Microvascular Corrosion Casting Research Articles

Inflammation and vascular remodeling in COVID-19 hearts

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + —macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

Morphomolecular motifs of pulmonary neoangiogenesis in interstitial lung diseases.

The pathogenetic role of angiogenesis in interstitial lung diseases (ILDs) is controversial. This study represents the first investigation of the spatial complexity and molecular motifs of microvascular architecture in important subsets of human ILD. The aim of our study was to identify specific variants of neoangiogenesis in three common pulmonary injury patterns in human ILD.We performed comprehensive and compartment-specific analysis of 24 human lung explants with usual intersitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP) and alveolar fibroelastosis (AFE) using histopathology, microvascular corrosion casting, micro-comupted tomography based volumetry and gene expression analysis using Nanostring as well as immunohistochemistry to assess remodelling-associated angiogenesis.Morphometrical assessment of vessel diameters and intervascular distances showed significant differences in neoangiogenesis in characteristically remodelled areas of UIP, NSIP and AFE lungs. Likewise, gene expression analysis revealed distinct and specific angiogenic profiles in UIP, NSIP and AFE lungs.Whereas UIP lungs showed a higher density of upstream vascularity and lower density in perifocal blood vessels, NSIP and AFE lungs revealed densely packed alveolar septal blood vessels. Vascular remodelling in NSIP and AFE is characterised by a prominent intussusceptive neoangiogenesis, in contrast to UIP, in which sprouting of new vessels into the fibrotic areas is characteristic. The molecular analyses of the gene expression provide a foundation for understanding these fundamental differences between AFE and UIP and give insight into the cellular functions involved.

Comprehensive three-dimensional morphology of neoangiogenesis in pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis.

Pulmonary veno‐occlusive disease (PVOD) is a rare lung disease characterized by fibrotic narrowing of pulmonary veins leading to pulmonary hypertension (PH) and finally to death by right heart failure. PVOD is often accompanied by pulmonary capillary hemangiomatosis (PCH), a marked abnormal proliferation of pulmonary capillaries. Both morphological patterns often occur together and are thought to be distinct manifestations of the same disease process and accordingly are classified together in group 1′ of the Nice classification of PH. The underlying mechanisms of these aberrant remodeling processes remain poorly understood. In this study, we investigated the three‐dimensional structure of these vascular lesions in the lung explant of a patient diagnosed with PVOD by μ‐computed tomography, microvascular corrosion casting, electron microscopy, immunohistochemistry, correlative light microscopy and gene expression analysis. We were able to describe multifocal intussusceptive neoangiogenesis and vascular sprouting as the three‐dimensional correlate of progressive PCH, a process dividing pre‐existing vessels by intravascular pillar formation previously only known from embryogenesis and tumor neoangiogenesis. Our findings suggest that venous occlusions in PVOD increase shear and stretching forces in the pulmonary capillary bloodstream and thereby induce intussusceptive neoangiogenesis. These findings can serve as a basis for novel approaches to the analysis of PVOD.

Vascular architecture in free flaps: Analysis of vessel morphology and morphometry in murine free flaps

The aim of this study was to analyze the development of vascular architecture as well as vascular morphometry and morphology of anastomosed microvascular free flaps.Free pectoral skin flaps were raised in 25 rats and anastomosed to the femoral vessels in the groin region. CD31 immunohistology was performed after 3, 7 and 12 d (each 5 animals each) to analyze microvessel density (MVD), microvessel area (MVA) and microvessel size (MVS). Microvascular corrosion casting was performed after 7 and 12 d (5 animals each) to analyze vessel diameter (VD), intervascular distance (IVD), interbranching distance (IBD), and branching angle (BA). Further on, sprout and pillar density as hallmarks of sprouting and intussusceptive angiogenesis were analyzed. Pectoral skin isles from the contralateral side served as controls.A significantly increased MVD was found after 7 and 12 d (p each <0.001). MVA was significantly increased after 3, 7 and 12 d (p each <0.001) and a significantly increased MVS was analyzed after 3 and 7 d (p each <0.001). VD and IVD were significantly increased after 7 and 12 d (p each <0.001). For IBD, a significantly increase was measured after 7 d (p < 0.001). For IBA, sprout and pillar density, no significant differences were found (p each ≥0.05).Significant changes in the vascular architecture of free flaps after successful microvascular anastomosis were seen. Since there was no evidence for sprout and pillar formation within the free flaps, the increased MVD and flap revascularization might be induced by the receiving site.

