LOX-1 mRNA Research Articles

Abstract 3072: GDF15 Represses The Interferon Response Early In Vascular Senescence

Senescent cells have been identified as major contributors of the progression of vascular diseases including atherosclerosis by increasing the senescence-associated secretory phenotype (SASP) and dysregulating vascular cells. Notably, senescence and the SASP can contribute to plaque rupture and atherosclerotic progression by promoting inflammation and plaque vulnerability. A time course microarray analysis of vascular smooth muscle cell (VSMC) senescence revealed p53-regulated GDF15 mRNA was increased early and remained elevated throughout senescence. Notably, GDF15 has been implicated as a biomarker for cardiovascular disease mortality and is part of the conserved SASP across cell types and tissues. Interestingly, intracellular or secreted GDF15 protein levels were increased in monkeys fed a high-fat diet or in mice fed a high-fat diet and in individuals with high Framingham risk scores, respectively. We sought to identify the role of GDF15 in senescent VSMCs. Using CRISPRi, we silenced GDF15 during early senescence and performed RNA-sequencing. Pathway analysis revealed a enrichment in interferon signaling with the loss of GDF15. Coinciding with the increase in interferon signaling, phosphorylation of STAT1 and CREB were increased by loss of GDF15 in early senescence, aiding in the elevation of interferon expression. Given the role of interferons in communication and the prominence of macrophages in atherosclerosis, we tested the effects of conditioned media, derived from either proliferating or senescent VSMCs, on the activation and the expression of scavenger receptors on human macrophages. Senescent conditioned media increased expression of ABCG1 , CD36 , LOX-1 , SCARF1 , and GDF15 mRNA as well as TNFα and CCL2 secretion in macrophages. We propose that elevated GDF15 levels in early senescence suppress interferon signaling, thus preventing macrophage chemotaxis and in turn leading to the accumulation of senescent cells in the vasculature. Thus, disrupting the senescent VSMC-macrophage communication through secreted proteins may offer promising avenues of therapeutic development to prevent or improve inflammatory vascular diseases.

LOX-1 acts as an N6-methyladenosine-regulated receptor for Helicobacter pylori by binding to the bacterial catalase.

The role of N6-methyladenosine (m6A) modification of host mRNA during bacterial infection is unclear. Here, we show that Helicobacter pylori infection upregulates host m6A methylases and increases m6A levels in gastric epithelial cells. Reducing m6A methylase activity via hemizygotic deletion of methylase-encoding gene Mettl3 in mice, or via small interfering RNAs targeting m6A methylases, enhances H. pylori colonization. We identify LOX-1 mRNA as a key m6A-regulated target during H. pylori infection. m6A modification destabilizes LOX-1 mRNA and reduces LOX-1 protein levels. LOX-1 acts as a membrane receptor for H. pylori catalase and contributes to bacterial adhesion. Pharmacological inhibition of LOX-1, or genetic ablation of Lox-1, reduces H. pylori colonization. Moreover, deletion of the bacterial catalase gene decreases adhesion of H. pylori to human gastric sections. Our results indicate that m6A modification of host LOX-1 mRNA contributes to protection against H. pylori infection by downregulating LOX-1 and thus reducing H. pylori adhesion.

PS-BPB01-1: SPARC IS INDUCED AT THE EARLY STAGE OF VASCULAR ENDOTHELIAL DYSFUNCTION TO COMPENSATE INFLAMMATION IN DOCA-SALT HYPERTENSIVE RATS

