Endothelial Cells Of Blood Vessels Research Articles

Cancer–stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis

Interactions between cancer cells and stromal cells, including blood vessel endothelial cells (BECs), lymphatic vessel endothelial cells (LECs), bone marrow-derived angiogenic cells (BMDACs) and other bone marrow-derived cells (BMDCs) play important roles in cancer progression. Intratumoral hypoxia, which affects both cancer and stromal cells, is associated with a significantly increased risk of metastasis and mortality in many human cancers. Recent studies have begun to delineate the molecular mechanisms underlying the effect of intratumoral hypoxia on cancer progression. Reduced O2 availability induces the activity of hypoxia-inducible factors (HIFs), which activate the transcription of target genes encoding proteins that play important roles in many critical aspects of cancer biology. Included among these are secreted factors, including angiopoietin 2, angiopoietin-like 4, placental growth factor, platelet-derived growth factor B, stem cell factor (kit ligand), stromal-derived factor 1, and vascular endothelial growth factor. These factors are produced by hypoxic cancer cells and directly mediate functional interactions with BECs, LECs, BMDACs and other BMDCs that promote angiogenesis, lymphangiogenesis, and metastasis. In addition, lysyl oxidase (LOX) and LOX-like proteins, which are secreted by hypoxic breast cancer cells, remodel extracellular matrix in the lungs, which leads to BMDC recruitment and metastatic niche formation.

The transmembrane chemokines CXCL16 and CX3CL1 and their receptors are expressed in human meningiomas

Meningiomas are common slowly growing benign tumors, however, anaplastic meningiomas have an aggressive biological and clinical behavior associated with high rates of recurrence and unfavorable prognosis. Since the molecular mechanisms involved in progression of meningiomas are not yet fully understood and recent investigations have suggested a possible role of chemokines in tumor biology, the aim of the study was to investigate the expression of CX3CL1/CX3CR1 and CXCL16/CXCR6 on mRNA and protein level in human meningiomas. Quantitative reverse-transcription polymerase chain reaction, immunohistochemistry and double immuno-staining techniques were used for the investigations. We showed that mRNA and protein expression of the chemokine/receptor pairs CX3CL1/CX3CR1 and CXCL16/CXCR6 were detectable in human meningioma samples. Double immunostaining revealed that the chemokines/receptors were predominantly expressed in the tumor cells themselves, in infiltrating microglia cells/macrophages and endothelial cells of blood vessels. Nevertheless, not all cells of different kinds were positive for different chemokine/receptors. Of note, in comparison to more benign meningioma samples, CX3CR1 and CXCL16 were found to be expressed at lower levels in anaplastic variants. Moreover, a positive correlation between expression levels of ligands and corresponding receptors could be observed for some malignancy grades. Taken together, these results showed that chemokines and their receptors are involved in the pathogenesis of human meningiomas. Our results provide an interesting basis for further investigations that should be performed to characterize the functional roles of chemokines and their receptors in human meningiomas, and also enhance future therapeutic design.

