Expression Of Inflammatory Adhesion Molecules Research Articles

Abstract 6791: Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment

Abstract Inflammation and pathological vasculature are common features of the tumor microenvironment (TME). Sex hormones potently modulate vascular physiology and immune cell functions, and there are established correlations with the outcomes of triple negative (TNBC) despite the defining feature of receptor negativity. Circulating estrogens and androgens have been hypothesized as drivers of TNBC through the actions exerted by receptor-positive cells in the TME. Here, we present an organ chip model of the TNBC microenvironment for quantifying the impact of configurable TME variables such as the ECM composition and the relative densities and phenotypes of tumor cells, cancer associated fibroblasts, and myeloid cells on endothelial barrier function. After establishing baselines of vascular permeability in the presence and absence of TNBC cells, we tested the effect of estradiol, estrone, and dihydrotestosterone on barrier permeability. Multilayer polydimethylsiloxane (PDMS) organ chips were fabricated using standard soft lithography. The layers of the device were separated with semi-permeable membranes that allow exchange of soluble factors. Once assembled, the upper channel was loaded with female (XX) human umbilical vein endothelial cells. Collagen hydrogels were injected into the tissue layer and the devices were cultured on a rocker to induce endothelial barrier alignment via application of an average shear stress of 5 dynes/cm2. We optimized rocking conditions by assessing endothelial layer alignment, junctional organization, and permeability. TNBC cells and XX mammary carcinoma fibroblasts were added to the hydrogel to model the effects of TME-derived signals on endothelial barrier permeability. 40 kDa FITC- Dextran was chosen to assay permeability as it can show leakage due to active disruption of the barrier. Staining for F-actin and ZO-1 allowed for quantification of junctional organization. Sex hormone modulation of proinflammatory gene expression was assessed by RT-qPCR. The presence of TNBC cells significantly increased transport of 40kDa FITC-Dextran across the endothelial barrier, disrupted intercellular junction quality, and induced the expression of inflammatory adhesion molecules including ICAM-1. Our results demonstrate that sex hormones modulate TNBC-associated changes in endothelial cell bioenergetics, junctional organization, and macromolecular barrier permeability. Ongoing work will investigate the effects of age-matched XX monocyte derived macrophages on barrier function in the model. This organ chip platform provides an assay for measuring altered vascular inflammation and permeability in the TNBC microenvironment and may provide a venue for investigating vascular-normalizing effects of various candidate therapeutics. Citation Format: Delia Carlino, Ashley Martier, Yasmin Maurice, G. Wills Kpeli, Matthew Burow, Mark Mondrinos. Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6791.

Nucleoporin93 limits Yap activity to prevent endothelial cell senescence.

As the innermost lining of the vasculature, endothelial cells (ECs) are constantly subjected to systemic inflammation and particularly vulnerable to aging. Endothelial health is hence vital to prevent age-related vascular disease. Healthy ECs rely on the proper localization of transcription factors via nuclear pore complexes (NPCs) to govern cellular behavior. Emerging studies report NPC degradation with natural aging, suggesting impaired nucleocytoplasmic transport in age-associated EC dysfunction. We herein identify nucleoporin93 (Nup93), a crucial structural NPC protein, as an indispensable player in vascular protection. Endothelial Nup93 protein levels are significantly reduced in the vasculature of aged mice, paralleling observations of Nup93 loss when using invitro models of EC senescence. The loss of Nup93 in human ECs induces cell senescence and promotes the expression of inflammatory adhesion molecules, where restoring Nup93 protein in senescent ECs reverses features of endothelial aging. Mechanistically, we find that both senescence and loss of Nup93 impair endothelial NPC transport, leading to nuclear accumulation of Yap and downstream inflammation. Pharmacological studies indicate Yap hyperactivation as the primary consequence of senescence and Nup93 loss in ECs. Collectively, our findings indicate that the maintenance of endothelial Nup93 is a key determinant of EC health, where aging targets endothelial Nup93 levels to impair NPC function as a novel mechanism of EC senescence and vascular aging.

