Dorsal Skinfold Chamber Research Articles

Early Host Tissue Response to Different Types of Vascular Prostheses Coated with Silver Acetate or Vaporized Metallic Silver

In vascular surgery, the infection of prosthetic vascular grafts represents a serious life-threatening complication. Due to the increasing resistance of hospital micro-organisms to standard antibiotic therapies, maximum effort should be put in the primary prevention of such infections. For this purpose, grafts may be coated with different antibacterial silver formulations. In the present study the different effects of silver acetate-coating and vaporized metallic silver-coating on the vascularization and perigraft inflammation during the initial phase after implantation of Intergard Silver (IS) and Silver Graft (SG) were compared. Silver acetate-coated IS and vaporized metallic silver-coated SG were implanted into the dorsal skinfold chamber of C57BL/6 mice (n = 8 per group) to study angiogenesis and leukocyte inflammation at the implantation site by means of repetitive intravital fluorescence microscopy over a 14-day period. At the end of the in vivo experiments, apoptosis and cell proliferation in the newly developed granulation tissue surrounding the implants was analyzed by immunohistochemistry. IS exhibited an improved vascularization, resulting in a significantly higher functional capillary density when compared to SG. Moreover, the leukocyte inflammatory response to IS was less pronounced, as indicated by a reduced number of adherent leukocytes in perigraft venules. This was associated with a higher proliferative activity of the granulation tissue incorporating the IS when compared to SG. The numbers of apoptotic cells in the perigraft tissue were low and did not differ between the two groups. Silver acetate-coated IS exhibits an improved vascularization and reduced perigraft inflammation during the first 14 days after implantation when compared to vaporized metallic silver-coated SG. This may contribute to reducing the risk of early perigraft seroma formation and subsequent infection.

Breast cancer cell homing to bone in vivo is modulated by zoledronic acid (676.6)

Cancer metastasis to the skeleton causes mortality in breast cancer, however the mechanisms involved in bone metastases development remain unclear. A metastatic initiating cell (MIC) phenotype CD44+/CD24‐/ALDHhigh has been identified in breast cancer. Zoledronic acid (zol), a bone anti‐resorptive bisphosphonate is currently the only agent with proven benefit in breast cancer. This study investigates whether zol modulates MIC homing in the early stages of breast cancer bone metastasis using the Dorsal Skinfold Chamber (DSC) metatarsal model in vivo.Following DSC metatarsal implantation, mice were treated weekly either before or after intra‐cardiac MDA‐MB‐231 tumour cell injection (1x105) for 4 weeks (Zol: 25µg/kg/injection=100μg/kg or PBS control). MDA‐MB‐231 cells were sorted into ALDHhigh and ALDHlow subpopulations using the ALDEFLUORkit and then injected into groups of mice (n=6), treated with zol before tumour cell injection. Chamber tissue and metatarsal recordings were made every 48hr for up to 4 weeks to identify tumour cell homing. At the end of the study, tissue was harvested and processed for µCT, multi‐photon analysis, histology and immunohistochemistry.Zol treatment reduces MDA‐MB‐231 cells homing to bone by 76% (before) and 62% (after) compared to control animals. ALDHhigh cells home to bone in significantly (p<0.05) higher numbers than ALDHlow cells (Day 25: 10.7±1.3 cells vs. 1.1±0.6 cells). Week zol administration (before tumour cell injection) does not reduce MDA‐MB‐231 cells bone‐homing.Weekly treatment with zol reduces breast tumour cell homing to bone, whereas ALDHhigh cell homing appears resistant to zol treatment in this model.

Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor- by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions

Is telmisartan effective in the treatment of endometriosis? Combined blockade of angiotensin II type 1 receptor (AT1R) and activation of peroxisome proliferator-activated receptor (PPAR)-γ by telmisartan inhibits vascularization and growth of murine endometriosis-like lesions. AT1R and PPAR-γ are involved in the regulation of inflammation, proliferation and angiogenesis. These processes are also crucial for the pathogenesis of endometriosis and both receptors are expressed in endometrial tissue. Telmisartan is a partial agonist of PPAR-γ, which additionally blocks AT1R. This was a randomized study in the mouse dorsal skinfold chamber and peritoneal model of endometriosis. Endometriosis-like lesions were induced in dorsal skinfold chambers of 21 female C57BL/6 mice, and in the peritoneal cavity of 15 additional animals, which were daily treated with an i.p. injection of pioglitazone (10 mg/kg, n = 12), telmisartan (10 mg/kg, n = 12) or vehicle (5% dimethyl sulfoxide (DMSO), n = 12) throughout an observation period of 14 and 28 days, respectively. The anti-angiogenic actions of pioglitazone, a full PPAR-γ agonist, and telmisartan were firstly assessed in vitro by an aortic ring assay. Endometriosis-like lesions were induced in the dorsal skinfold chamber or peritoneal cavity and the effects of telmisartan and pioglitazone on their vascularization, immune cell content and growth were studied by intravital fluorescence microscopy, high-resolution ultrasound imaging as well as histological, immunohistochemical and immunofluorescent analyses. Additional quantitative real-time polymerase chain reaction (qRT-PCR) arrays served for gene expression profiling of the lesions. To limit the role of chance, the experiments were conducted under standardized laboratory conditions with appropriate vehicle-treated controls. Statistical significance was accepted for a value of P < 0.05. Telmisartan inhibited vascular sprout formation of aortic rings more effectively than pioglitazone. Accordingly, endometriosis-like lesions in dorsal skinfold chambers of telmisartan-treated animals exhibited a markedly lower functional microvessel density and blood perfusion. High-resolution ultrasound analyses of peritoneal endometriosis-like lesions revealed that the compound inhibited the stromal tissue growth, resulting in a significantly reduced final lesion volume. In contrast, the development of cysts did not differ between the groups. Moreover, telmisartan induced an up-regulation of PPAR-γ and a down-regulation of AT1R proteins in endometriosis-like lesions, which was associated with a decreased density of CD31-positive microvessels, a reduced immune cell content and a lower number of Ki67-positive proliferating cells. qRT-PCR arrays further demonstrated an inhibitory action of telmisartan on the expression of several angiogenic and inflammatory genes. Endometriosis-like lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. This study demonstrates that telmisartan inhibits vascularization, immune cell content and growth of endometriosis-like lesions. Accordingly, the combined blockade of AT1R and activation of PPAR-γ represents a promising new concept in the development of novel compounds for the treatment of endometriosis. There was no specific funding of this study. The authors have no conflicts of interest to declare.

Antiangiogenic Therapy Inhibits the Recruitment of Vascular Accessory Cells to the Perivascular Niche in Glioma Angiogenesis

Objective: Antiangiogenic therapies could be limited by various escape mechanisms including bone marrow-derived myeloid cell-induced vasculogenesis. The recruitment of vascular accessory cells (VACs) to the tumor neovasculature is as a multistep process. However, the recruitment process of these cells during antiangiogenic treatment remains unknown. The aim of our study was to characterize the recruitment of VACs during antiangiogenic therapy using sunitinib. Methods: C6 glioma cells were implanted into dorsal skinfold chambers. Animals received antiangiogenic therapy intraperitoneally for 5 days prior to VAC application intra-arterially. Intravital microscopy was performed during VAC injection and 1 and 48 h after injection. Analyses included total (TVD) and functional vessel densities (FVD), the perfusion index (PI), microvascular permeability, blood flow rate (Q), microvascular diameter (D), red blood cell velocity (RBCV), wall shear rate (γ), wall shear stress (τ), first and firm adhesions of VACs, and accumulation in the perivascular niche. Results: Antiangiogenic therapy resulted in a significant reduction in TVD (365 ± 47 cm/cm² vs. 183 ± 37 cm/cm²) and FVD (227 ± 65 cm/cm² vs. 147 ± 25 cm/cm²) and an increase in PI, Q, D, and RBCV. γ and τ remained unaltered. Initial adhesion and firm adhesion were unaffected by antiangiogenic therapy; however, the accumulation in the perivascular niche was significantly diminished in treated tumors (53.7 ± 8% vs. 24.0 ± 17%). Conclusions: Antiangiogenic treatment inhibits the accumulation of VACs in the perivascular niche and therefore interferes with consecutive neovascularization.

