Prothrombotic effects of diclofenac on arteriolar platelet activation and thrombosis in vivo.

J Thromb Haemost. 2009 Oct;7(10):1727-35

Authors: Struthmann L, Hellwig N, Pircher J, Sohn HY, Buerkle MA, Klauss V, Mannell H, Pohl U, Krötz F

BACKGROUND: Diclofenac, like selective cyclooxygenase-2 inhibitors, which alter vascular levels of platelet active prostaglandins, has been reported to increase rates of acute myocardial infarction. OBJECTIVE: The study was performed to investigate, in an animal model of arterial thrombosis in vivo, whether diclofenac differentially influences platelet activation and thrombosis in vessels under non-stimulated conditions or during acute systemic inflammation, such as induced by tumor necrosis factor-alpha (TNF-alpha). METHODS: Platelet-vessel wall interaction (PVWI), firm platelet adhesion and arterial thrombosis following vessel injury were analyzed by intravital microscopy in arterioles of hamsters in the dorsal skinfold chamber model. Prostacyclin [prostaglandin I(2) (PGI(2))] and thromboxane A(2) (TxA(2)) metabolites were measured. In vitro, endothelial adhesion molecule expression in cultured human microvascular endothelial cells was analyzed. RESULTS: Under non-stimulated conditions, diclofenac (1 mg kg(-1)) enhanced PVWI, which was not mediated by increased adhesion molecule expression, but by decreased systemic PGI(2) levels. Following ferric chloride-induced endothelial injury, diclofenac accelerated thrombotic vessel occlusion time, an effect that was reversed by the stable PGI(2) analog iloprost. TNF-alpha, through induction of endothelial adhesion molecule expression, also enhanced PVWI, firm adhesion, and arterial thrombosis, but simultaneous treatment with TNF-alpha and diclofenac did not have an additive effect. CONCLUSIONS: By decreasing levels of PGI(2) without, at the same time, altering prothrombotic TxA(2) levels, diclofenac can exert prothrombotic effects. However, this is not the case when an inflammatory situation is created by TNF-alpha treatment. These data may explain the enhanced risk of acute myocardial infarction observed in patients taking diclofenac.

PMID: 19691487 [PubMed - indexed for MEDLINE]

Accelerated angiogenic host tissue response to PLGA- scaffolds by vitalization with osteoblast like cells.

Tissue Eng Part A. 2010 Feb 25;

Authors: Tavassol F, Schumann P, Lindhorst D, Sinikovic B, Voss A, Stühmer C, Kampmann A, Bormann KH, Carvalho C, Mülhaupt R, Harder Y, Laschke MW, Menger M, Gellrich NC, Rücker M

Bone substitutes should ideally promote rapid vascularization, which could be accelerated if these substitutes were vitalized by autologous cells. Although adequate engraftment of porous poly(L-lactide-co-glycolide) (PLGA) scaffolds has been demonstrated in the past, it has not yet been investigated how vascularization is influenced by vitalization or, more precisely, by seeding PLGA scaffolds with osteoblast-like cells (OLCs). For this reason, we conducted an in vivo study to assess host angiogenic and inflammatory responses after the implantation of PLGA scaffolds vitalized with isogeneic OLCs. OLCs were seeded on collagen-coated PLGA scaffolds that were implanted into dorsal skinfold chambers in BALB/c mice (n=8). Two further groups of animals received either collagen-coated (n=8) or uncoated PLGA scaffolds (n=8). Animals that received chambers without implants served as controls (n=8). Angiogenesis, neovascularization and leukocyte-endothelial cell interaction were analyzed during a 14-day period using intravital fluorescence microscopy. PLGA scaffolds with and without OLCs showed a temporary increase in leukocyte recruitment. At day 3 after implantation, a marked angiogenic host tissue response was observed in close vicinity of all scaffolds studied. At days 6 and 10, the angiogenic response was significantly higher (p<0.05) in PLGA scaffolds vitalized with OLCs than in uncoated or collagen-coated PLGA scaffolds. The majority of OLCs, however, died within 14 days after implantation. Our study demonstrates that PLGA scaffold vitalization with OLCs accelerates the angiogenic response in the surrounding host tissue. Bone substitutes created by tissue engineering may thus be superior to non-vitalized substitutes although the seeded cells do not survive for long periods of time.

