Expression Of Adhesion Molecules VCAM-1 Research Articles

MXRA7 is involved in monocyte-to-macrophage differentiation

Macrophages are critical in mediating immune and inflammatory responses, while monocyte-to-macrophage differentiation is one of the main macrophage resources that involves various matrix proteins. Matrix remodeling associated 7 (MXRA7) was recently discovered to affect a variety of physiological and pathological processes related to matrix biology. In the present study, we investigated the role of MXRA7 in monocyte-to-macrophage differentiation in vitro. We found that knockdown of MXRA7 inhibited the proliferation of THP-1 human monocytic cells. Knockdown of MXRA7 increased the adhesion ability of THP-1 cells through upregulation the expression of adhesion molecules VCAM-1 and ICAM1. Knockdown of MXRA7 alone could promoted the differentiation of THP-1 cells to macrophages. Furthermore, the MXRA7-knockdown THP-1 cells produced a more significant upregulation pattern with M1-type cytokines (TNF-α, IL-1β and IL-6) than with those M2-type molecules (TGF-β1 and IL-1RA) upon PMA stimulation, indicating that knockdown of MXRA7 facilitated THP-1 cells differentiation toward M1 macrophages. RNA sequencing analysis revealed the potential biological roles of MXRA7 in cell adhesion, macrophage and monocyte differentiation. Moreover, MXRA7 knockdown promoted the expression of NF-κB p52/p100, while PMA stimulation could increase the expression of NF-κB p52/p100 and activating MAPK signaling pathways in MXRA7 knockdown cells. In conclusion, MXRA7 affected the differentiation of THP-1 cells toward macrophages possibly through NF-κB signaling pathways.

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo-angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C-HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD-HUVECs), leading to a permanent pro-inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti-inflammatory and pro-angiogenic properties of OA are still under investigation. We tested OA on C- and GD-HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA-pre-treated C- and GD-HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD-HUVEC endothelium under OA pre-treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD-HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C- and GD-HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.

Identification and Quantitation of the Bioactive Components in Wasted Aralia elata Leaves Extract with Endothelial Protective Activity.

Aralia elata, a renowned medicinal plant with a rich history in traditional medicine, has gained attention for its potential therapeutic applications. However, the leaves of this plant have been largely overlooked and discarded due to limited knowledge of their biological activity and chemical composition. To bridge this gap, a comprehensive study was conducted to explore the therapeutic potential of the 70% ethanol extract derived from Aralia elata leaves (LAE) for the treatment of cardiovascular disease (CVD). Initially, the cytotoxic effects of LAE on human umbilical vein endothelial cells (HUVECs) were assessed, revealing no toxicity within concentrations up to 5 μg/mL. This suggests that LAE could serve as a safe raw material for the development of health supplements and drugs aimed at promoting cardiovascular well-being. Furthermore, the study found that LAE extract demonstrated anti-inflammatory properties in HUVECs by modulating the PI3K/Akt and MAPK signaling pathways. These findings are particularly significant as inflammation plays a crucial role in the progression of CVD. Moreover, LAE extract exhibited the ability to suppress the expression of adhesion molecules VCAM-1 and ICAM-1, which are pivotal in leukocyte migration to inflamed blood vessels observed in various pathological conditions. In conjunction with the investigation on therapeutic potential, the study also established an optimal HPLC-PDA-ESI-MS/MS method to identify and confirm the chemical constituents present in 24 samples collected from distinct regions in South Korea. Tentative identification revealed the presence of 14 saponins and nine phenolic compounds, while further analysis using PCA and PLS-DA allowed for the differentiation of samples based on their geographical origins. Notably, specific compounds such as chlorogenic acid, isochlorogenic acid A, and quercitrin emerged as marker compounds responsible for distinguishing samples from different regions. Overall, by unraveling its endothelial protective activity and identifying key chemical constituents, this research not only offers valuable insights for the development of novel treatments but also underscores the importance of utilizing and preserving natural resources efficiently.