Interaction of the chemokines I-TAC (CXCL11) and SDF-1 (CXCL12) in the regulation of tumor angiogenesis of colorectal cancer

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.

Correlative intravital imaging of cGMP signals and vasodilation in mice.

Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism.

In Vivo Evaluation of Wound Bed Reaction and Graft Performance After Cold Skin Graft Storage

Surplus harvested skin grafts are routinely stored at 4 to 6°C in saline for several days in plastic surgery. The purpose of this study was to evaluate the influence of storage on human skin graft performance in an in vivo intravital microscopic setting after transplantation. Freshly harvested human full-thickness skin grafts and split-thickness skin grafts (STSGs) after storage of 0, 3, or 7 days in moist saline at 4 to 6°C were transplanted into the modified dorsal skinfold chamber, and intravital microscopy was performed to evaluate vessel morphology and angiogenic change of the wound bed. The chamber tissue was harvested 10 days after transplantation for evaluation of tissue integrity and inflammation (hematoxylin and eosin) as well as for immunohistochemistry (human CD31, murine CD31, Ki67, Tdt-mediated dUTP-biotin nick-end labelling). Intravital microscopy results showed no differences in the host angiogenic response between fresh and preserved grafts. However, STSGs and full-thickness skin grafts exhibited a trend toward different timing and strength in capillary widening and capillary bud formation. Preservation had no influence on graft quality before transplantation, but fresh STSGs showed better quality 10 days after transplantation than 7-day preserved grafts. Proliferation and apoptosis as well as host capillary in-growth and graft capillary degeneration were equal in all groups. These results indicate that cells may activate protective mechanisms under cold conditions, allowing them to maintain function and morphology. However, rewarming may disclose underlying tissue damage. These findings could be translated to a new approach for the design of full-thickness skin substitutes.

Blocking Von Willebrand Factor for Treatment of Cutaneous Inflammation

Von Willebrand factor (VWF), a key player in hemostasis, is increasingly recognized as a proinflammatory protein. Here, we found a massive accumulation of VWF in skin biopsies of patients suffering from immune complex (IC)-mediated vasculitis (ICV). To clarify the impact of VWF on cutaneous inflammation, we induced experimental ICV either in mice treated with VWF-blocking antibodies or in VWF(-/-) mice. Interference with VWF led to a significant inhibition of the cutaneous inflammatory response. We confirmed the major findings in irritative contact dermatitis, a second model of cutaneous inflammation. In vivo imaging of cutaneous inflammation in the dorsal skinfold chamber revealed unaffected leukocyte rolling on anti-VWF treatment. However, we identified that reduced leukocyte recruitment is accompanied by reduced vascular permeability. Although VWF-mediated neutrophil recruitment to the peritoneum was described to require the VWF receptor on platelets (glycoprotein Ibα (GPIbα)), the VWF/GPIbα axis was dispensable for cutaneous inflammation. As assessed in tail bleeding assays, we could exclude interference of VWF blockade with hemostasis. Of particular importance, anti-VWF treatment was effective both in prophylactic and therapeutic administration. Thus, VWF represents a promising target for the treatment of cutaneous inflammation, e.g., leukocytoclastic vasculitis.

