Claudia Scheuer

Endothelial Progenitor Cells Contribute to the Vascularization of Endometriotic Lesions

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.

Epigallocatechin-3-gallate inhibits estrogen-induced activation of endometrial cells in vitro and causes regression of endometriotic lesions in vivo

BACKGROUND Epigallocatechin-3-gallate (EGCG), the major component of green tea, is a pleiotropic substance, which may inhibit tumor growth via multiple intracellular signaling pathways. Herein, we studied whether EGCG may also be effective in the treatment of endometriosis. METHODS We investigated the effect of EGCG on activation by estradiol (E(2)), proliferation and vascular endothelial growth factor (VEGF) expression of isolated hamster endometrial stromal cells and glandular cells in vitro using the water-soluble tetrazolium (WST)-1 colorimetric assay and western blot analysis. In the dorsal skinfold chamber model of Syrian golden hamsters, which were treated for 14 days with EGCG or vehicle, we further analyzed angiogenesis, blood perfusion and tissue integrity of both endometriotic lesions and ovarian follicles by intravital fluorescence microscopy and histology. RESULTS We found that EGCG suppresses E(2)-stimulated activation, proliferation and VEGF expression of endometrial cells in vitro (all P < 0.05). Furthermore, EGCG selectively inhibited angiogenesis and blood perfusion (P < 0.05) of endometriotic lesions in vivo without affecting blood vessel development in ovarian follicles. Histology confirmed that EGCG-treatment induces regression of the endometriotic lesions. CONCLUSIONS Our data indicate that EGCG might be a promising therapeutic agent in the treatment of endometriosis, preventing the establishment of new endometriotic lesions.

CXCR4 and CXCR7 regulate angiogenesis and CT26.WT tumor growth independent from SDF‐1

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.

Improvement of rat liver graft quality by pifithrin‐α–mediated inhibition of hepatocyte necrapoptosis

Early graft dysfunction due to ischemia reperfusion injury remains a major clinical challenge in liver transplantation. Because apoptosis may contribute to graft dysfunction, we studied whether transient inhibition of p53 is capable of improving graft quality by reducing apoptotic cell death. Rat livers were harvested and stored for 24 hours or 48 hours in a 4°C solution containing either pifithrin‐α (PFT‐α), a specific p53‐inhibitor, or the vehicle dimethyl‐sulfoxide. Storage was followed by 2‐hour reperfusion with 37°C Krebs‐Henseleit buffer in an isolated liver perfusion system. Besides caspase‐3 activation, apoptosis was quantified using fluorescence microscopy and hematoxylin‐eosin histology. Trypan blue allowed for assessment of cell membrane damage, indicating both secondary apoptosis and primary necrosis. Bile flow, oxygen consumption, K+‐excretion and enzyme release served as indicators of overall graft quality. Upon 2‐hour reperfusion, livers developed procaspase activation as well as a mixture of apoptotic and necrotic cell death, representing necrapoptosis. In livers that had been stored for 48 hours, necrapoptotic injury was more pronounced compared with that after 24‐hour storage. PFT‐α effectively attenuated caspase activation as well as hepatocellular apoptosis and necrosis. Attenuation of both modes of cell death by PFT‐α was associated with improved liver function, metabolism, and integrity. Experiments with the caspase inhibitor z‐VAD‐fmk confirmed that apoptosis is one mode of cell death in cold ischemia reperfusion. In conclusion, inhibition of p53‐dependent apoptosis by PFT‐α reduces hepatic preservation‐reperfusion injury and improves primary organ function and metabolism. Fortification of the preservation solution with PFT‐α may represent a promising and easily applicable approach to mitigate reperfusion injury in liver transplants. (HEPATOLOGY 2004;39:1553–1562.)

Macrophage Inflammatory Protein-2 Promotes Angiogenesis, Cell Migration, and Tumor Growth in Hepatic Metastasis