Objective: Extracellular matrix (ECM) regulates various cellular functions as well as provides structural support. SPARC (secreted protein acidic and rich in cysteine) is one of the ECM components, which participates in various cellular functions, especially during morphogenesis and tissue remodeling. We have reported that 1) SPARC, which increases in accordance with renal injury, causes collagen deposition through ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) production, and 2) renin-angiotensin system is placed in the upstream of SPARC overexpression in the hypertensive rat kidney. The present study investigated the roles for SPARC in vascular endothelial cell dysfunction and inflammation in the setting of hypertension. Design and method: Rats were treated with deoxycorticosterone acetate (DOCA) and 1% salt water for 0, 1, 2 or 3 weeks. Blood pressure was measured by tail-cuff methods, and thoracic aorta was isolated to examine acetylcholine-induced vasodilatory response, macrophage infiltration by immunohistochemistry, and protein expression of SPARC and ADAMTS1 by Western blotting. In the separate experiment, angiotensin II (10–7mol/L, 6hr) or SPARC recombinant protein (100nmol/L, 24hr) was treated to cultured rat aortic endothelial cells (RAECs) with/without siRNA targeting SPARC and ADAMTS1. MCP-1 and LOX-1 mRNA levels were measured by real-time RT-PCR. Result: DOCA-salt caused time-dependent increases in systolic blood pressure, impairment of endothelium-dependent vasodilation, and macrophage infiltration into the adventitial area of the aorta. The expression of SPARC in the DOCA-salt rat aorta was enhanced at week1 and week2 compared to week0. ADAMTS1 expression tended to increase from week1 and showed significant increases at week3. In RAECs, SPARC and ADAMTS1 gene knockdown aggravated angiotensin II-induced overexpression of LOX-1 and MCP-1 mRNA, indicating anti-inflammatory effects of SPARC. On the other hand, SPARC recombinant protein treatment induced the upregulation of LOX-1 and MCP-1. Conclusions: SPARC may be induced at the early stage of vascular injury to compensate inflammation under the existence of angiotensin II. We need further investigation into the diverse effects of SPARC and the relationship between SPARC and ADAMTS1.

Attenuation of streptozotocin induced high fat diet exacerbated dyslipidemia mediated hepatic and aortic injuries in male pigs by camel milk

ABSTRACT Dyslipidemia, diabetes, and the upregulation of receptors for oxidized low-density lipoprotein all contribute to hepatic and aortic endothelial dysfunction. The current research examined how camel milk could possibly decrease induced Streptozotocin-high fat diet-aggravated dyslipidemia-mediated damage in male pig hepatic and aortic tissues. Twenty-five pigs were separated into five groups of five pigs each, with five non-atherosclerotic pigs in the control group and twenty atherosclerotic pigs in the experimental groups. Groups 1 and 2 received distilled water as the standard control and atherosclerotic control groups, respectively, while Groups 3 and 4 received camel milk at 250 mL/day and 500 mL/day, respectively, and Group 5 received Metformin at 500 mg/day. The experiment lasted ten weeks. After 10 weeks, all of the pigs were euthanized. The lipid profile and liver enzyme levels were determined from the analysis of blood serum and the histology of the liver and aortic. When pigs administered, camel milk were compared to atherosclerotic control pigs, there was a significant decrease (p 0.05) in cholesterol, triglyceride, low density lipoprotein (LDL), and very low-density lipoprotein (VLDL) levels, while a significant increase in high density lipoprotein levels was observed in pigs that received camel milk. Also, pigs in groups that received camel milk had their liver enzyme parameters (ALP, AST, ALT, GGT, LDH, and CK-MB) significantly lowered when compared with atherosclerotic control pigs. Similarly, treatment with camel milk ameliorates aortic tissues from degradation and reduces histological liver steatosis, and it also decreases the expression of LOX-1 mRNA in the aorta of atherosclerotic pigs.

P-305 Oxidized-LDL promotes colorectal cancer progression and growth of human colonoides

