Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling

New blood vessels arise initially as blood islands in the process known as vasculogenesis or as new capillary segments produced through angiogenesis. Angiogenesis itself encompasses two broad processes, namely sprouting (SA) and intussusceptive (IA) angiogenesis. Primordial capillary plexuses expand through both SA and IA, but subsequent growth and remodeling are achieved through IA. The latter process proceeds through transluminal tissue pillar formation and subsequent vascular splitting, and the direction taken by the pillars delineates IA into overt phases, namely: intussusceptive microvascular growth, intussusceptive arborization, and intussusceptive branching remodeling. Intussusceptive microvascular growth circumscribes the process of initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced. In contrast, intussusceptive arborization entails formation of serried pillars that remodel the disorganized vascular meshwork into the typical tree-like arrangement. Optimization of local vascular branching geometry occurs through intussusceptive branching remodeling so that the vasculature is remodeled to meet the local demand. In addition, IA is important in creation of the local organ-specific angioarchitecture. While hemodynamic forces have proven direct effects on IA, with increase in blood flow resulting in initiation of pillars, the preponderant mechanisms are unclear. Molecular control of IA has so far not been unequivocally elucidated but interplay among several factors is probably involved. Future investigations are strongly encouraged to focus on interactions among angiogenic growth factors, angiopoetins, and related receptor

Ex vivo microangioCT: Advances in microvascular imaging

Therapeutic modulation of angiogenesis is believed to be a prospective powerful treatment strategy to modulate the microcirculation and therefore help millions of patients with cardiovascular and cancer diseases. The often-frustrating results from late-stage clinical studies indicate an urgent need for improved assessment of the pro- and anti-angiogenic compounds in preclinical stage of investigation. For such a proper assessment, detailed vascular visualization and adequate quantification are essential. Nowadays, there are few imaging modalities available, but none of them provides non-destructive 3D-visualization of the vasculature down to the capillary level. In many instances, the approaches cannot be combined with the subsequent histological or ultrastructural analysis. In this review, we address the latest developments in the microvascular imaging, namely, the microangioCT approach with a polymer-based contrast agent (μAngiofil). This approach allows time-efficient non-destructive 3D-imaging of the organ and its vasculature including the finest capillaries. Besides the superior visualization, the obtained detailed 3D information on the organ vasculature enables its 3D-skeletonization and further quantitative analysis. Probably the only significant limitation of the described approach is that it can be used only ex vivo, i.e., no longitudinal studies. In spite of this drawback, microangioCT with μAngiofil is a relatively simple and straightforward tool with a broad application range for studying physiological and pathological alterations in the microvasculature of any organ. It provides microvascular imaging at unprecedented level and enables correlative microscopy

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental mode

Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light Microscopy

A detailed vascular visualization and adequate quantification is essential for the proper assessment of novel angiomodulating strategies. Here, we introduce an ex vivo micro-computed tomography (microCT)-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level and rapid estimation of the vascular volume and vessel size distribution. After perfusion with μAngiofil®, a novel polymerizing contrast agent, low- and high-resolution scans (voxel side length: 2.58-0.66 μm) of the entire vasculature were acquired. Based on the microCT data, sites of interest were defined and samples further processed for correlative morphology. The solidified, autofluorescent μAngiofil® remained in the vasculature and allowed co-registering of the histological sections with the corresponding microCT-stack. The perfusion efficiency of μAngiofil® was validated based on lectin-stained histological sections: 98 ± 0.5% of the blood vessels were μAngiofil®-positive, whereas 93 ± 2.6% were lectin-positive. By applying this approach we analyzed the angiogenesis induced by the cell-based delivery of a controlled VEGF dose. Vascular density increased by 426% mainly through the augmentation of medium-sized vessels (20-40 μm). The introduced correlative and quantitative imaging approach is highly reproducible and allows a detailed 3D characterization of the vasculature and muscle tissue. Combined with histology, a broad range of complementary structural information can be obtained

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

Fossil Carychiidae (Eupulmonata, Ellobioidea) from the Lower Pleistocene Nashua Formation of Florida, with the description of a new species.