Bilateral Angiosarcoma of the Breast: Case Report and Review of the Literature

INTRODUCTION: The Angiosarcoma of the breast is a malignant mesenchymal neoplasia that has its origin at endothelial cells of blood vessels of the breast. It is a rare pathology, which represents 0. 04% of the primary tumors of the breast and about 8% of the sarcomas presented at the mammary gland. It occurs mainly in women under 40 years old. The incidence of synchronic contralateral breast Angiosarcoma is about 20%. The dissemination of the tumor is mainly hematogenic. It is usually presented as tumor, with a diameter of about 5 centimeters with soft consistence and badly bounded borders. The diagnosis is histopathological. The prognosis is unfavorable due to the high aggressiveness of the tumor, with recurrence taxes around 50% and a survival index of about 41% in 3 years. The usually recommended treatment is the surgical approach through mastectomy. CASE REPORT: SNPM, female, 44 years old. She was sent to Hospital Federal da Lagoa for having a touchable nodule in the left breast two months before the first interview. The physical examination showed medium-volume breasts, with a palpable tumor at the union of internal quadrants of the left breast with four centimeters in diameter, badly defined bounds, mobile, with a slightly hard consistence and negative papillary discharge bilaterally. Mammography presented with no suspicious lesions. Ultrasonography of the breasts showed at the union of the internal quadrants of the left breast a hypoechogenic, heterogeneous and irregular nodule with. This nodule measured 3. 2 x 1. 3 x 2. 8 centimeters category BI-RADS 4. We made then a core biopsy of this nodule, with a hystopathological report compatible with breast Angiosarcoma of a low degree. An MRI was performed after this diagnosis. The MRI revealed an irregular and heterogeneous nodule, with intense precocious impregnation by the contrast medium and signals of limited distribution at the union of medium quadrants of the left breast. The nodule measured 4. 7 x 3. 8 x 3.5 centimeters corresponding to what was found at the USG performed previously. The MRI showed also a small irregular nodule with intense heterogeneous impregnation of the contrast medium and ascending curve placed in the third medium of the union of the superior quadrants of the right breast classified as BI-RADS 4. At this point, we performed core biopsy of the nodule of the right breast, which made the same diagnosis of the left breast lump. Skin bilateral sparing mastectomy with expander placement bilaterally was performed as the surgical treatment. Histopathological examination of the surgical right breast revealed a low-grade breast angiosarcoma with free surgical margins. The result of the left breast surgical specimen was breast angiosarcoma of intermediate histological grade with foci of high grade, focal necrosis, angiolymphatic invasion was not observed, surgical margins were also free. CONCLUSION: This is a rare and very agressive disease. In the case reported, the patient is at the age when the most cases of this kind of sarcoma appear. The patient had a bilateral tumor. Although the prognosis is dark and there is a high recurrence of the disease, we have decided for the bilateral adenectomy that spare the skin and areolopapilar complexes with expander placement bilaterally, respecting the desire of a young and depressive patient.

Role of the blood–cerebrospinal fluid barrier transporter as a cerebral clearance system for prostaglandin E2 produced in the brain

An increasing level of prostaglandin (PG) E(2) is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE(2) under these pathological conditions appears to affect the concentration of PGE(2) in the cerebrospinal fluid (CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE(2) production via microsomal PGE synthetase-1 (mPGES-1), the inducible PGE(2) -generating enzyme, and PGE(2) elimination from the CSF via the blood-CSF barrier (BCSFB). Immunohistochemical analysis revealed that mPGES-1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE(2) in the CSF. The in vivo PGE(2) elimination clearance from the CSF was eightfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE(2) and β-lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 (OAT3). The characteristics of PGE(2) uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE(2) transport with a Michaelis-Menten constant of 4.24μM. These findings indicate that a system regulating the PGE(2) level in the CSF involves OAT3-mediated PGE(2) uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE(2) .

Orbital intravascular papillary endothelial hyperplasia in a Nigerian child: a case report and review of the literature

IntroductionIntravascular papillary endothelial hyperplasia is a reactive proliferative lesion of endothelial cells in blood vessels. It typically presents as a painless, reddish purple lesion in the sites affected. The orbit remains an uncommon site of affectation of this relatively common disease. It is noteworthy that this is the first reported case, to the best of our knowledge, of orbital intravascular papillary endothelial hyperplasia in a Nigerian child.Case presentationThe case reported here is an orbital intravascular papillary endothelial hyperplasia causing non-axial proptosis and loss of vision in a 14-year-old Nigerian boy. We describe the clinical and histological findings of intravascular papillary endothelial hyperplasia in the orbit of this 14-year-old boy. The key distinguishing features are discussed and relevant literature is reviewed.ConclusionAlthough unusual in presentation, intravascular papillary endothelial proliferation should be considered in the list of differentials of proptosis due to mass lesion in young Nigerians and, possibly, Africans.

Lipoprotein(a): Cellular Effects and Molecular Mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease (CVD). Indeed, individuals with plasma concentrations >20 mg/dL carry a 2-fold increased risk of developing CVD, accounting for ~25% of the population. Circulating levels of Lp(a) are remarkably resistant to common lipid lowering therapies, and there are currently no robust treatments available for reduction of Lp(a) apart from plasma apheresis, which is costly and labour intensive. The Lp(a) molecule is composed of two parts, an LDL/apoB-100 core and a unique glycoprotein, apolipoprotein(a) (apo(a)), both of which can interact with components of the coagulation cascade, inflammatory pathways, and cells of the blood vessel wall (smooth muscle cells (SMC) and endothelial cells (EC)). Therefore, it is of key importance to determine the molecular pathways by which Lp(a) exerts its influence on the vascular system in order to design therapeutics to target its cellular effects. This paper will summarise the role of Lp(a) in modulating cell behaviour in all aspects of the vascular system including platelets, monocytes, SMC, and EC.

