Publications by authors named "Holger Gerhardt"

130 Publications

A backward-mode optical-resolution photoacoustic microscope for 3D imaging using a planar Fabry-Pérot sensor.

Photoacoustics 2021 Dec 10;24:100293. Epub 2021 Aug 10.

Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, Von-danckelmann-platz 3, 06120, Halle (Saale), Germany.

Optical-resolution photoacoustic microscopy (OR-PAM) combines high spatial resolution and strong absorption-based contrast in tissue, which has enabled structural and spectroscopic imaging of endogenous chromophores, primarily hemoglobin. Conventional piezoelectric ultrasound transducers are typically placed far away from the photoacoustic source due to their opacity, which reduces acoustic sensitivity. Optical ultrasound sensors are an alternative as their transparency allows them to be positioned close to the sample with minimal source-detector distances. In this work, a backward-mode OR-PAM system based on a planar Fabry-Pérot ultrasound sensor and coaxially aligned excitation and interrogation beams was developed. Two 3D imaging modes, using raster-scanning for enhanced image quality and continuous-scanning for fast imaging, were implemented and tested on a leaf skeleton phantom. In fast imaging mode, a scan-rate of 100,000 A-lines/s was achieved. 3D images of a zebrafish embryo were acquired The transparency of the FP sensor in the visible and near-infrared wavelength region makes it suitable for combined functional and molecular imaging applications using OR-PAM and multi-photon fluorescence microscopy.
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http://dx.doi.org/10.1016/j.pacs.2021.100293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385441PMC
December 2021

Association between erythrocyte dynamics and vessel remodelling in developmental vascular networks.

J R Soc Interface 2021 06 23;18(179):20210113. Epub 2021 Jun 23.

Centre for Medical Informatics, Usher Institute, The University of Edinburgh, Edinburgh, UK.

Sprouting angiogenesis is an essential vascularization mechanism consisting of sprouting and remodelling. The remodelling phase is driven by rearrangements of endothelial cells (ECs) within the post-sprouting vascular plexus. Prior work has uncovered how ECs polarize and migrate in response to flow-induced wall shear stress (WSS). However, the question of how the presence of erythrocytes (widely known as red blood cells (RBCs)) and their impact on haemodynamics affect vascular remodelling remains unanswered. Here, we devise a computational framework to model cellular blood flow in developmental mouse retina. We demonstrate a previously unreported highly heterogeneous distribution of RBCs in primitive vasculature. Furthermore, we report a strong association between vessel regression and RBC hypoperfusion, and identify plasma skimming as the driving mechanism. Live imaging in a developmental zebrafish model confirms this association. Taken together, our results indicate that RBC dynamics are fundamental to establishing the regional WSS differences driving vascular remodelling via their ability to modulate effective viscosity.
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http://dx.doi.org/10.1098/rsif.2021.0113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220266PMC
June 2021

Remodeling of an microvessel exposed to cyclic mechanical stretch.

APL Bioeng 2021 Jun 2;5(2):026102. Epub 2021 Apr 2.

Integrative Vascular Biology Laboratory, Max-Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC),13092 Berlin, Germany.

In the lungs, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological responses remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lungs and thus assumed to be the same as the one acting on the alveolar epithelium. Existing models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature . We studied the effects of the cyclic strain on a perfusable 3D vasculature, made of either human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The 3D vessels underwent both -like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with the 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however, we observed several phenomena in the 3D model that the 2D model was unable to predict. Angiogenic sprouting induced by VEGF decreased significantly in the presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast to our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer, which truly recapitulates the situation.
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http://dx.doi.org/10.1063/5.0010159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019357PMC
June 2021

Endothelial Cell Orientation and Polarity Are Controlled by Shear Stress and VEGF Through Distinct Signaling Pathways.

Front Physiol 2020 2;11:623769. Epub 2021 Mar 2.

Integrative Vascular Biology Laboratory, Max Delbruck Center for Molecular Medicine, Berlin, Germany.

