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Objective: Chronic wounds represent a considerable burden for the affected patients and the health care system. To overcome this problem, effective treatment strategies are urgently required. In this study, we tested a novel approach by combining platelet-rich plasma (PRP) and microvascular fragments (MVF) to create a prevascularized gel dressing. Approach: MVF were enzymatically isolated from the epididymal fat pads of transgenic green fluorescent protein (GFP)+ C57BL/6J donor mice. Subsequently, 5,000 MVF were suspended in 10 µL murine PRP as carrier and transferred into full-thickness skin wounds within dorsal skinfold chambers of C57BL/6J wild-type mice (PRP+MVF). Wound healing in comparison to empty wounds (control) and wounds filled with PRP alone was repeatedly analyzed throughout 14 days by means of stereomicroscopy, histology, and immunohistochemistry. Results: Planimetric assessment of the wound size over time revealed a significantly accelerated and improved healing of PRP+MVF-treated wounds when compared with PRP-treated and empty control wounds. These wounds also exhibited a significantly higher density of blood and lymph vessels, which originated from the GFP+ MVF isolates and effectively promoted granulation tissue formation inside the skin defects. Innovation: This study is the first to combine PRP and MVF for the improvement of wound healing. Conclusion: The combination of PRP and MVF represents a promising approach for the future treatment of wounds that do not heal spontaneously due to poor wound-healing conditions.
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Ratones Endogámicos C57BL , Plasma Rico en Plaquetas , Cicatrización de Heridas , Animales , Cicatrización de Heridas/fisiología , Ratones , Masculino , Vendajes , Microvasos , Tejido de Granulación , Piel/lesiones , Ratones Transgénicos , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies in rodents exclusively use epididymal adipose tissue as a visceral fat source for MVF isolation. However, in future clinical practice, MVF may be rather isolated from liposuctioned subcutaneous fat tissue of patients. Therefore, we herein compared the vascularization characteristics of MVF isolates from visceral and subcutaneous fat tissue of murine origin. METHODS: MVF isolates were generated from visceral and subcutaneous fat tissue of donor mice using two different enzymatic procedures. For in vivo analyses, the MVF isolates were seeded onto collagen-glycosaminoglycan scaffolds and implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice. RESULTS: By means of the two isolation procedures, we isolated a higher number of MVF from visceral fat tissue when compared to subcutaneous fat tissue, while their length distribution, viability and cellular composition were comparable in both groups. Intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a significantly reduced vascularization of implanted scaffolds seeded with subcutaneous MVF isolates when compared to implants seeded with visceral MVF isolates. Light and scanning electron microscopy showed that this was due to high amounts of undigested connective tissue within the subcutaneous MVF isolates, which clogged the scaffold pores and prevented the interconnection of individual MVF into new microvascular networks. CONCLUSION: These findings indicate the need for improved protocols to generate connective tissue-free MVF isolates from subcutaneous fat tissue for future translational studies.
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Microvasos , Neovascularización Fisiológica , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Grasa Subcutánea , Ingeniería de Tejidos/métodosRESUMEN
Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies exclusively use epididymal fat tissue of male donor mice as a source for MVF isolation. However, in future clinical practice, MVF-based approaches may be applied in both male and female patients. Therefore, we herein compared the vascularization capacity of MVF isolated from the epididymal and peri-ovarian fat tissue of male and female donor mice. Freshly isolated MVF from male and female donors did not differ in their number, length distribution, viability and cellular composition. After their assembly into spheroids, they also exhibited a comparable in vitro sprouting activity. Moreover, they could be seeded onto collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects within mouse dorsal skinfold chambers. Repetitive intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a comparable vascularization and incorporation of implants seeded with MVF of male and female origin. Taken together, these findings demonstrate that the vascularization capacity of MVF is not gender-specific.
