Platelet-endothelial Cell Adhesion Molecule-1 Staining Research Articles

PECAM‐1‐targeted micron‐sized particles of iron oxide as MRI contrast agent for detection of vascular remodeling after cerebral ischemia

An increasing amount of studies have provided evidence for vascular remodeling, for example, angiogenesis, after cerebral ischemia, which may play a significant role in post-stroke brain plasticity and recovery. Molecular imaging can provide unique in vivo whole-brain information on alterations in the expression of specific endothelial markers. A possible target for molecular magnetic resonance imaging (MRI) of post-stroke (neo)vascularization is platelet endothelial cell adhesion molecule-1 (PECAM-1). Here we describe significantly increased PECAM-1 mRNA levels in ipsilesional brain tissue at 6 h, 24 h and 3 days after transient middle cerebral artery occlusion in mice, and elevated PECAM-1 staining throughout the lesion at 3, 7 and 21 days post-stroke. The potential of micron-sized particles of iron oxide (MPIO) conjugated with PECAM-1-targeted antibodies, that is, αPECAM-1-MPIO, to expose stroke-induced PECAM-1 upregulation with molecular MRI was assessed. In vitro studies demonstrated that PECAM-1-expressing brain endothelial cells could be effectively labeled with αPECAM-1-MPIO, giving rise to a fourfold increase in MRI relaxation rate R2. Injection of near-infrared fluorescent dye-labeled αPECAM-1 showed target specificity and dose efficiency of the antibody for detection of brain endothelial cells at 3 days post-stroke. However, in vivo molecular MRI at 3 and 7 days after stroke revealed no αPECAM-1-MPIO-based contrast enhancement, which was corroborated by the absence of αPECAM-1-MPIO in post mortem brain tissue. This indicates that this molecular MRI approach, which has been proven successful for in vivo detection of other types of cell adhesion molecules, is not invariably effective for MRI-based assessment of stroke-induced alterations in expression of cerebrovascular markers.

Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation

ObjectiveIn ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. Study designOvarian fragments from five patients were cut into pieces (∼0.5mm×1.0mm×1.0mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5ml insemination straws with 1.5M ethylene glycol+0.1M sucrose, with thawing in a 40°C water bath and step-wise removal of cryoprotectants at room temperature in 0.5M, 0.25M and 0.15M sucrose with gentle agitation (group B); tissue vitrified in 2.62M dimethylsulphoxide+2.6M acetamide+1.31M propylene glycol+0.0075M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20ml of 50% vitrification solution pre-warmed to 40°C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. ResultsNo PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P<0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P>0.5). ConclusionThe process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality.

14-3-3 protein regulates Ask1 signaling and protects against diabetic cardiomyopathy

Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. In order to study the pathogenic changes underlying diabetic cardiomyopathy, we examined the role of 14-3-3 protein and apoptosis signal-regulating kinase 1 (Ask1) signaling by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3η protein mutant (DN 14-3-3η) after induction of experimental diabetes. The elevation in blood glucose was comparable between wild type (WT) and DN 14-3-3η mice. However, a marked downregulation of thioredoxin reductase was apparent in DN 14-3-3η mice compared to WT mice after induction of diabetes. Significant Ask1 activation in DN 14-3-3η after diabetes induction was evidenced by pronounced de-phosphorylation at Ser-967 and intense immunofluorescence observed in left ventricular (LV) sections. Echocardiographic analysis revealed that cardiac functions were notably impaired in diabetic DN 14-3-3η mice compared to diabetic WT mice. Marked increases in myocardial apoptosis, cardiac hypertrophy, and fibrosis were observed with a corresponding up-regulation of atrial natriuretic peptide and galectin-3, as well as a downregulation of sarcoendoplasmic reticulum Ca 2+ ATPase2 expression. Furthermore, diabetic DN 14-3-3η mice displayed significant reductions of platelet-endothelial cell adhesion molecule-1 staining as well as endothelial nitric acid synthase and vascular endothelial growth factor expression. In conclusion, our data suggests that enhancement of 14-3-3 protein could provide a novel therapeutic strategy against hyperglycemia-induced left ventricular dysfunction and can limit the progression of diabetic cardiomyopathy by regulating Ask1 signaling.