PMID: 20184434 [PubMed - as supplied by publisher]

VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo.

Eur Cell Mater. 2010;19:30-40

Authors: Wernike E, Montjovent MO, Liu Y, Wismeijer D, Hunziker EB, Siebenrock KA, Hofstetter W, Klenke FM

Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. Angiogenic growth factors such as vascular endothelial growth factor (VEGF) were shown to promote biomaterial vascularization and enhance bone formation. However, high local concentrations of VEGF induce the formation of malformed, nonfunctional vessels. We hypothesized that a continuous delivery of low concentrations of VEGF from calcium phosphate ceramics may increase the efficacy of VEGF administration.VEGF was co-precipitated onto biphasic calcium phosphate (BCP) ceramics to achieve a sustained release of the growth factor. The co-precipitation efficacy and the release kinetics of the protein were investigated in vitro. For in vivo investigations BCP ceramics were implanted into critical size cranial defects in Balb/c mice. Angiogenesis and microvascularization were investigated over 28 days by means of intravital microscopy. The formation of new bone was determined histomorphometrically. Co-precipitation reduced the burst release of VEGF. Furthermore, a sustained, cell-mediated release of low concentrations of VEGF from BCP ceramics was mediated by resorbing osteoclasts. In vivo, sustained delivery of VEGF achieved by protein co-precipitation promoted biomaterial vascularization, osseointegration, and bone formation. Short-term release of VEGF following superficial adsorption resulted in a temporally restricted promotion of angiogenesis and did not enhance bone formation. The release kinetics of VEGF appears to be an important factor in the promotion of biomaterial vascularization and bone formation. Sustained release of VEGF increased the efficacy of VEGF delivery demonstrating that a prolonged bioavailability of low concentrations of VEGF is beneficial for bone regeneration.

PMID: 20178096 [PubMed - in process]

Uterine NK Cells, Spiral Artery Modification and the Regulation of Blood Pressure During Mouse Pregnancy.

Am J Reprod Immunol. 2010 Feb 17;

Authors: Burke SD, Barrette VF, Gravel J, Carter AL, Hatta K, Zhang J, Chen Z, Leno-Durán E, Bianco J, Leonard S, Murrant C, Adams MA, Anne Croy B

Citation Burke SD, Barrette VF, Gravel J, Carter ALI, Hatta K, Zhang J, Chen Z, Leno-Durán E, Bianco J, Leonard S, Murrant C, Adams MA, Anne Croy B. Uterine NK cells, spiral artery modification and the regulation of blood pressure during mouse pregnancy. Am J Reprod Immunol 2010 Reproductive success in mammals involves coordinated changes in the immune and cardiovascular as well as in the neuroendocrine and reproductive systems. This review addresses studies that identify potential links for NK cells and T cells with the local and systemic cardiovascular adaptations of pregnancy. The studies reviewed have utilized immunohistochemisty and in vivo analyses of vascular parameters by ultrasound, chronic monitoring of hemodynamics via radiotelemetric recording and intravital microscopy. At the uterine level, functional subsets of uterine natural killer cells were identified. These included subsets expressing molecules important for vasoregulation, in addition to those previously identified for angiogenesis. Spiral arteries showed conducted responses that could account for conceptus control of vasoactivity and mouse gestational blood pressure 5-phase pattern. Vascular immunology is an emerging transdisciplinary field, critical for both reproductive immunology and cardiovascular disease.

PMID: 20175772 [PubMed - as supplied by publisher]

Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth.

Proc Natl Acad Sci U S A. 2010 Feb 11;

Authors: Al-Jamal KT, Al-Jamal WT, Akerman S, Podesta JE, Yilmazer A, Turton JA, Bianco A, Vargesson N, Kanthou C, Florence AT, Tozer GM, Kostarelos K

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G(6)) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

PMID: 20150514 [PubMed - as supplied by publisher]

Magnetic Resonance Molecular Imaging of Thrombosis in an Arachidonic Acid Mouse Model Using an Activated Platelet Targeted Probe.