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Adipose tissue-derived microvascular fragments rapidly reassemble into microvascular networks within implanted scaffolds. Herein, we analyzed the contribution of macrophages to this process. C57BL/6 mice received clodronate (clo)-containing liposomes for macrophage depletion, whereas animals treated with phosphate-buffered-saline-containing liposomes served as controls. Microvascular fragments were isolated from clo- and phosphate-buffered-saline-treated donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of clo- and phosphate-buffered-saline-treated recipient mice. The implants' vascularization and incorporation were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. Compared to controls, matrices within clo-treated animals exhibited a significantly reduced functional microvessel density. Moreover, they contained a lower fraction of microvessels with an α-smooth muscle actin (SMA)+ cell layer, indicating impaired vessel maturation. This was associated with a deteriorated implant incorporation. These findings demonstrate that macrophages not only promote the reassembly of microvascular fragments into microvascular networks, but also improve their maturation during this process.
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Porous polyethylene (pPE) is a commonly used biomaterial in craniofacial reconstructive surgery. However, implant failure due to insufficient vascularization represents a major issue. To overcome this problem, we herein introduce an effective strategy to improve the vascularization and incorporation of pPE. Adipose tissue-derived microvascular fragments (MVF) from transgenic green fluorescent protein (GFP)+ mice were suspended in platelet-rich plasma (PRP) for the coating of pPE. PRP/MVF-coated pPE as well as PRP-coated and uncoated controls were subsequently implanted into the dorsal skinfold chamber and the flanks of GFP- wild-type mice to analyze their in vivo performance throughout 2, 4 and 8 weeks by means of intravital fluorescence microscopy, histology and immunohistochemistry. The GFP+/GFP- cross-over design allowed the identification of GFP+ MVF within the implants. Shortly after implantation, they rapidly reassembled into new blood-perfused microvascular networks, resulting in a significantly accelerated vascularization of PRP/MVF-coated pPE when compared to both controls. The overall numbers of rolling and adherent leukocytes within the microcirculation as well as macrophages, multi-nucleated giant cells and mast cells around the implants did not differ between the three groups. However, in contrast to uncoated controls, PRP/MVF-coated and PRP-coated pPE promoted pro-angiogenic M2 macrophage polarization at the implantation site. These findings demonstrate that PRP/MVF-coating represents a highly effective strategy to enhance the vascularization, biocompatibility and tissue incorporation of pPE. STATEMENT OF SIGNIFICANCE: The clinical in vivo performance of implanted biomaterials is crucially dependent on their adequate incorporation into the body. To achieve this, we herein introduce an effective biological coating strategy. Our results demonstrate that coating with PRP and MVF accelerates and enhances the vascularization, biocompatibility and tissue incorporation of porous polyethylene. Because this type of biological coating is easily applicable on any type of biomaterial, our approach may rapidly be translated into clinical practice to improve the outcome of various regenerative approaches.
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Plasma Rico en Plaquetas , Polietileno , Tejido Adiposo , Animales , Ratones , Neovascularización Fisiológica , PorosidadRESUMEN
BACKGROUND: The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach. RESULTS: SVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)+ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP- recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31+/GFP+ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time. CONCLUSIONS: The present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.
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The incorporation of endothelial progenitor cells (EPCs) into newly developing blood vessels contributes to the vascularization of endometriotic lesions. We analyzed whether cyclooxygenase (COX)-2 signaling regulates this vasculogenic process. Endometriotic lesions were surgically induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN [Tie2/green fluorescent protein (GFP)] 287 Sato mice. The animals received ß-estradiol 17-valerate once a week and were treated daily with the selective COX-2 inhibitor parecoxib (25 mg/kg) or vehicle (control) for 7 and 28 days. Analyses involved the determination of lesion growth, cyst formation, homing of GFP+/Tie2+ EPCs, numbers of circulating EPCs, vascularization, cell proliferation, apoptosis, and immune cell infiltration by means of high-resolution ultrasonography, caliper measurements, flow cytometry, histologic analysis, and immunohistochemical analysis. In parecoxib-treated mice, blood circulating EPCs were higher, but numbers of recruited EPCs in endometriotic lesions were significantly lower when compared with controls. This finding was associated with an impaired early vascularization and stromal tissue growth as well as reduced glandular secretory activity of the lesions. Parecoxib-treated lesions further contained less proliferating and more apoptotic cells and exhibited lower numbers of infiltrating macrophages and neutrophilic granulocytes. These findings demonstrate that the inhibition of COX-2 suppresses vasculogenesis in endometriotic lesions, which may contribute to an impaired lesion vascularization and growth.
