Dysferlin is a new marker for leaky brain blood vessels in multiple sclerosis

Dysferlin is a muscle protein involved in cell membrane repair and its deficiency is associated with muscular dystrophy. We describe that dysferlin is also expressed in leaky endothelial cells. In the normal central nervous system (CNS), dysferlin is only present in endothelial cells of circumventricular organs. In the inflamed CNS of patients with multiple sclerosis (MS) or in animals with experimental autoimmune encephalomyelitis, dysferlin reactivity is induced in endothelial cells and the expression is associated with vascular leakage of serum proteins. In MS, dysferlin expression in endothelial cells is not restricted to vessels with inflammatory cuffs but is also present in noninflamed vessels. In addition, many blood vessels with perivascular inflammatory infiltrates lack dysferlin expression in inactive lesions or in the normal-appearing white matter. In vitro, dysferlin can be induced in endothelial cells by stimulation with tumor necrosis factor-alpha. Hence, dysferlin is not only a marker for leaky brain vessels, but also reveals dissociation of perivascular inflammatory infiltrates and blood-brain barrier disturbance in multiple sclerosis.

Postmortem radiology of fatal hemorrhage: measurements of cross-sectional areas of major blood vessels and volumes of aorta and spleen on MDCT and volumes of heart chambers on MRI

OBJECTIVE: Autopsy determination of fatal hemorrhage as the cause of death is often a difficult diagnosis in forensic medicine. No quantitative system for accurately measuring the blood volume in a corpse has been developed. MATERIALS AND METHODS: This article describes the measurement and evaluation of the cross-sectional areas of major blood vessels, of the diameter of the right pulmonary artery, of the volumes of thoracic aorta and spleen on MDCT, and of the volumes of heart chambers on MRI in 65 autopsy-verified cases of fatal hemorrhage or no fatal hemorrhage. RESULTS: Most cases with a cause of death of "fatal hemorrhage" had collapsed vessels. The finding of a collapsed superior vena cava, main pulmonary artery, or right pulmonary artery was 100% specific for fatal hemorrhage. The mean volumes of the thoracic aorta and of each of the heart chambers and the mean cross-sectional areas of all vessels except the inferior vena cava and abdominal aorta were significantly smaller in fatal hemorrhage than in no fatal hemorrhage. CONCLUSION: For the quantitative differentiation of fatal hemorrhage from other causes of death, we propose a three-step algorithm with measurements of the diameter of the right pulmonary artery, the cross-sectional area of the main pulmonary artery, and the volume of the right atrium (specificity, 100%; sensitivity, 95%). However, this algorithm must be corroborated in a prospective study, which would eliminate the limitations of this study. Quantitative postmortem cross-sectional imaging might become a reliable objective method to assess the question of fatal hemorrhage in forensic medicine.

A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

Overexpression of gastrin-releasing peptide receptors in tumor-associated blood vessels of human ovarian neoplasms

BACKGROUND: Peptide receptors, overexpressed in specific cancers, represent new diagnostic and therapeutic targets. In this study, receptors for the gastrin-releasing peptide (GRP), and other members of the bombesin-family of peptides, were evaluated in ovarian neoplasms. METHODS: 75 primary, secondary and metastatic ovarian tumors were investigated for their bombesin-receptor subtype expression, incidence, localization and density using in vitro autoradiography on tissue sections with the universal radioligand (125)I-[D-Tyr(6), beta-Ala(11), Phe(13), Nle(14)]-bombesin(6-14) and the GRP-receptor subtype-preferring (125)I-[Tyr(4)]-bombesin. RESULTS: GRP-receptors were detected in 42/61 primary ovarian tumors; other bombesin-receptor subtypes (BB1, bb3) were rarely present (3/61). Two different tissue compartments expressed GRP-receptors: the tumoral vasculature was the predominant site of GRP-receptor expression (38/61), whereas neoplastic cells more rarely expressed GRP-receptors (14/61). GRP-receptor positive vessels were present in the various classes of ovarian tumors; generally, malignant tumors had a higher incidence of GRP-receptor positive vessels compared to their benign counterparts. The prevalence of such vessels was particularly high in ovarian carcinomas (16/19) and their metastases (5/5). The GRP-receptors were expressed in high density in the muscular vessel wall. Normal ovary (n=10) lacked GRP-receptors. CONCLUSIONS: The large amounts of GRP-receptors in ovarian tumor vessels suggest a role in tumoral vasculature and possibly angiogenesis. Further, these vessels might be targeted in vivo with bombesin analogs for diagnosis or for therapy.