Expression levels of the oxidized-LDL receptor LOX-1 have been related to cancer progression in prostatic, gastric, pancreatic, and colorectal cancer (CRC); however, the relationship among pro-inflammatory markers, ox-LDL/LOX-1 and CRC are not completely elucidated. We tested the hypothesis that stimulation of CRC cell lines with TNF-α and ox-LDL promotes tumor progression. We also explore the effect of ox-LDL on human colonoids growth. The colorectal cancer (CRC) cell lines HCT116 and COLO320 were treated with TNF-α and ox-LDL and tested for LOX-1 expression. The effect on cell proliferation of this proinflammatory signals were assayed by MTT and cell count with the exclusion dye trypan blue. LOX-1-negative cell lines were obtained by transduction with lentiviral vectors encoding a shRNA specific for LOX-1. The expression of genes involved in tumor progression were analyzed by qRT-PCR and Western blot in wild type and LOX-1 negative cells treated with TNF-α and ox-LDL. Cell migration and invasion was assayed in ox-LDL and TNF-α treated cells. Human colonoids were stimulated with ox-LDL in the presence of the NADPH oxidase inhibitors ML171, VAS2870 and ROS scavenger NAC. Surface area and survival were assessed by contrast microscopy and MTT. Proliferation was assessed by click-chemistry. LOX-1 receptor is expressed in the CRC cell lines HCT116 and COLO320. Additionally, we found in microarrays data stored in public databases that LOX-1 mRNA is over-expressed in colorectal carcinomas and adenocarcinomas compared with normal colon tissue. Treatment of CRC cell lines with TNF-α and ox-LDL augmented LOX-1 expression and LOX-1 silencing abrogated this effect. Moreover, both cell lines treated with ox-LDL proliferated in a dose-dependent manner. Importantly, ox-LDL stimulated human organoids proliferation and viability also in a dose-dependent manner, an effect partly mediated by ROS. The activation of LOX-1 by ox-LDL induced the expression of angiogenic markers such as MMP2, MMP7, MMP9 and VEGF. LOX-1 activation by oxLDL also promotes epithelial to mesenchymal transition of human colorectal cancer cells, characterized by reduced expression of the epithelial marker E-cadherin and increased expression of mesenchymal markers such as Vimentin, N-Cadherin, TWIST, ZEB1 and ZEB2. In addition, we determined that ox-LDL promotes invasion and migration of colorectal cancer cells. Nevertheless, co-treatment of colorectal cancer cells with TNF-α and ox-LDL did not show an additive effect. LOX-1 activation by ox-LDL induces a transcription reprogramming of genes involved in angiogenesis, invasion, migration, and cell proliferation. In the context of inflammation and obesity, we speculate that overexpression of LOX-1 provides additional stimulation accelerating tumor progression.

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells.

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors—classical LDLR and alternative LOX-1—in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Role of secreted protein acidic and rich in cysteine (SPARC) in vascular endothelial cell dysfunction and inflammation

Secreted protein acidic and rich in cysteine (SPARC) is reported to induce collagen deposition through a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) production and promote pro-inflammatory cytokine release in aging myocardium. The present study investigated the roles for SPARC in vascular endothelial cell dysfunction and inflammation using deoxycorticosterone acetate (DOCA)-salt hypertensive rats and cultured aortic endothelial cells. DOCA-salt treatment increased systolic blood pressure in 2 weeks and induced further hypertensin in 3 weeks. Acetylcholine-induced vasodilatory response impaired and macrophage infiltration increased in DOCA-salt rat aortas, both of which showed time-dependent changes. Immunoblot analysis revealed that SPARC and ADAMTS1 expression increased in DOCA-salt rats and peaked at 1 week. Angiotensin II treatment enhanced SPARC and ADAMTS1 protein expression in cultured aortic endothelial cells. Angiotensin II-induced overexpression of LOX-1 and MCP-1 mRNA was further augmented by SPARC or ADAMTS1 gene silencing. In conclusion, SPARC may be induced at the early stage of vascular injury to compensate inflammation. Further investigation into roles for SPARC in inflammation and relationship between SPARC and ADAMTS1 is needed.

The Role of SREC-Ⅰ in Innate Immunity to Aspergillus fumigatus Keratitis.

PurposeTo determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis.MethodsSREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1β, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1β, and TNF-α mRNA expression levels were tested before and after anti–SREC-Ⅰ treatment.ResultsSREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1β, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1β, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ–neutralizing antibody treatment.ConclusionsSREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.

Ketamine alleviates HMGB1-induced acute lung injury through TLR4 signaling pathway.

Acute lung injury (ALI) is a common critical respiratory disease that seriously threatens human health. Ketamine has good anti-inflammatory and immune-regulating properties that can delay the lung injury process. High mobility group box protein 1 (HMGB1) plays an important role in the occurrence, development and treatment of ALI. Toll-like receptor 4 (TLR4) is the receptor for HMGB1. The aim of this study was to determine the role of the HMGB1 TLR4 signaling pathway in the treatment of ALI using ketamine. A total of 30 healthy, male, 8-week-old Sprague-Dawley rats were randomly, equally divided into a control group, an lipopolysaccharide (LPS) group and a ketamine group. In order to establish a rat ALI model, 15 mg/kg of LPS was injected into the femoral veins. Ketamine was intravenously injected (10 mg/kg) into the experimental group rats. The rats were euthanized 24 h after modeling and lung tissue samples were collected. Western blot was used to test TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 protein expression in the lung tissue. Real-time polymerase chain reaction (RT-PCR) was performed to detect TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA levels. Compared with the controls, the LPS group had significantly higher TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA and protein levels (p < 0.05). These levels were significantly lower after ketamine intervention in comparison with the LPS group (p < 0.05). A positive correlation was found between TLR4 and HMGB1 expression in the LPS and ketamine groups (r = 0.952, p < 0.001; r = 0.941, p < 0.001). Ketamine attenuates HMGB1-induced ALI, possibly by regulating the TLR4 signaling pathway.