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Ciclooxigenasa 2/fisiología , Endometriosis/patología , Células Progenitoras Endoteliales/patología , Neovascularización Patológica/prevención & control , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Inhibidores de la Ciclooxigenasa 2/farmacología , Endometriosis/diagnóstico por imagen , Endometriosis/enzimología , Endometriosis/inmunología , Endometrio/irrigación sanguínea , Endometrio/patología , Endotelio Vascular/patología , Femenino , Isoxazoles/farmacología , Ratones Endogámicos , Microvasos/patología , Neovascularización Patológica/enzimología , Neovascularización Patológica/patología , Transducción de Señal/fisiología , UltrasonografíaRESUMEN
Micro (mi)RNAs are short, noncoding RNAs and deregulation of miRNAs and their targets are implicated in tumor generation and progression in many cancers. Meningiomas are mostly benign, slow growing tumors of the central nervous system with a small percentage showing a malignant phenotype.Following in silico prediction of potential targets of miR-34a-3p, SMAD4, FRAT1, and BCL2 have been confirmed as targets by dual luciferase assays with co-expression of miR-34a-3p and reporter gene constructs containing the respective 3'UTRs. Disruption of the miR-34a-3p binding sites in the 3'UTRs resulted in loss of responsiveness to miR-34a-3p overexpression. In meningioma cells, overexpression of miR-34a-3p resulted in decreased protein levels of SMAD4, FRAT1 and BCL2, while inhibition of miR-34a-3p led to increased levels of these proteins as confirmed by Western blotting. Furthermore, deregulation of miR-34a-3p altered cell proliferation and apoptosis of meningioma cells in vitro.We show that SMAD4, FRAT1 and BCL2 are direct targets of miR-34a-3p and that deregulation of miR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro. As part of their respective signaling pathways, which are known to play a role in meningioma genesis and progression, deregulation of SMAD4, FRAT1 and BCL2 might contribute to the aberrant activation of these signaling pathways leading to increased proliferation and inhibition of apoptosis in meningiomas.
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Apoptosis/genética , Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Smad4/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Simulación por Computador , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patología , Meningioma/genética , Meningioma/patología , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/genética , Proteína Smad4/genéticaRESUMEN
The incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 µg/kg ß-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein(+)/Tie2(+) EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis.
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Endometriosis/patología , Células Progenitoras Endoteliales/patología , Estradiol/análogos & derivados , Neovascularización Patológica/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Neovascularización Patológica/patología , Reacción en Cadena de la Polimerasa , TranscriptomaRESUMEN
In a previous study, we identified thioredoxin domain containing 16 (TXNDC16) as a meningioma-associated Ag by protein macroarray screening. Serological screening detected autoantibodies against TXNDC16 exclusively in meningioma patients' sera and not in sera of healthy controls. TXNDC16 was previously found to be an endoplasmic reticulum (ER)-luminal glycoprotein. In this study, we show an additional ER-associated localization of TXNDC16 in the cytosol by in vitro synthesis, molecular mass shift assay, and flow cytometry. We were able to show TXNDC16 secretion in different human cell lines due to masked and therefore nonfunctional ER retrieval motif. A previously indicated exosomal TXNDC16 secretion could not be confirmed in HEK293 cells. The secreted serum protein TXNDC16 is bound in circulating immune complexes, which were found both in meningioma and healthy blood donor sera. Employing a customized array with 163 overlapping TXNDC16 peptides and measuring autoantibody reactivity, we achieved discrimination of meningioma sera from healthy controls with an accuracy of 87.2% using a set of only five immunogenic TXNDC16 epitopes.