A novel small molecule inhibitor of vascular endothelin signaling and hypertension

Augmented endothelin-1 (ET-1) signaling causes profound vasoconstriction, smooth muscle proliferation, endothelial dysfunction, oxidative stress, and vascular inflammation and sensitivity, which are often present in resistant hypertension, salt-sensitive hypertension and hypertension among African Americans. Here, we sought to examine the role of indole-3 acetic acid (IAA) as a novel inhibitor of vascular ET-1 signaling and hypertension. IAA, popularly known as auxin, is the most potent plant growth hormone, which is also produced in the human body via tryptophan metabolism by gut microbiota. Although the role of IAA as a potent plant growth hormone has been characterized, little is known about the biological activity of this compound in humans. Importantly, studies on the role of IAA as a regulator of arterial contractility, vascular inflammation and hypertension remain completely unexplored. Our data demonstrates that IAA inhibits ET-1-evoked vasoconstriction in resistance mesenteric arteries, ROS production in human aortic SMCs, expression of adhesion molecule VCAM-1 on EC surface, leukocyte adhesion, and ECE-1 activity. IAA dose-dependently inhibits the activity of a purified human endothelin converting enzyme-1 (ECE-1) that converts the precursor big ET to its active form ET-1 and is upregulated in hypertension. Consistent with these findings, we show that treatment of Dahl salt-sensitive (DSS) hypertensive rats, a model of augmented ET-1 signaling, with an oral extended-release, in situ gel-based formulation of IAA (IAA-oragel) reduces blood pressure (BP) elevation, suggesting a key role for IAA in BP regulation. Taken together, this study presents a novel inquiry into regulation of ET-1 function by IAA and could identify IAA as the first dual action inhibitor of the ET system. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NIK-SIX1 signalling axis regulates high glucose-induced endothelial cell dysfunction and inflammation

Endothelial dysfunction and inflammation are the main manifestations of diabetes-associated atherosclerosis. This paper studied the roles of NF-κB-inducing kinase (NIK) and sine oculis homeobox homolog 1 (SIX1) in regulating high glucose-induced endothelial dysfunction and inflammation. The expression of NIK and SIX1 in human umbilical vein endothelial cells (HUVECs) was silenced by transfection with the specific shRNAs. HUVECs exposed to high glucose were considered as a cell model of endothelial dysfunction. Expression of NIK and SIX1 following transfection was measured by qRT-PCR and western blotting analysis. The proliferation, migration, and inflammation of HUVECs were evaluated by EdU staining, scratch test, ELISA, and western blotting. High glucose (30 mM) significantly decreased the proliferation and migration of HUVECs. High glucose-induced the expression of adhesion molecules VCAM-1 and ICAM-1. Moreover, high glucose increased the release of IL-1β, IL-6, TNF-α, and MCP-1. Transfection of cells with NIK shRNA significantly reversed the toxic effects of high glucose on HUVECs. Of contrast, SIX1 shRNA accelerated the effects of high glucose on HUVECs. NIK shRNA inhibited the accumulation of RelA, RelB, and p52. Meanwhile, NIK shRNA led to SIX1 downregulation which further induced the activation of the NF-κB pathway. NIK-SIX1 signalling axis was suggested to be critical in the regulation of high glucose-induced endothelial dysfunction and inflammation. SIX1 may function as an immunological gatekeeper to control the excessive inflammation mediated by NIK in diabetes-associated atherosclerosis.

Association of KLOTHO polymorphisms with clinical complications of sickle cell anemia.

The clinical and phenotypic heterogeneity of patients with sickle cell anemia (SCA) is influenced by environmental and genetic factors. Several genetic modifiers, such as the KLOTHO (KL) gene, have been associated with SCA clinical outcomes. The KL gene and its encoded proteins are implicated in important biological pathways, which affect the disease's pathophysiology, such as expression of adhesion molecules VCAM-1 and ICAM-1, oxidative stress, and nitric oxide biology. Here, we evaluated the clinical relevance of two polymorphisms found on the KL gene (rs685417 and rs211239) in 588 unrelated patients with SCA. Genotyping analyses were performed using the TaqMan system. The KL rs211239 was associated with increased number of vaso-occlusive crisis (VOCs) per year (P = 0.001), while KL rs685417 was associated with increased frequency of stroke (P = 0.034), priapism (P = 0.011), number of complications (P = 0.019), and with a lower incidence of priapism (P = 0.036). Additionally, the associations with VOCs, stroke, and priapism remained consistent in multivariate analyses (P < 0.05). Our data highlight the clinical importance of KL in SCA.