Vascular networks form, remodel and mature under the influence of multiple signals of mechanical or chemical nature. How endothelial cells read and interpret these signals, and how they integrate information when they are exposed to both simultaneously is poorly understood. Here, we show using flow-induced shear stress and VEGF-A treatment on endothelial cells , that the response to the magnitude of a mechanical stimulus is influenced by the concentration of a chemical stimulus, and vice versa. By combining different flow levels and different VEGF-A concentrations, front-rear polarity of endothelial cells against the flow direction was established in a flow and VEGF-A dose-response while their alignment with the flow displayed a biphasic response depending on the VEGF-A dose (perpendicular at physiological dose, aligned at no or pathological dose of VEGF-A). The effect of pharmaceutical inhibitors demonstrated that while VEGFR2 is essential for both polarity and orientation establishment in response to flow with and without VEGF-A, different downstream effectors were engaged depending on the presence of VEGF-A. Thus, Src family inhibition (c-Src, Yes, Fyn together) impaired alignment and polarity without VEGF-A while FAK inhibition modified polarity and alignment only when endothelial cells were exposed to VEGF-A. Studying endothelial cells in the aortas of VEGFR2 mutant mice and SRC mice confirmed the role of VEGFR2 and specified the role of c-SRC . Endothelial cells of VEGFR2 mutant mice lost their polarity and alignment while endothelial cells from SRC mice only showed reduced polarity. We propose here that VEGFR2 is a sensor able to integrate chemical and mechanical information simultaneously and that the underlying pathways and mechanisms activated will depend on the co-stimulation. Flow alone shifts VEGFR2 signaling toward a Src family pathway activation and a junctional effect (both and ) while flow and VEGF-A together shift VEGFR2 signaling toward focal adhesion activation () both modifying cell responses that govern orientation and polarity.
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http://dx.doi.org/10.3389/fphys.2020.623769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960671PMC
March 2021

Vasohibin 1 selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk.

Development 2021 02 19;148(4). Epub 2021 Feb 19.

Integrative Vascular Biology Laboratory, Max-Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rössle-Strasse 10, Berlin 13125, Germany

Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.
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http://dx.doi.org/10.1242/dev.194993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904002PMC
February 2021

On the preservation of vessel bifurcations during flow-mediated angiogenic remodelling.

PLoS Comput Biol 2021 02 4;17(2):e1007715. Epub 2021 Feb 4.

Centre for Medical Informatics, Usher Institute, The University of Edinburgh, Edinburgh, United Kingdom.

During developmental angiogenesis, endothelial cells respond to shear stress by migrating and remodelling the initially hyperbranched plexus, removing certain vessels whilst maintaining others. In this study, we argue that the key regulator of vessel preservation is cell decision behaviour at bifurcations. At flow-convergent bifurcations where migration paths diverge, cells must finely tune migration along both possible paths if the bifurcation is to persist. Experiments have demonstrated that disrupting the cells' ability to sense shear or the junction forces transmitted between cells impacts the preservation of bifurcations during the remodelling process. However, how these migratory cues integrate during cell decision making remains poorly understood. Therefore, we present the first agent-based model of endothelial cell flow-mediated migration suitable for interrogating the mechanisms behind bifurcation stability. The model simulates flow in a bifurcated vessel network composed of agents representing endothelial cells arranged into a lumen which migrate against flow. Upon approaching a bifurcation where more than one migration path exists, agents refer to a stochastic bifurcation rule which models the decision cells make as a combination of flow-based and collective-based migratory cues. With this rule, cells favour branches with relatively larger shear stress or cell number. We found that cells must integrate both cues nearly equally to maximise bifurcation stability. In simulations with stable bifurcations, we found competitive oscillations between flow and collective cues, and simulations that lost the bifurcation were unable to maintain these oscillations. The competition between these two cues is haemodynamic in origin, and demonstrates that a natural defence against bifurcation loss during remodelling exists: as vessel lumens narrow due to cell efflux, resistance to flow and shear stress increases, attracting new cells to enter and rescue the vessel from regression. Our work provides theoretical insight into the role of junction force transmission has in stabilising vasculature during remodelling and as an emergent mechanism to avoid functional shunting.
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http://dx.doi.org/10.1371/journal.pcbi.1007715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909651PMC
February 2021

Astrocyte-derived Wnt growth factors are required for endothelial blood-brain barrier maintenance.