BackgroundIn a mouse model of hepatic metastasis, we herein analyzed whether the CXC chemokine macrophage inflammatory protein (MIP)-2, a functional analogue of the human interleukin 8, stimulates tumor cell migration in vitro and angiogenesis and tumor growth in vivo.MethodsBy using chemotaxis chambers, CT26.WT colorectal tumor cell adhesion and migration were studied under stimulation with different concentrations of MIP-2. To evaluate angiogenesis and tumor growth in vivo, 1 × 105 CT26.WT cells were implanted into the left liver lobe of syngeneic BALB/c mice, and 10, 100, and 1000 nM of MIP-2 or phosphate-buffered saline (controls) was injected into the peritumoral area. After 7 days, angiogenesis, proliferation, tumor growth, apoptosis, cleaved caspase 3, and CXCR-2 expression were analyzed by using intravital fluorescence microscopy, histology, immunohistochemistry, and fluorescence-activated cell sorting.ResultsIn vitro, 98.8% of unstimulated CT26.WT cells showed CXCR-2 receptor expression. In the chemotaxis assays, MIP-2 provoked a dose-dependent increase of cell migration and a most pronounced cell adhesion at a dose of 100 nM. In vivo, MIP-2, in particular in a dose of 100 or 1000 nM, induced a significant increase of tumor capillary density and a marked widening of the angiogenic front at the tumor margin. Capillaries of the angiogenic front, but not of the tumor center, showed significant dilation, thus indicating a pronounced action of vascular endothelial growth factor. Tumor volume was significantly increased, in particular after 100 nM of MIP-2 stimulation, when compared with phosphate-buffered saline–treated controls, whereas only 1000 nM of MIP-2–treated animals additionally showed a higher frequency of apoptotic cell death within the tumor margin.ConclusionsOur study indicates for the first time that the CXC chemokine MIP-2 promotes angiogenesis and growth of colorectal CT26.WT hepatic metastasis.

Erythropoietin protects critically perfused flap tissue

Objective:The objective of this study was to analyze whether erythropoietin (EPO) protects from necrosis of critically perfused musculocutaneous tissue and the mechanisms by which this protection is achieved. Background:EPO is the regulator of erythropoiesis and is used to treat patients with anemia of different causes. Recent studies suggest that EPO has also other tissue-protective effects, irrespective of its erythropoietic properties. Material and Methods:C57BL/6-mice were treated with 3 doses of EPO at 500 IU/kg intraperitoneally. EPO was given either before (preconditioning, n = 7), before and after (overlapping treatment, n = 7), or after (treatment, n = 7) surgery. Animals receiving only saline served as controls (CON). Acute persistent ischemia was induced by elevating a randomly perfused flap in the back of the animals. This critically perfused tissue demonstrates an initial microvascular failure of ∼40%, resulting in ∼50% tissue necrosis if kept untreated. Repetitive fluorescence microscopy was performed over 10 days, assessing angiogenesis, functional capillary density, inflammatory leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis. Additional molecular tissue analyses included the determination of inducible nitric oxide synthase, erythropoietin receptor (EPO-R), and vascular endothelial growth factor (VEGF). Results:EPO preconditioning did not affect hematocrit and EPO-R expression, but increased inducible nitric oxide synthase in the critically perfused tissue. This correlated with a significant arteriolar dilation, which resulted in a maintained functional capillary density (CON: 0 ± 0 cm/cm2; preconditioning: 37 ± 21 cm/cm2; overlapping treatment: 72 ± 26 cm/cm2; P < 0.05). EPO pretreatment further significantly reduced microvascular leukocyte adhesion and apoptotic cell death. Moreover, EPO pretreatment induced an early VEGF upregulation, which resulted in new capillary network formation (CON: 0 ± 0 cm/cm2; preconditioning: 40 ± 3 cm/cm2; overlapping treatment: 33 ± 3 cm/cm2; P < 0.05). Accordingly, EPO pretreatment significantly reduced tissue necrosis (CON: 48% ± 2%; preconditioning: 26% ± 3%; overlapping treatment: 20% ± 3%; P < 0.05). Of interest, EPO treatment was only able to alleviate ischemia-induced inflammation but could not improve microvascular perfusion and tissue survival. Conclusions:EPO pretreatment improves survival of critically perfused tissue by nitric oxide -mediated arteriolar dilation, protection of capillary perfusion, and VEGF-initiated new blood vessel formation.

In vitro and in vivo evaluation of the anti-angiogenic actions of 4-hydroxybenzyl alcohol

4‐Hydroxybenzyl alcohol (HBA) is a phenolic plant compound, which has been shown to influence many cellular mechanisms. In the present study, we analysed in vitro and in vivo the anti‐angiogenic actions of this pleiotropic agent.