Abstract 285: Organ chip culture of vascularized triple negative breast cancer explant tissues with validated pathological and clinically-targetable properties of tumor vasculature

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of the classic targetable receptors. Targeting the TNBC microenvironment via the biophysical properties of the tumor vasculature is a promising approach. The objective of this study was to engineer a microphysiological model of vascularized TNBC with validated pathological properties of tumor vasculature for in vitro modeling of selective drug delivery via the enhanced permeability and retention effect. We engineered a method for standardized production of milliscale tissues housed in fluidic culture devices that are accessible only via a fully anastomosed and perfusable internal vasculature comprised of endothelial cells and organ specific fibroblasts. We first established a baseline of vascular morphometry, endothelial barrier function, inflammatory activation, and perivascular extracellular matrix (ECM) composition in vascularized tissues devoid of cancer cells. In parallel, we developed a novel approach for processing patient-derived TNBC tissues into injectable explants of disaggregated tumor imbibed in ECM hydrogel in less than one hour. These explant tissues cultured in our devices consistently maintain high viability and a Ki67 index around 70%, on par with the Ki67 indexes of source TNBC tumors. We hypothesized that TNBC derivatives would induce the formation of a pathological vasculature in our model. The TNBC derivatives were mixed with exogenous fibroblasts and endothelial cells at optimized ratios and the same workflow described above was used to form vascularized TNBC explant tissues. TNBC-associated vasculature exhibited a significantly altered vascular morphometry relative to cancer-free control tissues across multiple quantitative metrics including vessel length, vessel diameter, and network branch points. These morphological changes were consistent with known dysmorphic features of tumor vasculature. Twofold or greater increases in endothelial ICAM-1 expression in TNBC-associated vasculature was measured by multiple methods of analysis. Increased inflammatory adhesion molecule expression was accompanied by disorganized VE-cadherin junctions and disrupted patterns of perivascular cell coverage that suggested TNBC-associated vasculature should be significantly more permeable. FITC-dextran perfusion studies revealed widespread macromolecular leakage from TNBC-associated vasculature into the TNBC explant tissue interstitium by 5 minutes, whereas no leakage was observed from control vasculature for the duration of perfusion up to one hour. Ongoing work to be presented is focused on validating our model as a platform for studying drug delivery via the enhanced permeability and retention effect using Abraxane nanoparticles. Citation Format: Kevin M. Conrad, Charles E. Byrne, Matthew R. Burow, Mark Mondrinos. Organ chip culture of vascularized triple negative breast cancer explant tissues with validated pathological and clinically-targetable properties of tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 285.

High stretch induces endothelial dysfunction accompanied by oxidative stress and actin remodeling in human saphenous vein endothelial cells

The rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin’s nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

Pharmacological inhibition of focal adhesion kinase 1 (FAK1) and anaplastic lymphoma kinase (ALK) identified via kinome profile analysis attenuates lipopolysaccharide-induced endothelial inflammatory activation

Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies.

The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.-Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Ultrasonic Perfluorohexane‐Loaded Monocyte Imaging: Toward a Minimally Invasive Technique for Selective Detection of Liver Inflammation in Fatty Liver Disease