Arterioscler Thromb Vasc Biol. 2010 Feb 5;

Authors: Klink A, Lancelot E, Ballet S, Vucic E, Fabre JE, Gonzalez W, Medina C, Corot C, Mulder WJ, Mallat Z, Fayad ZA

OBJECTIVE: Atherosclerotic plaque rupture leads to acute thrombus formation and may trigger serious clinical events such as myocardial infarction or stroke. Therefore, it would be valuable to identify atherothrombosis and vulnerable plaques before the onset of such clinical events. We sought to determine whether the noninvasive in vivo visualization of activated platelets was effective when using a target-specific MRI contrast agent to identify thrombi, hallmarks of vulnerable or high-risk atherosclerotic plaques. METHODS AND RESULTS: Inflammatory thrombi were induced in mice via topical application of arachidonic acid on the carotid. Thrombus formation was imaged with intravital fluorescence microscopy and molecular MRI. To accomplish the latter, a paramagnetic contrast agent (P975) that targets the glycoprotein alphaIIbbeta3, expressed on activated platelets, was investigated. The specificity of P975 for activated platelets was studied in vitro. In vivo, high spatial-resolution MRI was performed at baseline and longitudinally over 2 hours after injecting P975 or a nonspecific agent. The contralateral carotid, a sham surgery group, and a competitive inhibition experiment served as controls. P975 showed a good affinity for activated platelets, with an IC50 (concentration of dose that produces 50% inhibition) value of 2.6 mumol/L. In thrombosed animals, P975 produced an immediate and sustained increase in MRI signal, whereas none of the control groups revealed any significant increase in MRI signal 2 hours after injection. More important, the competitive inhibition experiment with an alphaIIbbeta3 antagonist suppressed the MRI signal enhancement, which is indicative for the specificity of P975 for the activated platelets. CONCLUSIONS: P975 allowed in vivo target-specific noninvasive MRI of activated platelets.

PMID: 20139362 [PubMed - as supplied by publisher]

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke.

BMC Neurosci. 2010 Feb 2;11(1):12

Authors: Jin AY, Tuor UI, Rushforth D, Kaur J, Muller RN, Petterson JL, Boutry S, Barber PA

ABSTRACT: BACKGROUND: The link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Slex) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T1 maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G ( IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa). RESULTS: MRI confirmed similar infarct sizes and T1 values at 24 hours following stroke for both WT and KO animals. However, the blood to brain transfer constant for Gd DTPA (Kgd) demonstrated greater tissue extravasation of Gd DTPA in WT animals than KO mice (P<0.03). In the P selectin KO mice, Delta T1 stroke -Delta T1 contralateral control cortex, decreased significantly in the Gd-DTPA(sLex) group compared to Gd-DTPA, indicative of sLex mediated accumulation of the targeted contrast agent. Regarding BBB function, in the P-selectin KO mice compared to WT control mice, there was an attenuation in the extravasation of IgG (P<0.001), a trend for decreased FITC extravasation and less infiltration of PMN leukocytes (P<0.001) thereby supporting the observed increase in Kgd permeability in stroke brain of WT compared to KO mice. CONCLUSION: P-selectin expression contributes to enhanced BBB dysfunction at 24 hours after transient focal cerebral ischemia.

PMID: 20122276 [PubMed - as supplied by publisher]

Determination of Microcirculatory Changes and Angiogenesis in a Model of Frostbite Injury In Vivo.

