Celecoxib attenuates interleukin 33-induced expression of vascular cell adhesion molecule-1 in human ovarian endometriotic stromal cells.

OBJECTIVE: Endometriosis is an estrogen-dependent chronic inflammatory disease in women of reproductive age. A review of the literature revealed that cytokines and inflammatory factors are associated with endometriosis-associated infertility. Interleukin 33 (IL-33) is a strong inducer of other pro-inflammatory cytokines. Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in recruiting inflammatory cells, whose expression facilitates leukocyte adhesion and is rapidly induced by pro-inflammatory cytokines. Many studies have indicated that VCAM-1 expression is high in endometriosis; however, whether the expression of VCAM-1 is related to IL-33 is unclear. MATERIALS AND METHODS: Human ovarian endometriotic stromal cells (hOVEN-SCs) were treated with IL-33 to enable investigation of cell characterization, gene and protein expression, and signal pathways. Proliferation potential was measured using an MTT assay. Gene expression was analyzed using reverse transcription-polymerase chain reaction. Protein expression assay was performed using western blot analysis. RESULTS: This study investigated the effects of IL-33 on VCAM-1 and COX-2 expression in hOVEN-SCs. First, the results revealed that the IL-33/ST2/mitogen-activated protein kinase (MAPK) signaling pathway could increase the expression of VCAM-1 and COX-2 in hOVEN-SCs. Second, we discovered that COX-2 expression was essential for IL-33-induced VCAM-1 expression because the effects could be negated through NS398, a selective COX-2 inhibitor. Finally, treatment of IL-33-treated hOVEN-SCs with celecoxib significantly and dose-responsively decreased VCAM-1 expression. CONCLUSION: Taken together, these results indicate that IL-33 can upregulate VCAM-1 expression in hOVEN-SCs through the IL-33/ST2/MAPK/COX-2 signaling pathway and thereby contribute to endometriosis.

Pen Yan Jing Tablets Alleviates Pelvic Inflammatory Disease by Inhibiting Akt/NF-κB Pathway.

Purpose: Pen Yan Jing tablets (PYJ), a Chinese patent medicine, has being used for pelvic inflammatory disease (PID) effectively. This study was designed to explore the underlying mechanisms of PYJ for treating PID. Methods: A rat model of PID was established by mixed bacteria liquid plus mechanical damage. After PYJ treatment, the morphology of uteri and extent of pelvic adhesion were observed. The pathological changes were evaluated by hematoxylin-eosin (HE) staining. The protein expressions of CD68, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) were quantitated by immunohistochemistry. A cell model of lipopolysaccharide (LPS)-activated RAW 264.7 macrophages was performed. The cell proliferation and NO level were measured by CCK-8 and Griess method, respectively. The tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected by ELISA. The protein kinase B (Akt)/nuclear factor kappa-B (NF-κB) pathway-related protein expressions were assayed by western blot or immunofluorescence. Results: PYJ alleviated pelvic adhesion and inflammatory lesions of uteri in PID rats. PYJ down-regulated protein expressions of ICAM-1, VCAM-1, MCP-1, COX-2, p-Akt, p-IκB kinaseα/ß (p-IKKα/ß), p-IκBα, p65, and p-p65 in uteri of PID rats. Moreover, PYJ medicated serum inhibited abnormal cell proliferation, NO release, levels of TNF-α and IL-6, nuclear translocation of p65, and protein expressions of p-Akt, p-p65 and p-IκBα in LPS-activated RAW 264.7 macrophages. Conclusions: Taken together, PYJ may alleviates PID through inhibiting Akt/NF-κB pathway.

Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

Guanxinping ameliorates atherosclerosis via MAPK/NF-κB signaling pathway in ApoE-/- mice.

