Number Of CD31-positive Vessels Research Articles

Plasminogen activator inhibitor-1 is involved in glucocorticoid-induced decreases in angiogenesis during bone repair in mice.

Glucocorticoids delay fracture healing and induce osteoporosis. Angiogenesis plays an important role in bone repair after bone injury. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. However, the mechanisms by which glucocorticoids delay bone repair remain unclear. Therefore, we herein investigated the roles of PAI-1 and angiogenesis in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered dexamethasone (Dex). PAI-1 deficiency significantly attenuated Dex-induced decreases in the number of CD31-positive vessels at damaged sites 4days after femoral bone injury in mice. PAI-1 deficiency also significantly ameliorated Dex-induced decreases in the number of CD31- and endomucin-positive type H vessels and CD31-positive- and endomucin-negative vessels at damaged sites 4days after femoral bone injury. Moreover, PAI-1 deficiency significantly mitigated Dex-induced decreases in the expression of vascular endothelial growth factor as well as hypoxia inducible factor-1α, transforming growth factor-β1, and bone morphogenetic protein-2 at damaged sites 4days after femoral bone injury. The present results demonstrate that Dex-reduced angiogenesis at damaged sites during the early bone-repair phase after femoral bone injury partly through PAI-1 in mice.

Itaconic acid induces angiogenesis and suppresses apoptosis via Nrf2/autophagy to prolong the survival of multi-territory perforator flaps

BackgroundPerforator flaps are widely used in hand microsurgery to reconstruct and repair soft tissue injuries. However, ischemia and subsequent ischemia-reperfusion injury may cause distal necrosis of the flap. Itaconic acid (IA) is a modulator of macrophage function, which exerts anti-inflammatory effects in macrophage activation. MethodsThe necrotic area of the flap was detected by measuring the flap temperature with an infrared thermometer. Flap cell apoptosis was detected by TUNEL staining and Western blot analysis of the apoptosis-associated proteins Bcl-2 and Bax. HE staining was used to detect angiogenesis of the skin flaps. CD31 was detected to identify positive vascular expression, and the survival of choke vessels in different areas of the skin flap was assessed by arteriography. In addition, Western blot was performed to quantify the expressions of VEGF, Nrf2, LC3II, SQSTM1, and CTSD. ResultsItaconic acid raises VEGF protein levels in skin flaps and the number of CD31-positive vessels. The skin flaps in the IA treatment group exhibited higher neovascularization and less necrosis than those in the control group. The results of TUNEL staining and Western blot revealed that Itaconic acid attenuated apoptosis in the ischemic area of flap. The combination of itaconic acid and Nrf2 inhibitor ML385 reversed this beneficial effect. The results revealed that itaconic acid attenuated apoptosis, enhanced angiogenesis, and enhanced autophagy. ConclusionIn summary, our findings indicate that itaconic acid might be an effective treatment to reduce flap necrosis. Additionally, this study identified a novel mechanism for the effects of itaconic acid on flap survival.

Adipose-derived stem cells improve tendon repair and prevent ectopic ossification in tendinopathy by inhibiting inflammation and inducing neovascularization in the early stage of tendon healing.

IntroductionAchilles tendinopathy is characterized by scar formation or ectopic ossification, both of which result in pain and worsened physical function in athletes and older people. Although cell therapy using adipose-derived stem cells (ASCs) has been shown to be effective for tendinopathy, the underlying mechanisms by which ASCs result in tendon healing in vivo have not yet been fully clarified.MethodsASCs were obtained from the fat pads of EGFP transgenic mice by collagenase digestion. C57BL/6 mice were used in a collagenase-induced injury model. ASCs were transplanted into injury sites at 1 week after injury. Tendons were harvested at 9 days, 2 weeks, and 4 weeks after transplantation, and analyzed by histological examination and μCT scanning.ResultsHistological analysis and μCT scanning revealed greater recovery of collagen fibers and suppression of ectopic ossification in the ASC-treated group than in the control group at 2 and 4 weeks after injury. Immunohistochemical analysis identified transplanted ASCs in the tendon core close to peritenon and connective tissue at 2 days and 1 week after transplantation, but not at 3 weeks. Furthermore, while the expression levels of IL-1β, GLUT1, and CA9 were significantly reduced in the ASC group compared to the control group at 9 days after injury, those of VEGF and the number of CD31 positive vessels were significantly increased.ConclusionThe efficacy of ASCs for tendon repair and the prevention of ectopic ossification in Achilles tendinopathy were demonstrated. Our data suggest that ASCs can modulate inflammation and induce neovascularization in the early stage of tendon injury.