J Surg Res. 2009 Aug 11;

Authors: Goertz O, Baerreiter S, Ring A, Jettkant B, Hirsch T, Daigeler A, Steinau HU, Langer S

BACKGROUND: The breakdown of skin microcirculation and the leukocyte-endothelium interaction are assumed to play a key role in the pathophysiology of frostbite injuries. However, little is known as yet. The aim was to develop an in vivo frostbite model to monitor microcirculatory changes and angiogenesis after frostbite injury. MATERIALS AND METHODS: Deep partial thickness frostbite injuries were inflicted with a no-touch-technique to the ears of hairless mice (n=9). To this end, a gas jet of nitrogen vapor (T=-195,8+/-2.7 degrees C) was delivered onto an area of 1.9mm(2) for 1,5 s. Intravital fluorescent microscopy in combination with FITC-dextran and Rhodamin 6G as fluorescent dyes was used to assess microcirculatory changes, leukocyte behavior, and angiogenesis during the 14 d of wound healing. RESULTS: The area of no perfusion decreased significantly over the observed period, and perfusion was almost completely restored due to angiogenesis by d 14 (day 1: 1.89 [mm(2)]+/-0.44SEM, d 14: 0.02+/-0.01). No post-traumatic extension of the trauma could be observed. Edema formation increased significantly up to d 7. The number of adherent leukocytes showed a significant increase during the first 7 d. Functional vessel density showed a significant post-frostbite decrease to 60% of the baseline value. CONCLUSIONS: This novel frostbite model provides a simple and nonetheless highly effective technique of creating locally limited reproducible frostbite injuries using a no touch technique. Tissue damage can be fully attributed to the thermal trauma, and the model allows repetitive intravital fluorescent microscopy of the microcirculation, leukocyte-endothelium interaction, and angiogenesis.

PMID: 20097363 [PubMed - as supplied by publisher]

Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy.

J Gastroenterol. 2010 Jan 9;

Authors: Toiyama Y, Mizoguchi A, Okugawa Y, Koike Y, Morimoto Y, Araki T, Uchida K, Tanaka K, Nakashima H, Hibi M, Kimura K, Inoue Y, Miki C, Kusunoki M

BACKGROUND: Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging. MATERIALS AND METHODS: An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM. RESULTS AND CONCLUSIONS: We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.

PMID: 20058031 [PubMed - as supplied by publisher]

Photodynamic therapy induces selective extravasation of macromolecules: Insights using intravital microscopy.

J Photochem Photobiol B. 2009 Nov 26;

Authors: Debefve E, Cheng C, Schaefer SC, Yan H, Ballini JP, van den Bergh H, Lehr HA, Ruffieux C, Ris HB, Krueger T

Photodynamic therapy (PDT) with Visudyne((R)) acts by direct cellular phototoxicity and/or by an indirect vascular-mediated effect. Here, we demonstrate that the vessel integrity interruption by PDT can promote the extravasation of a macromolecular agent in normal tissue. To obtain extravasation in normal tissue PDT conditions were one order of magnitude more intensive than the ones in tissue containing neovessels reported in the literature. Fluorescein isothiocyanate dextran (FITC-D, 2000kDa), a macromolecular agent, was intravenously injected 10min before (LK0 group, n=14) or 2h (LK2 group, n=16) after Visudyne((R))-mediated PDT in nude mice bearing a dorsal skin fold chamber. Control animals had no PDT (CTRL group, n=8). The extravasation of FITC-D from blood vessels in striated muscle tissue was observed in both groups in real-time for up to 2500s after injection. We also monitored PDT-induced leukocyte rolling in vivo and assessed, by histology, the corresponding inflammatory reaction score in the dorsal skin fold chambers. In all animals, at the applied PDT conditions, FITC-D extravasation was significantly enhanced in the PDT-treated areas as compared to the surrounding non-treated areas (p<0.0001). There was no FITC-D leakage in the control animals. Animals from the LK0 group had significantly less FITC-D extravasation than those from the LK2 group (p=0.0002). In the LK0 group FITC-D leakage correlated significantly with the inflammation (p<0.001). At the selected conditions, Visudyne((R))-mediated PDT promotes vascular leakage and FITC-D extravasation into the interstitial space of normal tissue. The intensity of vascular leakage depends on the time interval between PDT and FITC-D injection. This concept could be used to locally modulate the delivery of macromolecules in vivo.

PMID: 20056552 [PubMed - as supplied by publisher]

Exogenous Nitric Oxide Donation Causes Desensitization of Arteriolar Relaxing Activity In Vivo: An Intravital Analysis in Mice.