BACKGROUND: Atherosclerosis (AS), one of the leading causes of deaths and disabilities, is a kind of vascular disease of lipid disorders and chronic inflammation. Guanxinping (GXP) has been administrated in the treatment of AS for nearly 20 years with satisfying clinical response. This study aimed to explore its underlying mechanisms of anti-atherosclerotic effect in AS. METHODS: Male ApoE-/- mice were randomized into five groups and fed with either standard diet (control group, CON) or high-fat diet (HFD) for 12 weeks. HFD mice were further divided randomly and either fed continually with HFD as a model group, or atorvastatin (ATO), or low-dose GXP (LGXP), or high-dose GXP (HGXP). After 12 weeks, the body weight, serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were detected. Moreover, serum inflammation cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) concentrations were measured. The structure of aortic tissues was examined by hematoxylin-eosin staining. The mRNA expression of TNF-α, IL-6, and IL-1ß were assessed by qPCR. The protein expressions of ICAM-1, VCAM-1, MCP-1, IL-6, IL-1ß, p38MAPK, ERK1/2, JNK, IκB-α, and NF-κBp65 in the aorta were also detected. RESULTS: GXP treatment reduced serum TG, TC, and LDL-c levels in ApoE-/- mice. Moreover, GXP reduced lipid accumulation in the aorta of ApoE-/- mice, induced by HFD. Furthermore, GXP ameliorated the aorta morphological damage and reduced the serum TNF-α, IL-6, and IL-1ß levels. GXP also attenuated the protein expression of ICAM-1, VCAM-1, MCP-1, IL-6, IL-1ß, p38MAPK, ERK1/2, JNK, and NF-κBp65, whereas it increased the IκBα level in aortic tissues of ApoE-/- mice. CONCLUSIONS: Our results show that GXP could ameliorate atherosclerosis, which is mediated by inhibition of the MAPK/NF-κB signaling pathway in ApoE-/- mice. This study provides evidence that GXP might be a promising drug for the treatment of AS.

Effects of a high-fat meal on inflammatory and endothelial injury biomarkers in accordance with adiposity status: a cross-sectional study.

BACKGROUND: It is known that consuming a high-fat meal (HFM) induces microvascular dysfunction (MD) in eutrophic women and aggravates it in those with obesity. Our purpose was to investigate if the MD observed after a single HFM intake is caused by endothelial damage or increased inflammatory state, both determined by blood biomarkers. METHODS: Nineteen women with obesity (BMI 30-34.9 kg/m2) and 18 eutrophic ones (BMI 20.0-24.9 kg/m2) were enrolled into two groups: Obese (OBG) and Control (CG), respectively. Blood samples were collected at five-time points: before (fasting state) and 30, 60, 120, and 180 min after HFM intake to determine levels of adipokines (adiponectin, leptin), non-esterified fatty acid (NEFA), inflammatory [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)] and endothelium damage [soluble E-selectin, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1)] biomarkers. RESULTS: Levels of soluble E-selectin, leptin, and PAI-1 were higher in OBG at all-time points (P < 0.05) compared to CG. In the fasting state, OBG had higher levels of NEFA compared to CG (P < 0.05). In intra-group analysis, no significant change in the levels of circulating inflammatory and endothelial injury biomarkers was observed after HFM intake, independently of the group. CONCLUSION: Our findings suggest that women with obesity have an increased pro-inflammatory state and more significant endothelial injury compared to eutrophic ones. However, the consumption of a HFM was not sufficient to change circulating levels of inflammatory and endothelial injury biomarkers in either group. REGISTRATION NUMBER FOR CLINICAL TRIALS: NCT01692327.

Inhibitory effects of 6'-sialyllactose on angiotensin II-induced proliferation, migration, and osteogenic switching in vascular smooth muscle cells.

Excessive production and migration of vascular smooth muscle cells (VSMCs) are associated with vascular remodeling that causes vascular diseases, such as restenosis and hypertension. Angiotensin II (Ang II) stimulation is a key factor in inducing abnormal VSMC function. This study aimed to investigate the effects of 6'-sialyllactose (6'SL), a human milk oligosaccharide, on Ang II-stimulated cell proliferation, migration and osteogenic switching in rat aortic smooth muscle cells (RASMCs) and human aortic smooth muscle cells (HASMCs). Compared with the control group, Ang II increased cell proliferation by activating MAPKs, including ERK1/2/p90RSK/Akt/mTOR and JNK pathways. However, 6'SL reversed Ang II-stimulated cell proliferation and the ERK1/2/p90RSK/Akt/mTOR pathways in RASMCs and HASMCs. Moreover, 6'SL suppressed Ang II-stimulated cell cycle progression from G0/G1 to S and G2/M phases in RASMCs. Furthermore, 6'SL effectively inhibited cell migration by downregulating NF-κB-mediated MMP2/9 and VCAM-1 expression levels. Interestingly, in RASMCs, 6'SL attenuated Ang II-induced osteogenic switching by reducing the production of p90RSK-mediated c-fos and JNK-mediated c-jun, leading to the downregulation of AP-1-mediated osteopontin production. Taken together, our data suggest that 6'SL inhibits Ang II-induced VSMC proliferation and migration by abolishing the ERK1/2/p90RSK-mediated Akt and NF-κB signaling pathways, respectively, and osteogenic switching by suppressing p90RSK- and JNK-mediated AP-1 activity.

