Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Insight into the molecular and cellular processes in blood-retinal barrier (BRB) development, including the contribution of paracellular and transcellular pathways, is still incomplete but may help to understand the inverse process of BRB loss in pathologic eye conditions. In this comprehensive observational study, we describe in detail the formation of the BRB at the molecular level in physiologic conditions, using mice from postnatal day (P)3 to P25. Our data indicate that immature blood vessels already have tight junctions at P5, before the formation of a functional BRB. Expression of the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), which is known to be involved in transcellular transport and associated with BRB permeability, decreased during development and was absent when a functional barrier was formed. Moreover, we show that PLVAP deficiency causes a transient delay in retinal vascular development and changes in mRNA expression levels of endothelial permeability pathway proteins.-Van der Wijk, A.-E., Wisniewska-Kruk, J., Vogels, I. M. C., van Veen, H. A., Ip, W. F., van der Wel, N. N., van Noorden, C. J. F., Schlingemann, R. O., Klaassen, I. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Klebsiella pneumoniae-Induced Pneumonia-Derived Sepsis.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

Plasmalemma Vesicle-Associated Protein Has a Key Role in Blood-Retinal Barrier Loss.

Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle-associated protein (PLVAP) in retinal capillaries associates with loss of BRB properties and correlates with increased vascular permeability in diabetic macular edema. In this study, we investigated whether absence of PLVAP protects the BRB from VEGF-induced permeability. We used lentiviral-delivered shRNA or siRNA to inhibit PLVAP expression. The barrier properties of in vitro BRB models were assessed by measuring transendothelial electrical resistance, permeability of differently sized tracers, and the presence of endothelial junction complexes. The effect of VEGF on caveolae formation was studied in human retinal explants. BRB loss in vivo was studied in the mouse oxygen-induced retinopathy model. The inhibition of PLVAP expression resulted in decreased VEGF-induced BRB permeability of fluorescent tracers, both in vivo and in vitro. PLVAP inhibition attenuated transendothelial electrical resistance reduction induced by VEGF in BRB models in vitro and significantly increased transendothelial electrical resistance of the nonbarrier human umbilical vein endothelial cells. Furthermore, PLVAP knockdown prevented VEGF-induced caveolae formation in retinal explants but did not rescue VEGF-induced alterations in endothelial junction complexes. In conclusion, PLVAP is an essential cofactor in VEGF-induced BRB permeability and may become an interesting novel target for diabetic macular edema therapy.

Bacillus subtilis Spore Inner Membrane Proteome.

The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.

Ultrastructural aspects of foreign body giant cells generated on different substrates.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.

Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms.

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout (Thbs4(-/-)) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4(-/-) mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4(-/-) mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4(-/-) mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4(-/-) mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides.

Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntington's disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

Hypersensitivity to acid is associated with impaired esophageal mucosal integrity in patients with gastroesophageal reflux disease with and without esophagitis.

Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus is associated with the expression of filaggrin.

Aminopeptidase-resistant peptides are targeted to lysosomes and subsequently degraded.

Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of the proteasome and the effect of its impairment on aggregation have been intensively studied, little is known about how cells deal with peptides that show resistance to degradation by aminopeptidases. Here, we introduced peptidase-resistant peptides into living cells and show that these peptides rapidly and irreversibly accumulate into puncta in the perinuclear region of the cell. Accumulation appears to be independent of peptide sequence but is less efficient for longer peptides. The puncta colocalize with autophagosomal and lysosomal markers, suggesting that these peptides end up within lysosomes via macroautophagy. Surprisingly, the peptides still accumulate within lysosomes when macroautophagy is impaired, suggesting a trafficking route independent of macroautophagy. Upon lysosomal uptake, peptides are degraded, suggesting that cells can clear peptidase-resistant proteasomal products by an alternative pathway, which targets them to lysosomes.

Heparin blocks transfer of extracellular vesicles between donor and recipient cells.

Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a role.

Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity.

Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

An ultrasoft X-ray multi-microbeam irradiation system for studies of DNA damage responses by fixed- and live-cell fluorescence microscopy.

Localized induction of DNA damage is a valuable tool for studying cellular DNA damage responses. In recent decades, methods have been developed to generate DNA damage using radiation of various types, including photons and charged particles. Here we describe a simple ultrasoft X-ray multi-microbeam system for high dose-rate, localized induction of DNA strand breaks in cells at spatially and geometrically adjustable sites. Our system can be combined with fixed- and live-cell microscopy to study responses of cells to DNA damage.

New Generation of Mesoporous Silica Membranes Prepared by a Stöber-Solution Pore-Growth Approach.

Membranes consisting of uniform and vertically organized mesopores are promising systems for molecular filtration because of the possibility to combine high-flux and high-rejection properties. In this work, a new generation of mesoporous silica membranes (MSMs) have been developed, in which an organized mesoporous layer is directly formed on top of a porous ceramic support via a Stöber-solution pore-growth approach. Relevant characterization methods have been used to demonstrate the growth of the membrane separation layer and the effect of reaction time and the concentration of the reactants on the microstructure of the membrane. Compared to previous studies using the evaporation-induced self-assembly method to prepare MSMs, an important increase in water permeability was observed (from 1.0 to at least 3.8 L m-2 h-1 bar-1), indicating an improved pore alignment. The water permeability, cyclohexane permporometry tests, and molecular cut-off measurements (MWCO ≈ 2300 Da) were consistent with membranes composed of 2-3 nm accessible pores.

The Challenge of Teaching Essential Immunology Laboratory Skills to Undergraduates in One Month-Experience of an Osteoimmunology Course on TLR Activation.

