Head-to-Head Comparison of Selected Extra- and Intracellular CO-Releasing Molecules on Their CO-Releasing and Anti-Inflammatory Properties.

Over the past decade, a variety of carbon monoxide releasing molecules (CORMs) have been developed and tested. Some CORMs spontaneously release CO once in solution, while others require a trigger mechanism to release the bound CO from its molecular complex. The modulation of biological systems by CORMs depends largely on the spatiotemporal release of CO, which likely differs among the different types of CORMs. In spontaneously releasing CORMs, CO is released extracellularly and crosses the cell membrane to interact with intracellular targets. Other CORMs can directly release CO intracellularly, which may be a more efficient method to modulate biological systems. In the present study, we compared the efficacy of extracellular and intracellular CO-releasing CORMs that either release CO spontaneously or require an enzymatic trigger. The efficacy of such CORMs to modulate HO-1 and VCAM-1 expression in TNF-α-stimulated human umbilical vein endothelial cells (HUVEC) was evaluated.

Protease-Activated Receptor 2 (PAR-2) Antagonist AZ3451 Mitigates Oxidized Low-Density Lipoprotein (Ox-LDL)-Induced Damage and Endothelial Inflammation.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction plays an important role in the initiation and development of cardiovascular diseases, especially atherosclerosis (AS). Protease-activated receptor 2 (PAR-2) is a receptor for inflammatory proteases. However, the biological function of PAR-2 in endothelial cells and the pathophysiological process of AS are still unknown. In the current study, we found that treatment with ox-LDL increased the gene and protein expressions of PAR-2 in EA.hy926 endothelial cells. Interestingly, we found that antagonism of PAR-2 with its specific antagonist AZ3451 could ameliorate ox-LDL-induced lactate dehydrogenase (LDH) release. Treatment with AZ3451 considerably improved the mitochondrial function by restoring the mitochondrial membrane potential and increasing the levels of intracellular adenosine triphosphate (ATP). Also, we found that AZ3451 attenuated ox-LDL-induced expression and production of pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8). Treatment with AZ3451 also mitigated the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Notably, our results demonstrated that the presence of AZ3451 alleviated ox-LDL-induced expression of the endothelial cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). Mechanistically, we found that AZ3451 attenuated ox-LDL-induced activation of nuclear factor-κB (NF-κB) by reducing the levels of intracellular NF-κB p65 and the luciferase activity of NF-κB promoter. Based on these findings, we conclude that PAR-2 might become a novel therapeutic target for the treatment of AS.

MiR-520b inhibits endothelial activation by targeting NF-κB p65-VCAM1 axis.

MiR-520b belongs to the miR-373/520 family, is expressed only in human and nonhuman primates. Previous reports indicated that the expression of miR-520b was repressed in human atherosclerotic plaque tissue compared with healthy vessels. However, the role of miR-520b in coronary artery disease still remains to be uncovered. In this study, we demonstrated that endothelial cells (ECs) in human atherosclerotic plaques expressed miR-520b and aimed to elucidate the impact of miR-520b on EC activation and inflammatory response. To determine the potential targets of miR-520b, we performed RNA-seq analysis by transfecting miR-520b mimics in ECs. The quantitative real-time PCR (qPCR) validation suggested that miR-520b over-expression reduced pro-inflammatory gene expression (e.g. ICAM1, VCAM1, SELE) while the inhibition of miR-520b induced their expression. By combining bioinformatics prediction and functional assays, we identified that RELA (Nuclear Factor-κB (NF-κB) Transcription Factor P65) was a direct target of miR-520b. Moreover, miR-520b mimics attenuated monocyte adhesion and monocyte trans-endothelial migration (the initial steps of atherosclerotic formation) in response to lipopolysaccharides (LPS) stimulation. Re-expression of a non-miR-targetable version of p65 could rescue the reduced monocyte cell attachment, suggesting that this process is NF-κB p65 dependent. MiR-520b reduced the abundance of NF-κB p65 in cytoplasmic fractions without corresponding increase in nuclear fractions, indicating that this regulation is independent of p65 translocation process. MiR-520b mimics attenuated the activity of VCAM-1 promoter, whereas miR-520b inhibitor activated its activity. However, miR-520b inhibitor had no effect on promoter activity containing the mutated NF-κB p65 binding sites, strongly demonstrating that the impact of miR-520b on VCAM1 gene is mediated by NF-κB p65. Thus, we concluded that miR-520b suppressed EC inflammation and the cross-talk between monocytes and ECs by down-regulating NF-κB p65-ICAM1/VCAM1 axis and might serve as a potential therapeutic target for EC dysfunction and atherosclerosis.