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas.

Background:Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells.Methods:The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT–PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells.Results:B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA121, 165, 189, and 165b in SK-MEL-25 and SCC-9 cells.Conclusion:FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.

Soluble VEGFR‐2 Expression in Head and Neck Malignant Tumors

Objective: 1) Define the spatial pattern of expression of soluble vascular endothelial growth factor receptor 2 (sVEGFR-2), a recently discovered receptor that has an important role in tumor lymphangiogenesis, in head and neck malignant tumors. 2) Examine the relationship between sVEGFR-2 expression and lymphatic vessel density in head and neck malignant tumors. Method: One hundred ten paraffin-embedded tissue samples from patients with malignant tumors were stained using immunohistochemistry for sVEGFR-2 and podoplanin, a marker for lymphatic vessels. Tissues studied included: squamous cell carcinoma of the larynx (61), oral cavity (16), oropharynx (20), hypopharynx (5), nasopharynx (1). Papillary (4), medullary (2), and follicular (1) thyroid cancers were also examined. Results: sVEGFR-2 expression was identified in all squamous cell carcinomas of the head and neck. Expression was specific to the endothelial cells in blood vessels of both malignant tissue as well as adjacent normal tissues. sVEGFR-2 was not expressed in lymphatic vessels. This secreted protein was also expressed in tissue stroma. Decreased expression of sVEGFR-2 was correlated with increased lymphatic vessel density in tumors, as measured by podoplanin expression. sVEGFR-2 was minimally expressed in normal thyroid tissue but was abundant in the basement membrane of papillary thyroid cancer cells. Conclusion: We have provided the first evidence of sVEGFR-2 expression in head and neck malignant tumors. Its expression correlates with lymphatic vessel density in head and neck malignant tumors and ongoing studies will reveal the precise function of sVEGFR-2 in nodal metastasis and disease progression.

MiR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia

The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body's response. We found miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia.

Functional impairment of P-glycoprotein by sodium nitroprusside pretreatment in mouse brain capillary endothelial cells

We examined whether pretreatment of mouse brain blood vessel endothelial cell clone 4 (MBEC4) cells with sodium nitroprusside (SNP), a NO(x) donor, as an in vitro model of the bloodbrain barrier could affect P-glycoprotein (P-gp) functional activity. Uptake into the cells and MBEC4 plasma membrane vesicles (MPMVs) in the presence or absence of SNP pretreatment was used to investigate functional changes. Increased accumulation of [(3)H]vincristine, a widely used substrate for P-gp, into MBEC4 was observed upon SNP pretreatment, likely due to impaired P-gp function. To better understand the mechanism of the impairment, MPMVs were prepared and characterized in terms of purity and Na(+)-dependent glucose uptake. [(3)H]daunomycin uptake into MPMVs was diminished after SNP pretreatment in the presence of an ATP-regenerating system, indicating that the functional activity of P-gp was impaired after exposure to SNP. Under conditions of excess ATP, daunomycin uptake into the vesicles was still decreased after SNP pretreatment, indicating that SNP interacted directly with the transport system, but not with the ATP-regenerating system. Together, these results suggest that NO or NO(x) functionally impairs P-gp in the in vitro blood-brain barrier model with SNP pretreatment.

Role of miR-132 in Angiogenesis after Ocular Infection with Herpes Simplex Virus

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.