Morphological Study of the Articular Disc and Capillary of the Retrodiscal Tissue in a Type 2 Spontaneous Diabetes Mellitus Rat Model.

The objective of this study was to evaluate morphological changes at the articular disc of the temporomandibular joint and capillary of the retrodiscal tissue in a rat model for type 2 spontaneous diabetes mellitus (DM) (i.e., Goto-Kakizaki [GK] rats) compared to normal Wistar rats. A total of 20 experimental rats were used in this study; the rats were categorized into the normal (n = 10 male 8-week-old Wistar rats) and DM (n = 10 male 8-week-old GK rats) groups. Hematoxylin-eosin stained specimens were obtained from 5 rats from each group. Image analyses of the hematoxylin-eosin stained specimens were conducted using light micrographs, which allowed comparisons of the thickness of the anterior, central, and posterior parts of the articular disc. Afterwards, the microvascular corrosion cast specimens were obtained from 5 rats from each group. The diameter of the capillary of the retrodiscal tissue was determined by analyzing scanning electron micrographs of the microvascular corrosion cast specimens. Student's t-test was used to test for statistical significant differences between the 2 groups. Differences were considered significant when p < 0.01. We found that the thickness of the anterior, central, and posterior parts of the articular disc, and the diameter of the capillary of the retrodiscal tissue was significantly lower in the DM vs. normal group. Therefore, we consider that DM-associated the hyperglycemia causes atrophy of the articular disc and microangiopathy of the capillary of the retrodiscal tissue in GK rats.

Abstract B09: Nintedanib inhibits tumor and vessel growth and leads to vascular normalization in A549-NSCLC-xenografts

Abstract Angiogenesis plays a major role in the growth and progression of non-small-cell lung cancer (NSCLC). The triple angiokinase inhibitor nintedanib is a potent inhibitor of the receptor tyrosine kinases FGFR-1, 2, 3, PDGFR-α and β, VEGFR-1, 2, 3. and Flt-3. as well as of non-receptor tyrosine kinases like Src, Lyn and Lck by occupying the intracellular ATP-binding pocket. In the pivotal LUME-Lung 1 trial Nintedanib plus docetaxel has proven an overall survival benefit over docetaxel monotherapy in second-line treatment of non-small-cell lung cancer of adenocarcinoma. The aim of this study was to analyse treatment induced vascular normalization of nintedanib, bevacizumab and docetaxel alone versus the respective combinations with docetaxel. Athymic NCRNU-M-F nude mice with subcutaneous A549-NSCLC xenografts were treated with nintedanib (50mg/kg), bevacizumab (10mg/kg) and docetaxel (10mg/kg) alone versus the combination of nintedanib (50mg/kg) + docetaxel (10mg/kg) or bevacizumab (10mg/kg) + docetaxel (10mg/kg)Treatment started seven days after tumor cell inoculation, when the mean tumor volume was ∼200 mm3, and lasted two weeks. The tumor sizes were measured three times a week with calipers. Vessel densities (anti-CD31), cellular proliferation (anti-Ki67) and lymphangiogenesis (anti-LYVE1) were assessed morphometrically by immunohistochemistry. Microvascular corrosion casting was used to analyze and evaluate two- and three-dimensionally the morphology and architecture of blood vessels. Tumor volumes were significantly reduced under nintedanib treatment whereas docetaxel resulted in non-significant tumor growth differences as compared to the controls. The bevacizumab-treated groups were just in-between. This was also true for the vessel densities: significant lower vascular densities were seen only in the nintedanib and combi treatment groups. Docetaxel alone had no significant impact on vessel densities. Microvascular corrosion casting and 3D-morphometry showed significantly qualitative and quantitative differences in the treatment groups. The intervascular distances and interbranching distances were significantly altered towards more control values, evident also in the microscopic appearance of a normalized microvascular architecture. Docetaxel alone had also an effect on these parameters, however, to a far lesser extent. The same is true for the vessel diameters. The absolute vascular volume was significantly lower in the nintedanib and combi treatment groups whereas the relative percentual vascular volume did not show significant differences. Quantitative analyses of anti-ki67 stained sections showed significantly less proliferation in the nintendanib single and combi treatment groups. In summary, we conclude a significant positive therapeutic effect of nintedanib on the microvascular architecture and tumor growth inhibition in A549-NSCLC-xenografts. The vascular architecture in nintedanib treated tumors was characterized by a strong normalization of vasculature. This emerging concept of vascular normalization might be mainly attributable for the significant anti-tumorigenic effects of combinatorial treatment with nintedanib and docetaxel in the NSCLC-xenografts. Citation Format: Maximilian Ackermann, Frank Hilberg, Moritz Anton Konerding. Nintedanib inhibits tumor and vessel growth and leads to vascular normalization in A549-NSCLC-xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B09.

