Dorsal Skinfold Chamber Model Research Articles

Absent in Melanoma (AIM)2 Promotes the Outcome of Islet Transplantation by Repressing Ischemia-Induced Interferon (IFN) Signaling.

Clinical islet transplantation is limited by ischemia-induced islet cell death. Recently, it has been reported that the absent in melanoma (AIM)2 inflammasome is upregulated by ischemic cell death due to recognition of aberrant cytoplasmic self-dsDNA. However, it is unknown whether AIM2 determines the outcome of islet transplantation. To investigate this, isolated wild type (WT) and AIM2-deficient (AIM2-/-) islets were exposed to oxygen-glucose deprivation to mimic ischemia, and their viability, endocrine function, and interferon (IFN) signaling were assessed. Moreover, the revascularization and endocrine function of grafted WT and AIM2-/- islets were analyzed in the mouse dorsal skinfold chamber model and the diabetic kidney capsule model. Ischemic WT and AIM2-/- islets did not differ in their viability. However, AIM2-/- islets exhibited a higher protein level of p202, a transcriptional regulator of IFN-β and IFN-γ gene expression. Accordingly, these cytokines were upregulated in AIM2-/- islets, resulting in a suppressed gene expression and secretion of insulin. Moreover, the revascularization of AIM2-/- islet grafts was deteriorated when compared to WT controls. Furthermore, transplantation of AIM2-/- islets in diabetic mice failed to restore physiological blood glucose levels. These findings indicate that AIM2 crucially determines the engraftment and endocrine function of transplanted islets by repressing IFN signaling.

Caloric Restriction: A Novel Conditioning Strategy to Improve the Survival of Ischemically Challenged Musculocutaneous Random Pattern Flaps.

Caloric restriction (CR) is a cost-effective and easy-to-perform approach to counteracting surgical stress. The present study therefore evaluates the tissue-protective effects of a 30% CR in musculocutaneous flaps undergoing ischemia. For this purpose, a well-established murine dorsal skinfold chamber model, in combination with random pattern musculocutaneous flaps, was used. C57BL/6N mice were divided at random into a CR group (n = 8) and a control group with unrestricted access to standard chow (n = 8). The CR animals were subjected to a 30% reduction in caloric intake for 10 days before flap elevation. Intravital fluorescence microscopy was carried out on days 1, 3, 5, 7 and 10 after flap elevation to assess the nutritive blood perfusion, angiogenesis and flap necrosis. Subsequently, the flap tissue was harvested for additional histological and immunohistochemical analyses. The CR-treated animals exhibited a significantly higher functional capillary density and more newly formed microvessels within the flap tissue when compared to the controls; this was associated with a significantly higher flap survival rate. Immunohistochemical analyses showed a decreased invasion of myeloperoxidase-positive neutrophilic granulocytes into the flap tissue of the CR-treated mice. Moreover, the detection of cleaved caspase-3 revealed fewer cells undergoing apoptosis in the transition zone between the vital and necrotic tissue in the flaps of the CR-treated mice. These results demonstrate that a CR of 30% effectively prevents flap necrosis by maintaining microperfusion on a capillary level and inhibiting inflammation under ischemic stress. Hence, CR represents a promising novel conditioning strategy for improving the survival of musculocutaneous flaps with random pattern perfusion.

An In Vivo Model to Study Cell Migration in XYZ-T Dimension Followed by Whole-Mount Re-evaluation.

Cell migration is a very dynamic process involving several chemical as well as biological interactions with other cells and the environment. Several models exist to study cell migration ranging from simple 2D in vitro cultures to more demanding 3D multicellular assays, to complex evaluation in animals. High-resolution 4D (XYZ, spatial + T, time dimension) intravital imaging using transgenic animals with a fluorescent label in cells of interest is a powerful tool to study cell migration in the correct environment. Here we describe an advanced dorsal skinfold chamber model to study endothelial cell and pericyte migration and association.

