A Comparison of Ramipril and Bevacizumab to Mitigate Radiation-Induced Brain Necrosis: An Experimental Study.

BACKGROUND: Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, is a new treatment approach for radionecrosis. In our study, we compared the prophylactic and therapeutic usage of a promising agent, ramipril (an angiotensin-converting enzyme inhibitor), with that of bevacizumab for reducing radiation-induced brain injury after high-dose stereotactic radiosurgery (SRS). METHODS: A total of 60 Wistar rats were used. The rats were irradiated with a single dose of 50 Gy using a Leksell Gamma Knife device. Bevacizumab and ramipril were administered in the prophylactic protocol (starting the first day of SRS) and in the therapeutic protocol (starting the fourth week of SRS). Their usage was continued until 12 weeks, and the right frontal lobes of the rats were examined histologically (hematoxylin and eosin stain) and immunohistochemically (hypoxia-inducible factor [HIF]-1α, VEGF, and CD31 antibody expression). RESULTS: The expression of VEGF, HIF-1α, and CD31 had significantly increased at 12 weeks after SRS compared with the control group. The addition of bevacizumab or ramipril to SRS significantly mitigated the histological severity of radiation injury and the expression of VEGF, HIF-1α, and CD31. However, the prophylactic use of bevacizumab and ramipril seemed to be more effective than therapeutic administration. Our results also revealed that the greatest benefit was achieved with the use of prophylactic administration of bevacizumab compared with other treatment protocols. CONCLUSIONS: Ramipril might be a promising agent for patients with radionecrosis. Clinical studies are required to investigate the effective and safe doses of ramipril, which is an inexpensive, well-tolerated drug that can cross the blood-brain barrier.

Apatinib Combined With Radiotherapy Enhances Antitumor Effects in an In Vivo Nasopharyngeal Carcinoma Model.

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are highly expressed in nasopharyngeal carcinoma; therefore, blocking the binding of VEGF and VEGFR may be a potential way to treat nasopharyngeal carcinoma. Apatinib inhibits tumor angiogenesis. Previous studies have suggested that treatment with apatinib has an antitumor effect on nasopharyngeal carcinoma. This study will investigate the effect of apatinib combined with radiotherapy. In this study, nude mice injected with CNE-2 nasopharyngeal carcinoma cells were randomly divided into 6 groups. Therapeutic effects were assessed by evaluating tumor inhibition rate, phosphorylation of VEGFR-2, CD31, partial oxygen pressure, and tumor metabolism. We found that the tumor inhibition of mice in the treated groups was better compared to that of the control group. In mice treated with apatinib alone, angiogenesis was prevented, and the tumor tissue partial oxygen pressure was reduced, thereby achieving an antitumor effect. Moreover, the tumor inhibitory effect of combined treatment was stronger than treatment with either apatinib or radiotherapy alone. Compared with monotherapy treatment, combined treatment better resisted angiogenesis. Apatinib combined with radiotherapy to treat nasopharyngeal carcinoma has synergistic effects, which may be related to enhanced antiangiogenesis.

Liposome-encapsulated statins reduce hypertrophic scarring through topical application.

Hypertrophic scar is an important clinical problem with limited therapeutic options. Aside from their roles as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins have also been demonstrated to decrease scarring by reducing connective tissue growth factor (CTGF) expression. However, poor penetrative ability limits their utility as topical treatments for hypertrophic scar. Here, we aim to develop novel statin formulations using liposomes to enhance dermal penetrative ability and to evaluate their efficacy against formation of hypertrophic scar utilizing our validated rabbit ear hypertrophic scar model. Liposomal simvastatin or pravastatin were compounded using a novel, flexible liposomal formulation and applied topically to rabbit ear hypertrophic scars daily from postoperation day (POD) 14 until POD 25. Scar color, including erythema and melanin, was measured using reflectance spectrophotometry on POD 28, and scar tissue was harvested for evaluation of scar elevation index as well as gene and protein expression. Human foreskin fibroblasts were also treated with statin formulations and CCN2 expression was determined by quantitative PCR. Both simvastatin and pravastatin were efficiently encapsulated in liposomes, forming nanometer-scale particles possessing highly negative charges. Topical treatment with liposomal simvastatin and pravastatin at 6.5% concentration significantly reduced scar elevation index and decreased type I/III collagen content and myofibroblast persistence in the wound. The erythema/vascularity of scars was reduced by liposomal statin treatment, with concomitant decrease of CD31 expression as measured histologically. Expression levels of transcripts encoding CTGF, collagen I, and collagen III collagen in scar tissue were also decreased by liposomal pravastatin treatment, as were myofibroblast persistence and the type I/III collagen ratio as assessed by immunofluorescence and picrosirus red staining, respectively. Treatment of human foreskin fibroblasts with simvastatin or with liposome-encapsulated pravastatin resulted in decreased expression of transcript encoding CTGF. Overall, our novel statin formulations encapsulated in liposomes were successfully delivered through topical application, significantly reducing hypertrophic scarring in a rabbit ear model.

Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells.

The use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

Growth Hormone Improves Cognitive Function After Experimental Stroke.

BACKGROUND AND PURPOSE: Cognitive impairment is a common outcome for stroke survivors. Growth hormone (GH) could represent a potential therapeutic option as this peptide hormone has been shown to improve cognition in various clinical conditions. In this study, we evaluated the effects of peripheral administration of GH at 48 hours poststroke for 28 days on cognitive function and the underlying mechanisms. METHODS: Experimental stroke was induced by photothrombotic occlusion in young adult mice. We assessed the associative memory cognitive domain using mouse touchscreen platform for paired-associate learning task. We also evaluated neural tissue loss, neurotrophic factors, and markers of neuroplasticity and cerebrovascular remodeling using biochemical and histology analyses. RESULTS: Our results show that GH-treated stroked mice made a significant improvement on the paired-associate learning task relative to non-GH-treated mice at the end of the study. Furthermore, we observed reduction of neural tissue loss in GH-treated stroked mice. We identified that GH treatment resulted in significantly higher levels of neurotrophic factors (IGF-1 [insulin-like growth factor-1] and VEGF [vascular endothelial growth factor]) in both the circulatory and peri-infarct regions. GH treatment in stroked mice not only promoted protein levels and density of presynaptic marker (SYN-1 [synapsin-1]) and marker of myelination (MBP [myelin basic protein]) but also increased the density and area coverage of 2 major vasculature markers (CD31 and collagen-IV), within the peri-infarct region. CONCLUSIONS: These findings provide compelling preclinical evidence for the usage of GH as a potential therapeutic tool in the recovery phase of patients after stroke.

Expression of genes encoding IGFBPs, SNARK, CD36, and PECAM1 in the liver of mice treated with chromium disilicide and titanium nitride nanoparticles.

OBJECTIVE: The aim of the present study was to examine the effect of chromium disilicide and titanium nitride nanoparticles on the expression level of genes encoding important regulatory factors (IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK/NUAK2, CD36, and PECAM1/CD31) in mouse liver for evaluation of possible toxic effects of these nanoparticles. METHODS: Male mice received 20 mg chromium disilicide nanoparticles (45 nm) and titanium nitride nanoparticles (20 nm) with food every working day for 2 months. The expression of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver was studied by quantitative polymerase chain reaction. RESULTS: Treatment of mice with chromium disilicide nanoparticles led to down-regulation of the expression of IGFBP2, IGFBP5, PECAM1, and SNARK genes in the liver in comparison with control mice, with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3 and CD36 genes was increased in mouse liver upon treatment with chromium disilicide nanoparticles. We have also shown that treatment with titanium nitride nanoparticles resulted in down-regulation of the expression of IGFBP2 and SNARK genes in the liver with more prominent changes for SNARK gene. At the same time, the expression of IGFBP3, IGFBP4, and CD36 genes was increased in the liver of mice treated with titanium nitride nanoparticles. Furthermore, the effect of chromium disilicide nanoparticles on IGFBP2 and CD36 genes expression was significantly stronger as compared to titanium nitride nanoparticles. CONCLUSIONS: The results of this study demonstrate that chromium disilicide and titanium nitride nanoparticles have variable effects on the expression of IGFBP2, IGFBP3, IGFBP4, IGFBP5, SNARK, CD36, and PECAM1 genes in mouse liver, which may reflect the genotoxic activities of the studied nanoparticles.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes.

Type 1 diabetes (T1D) originates from autoimmune ß-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate ß-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

Ligustrazine effect on lipopolysaccharide-induced pulmonary damage in rats.