To investigate the utility of ultrasonic (US) perfluorohexane (PFH)-loaded monocyte imaging for detection of liver inflammation in fatty liver disease. C57Bl6 mice were injected intraperitoneally with tumor necrosis factor α and assessed by US PFH-loaded monocyte imaging 3 hours later. Echogenic monocytes were injected intravenously, leading to a transient increase in liver tissue intensity on a US perfusion scan. The contrast wash-out time constant was hypothesized to reflect the degree of inflammation. Next, we evaluated US PFH-loaded monocyte imaging in Ldlr-/- mice fed a 1-week high-fat/high-cholesterol diet as model for early developing nonalcoholic steatohepatitis. Adjunct analyses included tissue markers of liver inflammation. Tumor necrosis factor α-injected mice showed a reduced wash-out time constant (mean ± SEM, 0.013 ± 0.003; n = 8) compared to controls (0.054 ± 0.009; n = 7; P = .0006), indicative of increased inflammatory adhesion molecule expression on the endothelium. The Ldlr-/- mice fed the high-fat/high-cholesterol diet showed liver inflammation, as reflected by increased (3- to 4-fold) infiltration of inflammatory cells and increased (3- to 4-fold) gene expression of tumor necrosis factor α, integrin αM, intracellular adhesion molecule, and vascular cell adhesion molecule. However, in these mice, no difference was detected in the wash-out time constant as assessed by US PFH-loaded monocyte imaging (high-fat/high-cholesterol, 0.050 ± 0.017; n = 5; chow, 0.048 ± 0.006; n = 6; P = .91). Our results indicate that US PFH-loaded monocyte imaging is able to detect vascularly expressed inflammatory adhesion molecules in the mouse liver on direct endothelial stimulation. However, in our mouse model of early developing nonalcoholic steatohepatitis, we did not detect inflammation by this method, which may suggest that the time-dependent relationship between parenchymal and endothelial inflammation remains a fundamental issue to be addressed.

Abstract 417: Trf2-Interacting Protein (Terf2ip) S205 Phosphorylation Mediates Atherosclerosis via Orchestrating Endothelial Senescence and Inflammation

Telomeric repeat-binding factor 2 (TRF2)-interacting protein (TERF2IP) S205 phosphorylation mediates atherosclerosis (AS) via orchestrating endothelial cell (EC) senescence (Sen) and inflammation. Nhat-Tu Le, Kyung-Sun Heo, Kyung Ae Ko, Yunting Tao,Tamlyn Thomas, Raymundo A Quintana Quezada, Keigi Fujiwara, and Jun-ichi Abe Department of Cardiology, UT MD Anderson Cancer Center Objective: Shortened telomere (TL) provokes EC Sen, which is associated with focalized EC inflammation and plaque formation in arteries exposed by disturbed blood flow (d-flow). Although d-flow is known to increase both EC inflammation and Sen, the exact mechanism by which d-flow induces these two different EC pathologies and subsequent AS is poorly understood. Methods & Results: TERF2IP, as a member of the sheltrin complex, is not only crucial for DNA double strand break repair at the TL, but also for activation of cytosolic IkB kinase (IKK) and subsequent NF-kB activity. TERF2IP and TRF2 form a stable heterodimer to protect the duplex region of TLs, while activating NF-kB, especially under the d-flow condition. We have discovered that inhibition of p90RSK reduces d-flow-induced EC Sen and inflammation via down-regulating TERF2IP S205 phosphorylation. The nuclear TERF2IP-TRF2 complex protects TLs by repairing DNA damages while the cytosolic complex plays a role in NF-kB activation. When we mutated the TERF2IP phosphorylation site (S205A) in ECs, the nuclear export of the TERF2IP-TRF2 complex was abolished, which inhibited d-flow-induced NF-kB activation and Sen. Both the depletion of TERF2IP by siRNA and S205A mutation inhibited d-flow-induced TERF2 nuclear export, and residual TRF2 at the TL inhibited d-flow-induced TL dysfunction. The mutation of TERF2IP S205A inhibited cytosolic IKK-NF-kB activation by eliminating the cytosolic TERF2IP-TERF2 complex. The inhibition of d-flow-induced inflammatory adhesion molecule expression and consequent AS were also confirmed in EC-specific TERF2IP knock out mice. Conclusion: These results suggest that S205 phosphorylation of TERF2IP induced by p90RSK activation is required for d-flow-induced TL shortening and NF-kB activation via regulating nuclear export of the TERF2IP-TERF2 complex.

Circulating microparticles in severe pulmonary arterial hypertension increase intercellular adhesion molecule-1 expression selectively in pulmonary artery endothelium.