Extracellular Vesicles from Sickle Red Blood Cells Activate Endothelial Cells and Trigger Von Willebrand Factor-Mediated Sickle Red Cell Adhesion

Introduction: The sickle mutation of hemoglobin (HbS) afflicts millions of people worldwide and is characterized by hemolytic anemia, inflammation, painful vaso-occlusive crises, significant morbidity, and early mortality. In sickle cell disease (SCD), HbS polymerization under deoxygenation increases red blood cell (RBC) rigidity, adhesivity and susceptibility to lysis. Products of intravascular hemolysis including heme and RBC-released extracellular vesicles (REVs) promote acute and chronic inflammation triggering endothelial activation and abnormal RBC adhesion. Extracellular vesicles (EVs) are nano- or micro-particles composed of a lipid bilayer, comprised of transmembrane proteins, and enclosing intracellular remnants, including cytosolic proteins and miRNAs. EVs are known to exchange biomaterial between cells, and may serve as surrogate markers for the activated state of the parent cell thus are of high clinical significance. Quantitatively, REVs are more abundant from sickle RBCs (SS RBCs) compared with normal HbA-containing RBCs (AA RBCs). SS REVs are known to promote endothelial cell (EC) activation through cell signaling and transcriptional regulation at longer terms. However, the SS REV-mediated short term non transcriptional response of EC is unclear. Methods: Here, we have developed the SCD-EV-BioChip using microfluidic technology. SCD-EV-BioChip assesses RBC adhesion as a biomarker for REV-mediated human pulmonary microvascular endothelial cells (HPMECs) dysfunction using functional biomarker of RBC adhesion. Utilizing SCD-EV-BioChip, Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 hours. For in vitro studies, SS REVs and AA REVs were collected from pooled human SS RBCs and AA RBCs activated using calcium ionophore. The generated REVs were characterized using scanning electron microscopy (SEM) and nanoparticle tracking analyzer (NTA). HPMECs were cultured in microfluidic channels under controlled shear stress for more than 72 hours then treated with REVs at 37 ºC for 2 hours. Von Willebrand factor (vWF) expression on REVs-treated HPMECs were quantified. RBC adhesion tests were conducted with and without vWF protease ADAMTS13 and heme-binding protein hemopexin under shear stress at 1 dyne/cm2 followed by a rinse step and quantification of adhered RBCs. For in vivo study, HbSS-Townes mice and a dorsal skin-fold chamber model was used to determine if ADAMTS13 would reduce microvascular stasis in response to hemin. Results: Within 2 hours, SS REVs, but not AA REVs, promoted increased levels of vWF expression on HPMECs, indicating HPMEC activation (Fig. 1A-C). VWF is known to trigger SRBC adhesion and vaso-occlusion. Using SCD-EV-BioChip, we found significantly increased SS RBC adhesion to HPMECs exposed to SREVs, compared to SRBC adhesion to HPMECs exposed to AA REVs, and AA RBC adhesion to HPMECs exposed to SS REVs (Fig. 1D-G). SS RBC adhesion was significantly reduced on HPMECs treated with SS REVs pre-incubated with hemopexin. ADAMTS13 is a regulator which is responsible for cleaving ultra large vWF. In this study, SS RBC adhesion was significantly reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs (Fig. 1G). Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13 (Fig. 1F). SRBC adhesion was variable within a total of 15 test subjects with SCD, and was higher with SRBCs from patients with increased markers of hemolysis (LDH and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Conclusions: This work demonstrated the acute, non-transcriptional contribution made by REVs to the microvascular EC activation and vWF-mediated SRBC adhesion. Results from SCD-EV-BioChip indicated the effect of hemopexin in mitigating SREV-mediated HPMEC activation. Additionally, results from in vitro SCD-EV BioChip tests agreed with in vivo stasis tests using SS mice, both of which indicated the effect of ADAMTS13 in mitigating SS RBC adhesion and vaso-occlusion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cold Exposure Induces Vaso-Occlusion and Hyperalgesia in Mice with Sickle Cell Disease That Is Dependent on Complement Activation