Isobel Quita Barber

Background: Collagenase and hyaluronidase are enzymes which degrade the extracellular matrix and increase the uptake and improve the distribution of therapeutic macromolecules in tumours. The purpose of the present work...

Novel Tubulin Antagonist Pretubulysin Displays Antivascular Properties In Vitro and In Vivo

Pretubulysin (PT) is a novel, synthetically accessible myxobacterial compound that acts as a tubulin-depolymerizing agent and inhibits cancer cell growth in vitro and in vivo. Moreover, PT was found to attenuate tumor angiogenesis. Here, we hypothesized that PT could exert antivascular activities on existing tumor vessels. We aimed to characterize the antivascular effects of PT and to elucidate the underlying mechanisms in endothelial cells. In vitro, PT rapidly induced endothelial hyperpermeability and a concentration-dependent disassembly of established endothelial tubes on Matrigel and in an ex vivo aortic ring model. It disrupted endothelial cell junctions and triggered F-actin stress fiber formation and cell contraction by the RhoA/Rho-associated protein kinase pathway without causing cell death. In vivo, using a hamster dorsal skinfold chamber preparation, PT significantly decreased blood flow and vessel diameter in hamster A-Mel-3 amelanotic melanoma tumors but not in the neighboring healthy tissue. In a second tumor model using mice with subcutaneous murine B16 melanoma tumors, a single dose of PT (10 mg/kg) caused a shut down of tumor blood flow and a strong central tumor cell necrosis within 24 hours. Repeated PT administration significantly decelerates tumor growth and seems to be well tolerated. In summary, we could show for the first time that the antitumor effect of PT is, at least in part, mediated via its antivascular activities on existing tumor vessels.

Fucosylation Inhibitor, 2-Fluorofucose, Inhibits NF-κB Activation and Vaso-Occlusion In Transgenic Sickle Mice

Polymorphonuclear cells (PMNs) roll along vessel walls by binding to E- and P-selectins on the endothelial surface. These vascular adhesion molecules are involved in the initial tethering of PMNs on endothelium rather than their firm adhesion. Endothelial cell–expressed P- and E-selectins interact with leukocyte counterreceptors P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1). These glycoprotein counterreceptors are active only when modified posttranslationally by fucosylated oligosaccharides represented by the sialyl Lewis x tetrasaccharide (sLex) or its structural variants. Because of the important role that fucose plays in protein glycosylation and cellular functions, specific inhibitors of its incorporation could have significant applications in basic research and therapy. One such inhibitor, 2-fluorofucose peracetate, was recently reported to block fucosylation of cellular proteins and to inhibit binding of PMNs to P- and E-selectin. One potential application is the inhibition of leukocyte recruitment in pro-inflammatory disease states such as sickle cell disease (SCD). P-selectin expression on endothelium has been demonstrated