Leukocyte Integrin Antagonists as a Novel Option to Treat Dry Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a complex multifactorial degenerative disease that leads to irreversible blindness. AMD affects the macula, the central part of the retina responsible for sharp central vision. Retinal pigment epithelium (RPE) is the main cellular type affected in dry AMD. RPE cells form a monolayer between the choroid and the neuroretina and are in close functional relationship with photoreceptors; moreover, RPE cells are part of the blood retina barrier that is disrupted in ocular diseases such as AMD. During ocular inflammation lymphocytes and macrophages are recruited, contact RPE and produce pro-inflammatory cytokines, which play an important role in AMD pathogenesis. The interaction between RPE and immune cells is mediated by leukocyte integrins, heterodimeric transmembrane receptors, and adhesion molecules, including VCAM-1 and ICAM-1. Within this frame, this study aimed to characterize RPE-leukocytes interaction and to investigate any potentially beneficial effects induced by integrin antagonists (DS-70, MN27 and SR714), developed in previous studies. ARPE-19 cells were co-cultured for different incubation times with Jurkat cells and apoptosis and necrosis levels were analyzed by flow cytometry. Moreover, we measured the mRNA levels of the pro-inflammatory cytokine IL-1β and the expression of adhesion molecules VCAM-1 and ICAM-1. We found that RPE-lymphocyte interaction increased apoptosis and necrosis levels in RPE cells and the expression of IL-1β. This interaction was mediated by the binding of α4β1 and αLβ2 integrins to VCAM-1 and ICAM-1, respectively. The blockade of RPE-lymphocyte interaction with blocking antibodies highlighted the pivotal role played by integrins. Therefore, α4β1 and αLβ2 integrin antagonists were employed to disrupt RPE-lymphocyte crosstalk. Small molecule integrin antagonists proved to be effective in reducing RPE cell death and expression of IL-1β, demonstrating that integrin antagonists could protect RPE cells from detrimental effects induced by the interaction with immune cells recruited to the retina. Overall, the leukocyte integrin antagonists employed in the present study may represent a novel opportunity to develop new drugs to fight dry AMD.

INCREASED C3 IN THE AGING BRAIN PROMOTES INFLAMMATORY TRANSITION IN ENDOTHELIAL CELLS

Innate immunity has been implicated in normal aging, and age-related disease. The connection between age-related neuroinflammation and change in brain vasculature prior to disease onset remains poorly understood. The complement pathway is an established mediator of neuroinflammation, and increased complement C3 is seen in the aging brain. Thus, we asked whether C3 can promote changes in brain vasculature. We found age dependent increase of brain C3 levels in C57BL/6J mice. Furthermore, we found an increase in expression of adhesion molecule VCAM-1 in endothelial cells (ECs) of the cortex and hippocampus, which was rescued in aged C3a receptor null (C3ar1-/-) mice and aged C3a receptor (C3aR) antagonist treated mice. We confirmed these results by qPCR analysis for Vcam1 in sorted ECs. Human brain microvascular endothelial cells (HBMECs) treated with C3a showed increased expression of VCAM-1, but not other adhesion molecules. Sorted ECs from C3ar1-/- mice challenged with LPS confirmed these findings. Furthermore, C3aR signaling in ECs showed increased blood-brain barrier (BBB) permeability using trans-endothelial electrical resistance (TEER), and BBB impermeable dye injections. HBMECs treated with C3a revealed mis-localization of VE-Cadherin, followed by reduction in protein level when analyzed by immunofluorescence, which promotes increased barrier permeability. As a functional consequence of VCAM-1 expression and increased BBB permeability we found aged mouse brains have increased peripheral lymphocyte (CD45+/CD11b-) infiltration, which was reduced in a C3aR dependent manner. In conclusion, our work suggests there is a strong relationship between C3 expression and vascular C3aR contributing to a functional transition in endothelial cells during aging.

Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells.

Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-κB (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-κB by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC.