Altered microglia and neurovasculature in the Alzheimer's disease cerebellum

Traditionally regarded to coordinate movement, the cerebellum also exerts non-motor functions including the regulation of cognitive and behavioral processing, suggesting a potential role in neurodegenerative conditions affecting cognition, such as Alzheimer's disease (AD). This study aims to investigate neuropathology and AD-related molecular changes within the neocerebellum using post-mortem human brain tissue microarrays (TMAs).Immunohistochemistry was conducted on neocerebellar paraffin-embedded TMAs from 24 AD and 24 matched control cases, and free-floating neocerebellar sections from 6 AD and 6 controls. Immunoreactivity was compared between control and AD groups for neuropathological hallmarks (amyloid-β, tau, ubiquitin), Purkinje cells (calbindin), microglia (IBA1, HLA-DR), astrocytes (GFAP) basement-membrane associated molecules (fibronectin, collagen IV), endothelial cells (CD31/PECAM-1) and mural cells (PDGFRβ, αSMA).Amyloid-β expression (total immunolabel intensity) and load (area of immunolabel) was increased by >4-fold within the AD cerebellum. Purkinje cell counts, ubiquitin and tau immunoreactivity were unchanged in AD. IBA1 expression and load was increased by 91% and 69%, respectively, in AD, with no change in IBA1-positive cell number. IBA1-positive cell process length and branching was reduced by 22% and 41%, respectively, in AD. HLA-DR and GFAP immunoreactivity was unchanged in AD. HLA-DR-positive cell process length and branching was reduced by 33% and 49%, respectively, in AD. Fibronectin expression was increased by 27% in AD. Collagen IV, PDGFRβ and αSMA immunoreactivity was unchanged in AD. The number of CD31-positive vessels was increased by 98% in AD, suggesting the increase in CD31 expression and load in AD is due to greater vessel number. The PDGFRβ/CD31 load ratio was reduced by 59% in AD.These findings provide evidence of molecular changes affecting microglia and the neurovasculature within the AD neocerebellum. These changes, occurring without overt neuropathology, support the hypothesis of microglial and neurovascular dysfunction as drivers of AD, which has implications on the neocerebellar contribution to AD symptomatology and pathophysiology.

18F-Fluoroethyl-tyrosine uptake is correlated with amino acid transport and neovascularization in treatment-naive glioblastomas.

To investigate the in vivo correlation between 18F-fluoroethyl-tyrosine (18F-FET) uptake and amino acid transporter expression and vascularization in treatment-naive glioblastomas. A total of 43 stereotactic biopsies were obtained from 13 patients with suspected glioblastoma prior to therapy. All patients underwent a dynamic 18F-FET PET/MRI scan before biopsy. Immunohistochemistry was performed using antibodies against SLC7A5 (amino acid transporter), MIB-1 (Ki67, proliferation), CD31 (vascularization) and CA-IX (hypoxia). The intensity of staining was correlated with 18F-FET uptake and the dynamic 18F-FET uptake slope at the biopsy target point. In all patients, the final diagnosis was IDH-wildtype glioblastoma, WHO grade IV. Static 18F-FET uptake was significantly correlated with SLC7A5 staining (r = 0.494, p = 0.001). While the dynamic 18F-FET uptake slope did not show a significant correlation with amino acid transporter expression, it was significantly correlated with the number of CD31-positive vessels (r = -0.350, p = 0.031), which is line with earlier results linking 18F-FET kinetics with vascularization and perfusion. Besides, static 18F-FET uptake also showed correlations with CA-IX staining (r = 0.394, p = 0.009) and CD31 positivity (r = 0.410, p = 0.006). While the correlation between static 18F-FET uptake and SLC7A5 staining was confirmed as significant in multivariate analysis, this was not the case for the correlation with CD31 positivity, most likely because of the lower effect size and the relatively low number of samples. No significant correlation between 18F-FET uptake and Ki67 proliferation index was observed in our cohort. Our results support the findings of preclinical studies suggesting that specific 18F-FET uptake in glioblastomas is mediated by amino acid transporters. As proposed previously, dynamic 18F-FET parameters might be more influenced by perfusion and therefore related to properties of the tumour neovascularization.

CD31 and VEGF are prognostic biomarkers in early-stage, but not in late-stage, laryngeal squamous cell carcinoma

BackgroundPatients suffering from squamous cell carcinoma of the larynx (LSCC) with lymphatic metastasis have a relatively poor prognosis and often require radical therapeutic management. The mechanisms which drive metastasis to the lymph nodes are largely unknown but may be promoted by a pro-angiogenic tumor microenvironment. In this study, we examined whether the number of microvessels and the expression level of vascular endothelial growth factor (VEGF) in the primary tumor are correlated with the degree of lymph node metastasis (N-stage), tumor staging (T) and survival time in LSCC patients.MethodsTissue-Microarrays of 97 LSCC patients were analyzed using immunohistochemistry. The expression of VEGF was scored as intensity of staining (low vs high) and the number of CD31-positive vessels (median </≥7 vessels per visual field) was counted manually. Scores were correlated with N-stage, T-stage and 5-year overall survival rate.ResultsA high expression of angiogenic biomarkers was not associated with poor overall survival in the overall cohort of patients. Instead high CD31 count was associated with early stage cancer (p = 0.004) and in this subgroup high VEGF expression correlated with poor survival (p = 0.032). Additionally, in early stage cancer a high vessel count was associated with an increased recurrence rate (p = 0.004).ConclusionOnly in the early stage subgroup a high expression of angiogenic biomarkers was associated with reduced survival and an increased rate of recurrence. Thus, biomarkers of angiogenesis may be useful to identify high risk patients specifically in early stage LSCC.

