Reversible photoregulation of cell-cell adhesions with opto-E-cadherin.

E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.

Platelets, Constant and Cooperative Companions of Sessile and Disseminating Tumor Cells, Crucially Contribute to the Tumor Microenvironment.

Although platelets and the coagulation factors are components of the blood system, they become part of and contribute to the tumor microenvironment (TME) not only within a solid tumor mass, but also within a hematogenous micrometastasis on its way through the blood stream to the metastatic niche. The latter basically consists of blood-borne cancer cells which are in close association with platelets. At the site of the primary tumor, the blood components reach the TME via leaky blood vessels, whose permeability is increased by tumor-secreted growth factors, by incomplete angiogenic sprouts or by vasculogenic mimicry (VM) vessels. As a consequence, platelets reach the primary tumor via several cell adhesion molecules (CAMs). Moreover, clotting factor VII from the blood associates with tissue factor (TF) that is abundantly expressed on cancer cells. This extrinsic tenase complex turns on the coagulation cascade, which encompasses the activation of thrombin and conversion of soluble fibrinogen into insoluble fibrin. The presence of platelets and their release of growth factors, as well as fibrin deposition changes the TME of a solid tumor mass substantially, thereby promoting tumor progression. Disseminating cancer cells that circulate in the blood stream also recruit platelets, primarily by direct cell-cell interactions via different receptor-counterreceptor pairs and indirectly by fibrin, which bridges the two cell types via different integrin receptors. These tumor cell-platelet aggregates are hematogenous micrometastases, in which platelets and fibrin constitute a particular TME in favor of the cancer cells. Even at the distant site of settlement, the accompanying platelets help the tumor cell to attach and to grow into metastases. Understanding the close liaison of cancer cells with platelets and coagulation factors that change the TME during tumor progression and spreading will help to curb different steps of the metastatic cascade and may help to reduce tumor-induced thrombosis.

Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts.

Cadherins mediate cohesive contacts between isotypic cells by homophilic interaction and prevent contact between heterotypic cells. Breast cancer cells neighboring endothelial cells (ECs) atypically express vascular endothelial (VE)-cadherin. To understand this EC-induced VE-cadherin expression in breast cancer cells, MCF7 and MDA-MB-231 cells expressing different endogenous cadherins were co-cultured with ECs and analyzed for VE-cadherin at the transcriptional level and by confocal microscopy, flow cytometry, and immunoblotting. After losing their endogenous cadherins and neo-expression of VE-cadherin, these cells integrated into an EC monolayer without compromising the barrier function instantly. However, they induced the death of nearby ECs. EC-derived extracellular vesicles (EVs) contained soluble and membrane-anchored forms of VE-cadherin. Only the latter was re-utilized by the cancer cells. In a reporter gene assay, EC-adjacent cancer cells also showed a juxtacrine but no paracrine activation of the endogenous VE-cadherin gene. This cadherin switch enabled intimate contact between cancer and endothelial cells in a chicken chorioallantoic membrane tumor model showing vasculogenic mimicry (VM). This EV-mediated, EC-induced cadherin switch in breast cancer cells and the neo-expression of VE-cadherin mechanistically explain the mutual communication in the tumor microenvironment. Hence, it may be a target to tackle VM, which is often found in breast cancers of poor prognosis.

Alterations in Tight- and Adherens-Junction Proteins Related to Glaucoma Mimicked in the Organotypically Cultivated Mouse Retina Under Elevated Pressure.