BACKROUND AND AIM: Vaso-occlusive crises (VOC) cause severe pain in patients with sickle cell disease (SCD). To date, epidemiological studies have shown seasonably colder temperatures exacerbate SCD-related pain. Although literature and clinical observations suggest a connection between cold and triggering of acute VOC pain, the biologic mechanisms underlying VOC-evoked pain in SCD patients are poorly understood. Repeated cold or painful events results in vasoconstriction and decreased perfusion, which increases red cell transit time, deoxygenation, and hemoglobin S polymerization [Veluswamy et al. Blood. 2020;136:1191]. SCD mice exhibit ischemia-reperfusion (I/R) pathophysiology in response to hypoxia-reoxygenation with a concomitant increase in hyperalgesia, inflammation, and complement activation. Because complement activation plays a crucial role in animal pain models, we used a new model of cold-evoked VOC and I/R in HbSS mice to examine complement C5a/C5aR signaling as a mechanistic link between vaso-occlusion and acute pain in SCD. MATERIALS AND METHODS: We used a dorsal skinfold chamber model to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paws to assess mechanical hyperalgesia (pain) in Townes HbSS and control HbAA mice. Non-hyperalgesic mice were exposed to cold (10oC/50oF) for 1 hour (h) and then returned to room temperature. Measurements of VOC and paw withdrawal threshold were collected at baseline before cold and at 1-4 h after cold exposure. After the 4 h stasis measurement, mice were sacrificed, and livers removed and frozen. Hepatic microsomes and nuclear extracts were isolated by differential centrifugation and liver inflammation markers were assessed on Western blots. Hepatic nuclear extracts were blotted with antibodies to NF-ĸB phospho-p65 and total p65. Hepatic microsomes were blotted with antibodies to VCAM-1 and ICAM-1. Plasma samples were blotted with antibodies to alternative pathway factor B activation fragment Bb and complement anaphylatoxin C5a. To examine the role of complement activation in stasis and hyperalgesia produced by cold-evoked VOC, HbSS mice were infused 30 minutes before cold exposure with an inhibitory monoclonal antibody (mAb) to murine C5 to prevent C5 cleavage or C5aR to prevent C5a binding to C5aR and subsequent C5a/C5aR signaling, or a control mAb (1.2 mg/kg body weight). RESULTS: Cold exposure induced more microvascular stasis (vaso-occlusion), 1, 2, 3, and 4 h after cold exposure in subcutaneous venules of HbSS mice as compared to HbAA mice exposed to cold and HbSS mice left at room temperature (p<0.001). Cold exposure also evoked mechanical hyperalgesia in HbSS mice 1, 2, and 24 h after cold as compared to baseline prior to cold (p<0.01) and cold exposed HbAA mice (p<0.05). Vaso-occlusion and mechanical hyperalgesia were accompanied by an increase in Bb and C5a levels in the plasma of HbSS, but not HbAA mice 4 h after cold exposure (p<0.01). In addition, there was an increase in NF-κB activation and VCAM-1 and ICAM-1 adhesion molecule expression in the livers of HbSS mice after cold as compared to HbAA mice exposed to cold and HbSS mice unexposed to cold (p<0.01). Importantly, pretreatment of HbSS mice before cold exposure with a blocking mAb to C5 or C5aR inhibited vaso-occlusion (Figure 1A), mechanical hyperalgesia (Figure 1B), and complement activation (Figure 1C) as compared to cold-exposed HbSS mice treated with a control mAb. CONCLUSION: This mouse model of cold-evoked VOC provides an opportunity to examine potential mediators between vaso-occlusion and pain in SCD. Our results demonstrate that in response to cold temperatures, complement activation contributes to vaso-occlusion and acute hyperalgesia, suggesting a linkage between complement activation, vaso-occlusion, and pain during cold-induced VOC. These studies provide a rationale for clinical use of complement inhibitors to prevent and treat pain in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Brassinin Promotes the Degradation of Tie2 and FGFR1 in Endothelial Cells and Inhibits Triple-Negative Breast Cancer Angiogenesis

