Upregulation Of Vascular Cell Adhesion Molecule-1 Research Articles

Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1β, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

SVCAM1 in the Hippocampus Contributes to Postoperative Cognitive Dysfunction in Mice by Inducing Microglial Activation Through the VLA-4 Receptor.

Postoperative cognitive dysfunction (POCD) is a severe postsurgical complication, but its underlying mechanisms remain unclear. Neuroinflammation mediated by microglial activation plays a major role in POCD pathophysiology. Upregulation of vascular cell adhesion molecule 1 (VCAM1) on brain endothelial cells is closely correlated with microglial activation in the mouse hippocampus. However, the role of VCAM1 upregulation in microglial activation remains unknown. Soluble VCAM1 (sVCAM1) activates the very late antigen-4 (VLA-4) receptor under inflammatory conditions. Therefore, we hypothesized that sVCAM1 which is shed from VCAM1 contributes to POCD by triggering hippocampal microglial activation through the VLA-4 receptor. We found that VCAM1 and sVCAM1 expression in the mouse hippocampus was upregulated after surgery, and the upregulation was accompanied by hippocampal microglial activation. sVCAM1 levels in mouse and human serum were increased after surgery. Anti-VCAM1 treatment inhibited microglial activation, proinflammatory cytokine production, VLA-4 expression and P38 mitogen-associated protein kinase (MAPK) pathway activation and attenuated hippocampal-dependent cognitive dysfunction. In vitro, recombinant sVCAM1 promoted M1 polarization in BV2 cells, increased VLA-4 expression and activated the P38 MAPK pathway. These effects were reversed by VLA-4 receptor blockade. Anti-VLA-4 treatment ameliorated hippocampal-dependent cognitive dysfunction after surgery by inhibiting microglial activation, proinflammatory cytokine production and P38 pathway activation. In conclusion, increased sVCAM1 in the hippocampus is involved in microglial activation and cognitive dysfunction induced by surgery. Inhibiting the sVCAM1-VLA-4 interaction in microglia may be a therapeutic strategy for POCD.

VCAM-1-targeted MRI Improves Detection of the Tumor-brain Interface.

Despite optimal local therapy, tumor cell invasion into normal brain parenchyma frequently results in recurrence in patients with solid tumors. The aim of this study was to determine whether microvascular inflammation can be targeted to better delineate the tumor-brain interface through vascular cell adhesion molecule-1 (VCAM-1)-targeted MRI. Intracerebral xenograft rat models of MDA231Br-GFP (breast cancer) brain metastasis and U87MG (glioblastoma) were used to histologically examine the tumor-brain interface and to test the efficacy of VCAM-1-targeted MRI in detecting this region. Human biopsy samples of the brain metastasis and glioblastoma margins were examined for endothelial VCAM-1 expression. The interface between tumor and surrounding normal brain tissue exhibited elevated endothelial VCAM-1 expression and increased microvessel density. Tumor proliferation and stemness markers were also significantly upregulated at the tumor rim in the brain metastasis model. T2*-weighted MRI, following intravenous administration of VCAM-MPIO, highlighted the tumor-brain interface of both tumor models more extensively than gadolinium-DTPA-enhanced T1-weighted MRI. Sites of VCAM-MPIO binding, evident as hypointense signals on MR images, correlated spatially with endothelial VCAM-1 upregulation and bound VCAM-MPIO beads detected histologically. These findings were further validated in an orthotopic medulloblastoma model. Finally, the tumor-brain interface in human brain metastasis and glioblastoma samples was similarly characterized by microvascular inflammation, extending beyond the region detectable using conventional MRI. This work illustrates the potential of VCAM-1-targeted MRI for improved delineation of the tumor-brain interface in both primary and secondary brain tumors.

Improved impedance to maladaptation and enhanced VCAM-1 upregulation with resistance-type training in the long-lived Snell dwarf (Pit1dw/dw) mouse.

