B, Reichel CA

Neutrophils represent the first immune cells recruited to the site of injury or infection, followed by monocytes and, later on, lymphocytes. In addition to their fundamental role in hemostasis, platelets are increasingly recognized to serve as critical modulators of immune responses. Whereas interactions of platelets with myeloid leukocytes have been extensively studied, the functional relevance of these cellular blood components for the regulation of lymphocyte trafficking remains poorly understood. Employing multi‐channel in vivo microscopy on the mouse cremaster muscle, platelets were found to instantly accumulate in the venular microvasculature upon induction of inflammation right before neutrophils, monocytes, and lymphocytes, but are progressively released from the endothelium into the bloodstream after extravasation of myeloid leukocytes. In this context, platelets divergently control the trafficking of myeloid leukocytes and lymphocytes to inflamed tissue as assessed by multi‐channel flow cytometry analyses in a mouse peritonitis assay: Whereas depletion of platelets severely compromised the extravasation of neutrophils and classical monocytes, the recruitment of CD4+ lymphocytes, CD8+ lymphocytes, and B lymphocytes to the perivascular space was dramatically enhanced. Importantly, depletion of neutrophils in these experiments completely abolished responses of classical monocytes, but did not affect lymphocyte trafficking. In this context, activated platelets were found to upregulate surface expression of adhesion/signaling molecules facilitating interactions with neutrophils such as CD40 and CD40L as well as of factors interfering with lymphocyte activation including programmed death‐ligand 1 (PD‐L1), CD80, and CD86. Blockade of these latter immune checkpoint molecules significantly enhanced lymphocyte responses to the level of platelet‐depleted animals. Thus, our experimental data identify a previously unappreciated functional role of platelets as ‚decision makers' in the immune system that critically regulate the onset of innate and adaptive immune cell responses in inflammatory reactions.Support or Funding InformationThis study was supported by Deutsche Forschungsgemeinschaft (DFG), Sonderforschungs‐bereich (SFB) 914.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation.To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets.To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation.To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

SummaryNeutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine “depot” in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Rolling neutrophils form tethers and slings under physiologic conditions in vivo.

Human and mouse neutrophils are known to form tethers when rolling on selectins in vitro. Tethers are ∼0.2 μm thin, ∼5-10 μm-long structures behind rolling cells that can swing around to form slings that serve as self-adhesive substrates. Here, we developed a mouse intravital imaging method, where the neutrophil surface is labeled by injecting fluorescently labeled mAb to Ly-6G. Venules in the cremaster muscle of live mice were imaged at a high frame rate using a confocal microscope equipped with a fast resonant scanner. We observed 270 tethers (median length 3.5 μm) and 31 slings (median length 6.9 µm) on 186 neutrophils of 15 mice. Out of 199 tether break events, 123 were followed by immediate acceleration of the rolling cell, which shows that tethers are load-bearing structures in vivo. In venules with a high wall shear stress (WSS; > 12 dyn/cm2 ), median rolling velocity was higher (19 μm/s), and 43% of rolling neutrophils had visible tethers. In venules with WSS < 12 dyn/cm2 , only 26% of rolling neutrophils had visible tethers. We conclude that neutrophil tethers are commonly present and stabilize rolling in vivo.

Rock Inhibitor Fasudil Reduces Leukocyte-Endothelium Interactions in the Microvasculature of a Sickle Cell Mouse Model of Allergic Inflammation

Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. DisclosuresConran:Bayer AG: Research Funding. Fertrin:Alexion Pharmaceuticals: Consultancy.

Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts

BackgroundSea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects.MethodsE. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats.ResultsOne molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10.ConclusionsOne isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.

Vascular sprouts induce local attraction of proangiogenic neutrophils.