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Complejo Antígeno-Anticuerpo/sangre , Antígenos de Neoplasias/inmunología , Glicoproteínas de Membrana/inmunología , Meningioma/inmunología , Secuencia de Aminoácidos , Autoanticuerpos/sangre , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Epítopos/inmunología , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia MolecularRESUMEN
BACKGROUND: Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens. Upon binding to these receptors, particles become internalized. An interaction of particles with macrophage surface receptors is accompanied by an increase in cytosolic calcium concentration. This calcium is provided by intracellular stores and also by an influx of external calcium upon activation of the calcium channels. Nevertheless, the role of calcium in phagocytosis remains controversial. Some researchers postulate the necessity of calcium in Fc-receptor-mediated phagocytosis and a calcium-independent phagocytosis of complement opsonized particles. Others refute the need for calcium in Fc-receptor-mediated phagocytosis by macrophages. METHODS: In this study, the influence of external calcium concentrations and thapsigargin on the phagocytosis of polystyrene latex beads by the macrophage-like cell lines MH-S (murine) and differentiated U937 (human) was analyzed. The phagocytosis efficiency was determined by flow cytometry and was evaluated statistically by ANOVA test and Dunett's significance test, or ANOVA and Bonferroni's Multiple Comparison. RESULTS: Acquired data revealed an external calcium-independent way of internalization of non-functionalized polystyrene latex beads at free calcium concentrations ranging from 0 mM to 3 mM. The phagocytosis efficiency of the cells is not significantly decreased by a complete lack of external calcium. Furthermore, the presence of thapsigargin, known to lead to an increase of cytosolic calcium levels, did not have a significant enhancing influence on bead uptake by MH-S cells and only an enhancing effect on bead uptake by macrophage-like U937 cells at an external calcium concentration of 4 mM. CONCLUSION: The calcium-independent phagocytosis process and the decrease of phagocytosis efficiency in the presence of complement receptor inhibitor staurosporine lead to the assumption that besides other calcium independent receptors, complement receptors are also involved in the uptake of polystyrene beads. The comparison of the phagocytosis efficiencies of both cell types in bivalent cation-free HBSS buffer and in cell medium, leads to the conclusion that it is more likely that other media ingredients such as magnesium are of greater importance for phagocytosis of non-functionalized polystyrene beads than calcium.
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Calcio/farmacología , Macrófagos/efectos de los fármacos , Poliestirenos/farmacología , Tapsigargina/farmacología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Línea Celular , Humanos , Macrófagos/fisiología , Ratones , Fagocitosis/efectos de los fármacos , Células U937RESUMEN
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 14 days. 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.
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Movimiento Celular , Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias Colorrectales/irrigación sanguínea , Neovascularización Patológica/patología , Animales , Apoptosis , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Neovascularización Patológica/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: We developed a model for direct assessment of BMC sequestration in the postischemic murine myocardium after direct antegrade intracoronary injection. METHODS: Modified syngeneic heterotopic heart transplantation was used as a basic model for global myocardial I/R injury in a total of n = 29 animals. IVM was employed to analyze the right ventricular subepicardial coronary microcirculation and for tracking fluorescently labeled BMCs. RESULTS: IVM allowed monitoring all segments of the coronary microcirculation including feeding arterioles, nutritive capillaries, and postcapillary venules. WI and generalized atherosclerosis induced profound reperfusion failure, particularly in nutritive myocardial capillaries. BMCs were found to exclusively sequester in myocardial capillaries, but not in coronary arterioles or postcapillary venules. The sequestration of BMCs in coronary capillaries occurred independent of WI, generalized atherosclerosis, or adhesion molecule function. CONCLUSIONS: This is the first study allowing direct assessment of BMC homing to the postischemic myocardium. Heterotopic heart transplantation and IVM are proper means to study the myocardial sequestration of BMCs after direct antegrade intracoronary injection in vivo. We show for the first time that intracoronarily injected BMCs sequester exclusively in nutritive myocardial capillaries.