Expression and localization of aquaporin 1, 2, 3, 4, and 5 in human nasal mucosa

Aquaporins (AQPs) are water-specific membrane channel proteins that regulate water homeostasis for cells and organisms. The purpose of this study was to elucidate the role of AQPs 1, 2, 3, 4, and 5 in normal and diseased human nasal mucosa. Nasal polyps were obtained from eight patients with chronic rhinosinusitis (CRS). Inferior turbinate tissue was obtained from five patients with allergic rhinitis (AR) and from six patients with septal deviation (controls). Expression of AQP1-5 mRNA was examined using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting, and tissue localization of AQP1-5 was studied by immunohistochemistry. Semiquantitative RT-PCR was used to investigate disease-specific changes in the expression in AQP1, -3, and -5 mRNA. Expression of AQP1, -3, -4, and -5 mRNA and AQP1-5 protein were confirmed in normal human nasal mucosa. Immunohistochemical studies revealed that AQP1 was localized in fibroblasts (especially in the subepithelial area) and endothelial cells of blood vessels, AQP2 was localized in the cytoplasm of epithelial cells and acinar cells, AQP3 was localized in the basolateral sites of epithelial cells and acinar cells, AQP4 was localized in the basolateral sites of acinar cells, and AQP5 was localized in the apical sites of epithelial cells and acinar cells. Semiquantitative RT-PCR revealed that there were no significant differences in the mRNA expression of AQP1, AQP3, and AQP5 among control, AR, and CRS patients. AQP1, -2, -3, -4, and -5 were present in normal human nasal mucosa. mRNA expression of AQP1, -3, and -5 did not change among control, AR, and CRS patients.

Abstract 5278: Suppression of angiogenic potential of breast cancer by the inhibition of SAF-1 transcription factor activity

Abstract Angiogenesis plays an important role in the tumor growth and metastasis by facilitating increased blood supply and thus fueling unwanted growth factors and nutrients to the growing tumor. Vascular endothelial growth factor (VEGF) is recognized as a multifunctional angiogenic stimulator that stimulates blood vessel formation and endothelial cell survival. Recent therapeutic trials with anti-VEGF antibody bevacizumab (Avastin), although produced beneficial effects on some cancers including colorectal and lung cancers, had very limited beneficial outcome in breast cancer. The diverse response of avastin raised questions about the efficacy of anti-VEGF antibody approach for breast cancer therapy. Despite the disappointing outcome, it cannot be ignored that regulation of angiogenesis plays a critical role in breast cancer. We hypothesized that angiogenesis may be better controlled by blocking the synthesis of VEGF protein as opposed to the present approach that attempts to block the functions of VEGF by using antibodies. Induction of VEGF by a plethora of physiological and environmental stimuli strongly suggests the presence of multiple routes of VEGF synthesis, which are incompletely understood. We present evidence for a novel mode of transcriptional induction of VEGF, regulated by an inflammation-responsive transcription factor, SAF-1. Incidentally, inflammatory breast cancer exhibits increased angiogenesis and has a higher metastatic potential than noninflammatory breast cancer, supporting the notion that chronic inflammation plays a strong role in angiogenesis and in the progression of cancer. SAF-1 is present in high abundance in human breast cancer tissues. We show that inhibition of endogenous SAF-1 by antisense shRNA can markedly inhibit VEGF expression and block cancer cell-supported vascular endothelial cell function and angiogenesis. Furthermore, SAF-1 inhibition by shRNA reduces tumor growth in mouse tumor model. We provide evidence that SAF-1-mediated induction of VEGF remains suppressed by KLF-4 transcription factor which is abundantly present in normal breast epithelial cells but absent in breast cancer cells. Mutually exclusive interaction of KLF-4 and SAF-1 at the VEGF promoter provides a mechanism for regulation of low-expression of VEGF in normal breast tissues. In correlation, lack of KLF-4 in breast cancer cells appears to be linked to un-restricted interaction of SAF-1 leading to the induction of VEGF expression. Together, our studies provide a new insight into the molecular mechanisms of VEGF expression regulated by SAF-1 and its impact in angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5278. doi:1538-7445.AM2012-5278

Abstract 3042: Glypican-1 (GPC1) promotes S-phase entry and DNA replication in human glioma cells