Effects of GABAB receptor positive allosteric modulator BHF177 and IRS-1 on apoptosis of hippocampal neurons in rats with refractory epilepsy via the PI3K/Akt pathway.

The present study was conducted to determine the effects of the γ-aminobutyric acid B (GABAB ) receptor positive allosteric modulator BHF177 on refractory epilepsy (RE). An RE rat model was initially established via treatment with lithium-pilocarpine. The RE rats were then treated with BHF177 or the GABAB receptor antagonist CGP46381, followed by recording of their seizure rate and assessment of their spatial learning in the Morris water maze test. Treatment of BHF177 reduced the seizure intensity, whereas this effect was revered upoj treatment with CGP46381. Immunohistochemistry revealed that BHF177 treatment diminished P-glycoprotein (P-gp) expression in the hippocampal tissues of RE rats. Next, we found that BHF177 activated GABAB receptor, resulting in upregulated expression of insulin receptor substrate 1 (IRS-1) and PI3K, as well as antiapoptotic factors (Bcl-2 and mTOR), along with suppression of the apoptosis factors Bax and cleaved caspase-3 in the hippocampal tissues. Further, activation of GABAB receptors by BHF177 alleviated the inflammatory response in hippocampal tissues of RE rats, as evidenced by reduced VCAM-1, ICAM-1, and tumor necrosis factor-α levels. Next, we treated primary cultured rat hippocampal neurons with BHF177 and the IRS-1 selective inhibitor NT157. BHF177 inhibited hippocampal apoptosis in rat hippocampal neurons by regulating the IRS-1/PI3K/Akt axis through crosstalk between GABAB and insulin-like growth factor-1 receptors. Collectively, our findings indicate that the BHF177 inhibited neuron apoptosis, thus protecting against RE through the IRS-1/PI3K/Akt axis, which may present a new therapeutic channel for RE.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

BACKGROUND AND OBJECTIVE: Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000 ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100 ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10 µΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10 µΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. RESULTS: LPS (at 10 ng/mL, 100 ng/mL and 1,000 ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100 ng/mL and 1,000 ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38 MAPK in HUVECs, which was blocked by mecamylamine. CONCLUSIONS: ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

Chondrocytes from osteoarthritic cartilage of obese patients show altered adiponectin receptors expression and response to adiponectin.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months' storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Explants-isolated human placenta and umbilical cord cells share characteristics of both epithelial and mesenchymal stem cells.