Prog Neurobiol 2021 04 28;199:101937. Epub 2020 Dec 28.

Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt Am Main, Germany; Excellence Cluster Cardio-Pulmonary Systems (ECCPS), Partner Site Frankfurt, Germany; German Center for Cardiovascular Research (DZHK), Partner Site, Frankfurt/Mainz, Frankfurt, Germany. Electronic address:

Maintenance of the endothelial blood-brain-barrier (BBB) through Wnt/β-catenin signalling is essential for neuronal function. The cells however, providing Wnt growth factors at the adult neurovascular unit (NVU) are poorly explored. Here we show by conditionally knocking out the evenness interrupted (Evi) gene in astrocytes (Evi) that astrocytic Wnt release is crucial for BBB and NVU integrity. Evi mice developed brain oedema and increased vascular tracer leakage. While brain vascularization and endothelial junctions were not altered in 10 and 40 week-old mice, endothelial caveolin(Cav)-1-mediated vesicle formation was increased in vivo and in vitro. Moreover, astrocytic end-feet were swollen, and aquaporin-4 distribution was disturbed, coinciding with decreased astrocytic Wnt activity. Vascular permeability correlated with increased neuronal activation by c-fos staining, indicative of altered neuronal function. Astrocyte-derived Wnts thus serve to maintain Wnt/β-catenin activity in endothelia and in astrocytes, thereby controlling Cav-1 expression, vesicular abundance, and end-feet integrity at the NVU.
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http://dx.doi.org/10.1016/j.pneurobio.2020.101937DOI Listing
April 2021

Do Disadvantageous Social Contexts Influence Food Choice? Evidence From Three Laboratory Experiments.

Front Psychol 2020 6;11:575170. Epub 2020 Nov 6.

Center for Economics and Neuroscience, University of Bonn, Bonn, Germany.

Increasing rates of obesity have fueled interest in the factors underlying food choice. While epidemiological studies report that disadvantaged social groups exhibit a higher incidence of obesity, causal evidence for an effect of social contexts on food choice remains scarce. To further our knowledge, we experimentally investigated the effect of disadvantageous social context on food choice in healthy, non-dieting participants. We used three established experimental methods to generate social contexts of different valence in controlled laboratory settings: (i) receiving varying amounts of money in a Dictator Game (DG; = 40), (ii) being included or excluded in a Cyberball Game (CBG; = 35), and (iii) performing well, average, or poorly in a response time ranking task (RTR; = 81). Following exposure to a particular social context, participants made pairwise choices between food items that involved a conflict between perceived taste and health attributes. In line with previous research, stronger dispositional self-control (assessed via a questionnaire) was associated with healthier food choices. As expected, being treated unfairly in the DG, being excluded in the CBG, and performing poorly in the RTR led to negative emotions. However, we did not find an effect of the induced social context on food choice in any of the experiments, even when taking into account individual differences in participants' responses to the social context. Our results suggest that-at least in controlled laboratory environments-the influence of disadvantageous social contexts on food choice is limited.
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http://dx.doi.org/10.3389/fpsyg.2020.575170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677191PMC
November 2020

Long-lived tumor-associated macrophages in glioma.

Neurooncol Adv 2020 Jan-Dec;2(1):vdaa127. Epub 2020 Sep 25.

Department: Cardiovascular and metabolic diseases, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.

Background: The tumor microenvironment plays a major tumor-supportive role in glioma. In particular, tumor-associated macrophages (TAMs), which can make up to one-third of the tumor mass, actively support tumor growth, invasion, and angiogenesis. Predominantly alternatively activated (M2-polarized) TAMs are found in late-stage glioma in both human and mouse tumors, as well as in relapse samples from patients. However, whether tumor-educated M2 TAMs can actively contribute to the emergence and growth of relapse is currently debated.