Tannic acid as a plant-derived polyphenol exerts vasoprotection via enhancing KLF2 expression in endothelial cells

The transcription factor Kruppel-like factor 2 (KLF2) is a critical anti-inflammatory and anti-atherogenic molecule in vascular endothelium. Enhancing KLF2 expression and activity improves endothelial function and prevents atherosclerosis. However, the pharmacological and molecular regulators for KLF2 are scarce. Using high-throughput luciferase reporter assay to screen for KLF2 activators, we have identified tannic acid (TA), a polyphenolic compound, as a potent KLF2 activator that attenuates endothelial inflammation. Mechanistic studies suggested that TA induced KLF2 expression in part through the ERK5/MEF2 pathway. Functionally, TA markedly decreased monocyte adhesion to ECs by reducing expression of adhesion molecule VCAM1. Using lung ECs isolated from Klf2+/+ and Klf2+/− mice, we showed that the anti-inflammatory effect of TA is dependent on KLF2. Collectively, our results demonstrate that TA is a potent KLF2 activator and TA attenuated endothelial inflammation through upregulation of KLF2. Our findings provide a novel mechanism for the well-established beneficial cardiovascular effects of TA and suggest that KLF2 could be a novel therapeutic target for atherosclerotic vascular disease.

Deleterious vascular effects of indoxyl sulfate and reversal by oral adsorbent AST-120

BackgroundIn chronic kidney disease (CKD), blood vessels are permanently exposed to uremic toxins such as indoxyl sulfate (IS). We hypothesized that IS could alter vascular tone and that reducing its serum concentration could be beneficial. DesignWe studied acute and longer-term effects of IS and AST-120, an oral charcoal adsorbent, on vascular reactivity, endothelium integrity and expression of adhesion molecules VCAM-1 and ICAM-1 in aortic rings of normal and uremic wild type (WT) mice in vitro, and the cardiovascular effects of AST-120 in both WT and apoE−/− mice with CKD in vivo. ResultsIn vitro, 1.0 mM IS acutely reduced vascular relaxation (64% for IS 1.0 mM vs. 80% for control, p < 0.05). The effect was more marked after 4 days exposure (39% for IS 1.0 mM 4 days; p < 0.001, prolonged vs. acute exposure), and was associated with endothelial cell loss and upregulation of ICAM-1/VCAM-1 expression. In vitro, AST-120 restored normal vascular function and prevented IS induced endothelial cell loss and ICAM-1/VCAM-1 upregulation. In vivo, AST-120 treatment of CKD mice (1) improved vascular relaxation (72% vs. 48% maximal relaxation in treated vs. untreated mice, p < 0.001), (2) reduced aortic VCAM-1 and ICAM-1 expression, (3) decreased aorta systolic expansion rate (9 ± 3% CKD vs. 14 ± 3% CKD + AST-120, p < 0.02), and (4) prevented the increase in pulse wave velocity (3.56 ± 0.17 m/s CKD vs. 3.10 ± 0.08 m/s CKD + AST-120, p < 0.006). Similar changes were observed in apoE−/− mice. ConclusionIS appears to be an important contributor to the vascular dysfunction associated with CKD. AST-120 treatment ameliorates this dysfunction, possibly via a decrease in serum IS concentration.

OP0265 Anti-At1r and Anti-Etar Autoantibodies from Patients with SSC and their Agonistic Effects

BackgroundFunctional autoantibodies to angiotensin II type 1 receptor (AT1R) and endothelin 1 type A receptor (ETAR) are found in elevated levels in systemic sclerosis (SSc) and they show an association...

OP0224 Anti-AT1R and Anti-ETAR Autoantibodies from Patients with SSC and their Agonistic Effects

BackgroundAutoantibodies reactive to angiotensin II type 1 receptor (AT1R) and endothelin 1 type A receptor (ETAR), anti-AT1R and anti-ETAR autoantibodies, are found in patients with Systemic sclerosis (SSc) as a...

FOLIC ACID REDUCES ADHESION MOLECULES VCAM-1 EXPRESSION ON ENDOTHELIUM IN RATS WITH HYPERHOMOCYSTEINEMIA

ObjectivesTo investigate the effects of folic acid supplementation on the expression of adhesion molecules VCAM·1 in aortic endothelium of rats with hyperhomocys teinemia (HHCY) induced by ingestion of excess methionine...