Simple SummaryBrassinin is a natural compound enriched in several commonly consumed vegetables, such as broccoli and cabbages. It shows potent anti-cancer activity against several cancers. However, its effects on triple-negative breast cancer (TNBC), an aggressive subtype with limited treatment options, remain elusive so far. Therefore, we investigated the effects of brassinin on TNBC angiogenesis and growth. Our results demonstrate that brassinin inhibits TNBC growth preferentially through inhibiting the angiogenic activity of endothelial cells (ECs). Additional in-vitro analyses revealed that this effect may be mediated by brassinin-stimulated degradation of two pivotal angiogenesis-related receptors in ECs: Tie2 and fibroblast growth factor receptor 1. These findings provide novel insights into the cellular and molecular mechanisms underlying the anti-cancer activity of brassinin and indicate that this phytochemical may be a promising lead compound or drug candidate for TNBC treatment.Brassinin, a phytoalexin derived from cruciferous vegetables, has been reported to exhibit anti-cancer activity in multiple cancer types. However, its effects on triple-negative breast cancer (TNBC) development and the underlying mechanisms have not been elucidated so far. In this study, we demonstrated in vitro that brassinin preferentially reduces the viability of endothelial cells (ECs) when compared to other cell types of the tumor microenvironment, including TNBC cells, pericytes, and fibroblasts. Moreover, brassinin at non-cytotoxic doses significantly suppressed the proliferation, migration, tube formation, and spheroid sprouting of ECs. It also efficiently inhibited angiogenesis in an ex-vivo aortic ring assay and an in-vivo Matrigel plug assay. Daily intraperitoneal injection of brassinin significantly reduced tumor size, microvessel density, as well as the perfusion of tumor microvessels in a dorsal skinfold chamber model of TNBC. Mechanistic analyses showed that brassinin selectively stimulates the degradation of Tie2 and fibroblast growth factor receptor 1 in ECs, leading to the down-regulation of the AKT and extracellular signal-regulated kinase pathways. These findings demonstrate a preferential and potent anti-angiogenic activity of brassinin, which may be the main mechanism of its anti-tumor action. Accordingly, this phytochemical represents a promising candidate for the future anti-angiogenic treatment of TNBC.

Boosting Tissue Vascularization: Nanofat as a Potential Source of Functional Microvessel Segments.

Nanofat is increasingly applied in plastic surgery for the improvement of scar quality and skin rejuvenation. However, little is known about the underlying regenerative mechanisms. Therefore, we herein investigated nanofat grafts in a murine dorsal skinfold chamber model. Nanofat generated from subcutaneous, inguinal adipose tissue of green fluorescent protein (GFP)+ C57BL/6 male and female donor mice was injected intracutaneously into dorsal skinfold chambers of gender-matched GFP− wild-type mice. The vascularization and tissue composition of the grafted nanofat were analyzed by means of intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. The freshly generated nanofat consisted of small fragments of perilipin+ adipocytes surrounded by Sirius red+ collagen fibers and still contained intact CD31+/GFP+ vessel segments. After transplantation into the dorsal skinfold chamber, these vessel segments survived and developed interconnections to the surrounding CD31+/GFP− host microvasculature. Accordingly, the grafted nanofat rapidly vascularized and formed new microvascular networks with a high functional microvessel density on day 14 without marked differences between male and female mice. Even though further research is needed to confirm these findings, the present study suggests that nanofat boosts tissue vascularization. Thus, nanofat may represent a versatile resource for many applications in tissue engineering and regenerative medicine.

Assessment of the In Vivo Response to Nanobody-Targeted PDT Through Intravital Microscopy.

Methods that allow real-time, longitudinal, intravital detection of the fluorescence distribution and the cellular and vascular responses within tumor and normal tissue are important tools to obtain valuable information when investigating new photosensitizers and photodynamic therapy (PDT) responses. Intravital confocal microscopy using the dorsal skinfold chamber model gives the opportunity to visualize and determine the distribution of photosensitizers within tumor and normal tissue. Next to that, it also allows the visualization of the effect of treatment with respect to changes in vascular diameter and blood flow, vascular leakage, and tissue necrosis, in the first days post-illumination. Here, we describe the preparation of the skinfold chamber model and the intravital microscopy techniques involved, for a strategy we recently introduced, that is, the nanobody-targeted PDT. In this particular approach, photosensitizers are conjugated to nanobodies to target these specifically to cancer cells.