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Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Circulación Coronaria , Microcirculación , Daño por Reperfusión Miocárdica , Células Madre/metabolismo , Animales , Células de la Médula Ósea/patología , Trasplante de Corazón , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Células Madre/patología , Trasplante Heterotópico , Trasplante IsogénicoRESUMEN
Endometriosis is a frequent gynecological disease that is characterized by the development of vascularized endometriotic lesions inside the peritoneal cavity. Herein, we analyzed whether circulating endothelial progenitor cells (EPCs) are recruited and incorporated into the microvasculature of these lesions. Intraperitoneal endometriotic lesions were surgically induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein [GFP]) 287 Sato mice. Vascularization and recruitment of GFP-positive EPCs in the lesions was analyzed by intravital fluorescence microscopy and immunohistochemistry over 4 weeks. The numbers of stem cell antigen-1 (Sca-1)/vascular endothelial growth factor receptor-2-positive EPCs in blood and hematopoietic organs of additional endometriotic and control mice were assessed by flow cytometry. We found that approximately 15% of the microvascular endothelium in engrafting endometriotic lesions consisted of incorporated GFP-positive EPCs. Recruitment of EPCs into the lesions coincided with the establishment of own blood supply and the expression of stromal cell-derived factor-1. Accordingly, treatment with the stromal cell-derived factor-1/chemokine receptor type 4 axis antagonist AMD3100 significantly decreased the number of recruited EPCs and the vascularization of endometriotic lesions. However, endometriosis did not induce increased levels of EPCs in the blood, bone marrow, and spleen of C57BL/6 mice. To our knowledge, our findings indicate for the first time that vasculogenesis (ie, de novo generation of blood vessels from EPCs) may represent an integral mechanism in the pathogenesis of endometriosis.
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Endometriosis/patología , Endotelio Vascular/patología , Neovascularización Patológica/patología , Células Madre/patología , Animales , Antígenos Ly/sangre , Antígenos Ly/metabolismo , Bencilaminas , Ciclamas , Endometriosis/sangre , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos/farmacología , Proteínas de la Membrana/sangre , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microvasos/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/sangre , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: This study was aimed to assess the pleiotropic effects of non-hematopoietic doses of erythropoietin (Epo) on post-ischemic microcirculatory dysfunction and inflammation in murine cardiac allografts. METHODS: Epo was given intraperitoneally 2 hours prior to explantation (Epo-donor) or 2 hours prior to the onset of reperfusion (Epo-recipient). Controls were not treated. Intravital fluorescence microscopy (IVM) was used to assess post-ischemic coronary microcirculatory dysfunction. RESULTS: IVM exhibited decreasing capillary blood flow velocities and functional capillary densities (FCD) in controls. Capillary diameters and venular blood flow characteristics showed no significant changes over time. Epo-treatment had no effect on coronary microhemodynamics. Post-ischemic inflammation was characterized by augmented macromolecular leakage. Microvascular permeability decreased in the Epo-donor group (p < 0.05). Leukocyte rolling in coronary post-capillary venules decreased during reperfusion from 64 ± 16 to 19 ± 16 cells/min/mm, whereas firm adhesion increased from 333 ± 135 to 479 ± 154 cells/mm(2) in controls. Capillary leukocyte plugging remained stationary over time with approximately 4 to 6 cells/HPF. Firm adhesion was inhibited in the Epo-recipient group, resulting in 84 ± 34 cells/mm(2) at 6 hours of reperfusion (p < 0.05). Capillary leukocyte plugging was also reduced in the Epo-recipient group (p < 0.05). Epo exerted no effect on leukocyte rolling. Histology revealed significant myocardial edema formation in allografts, without any alteration by Epo treatment. Immunohistochemistry indicated the involvement of resident cardiac mast cells. Allograft rejection was not affected by Epo treatment. CONCLUSIONS: We demonstrate that non-hematopoietic treatment with Epo inhibits acute post-ischemic myocardial leukocyte sequestration, without affecting microcirculatory dysfunction and allograft rejection.