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.

Effects of Carnoy's solution on blood vessels of the axillary fossa of rats

Carnoy's solution is applied to reduce the recurrence of odontogenic keratocysts and unicystic ameloblastomas. The deleterious action of this fixative on nerves has been studied but no attention has been paid to its effects on nearby vessels. The aim of this study was to investigate the effects of Carnoy's solution on blood vessels. The rat axillary artery and vein were surgically exposed, soaked with Carnoy's solution and kept in place for 2, 5 or 10 min, depending on the treatment group. The 5-min group was followed for 1, 2 and 3 weeks postoperatively. The vessels in the 2-min and 5-min exposure groups showed histological changes to the vessels, represented by focal loss of the endothelium and hyalinization of the wall. These alterations increased in the 10-min group. The vessels in the 3-week observation period revealed signs of recovery. It is concluded that Carnoy's solution can damage blood vessels but the process is reversible for exposure times less than 5 min.

Potential role of pre-existing blood vessels for vascularization and mineralization of osteochondral grafts: an intravital microscopic study in mice

BACKGROUND: The aim of this study was to develop an experimental model that allows to elude the potential role of the preexisting graft microvasculature for vascularization and mineralization of osteochondral grafts. ANIMALS AND METHODS: For that purpose, the II-IV metatarsals of fetal DDY-mice known to be nonvascularized at day 16 of gestation (M16) but vascularized at day 18 (M18) were freshly transplanted into dorsal skin fold chambers of adult DDY mice. Using intravital microscopy angiogenesis, leukocyte-endothelium interaction and mineralization were assessed for 12 days. RESULTS: Angiogenesis occurred at 32 hours in M18, but not before 57 hours in M16 (p = 0.002), with perfusion of these vessels at 42 hours (p = 0.005) and 65 hours (p = 0.1), respectively. Vessels reached a density three times as high as that of the recipient site at day 6, remaining constant until day 12 in M18, whereas in M16 vascular density increased from day 6 and reached that of M18 at day 12 (p = 0.04). Leukocyte-endothelium interaction showed sticker counts elevated by a factor of 4-5 in M18 as compared to M16. Mineralization of osteochondral grafts did not differ between M16 and M18, which significantly increased in both groups throughout the observation period. INTERPRETATION: We propose the faster kinetics in the angiogenic response to M18 and the elevated counts of sticking leukocytes to rest on the potential of establishing end-to-end anastomoses (inosculation) of the vascularized graft with recipient vessels.

Effects of microbeam radiation therapy on normal and tumoral blood vessels

Microbeam radiation therapy (MRT) is a new form of preclinical radiotherapy using quasi-parallel arrays of synchrotron X-ray microbeams. While the deposition of several hundred Grays in the microbeam paths, the normal brain tissues presents a high tolerance which is accompanied by the permanence of apparently normal vessels. Conversely, the efficiency of MRT on tumor growth control is thought to be related to a preferential damaging of tumor blood vessels. The high resistance of the healthy vascular network was demonstrated in different animal models by in vivo biphoton microscopy, magnetic resonance imaging, and histological studies. While a transient increase in permeability was shown, the structure of the vessels remained intact. The use of a chick chorioallantoic membrane at different stages of development showed that the damages induced by microbeams depend on vessel maturation. In vivo and ultrastructural observations showed negligible effects of microbeams on the mature vasculature at late stages of development; nevertheless a complete destruction of the immature capillary plexus was found in the microbeam paths. The use of MRT in rodent models revealed a preferential effect on tumor vessels. Although no major modification was observed in the vasculature of normal brain tissue, tumors showed a denudation of capillaries accompanied by transient increased permeability followed by reduced tumor perfusion and finally, a decrease in number of tumor vessels. Thus, MRT is a very promising treatment strategy with pronounced tumor control effects most likely based on the anti-vascular effects of MRT.

Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels

Intestinal bacterial flora may induce splanchnic hemodynamic and histological alterations that are associated with portal hypertension (PH). We hypothesized that experimental PH would be attenuated in the complete absence of intestinal bacteria. We induced prehepatic PH by partial portal vein ligation (PPVL) in germ-free (GF) or mice colonized with altered Schaedler's flora (ASF). After 2 or 7 days, we performed hemodynamic measurements, including portal pressure (PP) and portosystemic shunts (PSS), and collected tissues for histomorphology, microbiology, and gene expression studies. Mice colonized with intestinal microbiota presented significantly higher PP levels after PPVL, compared to GF, mice. Presence of bacterial flora was also associated with significantly increased PSS and spleen weight. However, there were no hemodynamic differences between sham-operated mice in the presence or absence of intestinal flora. Bacterial translocation to the spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and blood vessels were more abundant in colonized and in portal hypertensive mice, as compared to GF and sham-operated mice. Expression of the intestinal antimicrobial peptide, angiogenin-4, was suppressed in GF mice, but increased significantly after PPVL, whereas other angiogenic factors remained unchanged. Moreover, colonization of GF mice with ASF 2 days after PPVL led to a significant increase in intestinal blood vessels, compared to controls. The relative increase in PP after PPVL in ASF and specific pathogen-free mice was not significantly different. CONCLUSION In the complete absence of gut microbial flora PP is normal, but experimental PH is significantly attenuated. Intestinal mucosal lymphatic and blood vessels induced by bacterial colonization may contribute to development of PH.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Role of the extraosseus blood supply in osteoarthritic femoral heads

Blood perfusion to the femoral head might be endangered during the surgical approach or the preparation of the femoral head or both in hip resurfacing arthroplasty. The contribution of the intramedullary blood supply to the femoral head in osteoarthritis is questionable. Therefore, the contribution of the extraosseous blood supply to osteoarthritic femoral heads was measured intraoperatively to question if there is measurable blood flow between the epiphysis and metaphysis in osteoarthritic hips in case of extraosseus vessel damage. At defined points during surgery we acquired the epiphyseal and metaphyseal femoral head perfusion by high-energy laser Doppler flowmetry. Complete femoral neck osteotomy sparing the retinacular vessels to simulate intraosseous blood disruption showed unchanged epiphyseal blood flow compared to initial measurement after capsulotomy. The pulsatile signal disappeared after transection of the retinacular vessels. Based on these acute measurements, we conclude intramedullary blood vessels to the femoral head do not provide measurable blood supply to the epiphysis once the medial femoral circumflex artery or the retinacular vessels have been damaged. We recommend the use of a safe surgical approach for hip resurfacing and careful implantation of the femoral component to respect blood supply to the femoral head and neck region in hip resurfacing arthroplasty.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Pulse or hyaline ring granuloma. Review of the literature on etiopathogenesis of oral and extraoral lesions

Since the late 1950s, reports on an unusual giant-cell granulomatous lesion affecting the jaws, lungs, stomach and intestines have been published. Histopathologically, the lesions showed the presence of structureless hyaline rings with multinucleated giant cells. The aim of this review was to summarize the literature on the etiopathogenesis of the so-called oral and extraoral pulse or hyaline ring granuloma. Literature was searched using PubMed and Medline. In addition, hand search was performed. Search words were oral and extraoral hyaline ring granuloma, giant-cell hyaline angiopathy, pulse granuloma and chronic periostitis. Numerous terms for hyaline ring granuloma have been introduced over time (1971-2008). One hundred seventy-three cases of oral hyaline ring granuloma have been retrieved from the literature. In the mandible, 72.3% occurred . Two theories for etiopathogenesis have been proposed: (1) the origin of the hyaline rings is due to a foreign material (pulse and legumes) having penetrated the oral mucosa or gastrointestinal tract and lungs (exogenous theory) and (2) the rings are due to hyaline degenerative changes in walls of blood vessels (endogenous theory). Experimental production of oral and extraoral hyaline ring granulomas is consistent with the exogenous origin. Particles or remains of leguminous cells having been implanted or aspirated into human tissues whether located to the oral cavity or throughout the entire digestive tract and respiratory system are thought to be causative. Pulse or hyaline ring granulomas are rare but are well-defined oral and extraoral lesions due to implantation of the cellulose moiety of plant foods in contrast to the starch components.