Targeting sphingosine kinase-1 with the low MW inhibitor SKI-5C suppresses the development of endometriotic lesions in mice.

Limited evidence suggests that the sphingosine-1-phosphate/sphingosine kinase 1 (S1P/SPHK1) signalling pathway is involved in the pathogenesis of endometriosis. Therefore, we analyzed in this study whether the inhibition of SPHK1 and, consequently, decreased levels of S1P affected the vascularization and growth of endometriotic lesions. Endometriotic lesions were surgically induced in the peritoneal cavity and the dorsal skinfold chamber of female BALB/c mice. The animals received a daily dose of the SPHK1 inhibitor SKI-5C or vehicle (control). Analyses involved the determination of lesion growth, cyst formation, microvessel density and cell proliferation within peritoneal endometriotic lesions by means of high-resolution ultrasound imaging, caliper measurement, histology and immunohistochemistry. In the dorsal skinfold chamber model the development of newly formed microvascular networks and their microhemodynamic parameters within endometriotic lesions were investigated by means of intravital fluorescence microscopy. SKI-5C significantly inhibited the development and vascularization of peritoneal endometriotic lesions, as indicated by a reduced growth and cyst formation, a lower microvessel density and a suppressed cell proliferation, when compared to vehicle-treated controls. Endometriotic lesions in dorsal skinfold chambers of SKI-5C-treated animals exhibited a significantly smaller lesion size, lower functional microvessel density, smaller microvessel diameters and a reduced blood perfusion of the newly developing microvascular networks. SPHK1/S1P signalling promotes the establishment and progression of endometriotic lesions. The inhibition of this pathway suppresses the development of endometriotic lesions, suggesting SPHK1 as a potential novel target for future endometriosis therapy.

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

AimsThe exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization.MethodsFlow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets.ResultsWe found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls.ConclusionThese findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.

GBT1118 Diminishes Vaso-Occlusion in Sickle Cell Disease Mice

Recurrent, painful vaso-occlusive events are one of the most debilitating complications of sickle cell disease (SCD). The process involves occlusion of blood flow through small vessels by heterogeneous cell clusters comprised of red blood cells (RBCs), neutrophils, and platelets. Activated endothelial cells localize and facilitate vaso-occlusion. The central triggering event in the process is hemoglobin S (HbS) polymerization and the consequent damage to RBCs. Many recent clinical efforts to prevent vaso-occlusive events have focused on downstream events mediated by inflammatory molecules. The present work however studies the triggering event by assessing the direct impact of allosteric modulators of hemoglobin oxygen (Hb-O2) affinity on vaso-occlusion in a murine model of SCD.In this study, we used GBT1118 (a structural analog of GBT440) in a modified dorsal skin fold model in an HbSS knock-in mouse model of SCD (Townes; Wu et al., Blood 2006). GBT1118 binds covalently and reversibly via an imine intermediate to the N-terminal valine of the hemoglobin α chain, and allosterically increases the intracellular hemoglobin affinity for oxygen (Dufu et al., Am J Physiol Heart Circ Physiol 2017). The dorsal skin fold chamber model has been used previously to study vaso-occlusion in SCD mice (Kalambur et al., Am J Hematol 2004). In this present study, mice were treated acutely by oral gavage with GBT1118 or vehicle control in a blinded fashion. Two hours after drug treatment, a baseline of vascular patency at ambient air was established using intravital microscopy to count blood vessels with blood flow. Subsequently animals were exposed to 1 h hypoxia (7.0% oxygen) and 1 h reoxygenation at ambient air. At that time the previously observed blood vessels were monitored again, and the percentage of occluded vessels was recorded. We observed a significant reduction of vaso-occlusion in animals that had been treated with GBT1118 (p<0.05).In summary, while improved survival has been described in hypoxia-challenged SCD mice treated with allosteric modulators of hemoglobin (Abdulmalik et al., Br J Haematol 2005), our study demonstrates for the first time that a hemoglobin allosteric modulator can reduce vaso-occlusion in a mouse model of SCD. The protective effect elicited by GBT1118 may either be attributed to improved oxygenation or allosteric modulation of HbS. Both mechanisms may directly delay hypoxia-triggered polymerization of HbS, sufficiently for RBCs to transit the microcirculation without sickling, without adhering to vessel wall, and therefore without causing vaso-occlusion.The results of this study expand on the potential mechanisms by which allosteric modulators of Hb-O2 affinity may contribute to the treatment of SCD, in addition to previously described hematological benefits. DisclosuresAlt:Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Liu:Global Blood Therapeutics: Employment. Li:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Zou:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Kauser:Global Blood Therapeutics: Employment, Equity Ownership.