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Cardiotónicos/farmacología , Eritropoyetina/farmacología , Trasplante de Corazón/métodos , Isquemia Miocárdica/prevención & control , Animales , Adhesión Celular/efectos de los fármacos , Vasos Coronarios , Rechazo de Injerto , Leucocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microcirculación , Trasplante HomólogoRESUMEN
BACKGROUND: Liver insufficiency after major hepatectomy still represents a serious challenge in liver surgery. Although some previous studies indicate that erythropoietin (EPO) and its analogue darbepoetin-alpha (DPO) may improve liver function and liver regeneration, little is known on their effect on tumor growth after hepatectomy. Because EPO may promote tumor progression, we herein studied the effect of DPO on tumor growth after major hepatectomy. METHODS: CT26.WT colorectal cancer cells were implanted into the left liver lobe of BALB/c mice. Animals underwent 50% hepatectomy (Phx) and received 10 microg/kg DPO-treatment. Additional Phx animals received only saline treatment. Nonhepatectomized animals with DPO-treatment or saline treatment served as controls. One week after hepatectomy angiogenic blood vessel formation, leukocyte-endothelial cell interaction, tumor cell proliferation, apoptotic cell death, and tumor growth were studied using intravital fluorescence microscopy, histology, Immunohistochemistry, and Western blot analysis. RESULTS: Phx significantly enhanced the growth of liver metastases. This was associated with an increase of tumor capillary density and tumor cell proliferation. In nonhepatectomized animals, DPO only slightly affected metastatic growth. In hepatectomized animals, however, DPO significantly enhanced the Phx-induced stimulation of tumor growth. This was associated with an increased tumor capillary density, a decreased leukocyte-endothelial cell interaction, and a reduced cleaved caspase-3 expression of the CT26.WT cells. CONCLUSIONS: : Our data indicate that DPO significantly enhances the hepatectomy-induced stimulation of colorectal liver metastatic growth by increasing neovascularization, suppressing intratumoral leukocyte recruitment, and reducing tumor cell apoptosis. Thus, EPOs may not be used in patients undergoing hepatectomy for malignant tumor resection.
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Neoplasias Colorrectales/patología , Eritropoyetina/análogos & derivados , Hematínicos/efectos adversos , Hepatectomía/efectos adversos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/análisis , Darbepoetina alfa , Eritropoyetina/efectos adversos , Eritropoyetina/farmacología , Femenino , Citometría de Flujo , Hematínicos/farmacología , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica , Infiltración Neutrófila/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Recent studies have shown that the chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4 are involved in the metastatic process of colorectal cancer. The impact of SDF-1 on the stimulated metastatic growth during hepatectomy-associated liver regeneration is unknown. With the use of a heterotopic murine colon cancer model, we analyzed whether blockade of SDF-1 inhibits angiogenesis and extrahepatic growth of colorectal cancer after liver resection. Functional neutralization of SDF-1 by 1 mg/kg body weight anti-SDF-1 antibody only slightly delayed the initial tumor cell engraftment but also did not reduce the size of established extrahepatic tumors compared with controls. Tumor cell apoptosis was increased by anti-SDF-1 treatment only during the early 5-9-day period of tumor cell engraftment, but was found significantly decreased during the late phase of tumor growth. The initial delay of tumor cell engraftment was associated with an increase of tumor capillary density and microvascular permeability. This was associated with an increased vascular endothelial growth factor (VEGF) expression and an enhanced tumor cell invasion of the neighboring tissue. In contrast to the neutralization of SDF-1, blockade of the SDF-1 receptors CXCR4 and CXCR7 significantly reduced tumor capillary density and tumor growth. Thus, our study indicates that neutralization of SDF-1 after hepatectomy is not capable of inhibiting angiogenesis and growth of extrahepatic colorectal tumors, because it is counteracted by the compensatory actions through an alternative VEGF-dependent pathway.