Acquiring immunology laboratory skills during undergraduate studies is often a prerequisite for admission to Masters' programs. Many broad liberal arts and sciences honors degree colleges struggle in teaching these essentials since only limited time is usually reserved for this. Here, we describe a new 1-month-course developed to train a small group of honors students in 6 techniques that are useful for immunology research. In essence, 15 students were divided into 3 groups of 5 students where each student became involved in current osteoimmunology research. Osteoimmunology is a relatively new branch of the immunology tree, where the effects of inflammation and the immune system on bone formation and bone degradation is studied. A broad, 3 weeks experiment on the chronic effects of molecules that specifically activate toll-like receptors TLR2 and TLR4 on bone formation or osteoclast differentiation was performed just before the start of the course. Control samples and samples treated with TLR2 (group A), TLR4 (group B), or TLR2+TLR4 (group C) agonists were harvested and analyzed using quantitative PCR, ELISA, biochemistry, microscopy of enzyme-histochemically stained osteoclasts, scanning electron microscopy, and confocal microscopy. Each technique was taught for 2 days by a specialized instructor, who was present at all laboratory activities. The primary research question for each group was: how does the experimental condition affect bone formation or osteoclast formation? The secondary research question specified per technique was: how does this technique answer part of the primary research question? Pedagogically, students were encouraged to collaborate within the group to analyze the obtained data. Secondly, at the end of the course, a representative of each group collaborated to summarize the TLR activation modalities of a technique of choice. Thirdly, each group wrote a report, where introduction and discussion were graded as a group; each technique part was graded individually. The summary of the results from the 3 treatment modalities was presented orally. The student evaluation of the course was high, students remarked that the course had a curriculum overarching function, since it created an awareness and appreciation for both the joy and the blood-sweat-and-tears aspects of pipetting, and writing research articles, making interpretation of those easier.

Nanoparticle-Aided Characterization of Arterial Endothelial Architecture during Atherosclerosis Progression and Metabolic Therapy.

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in Apoe-/- mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated in vitro. Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis.

Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response.

BACKGROUND: Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI). METHODS: To elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays. Physiological analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP). RESULTS: The transcriptome analysis showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiological observations that TC19, TC84 and BP2 perturb the membrane of B. subtilis. Using B. subtilis mutants, we established that the cell wall stress response is due to the delocalization of essential membrane bound proteins involved in cell wall synthesis. Other essential membrane proteins, involved in cell membrane synthesis and metabolism, were also delocalized due to alterations caused by the AMPs. CONCLUSIONS: We showed that peptides TC19, TC84 and BP2 perturb the membrane causing essential proteins to delocalize, thus preventing the possible repair of the cell envelope after the initial interference with the membrane. GENERAL SIGNIFICANCE: These AMPs show potential for eventual clinical application against Gram-positive bacterial cells and merit further application-oriented investigation.

Spatial and temporal recruitment of the neurovascular unit during development of the mouse blood-retinal barrier.

The inner blood-retinal barrier (BRB) is made up by the neurovascular unit, consisting of endothelial cells, pericytes and glial cells. The BRB maintains homeostasis of the neural retina, but in pathological eye conditions the neurovascular unit is often disrupted, causing BRB loss. Here, we investigated in detail temporal and spatial recruitment of the neurovascular unit in the neonatal mouse retina from postnatal day (P)3 to P25 employing immunohistochemical staining of vascular endothelium (isolectin B4), pericytes (α-SMA and NG2) and astrocytes (GFAP). In addition, we investigated gene expression of polarized astrocytic end-feet markers aquaporin-4 and laminin α2 chain with qPCR. We observed GFAP-positive cells migrating ahead of the retinal vasculature during the first postnatal week, suggesting that the retinal vasculature follows an astrocytic meshwork. From P9 onwards, astrocytes acquired a mature phenotype, with a more stellate shape and increased expression of aquaporin-4. NG2-positive cells and tip cells co-localized at P5 and invaded the retina together as a vascular sprouting front. In summary, these data suggest that recruitment of the cell types of the neurovascular unit is a prerequisite for proper retinal vascularization and BRB formation.

Thrombospondin-4 mediates cardiovascular remodelling in angiotensin II-induced hypertension.

Thrombospondin 4 (TSP-4) expression is induced in the heart and vasculature under pathological conditions, including myocardial infarction, myocardial pressure overload, and hypertension. TSP-4 is linked to remodelling processes, where it may affect extracellular matrix protein organization. In previous work, we studied the role of TSP-4 in small arteries during hypertension using Ang II-treated Thrombospondin 4 knockout (Thbs4-/-) mice. We reported increased heart weight, as well as the occurrence of aortic aneurysms in the Ang II-treated Thbs4-/- animals. In the present study, we further characterized the hearts and aortas from these animals. Hypertrophy of cardiomyocytes, together with perivascular fibrosis and inflammation was observed in the Ang II-treated Thbs4-/- hearts. In the aortas, an increase in the aortic wall cross-sectional area (CSA) and wall thickness of the Ang II-treated Thbs4-/- mice was found. More detailed investigation of the Ang II-treated Thbs4-/- aortas also revealed the appearance of aortic dissections in the outer medial layer of the arteries, as well as pronounced inflammation. No differences were found in several other extracellular matrix-related parameters, such as number of elastin breaks or stress-strain relationships. However, at the ultrastructural level, collagen fibers showed alterations in diameter in the media and adventitia of the Ang II-treated Thbs4-/- mice, in the area prone to dissection. In conclusion, we identified TSP-4 as an important protein in the development of cardiac hypertrophy and aortic dissections in Ang II-induced hypertension.