We investigated the effectiveness of ligustrazine (tetramethylpyrazine, TMP) in alleviating pulmonary damage induced by lipopolysaccharide (LPS). Twenty-four healthy male Sprague-Dawley rats were randomly divided into three groups: the blank group, LPS group, and TMP treatment group (TMP group). The LPS group was intraperitoneally injected with LPS (20mg/kg), and the TMP group was intraperitoneally injected with LPS (20mg/kg) and ligustrazine (80mg/kg). Blood gas analysis, hematoxylin-eosin staining dye extravasation and diffuse alveolar damage (DAD) score, the wet/dry lung tissue (W/D) ratios, the expression of CD31+ vascular endothelial microparticles (EMPs), tumor necrosis factor alpha (TNF-α) levels, and the protein expression of Rho-associated coiled-coil-forming protein kinase (ROCK) II and Toll-like receptor 4 (TLR4) were analyzed. Compared with the blank group, the arterial partial pressure of oxygen (PaO2) declined in both 1 and 4h (P<0.01), the W/D ratio and DAD score increased (P<0.01), and the TNF-α levels in serum, CD31+ EMPs, and protein expression of ROCK II and TLR4 were significantly increased (P<0.01) in the LPS group. Compared with the LPS group, PaO2 significantly increased in the TMP group at 4h (P<0.05), while the W/D ratio and DAD score were significantly decreased in the TMP group (P<0.01). TNF-α levels, CD31+ EMPs, and protein expression of ROCK II and TLR4 were significantly decreased in the TMP group compared with the LPS group (P<0.01). This study demonstrated that TMP can alleviate LPS-induced pulmonary damage by attenuating pulmonary vascular permeability and CD31+ EMP levels in the plasma, reducing the release of the inflammatory mediator TNF-α and inhibiting the protein expression of ROCK II and TLR4.

[The effect of 7-ketocholesterol on surface ICAM-1 and PECAM-1 expression in human aortic endothelial cells]. / Ocena wplywu 7-ketocholesterolu na powierzchniowa ekspresje molekul adhezyjnych ICAM-1 i PECAM-1 w ludzkich komórkach sródblonka aortalnego.

UNLABELLED: Proatherogenic factors lead to activation of endothelial cells, which symptom is an increased expression of surface adhesion molecules enabling the initiation of a local inflammatory response. 7-ketocholesterol (7-KCH) is a product of oxidation of cholesterol with proven pro-apoptotic effect on the cells of the vessel wall. So far, however, the impact of 7-KCH on surface expression of adhesion molecules has not been assessed. The aim of this study was to evaluate the influence of 7-KCH on the surface expression of adhesion molecules--intracellular adhesion molecule 1 (ICAM-1, CD 54) and .platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31) on human aortic endothelial cells (HAECs). MATERIAL AND METHODS: After treatment with 7-KCH surface expression of adhesion molecules on HAEC was measured with antihuman CD31 and anti-human CD54 antibodies using flow cytometer. RESULTS: 7-KCH significantly increases percentages of CD 54 on viable HEAC, but does not affect expression of CD 31. CONCLUSION: 7-KCH may enhance the initiation of a local inflammatory response in atherosclerosis by increasing the expression of ICAM-1.

Magnolol inhibits angiogenesis by regulating ROS-mediated apoptosis and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

Magnolol, a neolignan from the traditional medicinal plant Magnolia obovata, has been shown to possess neuroprotective, anti-inflammatory, anticancer and anti-angiogenic activities. However, the precise mechanism of the anti-angiogenic activity of magnolol remains to be elucidated. In the present study, the anti-angiogenic effect of magnolol was evaluated in mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial-like cells. The endothelial-like cells were obtained by differentiation from mES/EB cells. Magnolol (20 µM) significantly suppressed the transcriptional and translational expression of platelet endothelial cell adhesion molecule (PECAM), an endothelial biomarker, in mES/EB-derived endothelial-like cells. To further understand the molecular mechanism of the suppression of PECAM expression, signaling pathways were analyzed in the mES/EB-derived endothelial-like cells. Magnolol induced the generation of reactive oxygen species (ROS) by mitochondria, a process that was associated with the induction of apoptosis as determined by positive Annexin V staining and the activation of cleaved caspase-3. The involvement of ROS generation by magnolol was confirmed by treatment with an antioxidant, N-acetyl-cysteine (NAC). NAC inhibited the magnolol-mediated induction of ROS generation and suppression of PECAM expression. In addition, magnolol suppressed the activation of MAPKs (ERK, JNK and p38) and the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells. Taken together, these findings demonstrate for the first time that the anti-angiogenic activity of magnolol may be associated with ROS-mediated apoptosis and the suppression of the PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

Angiogenic response of advanced glycation end products (AGEs) involves PPARgamma.