Angiogenesis, the growth of new blood vessels, is a complex process requiring the orchestration of numerous different cell types, growth factors, and chemokines. Some of the recently acknowledged actors in this process are immune cells. They accumulate at hypoxic sites, but the kinetics, dynamics, and regulation of that trafficking are unknown. In this study, we used intravital and live cell imaging to understand how neutrophils and macrophages migrate and behave at angiogenic sites. We developed two reproducible models of angiogenesis: one by transplanting isolated and hypoxic pancreatic islets into the cremaster muscles of mice, and another by in vitro coculturing of mouse aortic rings with neutrophils. In vivo imaging of the hypoxic site revealed recruitment of neutrophils and macrophages, which occurred in parallel, with depletion of one subset not affecting the accumulation of the other. We found, by cell tracking and statistical analyses, that neutrophils migrated in a directional manner to "angiogenic hotspots" around the islet where endothelial sprouting occurs, which was confirmed in the in vitro model of angiogenesis and is dependent on CXCL12 signaling. Intimate interactions between neutrophils and neovessels were prevalent, and neutrophil depletion greatly hampered vessel growth. Macrophages were less motile and attained supportive positions around the neovessels. Here, we present two novel in vivo and in vitro imaging models to study leukocyte behavior and actions during angiogenesis. These models unveiled that neutrophil migration at a hypoxic site was guided by signals emanating from sprouting endothelium where these immune cells gathered at "angiogenic hotspots" at which vascular growth occurred.

Dexamethasone-conjugated DNA nanotubes as anti-inflammatory agents invivo.

The biopolymer DNA allows to create nanoscale, biocompatible structures, which can be designed in a target-specific and stimuli-responsive manner. DNA carrier systems with these characteristics hold a great potential for nanomedical applications, such as for the treatment of inflammatory diseases. Here we used a DNA-based drug carrier system for the pH-dependent delivery of the glucocorticoid dexamethasone into macrophages, a cell type with a key role in the regulation of inflammation. Dexamethasone (Dex) nanotubes were internalized within minutes by MH-S macrophages invitro and by tissue resident macrophages in the mouse cremaster muscle invivo and localized in their endosomes. Treatment with Dex nanotubes invitro significantly reduced the LPS-induced TNF secretion by macrophages, as compared to equivalent amounts of free dexamethasone without affecting cell viability. Microinjection of Dex nanotubes into postischemic muscle tissue of anesthetized mice resulted in a marked reduction of ischemia-reperfusion-elicited leukocyte transmigration and diminished vascular expression of the endothelial adhesion molecules VCAM-1 and ICAM-1. Taken together, our results demonstrate that DNA nanotubes can be used as a platform for the targeted delivery of glucocorticoids and could thus foster the development of nanomedical therapeutics with reduced off-target effects.

Therapeutic progenitor cell application for tissue regeneration: Analyzing the impact of toll-like receptor signaling on c-kit+ cell migration following ischemia-reperfusion injury in vivo

ObjectivesToll-like-receptor (TLR) mediated immune response has been shown to regulate myocardial damage following cardiac ischemia-reperfusion (IR). It has not been described conclusively so far whether migration of therapeutically applied progenitor cells following an IR event depends on TLR-signaling. MethodsIn vivo migratory capacity murine c-kit+ cells following IR injury was quantified by intravital fluorescence microscopy, utilizing the mouse cremaster muscle model and analyzing early (rolling) and late (adhesion) c-kit+ cell interaction with the local endothelium. The role of TLR-2 and TLR-4, as well as MyD88 and TRIF was analyzed by applying specific knock-out models. ResultsA sequence of 15min ischemia followed by 15min of reperfusion induced firm endothelial c-kit+ cell adhesion (5.6±1.3cells/mm2 in Control vs. 30.2±10.1cells/mm2 in IR, p<0.05). Knock-out of TLR-2 and TLR-4 diminished both IR induced early c-kit+ cell-endothelial cell interactions (67.6±2.3% c-kit+ cell rolling in IR vs. 46.3±4.8% c-kit+ cell rolling in IR-TLR-2-ko vs. 45.3±4.8% c-kit+ cell rolling in IR-TLR-4-ko, p<0.05) as well as firm endothelial c-kit+ cell adhesion (30.2±10.1cells/mm2 in IR vs. 16.3±3.9cells/mm2 in IR-TLR-2-ko vs. 14.5±4.4cells/mm2 in IR-TLR-4-ko, p<0.05). Adaptor protein knock-out resulted in a significantly decreased firm endothelial c-kit+ cell adhesion only in MyD88 knock-out but not in TRIF knock-out (9.2±2.2cells/mm2 in IR-MyD88-ko vs. 30.1±9.9cells/mm2 in IR-WT, p<0.05). ConclusionArtificially applied c-kit+ cells interact with the target organ endothelium following IR injury. This interaction seems to depend on TLR-MyD88 signaling. Therapeutic blockade of TLR signaling for anti-inflammatory purposes might interfere with regenerative cell-based therapy protocols.

