High resolution imaging of human development: shedding light on contrast agents.

BACKGROUND: Visualizing (micro)vascular structures remains challenging for researchers and clinicians due to limitations in traditional radiological imaging methods. Exploring the role of vascular development in craniofacial malformations in experimental settings can enhance understanding of these processes, with the effectiveness of high-resolution imaging techniques being crucial for successful research in this field. Micro-CT imaging offers 3D microstructural insights, but requires contrast-enhancing staining agents (CESAs) for visualizing (micro)-vascular tissues, known as contrast-enhanced micro-CT (CECT). As effective contrast agents are crucial for optimal visualization, this review focuses on comparative studies investigating such agents for micro-vascular tissue imaging using micro-CT. Furthermore, we demonstrate the utilization of B-Lugol solution as a promising contrast agent for acquiring high-quality micro-CT images of (micro)vascular structures in human embryonic samples. METHOD: This scoping review followed Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols. PubMed database provided relevant articles, screened initially by title and abstract. Inclusion and exclusion criteria defined outcomes of interest. RESULTS: From an initial search, 273 records were identified, narrowed down to 9 articles after applying our criteria. Additionally, two articles were added through citation searching. This, a total of 11 articles were incorporated in this study. CONCLUSION: This micro-CT contrast agent review underscores the need for tailored choices based on research goals. Both Barium sulfate and Iodine-based agents showing excellent results, providing high resolution (micro) vascular content, especially in ex-vivo specimens. However, careful consideration of protocols and tissue characteristics remains imperative for optimizing the effectiveness of micro-CT imaging for the study of cranio-facial vascular development.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics.

VEGF-B Promotes Endocardium-Derived Coronary Vessel Development and Cardiac Regeneration.

BACKGROUND: Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction. METHODS: We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene-deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3-CreERT recombinase-mediated genetic cell lineage tracing and viral vector-mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2'-deoxyuridine-mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B-induced vessels in the heart. RESULTS: We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function. CONCLUSIONS: The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.

IgA Triggers Cell Death of Neutrophils When Primed by Inflammatory Mediators.

IVIG preparations consisting of pooled IgG are increasingly used for the treatment of autoimmune diseases. IVIG is known to regulate the viability of immune cells, including neutrophils. We report that plasma-derived IgA efficiently triggers death of neutrophils primed by cytokines or TLR agonists. IgA-mediated programmed neutrophil death was PI3K-, p38 MAPK-, and JNK-dependent and evoked anti-inflammatory cytokines in macrophage cocultures. Neutrophils from patients with acute Crohn's disease, rheumatoid arthritis, or sepsis were susceptible to both IgA- and IVIG-mediated death. In contrast to IVIG, IgA did not promote cell death of quiescent neutrophils. Our findings suggest that plasma-derived IgA might provide a therapeutic option for the treatment of neutrophil-associated inflammatory disorders.

Combination of µCT and light microscopy for generation-specific stereological analysis of pulmonary arterial branches: a proof-of-concept study.

Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.

Automated segmentation and description of the internal morphology of human permanent teeth by means of micro-CT.

High-resolution micro-computed tomography is a powerful tool to analyze and visualize the internal morphology of human permanent teeth. It is increasingly used for investigation of epidemiological questions to provide the dentist with the necessary information required for successful endodontic treatment. The aim of the present paper was to propose an image processing method to automate parts of the work needed to fully describe the internal morphology of human permanent teeth. One hundred and four human teeth were scanned on a high-resolution micro-CT scanner using an automatic specimen changer. Python code in a Jupyter notebook was used to verify and process the scans, prepare the datasets for description of the internal morphology and to measure the apical region of the tooth. The presented method offers an easy, non-destructive, rapid and efficient approach to scan, check and preview tomographic datasets of a large number of teeth. It is a helpful tool for the detailed description and characterization of the internal morphology of human permanent teeth using automated segmentation by means of micro-CT with full reproducibility and high standardization.

Anatomy of the female pelvic nerves: a macroscopic study of the hypogastric plexus and their relations and variations.