MP94-06 MORPHOLOGICAL AND FUNCTIONAL RESTORATION COMPARISON BETWEEN A NOVEL BILAYER CHITOSAN AND BLADDER ACELLULAR MATRIX GRAFT AS SCAFFOLDS IN A RAT BLADDER AUGMENTATION MODEL.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-06 MORPHOLOGICAL AND FUNCTIONAL RESTORATION COMPARISON BETWEEN A NOVEL BILAYER CHITOSAN AND BLADDER ACELLULAR MATRIX GRAFT AS SCAFFOLDS IN A RAT BLADDER AUGMENTATION MODEL. Dongdong Xiao, Qiong Wang, and Mujun LU Dongdong XiaoDongdong Xiao More articles by this author , Qiong WangQiong Wang More articles by this author , and Mujun LUMujun LU More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2909AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Confronted by the limitations of current biomaterials applied in bladder augmentation and urgent clinic needs of alternatives for traditional enterocystoplasty, this study compared the performance of a novel asymmetric bilayer chitosan scaffold with conventional bladder acellular matrix graft (BAMG) in the purpose to assess the feasibility of this strategy as a supplement or replacement for current bladder biomaterials. METHODS Twenty-four 4-week-old male Sprague-Dawley rats were randomly divided into 3 groups which were subjected to augmentation cystoplasty with BAMG, bilayer chitosan scaffolds and sham operation respectively, with 8 rats in each group. At the time-points of 21 days and 70 days post-implantation, basic epidemiological data, morphological, histological (hematoxylin and eosin, Masson's trichrome and immunofluorescence analyses) and functional evaluations were carried out to compare bladder regeneration comprehensively. RESULTS Bilayer chitosan scaffold exhibited an excellent combination of mechanical strength and flexibility, with minimal fibrosis and contracture after integrated into bladder defect. All rats survived during the experimental period, recovered to normal activities and were able to void spontaneously without significant difference in body weight, except that one rat in BAMG group (70 days group) died of urinary ascites within first week post-op. It was proved that bilayer chitosan scaffold had a better performance in both histological staining and morphological analyses of cystography. While the de novo cytokeratin positive urothelia appeared more abundant in BAMG group than chitosan group when compared to control group, the regeneration of a-smooth muscle actin (a-SMA) and SM22-a positive smooth muscle bundles, NeuN positive neural buttons, the mean number of CD31 positive vessels were more favorable in chitosan group than BAMG group, but still did not catch up with the normal level. The mean diameter of CD31 positive vessels revealed that the neo-vessels in BAMG and chitosan group were relatively immature. For both BAMG and chitosan groups, urodynamic analyses demonstrated that the bladder compliance was improved after augmentation, and the interval void cycle time, void volume and bladder capacity were elevated correspondingly, while the peak void pressure of BAMG was exceptionally high. CONCLUSIONS The asymmetric bilayer chitosan scaffold displayed a high potential in facilitating defect regeneration in the rat model of bladder augmentation, which still need further modifications in enhancing the regeneration of urothelia, accelerating re-innervation and promoting neo-angiogenesis. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1247 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Dongdong Xiao More articles by this author Qiong Wang More articles by this author Mujun LU More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of botulinum toxin A on vasoconstriction and sympathetic neurotransmitters in a murine random pattern skin flap model

Blood supply is the most important factor determining the survival of a skin flap. Botulinum toxin-A (Botox-A) is used as pharmacologic agent not only for aesthetic purposes, but also for its vasomotor actions. This study was conducted to establish whether local application of Botox-A increased survival of random pattern skin flaps in rats by changing the expression of neurotransmitters. Forty adult Sprague-Dawley rats with a caudally-based random pattern skin flap were divided into two groups: Botox-A group and saline group. Surviving flap area and cutaneous blood flow in the flap were evaluated on postoperative days 3 and 7. After injection of Botox-A, changes in vessels were analyzed using immunohistochemical staining. Levels of norepinephrine, neuropeptide-Y, nitric oxide, and endothelial nitric oxide synthase were analyzed quantitatively by high performance liquid chromatography, Western blot, and colorimetric assay. The survived area in the Botox-A group was significantly higher than that in the control group on postoperative days 3 and 7. Blood flow in the Botox-A group was significantly high in the proximal and middle areas immediately after the operation. The number of CD31-positive vessels in the Botox-A group was significant greater than that in the control group. Norepinephrine level in the Botox-A group decreased significantly immediately after flap elevation and at postoperative day 3. There were no significant differences in neuropeptide-Y level between the two groups. Nitric oxide level did not change significantly in either group despite the increase in endothelial nitric oxide synthase immediately after flap elevation and at 3 days postoperatively. In conclusion, Botox-A increased vascular blood flow and viable flap area in rats by reducing norepinephrine level. In contrast, neuropeptide-Y, another vasoconstrictor, was not affected by Botox-A. Nitric oxide, a vasodilator, was also not affected by Botox-A, despite the significant increase in endothelial nitric oxide synthase expression in the flaps.