Adhesive and Inflammatory Properties Are Increased in Blood Outgrowth Endothelial Cells From Patients with Sickle Cell Anemia

Abstract 2133The endothelium plays an important role in the pathophysiology of sickle cell anemia (SCA) due to interactions of endothelial cells with other cell types in the vaso-occlusive process and endothelial dysfunction caused by chronic inflammation and decreased nitric oxide bioavailability. Inflammatory molecules, such as cytokine TNF-α, may alter the endothelial cell surface and, thus, modulate the adhesion molecules that bind red blood cells (RBC). Damaged or activated endothelium produces cytokines and chemokines that result in the high levels of circulating inflammatory molecules found in the SCA. Blood Outgrowth Endothelial Cells (BOEC) are circulating endothelial cells isolated from the peripheral blood of adults, they have a high proliferative capacity in vitro, with the characteristic phenotype of mature cells. BOEC cultures were established to serve as a model for an in vitro study of endothelial properties and function in SCA, to evaluate RBC adhesion, expression of adhesion molecules and production of inflammatory cytokines in BOEC from healthy controls (Con) and SCA patients. Three cultures of BOEC from Con and three cultures from SCA patients in steady state were established and identification confirmed by flow cytometry (CD31+; CD146+; VEGF R2+; CD45-; CD133-). BOEC were used under basal conditions or following treatment with 10ng/ml TNF-α (3 H). RBC were isolated from Con, SCA and SCA patients on hydroxyurea treatment (SCAHU; 20–30 mg/Kg/day) and their adhesion to confluent Con and SCA BOEC was evaluated (4×106 cell/ml in EGM-2), using static adhesion assays. BOEC expression of adhesion molecules VCAM-1, E-selectin, P-selectin, CD36, the Vitronectin receptor and BCAM/Lu was evaluated by flow cytometry. The production and release of GM-CSF and IL-8 to the supernatant of BOEC in the presence and absence of an antiinflammatory stimulus (1μM Simvastatin, 3.5H) was quantified by ELISA. Notably, Con RBC adhered significantly more to SCA BOEC (16.6 ± 4.6%; n=14; p<0.001; Wilcoxon Test) than to Con BOEC (9.4 ± 3.2%; n=14). Furthermore, TNF-α stimulation further increased the adhesion of Con RBC to both Con BOEC (16.6 ± 5.6%; n=14 p<0.001 comp. to non-stimulated Con BOEC) and SCA BOEC (20.6 ±7.0%; n=14; p<0.05 comp. to non-stimulated SCA BOEC). Similarly, SCA RBC adhered significantly more to SCA BOEC (18.5 ± 4.8%; n=10; p<0.001; Wilcoxon Test) than to Con BOEC (14.3 ± 4.2%; n=10) and TNF-α stimulation further increased SCA RBC adhesion to Con BOEC (18.3 ± 5.4%; n=10; p<0.01) and SCA BOEC (20.9 ± 6.4%; n=10; p<0.05; Wilcoxon). SCAHU RBC also adhered more to SCA BOEC (19.5 ± 7.4%; p<0.01; Wilcoxon) compared to Con BOEC (14.3 ± 4.2%; n=10) and to TNF-α stimulated Con BOEC (19.1 ± 6.0%; n=10; p<0.01) and SCA BOEC (23.8 ± 6.3%; n=10; p<0.01), compared to respective non-stimulated BOEC. Con and SCA BOEC presented low-level expression of the P-selectin and CD36 adhesion molecules, even following TNF-α inflammatory stimulus; VCAM-1 was increased on both Con and SCA BOEC under TNF-α conditions, but no difference in expression was observed between the Con and SCA BOEC (data not shown); however, after TNF-α stimulus the expression of E-selectin on SCA BOEC was significantly higher (6613 ± 729 MFI; n=3; p=0.03; Unpaired t Test) than on Con BOEC (3645 ± 547 MFI; n=3). Vitronectin receptor and BCAM/Lu expression was slightly higher on SCA BOEC (6248.3 ± 552.9MFI; 1152 ± 51.4MFI respect; n=3; p<0.05, Unpaired t Test) than on Con BOEC (5059.7 ± 426.2MFI; 904.7 ± 70.9MFI; respect; n=3). Following TNF-α stimulus, SCA BOEC released significantly more GM-CSF (271.5 ± 8.7pg/ml; n=3; p<0.01; Unpaired t Test) than Con BOEC (175.2 ± 18.4pg/ml; n=3); where co-incubation with Simvastatin reduced the release of GM-CSF from TNF-α stimulated SCA (189.0 ± 7.1pg/ml; n=3; p<0.05 comp. to TNF-α SCA BOEC; Paired t Test) and Con BOEC (118.9 ± 21.9pg/ml; p<0.01; Paired t Test). SCA BOEC released more IL-8 (10977.4 ± 379.7pg/ml) than BOEC Con (8913.7 ± 234.7pg/ml) under TNF-α inflammatory conditions; however Simvastatin reduced the production of IL-8 only from Con BOEC (data not shown). Results suggest that SCA BOEC present inherent endothelial abnormalities that promote increased adhesion of red cells and augmented inflammatory properties, which are maintained during in vitro culture and following inflammatory stimulus. These cells may serve as a model for in vitro study of aberrant endothelial function in SCA.Support by FAPESP and CNPq Disclosures:No relevant conflicts of interest to declare.

