Nicole Huttary

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

The Cellular Lesion of Humoral Rejection: Predominant Recruitment of Monocytes to Peritubular and Glomerular Capillaries

Accumulation of inflammatory cells within capillaries is a common morphologic feature of humoral renal allograft rejection and is most easily appreciated if it occurs in glomeruli. The aim of our study was to determine the amount and composition of immune cells within glomeruli and peritubular capillaries (PTC) in cellular and humoral allograft rejection.

Posttransplant HLA Alloreactivity in Stable Kidney Transplant Recipients—Incidences and Impact on Long-Term Allograft Outcomes

Humoral alloreactivity is well established to predict adverse allograft outcomes. However, in some recipients, alloantibodies may also occur in the absence of graft dysfunction. We evaluated if and how often complement‐ and noncomplement‐fixing alloantibodies are detectable in stable recipients and whether, in this context, they affect long‐term outcomes. Sera obtained from 164 kidney transplant recipients at 2, 6 and 12 months were evaluated by FlowPRA screening and single‐antigen testing for detection of IgG‐ or C4d‐fixing HLA panel reactivity and donor‐specific antibodies (DSA). Applying stringent criteria, we selected 34 patients with an uneventful 1‐year course (no graft dysfunction or rejection) and excellent graft function at 12 months [estimated glomerular filtration rate (eGFR) ≥60 mL/min and proteinuria ≤0.5 g/24 h]. Nine (27%) and 5 (15%) of these recipients tested positive by [IgG] and [C4d]FlowPRA screening, respectively. In five cases, DSA were identified. Frequencies of positive test results and DSA binding intensities were not significantly lower than those documented for patients who did not fulfill the above criteria. In recipients with an excellent 1‐year course, FlowPRA reactivity was not associated with lower eGFR or increased protein excretion during 68‐month median follow‐up. Our results suggest cautious interpretation of antibody monitoring in patients with normal‐functioning grafts.

In Vitro Detection of C4d‐Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody‐mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody‐triggered C4d deposition to HLA antigen‐coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86–0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50–0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83–1.05] vs. 0.76 [0.56–0.97] calculated for C4d‐fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement‐fixing donor‐specific alloreactivities failed to improve the predictive value of FlowPRA‐based serology. In conclusion, our results suggest that detection of complement‐fixing HLA panel reactivity could provide a specific tool for monitoring of C4d‐positive AMR.

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects’ (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro

Background:Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups.Methods:Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by 14C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers.Results:In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21Cip/Waf and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread.Conclusion:These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 μmol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion.

Background:Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.Methods:Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of ‘circular chemorepellent-induced defects’ (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.Results:Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.Conclusion:Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

Alterations in fatty acid utilization and an impaired antioxidant defense mechanism are early events in podocyte injury: a proteomic analysis.

Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.