to play an important role in vaso-occlusion, a hallmark of SCD. Inhibition of P-selectin binding inhibits vaso-occlusion in transgenic sickle mice. Therefore, we hypothesized that 2-fluorofucose would inhibit tissue inflammation and vaso-occlusion in SCD mice. MethodsNY1DD sickle mice were given plain water or 20 or 100 mM 2-fluorofucose in their drinking water ad libitum. The first cohort of mice (n=8/group) was treated for 7 days. On day 7 the mice were sacrificed with CO2, an EDTA blood sample was collected from the heart and the liver was removed and frozen in liquid nitrogen. Total white blood cell counts and differentials were performed by manual counting using a hemocytometer and Wright-stained blood smears, respectively. Nuclear extracts were prepared from nuclei isolated from liver homogenates. Western blots of the nuclear extracts were immunostained with antibodies to NF-ĸB phospho-p65 a marker of NF-ĸB activation. The second cohort of mice (n=4/group) was also treated for 7 days. On day 4, dorsal skin fold chambers were implanted onto the mice. On day 7, flowing venules in the dorsal skin-fold chamber window were selected and mapped using intravital microscopy. Thereafter, the mice were infused via the tail vein with human stroma-free hemoglobin (0.32 µmols heme/kg), a known inducer of vascular stasis in SCD mice. At 1 and 4 hours after infusion the same venules were re-examined and the percentage of vessels that had become static (no flow) was recorded. A positive control group of NY1DD mice (n=3) with implanted dorsal skin-fold chambers was given water to drink and pretreated with hemin (40 µmols/kg X 3 days, i.p.), a known inhibitor of vascular stasis. ResultsWhite blood cells counts were 16.3±3.2 (K/µL, mean±SD) in SCD mice treated with water. The white counts increased to 22.1±5.2 in SCD mice treated with 20 mM 2-fluorofucose and 34.2±7.2 in SCD mice treated with 100 mM 2-fluorofucose, respectively (p<0.05 for all pairwise comparisons). NF-ĸB in liver and other organs is activated in SCD mice compared to normal C57BL/6 mice. Nuclear NF-ĸB phospho-p65 was partially diminished in mice treated with 20 mM 2-fluorofucose and markedly decreased in mice treated with 100 mM 2-fluorofucose or pretreated with heme (Fig. 1).Infusion of hemoglobin induced ∼30% microvascular stasis at 1 and 4 hours in SCD mice treated with water (Fig. 2). Treatment with 20 mM 2-fluorofucose partially inhibited stasis at 1 and 4 hours (p<0.05 compared to water). Treatment with 100 mM 2-fluorofucose or heme inhibited stasis to 6.7% or less at 1 and 4 hours (p<0.025 compared to water). [Display omitted] [Display omitted] ConclusionsOral administration of 2-fluorofucose has significant anti-inflammatory and anti-vaso-occlusive properties in SCD mice. Disclosures:Belcher:Seattle Genetics, Inc.: Research Funding. Chen:Seattle Genetics, Inc.: Research Funding. Nguyen:Seattle Genetics, Inc.: Research Funding. Okeley:Seattle Genetics, Inc.: Employment. Senter:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Vercellotti:Seattle Genetics, Inc.: Research Funding.