The autonomic nerves of the lesser pelvis are particularly prone to iatrogenic lesions due to their exposed position during manifold surgical interventions. Nevertheless, the cause of rectal and urinary incontinence or sexual dysfunctions, for example after rectal cancer resection or hysterectomy, remains largely understudied, particularly with regard to the female pelvic autonomic plexuses. This study focused on the macroscopic description of the superior hypogastric plexus, hypogastric nerves, inferior hypogastric plexus, the parasympathetic pelvic splanchnic nerves and the sympathetic fibres. Their arrangement is described in relation to commonly used surgical landmarks such as the sacral promontory, ureters, uterosacral ligaments, uterine and rectal blood vessels. Thirty-one embalmed female pelvises from 20 formalin-fixed and 11 Thiel-fixed cadavers were prepared. In all cases explored, the superior hypogastric plexus was situated anterior to the bifurcation of the abdominal aorta. In 60% of specimens, it reached the sacral promontory, whereas in 40% of specimens, it continued across the pelvic brim until S1. In about 25% of the subjects, we detected an accessory hypogastric nerve, which has not been systematically described so far. It originated medially from the inferior margin of the superior hypogastric plexus and continued medially into the presacral space. The existence of an accessory hypogastric nerve was confirmed during laparoscopy and by histological examination. The inferior hypogastric plexuses formed fan-shaped plexiform structures at the end of both hypogastric nerves, exactly at the junction of the ureter and the posterior wall of the uterine artery at the uterosacral ligament. In addition to the pelvic splanchnic nerves from S2-S4, which joined the inferior hypogastric plexus, 18% of the specimens in the present study revealed an additional pelvic splanchnic nerve originating from the S1 sacral root. In general, form, breadth and alignment of the autonomic nerves displayed large individual variations, which could also have a clinical impact on the postoperative function of the pelvic organs. The study serves as a basis for future investigations on the autonomic innervation of the female pelvic organs.

SDF-1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception.

The precise mechanisms of SDF-1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF-1 and CXCR4. In the current study, we demonstrated SDF-1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF-1 expression in wild-type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF-1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF-1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF-1 application and two times less after blocking this pathway. Bone marrow-derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF-1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF-1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.

Cutting-edge microangio-CT: new dimensions in vascular imaging and kidney morphometry.

In the last decades, the contrast-enhanced micro-computed tomography (micro-CT) imaging of a whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop micro-CT scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature and glomeruli number. This method is based on µAngiofil, a new polymerizing contrast agent with homogenous X-ray absorption, which provides continuous filling of the complete vasculature and enables correlative imaging approaches. For rapid and reliable kidney morphometry, the microangio-CT (µaCT) data sets from glial cell line-derived neurotrophic factor (GDNF)+/- mice and their wild-type littermates were used. The results were obtained much faster compared with the current gold standard, histology-based stereology, and without processing artifacts. The histology-based morphometry was done afterward on the same kidneys. Both approaches revealed that the GDNF+/- male mice had about 40% fewer glomeruli. Furthermore, our approach allows for the definition of sites of interest for further histological investigation, i.e., correlative morphology. The polymerized µAngiofil stays in perfused vessels and is autofluorescent, which is what greatly facilitates the matching of histological sections with µaCT data. The presented approach is a time-efficient, reliable, qualitative, and quantitative methodology. Besides glomerular morphometry, the µaCT data can be used for qualitative and quantitative analysis of the kidney vasculature and correlative morphology.

Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway, which is counterregulated by autophagy.

BACKGROUND: Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with release of intact granules, so-called clusters of free eosinophil granules. The mechanism underlying this activation-induced form of cell death in eosinophils has remained unclear. OBJECTIVE: We aimed to elucidate the molecular mechanism of eosinophil cytolysis. METHODS: Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. A morphologic characterization of the distinct stages of the proposed cascade was addressed by means of time-lapse automated fluorescence microscopy, electron microscopy, and immunohistochemistry. Experiments with pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses. RESULTS: After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-dependent manner. All these steps are required for cytoplasmic vacuolization and subsequent cytolysis to occur. Interestingly, triggering cytolysis is associated with an induction of autophagy in eosinophils, and additional stimulation of autophagy by means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions. CONCLUSION: We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy.

Human IgA Fc receptor FcαRI (CD89) triggers different forms of neutrophil death depending on the inflammatory microenvironment.