Polymorphisms in TNFα Are Associated with Cerebrovascular Events in Sickle Cell Disease.

Abstract 1540Poster Board I-563Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that stimulates phagocytosis, neutrophil recruitment, and expression of adhesion molecule VCAM-1. Plasma levels of TNFα have been found to be increased in sickle cell disease (SCD), and in vitro studies show that TNFα causes increased adherence of sickle red blood cells to human umbilical vein endothelial cells. A polymorphism in the promoter region of the TNFα gene has previously been associated with stroke in children with SCD (Hoppe et al., 2007). The current study was designed to identify associations of additional TNFα single nucleotide polymorphisms (SNPs) with SCD clinical complications. We analyzed five SNPs in the TNFα gene in 509 DNA samples of SCD patients from Duke University, University of North Carolina at Chapel Hill, and Emory University. In our data set, cerebrovascular events (CVEs), including overt stroke, seizures, and transient ischemic attacks, occurred in 133 out of 509 SCD patients (26.1%). SNP genotyping was performed using Taqman genotyping assays from Applied Biosystems. Due to low minor allele frequencies (<0.05) for all the SNPs examined, genetic associations with SCD clinical complications were examined by using allele tests. After controlling for age, gender, and use of hydroxyurea, two of the five TNFα SNPs, rs2228088 and rs3093665, were significantly associated with CVEs (p=0.013 and 0.029, respectively). The odds that SCD patients with a G allele at rs2228088 suffered from CVEs were 0.485 times that for patients with the T allele, suggesting that the G allele had a protective effect. The odds of having the A allele at rs3093665 and suffering from CVEs was also reduced, at 0.45 compared to the C allele. Neither SNP was found to be in linkage disequilibrium (LD) with any of the other SNPs analyzed (r2≤0.002). There was also strong association of SNP rs2228088 with acute chest syndrome (ACS; p=0.003), occurring in 382 out of 509 SCD patients (75%). However, in this analysis, the G allele was associated with increased risk for ACS (OR=2.313). In addition to the association with CVEs, the SNP rs3093665 was also significantly associated with priapism (p=0.03), reported by 86 of 223 male SCD patients (38.6%). In this analysis, the A allele was protective, as had been observed for CVE (OR=0.188). Additionally, we found no difference in steady state plasma TNFα levels between genotypes for the two SNPs. The functional significance of these SNPs is presently unknown. SNP rs2228088 is a synonymous SNP located in the coding region, and rs3093665 is located in the 3' untranslated region of the TNFα gene. While the G to T change at SNP rs2228088 does not translate to a change in amino acid sequence, the A to C change at SNP rs3093665 may affect mRNA stability due to its location. It is also possible that one or both of these SNPs is in LD with another functionally relevant SNP. Our findings thus support previous data implicating TNFα polymorphisms in risk for central nervous system events. Interestingly, ACS has been previously associated with seizures, stroke and altered mental status in adults and children with SCD (Vinchinsky et al., 2000) and with silent cerebral infarcts and reversible posterior leukoencephalopathy syndrome in children with SCD (Henderson et al., 2003). However, in our dataset, ACS and the occurrence of CVEs were not associated (p=0.24). Further studies are required to elucidate these and other factors that potentially correlate with SCD clinical complications. DisclosuresNo relevant conflicts of interest to declare.