Recent fossil shell mining for a new rail line in the Orlando area of Orange County, Florida has uncovered two species of the ellobioid genus Carychium O. F. Müller, 1773 in a bed of freshwater marl from the Lower Pleistocene Nashua Formation. To taxonomically interpret these finds, the well-preserved shells were imaged via high-resolution X-ray tomography (micro-CT) to view significant internal diagnostic characters such as the columellar configuration and the degree of lamellar sinuosity and their relationship in context to the entire shell. The image data are compared to that of type material and extant and fossil Carychium species inhabiting the SE USA, Mexico, Central America, and Jamaica. Based on these results, the species Carychiumfloridanum G. H. Clapp, 1918 and Carychiumnashuaensesp. nov. are identified from fossil shells dating from the Early Pleistocene. This work documents the first fossil members of C.floridanum and the first fossil Carychium from the SE USA

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 VEGF164 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 sproutin

Characterization of a B16-F10 melanoma model locally implanted into the ear pinnae of C57BL/6 mice

<div><p>The common experimental use of B16-F10 melanoma cells focuses on exploring their metastatic potential following intravenous injection into mice. In this study, B16-F10 cells are used to develop a primary tumor model by implanting them directly into the ears of C57BL/6J mice. The model represents a reproducible and easily traceable tool for local tumor growth and for making additional <i>in vivo</i> observations, due to the localization of the tumors. This model is relatively simple and involves (i) surgical opening of the ear skin, (ii) removal of a square-piece of cartilage followed by (iii) the implantation of tumor cells with fibrin gel. The remodeling of the fibrin gel within the cartilage chamber, accompanying tumor proliferation, results in the formation of blood vessels, lymphatics and tissue matrix that can be readily distinguished from the pre-existing skin structures. Moreover, this method avoids the injection-enforced artificial spread of cells into the pre-existing lymphatic vessels. The tumors have a highly reproducible exponential growth pattern with a tumor doubling time of around 1.8 days, reaching an average volume of 85mm³ 16 days after implantation. The melanomas are densely cellular with proliferative indices of between 60 and 80%. The induced angiogenesis and lymphangiogenesis resulted in the development of well-vascularized tumors. Different populations of immunologically active cells were also present in the tumor; the population of macrophages decreases with time while the population of T cells remained quasi constant. The B16-F10 tumors in the ear frequently metastasized to the cervical lymph nodes, reaching an incidence of 75% by day 16. This newly introduced B16-F10 melanoma model in the ear is a powerful tool that provides a new opportunity to study the local tumor growth and metastasis, the associated angiogenesis, lymphangiogenesis and tumor immune responses. It could potentially be used to test different treatment strategies.</p></div

Internal morphology of 101 mandibular canines of a Swiss-German population by means of micro-CT: An ex vivo study

The aim of this study was to investigate the root canal system morphology by means of a root canal configuration (RCC) classification described with a four-digit system, the physiological foramen geometry and accessory canal frequency and morphology, of 101 mandibular canines (MaCa) of a Swiss-German population by means of micro-computed tomography. Micro-CT examination of the MaCa was performed and the obtained images analyzed with a 3D imaging software. In single-rooted MaCas, the most frequently observed RCCs were 1-1-1/1 (74.5%) and 1-1-1/2 (14.3%). Seven other RCCs were less frequently observed with a frequency from 4.1 to 1.0%. One physiological foramen was observed in 80.6% of the MaCas, two in 16.3%, three in 1.0% and four in 2.0%. Accessory and connecting canals were apparent only in the middle and apical root thirds. Two-rooted MaCas occurred less frequently (n = 3). When one physiological foramen was present, the mean size of the narrow and wide diameters were 0.28 mm (±0.07) and 0.40 mm (±0.11), while the distance between physiological and anatomical foramen was 0.45 mm (±0.17). MaCas are predominantly single-rooted teeth with a 1-1-1/1 or 1-1-1/2 RCC. Most MaCas had one physiological foramen with an oval shape

Registration of serial sections: An evaluation method based on distortions of the ground truths

Registration of histological serial sections is a challenging task. Serial sections exhibit distortions and damage from sectioning. Missing information on how the tissue looked before cutting makes a realistic validation of 2D registrations extremely difficult. This work proposes methods for ground-truth-based evaluation of registrations. Firstly, we present a methodology to generate test data for registrations. We distort an innately registered image stack in the manner similar to the cutting distortion of serial sections. Test cases are generated from existing 3D data sets, thus the ground truth is known. Secondly, our test case generation premises evaluation of the registrations with known ground truths. Our methodology for such an evaluation technique distinguishes this work from other approaches. Both under- and over-registration become evident in our evaluations. We also survey existing validation efforts. We present a full-series evaluation across six different registration methods applied to our distorted 3D data sets of animal lungs. Our distorted and ground truth data sets are made publicly available.Comment: Supplemental data available under https://zenodo.org/record/428244