Diabetes is associated with increased formation of advanced glycation end products (AGEs), which have been implicated in micro and macrovascular complications of diabetes. Our earlier reports showed proangiogenic effect of AGE-bovine serum albumin (BSA). In order to understand the mechanism of AGE-mediated angiogenesis, the possibility of involvement of peroxisome prolifeator activated receptor (PPAR) gamma, a ligand activated transcription factor was examined. The angiogenic effect was studied in chick chorio allantoic membrane (CAM) and by analyzing angiogenic markers in human umbilical vein endothelial cells (HUVECs) in culture. The involvement of PPAR y was investigated using synthetic PPAR gamma agonist GW 1929 and antagonist GW 9662 and by RT-PCR. In CAM assay, PPAR gamma antagonist GW 9662 reversed the AGE-induced effect on vascularity. In HUVECs in culture, GW 9662 reversed the effect of AGE-BSA and decreased the expression of CD 31, E-Selectin and VEGF. RT-PCR analysis showed that treatment with AGE-BSA caused upregulation of PPAR gamma mRNA levels. The reversal of the effect of AGE on angiogenesis by treatment with PPAR gamma antagonists and up-regulation of PPAR gamma gene in HUVECs treated with AGE-BSA suggested the possible involvement of PPAR gamma-dependent downstream pathway in mediating the angiogenic effect of AGE.

Mechanisms involved in CD4 cell gains in HIV-infected patients switched to raltegravir.

BACKGROUND: CD4 gains in HIV patients on HAART result from release of T cells recently migrated from the thymus, redistribution from lymphoid tissues, proliferation in the periphery and/or reduced apoptosis. The relative contribution of each mechanism in CD4 restoration in patients with suppressed viremia switching antiretrovirals is unclear. METHODS: HIV patients with undetectable viremia on HAART were identified at our clinic. A subset switched to raltegravir was compared with another group that kept therapy unmodified. Naive and memory CD4 T-cells were measured by flow cytometry using CD45RA and CD27, respectively. Activation was examined using CD38 and recent thymic emigrants using CD31. Apoptosis was analyzed measuring soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). RESULTS: Thirty-seven patients were examined, 19 switched to raltegravir and 18 controls, after a median of 26 months of suppressed viremia. At 6 months, mean CD4 cell counts significantly increased in raltegravir patients from 322 to 448 cells/µl (P = 0.026) but not in controls (from 312 to 330 cells/µl; P  = 0.813). No significant changes were recognized in activation or CD31 expression in any group. In raltegravir patients, however, the proportion of naive CD4 T cells significantly increased (P = 0.014) as well as CD38 expression in these cells (P = 0.036). A positive correlation was found between CD38 and CD31 expression in naive CD4 T cells (R  = 0.51, P < 0.001). TRAIL and FasL did not decline significantly in any group. CONCLUSION: HIV patients with prolonged undetectable viremia on HAART experience more pronounced CD4 gains after raltegravir switching than keeping the same regimen. An increased production of naive CD4 T cells largely explains this effect.

Gallotannin inhibits NFĸB signaling and growth of human colon cancer xenografts.

Gallotannin (GT), the polyphenolic hydrolyzable tannin, exhibits anti-inflammatory and anticancer activities through mechanisms that are not fully understood. Several effects modulated by GT have been shown to be linked to interference with inflammatory mediators. Considering the central role of nuclear factor kappa B (NF-ĸB) in inflammation and cancer, we investigated the effect of GT on NF-ĸB signaling in HT-29 and HCT-116 human colon cancer cells. DNA binding assays revealed significant suppression of tumor necrosis factor (TNF-α)-induced NFĸB activation which correlated with the inhibition of IĸBα phosphorylation and degradation. Sequentially, p65 nuclear translocation and DNA binding were inhibited. GT also down-regulated the expression of NFĸB-regulated inflammatory cytokines (IL-8, TNF-α, IL-1α) and caused cell cycle arrest and accumulation of cells in pre-G 1 phase. In vivo, GT (25 mg/kg body weight) injected intraperitoneally (i.p.) prior to or after tumor inoculation significantly decreased the volume of human colon cancer xenografts in NOD/SCID mice. GT-treated xenografts showed significantly lower microvessel density (CD31) as well as lower mRNA expression levels of IL-6, TNF-α and IL-1α and of the proliferation (Ki-67) and angiogenesis (VEGFA) proteins, which may explain GTs in vivo anti-tumorigenic effects. Overall, our results indicate that the anti-inflammatory and antitumor activities of GT may be mediated in part through the suppression of NF-ĸB activation.