Interference With TNFα Using Long-Term Etanercept In S+SAntilles Sickle Transgenic Mice Ameliorates Abnormal Endothelial Activation, Vasoocclusion, and Pulmonary Hypertension Including Its Pulmonary Arterial Wall Remodeling

A systemic inflammatory state is a prominent feature of sickle cell anemia. Using sickle transgenic mice, we previously identified a blood monocyte-TNF-endothelium activation axis using multiple complementary approaches. For example, endothelial activation (identified by abnormal expression of VCAM-1 and tissue factor (TF) by pulmonary vessel endothelium) was evident in unstressed BERK and S+SAntilles sickle mice. In NY1DD sickle mice, this was triggered by transient enhanced sickling and vasoocclusion resulting from transient exposure to hypoxia/reoxygenation (H/R). Endothelial activation depended upon Egr-1 and NFkB(p50) in peripheral blood mononuclear cells, and transfusion experiments clarified that the peripheral blood monocytes (PBM) were the responsible effecter cells. Finally, interference with TNFa using etanercept (Etn) prevented this PBM-induced endothelial activation. Based on these background data, we have now tested the “clinical” efficacy of long-term, anti-inflammatory TNFa interference via treatment of S+SAntilles sickle mice with Etn (started at weaning, 3 mg/kg sc, once/wk x 6 wk). In parallel, littermate controls received equimolar scrambled peptide version of Etn's TNF-receptor moiety. In response we observed significant improvement in multiple parameters relevant to inflammation-induced vessel wall disease in this model, as follows (data shown in inset). Etn decreased blood markers of inflammation: plasma levels of sVCAM-1 and SAP (by ELISA), and MCP-1 and TNFa (by FACS cytobead assay). Etn also decreased the % of PBM positive for TNFa (by FACS). Etn diminished the abnormal pulmonary vessel endothelial expression of VCAM-1 and TF (by histochemical and immunofluorescent staining). Etn eliminated the enhanced vasoocclusion seen as dermal microvascular stasis in the live mouse (using the dorsal skin fold chamber) measured at 1 and 4 hours post-H/R (7%O2x1hr, followed by 21%O2x 1-4hr). Instructively, Etn did not significantly improve the similar stasis induced by heme infusion, a TNF-independent direct endothelial stressor. Finally, Etn diminished evidence for pulmonary hypertension, assessed via three of its hallmark features: elevated right ventricular mean systolic pressure (by RV catheterization), abnormal peri-vascular inflammatory cell aggregates (by immuno-histochemical staining for CD11b), and abnormal pulmonary arterial wall muscularization (by immuno-histochemical staining for SM actin). Remarkably, Etn actually normalized RVSP, and Etn's partial improvement in pulmonary arterial muscularization resulted not only from prevention of its progression over duration of this study but also from some reversal of the abnormal remodeling that had already developed by the time Etn was started. In parallel, Etn reduced the number of abnormal peri-vascular aggregates of CDllb+ cells in the pulmonary arterial system.These results enable several conclusions. In the S+SAntilles sickle model, endothelial activation and aspects of vascular wall pathobiology derive from TNFa produced by activated PBM, a monocyte-TNF-endothelial activation axis. Interference with TNFa using etanercept exerts multiple salubrious vascular effects that predict possible clinical benefit. And results suggest that inflammation involving TNFa plays a proximate, generative role in development of pulmonary hypertension in this model. Assuming these benefits are independently corroborated, a controlled test of etanercept's efficacy for aspects of human sickle vascular pathobiology perhaps should be considered. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Generation of co-culture spheroids as vascularisation units for bone tissue engineering

Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. After in vitro characterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to study in vivo their viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry. In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.

Long‐term Preconditioning with Erythropoietin Reduces Ischemia‐Induced Skin Necrosis

Recent findings have attested to EPO tissue-protective effects in ischemically challenged tissues. Therefore, the study aimed at elaborating the effect of systemic pre- and postconditioning using EPO in a mouse model of persistent ischemia of the skin. Three groups of nine C57Bl/6-mice each were analyzed. The experimental groups consisted of untreated controls, EPO preconditioning, and EPO postconditioning (500 IU EPO/kg bw/day for 10 days). Critically perfused skin flaps undergoing necrosis, if kept untreated, were mounted into dorsal skinfold chambers. Intravital epi-fluorescence microscopy was performed for 10 days to assess tissue necrosis, microcirculation, inflammation, and angiogenesis. Protein expression analysis of eNOS was performed. Hematocrit analyses were carried out separately in eight animals. Only EPO preconditioning was able to significantly reduce necrosis, when compared with controls. This correlated with a significantly increased CD in the critically perfused tissue. Administration of EPO only slightly increased eNOS expression at day 10, when compared with controls. EPO induced angiogenesis and increased hematocrit. Finally, EPO significantly reduced leukocytic inflammation in arterioles in all EPO receiving mice. EPO preconditioning effectively reduces skin necrosis predominantly by capillary maintenance and reperfusion, as well as improved tissue regeneration. Thus, EPO preconditioning might represent a promising, non-invasive approach to reduce complications in ischemically challenged skin.