BackgroundMicroparticles (MPs) stimulate inflammatory adhesion molecule expression in systemic vascular diseases, however it is unknown whether circulating MPs stimulate localized ICAM-1 expression in the heterogeneically distinct pulmonary endothelium during pulmonary arterial hypertension (PAH). Pulmonary vascular lesions with infiltrating inflammatory cells in PAH form in the pulmonary arteries and arterioles, but not the microcirculation. Therefore, we sought to determine whether circulating MPs from PAH stimulate pulmonary artery endothelial cell-selective ICAM-1 expression.ResultsPulmonary artery endothelial cells (PAECs) were exposed to MPs isolated from the circulation of a rat model of severe PAH. During late-stage (8-weeks) PAH, but not early-stage (3-weeks), an increase in ICAM-1 was observed. To determine whether PAH MP-induced ICAM-1 was selective for a specific segment of the pulmonary circulation, pulmonary microvascular endothelial cells (PMVECs) were exposed to late-stage PAH MPs and no increase in ICAM-1 was detected. A select population of circulating MPs, the late-stage endoglin + MPs, were used to assess their ability to stimulate ICAM-1 and it was determined that the endoglin + MPs were sufficient to promote ICAM-1 increases in the whole cell, but not surface only expression.ConclusionsLate-stage, but not early-stage, MPs in a model of severe PAH selectively induce ICAM-1 in pulmonary artery endothelium, but not pulmonary microcirculation. Further, the selected endoglin + PAH MPs, but not endoglin + MPs from control, are sufficient to promote whole cell ICAM-1 in PAECs. The implications of this work are that MPs in late-stage PAH are capable of inducing ICAM-1 expression selectively in the pulmonary artery. ICAM-1 likely plays a significant role in the observed inflammatory cell recruitment, specifically to vascular lesions in the pulmonary artery and not the pulmonary microcirculation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0445-1) contains supplementary material, which is available to authorized users.

PS 04-09 HIGH GLUCOSE AND PALMITATE INCREASES BONE MORPHOGENIC PROTEIN 4 EXPRESSION IN HUMAN ENDOTHELIAL CELLS

Objective: We investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) affect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. Design and Method: To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. Results: High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this effect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of inflammatory adhesion molecules and ROS production. Conclusions: Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL.

Extracellular granzyme K mediates endothelial activation through the cleavage of protease-activated receptor-1.

Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-apoptotic roles for granzymes, including granzyme K (GzK), have been proposed. As recent studies have observed elevated levels of GzK in the plasma of patients diagnosed with clinical sepsis, we hypothesized that extracellular GzK induces a proinflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated protease-activated receptor-1 leading to increased interleukin 6 and monocyte chemotactic protein 1 production in endothelial cells. Enhanced expression of intercellular adhesion molecule 1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the mitogen-activated protein kinase p38 pathway for intercellular adhesion molecule 1 expression, and both the p38 and the extracellular signal-regulated protein kinases 1 and 2 pathways in cytokine production. GzK also increased tumour necrosis factor α-induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, inter-α-inhibitor protein, significantly inhibited GzK activity in vitro. In summary, extracellular GzK promotes a proinflammatory response in endothelial cells.

A functional SNP in FLT1 increases risk of coronary artery disease in a Japanese population.

Coronary artery disease (CAD) including myocardial infarction is one of the leading causes of death in many countries. Similar to other common diseases, its pathogenesis is thought to result from complex interactions among multiple genetic and environmental factors. Recent large-scale genetic association analysis for CAD identified 15 new loci. We examined the reproducibility of these previous association findings with 7990 cases and 6582 controls in a Japanese population. We found a convincing association of rs9319428 in FLT1, encoding fms-related tyrosine kinase 1 (P=5.98 × 10(-8)). Fine mapping using tag single-nucleotide polymorphisms (SNPs) at FLT1 locus revealed that another SNP (rs74412485) showed more profound genetic effect for CAD (P=2.85 × 10(-12)). The SNP, located in intron 1 in FLT1, enhanced the transcriptional level of FLT1. RNA interference experiment against FLT1 showed that the suppression of FLT1 resulted in decreased expression of inflammatory adhesion molecules. Expression of FLT1 was observed in endothelial cells of human coronary artery. Our results indicate that the genetically coded increased expression of FLT1 by a functional SNP implicates activation in an inflammatory cascade that might eventually lead to CAD.

Antioxidants and vascular health.