Snell dwarf mice with the Pit1dw/dw mutation are deficient in growth hormone, prolactin, and thyroid stimulating hormone and exhibit >40% lifespan extension. This longevity is accompanied by compromised muscular performance. However, research regarding young (3-month-old) Snell dwarf mice demonstrate exceptional responsivity to resistance-type training especially in terms of a shifted fiber type distribution and increased protein levels of vascular cell adhesion molecule-1 (VCAM-1), a possible mediator of such remodeling. In the present study, we investigated whether this responsiveness persists at 12 months of age. Unlike 12-month-old control mice, age-matched Snell dwarf mice remained resistant to training-induced maladaptive decreases in performance and muscle mass. This was accompanied by retainment of the remodeling capacity in muscles of Snell dwarf mice to increase VCAM-1 protein levels and a shift in myosin heavy chain (MHC) isoform distribution with training. Even decreasing training frequency for control mice, an alteration which protected muscles from maladaptation at 12 months of age, did not result in the overt remodeling observed for Snell dwarf mice. The results demonstrate a distinct remodeling response to resistance-type exercise operative in the context of the Pit1dw/dw mutation of long-lived Snell dwarf mice.

Lysophosphatidylcholine promotes intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells via an orphan G protein receptor 2-mediated signaling pathway

ABSTRACT The oxLDL-based bioactive lipid lysophosphatidylcholine (LPC) is a key regulator of physiological processes including endothelial cell adhesion marker expression. This study explored the relationship between LPC and the human umbilical vein endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with a particular focus on the regulation of the LPC-G2A-ICAM-1/VCAM-1 pathway in this context. We explored the LPC-inducible role of orphan G protein receptor 2 (G2A) in associated regulatory processes by using human kidney epithelial (HEK293) cells that had been transfected with pET-G2A, human umbilical vein endothelial cells (HUVECs) in which an shRNA was used to knock down G2A, and western blotting and qPCR assays that were used to confirm changes in gene expression. For in vivo studies, a rabbit model of atherosclerosis was established, with serum biochemistry and histological staining approaches being used to assess pathological outcomes in these animals. The treatment of both HEK293 cells and HUVECs with LPC promoted ICAM-1 and VCAM-1 upregulation, while incubation at a pH of 6.8 suppressed such LPC-induced adhesion marker expression. Knocking down G2A by shRNA and inhibiting NF-κB activity yielded opposite outcomes. The application of a Gi protein inhibitor had no impact on LPC-induced ICAM-1/VCAM-1 expression. Atherosclerotic model exhibited high circulating LDL and LPC levels as well as high aortic wall ICAM-1/VCAM-1 expression. Overall, these results suggested that the LPC-G2A-ICAM-1/VCAM-1 pathway may contribute to the atherogenic activity of oxLDL, with NF-κB antagonists representing potentially viable therapeutic tools for the treatment of cardiovascular disease.

Type 2 Diabetes Mellitus Provokes Rat Immune Cells Recruitment into the Pulmonary Niche by Up-regulation of Endothelial Adhesion Molecules.

Purpose: Diabetes mellitus, especially type 2, is conceived as a devastating chronic metabolic disease globally. Due to the existence of an extensive vascular network in the pulmonary tissue, it is suggested that lungs are sensitive to the diabetic condition like other tissues. This study was designed to address the possible effect of type 2 diabetes mellitus on the promotion of pathological changes via vascular injury. Methods: Sixteen male Wistar rats were randomly allocated to the two of control and T2D groups. To induce type 2 diabetes (T2D), rats were received high-fat and a single dose of streptozotocin (STZ). On week 12, rats were euthanized and lungs samples were taken. Using hematoxylin and eosin (H&E) staining, the pathological changes were monitored. The expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and interleukin 10 (IL-10) was monitored using real-time PCR assay. The level of tumor necrosis factor-α (TNF-α) was detected using ELISA assay. Nitrosative stress was monitored using the Griess assay. Results: Pathological examination in bronchoalveolar discharge revealed the existence of mild to moderate interstitial bronchopneumonia and increased neutrophilic leukocytosis compared to the control. Enhanced ICAM-1 and VCAM-1 expression and suppression of IL-10 was found using real-time PCR analysis (P < 0.05). The levels of TNF-α and NO were increased with diabetic changes compared to the control rats (P < 0.05). Conclusion: T2D could promote pulmonary tissue injury via the production of TNF-α and up-regulation of vascular ICAM-1 and VCAM-1. The inflammatory status and vascular ICAM-1 and VCAM-1 increase immune cell recruitment into the pulmonary niche.