Therapeutic Activity of Peg Albumin in Transgenic Mouse Model of Sickle Cell Disease Is a Function of the Pattern of Pegylation

BackgroundConjugation of Albumin (Alb) with six copies of PEG 5K chains using extension arm chemistry (EAF) generates a novel semisynthetic supra perfusion resuscitation fluid (EAF P5K6 Alb) that performs better than the conventional colloidal plasma expanders in extreme hemodilution and hemorrhagic shock animal models. The superior activity of EAF P5K6 Alb is attributed to its property of increasing functional capillary density. This product has been shown to increase the efficacy of blood transfusion when given in combination with blood. The influence of pattern of surface decoration on SCD therapeutic activity of PEG Alb with one copy of PEG 30K (P30K1Alb) and six copies of PEG5K (EAF P5K6 Alb) has been investigated in the chronic mouse model of SCD, BERK low ϒ. Two important questions posed here are: 1) Does the pattern of PEGylation influence the increase in hydrodynamic volume and packing density of PEG shell in the PEG30KAlb adducts and 2) Does the P30K1Alb and EAF P5K6 Alb have the similar SCD therapeutic activity.MethodsP30K1 Alb was prepared by PEGylation at Cys34 of Alb using maleimidopropionamido PEG30K followed by ion exchange chromatography purification. EAF P5K6 Alb was purified by tangential flow filtration method. Intravital microscopy analysis for the assessment of therapeutic effects of both the PEG Alb adducts was examined in the cremaster muscle of transgenic SCD mice. Berk low ϒ mice were infused with 10 % top load of a 4 gm % PEG Alb solutions (either EAF P5K6 Alb or P30K1 Alb) by IP and the attenuation of vaso-occlusion, as well as improvement in blood flow has been compared.ResultsP30K1 Alb elutes as a very resolved material in ion exchange column and its 1 copy of PEG30K was confirmed by NMR studies. The hydrodynamic volume of P30K1 Alb is higher than EAF P5K6 Alb as reflected by size exclusion chromatography, even though the overall total mass to the Alb is same. This reflects that P30K1 is more flexible than EAF P5K6 Alb. The molecular radius and COP of EAFP5K6Alb is higher whereas viscosity is less than that of P30K1Alb (Table 1). Accordingly, plasma expander like properties of PEG Alb when surface decorated with a given total mass of PEG is a function of pattern of PEGylation. EAF P5K6 Alb exhibited a significant therapeutic activity after 3 hours (hrs) of treatment, as seen by the attenuation of the vaso-occlusion (Fig 1). This effect remained the same after 24 hrs. There was no noticeable change in the blood flow or RBC velocity in spite of significant reduction in leukocyte adhesion.Discussions and ConclusionsThe correlation of pattern of PEGylation of PEG Alb with the supra perfusion activity of EAF P5K6 Alb has been investigated by Tsai et. al. (unpublished results) by designing EAF P3K10. It was concluded that EAF P5K6 is the better pattern of PEGylation to induce the supra perfusion. Results suggest that the PEGylation induced increase in the hydrodynamic volume under flow is higher with a single copy of PEG30K than 6 copies of PEG 5K. Under flow conditions, PEG 30K and P30K1Alb exhibit comparable hydrodynamic volume, conjugating Alb to PEG 30K has not much influence on the overall hydrodynamic volume of PEGAlb. On the other hand, under static conditions, the packing density of its PEG shell is lower than that of EAF P5K6 Alb.There is significant reduction in adhesion of leucocytes in 3 hrs of treatment with EAF P5K6 Alb as seen in the intravital microscopy studies. However very little effect is seen in terms of changes in the blood flow. After 24 hrs of treatment, there is increase in emigration of leukocytes. Surprisingly, P30K1 has little or no SCD therapeutic activity. The amount of PEG Alb used here is only small (10 %) as compared to about 25 % in hemorrhagic shock models; accordingly, the contribution of viscosity is small, if any. We suggest that the SCD therapeutic activity of EAF P5K6 Alb is primarily related to the "parachute effect" of PEG Alb with multiple copies of PEG chains. We have also shown that EAF hexaPEGylation of Tempol (antioxidant) conjugated Alb, synergistically increased the antioxidant therapeutic activity Alb Tempol in attenuating the experimentally induced vaso-occlusive crisis in a transgenic mouse model of SCD, NY1DD. The P30K1 Alb lacks such a parachute-like effect. These results raise the important question whether the "mushroom" structure of EAF P3K10 Alb may show more SCD therapeutic activity than EAF P5K6 Alb and EAF P3K10 Alb conjugated with antioxidant. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Redesign of EAF Peg Hb As an Oxygen Transfer Catalyst for Improved Tissue Oxygenation of Hypoxic Areas: A Therapeutic for Anemia and Sickle Cell Disease