Brain angioarchitecture and intussusceptive microvascular growth in a murine model of Krabbe disease

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal β-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31(+) vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31(+) vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.

Imaging angiogenesis: Perspectives and opportunities in tumour research – A method display

The growth of solid tumours necessitates angiogenesis. The aim of this paper is the presentation and evaluation of different ex vivo methods for analysing tumour angiogenesis.Oral squamous cell carcinomas (SCC) were induced in mice by subcutaneous injection of tumour cells in the groin region and processed for histology and microvascular corrosion casting. Vascularization was analysed light microscopically using CD31 immunochemistry. Corrosion casts were analysed by scanning electron microscopy (SEM), micro computed tomography (μCT) and synchrotron radiation-based micro computed tomography (SRμCT).Immunochemistry allows for a simple and authentic detection and stereological quantification of the SCC vascularization. μCT imaging of the corrosion casts gives a high-quality overview over the three-dimensional (3D) microvascular architecture. SEM and SRμCT allow a high-definition display of the vessel morphology, providing magnificent detail recognition down to the capillary level enabling imaging of different forms of tumour angiogenesis, e.g., sprouting and intussusceptive angiogenesis.Immunochemistry and SEM are regarded as suitable for most of the morphometrical and morphological assessments because of the simple procedure and the high explanatory power, especially in combination with each other. High resolution SRμCT helps answering specialized questions, however, requires sophisticated data processing for visualization and is of limited availability.

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-013-9399-9) contains supplementary material, which is available to authorized users.

Acrylic resin injection method for blood vessel investigations

The injection of acrylic resin into vessels is an excellent method for macroscopically and microscopically observing their three-dimensional features. Conventional methods can be enhanced by removal of the polymerization inhibitor (hydroquinone) without requiring distillation, a consistent viscosity of polymerized resin, and a constant injection pressure and speed. As microvascular corrosion cast specimens are influenced by viscosity, pressure, and speed changes, injection into different specimens yields varying results. We devised a method to reduce those problems. Sodium hydroxide was used to remove hydroquinone from commercial methylmethacrylate. The solid polymer and the liquid monomer were mixed using a 1 : 9 ratio (low-viscosity acrylic resin, 9.07 ± 0.52 mPa•s) or a 3:7 ratio (high-viscosity resin, 1036.33 ± 144.02 mPa•s). To polymerize the acrylic resin for injection, a polymerization promoter (1.0% benzoyl peroxide) was mixed with a polymerization initiator (0.5%, N, N-dimethylaniline). The acrylic resins were injected using a precise syringe pump, with a 5-mL/min injection speed and 11.17 ± 1.60 mPa injection pressure (low-viscosity resin) and a 1-mL/min injection speed and 58.50 ± 5.75 mPa injection pressure (high-viscosity resin). Using the aforementioned conditions, scanning electron microscopy indicated that sufficient resin could be injected into the capillaries of the microvascular corrosion cast specimens.

Morphological Study of the Palatal Gingiva of the Maxillary First Molar in the Type 2 Diabetes Mellitus Model Rat