Methods: To investigate whether tumor-educated stromal cells remaining in the brain after surgical removal of the primary tumor can be long-lived and retain their tumor-supporting function, we developed a transplantation mouse model and performed lineage-tracing.

Results: We discovered that macrophages can survive transplantation and stay present in the tumor much longer than previously suggested, while sustaining an M2-polarized protumorigenic phenotype. Transplanted tumors showed a more aggressive growth and faster polarization of the TAMs toward an M2 phenotype compared with primary tumors, a process dependent on the presence of few cotransplanted macrophages.

Conclusions: Overall, we propose a new way for tumor-educated TAMs to contribute to glioma aggressiveness by long survival and stable protumorigenic features. These properties could have a relapse-supporting effect.
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http://dx.doi.org/10.1093/noajnl/vdaa127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649962PMC
September 2020

Intron with transgenic marker (InTraM) facilitates high-throughput screening of endogenous gene reporter lines.

Genesis 2020 11 11;58(10-11):e23391. Epub 2020 Aug 11.

Integrative Vascular Biology Lab, Max-Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.

The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.
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http://dx.doi.org/10.1002/dvg.23391DOI Listing
November 2020

Fear-induced increases in loss aversion are linked to increased neural negative-value coding.

Soc Cogn Affect Neurosci 2020 07;15(6):661-670

Department of Education and Psychology, Freie Universität Berlin, 14195 Berlin, Germany.

Human decisions are often influenced by emotions. An economically relevant example is the role of fear in generating loss aversion. Previous research implicates the amygdala as a key brain structure in the experience of fear and loss aversion. The neural mechanism behind emotional influences on loss aversion is, however, unclear. To address this, we measured brain activation with functional magnetic resonance imaging (fMRI) while participants made decisions about monetary gambles after viewing fearful or neutral faces. We observed that loss aversion following the presentation of neutral faces was mainly predicted by greater deactivations for prospective losses (relative to activations for prospective gains) in several brain regions, including the amygdala. By contrast, increases in loss aversion following the presentation of fearful faces were mainly predicted by greater activations for prospective losses. These findings suggest a fear-induced shift from positive to negative value coding that reflects a context-dependent involvement of distinct valuation processes.
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http://dx.doi.org/10.1093/scan/nsaa091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438956PMC
July 2020

Opposite Macrophage Polarization in Different Subsets of Ovarian Cancer: Observation from a Pilot Study.

Cells 2020 01 27;9(2). Epub 2020 Jan 27.

ImmunOvar Research Group, Laboratory of Tumor Immunology and Immunotherapy, Department of Oncology, KU Leuven, 3000 Leuven, Belgium.

The role of the innate immune system in ovarian cancer is gaining importance. The relevance of tumor-associated macrophages (TAM) is insufficiently understood. In this pilot project, comprising the immunofluorescent staining of 30 biopsies taken from 24 patients with ovarian cancer, we evaluated the presence of total TAM (cluster of differentiation (CD) 68 expression), M1 (major histocompatibility complex (MHC) II expression), and M2 (anti-mannose receptor C type 1 (MRC1) expression), and the blood vessel diameter. We observed a high M1/M2 ratio in low-grade ovarian cancer compared to high-grade tumors, more total TAM and M2 in metastatic biopsies, and a further increase in total TAM and M2 at interval debulking, without beneficial effects of bevacizumab. The blood vessel diameter was indicative for M2 tumor infiltration (Spearman correlation coefficient of 0.65). These data mainly reveal an immune beneficial environment in low-grade ovarian cancer in contrast to high-grade serous ovarian cancer, where immune suppression is not altered by neoadjuvant therapy.
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http://dx.doi.org/10.3390/cells9020305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072171PMC
January 2020

Lymphoma Angiogenesis Is Orchestrated by Noncanonical Signaling Pathways.