Quercetin inhibits TNF-α induced HUVECs apoptosis and inflammation via downregulating NF-kB and AP-1 signaling pathway in vitro.

Background:Quercetin, a major flavonol, wildly exists in plantage, which has been reported to have an anti-apoptosis and anti-inflammation effects on vascular endothelial cells, but its underlying molecular mechanisms remain unclear.Objective:The aim of this study was to investigate the mechanisms of how quercetin inhibits tumor necrosis factor alpha (TNF-α) induced human umbilical vein endothelial cells (HUVECs) apoptosis and inflammation.Methods and Results:HUVECs were preconditioned with quercetin for 18 hours, and subsequently treated with TNF-α for 6 hours to induce apoptosis. The expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, β-actin mRNA was then detected by RT-PCR. Flow cytometry was used to estimate the apoptosis rates, and the expression of activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) was measured by Western blot. TNF-α induced elevated apoptosis rates and upregulation of VCAM-1, ICAM-1, and E-selectin were meaningfully reduced in HUVECs by pretreatment with quercetin. In addition, quercetin also inhibited the activation of AP-1and NF-κB.Conclusion:Results indicate that quercetin could suppress TNF-α induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling pathway in HUVECs, which might be one of the underlying mechanisms in treatment of coronary heart disease.

Resveratrol prevents TNF-α-induced VCAM-1 and ICAM-1 upregulation in endothelial progenitor cells via reduction of NF-κB activation.

ObjectiveTo assess the effects of resveratrol (RSV) on expression of adhesion molecules in endothelial progenitor cells (EPCs) following tumor necrosis factor-α (TNF-α) stimulation.MethodsEPCs were treated with RSV and stimulated with TNF-α. A mononuclear cell (MNC) adhesion assay was used to assess the effects of RSV on TNF-α-induced MNC adhesion. Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression levels and nuclear factor κB (NF-κB) activation were assessed by immunoblotting.ResultsMNC adhesion to TNF-α-treated EPCs and VCAM-1/ICAM-1/E-selectin levels in EPCs were increased following TNF-α stimulation and decreased following RSV treatment. TNF-α enhanced NF-κB inhibitor α (IκB-α) phosphorylation in the cytosol as well as nuclear NF-κB p65 levels, both of which were decreased by RSV.ConclusionsThese findings provide new insights into RSV’s anti-inflammatory and anti-atherosclerotic effects. RSV’s mechanism of action might involve downregulation of VCAM-1, ICAM-1 and E-selectin by partial blockade of TNF-α-induced NF-κB activation and IκB-α phosphorylation in EPCs.

Chemokine-like factor 1 (CKLF1) aggravates neointimal hyperplasia through activating the NF-κB /VCAM-1 pathway.

Neointimal hyperplasia (NIH) is a complicated inflammatory process contributing to vascular restenosis. The present study aimed to explore whether chemokine-like factor 1 (CKLF1) aggravates NIH via the nuclear factor-kappa B (NF-κB)/vascular cell adhesion molecule-1 (VCAM-1) pathway. We found the expression of CKLF1 and VCAM-1 significantly increased in human carotid plaques compared to the control. Invivo, CKLF1 overexpression induced a thicker neointimal formation and VCAM-1 expression was correspondingly upregulated. Invitro, CKLF1 activated NF-κB and induced VCAM-1 upregulation in human aortic smooth muscle cells (HASMCs). Functional experiments demonstrated that CKLF1 promoted monocyte adhesion and HASMC migration via VCAM-1. These results suggest CKLF1 accelerates NIH by promoting monocyte adhesion and HASMC migration via the NF-κB/VCAM-1 pathway. Our findings contribute to a better understanding of the mechanisms underlying the causality of CKLF1 on NIH and could prove beneficial in designing therapeutic modalities with a focus on CKLF1.

Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice.

Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE-/-Fbn1C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE-/-Fbn1C1039G+/- mice after 10 weeks of treatment. Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.

Abstract 86: Pre-existing neurovascular inflammation increases the occurrence of brain metastases

Abstract BACKGROUND: Brain metastases (BM) are the most prevalent intracranial neoplasm. Inflammation is central to the development of cancer. While an intra-tumoral inflammatory microenvironment contributes to the acquisition of malignant phenotypes and leads to the release of circulating tumor cells (CTCs), pre-existing inflammation at distant sites facilitates the adhesion of CTCs to the activated vascular endothelium and the consequent formation of metastases. Cell adhesion molecules expressed by activated endothelial cells contribute to metastatic spread outside of the brain and the vascular cell adhesion molecule-1 (VCAM-1) is a key mediator of inflammation. Herein we assessed if cancer cells entry into the brain is aided by VCAM-1 upregulation with inflammation. METHODS: Stereotaxic lipopolysaccharide (LPS, 1 µg) injection into the right hemisphere was used to induce neurovascular inflammation in Balb/c mice. The distribution of VCAM-1 was semi-quantified 24 h post LPS injection using molecular magnetic resonance imaging (MRI) with microparticles of iron oxide (MPIOs) functionalized with VCAM-1 antibody (MPIO-VCAM-1). Mice injected with saline served as control. VCAM-1 is also expressed on vessels associated with metastases, MPIO-VCAM-1 were therefore used to detect metastases. Basal level of metastases in animal brains was measured in a group of mice intracardially injected with cancer cells without LPS intra-cortical injection. To study the impact of pre-existing inflammation on tumor cell entry into the brain, mice were injected with LPS (or saline as above) 24 h prior to intracardiac injection of 4T1 breast cancer cells (105 cells in 100 µL PBS). Metastases imaging was performed 18 days post cancer cells injection. To assess if blocking VCAM-1 would affect metastases implantation, an extra LPS-injected group of mice was injected intravenously with MPIO-VCAM-1 4 hours before 4T1 cells injection. In this group, MRI was performed 3 hours after MPIO-VCAM-1 injection (VCAM-1 imaging), and on day 18 post tumor cell injection (metastases imaging). All MRI experiments were conducted on a small animal 7T scanner (Varian Inc.) with a dedicated mouse head-coil (RAPID MR International) using a T2*-weighted sequence. Following the final imaging session brains were extracted for histological analysis. RESULTS: Both MR and histological data reveal that the metastatic burden significantly increases in the LPS-injected group compared to control conditions. In inflamed animals, blocking VCAM-1 with MPIO-VCAM-1 reduces the metastatic burden back to control values. This suggests that (1) a pre-existing inflammation increases the occurrence of brain metastases and (2) blocking VCAM-1 reduces this effect. CONCLUSION: We demonstrate that inflammation-induced VCAM-1 contributes to tumor cells adhesion in the brain. Therefore, VCAM-1 may represent an attractive therapeutic target to reduce risks of metastasis. Citation Format: Dina Sikpa, Lisa Whittingstall, Jérémie P. Fouquet, Luc Tremblay, Réjean Lebel, Martin Lepage. Pre-existing neurovascular inflammation increases the occurrence of brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 86.

Visfatin Promotes Monocyte Adhesion by Upregulating ICAM-1 and VCAM-1 Expression in Endothelial Cells via Activation of p38-PI3K-Akt Signaling and Subsequent ROS Production and IKK/NF-κB Activation.