J Surg Res. 2009 Aug 5;

Authors: Ring A, Mullershausen F, Goertz O, Koesling D, Muhr G, Steinau HU, Steinstraesser L, Langer S

BACKGROUND: The NO/cGMP pathway plays a crucial role in regulation of tissue perfusion. However, a NO-induced desensitization of cGMP-mediated relaxation has been reported in isolated tissue. To examine whether a similar phenomenon can be detected in vivo, we analyzed relaxations of microvessels in response to repeated applications of NO. MATERIALS AND METHODS: The investigations were performed by means of dynamic intravital fluorescence microscopy in the dorsal skinfold chamber of female balb/mice. First, the microvasculature was maximally preconstricted by the application of the vasoconstrictor 5-hydroxytryptamine. Subsequently, relaxation was induced by applying an NO-donator, the S-nitrosoglutathione, to the contracted vessels. Following buffer exchange, constriction and relaxation were repeated. Drugs were given topically into the chamber, directly onto the skin muscle. The response of arterioles to topical administration of vasoactive drugs was determined as the change of the diameter, and quantified using standard software. RESULTS: The relaxation of arterioles was reduced after repetitive application. The short pretreatment with NO-donor entailed a reduced relaxation of arterioles in response to following application. The absolute change in vessel diameter induced by S-nitrosoglutathione was significantly reduced from 21 mum to 16 mum after the first and the second application, respectively. However, the data also revealed a noticeable reduction of the constricting activity of 5-hydroxytryptamine during the second application, indicating a possible desensitization of the 5-hydroxytryptamine response or a humoral and/or neuronal compensatory mechanisms. CONCLUSIONS: The NO-induced cGMP-mediated relaxation of microvessels was quantified, and the phenomenon of desensitization visualized in vivo by means of dynamic fluorescence microscopy.

PMID: 20056246 [PubMed - as supplied by publisher]

Unbiased discovery of in vivo imaging probes through in vitro profiling of nanoparticle libraries.

Integr Biol (Camb). 2009 Apr;1(4):311-317

Authors: Kelly KA, Shaw SY, Nahrendorf M, Kristoff K, Aikawa E, Schreiber SL, Clemons PA, Weissleder R

In vivo imaging reveals how proteins and cells function as part of complex regulatory networks in intact organisms, and thereby contributes to a systems-level understanding of biological processes. However, the development of novel in vivo imaging probes remains challenging. Most probes are directed against a limited number of pre-specified protein targets; cell-based screens for imaging probes have shown promise, but raise concerns over whether in vitro surrogate cell models recapitulate in vivo phenotypes. Here, we rapidly profile the in vitro binding of nanoparticle imaging probes in multiple samples of defined target vs. background cell types, using primary cell isolates. This approach selects for nanoparticles that show desired targeting effects across all tested members of a class of cells, and decreases the likelihood that an idiosyncratic cell line will unduly skew screening results. To adjust for multiple hypothesis testing, we use permutation methods to identify nanoparticles that best differentiate between the target and background cell classes. (This approach is conceptually analogous to one used for high-dimensionality datasets of genome-wide gene expression, e.g. to identify gene expression signatures that discriminate subclasses of cancer.) We apply this approach to the identification of nanoparticle imaging probes that bind endothelial cells, and validate our in vitro findings in human arterial samples, and by in vivo intravital microscopy in mice. Overall, this work presents a generalizable approach to the unbiased discovery of in vivo imaging probes, and may guide the further development of novel endothelial imaging probes.

PMID: 20023731 [PubMed - as supplied by publisher]

Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy.

Opt Lett. 2009 Dec 15;34(24):3899-3901

Authors: Hu S, Yan P, Maslov K, Lee JM, Wang LV

We report optical-resolution photoacoustic microscopy (OR-PAM) for in vivo imaging of amyloid plaques in an Alzheimer's disease mouse model. Validation using conventional fluorescence microscopy and multiphoton microscopy shows that OR-PAM has sufficient sensitivity and spatial resolution to identify amyloid plaques in living brains. In addition, with dual-wavelength OR-PAM, the three-dimensional morphology of amyloid plaques and the surrounding microvasculature are imaged simultaneously through a cranial window without angiographic contrast agents. OR-PAM, capable of providing both exogenous molecular contrast and endogenous hemoglobin contrast, has the potential to serve as a new technology for in vivo microscopic observations of cerebral plaque deposits.