Cartilage Oligometric Matrix Protein-Angiopoietin-1 Promotes Revascularization Through Increased Survivin Expression in Dermal Endothelial Cells of Skin Grafts in Mice

The present study examined the effects of cartilage oligometric matrix protein angiopoietin-1 (COMP-Ang1) on the revascularization of mice skin grafts. Full-thickness skin grafts were autotransferred into BALB/c mice. The donor grafts were soaked in COMP-Ang1 protein (50 mug/ml, n = 10) or in bovine serum albumin (BSA) (50 mug/ml, n = 10) dissolved in 1 ml of sterile, phosphate-buffered saline for 5 minutes before transfer. Revascularization of the grafts was monitored using an intravital microscope on postoperative days 3, 4, and 5. Morphological and immunohistochemical analyses were performed to evaluate platelet-endothelial cell adhesion molecule-1 and survivin expression and apoptotic signal in the transplanted grafts. Grafts soaked in COMP-Ang1 (COMP-Ang1 group) showed significantly increased revascularization compared with grafts soaked in BSA (BSA group) on intravital microscopy and platelet-endothelial cell adhesion molecule-1 staining. The COMP-Ang1 group showed a significant increase of survivin expression in the endothelial cells and a reduction of apoptotic signal in comparison to the BSA group. Therefore, we believe that COMP-Ang1 provides the therapeutic benefit of enhancing the survival of vascular endothelial cells during transplantation of skin graft.

Null mutation for Macrophage Migration Inhibitory Factor (MIF) is associated with less aggressive bladder cancer in mice

BackgroundInflammatory cytokines may promote tumorigenesis. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with regulatory properties over tumor suppressor proteins involved in bladder cancer. We studied the development of bladder cancer in wild type (WT) and MIF knockout (KO) mice given N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a known carcinogen, to determine the role of MIF in bladder cancer initiation and progression.Methods5-month old male C57Bl/6 MIF WT and KO mice were treated with and without BBN. Animals were sacrificed at intervals up to 23 weeks of treatment. Bladder tumor stage and grade were evaluated by H&E. Immunohistochemical (IHC) analysis was performed for MIF and platelet/endothelial cell adhesion molecule 1 (PECAM-1), a measure of vascularization. MIF mRNA was analyzed by quantitative real-time polymerase chain reaction.ResultsPoorly differentiated carcinoma developed in all BBN treated mice by week 20. MIF WT animals developed T2 disease, while KO animals developed only T1 disease. MIF IHC revealed predominantly urothelial cytoplasmic staining in the WT control animals and a shift toward nuclear staining in WT BBN treated animals. MIF mRNA levels were 3-fold higher in BBN treated animals relative to controls when invasive cancer was present. PECAM-1 staining revealed significantly more stromal vessels in the tumors in WT animals when compared to KOs.ConclusionMuscle invasive bladder cancer with increased stromal vascularity was associated with increased MIF mRNA levels and nuclear redistribution. Consistently lower stage tumors were seen in MIF KO compared to WT mice. These data suggest that MIF may play a role in the progression to invasive bladder cancer.

Adenosine A2A analogue ATL-146e reduces systemic tumor necrosing factor-α and spinal cord capillary platelet-endothelial cell adhesion molecule-1 expression after spinal cord ischemia

Objective: Inflammation is likely a major contributor to spinal cord reperfusion injury after aortic reconstruction. Systemic 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL-146e), a selective adenosine A2A agonist, has been shown to reduce paralysis after spinal cord ischemia. We hypothesized that ATL-146e reduces cytokine production during spinal cord reperfusion, curtailing inflammation and decreasing spinal cord capillary platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression. Study design: New Zealand White rabbits sustained spinal cord ischemia with 45-minute cross-clamping of the infrarenal aorta. One group of animals received intravenous ATL-146e at 0.06 μg/kg/min for 3 hours during reperfusion, beginning after 30 minutes of ischemia. A second group received saline solution vehicle alone for 3 hours, serving as an ischemic control. A third group served as sham-operated animals, undergoing laparotomy with anesthesia. Serum was assayed with enzyme-linked immunosorbent assay for tumor necrosing factor-α (TNF-α). Animals were allowed to recover for 48 hours and were evaluated for hind-limb motor function with the Tarlov (0 to 5) scoring system. At necropsy, animals from each group yielded spinal cords for immunohistochemical staining for PECAM-1. Data are expressed as mean ± standard error of the mean, with statistical analysis with Student t test and Kruskal-Wallis nonparametric test. Results: Markedly improved Tarlov scores were seen in rabbits with ATL-146e (P <.001) during spinal cord reperfusion as compared with ischemic control animals. A significant reduction was found in TNF-α in the sera of rabbits with ATL-146e infusion (P <.01) as compared with ischemic control animals. Significantly reduced endothelial PECAM-1 staining intensity (P <.05) was seen in microscopic spinal cord sections from rabbits with ATL-146e. Conclusion: ATL-146e, an adenosine A2A agonist, reduces spinal cord reperfusion injury. The mechanism of the protection may involve a reduction in circulating TNF-α during a critical 3-hour reperfusion interval and reduction in spinal cord endothelial PECAM-1 upregulation. (J Vasc Surg 2002;35:994-8.)