Molecular imaging reveals time course of matrix metalloproteinase activity in acute cutaneous vasculitis in vivo

Matrix metalloproteinases (MMPs) play a critical role in various pathological conditions including cutaneous inflammation. Thus far, serial assessment of MMP activity in ongoing inflammation is hampered due to technical limitations. Here, we present an innovative method for longitudinal detection of MMP activity by in vivo imaging. First, we analysed skin sections from patients suffering from leucocytoclastic vasculitis (LcV) and detected a significant MMP signal via immunofluorescence staining. Then, we mimicked LcV in mice in a well-studied model of immune complex-mediated vasculitis (ICV). This acute inflammatory process was serially visualized in vivo using the fluorescence-labelled MMP tracer Cy5.5-AF443. The deposition of fluorescence-labelled immune complexes and MMP tracer distribution was visualized repeatedly and non-invasively by fluorescence reflectance imaging. In correlation with the presence of MMP-2 and MMP-9 in immunofluorescence stainings, Cy5.5-AF443 accumulated in ICV spots in the skin of C57BL/6 mice. This tracer accumulation could also be observed in mice equipped with a dorsal skinfold chamber, where microscopic observations revealed an increased recruitment of fluorescence-labelled leucocytes during ICV. The specificity of the MMP tracer was supported by (i) analysis of mice deficient in functional β2 -integrins (CD18(-/-) ) and (ii) subsequent MMP immunofluorescence staining. These findings let us conclude that MMP accumulation in the acute phase of ICV depends on β2 -mediated leucocyte recruitment. In summary, we show that MMPs are involved in ICV as determined by Cy5.5-AF443, a new optical marker to longitudinally and non-invasively follow MMP activity in acute skin inflammation in vivo.

Microdistribution of MC1R-targeted polyplexes in murine melanoma tumor tissue

Targeted sodium-iodide symporter (NIS) gene transfer can be considered as a promising approach for diagnostics of specific types of cancer. For this purpose we used targeted polyplexes based on PEI–PEG–MC1SP block-copolymer containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (MC1R) overexpressed on melanoma cells. Targeted polyplexes demonstrated enhanced NIS gene transfer compared to non-targeted (lacking MC1SP) ones in vitro. Using dorsal skinfold chamber and intravital microscopy we evaluated accumulation and microdistribution of quantum dot-labeled polyplexes in tumor and normal subcutaneous tissues up to 4 h after intravenous injection. Polyplexes demonstrated significantly higher total accumulation in tumor tissue in comparison with subcutaneous ones (control). Targeted and non-targeted polyplexes extravasated and penetrated into the tumor tissue up to 20 μm from the vessel walls. In contrast, in normal subcutaneous tissue polyplexes penetrated not more than 3 μm from the vessel walls with the level of extravasated polyplexes 400-fold less than in tumor. Accumulated polyplexes in tumor tissue caused NIS gene expression. Subsequent 123I− intravenous injection resulted in 6.8 ± 1.1 and 4.5 ± 0.8% ID/g (p < 0.001) iodide accumulation in tumors in the case of targeted and non-targeted polyplexes, respectively, as was shown using SPECT/CT.

Inhibition of angiogenesis

Arctigenin, a functional ingredient of several traditional Chinese herbs, has been reported to have potential antitumor activity. However, its mechanisms of action are still not well elucidated. Because the establishment and metastatic spread of tumors is crucially dependent on angiogenesis, here we investigated whether arctigenin inhibits tumor growth by disturbing blood vessel formation. For this purpose, human dermal microvascular endothelial cells were exposed to different arctigenin doses to study their viability, proliferation, protein expression, migration, and tube formation compared with vehicle-treated controls. In addition, arctigenin action on vascular sprouting was analyzed in an aortic ring assay. Furthermore, we studied direct arctigenin effects on CT26.WT colon carcinoma cells. Spheroids of these tumor cells were transplanted into the dorsal skinfold chamber of arctigenin-treated and vehicle-treated BALB/c mice for the in-vivo analysis of tumor vascularization and growth by intravital fluorescence microscopy, histology, and immunohistochemistry. We found that noncytotoxic doses of arctigenin dose dependently reduced the proliferation of human dermal microvascular endothelial cells without affecting their migratory and tube-forming capacity. Arctigenin treatment also resulted in a decreased cellular expression of phosphorylated serine/threonine protein kinase AKT, vascular endothelial growth factor receptor 2, and proliferating cell nuclear antigen and inhibited vascular sprouting from aortic rings. In addition, proliferation, but not secretion of vascular endothelial growth factor, was decreased in arctigenin-treated tumor cells. Finally, arctigenin suppressed the vascularization and growth of engrafting CT26.WT tumors in the dorsal skinfold chamber model. Taken together, these results show for the first time an antiangiogenic action of arctigenin, which may contribute considerably toward its antitumor activity.