Oxygen free radicals and other reactive oxygen species (ROS) are common products of normal aerobic cellular metabolism, but high levels of ROS lead to oxidative stress and cellular damage. Increased production of ROS favors vascular dysfunction, inducing altered vascular permeability and inflammation, accompanied by the loss of vascular modulatory function, the imbalance between vasorelaxation and vasoconstriction, and the aberrant expression of inflammatory adhesion molecules. Inflammatory stimuli promote oxidative stress generated from the increased activity of mitochondrial nicotinamide adenine dinucleotide phosphate oxidase, particularly of the Nox4 isoform, with the consequent impairment of mitochondrial β-oxidation. Vascular dysfunction due to the increase in Nox4 activity and ROS overproduction leads to the progression of cardiovascular diseases, diabetes, inflammatory bowel disease, and neurological disorders. Considerable research into the development of effective antioxidant therapies using natural derivatives or new synthetic molecules has been conducted. Antioxidants may prevent cellular damage by reducing ROS overproduction or interfering in reactions that involve ROS. Vitamin E and ascorbic acid are well known as natural antioxidants that counteract lipid peroxidative damage by scavenging oxygen-derived free radicals, thus restoring vascular function. Recently, preliminary studies on natural antioxidants such as goji berries, thymus, rosemary, green tea ginseng, and garlic have been conducted for their efficacy in preventing vascular damage. N-acetyl-cysteine and propionyl-L-carnitine are synthetic compounds that regulate ROS production by replacing endogenous antioxidants in both endothelial and smooth muscle cells. In this review, we consider the molecular mechanisms underlying the generation of oxidative stress-induced vascular dysfunction as well as the beneficial effects of antioxidant therapies.

Protective activity ethanol extract of the fruits of Illicium verum against atherogenesis in apolipoprotein E knockout mice.

BackgroundIllicium verum Hook. fil. Illiciaceae (Illicium v.) has been traditionally used in herbal medicine for treating many inflammatory diseases, including skin inflammation and rheumatism. We investigated its use as a preventive agent against inflammatory and vascular diseases in a murine model of atherosclerosis using apolipoprotein E-knockout (ApoE−/−) mice fed on a high-fat diet (HFD).MethodsWe investigated the effect of Illicium v. on cytotoxicity, NF-κB activity, and adhesion molecule expression in TNF-α – stimulated HASMCs (Human Aortic smooth muscle cells). ApoE−/−mice, fed a HFD and treated daily for 12 weeks by oral administration of either Illicium v. (100 or 200 mg/kg) or atorvastatin (10 mg/kg), were evaluated for atherosclerotic lesions and inflammatory responses by performing Oil red O and iNOS staining, respectively. Expression of inflammatory cytokines (i.e., NF-κB, TNF-α, IL-1β, COX, IκB-α, Iκκ-α/β) and adhesion molecules in the aorta were measured by western blot analysis.ResultsIn TNF-α-stimulated HASMCs, Illicium v. treatment decreased NF-κB transcriptional activity, and NF-κB protein levels were reduced in a dose-dependent manner over a range of 10–100 μg/mL Illicium v. Also, Illicium v. attenuated the expression of adhesion molecules that are responsible for inflammation in these cells. In animal experiments, treatment with Illicium v. or atorvastatin counteracted the characteristic changes in body weight, blood pressure, and lipid levels seen in HFD-fed ApoE−/− mice. In addition, Illicium v. treatment reduced aortic atherosclerotic plaque lesions and the immunoreactivity of iNOS activation. The aortic expression of inflammatory adhesion molecules and cytokines (TNF-α, IL-1β, NF-κB, COX, IκB-α, Iκκ-α/β), which is characteristic of HFD-fed ApoE−/− mice, was attenuated by 12-week treatment with daily oral administration of Illicium v. or atorvastatin, and the most potent effect was seen with the herbal tincture.ConclusionsThe beneficial effects of Illicium v. are consistent with a significant decrease in the iNOS-mediated inflammatory response, resulting in reduction of inflammation-associated gene expression. Treatment with Illicium v. may be the basis of a novel therapeutic strategy for hyperlipidemia-atherosclerosis.