Effects of four medicinal herbs on human vascular endothelial cells in culture

Background Danshen (DS, Salvia miltiorrhiza), Shanchi (SQ, Panax notoginseng), Shanzai (SZ, Hawthorn) and Heshouwu (HSW, Polygonum multiflorum Thunb) are four medicinal herbs commonly used in traditional Chinese medicine and previously shown to have activity that may contribute to cardiovascular protection. This study aims to investigate effects of these herbs on vascular endothelial cells with respect to cell viability and expression of cellular adhesion molecules under inflammatory conditions. Methods Herbal extracts were prepared by an established industrial manufacturing process. Human umbilical vein endothelial cells (HUVEC) were incubated with the herbal extract under normal or serum-free culture and tumor necrosis factor (TNF) α stimulation. Cell apoptosis, apoptosis-associated gene expression, expression of cellular adhesion molecules, DNA synthesis, and growth were assessed via morphological examination, Annexin-V staining, Western blotting analysis, Flow-Cytometry, [ 3H]-thymidine incorporation assay, and cell number study. Results SZ and HSW significantly inhibited apoptosis in HUVEC undergoing serum deprivation and TNFα stimulation, accompanied by down-regulation of caspase-3 gene expression. DS and SQ significantly attenuated TNFα-induced expression of adhesion molecule VCAM-1 and also ICAM-1 by DS in the cells. All four herbs at therapeutic concentrations (100 μg/mL) inhibited DNA synthesis (10–42% decrease in [ 3H]-thymidine incorporation rates) and growth (5–10% decrease in cell numbers) in HUVEC under normal cultures. Conclusion DS, SQ, SZ and HSW are physiologically active on human vascular endothelial cells. The actions by HSW and SZ to reduce apoptosis and DS and SQ to inhibit adhesion molecule expression may help protect endothelial function and inhibit atherogenesis, while their actions to inhibit DNA synthesis and cell growth may weaken the ability of endothelial repair. Further studies are needed to identify the chemical compounds responsible to these physiological effects by these herbs.

Placental apoptosis and adhesion molecules expression in the placenta and the maternal placental bed of pregnancies complicated by fetal growth restriction with and without pre-eclampsia

SummaryThe aim of the study was to examine the expression of adhesion molecules VCAM-1 and ICAM-3 in placental tissue samples and placental bed (maternal decidual tissue) biopsies of pregnancies complicated by pre-eclampsia (PE) and fetal growth restriction (FGR), and to determine whether PE and FGR are associated with an increase in placental apoptosis. We studied placentas and placental bed samples of 49 third trimester pregnancies complicated by FGR (26 with associated PE, 23 without PE) and 25 normotensive healthy pregnant women. Placental apoptosis was assessed by the TUNEL method. Immunohistochemistry was used to assess expression of the VCAM-1 and ICAM-3. There was no significant difference in the staining intensity of VCAM-1 in placentas ( p = 0.472) and placental bed biopsies ( p = 0.754) of women delivering appropriate for gestational age and growth restricted fetuses (with and without associated PE). The amount of lymphocytes staining positively with ICAM-3 was significantly higher in both placental and placental bed biopsies of women delivering growth restricted fetuses compared with control pregnancies ( p < 0.001). Fetal growth restricted pregnancies with associated PE showed higher staining of ICAM-3 in placental compared with placental bed samples ( p = 0.049). In fetal growth restricted placentas, apoptotic nuclei were more abundant compared with control placentas ( p < 0.001). Increased expression of ICAM-3 on lymphocyte surface of both maternal and fetal side, suggests lymphocyte overactivation in PE and FGR. Increased placental apoptosis may play an important role in the pathogenesis or sequelae of PE.

Folic acid reduces adhesion molecules VCAM-1 expession in aortic of rats with hyperhomocysteinemia