Xanthohumol inhibits growth and vascularization of developing endometriotic lesions

BACKGROUND Xanthohumol is a prenylated flavonoid isolated from hops, which is known to act as a pleiotropic cancer chemopreventive agent owing to its anti-proliferative, anti-inflammatory and anti-angiogenic properties. In the present study, we analyzed, for the first time, whether this dietary compound may also be used for the treatment of endometriosis. METHODS Peritoneal and mesenteric endometriotic lesions were surgically induced in BALB/c mice by uterine tissue transplantation into the abdominal cavity. The animals were treated daily with 100 µM xanthohumol (n= 8) or vehicle (control, n= 8) via the drinking water, starting 3 days before tissue transplantations. Lesion growth, cyst formation and vascularization were subsequently analyzed by means of high-resolution ultrasound imaging (at Day 0 and then once per week for 28 days), caliper measurements, western blotting, histology and immunohistochemistry over 4 weeks. RESULTS In the treatment and control groups, uterine grafts developed typical endometriotic lesions with cyst-like dilated glands surrounded by a vascularized endometrial stroma. However, xanthohumol efficiently decreased the size of these lesions at Day 28, independent of their localization within the peritoneal cavity, compared with control (peritoneal: P =0.041; mesenteric: P =0.038). This was associated with a reduced level of phosphoinositide 3-kinase protein. Moreover, vascularization of xanthohumol-treated lesions was suppressed, as indicated by a significantly lower microvessel density at Day 28 when compared with vehicle-treated controls (peritoneal: P =0.026; mesenteric: P =0.004). Additional analyses revealed that treatment with xanthohumol did not affect the histomorphology, proliferation and vascularization of the uterine horns and ovaries. CONCLUSIONS Taken together, these experimental findings suggest that xanthohumol inhibits the development of endometriotic lesions in mice without inducing serious side effects in the reproductive organs. Thus, xanthohumol represents a promising dietary phytochemical that, after further testing, may be considered for the use in the selective treatment of endometriotic lesions.

Multidrug donor preconditioning protects steatotic liver grafts against ischemia-reperfusion injury

BACKGROUND Graft dysfunction of steatotic livers (SL) still remains a major challenge in liver transplantation. Different mechanisms are thought to be involved in the impaired tolerance of SL to ischemia-reperfusion injury. Thus, different pharmacologic strategies may need to be combined to effectively protect SL and to reduce graft dysfunction after transplantation. Therefore, we analyzed the effectiveness of a multidrug donor preconditioning (MDDP) procedure to protect SL from cold ischemia-reperfusion injury. METHODS Liver steatosis was induced by a high-carbohydrate, fat-free diet. A total of 24 Sprague-Dawley rats were divided into 3 groups (n = 8 each), including a control group with nonsteatotic livers (Con), a vehicle-treated SL group (SL-Con), and a SL group undergoing MDDP (SL-MDDP), including pentoxyphylline, glycine, deferoxamine, N-acetylcysteine, erythropoietin, melatonin, and simvastatin. MDDP was applied before liver perfusion with 4°C histidine-tryptophan-ketoglutarate (HTK) solution and organ harvest. After 24 hours of cold storage in HTK, postischemic reperfusion was performed in an isolated liver reperfusion model using 37°C Krebs-Henseleit bicarbonate buffer. RESULTS After 60 minutes of reperfusion, SL showed a significant reduction of bile flow as well as a marked increase of liver enzyme levels and apoptotic cell death compared with Con. This was associated with an increased malondialdehyde formation, interleukin-1 production, and leukocytic tissue infiltration. MDDP completely abolished the inflammatory response and was capable of significantly reducing parenchymal dysfunction and injury. CONCLUSIONS MDDP decreases SL injury after cold storage and reperfusion. The concept of MDDP as a simple and safe preoperative regime, thus may be of interest in clinical use, expanding the donor pool from marginal donors.

Angiogenic and Inflammatory Host Response to Surgical Meshes of Different Mesh Architecture and Polymer Composition

One of the major challenges in hernia repair is the development of surgical meshes that guarantee an optimal biocompatibility and incorporation into the host tissue. For this purpose, it is necessary to study the impact of different mesh types on angiogenesis, inflammation, and tissue formation. In the present study, we analyzed the host tissue reaction to Prolene, Ultrapro, and Vicryl meshes over a 14-day period after implantation into the hamster dorsal skinfold chamber using intravital fluorescence microscopy and histology. Our results demonstrate that compared with the Prolene and Ultrapro mesh, the Vicryl mesh induced a more pronounced angiogenic and inflammatory host tissue response with formation of a densely vascularized granulation tissue, which was infiltrated by many inflammatory cells. However, the strong foreign body reaction was not associated with a better mesh incorporation, but resulted in a significantly reduced explantation strength when compared with that of Prolene and Ultrapro meshes. Histological examinations revealed that the granulation tissue surrounding the Vicryl mesh was instable, because of low collagen content and massive infiltration of inflammatory cells. Thus, our data demonstrate that a stronger angiogenic and inflammatory response to an implanted mesh does not necessarily result in a better incorporation into the host tissue.