Prolonged cold preservation augments vascular injury independent of renal transplant immunogenicity and function

While prolonged cold ischemia has detrimental effects on graft survival, the mechanisms remain unclear. We tested whether or not cold preservation enhances intragraft inflammatory responses and vascular injury. Rat renal grafts were cold preserved in University of Wisconsin solution for 2, 4, 6, 12, 24, and 48 hours, and then transplanted into syngeneic recipients and harvested after 24 hours. Frozen sections were examined histologically and stained for vascular cellular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), major histocompatibility complex (MHC) class II, tissue factor, leukocyte function associated molecule-1 (LFA-1), very late antigen-4 (VLA-4), as well as for inflammatory cells. Function did not differ between isografts preserved for shorter (2 to 6 hours) or longer times (12 to 24 hours). Neutrophil influx and that of LFA-1-positive cells showed similar increases in all groups. Compared with short preservation groups, the long preserved grafts had more VLA-4-positive ED-1+ monocytic infiltrates adjacent to vessels expressing VCAM-1 (P < or = 0.001). Increased preservation duration had no effect on infiltration with recipient ED-2+ macrophages, MHC class II-positive cells, or dendritic cells. Decreased color intensity and continuity of PECAM-1 staining indicated loss of endothelial integrity in grafts preserved for longer than six hours. Intensity in VCAM-1 staining increased progressively in grafts preserved for more than six hours and was localized predominantly on the endothelium of elastic vessels. Endothelial cells, vascular smooth muscle cells, and monocytes expressed increasingly more tissue factor in grafts preserved for more than six hours, revealing enhanced intragraft procoagulant capacity. Furthermore, grafts with preservation times of more than six hours developed more severe vascular endothelial injury and worse tubular necrosis scores (P < or = 0.001) compared with grafts with shorter preservation times. Because of the prominent vascular injury, strategies for endothelial protection should be attempted in grafts with long preservation times in clinical renal transplantation.

Regulation of PECAM-1 in endothelial cells during cell growth and migration.

Endothelial cells (EC) that form the inner lining of blood vessels remain quiescent in the normal adult vasculature except during angiogenesis and reendothelialization, which result in EC proliferation and migration. EC placed in culture at subconfluent density also undergo cell multiplication and movement. This report demonstrates that whereas in confluent EC in a compact monolayer, the EC-EC adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1) is strongly expressed at cell borders, little or no PECAM-1 immunostaining is detected in sparse or migrating cultured EC. Consistent with this observation, steady-state PECAM-1 mRNA expression was much lower in subconfluent EC than in confluent EC. The absence of PECAM-1 expression in sparse EC appeared not to be linked to ability to proliferate, since PECAM-1 expression remained low even in the presence of nitric oxide (NO) or mitomycin C, agents that inhibit EC growth. However, another growth-inhibitory agent, TGF-beta 1, did not alter PECAM-1 staining. Based on these observations, it is hypothesized that cell-associated mechanical forces underlying cell tensegrity regulate PECAM-1 expression.

Fibroblast Growth Factor 2 Activation of Stromal Cell Vascular Endothelial Growth Factor Expression and Angiogenesis

Angiogenesis is a key component of human cancer progression and metastasis. In an effort to recapitulate early events in tumor-induced angiogenesis, we have employed a subcutaneous Matrigel implant model using immunodeficient mice as hosts. Matrigel-containing fibroblast growth factor 2 (FGF-2; 1.2 μg/ml) induced stromal cell infiltration into the Matrigel/skin interface within 4 days and maximal neovascularization at 7 days. Cells staining positive for the endothelial cell marker, platelet-endothelial cell adhesion molecule 1 (PECAM-1), were present in neovessels and in isolated cells within the Matrigel matrix. Immunohistochemical analysis revealed high levels of vascular endothelial growth factor (VEGF) deposited in the stromal interface present only in the FGF-2–containing but not in control Matrigel implants. VEGF expression was confirmed with in situ hybridization. High VEGF mRNA levels were observed in the infiltrating stromal cells but not in endothelial or endothelial precursors as defined by PECAM-1 staining. In vitro analysis of FGF-2–treated embryonic fibroblasts, Balb/c 3T3 cells, showed an induction of VEGF transcription, mRNA synthesis, and protein secretion as defined by transcriptional reporter, Northern blot, and ELISA assays. The FGF-2–induced VEGF expression was not dependent on select matrix adherence or signaling components because VEGF mRNA expression induced by FGF-2 was equally activated on serum, basement membrane, and fibronectin matrix substrates. Systemic application of anti-VEGF antibodies significantly repressed FGF-2–induced angiogenesis over control antibody by 88% (p < 0.001). These data support an FGF-2 angiogenic model that is dependent on endothelial cell activation, stromal cell infiltration, and VEGF expression by the infiltrating stromal cell population.