Abstract 371: TNFα Drives Endothelial Dysfunction and Oxidative Stress via eNOS Uncoupling in Aortic Valve Disease

Objective: Oxidative stress (OxS) is known to contribute to late stages of aortic valve disease (AVD), but its role in early-stage valve inflammation and as a driver of valve pathology has not been investigated. Here we show that inflammation drives increased OxS via eNOS uncoupling. Using an ex vivo model of AVD, we show that targeting eNOS uncoupling with tetrahydrobiopterin or superoxide dismutase can mitigate later-stage valve degeneration by blocking the downstream effects of eNOS uncoupling. Methods and Results: We first observed evidence of eNOS uncoupling in the endothelium of calcified human aortic valve leaflets, which showed increased superoxide co-localized with cells positive for CD31 and VCAM-1, suggesting a connection between endothelial inflammatory activation and increased oxidative stress. We then showed that inflammatory cytokine TNF-α caused increased superoxide, free radicals, and hydrogen peroxide in aortic valve endothelial cells, as soon as 15 minutes after treatment. TNF-α did not cause increased oxidative stress in endothelial cells from the aorta, pointing to a valve-specific mechanism. TNF-α-driven oxidative stress decreased valve endothelial cell nitric oxide secretion, lowered eNOS and VE-cadherin protein levels, and increased expression of inflammatory adhesion molecules (VCAM1, ICAM1). Addition of eNOS co-factor tetrahydrobiopterin (BH4) mitigated the increases in OxS caused by TNF-α, demonstrating that eNOS uncoupling was the primary pathophysiological mechanism at play. Ex vivo, we used porcine aortic valve leaflets to screen the ability of BH4, catalase, and superoxide dismutase to protect against the downstream effects of eNOS uncoupling. We found that all three antioxidants mitigated increases in valve OxS, improved endothelial function, and protected against extracellular matrix remodeling. TNF-α+SOD also protected against increases in myofibroblastic protein α-smooth muscle actin and chronic elevation of VCAM1. Conclusions: These results present endothelial inflammatory oxidative stress as a new mechanism that connects early valve pathology with later stages of degeneration. Targeting these mechanisms via tailored antioxidant therapy could provide new avenues for treatment of CAVD.

Superoxide dismutase protects against effects of eNOS uncoupling in early‐stage aortic valve disease (278.6)

Objective: Oxidative stress (OxS) is known to contribute to late stages of aortic valve disease (AVD), but its role in early‐stage valve inflammation and as a driver of valve pathology has not been investigated. Here we show that inflammation drives increased OxS via eNOS uncoupling. Using an ex vivo model of AVD, we show that superoxide dismutase can block the downstream effects of eNOS uncoupling and mitigate later‐stage valve degeneration.Methods and Results: We first observed evidence of eNOS uncoupling in the endothelium of calcified human aortic valve leaflets, which had elevated dihydroethidium staining for superoxide in endothelial cells both near and distal to the primary nodule. We then used a 3‐D in vitro system to show that inflammatory cytokine TNF‐α caused increased superoxide, free radicals, and hydrogen peroxide in aortic valve endothelial cells, as soon as 15 minutes after treatment. The excess oxidative stress due to TNF‐α decreased VEC nitric oxide secretion, lowered eNOS and VE‐cadherin protein levels, and increased expression of inflammatory adhesion molecules (VCAM1, ICAM1). Addition of eNOS co‐factor tetrahydrobiopterin (BH4) mitigated the increases in OxS caused by TNF‐α, demonstrating the eNOS uncoupling was the primary pathophysiological mechanism at play. Using an ex vivo system of porcine aortic valve leaflets cultured for 21 days, we screened the ability of BH4, catalase, and superoxide dismutase (SOD) to decrease the endothelial OxS effects caused by TNF‐α. We found that all three antioxidants mitigated the effects of TNF‐α and improved endothelial function, but SOD showed the most significant reduction in protein levels of myofibroblastic protein α‐smooth muscle actin and VCAM1.Conclusions: These results present endothelial inflammatory oxidative stress as a new mechanism that connects early valve pathology with later stages of degeneration. Targeting these mechanisms via tailored antioxidant therapy could provide new avenues for treatment of CAVD.Grant Funding Source: Supported by NIH HL110328, NSF CBET‐0955172

Human Bone Marrow Derived Mesenchymal Stem Cells Regulate Leukocyte-Endothelial Interactions and Activation of Transcription Factor NF-Kappa B.