Visfatin is known to act as a mediator in several metabolic disorders, such as obesity, diabetes, and cardiovascular diseases. This study aimed to investigate the effect of visfatin on the adhesion of THP-1 monocytes to human vascular endothelial cells and the underlying mechanism. Monocytes adhesion to endothelial cells was determined by using fluorescence-labeled monocytes. ICAM-1 and VCAM-1 expression in endothelial cells were measured by western blotting. Production of reactive oxygen species (ROS) was measured by using a fluorescent dye. The amounts of nuclear factor-kappa B (NF-κB) and phosphorylation of inhibitory factor of NF-κB (IκB) were determined by using western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined by using immunofluorescence. Here we showed that visfatin significantly caused the upregulation of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, as well as enhanced monocyte adhesion to endothelial cells. Moreover, we found that inhibition of PI3K, Akt, and p38 MAPK activation significantly prevented visfatin-enhanced expression of ICAM-1 and VCAM-1 and monocyte adhesion to endothelial cells. Visfatin enhanced ROS production and IKK/NF-кB activation and then led to upregulation of ICAM-1 and VCAM-1 and enhanced monocyte adhesion to endothelial cells. These effects were also p38/PI3K/Akt-dependent. These results demonstrated that visfatin promoted monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression via the activation of p38/PI3K/Akt signaling and downstream ROS production and IKK/NF-кB activation.

VCAM-1-targeted MRI Enables Detection of Brain Micrometastases from Different Primary Tumors.

A major issue for the effective treatment of brain metastasis is the late stage of diagnosis with existing clinical tools. The aim of this study was to evaluate the potential of vascular cell adhesion molecule 1 (VCAM-1)-targeted MRI for early detection of brain micrometastases in mouse models across multiple primary tumor types.Experimental Design: Xenograft models of brain micrometastasis for human breast carcinoma (MDA231Br-GFP), lung adenocarcinoma (SEBTA-001), and melanoma (H1_DL2) were established via intracardiac injection in mice. Animals (n = 5-6/group) were injected intravenously with VCAM-1-targeted microparticles of iron oxide (VCAM-MPIO) and, subsequently, underwent T 2*-weighted MRI. Control groups of naïve mice injected with VCAM-MPIO and tumor-bearing mice injected with nontargeting IgG-MPIO were included. All models showed disseminated micrometastases in the brain, together with endothelial VCAM-1 upregulation across the time course. T 2*-weighted MRI of all tumor-bearing mice injected with VCAM-MPIO showed significantly more signal hypointensities (P < 0.001; two-sided) than control cohorts, despite a lack of blood-brain barrier (BBB) impairment. Specific MPIO binding to VCAM-1-positive tumor-associated vessels was confirmed histologically. VCAM-1 expression was demonstrated in human brain metastasis samples, across all three primary tumor types. VCAM-1-targeted MRI enables the detection of brain micrometastases from the three primary tumor types known to cause the majority of clinical cases. These findings represent an important step forward in the development of a broadly applicable and clinically relevant imaging technique for early diagnosis of brain metastasis, with significant implications for improved patient survival.

Histone H3 Serine 10 Phosphorylation Facilitates Endothelial Activation in Diabetic Kidney Disease

Post-translational modification of histone proteins epigenetically affects gene transcription. The histone modifications that have been most extensively linked to the development of diabetes complications are histone (de)methylation and (de)acetylation, whereas other modifications have been comparatively overlooked. Here, by examining the means by which podocytes may influence the glomerular endothelial phenotype, we identified a role for the phosphorylation of histone H3 on serine residue 10 (H3Ser10) in mediating endothelial activation in diabetes. Culture media conditioned by high glucose-exposed podocytes induced glomerular endothelial vascular cell adhesion molecule-1 (VCAM-1) upregulation and was enriched for the chemokine CCL2 in a JAK2-dependent manner. Antagonism of CCR2, the cognate receptor for CCL2, decreased the endothelial VCAM-1 upregulation that was induced either by conditioned medium or by recombinant CCL2, whereas CCR2 knockout mice exhibited diminished glomerular VCAM-1 upregulation when challenged by diabetes. Recombinant CCL2 induced glomerular endothelial VCAM-1 upregulation through a pathway mediated by p38 MAPK, its nuclear substrates MSK1/2 and subsequent H3Ser10 phosphorylation, which was enriched at the VCAM-1 promoter. H3Ser10 phosphorylation levels were elevated in the kidneys of streptozotocin-diabetic endothelial nitric oxide synthase knockout (STZ-eNOS-/-) mice and in the glomeruli of humans with diabetic kidney disease. Finally, treatment of STZ-eNOS-/- mice with an inhibitor of JAK2 decreased both urinary CCL2 excretion and renal H3Ser10 phosphorylation. Collectively, these findings indicate that histone phosphorylation promotes endothelial activation in diabetes. Histone protein phosphorylation should be set alongside other more canonical histone modifications when considering chromatin modifications that influence diabetic kidney disease and that may be amenable to therapeutic intervention. Disclosure T. Alghamdi: None. S. Batchu: None. M.J. Hadden: None. Y. Liu: None. B. Bowskill: None. L. Geldenhuys: None. F.S. Siddiqi: Research Support; Self; AstraZeneca. S. Majumder: None. A. Advani: Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH. Other Relationship; Self; Boehringer Ingelheim GmbH.