Crocin treatment decreased pancreatic atrophy, LOX-1 and RAGE mRNA expression of pancreas tissue in cholesterol-fed and streptozotocin-induced diabetic rats.

Objective Oxidative stress in diabetic mellitus is a consequence of oxidative stress, which plays a critical role in the pathogenesis of diabetic tissue damage. Receptors for advanced glycation end products and for oxidized low-density lipoproteins (LDL) have critical contribution in oxidative tissue damage. The present study investigated whether anti-diabetic effects of Crocin via modulation of mRNA expression of RAGE and LOX-1 receptors in diabetic rats. Methods In the current study, high-fat cholesterol (HFC) and streptozotocin (40 mg/kg) used to induce type II diabetes. Experimental groups as follows: (Group 1: control); (Group 2: control treatment [Crocin]); (Group 3: DM [STZ]); (Group 4: DM treatment [STZ + Crocin]); (Group 5; DM + HFC [STZ + HFC]); (Group 6; DM + HFC treatment [STZ + HFC + Crocin]). Crocin (20 mg/kg/day, i.p.) administered in treatment groups for 60 days. Serum glucose and cholesterol levels evaluated on days 5, 30 and 60 after induction of DM. Pancreatic tissue from all group removed on day 60 for histological and RT-PCR analysis. Results Application of Crocin significantly decreased serum cholesterol levels on day 60 after induction of DM in diabetic + HFC rats. Moreover, Crocin significantly decreased serum glucose levels on days 30 and 60 both in diabetic and diabetic + HFC rats. Crocin partially prevented the atrophic effects of STZ on both exocrine and endocrine parts of pancreas. Additionally, Crocin significantly decreased LOX-1 and RAGE mRNA expression OF pancreas in diabetic rats. Conclusion The current study suggested that Crocin suppressed atrophic change of the pancreas by decrease of LOX-1 and RAGE mRNA expression in diabetic rats.

Hydrogen sulfide stabilizes atherosclerotic plaques in apolipoprotein E knockout mice

Hydrogen sulfide gas (H2S) has protective effects in the cardiovascular system that includes preventing the development of atherosclerosis when tested in several in vivo models. Plaque instability is a major risk factor for thromboembolism, myocardial infarction, and stroke, so we examined if H2S can promote plaque stability and the potential underlying mechanisms. Apolipoprotein E knockout mice fed an atherogenic diet were administered the exogenous H2S donor sodium hydrosulfide (NaHS) or pravastatin as a positive control daily for 14 weeks. NaHS significantly enhanced plaque stability by increasing fibrous cap thickness and collagen content compared to vehicle-treated controls. NaHS treatment also reduced blood lipid levels and plaque formation. Preservation of plaque stability by NaHS was associated with reductions in vascular smooth muscle cells (VSMCs) apoptosis and expression of the collagen-degrading enzyme matrix metallopeptidase-9 (MMP-9) in plaque. While pravastatin also increased fibrous cap thickness and reduced VSMC apoptosis, but did not enhance plaque collagen or reduce MMP-9 significantly, suggesting distinct mechanisms of plaque stabilization. in vitro, NaHS also decreased MMP-9 expression in macrophages stimulated with tumor necrosis factor-α by inhibiting ERK/JNK phosphorylation and activator protein 1 nuclear translocation. Moreover, H2S reduced caspase-3/9 activity, Bax/Bcl-2 ratio, and LOX-1 mRNA expression in VSMCs stimulated with oxidized low-density lipoprotein. These results suggest that H2S enhances plaque stability and protects against atherogenesis by increasing plaque collagen content and VSMC count. In conclusion, H2S exerts protective effects against atherogenesis at least partly by stabilizing atherosclerotic plaque.