Efficacy and possible mechanisms of topical tranexamic acid in melasma.

Tranexamic acid (TA) has been suggested as an effective treatment for melasma. To investigate the effects and mechanism of action of topical TA in the treatment of melasma. In this study, 23 participants with melasma applied a 2% TA formulation to the whole face for 12 weeks. Clinical effects were evaluated using the modified Melasma Area and Severity Index (mMASI) and a chromameter. Skin biopsies were obtained from 10 participants to evaluate pigmentation, vascularity and the expression levels of possible paracrine factors contributing to the effect of TA. Most of the participants had mild melasma, with mMASI of < 5. The mMASI scores significantly improved in 22 of 23 participants after application. The L* values were increased and the a* values were decreased in both lesional and perilesional normal skin. Fontana-Masson staining showed a significant decrease in melanin content in the epidermis. The number of CD31-positive vessels and the expression of the vascular endothelial growth factor both tended to decrease. Endothelin (ET)-1 was found to be downregulated with TA. Topical TA is effective for melasma. This immunohistochemical study found that suppression of ET-1 could be one of the mechanisms of action of TA on melasma.

Abstract 407: Modulation of Delta-Like Ligand-4 Signaling Improves Myocardial Revascularization Following Coronary Ischemia

Delta-like ligand-4 (DLL4) is an arterial-specific Notch ligand and balanced signaling by DLL4 is required for functional neovascularization. We hypothesized that modest inhibition of DLL4 signaling improves myocardial revascularization following coronary ischemia. Myocardial infarction was induced by ligation of mid-left anterior descending (LAD) artery via rapid left thoracotomy in 6-8 week-old male mice with inducible endothelial-specific knockout or overexpression of DLL4. DLL4 silencing in homozygous mice on the day of or 5 days prior to LAD ligation resulted in significant reduction in revascularization. At four weeks, explanted hearts showed 1.8-fold fewer vessels in LAD territory, 3-fold greater myocardial hypoxia, 1.7-fold larger fibrotic scar and 17% increased perfusion defect in the left ventricle (LV) by scintigraphy. CT angiography confirmed fewer collateral vessels. Echocardiography showed increased LV dilation (37% higher end-diastolic volume) and 12% greater reduction in ejection fraction (EF) compared to baseline (all p&lt;0.001). Overexpression of DLL4 showed a similarly worse outcome. Conversely, partial DLL4 knockout in heterozygous animals resulted in improved outcomes in all parameters. For translational application, animals were systemically administered 1.5mg/kg or 3mg/kg DLL4-Fc intraperitoneally three times a week beginning the day of ligation. RT-PCR analysis of downstream molecules confirmed that systemic DLL4-Fc partially inhibits Notch signaling in endothelial cells in the ischemic LV. When LAD was ligated very proximally, DLL4-Fc improved survival. With mid-LAD ligation, DLL4-Fc induced a dose-dependent increase in number of CD 31-positive vessels and by CT angiography. There was a dose-dependent reduction in hypoxic myocardial area, scar burden, and scintigraphic perfusion defect. DLL4-Fc treated mice had lower end-diastolic LV volume and preserved or improved EF. In an ischemia-reperfusion model, DLL4-Fc increased the number of vessels in the ischemic zone. Our data suggests that balanced DLL4 signaling is crucially required for myocardial angiogenic recovery following coronary ischemia. Modulation of DLL4 signaling has translational therapeutic potential.

Massage Increases Muscle Fiber Regeneration And Angiogenesis And Decreases Fibrosis Following Eccentric Exercise Injury

Muscle injury is a common problem in sports medicine. Myofiber regeneration and repair is often hindered by the development of fibrotic tissue, which results in incomplete functional recovery and, likely in part, a reason for re-injury. A common treatment modality following intense eccentric exercise (EEC) is massage; however, the biologic mechanisms of action for massage therapies are still unknown. Studies have shown that mechanically stimulating muscle stem cells before implantation fosters muscle healing. We propose that massage accelerates muscle healing by increasing myofiber regeneration and reducing tissue fibrosis. PURPOSE: To determine effects of massage on regeneration and repair following EEC. METHODS: In this pilot study, seven female New Zealand White rabbits were instrumented with bilateral peroneal nerve cuffs for stimulation of the tibialis anterior (TA) muscle. One hindlimb was randomly selected to undergo a damaging bout of EEC. Animals were randomly selected for massage application immediately following EEC, 24 hours after EEC, or 48 hours after EEC. One animal was designated as an exercised, non-massaged control. Massage was applied using a customized device for 4 consecutive days. At the end of four days of massage, animals were euthanized and the TAs excised and frozen for immunohistochemical analysis. H&E and Masson’s trichrome staining was used to determine the amount of muscle regeneration (centronucleated fibers) and fibrosis. Angiogenesis was determined by the number of CD31 positive blood vessels. RESULTS: Massage resulted in an average 14% increase (normalized to contralateral limb) in the number of CD31 positive vessels. The number of regenerating fibers increased 3.5% with massage compared to an average 0.7% in the contralateral limb. Immediate massage was more effective than delayed massage in promoting regeneration and angiogenesis. Immediate massage was also most effective in reducing tissue fibrosis, resulting in 7% fibrotic area compared to 15% in the control. CONCLUSIONS: Massage increases the percentage of regenerating muscle fibers, promotes angiogenesis, and decreases fibrosis following EEC injury. Immediate massage was more effective than massage delayed 48 hours for promoting tissue regeneration and reducing fibrosis. Supported by NIH R01 AT004922 [TMB]