Monitoring inflammation injuries in the progression of atherosclerosis with contrast enhanced ultrasound molecular imaging.

PurposeThe upregulation of vascular cell adhesion molecule-1(VCAM-1) on vascular endothelium plays a great role in the progression of atherosclerosis (AS). In this study, ultrasound molecular imaging was performed to monitor the inflammation injuries in the onset and progression of atherosclerosis with microbubbles targeted to VCAM-1.MethodsMice deficient for the apolipoprotein E (ApoE-/-mice) with high-cholesterol diet were studied as an age-dependent model of atherosclerosis. At 8, 16, 24, and 32 weeks of age, contrast enhanced ultrasound (CEU) molecular imaging of proximal ascending aorta was performed with microbubbles targeted to VCAM-1. Plaque size, monocytes infiltration and the expression of VCAM-1 in the proximal ascending aorta were assessed by histology and western blot analysis, separately.ResultsIn ApoE-/- mice, molecular imaging for VCAM-1 detected selective signal enhancement (P<0.01 versus non-targeted microbubbles) at all ages of ApoE-/- mice. Moreover, signals from targeted microbubbles increased from 8wks to 32wks age (P<0.05 for trend) in ApoE-/- mice, indicating the upregulation of VCAM-1 with the progression of atherosclerosis. Consistent with CEU imaging results, both western blot analysis and immunohistochemistry revealed the expression of VCAM-1 and monocytes infiltration were age-dependent in ApoE-/- mice.ConclusionsCEU molecular imaging can be used to noninvasively detect the VCAM-1 expression on the endothelium in the progression of atherosclerosis. By investigating specific molecular biomarkers, it could help to monitor the inflammation and the progression of AS, which may in some extent contribute to the prediction of vulnerable plaque.

MiR-181b modulates EGFR-dependent VCAM-1 expression and monocyte adhesion in glioblastoma

Tumor-associated macrophages (TAMs) originate as circulating monocytes, and are recruited to gliomas, where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells, which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here, we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM, and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly, upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression, and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover, GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures, we found that monocyte adhesion to GBM occurs via integrin α4β1, which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach, we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family, and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation, which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM, which raises the possibility of developing effective and improved therapies for GBM.

Abstract 543: Myeloperoxidase Induces Endothelial Dysfunction via Activation of Calpain

