Ischaemic Preconditioning and Intermittent Clamping Does not Influence Mediators of Liver Regeneration in a Human Liver Sinusoidal Endothelial Cell Model of Ischaemia-Reperfusion Injury.

BACKGROUND: The role of surgical technique on liver regeneration following surgery remains inconclusive. The aim of the study was to assess the effect of ischaemic preconditioning (IPC) and intermittent clamping (IC) on mediators of regeneration produced by human liver sinusoidal endothelial cells (SECs), using an in vitro hypoxia-reoxygenation model to mimic ischaemia-reperfusion injury (IRI). METHODS: Following extraction from samples obtained from liver resection (n = 5), confluent culture flasks of SECs were subjected to IRI (1 hour hypoxia + 1 hour reoxygenation), IPC prior to IRI (10 minutes hypoxia + 10 minutes reoxygenation + 1 hour hypoxia + 1 hour reoxygenation), IC (15 minutes hypoxia + 5 minutes reoxygenation x 3 + 1 hour reoxygenation) and compared to controls. The production of various mediators was determined over 48 hours. RESULTS: Interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) were produced by SECs. Both IPC and IC did not significantly influence the profile of IL-6, IL-8, G-CSF and HGF by SECs compared to IRI over the study period. CONCLUSION: IPC and IC did not influence the production of pro-regenerative mediators in a SECs model of IRI. The role of surgical technique on liver regeneration remains to be determined.

An in vitro model of warm hypoxia-reoxygenation injury in human liver endothelial cells.

BACKGROUND: Ischemia-reperfusion or hypoxia-reoxygenation (H-R) injury adversely affects hepatic function following transplantation and major resection; the death of human sinusoidal endothelial cells (SECs) by apoptosis may play a central role in this process. Caspase-3 is an important intracellular protease in the intrinsic and extrinsic pathways of apoptosis. MATERIALS AND METHODS: SECs and EAhy926 cells were exposed to warm hypoxia at 37°C, followed by reoxygenation at 37°C. Activity of caspase-3 was quantified using Western blotting and colorimetric kinase assays. RESULTS: H-R caused a significant increase in caspase-3 activity compared with controls in both cell types. CONCLUSIONS: Warm H-R injury causes apoptotic cell death of SECs and immortalized cells, but with differing patterns of caspase activity.

Differential expression of hypoxia and (lymph)angiogenesis-related genes at different metastatic sites in breast cancer.

INTRODUCTION: Breast cancer can metastasize via lymphatic and hematogenous pathways. Hypoxia and (lymph)angiogenesis are closely related processes that play a pivotal role in the tumor progression and metastasis. The aim of this study was to compare expression of hypoxia and (lymph)angiogenesis-related genes between primary breast tumors and metastases in different tissues. MATERIALS AND METHODS: A gene list of 269 hypoxia and (lymph)angiogenesis-related genes was composed and validated using Onto-Express, Pathway-express and Ingenuity software. The expression of these genes was compared in microarray data of 62 samples of primary tumors and metastases of 31 patients with breast cancer retrieved from Gene Expression Omnibus. Similarity between samples was investigated using unsupervised hierarchical clustering analysis, principal component analysis and permutation testing. Differential gene expression between primary tumors and metastases and between metastases from different organs was analyzed using Kruskall-Wallis and Mann-Whitney statistics. RESULTS: Unsupervised hierarchical cluster analysis demonstrated that hypoxia and (lymph)angiogenesis-related gene expression was more similar between samples from the same patient, than between samples from the same organ. Principal component analysis indicated that 22.7% and 7.0% of the total variation in the gene list was respectively patient and organ related. When differences in gene expression were studied between different organs, liver metastases seemed to differ most from the other secondary sites. Some of the best characterized molecules differentially expressed were VEGFA, PDGFRB, FGF4, TIMP1, TGFB-R1 and collagen 18A1 (precursor of endostatin). To confirm the results of these experiments at the protein level, immunohistochemical experiments were performed with antibodies for VEGFA and MMP-2. CONCLUSIONS: Our results suggest that hypoxia and (lymph)angiogenesis-related gene expression is more dependent on the characteristics of the primary tumor than on the characteristics of the organs that bear the metastasis. However, when different organs are compared, the expression in liver metastases differs most from other metastatic sites and primary tumors, possibly due to organ-specific angiogenic and lymphangiogenic responses to metastasis-related hypoxia.

A potential role for TGFbeta in the regulation of uveal melanoma adhesive interactions with the hepatic endothelium.

PURPOSE: TGFbeta has been shown to have a regulatory effect on uveal melanoma invasion, but it is not known which processes are specifically influenced. The purpose of this study was to analyze the effect of TGFbeta stimulation on the adhesive interactions of uveal melanomas with the extracellular matrix (ECM) and endothelium and, in addition, its effect on the secretion of collagenases. METHODS: Invasive and a noninvasive uveal melanoma cell lines, supported by short-term primary uveal melanoma cultures, were used to assess the effect of TGFbeta on ECM and endothelial adhesion and degradation of the ECM. Changes in cell adhesion molecule expression were assessed by flow cytometry, and conditioned media were analyzed by gelatin zymography. Assays of adhesion to ECM substrates and endothelial cells were also performed. RESULTS: Treatment with TGFbeta increased low basal levels of adhesion molecule and latent MMP-2 expression, as well as adhesion to hepatic endothelial cells by the noninvasive cell line. Conversely, TGFbeta reduced adhesion to laminin and a laminin-binding integrin by invasive cells but had no effect on their adhesion to the endothelium. CONCLUSIONS: In this preliminary study, TGFbeta was found to upregulate levels of MMP-2, reduce adhesion to laminin, and downregulate expression of laminin-binding integrins. Specifically, TGFbeta was found to increase adhesion of noninvasive uveal melanoma cells to the hepatic, but not the dermal, endothelium and may therefore contribute to the preferential targeting of the liver by uveal melanomas.

Approaches to improve angiogenesis in tissue-engineered skin.

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Development of a reconstructed human skin model for angiogenesis.

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.