P34 Angiogenesis at the primary tumor site is a potential driver of lymphatic metastasis in squamous cell carcinoma of the larynx

Introduction Tumors with lymphatic metastasis require radical therapeutic management and have a poor prognosis. The mechanisms which drive metastasis to the lymph node in squamous cell carcinoma of the larynx are largely unknown. A pro-angiogenic tumor microenvironment may promote lymphatic metastasis. In this study we tested a possible correlation between markers of angiogenesis in the primary tumor and the degree of metastasis to the lymph nodes in a cohort of patients with laryngeal carcinoma. Methods The expression of CD31, VEGF and the number of CD31-positive vessels were analyzed by immunohistochemistry and scored in 83 patients with larynx carcinoma. Scores were correlated with N-stage, T-stage and overall survival. Results High VEGF expression correlated with high CD31 scores. High expression of both VEGF and CD31 correlated with high N-stage (N2 and N3). In patients with positive N status the subgroups of patients with high VEGF scores or a high number of CD31 positive vessels showed reduced overall survival. Conversely, patients with negative node status showed improved survival if VEGF score or the number of vessels were low. CD31 and VEGF scores did not correlate with T stage. Conclusion Our data suggest a possible link between high expression of pro-angiogenic markers in the primary tumor and lymphatic metastasis. Expression of VEGF and CD31 may predict poor prognosis in patients with positive lymph node status.

Gene therapy using hepatocyte growth factor expressing adenovirus improves skin flap survival in a rat model.

Hepatocyte growth factor (HGF) is a potent angiogenic factor that can stimulate the production of blood vessels in ischemic tissue. We investigated whether gene therapy using HGF-expressing adenovirus could enhance skin flap survival. Sprague-Dawley rats were randomly divided into three groups. Rats were subdermally injected with HGF-expressing adenovirus (HGF virus group), recombinant HGF (rhHGF group), or phosphate buffered saline (PBS group) 2 days before and immediately after 3 × 9 cm caudal flap elevation. The survival area of the skin flap, the ratio of blood flow, CD31-positive vessels and, VEGF expression were examined. Skin flap viability was significantly increased in the HGF virus group compared to the rhHGF and PBS groups (71.4% ± 5.9%, 63.8%± 6.4%, and 39.2% ± 13.0%, respectively) (P = 0.025). Furthermore, the blood flow ratio was significantly increased in the HGF virus group. In the HGF virus group, the number of CD31-positive vessels and vascular endothelial growth factor (VEGF) expression were significantly increased. Gene therapy using HGF-expressing adenovirus increase VEGF expression, the number of viable capillaries, and blood flow to the flap, thereby improving skin flap survival.