Overexpression of scavenger receptor and infiltration of macrophage in epicardial adipose tissue of patients with ischemic heart disease and diabetes

BackgroundOxidized low-density lipoproteins and scavenger receptors (SRs) play an important role in the formation and development of atherosclerotic plaques. However, little is known about their presence in epicardial adipose tissue (EAT). The objective of the study was to evaluate the mRNA expression of different SRs in EAT of patients with ischemic heart disease (IHD), stratifying by diabetes status and its association with clinical and biochemical variables.MethodsWe analyzed the mRNA expression of SRs (LOX-1, MSR1, CXCL16, CD36 and CL-P1) and macrophage markers (CD68, CD11c and CD206) in EAT from 45 patients with IHD (23 with type 2 diabetes mellitus (T2DM) and 22 without T2DM) and 23 controls without IHD or T2DM.ResultsLOX-1, CL-P1, CD68 and CD11c mRNA expression were significantly higher in diabetic patients with IHD when compared with those without T2DM and control patients. MSR1, CXCL16, CD36 and CD206 showed no significant differences. In IHD patients, LOX-1 (OR 2.9; 95% CI 1.6–6.7; P = 0.019) and CD68 mRNA expression (OR 1.7; 95% CI 0.98–4.5; P = 0.049) were identified as independent risk factors associated with T2DM. Glucose and glycated hemoglobin were also shown to be risk factors.ConclusionsSRs mRNA expression is found in EAT. LOX-1 and CD68 and were higher in IHD patients with T2DM and were identified as a cardiovascular risk factor of T2DM. This study suggests the importance of EAT in coronary atherosclerosis among patients with T2DM.

Azelnidipine Inhibits the Differentiation and Activation of THP-1 Macrophages through the L-Type Calcium Channel

Aim: Recently, calcium channel blockers (CCBs) have been reported to reduce atherosclerosis with anti-inflammatory or antiatherosclerotic effects in vivo. It is well established that monocytes and macrophages play important roles in promoting atherosclerosis. However, the effects of CCBs on macrophage activation remain unclear. The aim of this study was to evaluate the effects of azelnidipine, a dihydropyridine L-type CCB, on the activation of macrophages and to clarify the mechanisms of the effects of CCBs on atherosclerosis.Methods: THP-1 monocytes, a human leukemic cell line, were stimulated with 50 ng/mL of phorbol-12-myristate-13-acetate (PMA) 1 h after pretreatment with 10 µM azelnidipine or dimethyl sulfoxide (DMSO), and harvested.Results: Azelnidipine blocked the expression of intercellular adhesion molecule-1 quantified by FACS analysis. The expression levels of Apo E and MMP9, which are markers of macrophage differentiation, were inhibited by azelnidipine as evaluated by quantitative RT-PCR. The level of LOX-1 mRNA, a scavenger receptor, was also reduced significantly by pretreatment with 10 µM azelnidipine. Azelnidipine also lowered the uptake of acetylated LDL. The expression of the L-type calcium channel Cav1.2 was 10-fold higher after 24 h of PMA stimulation. A knockdown of the CACNA1C gene, which encodes Cav1.2 protein in humans, with siRNA blocked the effect of reducing adhesion by azelnidipine, indicating that the effects of azelnidipine on macrophage differentiation were expressed through the CACNA1C gene.Conclusion: Our results suggest that azelnidipine has potent antiatherosclerotic properties by inhibition of macrophage activation through Cav1.2.

Expression profiles of microRNAs in oxidized low-density lipoprotein-stimulated RAW 264.7 cells.

Macrophage-derived foam cells were one of the hallmarks of atherosclerosis, and microRNAs played an important role in the formation of foam cells. In order to explore the roles of miRNA in the formation of foam cells, we investigated miRNA expression profiles in foam cells through high-throughput sequencing technology. A total of 84 miRNAs were differentially expressed between RAW 264.7 macrophages and foam cells induced by ox-LDL. Thirty miRNAs were upregulated and 54 miRNAs were downregulated. GO terms and KEGG pathways analysis revealed that the target genes of most of DE miRNAs were mainly enriched in "cell differentiation," "endocytosis," "MAPK signaling pathway," and "FoxO signaling pathway." The target genes of some DE miRNAs were enriched in "Insulin signaling pathway," "Hippo signaling pathway," "TNF signaling pathway," "NF-kappa B signaling pathway," and "cell death." Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-28a-5p and miR-30c-1-3p directly inhibited LRAD3 and LOX-1 mRNA expression through targeting the 3'UTR of LRAD3 and LOX-1 mRNA, respectively. Our study indicates that miRNAs are extensively involved in the formation of foam cells, and provides a valuable resource for further study the role of miRNAs in atherosclerosis.