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Quimiocina CXCL12/metabolismo , Neoplasias Colorrectales/metabolismo , Neovascularización Patológica/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/inmunología , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/prevención & control , Femenino , Citometría de Flujo , Hepatectomía , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Receptores CXCR/inmunología , Receptores CXCR4/inmunología , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ebselen, a seleno-organic compound showing glutathione per-oxidase-like activity, has potent anti-inflammatory and anti-oxidant effects. Since selenium deficiency is thought to be associated with an increased incidence of vascular thrombosis, we studied the effect of ebselen on blood cell aggregate formation and vessel occlusion in vivo. In individual microvessels of rat cremaster muscle preparations, photochemically induced thrombus formation was analyzed in detail using intravital fluorescence microscopy. In ebselen-pretreated animals (30 mg/kg ip), venular thrombus formation was significantly delayed (50% vessel occlusion: 535+/-34 s; initial stasis: 872+/-82 s; complete occlusion: 908+/-87 s) as compared to vehicle-treated controls (416+/-42; 612+/-49; 647+/-51). Moreover, ebselen significantly prolonged the kinetics of arteriolar thrombus formation and even completely prevented blood cell aggregate and thrombus formation in 88.9% of all arterioles studied (p<0.05 vs controls: 37.5%). Anti-thrombotic properties of ebselen could also be observed in a model of ferric chloride-induced microvascular thrombosis, with a low dose (5 mg/kg ip) being as effective as a high dose pretreatment (30 mg/kg ip). As assessed by flow cytometry of platelet P-selectin immunfluorescence, whole blood isolated from ebselen-treated animals revealed a significantly lower fraction of P-selectin expressing platelets when compared with that of DMSO-treated controls. In addition, oxidant stress-induced upregulation of P-selectin on isolated platelets was found dose-dependently inhibited by increasing concentrations of ebselen (10-100 micro M). Moreover, ebselen dose-dependently inhibited H(2)O(2)-induced platelet-leukocyte aggregate formation in whole blood in vitro, suggesting that the anti-thrombotic effect of ebselen is achieved by attenuation of P-selectin dependent platelet-leukocyte aggregation. Thus, ebselen represents preventive and therapeutic value for disorders with increased risk for oxidant stress-associated thrombotic events.