Endothelial cells: new targets for MSCs-mediated immunomodulation in mouse

Event Abstract Back to Event Endothelial cells: new targets for MSCs-mediated immunomodulation in mouse Lucia Zanotti1*, Cristiana Soldani1, Cristina Ploia1, Federica Moalli2, Erica Dander3, Giovanna D'Amico3, Adelaida Sarukhan1 and Antonella Viola1 1 Humanitas Clinical and Research Center, Italy 2 Theodor Kocher Institute, University of Bern, Switzerland 3 Centro Ricerca “M. Tettamanti,” Clinica Pediatrica Università degli Studi di Milano Bicocca, Italy Chronic inflammation is involved in the pathogenesis of several human diseases, such as atherosclerosis, allergy and autoimmune diseases. Several anti-inflammatory pharmacological drugs are currently available but with limited efficacy and deleterious long-term consequences. Long lasting tolerance induction may represent an effective resolution for these diseases and scientists have become interested in therapies based on cells with immunomodulatory activity. It has been demonstrated that Mesenchymal Stem Cells (MSCs) exert potent immunosuppressive effect both in vitro and in vivo, although the results of clinical trials are still disappointing and the biological mechanisms responsible for MSC effect remain poorly defined. The aim of our study is to obtain a deeper understanding on MSC immunomodulatory mechanisms in vivo. By transplanting alginate-encapsulated MSCs, we have recently shown that their therapeutic effect is completely independent of migration capacity or cell to cell contacts. Using TCR transgenic mice, we have found that encapsulated MSCs hamper recruitment of leukocytes and dendritic cells into inflamed lymph nodes. We speculated that this could be due to a direct effect on endothelial activation. Here we show that, in vivo, MSC hamper the up-regulation of VCAM and the increase in the number of CD31 positive vessels in inflamed lymph nodes. Furthermore, MSC-conditioned medium is able to down regulate the expression of VCAM1 and ICAM1 on endothelial cell lines in vitro. Altogether, these data strongly suggest that soluble factors secreted by MSC can modulate immune responses by directly inhibiting endothelial cell activation. The precise molecules involved in this process are currently under investigation. Keywords: Mesenchymal Stem Cells, Immunomodulation, Stem Cell Transplantation, Endothelium, Inflammation Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Zanotti L, Soldani C, Ploia C, Moalli F, Dander E, D'Amico G, Sarukhan A and Viola A (2013). Endothelial cells: new targets for MSCs-mediated immunomodulation in mouse. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00373 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Lucia Zanotti, Humanitas Clinical and Research Center, Rozzano, Milano, Italy, lucia.zanotti@humanitasresearch.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lucia Zanotti Cristiana Soldani Cristina Ploia Federica Moalli Erica Dander Giovanna D'Amico Adelaida Sarukhan Antonella Viola Google Lucia Zanotti Cristiana Soldani Cristina Ploia Federica Moalli Erica Dander Giovanna D'Amico Adelaida Sarukhan Antonella Viola Google Scholar Lucia Zanotti Cristiana Soldani Cristina Ploia Federica Moalli Erica Dander Giovanna D'Amico Adelaida Sarukhan Antonella Viola PubMed Lucia Zanotti Cristiana Soldani Cristina Ploia Federica Moalli Erica Dander Giovanna D'Amico Adelaida Sarukhan Antonella Viola Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract P1-06-02: A newly angiogenic biomarker Vasohibin-1 expression in ductal carcinoma in situ of the breast.

Abstract Vasohibin-1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF-A and bFGF. Our previous study was the first study demonstrating the status of vasohibin-1 in human breast lesions, which indicates that vasohibin-1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. In this study, we first evaluated mRNA expression of vasohibin-1 and CD31 in 39 Japanese emale breast carcinoma specimens including 22 invasive ductal arcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real-time quantitative RT-PCR (QRT-PCR) with LightCycler system. Table shows the primer sequences used in real-time PCR in this study. In addition, we also immunolocalized vasohibin-1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin-1 mRNA expression and the number of vasohibin-1 positive vessels in DCIS and IDC (P = 0.022 and P &amp;lt; 0.001, respectively). There was a significant positive correlation between vasohibin-1 mRNA level and Ki-67 labeling index in DCIS (r2 = 0.293, P &amp;lt; 0.001). In addition, vasohibin-1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin-1 positive vessels and Ki-67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P &amp;lt;0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin-1 immunoreactivity. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-06-02.

Response of HT29 colorectal xenograft model to cediranib assessed with 18F‐fluoromisonidazole positron emission tomography, dynamic contrast‐enhanced and diffusion‐weighted MRI

Cediranib is a small-molecule pan-vascular endothelial growth factor receptor inhibitor. The tumor response to short-term cediranib treatment was studied using dynamic contrast-enhanced and diffusion-weighted MRI at 7 T, as well as (18) F-fluoromisonidazole positron emission tomography and histological markers. Rats bearing subcutaneous HT29 human colorectal tumors were imaged at baseline; they then received three doses of cediranib (3 mg/kg per dose daily) or vehicle (dosed daily), with follow-up imaging performed 2 h after the final cediranib or vehicle dose. Tumors were excised and evaluated for the perfusion marker Hoechst 33342, the endothelial cell marker CD31, smooth muscle actin, intercapillary distance and tumor necrosis. Dynamic contrast-enhanced MRI-derived parameters decreased significantly in cediranib-treated tumors relative to pretreatment values [the muscle-normalized initial area under the gadolinium concentration curve decreased by 48% (p=0.002), the enhancing fraction by 43% (p=0.003) and K(trans) by 57% (p=0.003)], but remained unchanged in controls. No change between the pre- and post-treatment tumor apparent diffusion coefficients in either the cediranib- or vehicle-treated group was observed over the course of this study. The (18) F-fluoromisonidazole mean standardized uptake value decreased by 33% (p=0.008) in the cediranib group, but showed no significant change in the control group. Histological analysis showed that the number of CD31-positive vessels (59 per mm(2) ), the fraction of smooth muscle actin-positive vessels (80-87%) and the intercapillary distance (0.17 mm) were similar in cediranib- and vehicle-treated groups. The fraction of perfused blood vessels in cediranib-treated tumors (81 ± 7%) was lower than that in vehicle controls (91 ± 3%, p=0.02). The necrotic fraction was slightly higher in cediranib-treated rats (34 ± 12%) than in controls (26 ± 10%, p=0.23). These findings suggest that short-term treatment with cediranib causes a decrease in tumor perfusion/permeability across the tumor cross-section, but changes in vascular morphology, vessel density or tumor cellularity are not manifested at this early time point.