BackgroundSurface decoration of Hb with PEG chains has been advanced as an approach to attenuate the vasoconstrictive activity of acellular Hb in circulation. PEGylated Hbs with higher oxygen affinities relative to unmodified Hb has an advantage when designing blood substitutes in achieving targeted transport of O2 from lungs to tissues. Extension Arm Facilitated (EAF) PEGylation of Hb with six copies of PEG 5K (EAF P5K6 Hb) was advanced as an optimum level of PEGylation that does not induce weakening of interdimeric interactions in the quaternary structure of Hb. EAF P5K6 Hb at 4 gm % is an O2 carrying colloidal plasma expander. However, a prototype of EAF P5K6 Hb, developed by Sangart as MP4, did not increase microvascular oxygen delivery or improve tissue oxygenation in models of extreme hemodilution,whereas the EAF P5K6 Hb and P5K2 did better than the non-oxygen carrying semisynthetic colloidal plasma expander and EAF P5K6 albumin.Also, P5K2 Hb does a better tissue oxygenation even though its oxygen affinity is comparable to that of EAF P5K6 Hb1.As PEG chains increases from 2 to 10 copies, there is a lesser effect in the oxygen affinity of PEG Hbs in the presence of allosteric effectors like IHP and L-35. This suggests that higher degree of PEGylation preferentially stabilize the oxy conformation of PEG Hb, decreasing the efficacy of O2 release in the circulation. We attributed this decreased efficiency to the larger PEG shell which might lower the diffusion of O2out to the plasma. We reasoned that we could reduce the size of the PEG shell using six copies of PEG 3K and that we could use this new PEG Hb (EAF P3K6 Hb) at 6 gm% to get nearly the same viscosity as 4 gm % EAF P5K6 Hb. We compared tissue oxygenation by EAF P3K6 Hb with EAF P5K6 Hb and P5K2 Hb in an experimentally induced anemia model and tested its therapeutic efficacy in attenuating the experimentally induced acute crisis in a mild murine model of SCD, NY1DD.MethodsEAF P3K6 Hb was prepared with PEG3K and characterized as EAF P5K6 Hb2. Molecular models and the shapes of PEG Hbs have been developed using InsightII program (Accelrys). Tissue oxygenation studies were carried out in the extreme hemodilution hamster model as described previously.1 Acute vaso-occlusion was studied using intravital microscopy in the venules of cremaster muscles of NY1DD SCD mice subjected to hypoxia-reoxygenation (18 hrs of hypoxia with 8% O2 and then normoxia).3The efficacy was measured after treatment with 10% top load of 6 gm % solution of EAF P3K6 Hb, given at the transitioning stage from hypoxia to normoxiaResultsPEG 3K chains reduced the molecular radius and volume while increased the packing density of PEG shell of EAF P3K6 Hb as compared to EAF P5K6 Hb (Table 1). The packing of the PEG chains induced a globular shape to the EAF P3K6 Hb vs ellipsoidal shape to EAF P5K6 Hb (Fig 1). Oxygen affinity of EAF P3K6 was comparable to that of EAF P5K6 Hb. In the extreme hemodilution hamster model of anemia, the efficacy of EAF P3K6 Hb to increase tissue oxygenation was comparable to that of P5K2 Hb (Fig 2). From the intravital micrographs of cremaster muscles (Fig 3), P3K6 Hb markedly attenuated the induced vaso-occlusive crisis in NY1DD mice.Discussion and ConclusionsWe have previously shown that EAF P3K6 Hb is effective in normalizing the chronic state of vaso-occlusion in BERK sickle mice. We demonstrate here that EAF P3K6 Hb protects NY1DD sickle mice from developing acute vaso-occlusion. These studies suggest that a smaller compact PEG shell around Hb increases the ability of the PEG Hb to oxygenate the tissues. NY1DD mice are not very anemic (Hb > 10 gm/dl); a 10% top load of 6 gm% high oxygen affinity Hb represents no significant increase in Hb level (≈0.06 gm %). We suggest that the ability of such a miniscule amount of PEG Hb in the plasma to protect the animal from hypoxia is due to its enhanced ability to deliver O2 under anemic and hypoxic conditions. We suggest that EAF P3K6 Hb facilitates improved extraction of O2 from RBC to plasma and to the subsequent diffusion to tissues, i.e. small amount of PEG Hb in plasma acts as a catalyst for O2 transfer while the oxyHb in RBC serves as the local reservoir for O2. This improved O2 extractionmay be effective in anemia in general, and have a unique application in sickle cell disease.