Purpose: To scrutinize alterations in cellular interactions and cell signaling in the glaucomatous retina, mouse retinal explants were exposed to elevated pressure. Methods: Retinal explants were prepared from C57bl6 mice and cultivated in a pressure chamber under normotensive (atmospheric pressure + 0 mm Hg), moderately elevated (30 mm Hg), and highly elevated (60 mm Hg) pressure conditions. The expression levels of proteins involved in the formation of tight junctions (zonula occludens 1 [ZO-1], occludin, and claudin-5) and adherens junctions (VE-cadherin and ß-catenin) and in cell-signaling cascades (Cdc42 and activated Cdc42 kinase 1 [ACK1]), as well as the expression levels of the growth-factor receptors platelet-derived growth factor receptor beta and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and of diverse intracellular proteins (ß-III-tubulin, glial fibrillary acidic protein transcript variant 1, α-smooth muscle actin, vimentin, and von Willebrand factor VIII), were analyzed using immunohistochemistry, western blotting, and quantitative real-time polymerase chain reactions. Results: The retinal explants were well preserved when cultured in the pressure chambers used in this study. The responses to pressure elevation varied among diverse retinal cells. Under elevated pressure, the expression of ZO-1 increased in the large vessels, neuronal cells began to express VEGFR-1, and the Cdc42 expression in the optic nerve head was downregulated. Overall we found significant transcriptional downregulation of VE-cadherin, ß-catenin, VEGFR-1, VEGFR-2, vimentin, Cdc42, and ACK1. Western blotting and immunohistochemistry indicated a loss of VE-cadherin with pressure elevation, whereas the protein levels of ZO-1, occludin, VEGFR-1, and ACK1 increased. Conclusions: The pressure chamber used for cultivating mouse retinal explants can serve as an in vitro model system for investigating molecular alterations in glaucoma. In this system, responses of the entire retinal cells toward elevated pressure with conspicuous changes in the vasculature and the optic nerve head can be seen. In particular, our investigations indicate that changes in the blood-retina barrier and in cellular signaling are induced by pressure elevation.

[Morphological and Quantitative Changes in Retinal and Optic Nerve Vessels in Experimental Glaucoma Model with Elevated IOP for 7 Weeks]. / Erhöhter Augeninnendruck für 7 Wochen induziert lokale Gefäßveränderungen im experimentellen Glaukommodell in vivo.

INTRODUCTION: Glaucoma is characterised by progressive loss of retinal ganglion cells and axons. Experimental research has concentrated on understanding the pathophysiological mechanisms involved in glaucomatous damage. It is still a matter of debate whether neurons or capillaries are primarily damaged by elevated intraocular pressure (IOP). The aim of this study was to detect IOP-induced vascular changes in the vessels of the optic nerve head and the main vessels of the retina in vivo. METHODS: Experimental glaucoma was induced in adult Sprague Dawley rats by cauterisation of three episcleral veins of the left eye (n = 3). In vivo, retinal vessel calibre was measured manually using a peripapillary scan with SD-OCT (Heidelberg Engineering) at baseline and after seven weeks of IOP elevation. The animals were then sacrificed and the optic nerve was fixed with 30% glutaraldehyde and cross-sections stained with paraphenylene diamine to mark the vessels. Contralateral eyes served as controls. Pictures were taken and number of vessels, vessel calibre and area were calculated and compared. RESULTS: IOP was significantly elevated (p < 0.001). In optic nerve cross sections, the number of capillaries did not differ significantly between animals with elevated IOP and controls. However, vessel calibre and area were significantly reduced (p < 0.001) in glaucomatous optic nerves. The calibre of the retinal vessels was significantly lowered - by 9.22% (p = 0.021). CONCLUSION: Retinal arterioles and optic nerve capillaries respond sensitively to abnormal pressure elevation in vivo, showing high and early vulnerability. The vascular responses may influence secondary neuronal responses, which culminate in the death of ganglion cells and blindness, as occurs in clinical glaucoma.

[Endothelial Cell Reaction to Elevated Hydrostatic Pressure and Oxidative Stress in Vitro]. / Reaktion der Endothelzellen auf kurzzeitig physiologisch erhöhte hydrostatische Drücke und oxidativen Stress in vitro.