LOX-1: A potential target for therapy in atherosclerosis; an in vitro study

Pro-inflammatory signal generated from the interaction of oxLDL with its cognate receptor LOX-1 has been attenuated successfully by a novel combination siRNA (siLOX-1Ω) targeting unique regions of Homo sapien LOX-1 mRNA. Signalling via LOX-1R was studied in a potentially pro-atherogenic arena recreated in a metabolic, pulse-chase set up. An initial pulse of oxLDL (20μg/mL;5h) was chased (without oxLDL) on a temporal scale upto 72h. Our study shows that the pro-inflammatory signal generated via oxLDL-LOX-1R interaction was mediated in two rungs, an initial sustained increase in LOX-1R expression up to 12h, and a renewal after 48h. TNF-α acted as a primary mediator of LOX-1R signalling, presumably also stimulating CD40 and MMP-9. Both TNF-α and IL-6 were involved in the second rung of LOX-1R signalling; maximum secretion of both was detected at 48h. Our study suggests a temporal sustenance of LOX-1R signalling by pro-inflammatory cytokines even on withdrawal of oxLDL. Also, siLOX-1Ω successfully abated LOX-1R expression along with its signalling intermediates, NO and NF-kB. Overall, LOX-1 signalling and the crucial role of cytokines in sustaining it is reported. Attenuation of this receptor may be of therapeutic value in atherosclerosis.

Dihydrotestosterone inhibits foam cell formation via a lectin-like ox-low-density lipoprotein receptor mediated mechanism in J774.1 cell line

Objective: To investigate the effect of dihydrotestosterone (DHT) on lectin-like ox- low-density lipoprotein (LDL) receptor(LOX-1)expression and foam cell formation in the female macrophage cell line J774.1. Methods: In cultured J774.1 cells, after pretreated with DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L, ox-LDL-induced LOX-1 expression and foam cell formation were investigated by quantitative real-time PCR, Western blotting, and oil-red O staining. Results: DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L inhibited ox-LDL-induced LOX-1 mRNA (2.81±0.46 and 2.29±0.21 vs 4.71±0.31, both P<0.01) and protein expression (1.35±0.06 and 1.09±0.04 vs 1.75±0.11, both P<0.05). The effect was partly reversed by the androgen receptor (AR) blocker flutamide (87.6%, P=0.004). Oil-red O staining also revealed that DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L suppressed ox-LDL-induced foam cell formation as quantified by the number of foam cells per high-power field (HPF) (36.0±3.0 and 29.1±1.3 vs 45.9±3.7, both P<0.05) and by the area of oil-red O stained particles per HPF (7 983±1 035 and 4 060±390 vs 14 750±2 489, both P<0.05). Conclusion: DHT at concentrations of 1×10-9 mol/L and 1×10-8 mol/L decreases LOX-1 expression and foam cell formation via AR.

Porphyromonas gingivalis promotes low-density lipoprotein oxidation and atherosclerosis

ObjectivesRecent studies have reported that periodontal disease increases the risk of atherosclerosis. We previously reported that Porphyromonas gingivalis (Pg) accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice by initiating inflammation. Because the oxidative modification of lipoprotein plays a major role in atherosclerosis, we characterized the reactive oxygen species (ROS) produced by Pg and its ability to oxidize low-density lipoprotein (LDL). MethodsAtherosclerotic plaque formation in the aortic sinuses of Apoeshl mice injected intravenously with Pg 381 was assessed by Oil Red O staining. Anti-mouse antibodies to HOCl-oxidized LDL and 4-HNE, PLA2, MPO, and CD36 were used for immunohistochemistry. Intracellular ROS detection was performed using 2′,7′-dichlorodihydrofluorescein diacetate. Quantitative reverse transcription polymerase chain reaction was performed using primers specific for TLR-2, TLR-4, TLR-9, NOD-1, LOX-1, NOX-2, NOX-4, p22phox, and p47phox. ResultsPg challenge significantly induced ox-LDL and 4-HNE-, PLA2-, MPO-, and CD36-positive areas in proximal aortic lesions. TLR-2, TLR-4, NOD-1, LOX-1, and NADPH oxidase subunit-specific mRNA levels in the aorta were significantly increased. Furthermore, Pg significantly induced ROS production in monocytes. ConclusionsThese results suggest that Pg promotes LDL oxidation and contributes to atheroma development.