We studied morphological changes at the maxillary first molar in a model rat for type 2 spontaneous diabetes mellitus (DM), the Goto-Kazizaki (GK) rat, vs. the normal 8-week-old Wistar rat. Serial frontal sections of the gingiva of the maxilla with the bone were prepared from the rats. Image analyses, performed on light micrographs of the hematoxylin-eosin stained specimens, allowed comparison of the thickness of the keratinized, granular, prickle, and basal layers. In addition, the cell population of the granular and prickle layers and the cross-sectional area of the connective tissue beneath the mucosal epithelium were examined. The thickness of the capillary of the maxillary first molar was determined by image analysis of scanning electron micrographs of microvascular corrosion cast specimens. We found that the thickness of the keratinized, granular, and prickle layers was significantly higher in the DM vs. normal group, as were the cell population of the granular and prickle layers. In contrast, the cross-sectional area of the connective tissue beneath the mucosal epithelium, and the thickness of the capillary were significantly lower in the DM vs. normal sections. Therefore, we consider that the DM-associated hyperglycemia causes hypertrophy of the mucosal epithelium, atrophy of the connective tissue beneath the mucosal epithelium, and microangiopathy of the capillary of the palatal gingiva of the maxillary first molar in the GK rat.

Antitumor effect of the vascular-disrupting agent ZD6126 in a murine renal cell carcinoma model

ZD6126 is a vascular-disrupting agent that affects the endothelial tubulin cytoskeleton causing selective occlusion of tumor vasculature and extensive tumor cell necrosis. The present study evaluated the antitumor and antivascular activities of ZD6126 in the clinically relevant murine renal cell carcinoma (RENCA) model and also evaluated biological response to therapy using color Doppler imaging as biomarker. Mice were implanted with RENCA tumor cells (day 0) and established tumors were treated with ZD6126 (100 mg/kg i.p.) or vehicle with repeated intermittent doses on day 10, 14 and 18. ZD6126 treatment led to a significant reduction in tumor size and was associated with extensive tumor necrosis and a reduction in tumor blood flow versus controls. MVD increased with intermittent treatment (day 10, 14 and 18). In an additional study, animals were treated at day 19 and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Corrosion casting showed that tumor vessel architecture is affected by treatment, whereas pre-existing vessels in control tissues are practically not affected. Inter-vessel and inter-branch distances as well as vessel diameters are influenced by treatment. In conclusion, ZD6126 showed potent antitumor efficacy in the RENCA model and our data suggest that decrease in tumor blood flow may be a useful surrogate marker of treatment effect.

Maternal and fetal microvasculature in sheep placenta at several stages of gestation

Maternal and fetal microvasculature was studied in ewes at days 50, 90 and 130 of gestation using microvascular corrosion casting and scanning electron microscopy. Microvascular corrosion casts of caruncles at day 50 were cup-shaped with a centrally located cavity. Branches of radial arteries entered the caruncle from its base and ramified on the maternal surface of the caruncle. Stem arteries broke into an extensive mesh of capillaries forming crypts on the fetal surface. The architecture of the caruncle at day 90 was similar to what was found at day 50 but the vascularity and the depth of the crypts increased in correspondence to increased branching of fetal villi. The substance of the caruncle was thicker at day 130 compared with day 50, with no remarkable difference compared with day 90. Capillary sinusoids of irregular form and diameter were observed on the fetal surface of the caruncle at all stages. These sinusoids may reduce blood flow resistance and subsequently increase transplacental exchange capacity. A microvascular corrosion cast of the cotyledon was cup-shaped with wide and narrow sides. Cotyledonary vessels entered and left the cotyledon from the narrow side. A cotyledonary artery gave proximal collateral branches immediately after entering the cotyledon and then further branched to supply the remaining portion of the cotyledon. Vessel branches broke into a mesh of capillaries forming the fetal vascular villi. Fetal villi that were nearest to the center of the cotyledon were the longest. Capillaries forming villi were in the form of a web-like mesh, were irregular in size and had sinusoidal dilations. The architecture of the cotyledon at day 90 was similar to day 50, but the vascularity increased. Branching of the fetal villi became more abundant. This extensive branching presumably allows a higher degree of invasion and surface contact to maternal tissues. At day 130, the distal portions of the fetal villi showed low ridges and troughs to increase the surface area for diffusion. Branching of fetal villi appears to influence the elaboration of maternal crypts in all stages of gestation. However, correspondence between crypts and villi is restricted to distal portions of fetal villi.

