Sigurd Krieger

Lymphatic Neoangiogenesis in Human Kidney Transplants Is Associated with Immunologically Active Lymphocytic Infiltrates

Renal transplant rejection is caused by a lymphocyte-rich inflammatory infiltrate that attacks cortical tubules and endothelial cells. Immunosuppressive therapy reduces the number of infiltrating cells; however, their exit routes are not known. Here a >50-fold increase of lymphatic vessel density over normal kidneys in grafts with nodular mononuclear infiltrates is demonstrated by immunohistochemistry on human renal transplant biopsies using antibodies to the lymphatic endothelial marker protein podoplanin. Nodular infiltrates are constantly associated with newly formed, Ki-67-expressing lymphatic vessels and contain the entire repertoire of T and B lymphocytes to provide specific cellular and humoral alloantigenic immune responses, including Ki-67(+) CD4(+) and CD8(+) T lymphocytes, S100(+) dendritic cells, and Ki-67(+)CD20(+) B lymphocytes and lambda- and kappa-chain-expressing plasmacytoid cells. Numerous chemokine receptor CCR7(+) cells within the nodular infiltrates seemed to be attracted by secondary lymphatic chemokine (SLC/CCL21) that is produced and released by lymphatic endothelial cells in a complex with podoplanin. From these results, it is speculated that lymphatic neoangiogenesis not only contributes to the export of the rejection infiltrate but also is involved in the maintenance of a potentially detrimental alloreactive immune response in renal transplants and provides a novel therapeutic target.

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.

Lymphatic Microvessels in the Rat Remnant Kidney Model of Renal Fibrosis: Aminopeptidase P and Podoplanin Are Discriminatory Markers for Endothelial Cells of Blood and Lymphatic Vessels

ABSTRACT. Rat remnant kidney is an established model of renal tubulointerstitial fibrosis and progression to end-stage renal failure. The morphologic lesions comprise nephron loss and regeneratory tubular hypertrophy, interstitial infiltration, predominately by macrophages, and progressive fibrosis. A critical role in this complex pathology was assigned to tubulointerstitial blood microvessels that regulate the supply of oxygen and nutrients of tubuli. Whereas some investigations reported a rarefaction of the vascular network in association with the degenerative cortical changes, others observed an increase in vascularization. Here these discrepant findings are addressed by reinvestigation of the vascularization of rat remnant kidneys by the use of two novel endothelial lineage specific, discriminatory markers, i.e., the membrane mucoprotein podoplanin with specificity for lymphatic endothelia, and the glycosyl-phosphatidylinositol (GPI)-anchored membrane enzyme aminopeptidase P that is recognized by a monoclonal antibody designated JG12 and that is specifically expressed by endothelial cells of blood vessels only. The results obtained confirm a regional rarefaction of aminopeptidase P-positive blood microvessels; they also establish major changes in the renal lymphatic vasculature. Massive proliferation of lymphatic vessels was observed in fibrotic tubulointerstitial regions, whereas in kidneys of sham-operated rats, only a few lymphatic vessels were found adjoined with arteries. The lymphatic vessels frequently contained mononuclear cells that were also encountered in the interstitial spaces and expressed relative large amounts of vascular endothelial growth factor-C mRNA by in situ hybridization. Collectively, these results indicate that a large proportion of the microvessels encountered in the cortex of remnant kidneys are of lymphatic origin and cannot be discriminated by common endothelial markers, such as CD34, that are expressed by both lymphatic and blood endothelia cells. As lymphatic endothelial cells secrete chemokines that attract dendritic cells, it is possible that the increase in lymphatic vascularization could enhance the immunologic surveillance of remnant kidneys.

Lymphatic Precollectors Contain a Novel, Specialized Subpopulation of Podoplaninlow, CCL27-Expressing Lymphatic Endothelial Cells

Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC podo-low and LEC podo-high. LEC podo-low is restricted to lymphatic precollector vessels that originate from initial LEC podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10+ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10+ T cells accumulate preferentially both around and within CCL27+ LEC podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10+ T lymphocytes are chemotactically attracted by LEC podo-low in a CCL27-dependent fashion, but not by LEC podo-high. These observations indicate that LEC podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC podo-low-containing vessels are involved in the trafficking of CCR10+ T cells during skin inflammation.

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.

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.

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis

Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce “circular chemorepellent-induced defects” (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.

In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen

Background:As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration.Methods:A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called ‘circular chemorepellent induced defects’ (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis.Results:Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation.Conclusion:The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Short 42°C heat shock induces phosphorylation and degradation of Cdc25A which depends on p38MAPK, Chk2 and 14.3.3

The effects of heat shock (HS; 42 degrees C) on the cell cycle and underlying molecular mechanisms are astonishingly unexplored. Here, we show that HS caused rapid Cdc25A degradation and a reduction of cell cycle progression. Cdc25A degradation depended on Ser75-Cdc25A phosphorylation caused by p38MAPK and Chk2, which phosphorylated Ser177-Cdc25A that is specific for 14.3.3 binding. Upon HS, Cdc25A rapidly co-localized with 14.3.3 in the perinuclear space that was accompanied with a decrease of nuclear Cdc25A protein levels. Consistently, a 14.3.3 binding-deficient Cdc25A double mutant (Ser177/Ala-Tyr507/Ala) was not degraded in response to HS and there was no evidence for an increased co-localization of Cdc25A with 14.3.3 in the cytosol. Therefore, upon HS, p38, Chk2 and 14.3.3 were antagonists of Cdc25A stability. On the other hand, Cdc25A was protected by Hsp90 in HEK293 cells because the specific inhibition of Hsp90 with Geldanamycin caused Cdc25A degradation in HEK293 implicating that Cdc25A is an Hsp90 client. Specific inhibition of Hsp90 together with HS caused and accelerated degradation of Cdc25A and was highly cytotoxic. The results presented here show for the first time that Cdc25A is degraded by moderate heat shock and protected by Hsp90. We describe the mechanisms explaining HS-induced cell cycle retardation and provide a rationale for a targeted hyperthermia cancer therapy.