Myeloperoxidase (MPO) is a peroxidase enzyme secreted by activated leukocytes, which has been associated with endothelial dysfunction and insulin resistance. The calcium dependent protease calpain has also been linked to vascular disease in insulin resistance and type 2 diabetes. Accordingly, we tested the hypothesis that endothelial expressed calpains play a role in MPO-induced endothelial dysfunction and vascular inflammation. Mouse lung microvascular endothelial cells (MMVEC) were stimulated with 10 nM MPO for 30, 60, 120, 180, and 240 minutes. Expression levels of Vascular Cell Adhesion Molecule 1 (VCAM-1), 5’ AMP Activated Protein Kinase (AMPK), Protein Kinase B (PKB) (Akt), endothelial Nitric Oxide (eNOS) phosphorylation at serine 1177 (Se1177), Protein Phosphatase 2 (PP2A), and calpains were measured by immunoblot analyses. MPO time dependently activated μ-calpain (P&lt;0.0001 vs control) but not m-calpain. MPO also significantly increased PP2A protein levels (P&lt;0.001), decreased AMPK phosphorylation (P&lt;0.01), AKT phosphorylation (P&lt;0.05), and eNOS phosphorylation at (Se1177) (P&lt;0.01), while significantly increased VCAM-1 protein levels (P&lt;0.01). Pretreatment of MMVECs with the selective calpain inhibitor ZLLal (100 μM) prevent MPO-induced calpain activation (P&lt;0.0001 vs MPO alone). Inhibition of calpain activity also preserved AMPK phosphorylation, eNOS phosphorylation, and PP2A levels, in the face of MPO (P&lt;0.001 versus MPO alone). Calpain inhibition also prevented upregulation of VCAM-1. Using an ex vivo leukocyte adhesion assay we also found leukocytes failed to adhere to the vascular endothelium of MPO treated aortas isolated from μ-calpain deficient mice (p&lt;0.001 versus aortas isolated from WT mice). Taken together, our data first demonstrate a primary role for μ-calpain in endothelial dysfunction induced by leukocyte derived MPO.

Abstract 164: Endothelial Gab1 Limits Vascular Remodeling and Atherosclerosis Through Inhibition of Endothelial Inflammation

Objective: We have previously shown that docking protein Grb2-associated binder 1 (Gab1) is a mechano-effector protein in response to fluid shear stress and regulates postnatal angiogenesis. The aim of this study was to determine the in vivo role of endothelial Gab1 in flow-mediated vascular remodeling and atherosclerosis and explore the underlying mechanisms. Methods and Results: To determine the role of endothelial Gab1 in disturbed flow-induced vascular remodeling in vivo, we performed partial carotid artery ligation in Gab1 endothelium-restricted knockout (Gab1-ecKO) mice and wild-type (WT) littermates, and we observed that Gab1-ecKO mice resulted in increased intima-media thickness. To examine the role of endothelial Gab1 in atherosclerosis, we next crossed Gab1-ecKO mice with ApoE KO mice. After partial ligation, Gab1-ecKO;ApoE KO mice under high fat diet showed increased atherosclerotic lesion size compared to Gab1-WT;ApoE KO mice. The levels of proatherogenic genes intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and leukocyte infiltration were all increased in Gab1-ecKO;ApoE KO mice. Using loss- and gain-of-function studies in cultured human endothelial cells (ECs), we found that Gab1 depletion by siRNA augmented monocyte adhesion to ECs by increasing ICAM-1 and VCAM-1 expression in response to the proinflammatory cytokine TNF-α. Conversely, adenoviral overexpression of Gab1 inhibited TNFα-induced monocyte adhesion to ECs and upregulation of ICAM-1 and VCAM-1 in ECs. Conclusions: These results demonstrate that endothelial Gab1 represses disturbed flow-induced vascular remodeling and atherogenesis through inhibition of vascular inflammation. Our findings suggest that Gab1 activation might represent novel approaches for the treatment of vascular diseases, including intimal hyperplasia and atherosclerosis.

Salusin-β induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway.

Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-β is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-β increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-β increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-β-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-β-induced monocyte adhesion in VSMCs. Salusin-β caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-β-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-β increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-β induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs.

Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor.

Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein with redox activity and is proved to be secreted from stimulated cells. The aim of this study was to evaluate the functions of extracellular APE1/Ref-1 with respect to leading anti-inflammatory signaling in TNF-α-stimulated endothelial cells in response to acetylation. Treatment of TNF-α-stimulated endothelial cells with an inhibitor of deacetylase that causes intracellular acetylation, considerably suppressed vascular cell adhesion molecule-1 (VCAM-1). During TSA-mediated acetylation in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human (rh) APE1/Ref-1 with reducing activity induced a conformational change in rh TNF-α receptor 1 (TNFR1) by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1, in accordance with the activation of p66shc and p38 MAPK. These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits TNF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.