Abstract 3840: Evaluation of the synergetic effect of a combination of dehydroxymethylepoxyquinomicin and gemcitabine on liver metastasis of pancreatic cancer

Abstract Activation of the nuclear transcription factor-kappaB (NF-κB) has been implicated in the progression and metastasis of pancreatic cancer. This study was designed to evaluate the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in inhibition of liver metastasis of pancreatic cancer in a clinical liver metastasis-mimicking mouse model in which cancer cells were intra-portal injected under small laparotomy. Moreover, the synergistic effect of combination therapy with DHMEQ and gemcitabine (GEM) was investigated. Cytotoxicity of DHMEQ and GEM was evaluated by cell growth inhibition assay. Both DHMEQ and GEM monotherapies inhibited cell growth of the human pancreas cell line AsPC-1. However, there was no synergistic effect in the combination therapy. For the in vivo study, male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1. Mice were divided into four groups: 1) control (without any treatment), 2) GEM-treated group, 3) DHMEQ-treated group, and 4) combined treatment group (GEM+DHMEQ). Mice were fed for four weeks, and the number of liver metastases, induction of apoptosis in metastasis focuses and angiogenesis were investigated after sacrifice. The number of liver metastasis was decreased significantly in GEM group, DHMEQ group and GEM+DHMEQ group compared with control group. Significant inductions of apoptosis were revealed in GEM group and DHMEQ group compared with control group, in which apoptosis bodies in metastasis sections were counted by using the TUNEL method. Moreover, combination therapy of DHMEQ+GEM resulted in the strongest inductions of apoptosis. Tumor vessels were identified by immunohistochemical staining of tumor metastasis sections with an antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of DHMEQ or/and GEM-treated mice as compared with control group. However, there was an inconsistency in the results of cell growth inhibition assay (in vitro) and mice metastasis model (in vivo). Thus we evaluated effect of combination therapy in the invasive potential of tumor cells by using chemoinvasion assay. The invasion ability was significantly inhibited in the GEM group, DHMEQ group and GEM+DHMEQ group. Furthermore, real-time PCR was performed to evaluate expression of mRNA of MMP-9. The expression levels of MMP-9 were inhibited in respective groups in the same order. In conclusion, the anti-tumor effects of combination therapy were significant, as evidenced by angiogenesis inhibition, apoptosis induction and invasiveness inhibition rather than cell growth inhibition. 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 3840. doi:1538-7445.AM2012-3840

Abstract 17828: Preconditioning of Mice with a Prolyl Hydroxylase Inhibitor, Dimethyloxalylglycine, Prevents Skin Flap Necrosis

Background- Local skin flaps often present with flap necrosis caused by critical disturbance of blood supply. Because hypoxia-inducible factor 1 (HIF-1) has a pivotal role in ischemic vascular responses, we assessed the hypothesis that preoperative stabilization of HIFs using prolyl hydroxylases (PHD) inhibitor contributes to improvement of skin flap survival. Methods and Results- Mice with ischemic skin flap on the dorsum were treated with PHD inhibitor dimethyloxalylglycine (DMOG) 48hr prior to operation by intraperitoneal administration. Immunoblot assay revealed sustained up-regulation of HIF-1α protein at pre- and postoperative states in the skin. Survival area of flap and the number of CD31 positive vessels at proximal part of flaps in DMOG-treated mice were significantly increased at day seven (20.6±4.9% vs. 55.5±13.2%; P&lt;0.001 and 11.3±1.7 vs. 17.3±3.3; P&lt;0.05). Serum vascular endothelial growth factor (VEGF) protein at the time of operation and its receptor Flk-1 protein at proximal part of flap on day one were significantly increased in DMOG-treated mice. Furthermore, flow cytometric analysis revealed a marked induction of circulating endothelial progenitor cells (EPCs) in DMOG-treated mice on day one (0.75±0.01% vs. 1.49±0.11% of CD45 gated CD34/CD133 positive cells; P&lt;0.005). Of interest, flow cytometric bromodeoxyuridine (BrdU)/DNA analysis showed a marked induction of proliferative progenitor cells in bone marrow 48hr after DMOG treatment. In addition, TUNEL staining at distal part of flaps revealed significantly decreasing apoptotic cells accompanied by increasing protein level of hexokinase II and decreasing Bax protein expression in DMOG-treated mice. HIF-1α heterozygous-null mice showed the reduction of flap survival areas, decreasing the number of circulating EPCs, and increasing the number of apoptotic cells, but these mutant mice with DMOG pretreatment demonstrated complete recovery of these parameters. Conclusion- These findings indicate that prevention of flap necrosis by pretreatment of DMOG is due to enhancement of both anti-apoptotic effects and angiogenesis at the flap region via HIF-1 functions, and that pretreatment of DMOG could be a potential therapeutic strategy for prevention of flap necrosis.