Abstract 352: Real-time Intravital Optical Imaging Reflects the Dynamic Changes of Oxidative Stress Induced by Cigarette Smoking in Vasculatures

Background: Reactive oxygen species (ROS) play a central role in cigarette smoking-induced atherogenesis. The present study aims to assess the smoking-induced acute oxidative stress within vasculatures, and evaluates whether the resveratrol, a natural polyphenol antioxidant, can counteract this ROS production, using a customized, high resolution intravital optical imaging in real-time. Methods and Results: 20-week-old male C57BL/6 mice were divided into four groups according to the preceding administration of resveratrol (R) (25mg/kg via gavage, for 7 days) and exposure to cigarette smoke (CS). To in vivo assess acute oxidative stress in blood vessels, dihydroethidium, which forms a red fluorescence (ethidium, excitation/emission: 520nm/610nm) upon reaction with ROS, was injected intraperitoneally. During CS exposure, temporal changes of fluorescence signals from the mouse cremaster muscle including vasculatures were assessed by intravital optical imaging for 15 minutes. Fluorescence signals were much more pronounced in CS exposed mice than controls (p&lt;0.001). Resveratrol p.o. significantly reduced the CS-induced ROS signals compared to the non-treated group (fluorescence signal to noise ratio, SNR, 2.51±0.09 vs. 12.52±2.116, p=0.0002) (Figure A). Without CS exposure, fluorescence signals in targeted vasculatures were very low showing no difference between groups (SNR, 1.65±0.19 vs. 1.53±0.07, p=0.80) (Figure A). Lipid peroxidation was increased in CS group and significantly attenuated in resveratrol-treated mice (Figure B). Fluorescence microscopy and immunostainings corroborated the in vivo findings. Conclusions: The intravital optical imaging was able to in vivo estimate the dynamic changes of ROS production by CS exposure. Our data demonstrated that even a brief exposure to CS increased oxidative stress in vasculatures promptly, and the resveratrol exerts protective effects against the CS-induced acute oxidative stress.