In recent years, identification of new sources of adult stem cells developed rapidly, pursuing to find easily available tissues, which will give rise to homogenous stem cells populations. Up to present, bone marrow-derived mesenchymal stem cells (BM-MSCs) are unanimously considered to fulfill the criteria for being used in clinical settings, but adipose stem cells, placental and umbilical cord stem cells, and other tissue-derived stem cells are making their way to being used at least in autologous transplantation. We isolated cellular populations from placental tissue and umbilical cord using the explants method. The placental (PL) and umbilical cord (UC)-derived cells were cultured and expanded in appropriate conditions for generation of stem cells. We assessed the stemness characteristics of the tissue-isolated cells and compared them to an established MSCs line. For this purpose, we determined the immunophenotype, morphological and ultrastructural characteristics, as well as functional abilities of PL- and UC-derived cells. Flow cytometric evaluation of cells revealed presence of CD90, CD73, and CD105 stem cells markers, while the cells were negative for CD34, CD45 and HLA-DR. Immunocytochemical staining showed that 100% of PL- and UC-derived cells are positive for vimentin and CD105 expression, while cytokeratin was revealed in less than 10% in both tissue-isolated cells. Morphological and ultrastructural characteristics of cells exposed analogous cellular size and intracellular organization, similar to MSCs, but detailed view of UC-derived cells by transmission electron microscopy (TEM) demonstrated presence of intercellular junctions-desmosomes, similar to epithelial cells. Both PL- and UC-derived cells confirmed their trilineage potential, being able to differentiate into adipocytes, osteoblasts, and chondrocytes in different proportions. Flow chamber in vitro assay was used to determine to what extent PL- and UC-derived cells are able to adhere to substrates (VCAM and ICAM) and we showed progressively decreased adhesion of both cellular types, inversely proportional to the generated shear stress. We may conclude that explants-isolated placental and umbilical cord cells are endowed with characteristics of both epithelial and mesenchymal stem cells, and purification procedures are additionally required for safe use of these cells in diverse clinical applications.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2.

BACKGROUND: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. METHODS: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. RESULTS: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion- and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFα induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. CONCLUSIONS: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion- and proliferation-stimulating effects of TNFα, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing melanoma cells. These data suggest COX-2 neutralization as a potential anti-metastatic therapy in melanoma patients at high risk of systemic and bone dissemination due to intercurrent infectious and inflammatory diseases.

HUVEC ICAM-1 and VCAM-1 synthesis in response to potentially athero-prone and athero-protective mechanical and nicotine chemical stimuli.

Previous mechano-transduction studies have investigated the endothelial cell (EC) morphological response to mechanical stimuli; generally consisting of a wall shear stress (WSS) and a cyclic tensile hoop strain (THS). More recent studies have investigated the EC biochemical response (intercellular adhesion molecule, ICAM-1, and vascular cellular adhesion molecule, VCAM-1, expression) to idealized mechanical stimuli. However, current literature is lacking in the area of EC biochemical response to combinations of physiological WSS and THS mechanical stimuli. The objective of this study is to investigate the EC response to physiological WSS and THS stimuli and to compare this response to that of ECs exposed to idealized steady WSS and cyclic THS of the same magnitudes. This study also investigated the EC response to a nicotine chemical stimulus combined with a suspected athero-prone physiological mechanical stimulus. A bioreactor was designed to apply a range of combinations of physiological WSS and THS waveforms. The bioreactor was calibrated and validated using computational fluid dynamics and video extensometry techniques. The bioreactor was used to investigated the biochemical response exhibited by human umbilical vein endothelial cells (HUVECs) exposed to physiological athero-protective (first bioreactor test case, pulsatile WSS combined with pulsatile THS) and athero-prone (second bioreactor test case, oscillating WSS combined with pulsatile THS) mechanical environments. The final testing environment (third bioreactor test case) combined a nicotine chemical stimulus with the mechanical stimuli of the second bioreactor test case. In first and second bioreactor test cases, the addition of a pulsatile THS to the WSS resulted in opposite trends of ICAM-1 down-regulation and up-regulation, respectively. This outcome suggests that the effect of the additional pulsatile THS depends on the state of the applied WSS waveform. Similarly, in first and second bioreactor test cases, the addition of a pulsatile THS to the WSS resulted in a VCAM-1 up-regulation. However, it has been previously shown that the addition of a cyclic THS to a high- or low-steady WSS resulted in a VCAM-1 down-regulation, indicating that the EC response to idealized mechanical stimuli (steady WSS and cyclic THS) is not comparable to physiological mechanical stimuli (unsteady WSS and pulsatile THS), even though in both situations the average magnitude of WSS and THS applied were similar. In third bioreactor test case, a nicotine chemical stimulus induced a substantial VCAM-1 up-regulation and a moderate ICAM-1 up-regulation. The addition of the mechanical stimuli of the second bioreactors test case resulted in a greater VCAM-1 up-regulation than what was expected, considering the observations of the previous second bioreactor test case alone. This study found that the EC biochemical response to physiological mechanical stimuli is not comparable to the previously observed EC response to idealized mechanical stimuli, even though in both environments the mechanical stimuli were of a similar magnitude. Also, the level of VCAM-1 expressed by the nicotine stimulated ECs showed an elevated level of sensitivity to the athero-prone mechanical stimuli.