Abstract Glypican-1 (GPC1), the most ubiquitously expressed member of the glypican family of heparan sulfate proteoglycans (HSPGs), is highly expressed in the developing brain and gliomas. In gliomas, GPC1 is overexpressed in both tumor blood vessel endothelial cells (ECs) and the neoplastic cells. Prior work by us in ECs indicates that GPC1 is overall mitogenic and essential for cell cycle progression and proliferation, implying a role in glioma angiogenesis. The present study focused on the role of GPC1 in glioma cells to explore potential roles in tumorigenesis and growth. The expression of GPC1 in U87 cells, a human glioma cell line with a relatively low basal level of GPC1, was titrated by adenoviral transduction. BrdU pulse-labeling showed a robust induction of S-phase entry and DNA replication by expression of GPC1 in a dose-dependent manner. GPC1 overexpression ultimately led to DNA re-replication and aneuploidy as well as enlarged nuclei and cell rounding. Consistent with these cell cycle effects, ectopic expression of GPC1 significantly induced downregulation of pRb, p21Cip1 and p27Kip1 and upregulation of cyclin E, CDK2 and E2F transactivation activity. These cellular and molecular activities of GPC1 were independent of serum concentrations in the growth medium, suggesting that they do not require exogenous growth factors. This activity required intact GPC1 as neither unglycanated GPC1 core protein nor isolated GPC1 HS chains induced the cell cycle and molecular alterations seen after intact GPC1 was added. The more general relevance of our observation to other cell types and the HS-dependency of the GPC1 activity were confirmed by GPC1 transduction of wild-type and HS synthesis-defective CHO cells. Overexpression of GPC1 induced dramatic S-phase entry in wild-type but not in HS-deficient mutant CHO cells. In summary, this work reveals a potent S-phase promoting activity of GPC1 in glioma cells, which may lead to accelerated cell growth and/or increased genomic instability and, therefore, implies a role for GPC1 in glioma tumorigenesis and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3042. doi:1538-7445.AM2012-3042

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 102, 103, 104, 105, and 106 doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the103 dose, and also heart, liver, brain and CA of the 104 dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 102 tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 103 and 104 doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.

Endothelial cell activation and proliferation in ovarian tumors: Two distinct steps as potential markers for antiangiogenic therapy response

Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Since most patients are diagnosed in advanced disease stages, the starting point, early steps and molecular mechanisms of ovarian cancer angiogenesis are still incompletely characterized. Most immunohistochemical studies for assessment of microvessel density (MVD) in ovarian tumors are based on CD31, CD34 and CD105 immunostaining of tumor blood vessels. Yet, the proliferative status of tumor blood vessel endothelial cells has not as yet been used in the assessment of tumor blood vessels. The present study investigated the Ki67 proliferative index of tumor blood vessel endothelial cells highlighted with the CD34 panendothelial marker and the CD105 endothelial marker in ovarian cancers by antigen co-localization with a doublestaining immunohistochemical method. Lack of co-localization of CD105 and Ki67 in all types of ovarian tumors together with the presence of CD34+/Ki67-positive endothelial cells suggest that endothelial cell activation and proliferation are distinct steps in ovarian tumors. Differences in the proliferation index were observed in endothelial cells from blood vessels of the tumor core and those of the tumor peripheral zones. Potential specific targeting of activated and proliferating tumor blood vessels may provide clues for improving antiangiogenic therapy efficiency.

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.

Arteriovenous Malformations in Five Dimensions

A tightly controlled Notch4 molecular switch in blood vessel endothelial cells induces regression of arteriovenous malformations in mice.

Over-expression of Slit2 induces vessel formation and changes blood vessel permeability in mouse brain

To investigate the effect of the axon guidance cue Slit2 on the density of blood vessels and permeability of the blood-brain barrier in mouse brain. hSlit2 transgenic mouse line was constructed, and the phenotypes of the mice were compared with wild-type mice in respect to the lateral ventricle (LV), ventricle pressure, and the choroids plexus. An in vivo Miles permeability assay and an amyloid-β permeability assay were used to assess the permeability of brain blood vessels. Brain vessel casting and intracerebral hemorrhage models were built to investigate vessel density in the transgenic mice. An in vitro permeability assay was used to test whether Slit2 could change the permeability and tight junctions of blood vessel endothelial cells. Hydrocephalus occurred in some transgenic mice, and a significantly larger lateral ventricle area and significantly higher ventricle pressure were observed in the transgenic mice. The transgenic mice displayed changed construction of the choroids plexus, which had more micro vessels, dilated vessels, gaps between epithelial cells and endothelial cells than wild-type mice. Slit2 significantly increased brain vessel density and the permeability of brain vessels to large molecules. These blood vessels were more sensitive to cues that induce brain hemorrhage. At the cellular level, Slit2 disturbed the integrity of tight junctions in blood vessel endothelial cells and improved the permeability of the endothelial cell layer. Thus, it promoted the entry of amyloid-β peptides from the serum into the central nervous system, where they bound to neurons. Slit2 increases vessel density and permeability in the brains of transgenic mice. Thus, Slit2 induces numerous changes in brain vessels and the barrier system.