INTRODUCTION: Endothelial dysfunction has become a strongly discussed factor regarding glaucoma pathogenesis. In addition to peripapillary bleedings as signs of vascular damage, there is a definite correlation between glaucoma and vascular dysregulation syndrome. The aim of this study was to evaluate endothelial cell reaction to moderately elevated hydrostatic pressure and oxidative stress in vitro. METHODS: In vitro, primarily dissociated brain microvascular endothelial cells (BMECs) were exposed to moderately elevated hydrostatic pressure (60 and 120 mmHg) in a special pressure chamber. Additionally, cells primarily exposed to pressure, and cells not exposed to pressure, were incubated with low amounts of H2O2. A live/dead assay was performed to evaluate cell viability. Immunohistochemical staining against actin was used for morphological evaluation. RESULTS: Neither 60 nor 120 mmHg of elevated pressure had a viability changing effect on primary endothelial cells. Secondary, no big morphological changes could be discovered. However, against a low concentration of oxidative stress, BMECs showed high vulnerability. A difference in reaction to cells stressed with high pressure before could not be shown. CONCLUSION: Direct effects, in terms of higher vulnerability or morphological changes of moderately elevated high pressure on endothelial cells, could not be shown. However, the reaction to low amounts of oxidative stress indicates the involvement of endothelial cells in the pathogenesis of glaucoma and the special role of oxidative stress when referring to endothelial dysfunction in glaucomatous disease.

Age-related Beta-synuclein Alters the p53/Mdm2 Pathway and Induces the Apoptosis of Brain Microvascular Endothelial Cells In Vitro.

Increased ß-synuclein (Sncb) expression has been described in the aging visual system. Sncb functions as the physiological antagonist of α-synuclein (Snca), which is involved in the development of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases. However, the exact function of Sncb remains unknown. The aim of this study was to elucidate the age-dependent role of Sncb in brain microvascular endothelial cells (BMECs). BMECs were isolated from the cortices of 5- to 9-d-old Sprague-Dawley rats and were cultured with different concentrations of recombinant Sncb (rSncb) up to 72 h resembling to some degree age-related as well as pathophysiological conditions. Viability, apoptosis, expression levels of Snca, and the members of phospholipase D2 (Pld2)/ p53/ Mouse double minute 2 homolog (Mdm2)/p19(Arf) pathway, response in RAC-alpha serine/threonine-protein kinase (Akt), and stress-mediating factors such as heme oxygenase (decycling) 1 (Hmox) and Nicotinamide adenine dinucleotide phosphate oxygenase 4 (Nox4) were examined. rSncb-induced effects were confirmed through Sncb small interfering RNA (siRNA) knockdown in BMECs. We demonstrated that the viability decreases, while the rate of apoptosis underly dose-dependent alterations. For example, apoptosis increases in BMECs following the treatment with higher dosed rSncb. Furthermore, we observed a decrease in Snca immunostaining and messenger RNA (mRNA) levels following the exposure to higher rScnb concentrations. Akt was shown to be downregulated and pAkt upregulated by this treatment, which was accompanied by a dose-independent increase in p19(Arf) levels and enhanced intracellular Mdm2 translocation in contrast to a dose-dependent p53 activation. Moreover, Pld2 activity was shown to be induced in rSncb-treated BMECs. The expression of Hmox and Nox4 after Sncb treatment was altered on BEMCs. The obtained results demonstrate dose-dependent effects of Sncb on BMECs in vitro. For example, the p53-mediated and Akt-independent apoptosis together with the stress-mediated response of BMECs related to exposure of higher SNCB concentrations may reflect the increase in Sncb with duration of culture as well as its impact on cell decay. Further studies, expanding on the role of Sncb, may help understand its role in the neurodegenerative diseases.

[Elevated Intraocular Pressure Induces Cellular Responses in the Retinal Capillaries]. / Erhöhter Intraokulardruck induziert primär zelluläre Reaktionen in den retinalen Kapillaren.

Background In the early diagnosis of clinical glaucoma, peripapillary bleedings were almost pathognomonic for a capillary insult. In the perfusion diagnostics, it is predominantly accepted that perfusion imbalances and IOP-induced changes occur and play a crucial role. Biomechanical peculiarities of the optic nerve head and cellular responses to astrocytes are also likely involved. Material and Methods We present in vivo and ex vivo models of IOP-elevation to enhance the resolution of examining cellular and molecular changes and to understand the mechanisms of capillary changes due to IOP-elevation. Results The in vivo model consists of cauterization-caused elevation of IOP in rat eyes. Two to 3 veins were cauterized to increase outflow resistance. The retinas were analyzed several weeks later and we found an abnormal expression of the neuron-specific molecule beta-III-tubulin in the capillary endothelium cells and in the vascular pericytes. Combined immunohistochemical stainings with different markers for various retinal cells confirmed the findings. The isolation of capillary endothelium cells and pericytes from rat brains (BMECs) and retinas (RMECs), and their cultivation under elevated IOP in vitro, confirmed the in vivo results. Conclusion The unexpected capillary response to elevation of IOP in vivo and in vitro could be seen as an early response of cells with expression of abnormal proteins. This result may explain clinical observations which dominate as peripapillary bleedings or microinfarctions and are likely associated with the glaucoma-induced opticopathy.