Cancer Res 2020 03 13;80(6):1316-1329. Epub 2020 Jan 13.

Translational Tumorimmunology, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed -driven B-cell lymphoma-induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis. SIGNIFICANCE: In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade. http://cancerres.aacrjournals.org/content/canres/80/6/1316/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1493DOI Listing
March 2020

Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1.

Elife 2019 10 3;8. Epub 2019 Oct 3.

Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven, Belgium.

The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy.
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http://dx.doi.org/10.7554/eLife.46380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797479PMC
October 2019

Artery-vein specification in the zebrafish trunk is pre-patterned by heterogeneous Notch activity and balanced by flow-mediated fine-tuning.

Development 2019 08 27;146(16). Epub 2019 Aug 27.

Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven B-3000, Belgium

How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep 'count'? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.
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http://dx.doi.org/10.1242/dev.181024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737902PMC
August 2019

ATTRACT.

Circ Res 2019 07 18;125(3):262-264. Epub 2019 Jul 18.

From the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany (P.B.G., H.G.).

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http://dx.doi.org/10.1161/CIRCRESAHA.119.315198DOI Listing
July 2019

Myeloid Derived Suppressor Cells: Key Drivers of Immunosuppression in Ovarian Cancer.

Front Immunol 2019 4;10:1273. Epub 2019 Jun 4.

ImmunOvar Research Group, Laboratory of Tumor Immunology and Immunotherapy, Department of Oncology, KU Leuven, Leuven, Belgium.

The presence of tumor infiltrating lymphocytes (TILs) is associated with a longer overall survival in advanced stage epithelial ovarian cancer. Despite the prognostic impact of TILs, response to checkpoint-inhibitors and antigen-specific active immunotherapy is limited in ovarian cancer. The goal of our study was to investigate the interaction between ovarian cancer and the innate and adaptive immune system in the ID8-fLuc syngeneic ovarian cancer mouse model. For the experiments C57BL/6, B6.129S7-Rag1/J, and B6.129P2(SJL)-Myd88/J mice were inoculated with ID8-fLuc. depletion experiments were performed using clodronate liposomes (CL), anti-CD8a, anti-GR1, anti-colony stimulating factor 1 (anti-CSF1), and TMβ1 (anti-CD122). Immune read out was performed by fluorescent activated cell sorting analysis for effector T cells, regulatory T cells, natural killer cells, B cells, macrophages, and myeloid derived suppressor cells (MDSC), immunohistochemistry for MDSC and tumor-associated macrophages (TAM) and immunofluorescence for M1 and M2 TAM in the vascular context. The effect of MDSC on T cell proliferation and phenotype were studied . We discovered that the absence of T and B cells did not influence tumor growth or survival of B6.129S7-Rag1/J mice compared to immunocompetent C57BL/6 mice. CL-induced macrophage depletion promoted tumor proliferation and shortened survival in C57BL/6 mice ( = 0.004) and in B6.129S7-Rag1/J mice ( = 0.0005). During CL treatment, we observed a clear increase of pro-inflammatory cytokines ( ≤ 0.02) and monocytic MDSC ( ≤ 0.01). Selective depletion of MDSC by anti-GR1 improved survival, certainly in comparison to mice treated with anti-CSF1 ( = 0.01-median survival 91 vs. 67.5 days). B6.129P2(SJL)-Myd88/J mice displayed to a longer median survival compared to C57BL/6 mice (90 vs. 76 days). MDSC activated by ID8-fLuc conditioned medium or ascites of tumor-bearing mice showed T cell suppressive functions . Based on these findings, we conclude that the adaptive immune system does not efficiently control tumor growth in the ID8-fLuc model. In addition, we discovered a prominent role for MDSC as the driver of immunosuppression in the ID8-fLuc ovarian cancer mouse model.
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http://dx.doi.org/10.3389/fimmu.2019.01273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558014PMC
October 2020

GPIHBP1 expression in gliomas promotes utilization of lipoprotein-derived nutrients.

Elife 2019 06 6;8. Epub 2019 Jun 6.

Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States.

GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) within the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1-bound LPL is essential for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. In peripheral tissues, the intravascular processing of TRLs by the GPIHBP1-LPL complex is crucial for the generation of lipid nutrients for adjacent parenchymal cells. GPIHBP1 is absent from the capillaries of the brain, which uses glucose for fuel; however, GPIHBP1 is expressed in the capillaries of mouse and human gliomas. Importantly, the GPIHBP1 in glioma capillaries captures locally produced LPL. We use NanoSIMS imaging to show that TRLs marginate along glioma capillaries and that there is uptake of TRL-derived lipid nutrients by surrounding glioma cells. Thus, GPIHBP1 expression in gliomas facilitates TRL processing and provides a source of lipid nutrients for glioma cells.
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http://dx.doi.org/10.7554/eLife.47178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594755PMC
June 2019

Endothelial Calcineurin Signaling Restrains Metastatic Outgrowth by Regulating Bmp2.

Cell Rep 2019 01;26(5):1227-1241.e6

Department of Oncology, University of Lausanne, Lausanne 1011, Switzerland; Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland; Division of Experimental Pathology, CHUV, Epalinges 1066, Switzerland; Swiss Institute for Cancer Research, École Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland. Electronic address:

Calcineurin/NFAT signaling is active in endothelial cells and is proposed to be an essential component of the tumor angiogenic response. Here, we investigated the role of endothelial calcineurin signaling in vivo in physiological and pathological angiogenesis and tumor metastasis. We show that this pathway is dispensable for retinal and tumor angiogenesis, but it is implicated in vessel stabilization. While ablation of endothelial calcineurin does not affect the progression of primary tumors or tumor cell extravasation, it does potentiate the outgrowth of lung metastases. We identify Bmp2 as a downstream target of the calcineurin/NFAT pathway in lung endothelium, potently inhibiting cancer cell growth by stimulating differentiation. We reveal a dual role of calcineurin/NFAT signaling in vascular regression or stabilization and in the tissue-specific production of an angiocrine factor restraining cancer cell outgrowth. Our results suggest that, besides targeting the immune system, post-transplantation immunosuppressive therapy with calcineurin inhibitors directly targets the endothelium, contributing to aggressive cancer progression.
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http://dx.doi.org/10.1016/j.celrep.2019.01.016DOI Listing
January 2019

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility.

Nat Commun 2018 11 16;9(1):4826. Epub 2018 Nov 16.

Vascular Signalling Laboratory, ProCURE, Oncobell Program, Institut d´Investigació Biomèdica de Bellvitge (IDIBELL), Gran Via de l'Hospitalet 199, 08908, L´Hospitalet de Llobregat, Barcelona, Spain.

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.
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http://dx.doi.org/10.1038/s41467-018-07172-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240100PMC
November 2018

Imaging Glioma Progression by Intravital Microscopy.

Methods Mol Biol 2019 ;1862:227-243

VIB-KU Leuven Center for Cancer Biology (CCB), Leuven, Belgium.

We describe here a method for generating mouse orthotopic gliomas in order to follow their progression over time by multi-photon laser scanning microscopy. After craniotomy of the parietal bone, glioma cells are implanted in the brain cortex and a glass window is cemented atop, allowing chronical imaging of the tumor. The expression of different fluorescent proteins in tumor cells and in specific cell types of a number of currently available transgenic mouse strains allows obtaining multicolor 3D images of the tumor over time. This technique is suitable both to evaluate the effect of pharmacological treatments and to unravel basic mechanisms of tumor-host interactions.
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http://dx.doi.org/10.1007/978-1-4939-8769-6_16DOI Listing
June 2019

Imaging of Endothelial Cell Dynamic Behavior in Zebrafish.

Methods Mol Biol 2018 ;1846:181-195

Integrative Vascular Biology Laboratory, Max-Delbrück Center for Molecular Medicine (MDC), Berlin, Germany.