Differences in growth and vascularization of ectopic menstrual and non-menstrual endometrial tissue in mouse models of endometriosis.

Is there a difference in the growth and vascularization between murine endometriotic lesions originating from menstrual or non-menstrual endometrial fragments? Endometriotic lesions developing from menstrual and non-menstrual tissue fragments share many similarities, but also exhibit distinct differences in growth and vascularization, particularly under exogenous estrogen stimulation. Mouse models are increasingly used in endometriosis research. For this purpose, menstrual or non-menstrual endometrial fragments serve for the induction of endometriotic lesions. So far, these two fragment types have never been directly compared under identical experimental conditions. This was a prospective experimental study in a murine peritoneal and dorsal skinfold chamber model of endometriosis. Endometrial tissue fragments from menstruated (n = 15) and non-menstruated (n = 21) C57BL/6 mice were simultaneously transplanted into the peritoneal cavity or dorsal skinfold chamber of non-ovariectomized (non-ovx, n = 17), ovariectomized (ovx, n = 17) and ovariectomized, estrogen-substituted (ovx+E2, n = 17) recipient animals and analyzed throughout an observation period of 28 and 14 days, respectively. The engraftment, growth and vascularization of the newly developing endometriotic lesions were analyzed by means of high-resolution ultrasound imaging, intravital fluorescence microscopy, histology and immunohistochemistry. Menstrual and non-menstrual tissue fragments developed into peritoneal endometriotic lesions without differences in growth, microvessel density and cell proliferation in non-ovx mice. Lesion formation out of both fragment types was markedly suppressed in ovx mice. In case of non-menstrual tissue fragments, this effect could be reversed by estrogen supplementation. In contrast, endometriotic lesions originating from menstrual tissue fragments exhibited a significantly smaller volume in ovx+E2 mice, which may be due to a reduced hormone sensitivity. Moreover, menstrual tissue fragments showed a delayed vascularization and a reduced blood perfusion after transplantation into dorsal skinfold chambers when compared to non-menstrual tissue fragments, indicating different vascularization modes of the two fragment types. To limit the role of chance, the experiments were conducted under standardized laboratory conditions. Statistical significance was accepted for a value of P < 0.05. N/A. Endometriotic 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. The present study significantly contributes to the characterization of common murine endometriosis models. These models represent important tools for studies focusing on the basic mechanisms of endometriosis and the development of novel therapeutic strategies for the treatment of this frequent gynecological disease. The presented findings indicate that the combination of different experimental models and approaches may be the most appropriate strategy to study the pathophysiology and drug sensitivity of a complex disease such as endometriosis under preclinical conditions. There was no specific funding of this study. The authors have no conflicts of interest to declare.

Feasibility study of incident dark-field video microscope for measuring microcirculatory variables in the mouse dorsal skinfold chamber model.