Abstract 1554: Claudin 3 (CLDN3) and claudin 4 (CLDN4) control the growth rate of human ovarian cancer cells in vivo

Abstract Claudins are the major integral membrane proteins forming the backbone of tight junctions between epithelial cells. Using gene expression profiling, we and others have found that the genes coding for claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in ovarian cancers. However, the role of the claudins and their functional importance for the growth of ovarian cancer remain unclear. We compared the in vivo growth rate of human ovarian cancer 2008 cells and sublines in which either CLDN3 or CLDN4 was knocked down, and investigated the mechanisms by which they control the growth rate of human ovarian cancers. Although no difference in growth rate was detected in vitro, strikingly the knockdown tumors grew much faster than the parental 2008 tumors. The tumor volume of the claudin knockdown 2008 xenografts was persistently larger than that of the parental 2008 tumors throughout the whole experimental period. To examine the basis for the difference in growth rate, 2008 and the CLDN3 and CLDN4 knockdown tumors were analyzed by immunohistochemistry for markers of tumor cell proliferation (Ki67), apoptosis (TUNEL) and vascular density (CD31). The effect on these parameters was similar for the CLDN3 and CLDN4 knockdown tumors. The CLDN3 and CLDN4 tumors, respectively, contained 2.9- and 6.8-fold more Ki67-positive cells than the parental 2008 tumors indicating that these knockdown cells possess higher proliferative capacity when grown in vivo. They contained 2.1- and 3.7-fold lower frequency of apoptotic cells suggesting that the death rate of tumor cells was decreased when either CLDN3 or CLDN4 was knocked down. The number of CD31-positive vessels per square millimeter was 130 and 136 in the 2008-CLDN3- and CLDN4- knockdown tumors but only 62 in the parental 2008 tumors reflecting a substantial effect of CLDN3 and CLDN4 on tumor vessel formation. A reduction in the transepithelial electrical resistance across a confluent monolayer confirmed that the knockdown of CLDN3 and CLDN4 impaired the formation of tight junctions. The effect of CLDN3 and CLDN4 knockdown on the expression of other proteins that participate in tight junction formation was determined by Western blot analysis but there were no significant changes in the expression levels of occludin, JAM-1, ZO-1 or E-cadherin when either CLDN3 or CLDN4 was knocked down. Interestingly, knockdown of CLDN3 reduced the expression of CLDN4 at both mRNA and protein level, and knockdown of CLDN4 had the same effect on CLDN3. Taken together, our results indicate that CLDN3 and CLDN4 control ovarian tumor growth in vivo through modulation of the birth rate and death rate of the tumor cells and vascular density within the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2011-1554

Abstract 4175: Anticancer effects of heparin in the experimental metastasis of the pancreatic cancer model with enhanced potency of gemcitabine

Abstract Preclinical evidence has suggested that anticoagulants, including the unfractionated heparin (UFH), exert an anti-tumor effect, whereas the role of anticoagulants in tumor development and metastatic processes has not been fully elucidated yet. Heparin is the major inhibitor of heparanase, which cleaves heparan sulfate and hence participates in degradation of the extracellular matrix. This enzyme also releases a lot of biologically active substance including some growth factors, cytokines, adhesion molecules, and angiogenic factors. Thus, heparin can affect proliferation, migration, and invasion of cancer cells. Moreover, heparin can interfere with adherence of cancer cells to vascular endothelium. This study was designed to evaluate the effects of UFH in the inhibition of liver metastasis of pancreatic cancer. We made a clinical liver metastasis-simulating mouse model, in which cancer cells were intra-portal injected via small laparotomy. Moreover, synergistic effect of combination therapy with UFH and gemcitabine was investigated. Male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1, a human pancreatic adenocarcinoma cell line. Mice were divided randomly into four groups; 1) positive control (PC) without any treatment, 2) gemcitabine treated group (GEM), 3) UFH treated group, and 4) combined treatment group (GEM+UFH). All mice were sacrificed on 28th day after transplantation of cells. The liver weight, the number of liver metastases, induction angiogenesis in metastasis foci were investigated. Heparan degrading enzyme, heparanase, were activated in liver metastasis, in which activity were measured by using the Heparan Degrading Enzyme Assay Kit. Gemcitabine inhibited tumor proliferation, whereas UFH little inhibited in vitro (WST assay). The number of liver metastases decreased significantly in the GEM (17.2±11.5) group, UFH (18.3±3.8) group and GEM+UFH (9.3±2.5) group when compared with the PC group (23.3±7.0). Liver/body weight ratio was lower in the GEM (7.44±1.36 %) group, UFH (7.56±1.47 %) group and GEM+UFH (6.98±0.63 %) group when compared with the PC group (7.99±0.94 %). Tumor vessels were identified by immunohistochemical staining of tumor metastasis sections with antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of UFH or/and GEM-treated mice as compared with control group. UFH exhibited anti-tumor effect by inhibiting angiogenesis, and invasiveness of tumor cells. Furthermore, when UFH and gemcitabine were administered simultaneously, our results suggested the possibility of a synergistic effect. Thus UFH might be a potent drug for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4175.