Bone marrow derived mesenchymal stem cells (MSCs) have been shown to demonstrate benefit in multiple disease models characterized by inflammation such as sepsis and acute lung injury. Mechanistically we hypothesized that MSCs exhibit these properties through inhibition of leukocyte activation and modulation of leukocyte-endothelial interactions; key interlinked processes involved in the deleterious effects of injury and inflammation. In this paper we found that MSCs co-cultured with a monocytoid line, U937, inhibit U937 binding to pulmonary endothelial cells (PECs) stimulated with the inflammatory cytokine TNFα. Furthermore, we show that these effects on functional adhesion are not due to changes in inflammatory adhesion molecule expression on U937s. No changes were found in CD62L, CD29, CD11b and CD18 expression on U937s co-cultured with MSCs. To determine if the effects of MSCs on leukocyte-endothelial interactions are due to the effects of MSCs on leukocyte activation, we investigated whether MSCs affect functional activation of the transcription factor NF-Kappa B. We found that MSCs significantly inhibit transcriptional activation of NF-kappa B in U937s. We also found that MSCs inhibit DNA binding of NF-kappa B subunits p50 and p65 to putative NF-kappa B DNA binding sites. Concomitant with a decrease in NF-kappa B activation was a significant increase in IL-10, an anti-inflammatory cytokine known to inhibit activation of NF-kappa B. Taken together, these findings show that MSCs have potent effects on leukocyte-endothelial interactions which may be due to the direct effects of MSCs on IL-10 and NF-kB. These findings suggest a potential therapeutic role for MSCs in diseases characterized by inflammation such as acute lung injury or multi-organ failure induced by traumatic injury.

Alpha-lipoic acid attenuates cisplatin-induced acute kidney injury in mice by suppressing renal inflammation

Cisplatin is a chemotherapeutic agent used in treatment of malignant tumours. However, cisplatin produces various side effects, such as nephrotoxicity, neurotoxicity, emetogenesis and ototoxicity. Inflammation is an important mechanism of cisplatin nephrotoxicity. Alpha-lipoic acid (alpha-LA) has anti-inflammatory effects that inhibit both adhesion molecule expression in human endothelial cells and monocyte adhesion by suppressing the nuclear factor-kappaB (NF-kappaB) signalling pathway. The goals of this study were to investigate the anti-inflammatory effects of alpha-LA during cisplatin-induced renal injury and to examine the mechanisms of protection. C57BL/6 mice were given cisplatin (20 mg/kg) with or without alpha-LA treatment (100 mg/kg for 3 days). Renal function, histological changes, adhesion molecule expression and inflammatory cell infiltration were examined. The effect of alpha-LA on NF-kappaB activity was evaluated by examining nuclear translocation and phosphorylation of NF-kappaB p65 subunits in kidney tissue. Cisplatin-induced decreases in renal function, measured by blood urea nitrogen, serum creatinine level and renal tubular injury scores, were attenuated by alpha-LA treatment. alpha-LA decreased the tissue levels of tumour necrosis factor-alpha, the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), and suppressed the infiltration of CD11b-positive macrophages. alpha-LA also attenuated the cisplatin-induced increases in the phosphorylation and nuclear translocation of NF- kappaB p65 subunits in kidney tissue. These results suggest that alpha-LA treatment ameliorates cisplatin-induced acute kidney injury by reducing inflammatory adhesion molecule expression and NF-kappaB activity.