BackgroundDespite the importance of microcirculation in organ function, monitoring microcirculation is not a routine practice. With developments in microscopic technology, incident dark field (IDF) microscopy (Cytocam) has allowed visualization of the microcirculation. Dorsal skinfold chamber (DSC) mouse model has been used to investigate microcirculation physiology. By employing Cytocam-IDF imaging with DSC model to assess microcirculatory alteration in lipopolysaccharide (LPS)-induced endotoxemia, we attempted to validate availability of Cytocam-IDF imaging of microcirculation.MethodsDSC was implanted in eight BALB/c mice for each group; control and sepsis. Both groups were given 72 hours to recover from surgery. The sepsis group had an additional 24-hour period of recovery post-LPS injection (4 mg/kg). Subsequently, a video of the microcirculation was recorded using Cytocam. Data on microcirculatory variables were obtained. Electron microscopy was implemented using lanthanum fixation to detect endothelial glycocalyx degradation.ResultsThe microcirculatory flow index was significantly lower (control, 2.8±0.3; sepsis, 2.1±0.8; P=0.033) and heterogeneity index was considerably higher (control, 0.10±0.15; sepsis, 0.53±0.48; P=0.044) in the sepsis group than in the control group. Electron microscopy revealed glycocalyx demolishment in the sepsis group.ConclusionsCytocam showed reliable ability for observing changes in the microcirculation under septic conditions in the DSC model. The convenience and good imaging quality and the automatic analysis software available for Cytocam-IDF imaging, along with the ability to perform real-time in vivo experiments in the DSC model, are expected to be helpful in future microcirculation investigations.

New model in diabetic mice to evaluate the effects of insulin therapy on biofilm development in wounds.

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7m +/+ Leprdb/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with Staphylococcus aureus ATCC 49230 (106 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence in situ hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19).Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and S. aureus-induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation.

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs.

Junctional Adhesion Molecule-C Mediates the Recruitment of Embryonic-Endothelial Progenitor Cells to the Perivascular Niche during Tumor Angiogenesis.

The homing of Endothelial Progenitor Cells (EPCs) to tumor angiogenic sites has been described as a multistep process, involving adhesion, migration, incorporation and sprouting, for which the underlying molecular and cellular mechanisms are yet to be fully defined. Here, we studied the expression of Junctional Adhesion Molecule-C (JAM-C) by EPCs and its role in EPC homing to tumor angiogenic vessels. For this, we used mouse embryonic-Endothelial Progenitor Cells (e-EPCs), intravital multi-fluorescence microscopy techniques and the dorsal skin-fold chamber model. JAM-C was found to be expressed by e-EPCs and endothelial cells. Blocking JAM-C did not affect adhesion of e-EPCs to endothelial monolayers in vitro but, interestingly, it did reduce their adhesion to tumor endothelium in vivo. The most striking effect of JAM-C blocking was on tube formation on matrigel in vitro and the incorporation and sprouting of e-EPCs to tumor endothelium in vivo. Our results demonstrate that JAM-C mediates e-EPC recruitment to tumor angiogenic sites, i.e., coordinated homing of EPCs to the perivascular niche, where they cluster and interact with tumor blood vessels. This suggests that JAM-C plays a critical role in the process of vascular assembly and may represent a potential therapeutic target to control tumor angiogenesis.

Lipoconstruct surface topography grating size influences vascularization onset in the dorsal skinfold chamber model

After skin tissue injury or pathological removal, vascularization timing is paramount in graft survival. As full thickness skin grafts often fail to become perfused over larger surfaces, split-thickness grafts are preferred and can be used together with biomaterials, which themselves are non-angiogenic. One way of promoting vascular ingrowth is to “pre-vascularize” an engineered substitute by introducing endothelial cells (ECs). Since it has been previously demonstrated that surface structured biomaterials have an effect on wound healing, skin regeneration, and fibrosis reduction, we proposed that a microvascular-rich lipoconstruct with anisotropic topographical cues could be a clinically translatable vascularization approach. Murine lipofragments were formed with three polydimethylsiloxane molds (flat, 5 µm, and 50 µm parallel gratings) and implanted into the dorsal skinfold chamber of male C57BL/6 mice. Vascular ingrowth was observed through intravital microscopy over 21 days and further assessed by histology and protein identification. Our investigation revealed that topographical feature size influenced the commencement of neovascular ingrowth, with 5 µm gratings exhibiting early construct perfusion at 3 days post-operation, and 50 µm being delayed until day 5. We therefore postulate that surface structured lipoconstructs may serve as an easily obtained and produced construct suitable for providing soft tissue and ECs to tissue defects. Statement of SignificanceSkin graft failures due to inadequate or uneven perfusion frequently occur and can be even more complicated in deep, difficult to heal wounds, or bone coverage. In complex injuries, biomaterials are often used to cover bone structures with a standard split thickness graft; however, perfusion can take up to 3 weeks. Thus, any means to promote faster and uniform vascularization could significantly reduce healing time, as well as lower patient down-time. As pre-vascularized constructs have reported success in research, we created a cost-efficient, translatable method with no additional laboratory time as adipose tissue can be harvested and used immediately. We further used surface topography as an aspect to modulate construct perfusion, which has been reported for the first time here.