Anti-VCAM-1 and Anti-IL4Rα Aptamer-Conjugated Super Paramagnetic Iron Oxide Nanoparticles for Enhanced Breast Cancer Diagnosis and Therapy.

The surface protein overexpressed on cancer cells can be used as biomarkers for early detection of specific diseases. Anti-VCAM-1 and anti-IL4Rα DNA aptamers specific to VCAM-1 and IL4Rα receptors that are overexpressed in 4T1 tumor-bearing mice could be used as potential biomarker for both diagnostic and therapeutic applications in cancer biology. Cell Viability and luciferase assay of 4T1-Luc2 cancer cells in the presence of anti-VCAM-1 ssDNA or anti-IL4Rα RNA aptamers was assessed by monitoring the changes in the absorbance and the fluorescence of Alamar blue dye. The aptamer-conjugated SPIO magnetic beads, used for the selective targeting to tumor sites, were monitored using noninvasive MRI and Bioluminescence imaging (BLI). Cell viability and luciferase assays showed that both anti-VCAM-1 and anti-IL4Rα aptamers favor the depletion of cancer cells and limit tumor progression. Microscopic analyses confirmed that the target specific aptamers significantly trigger tumor cell apoptosis and limit cancer cell growth in vitro. The intravenous injection of SPIO nanoparticle-conjugated aptamers were further confirmed using noninvasive MRI and Bioluminescence imaging. Anti-VCAM1 and anti-IL4Rα aptamers, specific to VCAM-1 and IL4Rα receptors overexpressed in 4T1-Luc2 tumor-bearing mice, were used as diagnostic and therapeutic tools.

Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression.

The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of Phc2 in mice causes a severe defect in HSPC mobilization through the derepression of Vcam1 in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 in Phc2-deficient mice reverses the symptoms. We further determine that Phc2-dependent Vcam1 repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.

FAK inhibition reduces metastasis of α4 integrin-expressing melanoma to lymph nodes by targeting lymphatic VCAM-1 expression.

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1). As VCAM-1 is a major receptor for α4 integrin and plays a key role in leukocyte recruitment, we reasoned that inhibition of FAK activity may reduce VCAM-1 expression within LNs and thus reduce metastasis of α4 integrin-expressing melanoma to LNs. First, we found that a pharmacological FAK inhibitor, PF-271, blocked tumor necrosis factor-α (TNF-α)-mediated VCAM-1 expression on human dermal lymphatic endothelial cells (HDLECs). In vitro, PF-271 significantly decreased B16F10 melanoma adhesion to and transmigration through HDLECs compared to TNF-α treated cells. Furthermore, in vivo FAK inhibition by oral PF-271 administration reduced VCAM-1 expression in inguinal, cervical, and popliteal LNs compared to vehicle treated mice. Finally, in a footpad metastasis model, B16F10 melanoma cells were injected into the right footpad of C57BL/6 mice, and PF-271 (50 mg/kg, twice daily for 6 days) was orally administrated after 1 week of tumor transplantation. While untreated mice exhibited significant metastatic melanoma lesions in popliteal LNs, PF-271 treated mice showed only marginal melanoma metastasis. These results support the possibility that FAK inhibitors may be a novel preventative option in melanoma metastasis by blocking VCAM-1 expression in LNs.

Comparison of sVCAM-1 and sICAM-1 levels in maternal serum and vaginal secretion between pregnant women with preterm prelabour ruptures of membranes and healthy pregnant women.