Carbamylated Low-Density Lipoproteins Induce a Prothrombotic State Via LOX-1: Impact on Arterial Thrombus Formation In Vivo

BackgroundCarbamylation alters low-density lipoprotein (LDL) structure and is thought to promote vascular inflammation and dysfunction in patients with chronic kidney disease (CKD). ObjectivesThis study sought to determine whether carbamylated LDL (cLDL) exerts prothrombotic effects in vascular cells and platelets and whether cLDL enhances arterial thrombus formation in vivo. MethodsLDL was isolated from healthy subjects or patients with CKD by sequential ultracentrifugation. Ex vivo carbamylation of LDL from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analyzed in a murine carotid artery photochemical injury model. Protein expression and mRNA levels were analyzed by Western blotting, flow cytometry, and real-time PCR. Platelet aggregation was measured by impedance aggregometry. ResultsIntravenous administration of cLDL in mice accelerated arterial thrombus formation compared to treatment with native LDL (nLDL) or vehicle. Tissue lysates of mouse carotid arteries revealed that cLDL induced the expression of TF, PAI-1, and LOX-1 mRNA in vascular cells. In human aortic smooth muscle and endothelial cells, cLDL induced TF and PAI-1 expression. In contrast, nLDL had no effect on either cell type. While nLDL and cLDL had no aggregatory effect on resting platelets, cLDL enhanced platelet aggregation in response to different agonists. This effect was mediated by mitogen-activated protein kinase p38 phosphorylation and LOX-1 translocation to the surface. LDL isolated from patients with CKD mimicked the prothrombotic effects of cLDL on vascular cells, platelets, and thrombus formation in vivo. ConclusionsWe found that cLDL induces prothrombotic effects in vascular cells and platelets by activation of the LOX-1 receptor and enhances thrombus formation in vivo. This observation reveals a new mechanism underlying the increased incidence of acute thrombotic events observed in patients with CKD and may lead to the development of new lipid-targeting therapies in this population.

Protection against vascular endothelial dysfunction by polyphenols in sea buckthorn berries in rats with hyperlipidemia.

Chronic hyperlipemia increases the incidence of vascular endothelial dysfunction and can even induce cardiovascular disease. Sea buckthorn contains a host of bioactives such as flavonoids and polyphenols that can prevent the development of cardiovascular disease. The current study isolated active ingredients, polyphenols, from sea buckthorn berries (SVP) and orally administered SVP at a dose of 7-28 mg/kg. This treatment significantly reduced serum lipids, it enhanced the activity of antioxidant enzymes, and it decreased the level of serum TNF-α and IL-6. SVP also alleviate vascular impairment by decreasing the expression of eNOS, ICAM-1, and LOX-1 mRNA and proteins in aortas of rats with hyperlipidemia. Based on these findings, SVP has antioxidant action and it protects endothelium.

Effects of resveratrol on the alterations of cavernosal eNOS and LOX-1 expression in the hypercholesterolemic condition: a preliminary study.

The aim of this study was to determine the effects of resveratrol on the alterations of cavernosal eNOS and LOX-1 mRNA expression in the hypercholesterolemic condition. Twenty-one New Zealand white male rabbits were separated into three groups. Rabbits were fed with a normal dietary intake for the control group and a 2% cholesterol diet for the hypercholesterolemia and resveratrol groups for 6 weeks. Resveratrol 4 mg/kg daily was administered for the resveratrol group. Cavernosal LOX-1 and eNOS mRNA expressions were determined with real-time RT-PCR in all groups. The statistical analysis was performed with the Kruskal-Wallis and Mann-Whitney U tests. We found no difference between mean LOX-1 mRNA expression levels in the three groups. Lower mean eNOS mRNA expression level was determined in the hypercholesterolemia group when compared with the control group (P = 0.011). Mean eNOS mRNA expression level in the resveratrol group was similar to that in the control group but significantly higher than that in the hypercholesterolemia group (P < 0.001). This preliminary study demonstrates the beneficial effects of resveratrol on cavernosal eNOS expression. The presence of cavernosal LOX-1 expression was also shown for the first time. Resveratrol may be an alternative option in hypercholesterolemic erectile dysfunction with further studies supporting its beneficial effects on the corpus cavernosum.