PMID: 20016651 [PubMed - as supplied by publisher]

Epifluorescence intravital microscopy of murine corneal dendritic cells.

Invest Ophthalmol Vis Sci. 2009 Dec 10;

Authors: Lee E, Rosenbaum JT, Planck SR

Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study we examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. We used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the response of resident central and peripheral corneal DCs to: 1) silver nitrate injury; 2) lipopolysaccharide (LPS); 3) microspheres; 4) tumor necrosis factor (TNF-alpha). In some experiments, TNF-alpha injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently re-injected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and more dense in the periphery, with epithelial-level DCs having extensions extending into the epithelium. Video microscopy showed that while cell processes were in continuous movement, cells generally did not migrate. Within the first 6 h after stimulation, neither central nor peripheral corneal DC exhibited significant lateral migration, but central corneal DCs assumed extreme morphological changes. An increased number of DCs in the TNF-alpha-stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-alpha injection are more likely to respond to a secondary insult with lateral migration.

PMID: 20007837 [PubMed - as supplied by publisher]

Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers.

Biomaterials. 2009 Oct;30(31):6202-12

Authors: Ghanaati S, Webber MJ, Unger RE, Orth C, Hulvat JF, Kiehna SE, Barbeck M, Rasic A, Stupp SI, Kirkpatrick CJ

Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the static analysis, the heparan sulfate-containing nanofiber gels were found to persist in the tissue for up to 30 days and revealed excellent biocompatibility. Strikingly, as the nanofiber gel biodegraded, we observed the formation of a de novo vascularized connective tissue. In the dynamic experiments using the dorsal skinfold chamber, the material again demonstrated good biocompatibility, with minimal dilation of the microcirculation and only a few adherent leukocytes, monitored through intravital fluorescence microscopy. The new application of the dorsal skinfold model corroborated our findings from the traditional static histology, demonstrating the potential use of this technique to dynamically evaluate the biocompatibility of materials. The observed biocompatibility and development of new vascularized tissue using both techniques demonstrates the potential of these angiogenesis-promoting materials for a host of regenerative strategies.

PMID: 19683342 [PubMed - indexed for MEDLINE]

Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model.

PLoS One. 2009;4(11):e8053

Authors: Thurber GM, Figueiredo JL, Weissleder R

BACKGROUND: Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time. METHODOLOGY/PRINCIPAL FINDINGS: Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry. CONCLUSIONS/SIGNIFICANCE: Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection.

PMID: 19956597 [PubMed - in process]

TLR4 signaling augments B lymphocyte migration and overcomes the restriction that limits access to germinal center dark zones.

J Exp Med. 2009 Nov 16;

Authors: Hwang IY, Park C, Harrison K, Kehrl JH

B lymphocyte-intrinsic Toll-like receptor (TLR) signals amplify humoral immunity and can exacerbate autoimmune diseases. We identify a new mechanism by which TLR signals may contribute to autoimmunity and chronic inflammation. We show that TLR4 signaling enhances B lymphocyte trafficking into lymph nodes (LNs), induces B lymphocyte clustering and interactions within LN follicles, leads to sustained in vivo B cell proliferation, overcomes the restriction that limits the access of nonantigen-activated B cells to germinal center dark zones, and enhances the generation of memory and plasma cells. Intravital microscopy and in vivo tracking studies of B cells transferred to recipient mice revealed that TLR4-activated, but not nonstimulated, B cells accumulated within the dark zones of preexisting germinal centers even when transferred with antigen-specific B cells. The TLR4-activated cells persist much better than nonstimulated cells, expanding both within the memory and plasma cell compartments. TLR-mediated activation of B cells may help to feed and stabilize the spontaneous and ectopic germinal centers that are so commonly found in autoimmune individuals and that accompany chronic inflammation.

PMID: 19917774 [PubMed - as supplied by publisher]