Eosinophil superoxide anion generation induced by adhesion molecules and leukotriene D4.

RATIONALE: Eosinophils preferentially accumulate at sites of inflammation in the asthmatic airway. Participation of circulating eosinophils in the airway inflammation in asthma involves their interaction with adhesion molecules expressed on the endothelial cell surface and exposure to inflammatory mediators, such as cysteinyl leukotrienes (cysLTs). OBJECTIVE: To investigate whether interaction of eosinophils with adhesion molecules modifies the functions of these cells induced by cysLTs. METHODS: Eosinophils were isolated from the blood of healthy donors, incubated in the EIA plates coated with adhesion proteins, and then exposed to LTD4. The generation of superoxide anion (O2-), adhesion to the plates, and release of eosinophil-derived neutrotoxin (EDN) were evaluated. RESULTS: Neither VCAM-1 nor LTD4 (100 nM) independently induced eosinophil O2- generation, however, combined exposure to the two molecules synergistically induced eosinophil O2- generation. ICAM-1 by itself induced eosinophil O2- generation, which was enhanced by LTD4. On the contrary, P-selectin did not induce O2- generation, either in the presence or absence of LTD4. LTD4 significantly enhanced eosinophil adhesion to rh-VCAM-1 and rh-ICAM-1, but not to rh-P-selectin. Finally, we observed that combined exposure of eosinophils to LTD4 and VCAM-1 induced the release of EDN. CONCLUSION: Combined exposure to VCAM-1 or ICAM-1 and cysLT effectively induces the effector functions of eosinophils. Eosinophil adhesion to and migration across endothelial cells via these specific adhesion proteins and subsequent exposure to cysLTs may be mechanisms underlying activation of the effector functions of eosinophils in the asthmatic airway.

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes.

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.

Nitric oxide reduces T lymphocyte adhesion to human brain microvessel endothelial cells via a cGMP-dependent pathway.

The entry of lymphocytes into the brain is normally limited by the blood-brain barrier, however, during inflammation prominent lymphocytic infiltration occurs. In this study, we investigated the effects of nitric oxide (NO) on the adhesion of T cells to cultured human brain microvessel endothelial cells. T cell adhesion to unstimulated or tumor necrosis factor-alpha (TNF-alpha)-treated cells was quantified by counting the number of lymphocytes bound to the monolayer by light microscopy. TNF-alpha increased T cell adhesion in a time-dependent manner. Incubation of monolayers with NO donors decreased adhesion. This effect was blocked by a guanylyl cyclase inhibitor and mimicked by a cGMP agonist, and was thus dependent on the generation of cGMP. NO did not modulate adhesion molecule expression in the endothelial cells, suggesting an action on the T cells. Pre-treatment of T cells with NO or a cGMP agonist decreased binding to recombinant endothelial adhesion molecules. These findings suggest that NO can modulate the adhesion of T cells to human brain microvessel endothelial cells via a cGMP-dependent mechanism, and may thus regulate lymphocyte traffic during central nervous system inflammation.

The extracellular matrix differentially regulates the expression of PTHrP and the PTH/PTHrP receptor in FG pancreatic cancer cells.

OBJECTIVES: Previous studies by our laboratory have demonstrated that parathyroid hormone-related protein (PTHrP) and its receptor (PTH/PTHrP receptor) are commonly expressed in pancreatic cancer and suggest their participation in the progression of this devastating disease. It has also been demonstrated that one of the major hallmarks of pancreatic adenocarcinoma is an increased production of the extracellular matrix (ECM), a critical regulator of diverse cellular processes such as differentiation, proliferation, and angiogenesis. The present study focused on the relationship between the PTHrP and ECM axes in the pathobiology of pancreatic cancer. METHOD AND RESULTS: Using the FG pancreatic adenocarcinoma cell line, we demonstrate a significant inverse correlation between FG cell proliferation and PTHrP expression that depended on the ECM protein on which the cells were cultured (P < 0.05). Generally, ECM proteins that promoted the strongest proliferation, including type I collagen, type IV collagen, and laminin, resulted in decreased expression of PTHrP. Conversely, ECM proteins that promoted the weakest proliferation, including fibronectin, vitronectin, and BSA, resulted in increased expression of PTHrP. A similar trend was found between FG cell proliferation and the PTH/PTHrP receptor expression, with Pearson correlation coefficients of 0.480 (mRNA) and -0.591 (protein). CONCLUSION: These observations demonstrate a unique functional relationship between the ECM and PTHrP axes and have important implications for our understanding of the complex mechanisms responsible for the progression of pancreatic cancer and its metastases.