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Antioxidantes/farmacología , Azoles/farmacología , Compuestos de Organoselenio/farmacología , Selectina-P/efectos de los fármacos , Trombosis/prevención & control , Animales , Plaquetas/química , Plaquetas/fisiología , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Isoindoles , Cinética , Microcirculación , Microscopía por Video , Músculos/irrigación sanguínea , Estrés Oxidativo , Selectina-P/análisis , Ratas , Trombosis/tratamiento farmacológicoRESUMEN
OBJECTIVE: In vitro and in vivo studies using isolated platelets require that the cells used for testing are not activated by the isolation procedure. This ensures that the effects measured by the test are the result of the environment or the applied stimulus, but is not an artifact resulting from activation by cell isolation. METHODS: Herein, we analyzed two different platelet isolation procedures (i.e., a Sepharose column versus density gradient centrifugation) with special emphasis on cell activation, including flow cytometric analysis of P-selectin expression, functional quantification of mechanical platelet retention, light microscopic assessment of platelet aggregation, and fluorescence microscopic determination of in vivo rat liver platelet-endothelium cell interaction. RESULTS: Under resting conditions, Sepharose column-isolated platelets showed a negligible fraction of only 2.7 +/- 3.3% cells (mean +/- SEM) with P-selectin expression, and an appropriate response (i.e., a 33-fold increase) upon activation with thrombin receptor-activating peptide (TRAP). In contrast, density gradient centrifugation resulted in P-selectin expression under resting conditions of approximately 50% of the isolated cells and only a 1.6-fold increase on further TRAP stimulation. In addition, density gradient-isolated platelets, but not Sepharose column-isolated platelets, showed increased mechanical retention and agglutination/aggregation in vitro, as well as pronounced adhesion to hepatic venular endothelium in vivo. Interestingly, density gradient-isolated platelets additionally induced in vivo an increase of colocalization of platelets with adherent leukocytes, indicating a generalized microvascular inflammatory response that is comparable to that observed after a 60-minute ischemia/30-minute reperfusion insult. CONCLUSION: Density gradient centrifugation-isolated platelets, but not Sepharose column-isolated platelets, are activated already under resting conditions and induce in vivo a platelet-leukocyte-endothelial cell-associated inflammatory response. Thus, we propose that the method of platelet isolation using the Sepharose column is superior to the density gradient centrifugation technique and might therefore be preferred for in vitro and in vivo assays to study platelet function.
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Plaquetas/citología , Separación Celular/métodos , Separación Celular/normas , Centrifugación por Gradiente de Densidad/métodos , Cromatografía en Agarosa/métodos , Endotelio Vascular/citología , Venas Hepáticas/citología , Humanos , Microcirculación/citología , Fragmentos de Péptidos/farmacología , Activación Plaquetaria , Adhesividad PlaquetariaRESUMEN
Elderly patients have a higher incidence of morbidity and mortality due to infectious diseases. Because most immune functions in the elderly differ compared with those in younger subjects, we studied the effect of the immunomodulating agent G-CSF on endotoxic liver injury and cytokine release in an aging animal model of acute sepsis. Young (3-month-old), mature (12-month-old), and senescent (24-month-old) male Sprague-Dawley rats (n = 6 each) were treated with bacterial endotoxin for 6 h. Age-matched groups of animals (n = 6 each) received G-CSF (200 microg/kg i.v.) 1 h prior to endotoxin. LPS-induced hepatic toxicity, as assessed in vivo by nutritive perfusion failure, intravascular leukocyte accumulation, biliary excretion dysfunction, and liver enzyme release, was significantly more pronounced in mature and old animals when compared with young animals. Concomitantly, mature but, in particular, senescent endotoxic animals exhibited 2- to 14-fold higher plasma levels of TNF-alpha, IL-1beta, IL-6, IL-10, and Rantes than young endotoxic rats. The percentage of G-CSF receptor surface expression on neutrophils did not significantly differ between young, mature, and senescent animals (36%-46%) and was found comparably down-regulated at 6 h after LPS exposure (15%-19%). Kupffer cell activation, i.e., clearance of intravascularly applied fluorescent latex beads, did not differ between the LPS-exposed age groups. G-CSF dampened LPS-induced Kupffer cell activation, and significantly reduced plasma cytokine levels in young and mature, but not in senescent animals. Thereby, G-CSF caused attenuation of hepatic tissue injury in all except the senescent endotoxic animals. In summary, our results show an age-dependent increase in hepatic LPS toxicity. Because flow cytometric analysis of G-CSF receptor expression disproved that cytokinemia-induced down-regulation of G-CSF receptors might account for the unresponsiveness to G-CSF in the senescent animals, other homeostatic regulatory mechanisms appear to undergo changes with age that bring out a disrupted balance between inflammatory cytokines with unresponsiveness to G-CSF and, thus, compromised host defense mechanisms in the elderly.