Vascularization and biocompatibility of poly(ε-caprolactone) fiber mats for rotator cuff tear repair.

Rotator cuff tear is the most frequent tendon injury in the adult population. Despite current improvements in surgical techniques and the development of grafts, failure rates following tendon reconstruction remain high. New therapies, which aim to restore the topology and functionality of the interface between muscle, tendon and bone, are essentially required. One of the key factors for a successful incorporation of tissue engineered constructs is a rapid ingrowth of cells and tissues, which is dependent on a fast vascularization. The dorsal skinfold chamber model in female BALB/cJZtm mice allows the observation of microhemodynamic parameters in repeated measurements in vivo and therefore the description of the vascularization of different implant materials. In order to promote vascularization of implant material, we compared a porous polymer patch (a commercially available porous polyurethane based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fiber mats and chitosan-graft-PCL coated electrospun PCL (CS-g-PCL) fiber mats in vivo. Using intravital fluorescence microscopy microcirculatory parameters were analyzed repetitively over 14 days. Vascularization was significantly increased in CS-g-PCL fiber mats at day 14 compared to the porous polymer patch and uncoated PCL fiber mats. Furthermore CS-g-PCL fiber mats showed also a reduced activation of immune cells. Clinically, these are important findings as they indicate that the CS-g-PCL improves the formation of vascularized tissue and the ingrowth of cells into electrospun PCL scaffolds. Especially the combination of enhanced vascularization and the reduction in immune cell activation at the later time points of our study points to an improved clinical outcome after rotator cuff tear repair.

High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments.

High glucose concentrations have been shown to activate endothelial cells and promote angiogenesis. In the present study, it was investigated whether high glucose concentrations could improve the vascularisation capacity of adipose-tissue-derived microvascular fragments (ad-MVF). Ad-MVF were isolated from the epididymal fat pads of donor mice and cultivated for 24 h in University of Wisconsin (UW) solution supplemented with vehicle or 30 mM glucose. Protein expression, morphology, viability and proliferation of the cultivated ad-MVF were analysed by means of proteome profiler mouse angiogenesis array, scanning electron microscopy and immunohistochemistry. Additional cultivated ad-MVF were seeded on to collagen-glycosaminoglycan scaffolds to study their in vivo vascularisation capacity in the dorsal skinfold chamber model by intravital fluorescence microscopy, histology and immunohistochemistry. In vitro, high glucose exposure changed the protein expression pattern of ad-MVF with endoglin, interleukin (IL)-1β and monocyte chemoattractant protein (MCP)-1 as the most up-regulated pro-angiogenic factors. Moreover, high glucose exposure induced the formation of nanopores in the ad-MVF wall. In addition, ad-MVF contained significantly larger numbers of proliferating endothelial and perivascular cells while exhibiting a comparable number of apoptotic cells when compared to vehicle-treated controls. In vivo, scaffolds seeded with high-glucose-exposed ad-MVF exhibited an improved vascularisation and tissue incorporation. These findings demonstrated that the exposure of cultivated ad-MVF to high glucose concentrations is a promising approach to improve their in vivo performance as vascularisation units for tissue engineering and regenerative medicine.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the β3 subunit of voltage-gated calcium channels (Cavβ3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cavβ3-deficient mice (Cavβ3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cavβ3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cavβ3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cavβ3-deficient mice or cells might also be applicable for specific molecules other than Cavβ3.