(-)-Oleocanthal as a c-Met inhibitor for the control of metastatic breast and prostate cancers.

The proto-oncogene receptor tyrosine kinase c-Met encodes the high-affinity receptor for hepatocyte growth factor (HGF). Dysregulation of the HGF-c-Met pathway plays a significant oncogenic role in many tumors. Overexpression of c-Met is a prognostic indicator for some transitional cell carcinomas. Extra-virgin olive oil (EVOO) provides a variety of minor phenolic compounds with beneficial properties. (-)-Oleocanthal (1) is a naturally occurring minor secoiridoid isolated from EVOO, which showed potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2. It altered the structure of neurotoxic proteins believed to contribute to the debilitating effects of Alzheimer's disease. Computer-Assisted Molecular Design (CAMD) identified 1 as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with an IC (50) range of 10-20 µM, and demonstrated anti-angiogenic activity via downregulating the expression of the microvessel density marker CD31 in endothelial colony forming cells with an IC (50) of 4.4 µM. It inhibited the phosphorylation of c-Met kinase IN VITRO in the Z'-LYTE™ assay, with an IC (50) value of 4.8 µM. (-)-Oleocanthal and EVOO can have potential therapeutic use for the control of c-Met-dependent malignancies.

Intracavernous delivery of synthetic angiopoietin-1 protein as a novel therapeutic strategy for erectile dysfunction in the type II diabetic db/db mouse.

INTRODUCTION: Patients with erectile dysfunction (ED) associated with type II diabetes often have impaired endothelial function and tend to respond poorly to oral phosphodiesterase type 5 inhibitors. Therefore, neovascularization is a promising strategy for curing diabetic ED. AIM: To determine the effectiveness of a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous angiogenesis and erectile function in a mouse model of type II diabetic ED. Methods. Sixteen-week-old male db/db mice (in which obesity and type II diabetes are caused by a mutation in the leptin receptor) and control C57BL/6J mice were used and divided into four groups (N=14 per group): age-matched controls; db/db mice receiving two successive intracavernous injections of phosphate-buffered saline (PBS) (days -3 and 0; 20 µL); db/db mice receiving a single intracavernous injection of COMP-Ang1 protein (day 0; 5.8 µg/20 µL); and db/db mice receiving two successive intracavernous injections of COMP-Ang1 protein (days -3 and 0; 5.8 µg/20 µL). MAIN OUTCOME MEASURES: Two weeks later, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with antibodies to platelet/endothelial cell adhesion molecule-1 (PECAM-1) (endothelial cell marker), phosphohistone H3 (PH3, a nuclear protein indicative of cell proliferation), phospho-endothelial nitric oxide synthase (eNOS), and eNOS. Penis specimens from a separate group of animals were used for cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) quantification. RESULTS: Local delivery of COMP-Ang1 protein significantly increased eNOS phosphorylation and cGMP and cAMP expression compared with that in the group treated with PBS. Repeated intracavernous injections of COMP-Ang1 protein completely restored erectile function and cavernous endothelial content through enhanced cavernous neoangiogenesis as evaluated by PECAM-1 and PH3 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, whereas a single injection of COMP-Ang1 protein elicited partial improvement. CONCLUSION: Cavernous neovascularization using recombinant Ang1 protein is a novel therapeutic strategy for the treatment of ED resulting from type II diabetes.

Blocking platelet/endothelial cell adhesion molecule 1 (PECAM) inhibits disease progression and prevents joint erosion in established collagen antibody-induced arthritis.