Role of alpha4 integrin and its ligand VCAM-1 in the specific extravasation of a tumor-specific TH2 clone into tumor tissue that initiates its rejection.

The effectiveness of anticancer immunotherapeutic strategies involving the transfer of tumor-specific T cells depends on appropriate lymphocyte-endothelial cell interactions that facilitate the migration of lymphocytes into tumor. Here, we investigated the molecular mechanisms underlying the migration of the antigen-specific Th2 CD4(+) T-cell clone YS1093 into S1509a tumor tissue. YS1093 is specific for the S1509a tumor but does not recognize the S713a tumor. Transfer of YS1093 cells into mice bearing both S1509a and S713a tumors caused only the S1509a tumor to regress. This regression was markedly inhibited by pretreating YS1093 cells with an anti-alpha4 integrin MAb and administering an anti-VCAM-1 MAb at T-cell transfer. Since vascular endothelial cells in S1509a tumor tissues express VCAM-1 and the MHC class II (I-E(k)) molecule restricting YS1093 activity, labeled YS1093 cells migrated specifically into the S1509a tumor, and this migration was also blocked by the anti-TCRbeta F(ab')(2) and anti-I-E(k) MAbs. Furthermore, in vitro assays revealed that anti-CD3 MAb-mediated TCR cross-linkage initiated the binding of alpha4 integrin on YS1093 cells to VCAM-1. This adhesive activity was completely blocked by the anti-alpha4 integrin MAb. These results strongly suggest that i.v.-transferred YS1093 cells act in tumor regression by specifically recognizing their tumor antigen peptide in the context of I-E(k) on vascular endothelial cells in the S1509a tumor, which activates the binding of alpha4 integrin to VCAM-1 on the endothelial cells, facilitating YS1093 extravasation into the tumor. It is likely that this initial migration of specific CD4(+) T cells into tumor tissues promotes the subsequent infiltration into the tumor of other immunocytes that effect tumor destruction.

Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo.

We examined the function of alpha4beta1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. Alpha4beta1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by alpha4beta1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an alpha4beta1-dependent manner. Soluble alpha4beta1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1-/- mice. Antagonizing alpha4beta1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the alpha4beta1 binding sequence stimulate angiogenesis in vivo. Therefore, alpha4beta1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Integrin alpha4beta1 (VLA-4) expression and activity in retinal and peripheral neurons.

The integrin alpha4beta1 fulfills important roles in inflammation and hematopoesis, but its functions in neurons are not well understood. Here we show that the alpha4 subunit is expressed on mouse retinal ganglion cells (RGCs) and undifferentiated retinal neuroblasts during the period of axon extension and migration. To determine if alpha4 integrins expressed by retinal neurons were active, neurons were cultured on known alpha4 ligands in vitro. Recombinant soluble vascular cell adhesion molecule 1 (rsVCAM-1), fibronectin, and osteopontin (OPN) induced neurite outgrowth that was diminished by function blocking antibodies specific for alpha4. Neurite outgrowth on OPN was also blocked by antibodies to the integrin beta1 subunit, implicating the alpha4beta1 heterodimer as one integrin receptor mediating outgrowth on OPN. OPN immunoreactivity was detected in the RGC fiber layer and optic nerve, suggesting that it may act as an alpha4 ligand in vivo. Neurons from chick lumbar sympathetic ganglia, chick dorsal root ganglia, and mouse superior cervical ganglia also extended neurites on rsVCAM-1, suggesting that integrin alpha4beta1 may play a role in the development of multiple neuronal cell types.