Is Angiostatin Involved in Physiological Foveal Avascularity?

Purpose: The primate central retina is characterized by an avascular fovea and well-defined perifoveal capillary plexus. Neither blood vessels nor their accompanying astrocytes enter the fovea during any stage of retinal development; a balance of angiogenic and angiostatic factors probably maintains foveal avascularity throughout life. The aim of this study was to identify potentially angiorepulsive factors involved in the development of the avascular primate retinal fovea. Methods: Retinas of newborn, juvenile, and adult Callithrix jacchus and Macaca fascicularis monkeys and control human retinas were studied to determine the localization of angiostatin relative to III ß-tubulin, glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule-1 (PECAM), and the angiostatin receptor αvß3-integrin in the foveal, macular, and peripheral retina. Expression studies were performed using immunohistochemistry (IHC) on retinal whole-mount and paraffin sections, and Western blotting on frozen material. The complex network of the main retinal cell types was identified by IHC of retinal whole mounts. Results: In general, lifetime expression of angiostatin was found in all retinas. Colabeling with different markers revealed retinal ganglion cells as the main source of angiostatin expression in the primate retina, whereas PECAM-immunopositive blood capillaries expressed the angiostatin receptor αvß3-integrin, and capillary-associated astrocytes expressed VEGF. Conclusions: This study provides the first evidence of angiostatin expression in the primate retina; the expression of angiostatin in the avascular foveal region and the peripheral retina suggests that angiostatin may play a role in the regulation of retinal vascularization, providing a possible explanation for the development and persistence of an avascular fovea.

The pro-inflammatory role of high-mobility group box 1 protein (HMGB-1) in photoreceptors and retinal explants exposed to elevated pressure.

To determine the role of high-mobility group box 1 protein (HMGB-1) in cellular and tissue models of elevated pressure-induced neurodegeneration, regeneration, and inflammation. Mouse retinal photoreceptor-derived cells (661W) and retinal explants were incubated either under elevated pressure or in the presence of recombinant HMGB-1 (rHMGB-1) to investigate the mechanisms of response of photoreceptors. Immunohistochemistry, western blotting, and the quantitative real-time PCR were used to examine the expression levels of immunological factors (eg, HMGB-1, receptor for advanced glycation end products (RAGE)), Toll-like receptors 2 and 4 (TLR-2, TLR-4), apoptosis-related factors (eg, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad)) as well as cytokine expression (eg, tumor necrosis factor alpha (TNF-α), interleukin (IL)-4, IL-6, and vascular endothelial growth factor (VEGF)). The data revealed increased the expression of HMGB-1 and its receptors RAGE, TLR-2, and TLR-4, and TNF-α as well as pro-apoptotic factors (eg, Bad) as well as apoptosis in 661W cells exposed to elevated pressure. Co-cultivation of 661W cells with rHMGB-1 increased the expression levels of pro-apoptotic Bad and cleaved Caspase-3 resulting in apoptosis. Cytokine array studies revealed an increased release of TNF-α, IL-4, IL-6, and VEGF after incubation of 661W cells with rHMGB-1. Upregulation of HMGB-1, TLR-2, and RAGE as well as anti-apoptotic Bcl-2 expression levels was found in the retinal explants exposed to rHMGB-1 or elevated pressure. The results suggest that HMGB-1 promotes an inflammatory response and mediates apoptosis in the pathology of photoreceptors and retinal homeostasis. HMGB-1 may have a key role in ongoing damage of retinal cells under conditions of elevated intraocular pressure.