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

NADPH oxidase-independent formation of extracellular DNA traps by basophils.

Basophils are primarily associated with a proinflammatory and immunoregulatory role in allergic diseases and parasitic infections. Recent studies have shown that basophils can also bind various bacteria both in the presence and the absence of opsonizing Abs. In this report, we show that both human and mouse basophils are able to produce mitochondrial reactive oxygen species and to form extracellular DNA traps upon IL-3 priming and subsequent activation of the complement factor 5 a receptor or FcεRI. Such basophil extracellular traps (BETs) contain mitochondrial, but not nuclear DNA, as well as the granule proteins basogranulin and mouse mast cell protease 8. BET formation occurs despite the absence of any functional NADPH oxidase in basophils. BETs can be found in both human and mouse inflamed tissues, suggesting that they also play a role under in vivo inflammatory conditions. Taken together, these findings suggest that basophils exert direct innate immune effector functions in the extracellular space.

Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model.

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.

Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma.

The aim of this study was to investigate whether there is a correlation between the expressions of four matrix metalloproteinases (MMPs): MMP-2, MMP-7, MMP-9 and MMP-13, and the TNM (tumour-node-metastasis) stages of oral squamous cell carcinoma (OSCC); and to explore the implication of these MMPs in OSCC dissemination. Samples from 61 patients diagnosed with oropharyngeal tumour were studied by immunohistochemistry against MMP-2, MMP-7, MMP-9 and MMP-13. The assessment of immunoreactivity was semi-quantitative. The results showed that MMP-2 and MMP-9 had similar expression patterns in the tumour cells with no changes in the immunoreactivity during tumour progression. MMP-9 always had the highest expression, whereas that of MMP-2 was moderate. MMP-7 showed a significant decrease in expression levels during tumour evolution. MMP-13 had constant expression levels within stage T2 and T3, but showed a remarkable decline in immunoreactivity in stage T4. No significant differences in the MMPs immunoreactivity between tumour cells and stroma were observed. Although strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired, we believe that combining patients' MMPs expression intensity and clinical features may improve the diagnosis and prognosis. Strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired. Application of MMPs as prognostic indicators for the malignancy potential of OSCC might be considered in every case of tumour examination. We believe that combining patients' MMPs expression intensity and clinical features may improve the process of making diagnosis and prognosis.

Revealing the complexity of meniscus microvasculature through 3D visualization and analysis.

Three-dimensional information is essential for a proper understanding of the healing potential of the menisci and their overall role in the knee joint. However, to date, the study of meniscal vascularity has relied primarily on two-dimensional imaging techniques. Here we present a method to elucidate the intricate 3D meniscal vascular network, revealing its spatial arrangement, connectivity and density. A polymerizing contrast agent was injected into the femoral artery of human cadaver legs, and the meniscal microvasculature was examined using micro-computed tomography at different levels of detail and resolution. The 3D vascular network was quantitatively assessed in a zone-base analysis using parameters such as diameter, length, tortuosity, and branching patterns. The results of this study revealed distinct vascular patterns within the meniscus, with the highest vascular volume found in the outer perimeniscal zone. Variations in vascular parameters were found between the different circumferential and radial meniscal zones. Moreover, through state-of-the-art 3D visualization using micro-CT, this study highlighted the importance of spatial resolution in accurately characterizing the vascular network. These findings, both from this study and from future research using this technique, improve our understanding of microvascular distribution, which may lead to improved therapeutic strategies.

An anatomical study of the subarachnoid space surrounding the trigeminal ganglion in horses-in preparation for a controlled glycerol rhizotomy in equids.