OBJECTIVE: The study aims to evaluate the maternal serum and the vaginal fluid levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecular (sICAM-1) in pregnant women complicated by preterm prelabour ruptures of membranes (PPROM). MATERIALS AND METHODS: The prospective case control study included 34 pregnant women with PPROM and 34 healthy pregnant women. Patients with additional diseases, a smoking habit and vaginal bleeding, as well as those using antibiotics, during the study period were not included in the study. Cervicovaginal fluid and serum samples were taken during the patients' admission. The demographic data, maternal serum and vaginal fluid sVCAM-1 and sICAM-1, C reactive protein (CRP) and leukocyte counts were noted for all pregnant women included in the study. The sVCAM-1 and sICAM-1 levels were measured by enzyme-linked immunosorbent assay kits. RESULTS: In pregnant women with PPROM, the serum leukocyte (mean ± SD =11.41 ± 1.067 versus 9.18 ± 1.56, p < .0001), serum sVCAM-1 (median 771.20 versus 704.60 ng/ml, p < .001), sICAM-1 (mean ± SD 213.10 ± 35.59 ng/ml versus 188.11 ± 37.35 ng/ml, p = .06), vaginal sVCAM-1 (median 208.00 versus 140.20 ng/ml, p = .014) and sICAM-1 (mean ± SD 32.32 ± 6.49 ng/ml versus 24.87 ± 6.79 ng/ml, p < .001) values were found to be significantly higher in pregnant women with PPROM than in healthy pregnant women. A positive and significant correlation was observed between the leukocyte count and the vaginal sVCAM-1 level (r = 0.850; p < .001). CONCLUSION: To the best of our knowledge, this is the first study evaluating the levels of sICAM-1 in maternal serum in pregnant women with PPROM. The maternal serum and vaginal fluid sVCAM-1 and sICAM-1 levels can be used as biochemical markers supporting the PPROM diagnosis because of the increase in both maternal serum and vaginal fluid sVCAM-1 and sICAM-1 levels in pregnant women with PPROM.

New Lignans from the Flower of Forsythia koreana and Their Suppression Effect on VCAM-1 Expression in MOVAS Cells.

Six lignans including two new lignans were obtained as the principal components of the Forsythia koreana flowers via silica gel (SiO2 ), octadecyl SiO2 (ODS) as well as Sephadex LH-20 column chromatography. In addition to two new lignans, named koreanaside A ((7R,8S,7'R,8'S)-7,7'-diepoxy-5'-hydroxy-3,3'-dimethoxylignan 4-O-ß-d-glucopyranoside) and koreanaside B ((7R,8S,7'S,8'R)-7,9'-epoxy-9,5',7'-trihydroxy-3,3'-dimethoxylignan 4-O-ß-d-glucopyranoside), four known lignans were identified to be (+)-phylligenin, (-)-epipinoresinol, pinoresinol, and tinosposide A. The structures and absolute configurations of koreanasides A and B were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD), whereas the structures of known lignans were identified by comparison their NMR and MS values with those in the reported literature. Their chemical structures including configuration were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD) but also comparison of their NMR and MS values with those in the reported literature. This is the first article for isolation of six lignans of F. koreana flowers. Koreanasides A and B showed high radical scavenging activity with oxygen radical absorbance capacity (ORAC) values of 0.97 ± 0.01 and 1.02 ± 0.01, respectively. Koreanaside A also prohibited expressing VCAM-1 in MOVAS cells with 80.5% at 25 mg/mL.

The Expanding Therapeutic Armamentarium for Inflammatory Bowel Disease: How to Choose the Right Drug[s] for Our Patients?

The therapeutic landscape for inflammatory bowel disease [IBD] is rapidly evolving. Two new biologic drugs, vedolizumab and ustekinumab, have recently entered the marketplace, the first biosimilars have been introduced, and several other agents are at an advanced stage of clinical development. In parallel, therapeutic goals have shifted from symptom control towards mucosal healing and prevention of bowel damage. In the coming years, gastroenterologists will be faced with unprecedented choices when selecting the best treatment for their patients with IBD. In this article, we review existing data on the mechanisms of action, efficacy, and safety of recently approved and late-stage pipeline therapies, and use this information to speculate on the positioning of these drugs, alone or in combination, in therapeutic algorithms for Crohn's disease and ulcerative colitis.

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells.

Chronic inflammation is an underlying contributor to various chronic diseases. The objectives of this study were to investigate the anti-inflammatory activity of zein hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption and to identify novel anti-inflammatory peptides after transport across Caco-2 cells. Three zein hydrolysates were prepared and further digested using gastrointestinal proteases; their transports were studied in Caco-2 cells. Anti-inflammatory activity was studied in endothelial EA.hy926 cells. Three zein hydrolysates and their digests significantly decreased the expression of tumor necrosis factor-α (TNF-α) induced pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) by 37.3-66.0%. Eleven novel peptides with 5-9 amino acid residues were sequenced; three peptides showed strong anti-inflammatory activity by inhibiting the VCAM-1 by 54-38.9% and intercellular cell adhesion molecule-1 (ICAM-1) by 36.5-28.6% at 0.2 mM. A new approach to identify novel anti-inflammatory peptides that could survive gastrointestinal digestion and absorption was developed.