Predicting Response to Radioimmunotherapy from the Tumor Microenvironment of Colorectal Carcinomas

Solid tumors have a heterogeneous pathophysiology, which directly affects antibody-targeted therapies. Here, we consider the influence of selected tumor parameters on radioimmunotherapy, by comparing the gross biodistribution, microdistribution, and therapeutic efficacy of either radiolabeled or fluorescently labeled antibodies (A5B7 anti-carcinoembryonic antigen antibody and a nonspecific control) after i.v. injection in two contrasting human colorectal xenografts in MF1 nude mice. The LS174T is moderately/poorly differentiated, whereas SW1222 has a well-differentiated glandular structure. Biodistribution studies (1.8 MBq (131)I-labeled A5B7, four mice per group) showed similar gross tumor uptake at 48 h in the two models (25.1% and 24.0% injected dose per gram, respectively). However, in therapy studies (six mice per group), LS174T required a 3-fold increase in dose (18 versus 6 MBq) to equal SW1222 growth inhibition ( approximately 55 versus approximately 60 days, respectively). To investigate the basis of this discrepancy, high-resolution multifluorescence microscopy was used to study antibody localization in relation to tumor parameters (5 min, 1 and 24 h, four mice per time point). Three-dimensional microvascular corrosion casting and transmission electron microscopy showed further structural differences between xenografts. Vascular supply, overall antigen distribution, and tumor structure varied greatly between models, and were principally responsible for major differences in antibody localization and subsequent therapeutic efficacy. The study shows that multiparameter, high-resolution imaging of both therapeutic and tumor microenvironment is required to comprehend complex antibody-tumor interactions, and to determine which tumor regions are being successfully treated. This will inform the design of optimized clinical trials of single and combined agents, and aid individual patient selection for antibody-targeted therapies.

Prenatal Development of the Caudal Vena Cava in Mammals: Review of the Different Theories with Special Reference to the Dog

Several items of the development of the caudal vena cava in domestic animals are still controversial. In particular, the origin of the lumbar segment of the caudal vena cava is equivocally described. According to different theories it originates from the right-sided supracardinal, caudal cardinal, sacrocardinal, lateral sympathetic or subcardinal veins. In this review, all five theories are compared with each other and discussed in the light of anatomical variations observed in eight dogs. Species-specific diversity, erratic observations because of technical artefacts, and biased interpretation of the original data are three major reasons for the dissimilarity between the five theories, but they cannot explain all differences. Further studies by using modern techniques such as microvascular corrosion casting and non-invasive three-dimensional microtomography are necessary to better understand the normal development and to explain the variations in domestic animal species.

The VEGF receptor tyrosine kinase inhibitor, ZD6474, inhibits angiogenesis and affects microvascular architecture within an orthotopically implanted renal cell carcinoma

The proangiogenic vascular endothelial growth factor-A (VEGF) is essential for the development of new tumor vessels. ZD6474 is a novel inhibitor of VEGF receptor-2 (VEGFR-2) tyrosine kinase activity, which also has additional activity against epidermal growth factor (EGF) receptor tyrosine kinase. The antitumor activity of different schedules of ZD6474 in a clinically relevant, metastasizing, murine renal cell carcinoma (RENCA) model was evaluated in this study. RENCA cells were inoculated into the left kidney of 24 mice (day 0). Daily ZD6474 (50 mg/kg p.o.) treatment was initiated 1 day or 10 days after tumor cell inoculation and continued until day 21. Following treatment, kidney weight and volume were assessed and blood vessel density determined by CD31 staining. Visible metastases in the lungs, spleen, and lymph nodes were quantified using a dissection microscope. In an additional study, animals were treated according to the same regimen and quantitative three-dimensional microvascular corrosion casting was performed to enable detailed assessment of the tumor vascular architecture. Therapy initiated on day 1 or day 10 resulted in a 79% and 59% reduction in primary tumor volume, a 79% and 60% reduction in the number of lung metastases, and a 58% and 59% reduction in vessel density of primary tumors compared with the control group, respectively. Corrosion casting proved a 5.4- and 3.2-fold lower vascular volume compared with untreated tumors, observations that paralleled with significant architectural alterations. In this RENCA model, ZD6474 was a highly active inhibitor of tumor angiogenesis, primary tumor growth and tumor metastasis.

Effect of interstitial laser hyperthermia in a murine model of colorectal liver metastases: scanning electron microscopic study.