Allopurinol Use Yields Potentially Beneficial Effects on Inflammatory Indices in Those With Recent Ischemic Stroke

Elevated serum uric acid level is associated with poor outcome and increased risk of recurrent events after stroke. The xanthine oxidase inhibitor allopurinol lowers uric acid but also attenuates expression of inflammatory adhesion molecules in murine models, reduces oxidative stress in the vasculature, and improves endothelial function. We sought to investigate whether allopurinol alters expression of inflammatory markers after acute ischemic stroke. We performed a randomized, double-blind, placebo-controlled trial to investigate the safety, tolerability, and effect of 6 weeks' treatment with high- (300 mg once a day) or low- (100 mg once a day) dose allopurinol on levels of uric acid and circulating inflammatory markers after ischemic stroke. We enrolled 50 patients with acute ischemic stroke (17, 17, and 16 in the high, low, and placebo groups, respectively). Mean (+/-SD) age was 70 (+/-13) years. Groups had similar characteristics at baseline. There were no serious adverse events. Uric acid levels were significantly reduced at both 7 days and 6 weeks in the high-dose group (by 0.14 mmol/L at 6 weeks, P=0.002). Intercellular adhesion molecule-1 concentration (ng/mL) rose by 51.2 in the placebo group, rose slightly (by 10.6) in the low-dose allopurinol group, but fell in the high-dose group (by 2.6; difference between groups P=0.012, Kruskal-Wallis test). Allopurinol treatment is well tolerated and attenuates the rise in intercellular adhesion molecule-1 levels seen after stroke. Uric acid levels were lowered with high doses. These findings support further evaluation of allopurinol as a preventive measure after stroke.

Protective Role of Angiopoietin-1 in Endotoxic Shock

Angiopoietin-1 (Ang1) plays an essential role in embryonic vasculature development, protects the adult peripheral vasculature from leakage, and has antiinflammatory properties. Because endotoxin-induced shock is a condition with microvascular leakage resulting from inflammation, we examined the potential therapeutic benefit of Ang1 in a murine model of lipopolysaccharide (LPS)-induced endotoxic shock. To induce endotoxic shock, LPS was injected intraperitoneally into C57BL/6 mice. Half of the mice received an intravenous application of 1.0x10(9) plaque-forming units of an adenoviral construct expressing human Ang1 (AdhAng1); in the other half an identical vector expressing green fluorescent protein (AdGFP) was injected as a control. In the AdhAng1-treated mice, hepatic transfection and high expression of circulating Ang1 protein were observed. Whereas in LPS-treated control mice, hemodynamic function was severely depressed 12 hours after LPS injection (decrease of blood pressure from 91+/-3 to 49+/-7 mm Hg, dP/dt(max) from 7284+/-550 to 2699+/-233 mm Hg/s, cardiac output from 11.3+/-1.2 to 2.8+/-0.8 mL/min; P<0.0005), in LPS-treated AdhAng1 mice blood pressure fell only to 76+/-3 mm Hg, dP/dt(max) to 5091+/-489 mm Hg/s, and cardiac output to 6.7+/-1.4 mL/min (P<0.05). This resistance to LPS-induced hemodynamic changes was reflected by an improved Kaplan-Meier survival rate of the AdhAng1 mice. Histological analysis revealed that lung injury after LPS injection was markedly attenuated in AdhAng1 mice. In addition, LPS-induced increase in lung water content and pulmonary myeloperoxidase activity was significantly reduced. Furthermore, LPS-induced increases in the expression level of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin protein in the lungs were markedly lower in AdhAng1 mice than in control mice. Finally, in the mice overexpressing Ang1, pulmonary endothelial NO synthase (eNOS) expression and activity remained preserved after LPS challenge, providing evidence that the beneficial effect of Ang1 in endotoxic shock is mediated by eNOS-derived NO. Our study demonstrates an improved mortality rate in mice with endotoxic shock pretreated with an adenoviral construct encoding Ang1. The enhanced survival rate induced by Ang1 was accompanied by an improvement in hemodynamic function, reduced lung injury, a lower expression of inflammatory adhesion molecules, and preserved eNOS activity in the lung tissue. Ang1 may therefore have utility as an adjunctive agent for the treatment of septic shock condition.