Nanoscale Tuning of VCAM-1 Determines VLA-4-Dependent Melanoma Cell Plasticity on RGD Motifs.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/µm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

Quinic acid inhibits vascular inflammation in TNF-α-stimulated vascular smooth muscle cells.

Atherosclerosis is a chronic inflammatory disease, and the increased expression of adhesion molecules on vascular smooth muscle cells contributes to the progression of vascular disease. Quinic acid (QA) has been shown to possess radioprotection, anti-neuroinflammatory, and anti-oxidant activities; however, an anti-vascular inflammatory effect has not been reported. This study investigated the effect of QA on the expression of vascular cell adhesion molecule-1 (VCAM-1) stimulated by TNF-α in MOVAS cells. Pre-incubation of MOVAS cells, the mouse vascular smooth muscle cell line for 2h with QA (0.1, 1 and 10 µg/mL) dose-dependently inhibits TNF-α-induced mRNA and protein expression of VCAM-1 and monocyte adhesion. QA inhibits TNF-α-stimulated phosphorylation of MAP kinase and NK-κB activation. Our results indicate that QA inhibits the TNF-α-stimulated induction of VCAM-1 in VSMC by inhibiting the MAP kinase and NF-κB signaling pathways and the adhesion capacity of VSMC, which may explain the ability of QA to inhibit vascular inflammation such as atherosclerosis.

Myricitrin inhibits vascular adhesion molecule expression in TNF­α­stimulated vascular smooth muscle cells.

Increased expression of adhesion molecules is thought to serve an important role in the pathogenesis of atherosclerosis. Myricitrin, a bioactive compound of Myrica cerifera, has been demonstrated to exhibit anti­atherogenic effects. However, the effect of myricitrin on the expression of adhesion molecules in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, the aim of the present study was to evaluate the inhibitory effects of myricitrin on tumor necrosis factor­α (TNF­α)­induced expression of adhesion molecules in VSMCs in vitro. The results revealed that myricitrin inhibited the adhesion of human THP­1 monocyte cells to TNF­α­stimulated mouse MOVAS­1 VSMC cells, and reduced the expression of adhesion molecules in TNF­α­stimulated MOVAS­1 cells. In addition, myricitrin significantly inhibited the TNF­α­induced expression of nuclear factor (NF)­κB p65, and prevented the TNF­α­induced degradation of nuclear factor of κ light chain enhancer in B­cells inhibitor α. Furthermore, myricitrin inhibited the production of intracellular reactive oxygen species in TNF­α­stimulated MOVAS­1 cells. In conclusion, the results of the present study indicated that myricitrin inhibits the expression of vascular cell adhesion protein­1 and intercellular adhesion molecule­1 in TNF­α­stimulated MOVAS­1 cells potentially via the NF­κB signaling pathway. Therefore, myricitrin may be an effective pharmacological agent for the prevention or treatment of atherosclerosis.

Signatures of anthocyanin metabolites identified in humans inhibit biomarkers of vascular inflammation in human endothelial cells.

SCOPE: The physiological relevance of contemporary cell culture studies is often perplexing, given the use of unmetabolized phytochemicals at supraphysiological concentrations. We investigated the activity of physiologically relevant anthocyanin metabolite signatures, derived from a previous pharmacokinetics study of 500 mg 13 C5 -cyanidin-3-glucoside in eight healthy participants, on soluble vascular adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6) in human endothelial cells. METHODS AND RESULTS: Signatures of peak metabolites (previously identified at 1, 6, and 24 h post-bolus) were reproduced using pure standards and effects were investigated across concentrations ten-fold lower and higher than observed mean (<5 µM) serum levels. Tumor necrosis factor-α (TNF-α)-stimulated VCAM-1 was reduced in response to all treatments, with maximal effects observed for the 6 and 24 h profiles. Profiles tested at ten-fold below mean serum concentrations (0.19-0.44 µM) remained active. IL-6 was reduced in response to 1, 6, and 24 h profiles, with maximal effects observed for 6 h and 24 h profiles at concentrations above 2 µM. Protein responses were reflected by reductions in VCAM-1 and IL-6 mRNA, however there was no effect on phosphorylated NFκB-p65 expression. CONCLUSION: Signatures of anthocyanin metabolites following dietary consumption reduce VCAM-1 and IL-6 production, providing evidence of physiologically relevant biological activity.

Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis.

Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell-cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.