To investigate effects of supplementation of folic acid on the expression of adhesion molecules VCAM-1 in the aortas of rats with hyperhomocysteinemia. Thirty male SD rats (200 ± 20 g) were invided into 3 groups ( n = 10 for each group): control group(Control), high Met group(Met) and Met plus Folate group(Met + Folate), fed. for 45 days. Plasma Hcy levels were higher with the high-methionine diet (140.68 ± 36.87 μmol/L vs 6.47 ± 1.10 μmol/L in control rats) an effect which was reduced by folate. Respectively, the aortic expression of adhesion molecules VCAM-1 at protein and mRNA levels were higher in the Met groups than those in the control groups or the Met + Folate groups. A high methionine diet for 45 days was sufficient to induce hyperhomocysteinemia. Folate supplementation prevented elevation of Hcy levels in the blood, and reduced expression of the adhesion molecule VCAM-1. Hyperhomocysteinemia is now regarded as one of the important risk factors for cardiovascular and cerebralvascular disorders.[Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998; 38(15):1042-50.] Several plausible mechanisms for Hcy-induecd atherosclerosis have been proposed. These include endothelial dysfunction, enhancement of oxidative stress, reduction in NO bioavailability, and augmentation of thrombus formation.[Holven KB, Holm T, Aukrust P, et al. Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects . Am J Med 2001;110(7):536-42; Guba SC, Fonseca V, Fink LM. Hyperhomocysteinemia and thrombosis. Semin Thromb Hemost 1999;25(3):291-309.] However, the precise molecular mechanism is still unclear. Recent reports have suggested a role for inflammatory processes in the pathogenesis of atherosclerosis.[Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001;2(2):108-15.] Dysfunction of endothelial cells is the key process promoting inflammatory reactions. On injury, endothlial cells are capable of producing various cytokines that participate in inflammatory reactions in the arterial wall. Although results from in vitro studies suggest that Hcy, at pathophysiological concentrations, stimulates chemokine expression in vascular cells, it is unknown whether hyperhomocysteinemia can initiate similar changes, leading to enhanced momocyte adhesion/binding to the vascular endothelium in vivo.[Zeng X, Dai J, Remick DG, Wang X. Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes. Circ Res 2003;93(4):311-20.] On the basis of the potential pathogenic role of chemokines in atherogenesis, the objective of the present study was to investigate that homocsteine may exert its effect in part though adhesion molecules VCAM-1 and that folic acid supplementation may downregulate these inflammatory responses. Male Sprague–Dawley rats (bred from animal centers of Tongji Medical College, Huazhong Science and Technology University) aged 8 weeks were divided into 3 groups( n = 10 for each group) and maintained for 45 days on the following diets before the experiments: (1) regular diet; (2) high-metheionine diet, consisting of regular diet plus 1.7% methionine; and (3) high-methionine plus folate -rich diet, consisting of regular diet plus 1.7% methionine and 0.006% folate.[Boisvert WA, Curtiss LK, Terkeltaub RA. Interleukin-8 and its receptor CXCR2 in atherosclerosis. Immunol Res 2000;21(2-3):129-d37.] Plasma and serum samples wee colleced and stored at − 80 °C after 45 days until analysis. The plasma homocysteine concentration of rats in three groups were determined by high-pressue liquid chromatography. To detect the endothelial expression of adhesion molecules VCAM-1, the thoracic aorta was isolated and dived into segments. These segments were immersion-fixed in 10% neutral-buffered formalin overlight and then embedded in paraffin. Sequential 5 μm paraffin-embedded cross sections were prepared. Immunohistochemical analyisis was performed to detect vascular cell adhesion molecule(VCAM)-1, The fixed cryosections were immediately blcked in 10% horse serum and phosphate baffered saline(PBS) at room temperature for 30 min. Goat polyclonal andibodies against rat VCAM-1(Santa Cruz Biotechnology) were diluted 1 : 100 in PBS and incubated with the cryosections for 1 h of room temperature. After three washes, the sections were incubated with biotin-conjugated rabbit anti-goat immunoglobulins(Dako) at 1 : 250 dilution in PBS. After three washes, the samples were mounted in 90% glycerol-PBS. Photographs were taken by use of a light microscope at a mignification of × 200.

IL-17 reduces TNF-induced Rantes and VCAM-1 expression

Functional diversity of the memory T-cell-derived cytokine IL-17 was explored at the receptor level. IL-17 inhibited TNF-induced chemokine Rantes expression in human synovial fibroblasts and mouse lung fibroblasts. This inhibitory activity of IL-17 (IC50 = 0.2 ng/ml) was 6-fold more potent than its stimulatory activity on TNF-α-induced IL-6 secretion (ED50 = 1.2 ng/ml), measured in the same cells. IL-17 also inhibited the TNF-mediated expression of adhesion molecule VCAM-1, and the NF-κB binding to the VCAM-1 promoter-specific site, along with the inhibitor of NF-κB, IκB-β. Neutralization of the human IL-17 receptor (IL-17R) by M202 antibody competitively reverses the IL-17-induced IL-6 upregulation. However, M202 only partially affected the inhibitions by IL-17. Yet, IL-17R was essential for the Rantes inhibition, as assessed in lung-derived fibroblasts from IL-17R gene deficient mice. Therefore, inhibitory and stimulatory functions of IL-17 involve receptor IL-17R but show distinct dose–responses and in turn different sensitivities to an IL-17R antagonizing antibody.