Interstitial laser hyperthermia (ILH) is an in situ ablative technique used to treat colorectal liver metastases. The relatively high recurrence of tumor after treatment by ILH may be related to incomplete destruction. Little is known about the effectiveness of ILH for destroying tumor microvasculature. The aim of this study was to define the changes to the microvascular architecture of tumors after treatment with ILH, specifically focusing on the completeness of tumor vasculature destruction. An intrasplenic induction model of liver metastases in 4- to 6-week-old male inbred CBA mice was used. Laser hyperthermia was applied to liver and tumor tissue using a bare optical quartz fiber from a Medilas Fibertom 4100 Nd:YAG surgical laser generator. The animals underwent microvascular corrosion casting of the livers immediately after application of ILH. Microvascular casts were then prepared and studied by scanning electron microscopy. ILH produced complete, uniform destruction of the tumor microvasculature with compete hemostasis. Blood flow in vessels larger than 100 microm diameter had a relatively protective effect, although ILH was able to overcome this barrier effectively by increasing the energy applied. ILH produces complete destruction of tumor microvasculature with hemostasis. The protective effect of blood flow in larger vessels can be overcome by the appropriate use of higher energy levels.

Angiogenesis after sintered bone implantation in rat parietal bone.

We studied the effect of bone substitutes on revascularization and the restart of blood supply after sintered bone implantation in comparison with synthetic hydroxyapatite implantation and fresh autogenous bone transplantation (control) in rat parietal bones. Methods for the study included the microvascular corrosion cast method and immunohistochemical techniques were also used. The revascularization of the control group was the same as that for usual wound healing in the observations of the microvascular corrosion casts. The sintered bone implantation group was quite similar to that of the control group. In the synthetic hydroxyapatite group, immature newly-formed blood vessels existed even on the 21st day after implantation and the physiological process of angiogenesis was interrupted. Immunohistochemically, vascular endothelial growth factor (VEGF), which activates angiogenesis, appeared at the early stages of both the control group and the sintered bone implantation group. VEGF reduced parallel with the appearance of the transforming growth factor factor-beta-1 (TGF-beta-1), which obstructs angiogenesis, and the angiogenesis passed gradually into the mature stage. In the hydroxyapatite implantation group, TGF-beta-1 appeared at the early stage of the implants. The appearance of VEGF lagged and it existed around the pores of hydroxyapatite even on the 21st day of the implantation. Proliferation and wandering of endothelial cells continued without any maturing of the vessels. These findings suggest that the structure and the components of the implant material affect angiogenesis after implantation as well as new bone formation.

Peripapillary microvascular study of human eyes with primary open-angle glaucoma

To identify peripapillary microvascular changes in eyes with primary open-angle glaucoma, the author studied flat-mount retina using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and selective microvascular corrosion casting of human eye-bank eyes. Decreased densities of peripapillary microvasculature were found in glaucomatous eyes. Regional vessel losses of the peripapillary microvasculature could be related to the changes of visual function in primary open-angle glaucoma.

How a Highly Complex Three-Dimensional Network of Blood Vessels Regresses: The Gill Blood Vascular System of Tadpoles of Xenopus during Metamorphosis. A SEM Study on Microvascular Corrosion Casts

The morphology and vasculature of filter plates and filter rows of tadpoles of Xenopus laevis Daudin were studied from prometamorphosis (stage 56) to metamorphic climax (stage 62) by scanning electron microscopy (SEM) of (i) critical point-dried specimens and (ii) microvascular corrosion casts. The number and frequencies of the highest branching orders of venous filter row vessels, which reach their maximum (6) in stage 58, as well as vessel diameters, interbranching distances, and branching angles were measured. Branching orders continuously decrease until stage 62, so that only two are left. Vessel (luminal) diameters increased from 16.9 ± 0.65 μm (SEM) (stage 56) to 19.3 ± 0.61 μm (stage 58) (branching order 0) and then decreased to 16.7 ± 0.91 μm (stage 62). Interbranching distances were largest in stage 59 (57.5 ± 2.07 μm; branching order 0). Branching angles were highest in stage 62 (91.4° ± 8.82°; branching order 2) and lowest in stage 56 (76.0° ± 4.64°; branching order 4). From the morphology of cast vessels we conclude that vascular regression is organized by progressive “fusion” of opposite vessels in the highest branching orders (“miniaturization”) until only a single capillary loop connects the terminal arteriole and the main filter fold venule. With obliteration of the single capillary loop, the regression of the gill vasculature is terminated. It is noted that the “fusion” of gill vessels is reminiscent of “reversed” intussusceptive microvascular growth.