Vascular disrupting effects of combretastatin A4 phosphate on murine endometriotic lesions

To study the effect of combretastatin A4 phosphate (CA4P) on the vascularization of endometriotic lesions. Intravital microscopic, histologic, and immunohistochemical study. University institute. BALB/c mice. Murine endometriotic lesions were induced by syngeneic transplantation of endometrium into dorsal skinfold chambers. After 6 days, the mice received an intraperitoneal injection of 80 mg/kg CA4P or vehicle. Vascularization of the lesions and the surrounding tissue was analyzed by intravital fluorescence microscopy over 8 days. Lesion morphology, vessel maturation, viability, and proliferation of endometrial glands and stroma were assessed by histology and immunohistochemistry. All lesions were initially well vascularized, containing immature and mature microvessels. Injection of CA4P induced a selective vessel collapse in the lesions without affecting the surrounding microvasculature. This resulted in a decreased functional capillary density and blood perfusion of CA4P-treated lesions after 2 hours when compared with controls. However, the vascularization of the lesions progressively normalized, and their numbers of proliferating and apoptotic cells did not differ from those of controls. This study demonstrates a selective vascular disrupting effect of CA4P on endometriotic lesions, indicating that vascular disrupting agents may be suitable for endometriosis therapy.

Opposing effect of amniotic membrane on angiogenesis originating from amniotic epithelial cells

There are controversies about the effects of amniotic membrane (AM) on angiogenesis. It was recently hypothesised that denuded AM (without epithelial cells) can induce angiogenesis. The results from the dorsal skinfold chamber method in an animal model and aortic ring assay showed that the epithelial side of the AM inhibits vessel sprouting and reduces capillary numbers, while the mesenchymal side of the AM increases angiogenesis. These data answered the controversies about the angiogenic capability of the AM and raised some questions about the mechanisms by which the AM affects angiogenesis. In this article, mechanism(s) by which the AM affects angiogenesis and viability state of amniotic cells and its effect on angiogenesis have been propounded.

Effects of systemic pretreatment with CpG oligodeoxynucleotides on skin wound healing in mice

Unmethylated CpG oligodeoxynucleotides (ODN) bind to the Toll-like receptor 9, thus stimulating the immune system. To study the effects of systemic pretreatment with CpG ODN on dermal regeneration, C57BL6/J Tyr mice were treated with CpG or control ODN 6 days prior to implantation of a dorsal skinfold chamber and skin wounding. Wound epithelialization was analyzed by planimetric microscopy. On day 18, wound tissues were taken for (immuno)histochemical staining. CpG ODN increased epithelialization compared with control ODN treatment. Histological analysis revealed reduced capillary density, reduced wound cellularity, and reduced numbers of infiltrating leukocytes, as well as reduced F4/80-positive macrophages, but increased numbers of RELM-α-positive M2 macrophages after CpG ODN treatment, reflecting a better quality of wound healing on day 18 compared with control ODN treatment. Reverse transcription-polymerase chain reaction analysis of Toll-like receptor 9 showed the receptor expression on both fibroblasts and keratinocytes. Fibroblasts showed an increase of migration upon increasing dosages of CpG and not control ODN, reaching ∼50% of the response of basic fibroblast growth factor-exposed cells. Keratinocytes dose-dependently responded to both CpG and control ODN up to values found in keratinocyte growth factor-exposed cells. In summary, CpG ODN support late tissue-remodeling processes that contribute to resolution of inflammation and solid wounds during skin regeneration.