Introduction: Equine trigeminal-mediated headshaking is a painful neuropathic disorder comparable to trigeminal neuralgia in humans. The selective destruction of pain fibers within the trigeminal ganglion, called rhizotomy, is the surgical treatment of choice for idiopathic trigeminal neuralgia refractory to medical treatment in humans. The human trigeminal ganglion is enclosed by a dural recess called the Meckel's or trigeminal cave, in which the ganglion is surrounded by a cerebrospinal fluid (CSF)-filled subarachnoid space. During glycerol rhizotomy, glycerol is percutaneously injected in this CSF-filled space. Until now, information about the anatomy of the dural recess and the subarachnoid space surrounding the trigeminal ganglion is lacking in horses. The aim of this study was to explore if a CSF-filled subarachnoid space around the trigeminal ganglion exists in horses. Materials and methods: Six equine cadaver heads were investigated for CSF accumulation around the ganglion with a 3 Tesla MRI. After anatomical dissection to expose the trigeminal root, a polymer-based radiopaque contrast agent was injected through the porus trigeminus into the subarachnoid space (cisternography). The exact delineation and the volume of the contrast agent accumulation were determined on subsequent micro-computed tomographic scans and segmentation. Finally, the distribution of the contrast agent within the subarachnoid space was examined histologically in three specimens. Results: In all 12 specimens included in this study, the trigeminal ganglion was surrounded by a subarachnoid space forming a trigeminal cistern. The mean volume of the trigeminal cave in this study was 0.31 mL (±SD: 0.11 mL). Distribution of the contrast agent along the peripheral nerves (i.e., ophthalmic, maxillary and/or mandibular nerve) was observed in 7 out of 12 specimens. Discussion/conclusion: A subarachnoid space surrounding the trigeminal ganglion exists in the horse and could be targeted for glycerol rhizotomy in horses suffering from trigeminal-mediated headshaking. However, the clinical relevance of contrast agent distribution along the peripheral nerves remains to be assessed.

Cox7a1 controls skeletal muscle physiology and heart regeneration through complex IV dimerization.

The oxidative phosphorylation (OXPHOS) system is intricately organized, with respiratory complexes forming super-assembled quaternary structures whose assembly mechanisms and physiological roles remain under investigation. Cox7a2l, also known as Scaf1, facilitates complex III and complex IV (CIII-CIV) super-assembly, enhancing energetic efficiency in various species. We examined the role of Cox7a1, another Cox7a family member, in supercomplex assembly and muscle physiology. Zebrafish lacking Cox7a1 exhibited reduced CIV2 formation, metabolic alterations, and non-pathological muscle performance decline. Additionally, cox7a1-/- hearts displayed a pro-regenerative metabolic profile, impacting cardiac regenerative response. The distinct phenotypic effects of cox7a1-/- and cox7a2l-/- underscore the diverse metabolic and physiological consequences of impaired supercomplex formation, emphasizing the significance of Cox7a1 in muscle maturation within the OXPHOS system.

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting.

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

Disruption of Notch1 induces vascular remodeling, intussusceptive angiogenesis, and angiosarcomas in livers of mice.

BACKGROUND & AIMS: Notch signaling mediates embryonic vascular development and normal vascular remodeling; Notch1 knockout mice develop nodular regenerative hyperplasia (NRH). The pathogenesis of NRH is unclear, but has been associated with vascular injury in the liver sinusoids in clinical studies. We investigated the role of Notch1 signaling in liver sinusoidal endothelial cells (LSECs). METHODS: We studied MxCre Notch1(lox/lox) mice (conditional knockout mice without tissue-specific disruption of Notch1); mice with hepatocyte-specific knockout were created by crossing Notch1(lox/lox) with AlbCre(+/-) mice. Portal vein pressure was measured; morphology of the hepatic vasculature was assessed by histologic and scanning electron microscopy analyses. We performed functional and expression analyses of isolated liver cells. RESULTS: MxCre-induced knockout of Notch1 led to NRH, in the absence of fibrosis, with a persistent increase in proliferation of LSECs. Notch1 deletion led to de-differentiation, vascular remodeling of the hepatic sinusoidal microvasculature, intussusceptive angiogenesis, and dysregulation of ephrinB2/EphB4 and endothelial tyrosine kinase. Time-course experiments revealed that vascular changes preceded node transformation. MxCre Notch1(lox/lox) mice had reduced endothelial fenestrae and developed portal hypertension and hepatic angiosarcoma over time. In contrast, mice with hepatocyte-specific disruption of Notch1 had a normal phenotype. CONCLUSIONS: Notch1 signaling is required for vascular homeostasis of hepatic sinusoids; it maintains quiescence and differentiation of LSECs in adult mice. Disruption of Notch1 signaling in LSECs leads to spontaneous formation of angiosarcoma, indicating its role as a tumor suppressor in the liver endothelium.