Collagen antibody-induced arthritis is a robust murine model of arthritis that histologically recapitulates the inflammatory characteristics of rheumatoid arthritis including pannus formation and destruction of articular cartilage and bone. PECAM is a molecule expressed by both leukocytes and endothelial cells that has been shown to play a major role in the extravasation of leukocytes into sites of inflammation. Genetic deletion of many molecules will blunt the onset and progression of arthritis in murine models, as will administration of various anti-inflammatory therapies given prior to the onset of disease. However, patients seek medical attention when symptomatic, which means that the disease is well established. We investigated whether blocking PECAM interactions would inhibit progression of established disease in the collagen antibody-induced arthritis model. We report that treatment of symptomatic mice with a PECAM-Fc chimera significantly reduced inflammation and virtually eliminated cartilage and bone destruction. The results suggest that therapies that block PECAM function may be beneficial in the treatment of established arthritis.

Effects of niacin on cell adhesion and early atherogenesis: biochemical and functional findings in endothelial cells.

Atherosclerotic cardiovascular disease is a major cause of morbidity and mortality. Increased expression of cell adhesion molecules (CAM) and their ligands mediate essential processes in atherogenesis. Niacin reduces atherosclerotic cardiovascular complications and total mortality. Further understanding is needed on effects of niacin on CAM, and its functional consequences. The aim of this study was to evaluate the effects of niacin on CAM expression and monocyte adhesion in endothelial cells. Endothelial cells (HUVEC) were cultured to reach 80-90% confluence before experiments were initiated. Cells were exposed to DME/F12 with selected concentrations of niacin. To elicit the expression of CAM, cells were stimulated by addition of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 or interferon-gamma. Lysate from the conditioned media was assayed for CAM. The effect of niacin on mRNA expression of ICAM-1 was studied using semi-quantitative analysis of ICAM-1 mRNA. Adhesion assays were performed with flow cytometry to study the functional significance of the effects niacin on CAM expression. Niacin significantly reduced ICAM-1 and PECAM-1 protein levels basally, and reduced the cytokine-induced rise in ICAM-1, with a similar effect for TNF-alpha-induced PECAM-1 rise. The decrease in TNF-alpha-induced rise in ICAM-1 level was associated with a reduction of NF-kappaB activation, a reduction in mRNA expression of ICAM-1, and a functional reduction in monocyte adhesion to cultured endothelial cells. Niacin reduces CAM expression and monocyte adhesion to endothelial cells. Apart from its lipid-modifying effects, these pleiotropic effects of niacin may potentially contribute to the beneficial effects of risk reduction for atherosclerotic disease.

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent.

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.

PECAM-1 modulates thrombin-induced tissue factor expression on endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is known to inhibit platelet function and thrombus formation. The mechanisms involved in PECAM-1's roles as a modulator of hemostasis are still not completely understood. We examined the role of PECAM-1 as a regulator of tissue factor (TF) expression, a known important inducer of thrombosis. Wildtype and CD31KO mice underwent transient (30 min) renal ischemia followed by 24 h re-perfusion and their kidneys assessed for apoptosis, fibrin formation, and tissue factor expression. CD31KO mice exhibited increased tubular epithelial and endothelial apoptosis, increased fibrin deposition, and tissue factor expression. Human umbilical vein endothelial cells (HUVEC) transfected with antisense (AS) PECAM-1 oligonucleotides to downregulate PECAM-1 expression, exhibited greater induction of TF mRNA and protein expression as well as increased expression and nuclear localization of the transcription factor Egr-1 compared to scrambled AS PECAM-1 (Scr)-treated HUVEC following thrombin stimulation. TF induction was found to be mediated through thrombin receptor PAR-1 and the Galphai/o subunit of G-protein, confirmed by PAR-1 antagonist and pertussis toxin inhibition respectively. Thrombin-mediated TF induction was dependent on Rho Kinase activity, phosphorylation of p38(MAPK) and p85 & Akt dephosphorylation. The inverse correlation of PI3K-Akt phosphorylation with p38 (MAPK) phosphorylation was confirmed by pharmacological inhibition. These studies suggest that PECAM-1 is involved in regulating a signaling pathway, affecting PI3K and Akt activation, p38 (MAPK) phosphorylation, which in turn, affects Egr-1 expression and nuclear translocation, ultimately affecting TF expression. These findings provide new insights into the action of PECAM-1 as a modulator of thrombosis.