In recent years, use of the zebrafish embryo as a model organism to study vascular development in vivo has provided valuable insights into the genetic and cellular events shaping the embryonic vasculature. In this chapter, we aim to present the methods for the measurement of some of the most commonly investigated dynamic parameters in endothelial cells during developmental angiogenesis, namely, migration speed and acceleration, filopodia extension, front-rear polarity, cell cycle progression, membrane deformations, and junctional rearrangements. We also offer suggestions on how to deal with the most common imaging and quantifications challenges faced when acquiring and quantifying endothelial cell dynamic behavior in vivo.We intend this section to serve as an experience-based imaging primer for scientists interested in endothelial cell imaging in the zebrafish embryo.
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http://dx.doi.org/10.1007/978-1-4939-8712-2_12DOI Listing
May 2019

NanoSIMS imaging reveals unexpected heterogeneity in nutrient uptake by brown adipocytes.

Biochem Biophys Res Commun 2018 10 15;504(4):899-902. Epub 2018 Sep 15.

Departments of Medicine, University of California, Los Angeles, CA, 90095, USA; School of Molecular Sciences and Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth, 6009, Australia. Electronic address:

Heterogeneity in the metabolic properties of adipocytes in white adipose tissue has been well documented. We sought to investigate metabolic heterogeneity in adipocytes of brown adipose tissue (BAT), focusing on heterogeneity in nutrient uptake. To explore the possibility of metabolic heterogeneity in brown adipocytes, we used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify uptake of lipids in adipocytes interscapular BAT and perivascular adipose tissue (PVAT) after an intravenous injection of triglyceride-rich lipoproteins (TRLs) containing [H]triglycerides (H-TRLs). The uptake of deuterated lipids into brown adipocytes was quantified by NanoSIMS. We also examined C enrichment in brown adipocytes after administering [C]glucose or C-labeled mixed fatty acids by gastric gavage. The uptake of H-TRLs-derived lipids into brown adipocytes was heterogeneous, with H enrichment in adjacent adipocytes varying by more than fourfold. We also observed substantial heterogeneity in C enrichment in adjacent brown adipocytes after administering [C]glucose or [C]fatty acids by gastric gavage. The uptake of nutrients by adjacent brown adipocytes within a single depot is variable, suggesting that there is heterogeneity in the metabolic properties of brown adipocytes.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476570PMC
October 2018

Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs.

Cell 2018 09 6;175(1):239-253.e17. Epub 2018 Sep 6.

Proteome Dynamics, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rössle-Str. 10, 13125 Berlin, Germany; Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany. Electronic address:

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."
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http://dx.doi.org/10.1016/j.cell.2018.08.019DOI Listing
September 2018

PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow.

EMBO Rep 2018 09 17;19(9). Epub 2018 Jul 17.

Laboratory for Cell Polarity and Organogenesis, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3β activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3β complexes controls local GSK3β activity and thereby regulates endothelial planar polarity. The spatial information for GSK3β activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.
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http://dx.doi.org/10.15252/embr.201745253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123654PMC
September 2018

PolNet: A Tool to Quantify Network-Level Cell Polarity and Blood Flow in Vascular Remodeling.

Biophys J 2018 05;114(9):2052-2058

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. Electronic address:

In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.
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http://dx.doi.org/10.1016/j.bpj.2018.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961748PMC
May 2018

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression.

J Cell Biol 2018 05 2;217(5):1651-1665. Epub 2018 Mar 2.

Max Delbrück Center for Molecular Medicine, Berlin, Germany

Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress-mediated remodeling.
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http://dx.doi.org/10.1083/jcb.201706151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940299PMC
May 2018

YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development.

Elife 2018 02 5;7. Epub 2018 Feb 5.

Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.
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http://dx.doi.org/10.7554/eLife.31037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814147PMC
February 2018

Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth.

EMBO Mol Med 2017 12;9(12):1629-1645

Vascular Patterning Lab, Center for Cancer Biology, VIB, Leuven, Belgium

Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences.
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http://dx.doi.org/10.15252/emmm.201607445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709745PMC
December 2017
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