Cytokine Circuits in Cardiovascular Disease.

Arterial inflammation is a hallmark of atherosclerosis, and appropriate management of this inflammation represents a major unmet therapeutic need for cardiovascular disease patients. Here, we review the diverse contributions of immune cells to atherosclerosis, the mechanisms of immune cell activation in this context, and the cytokine circuits that underlie disease progression. We discuss the recent application of these insights in the form of immunotherapy to treat cardiovascular disease and highlight how studies on the cardiovascular co-morbidity that arises in autoimmunity might reveal additional roles for cytokines in atherosclerosis. Currently, data point to interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and IL-17 as cytokines that, at least in some settings, are effective targets to reduce cardiovascular disease progression.

CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Exploring the causal role of immune cells in cerebral aneurysm through single-cell transcriptomics and Mendelian randomization analysis.

Cerebral aneurysm (CA) represents a significant clinical challenge, characterized by pathological dilation of cerebral arteries. Recent evidence underscores the crucial involvement of immune cells in CA pathogenesis. This study aims to explore the complex interplay between immune cells and CA formation. We analyzed single-cell RNA sequencing data from the GSE193533 dataset, focusing on unruptured CA and their controls. Comprehensive cell-type identification and pseudo-time trajectory analyses were conducted to delineate the dynamic shifts in immune cell populations. Additionally, a two-sample Mendelian randomization (MR) approach was employed to investigate the causal influence of various immunophenotypes on CA susceptibility and the reciprocal effect of CA formation on immune phenotypes. Single-cell transcriptomic analysis revealed a progressive loss of vascular smooth muscle cells (VSMCs) and an increase in monocytes/macrophages (Mo/MΦ) and other immune cells, signifying a shift from a structural to an inflammatory milieu in CA evolution. MR analysis identified some vital immunophenotypes, such as CD64 on CD14+ CD16+ monocytes (OR: 1.236, 95% CI: 1.064-1.435, P = 0.006), as potential risk factors for CA development, while others, like CD28- CD8br %CD8br (OR: 0.883, 95% CI: 0.789-0.988, P = 0.030), appeared protective. Reverse MR analysis demonstrated that CA formation could modulate specific immunophenotypic expressions, highlighting a complex bidirectional interaction between CA pathology and immune response. This study underscores the pivotal role of immune cells in this process through the integration of single-cell transcriptomics with MR analysis, offering a comprehensive perspective on CA pathogenesis, and potentially guiding future therapeutic strategies targeting specific immune pathways.

Easy recognition and high autoimmune hepatitis specificity of smooth muscle antibodies giving an actin microfilament immunofluorescent pattern on embryonal vascular smooth muscle cells.

Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney tissue, is restricted by operator-dependent interpretation. A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal aorta vascular smooth muscle (VSM) cell line-based assay with those of the rodent tissue-based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis, 40 primary sclerosing cholangitis, 50 chronic viral hepatitis, 20 alcohol-related liver disease, 40 steatotic liver disease, and 40 healthy controls) were assayed for MF-SMA and SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96 (70%) and 87 (63%) AIH-1 patients, and 2 controls (P < 0.0001). Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%, P = ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870, [0.789-0.952]). MF-SMA were associated with higher serum γ-globulin [26 (12-55) vs 20 g/l (13-34), P < 0.005] and immunoglobulin G (IgG) levels [3155 (1296-7344) vs 2050 mg/dl (1377-3357), P < 0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA.

Hexarelin attenuates abdominal aortic aneurysm formation by inhibiting SMC phenotype switch and inflammasome activation.

Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200 µg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response.

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

OBJECTIVE: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. CONCLUSIONS: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

Circ_0002984 induces proliferation, migration and inflammation response of VSMCs induced by ox-LDL through miR-326-3p/VAMP3 axis in atherosclerosis.

Atherosclerosis can result in multiple cardiovascular diseases. Circular RNAs (CircRNAs) have been reported as significant non-coding RNAs in atherosclerosis progression. Dysfunction of vascular smooth muscle cells (VSMCs) is involved in atherosclerosis. However, up to now, the effect of circ_0002984 in atherosclerosis is still unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low-density lipoprotein (ox-LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNF-α and IL-6 levels. The data implied that lack of circ_0002984 obviously repressed ox-LDL-stimulated VSMC inflammation. Meanwhile, miR-326-3p, which was predicted as a target of circ_0002984, was obviously down-regulated in VSMCs treated by ox-LDL. Additionally, after overexpression circ_0002984 in VSMCs, a decrease in miR-326-3p was observed. Subsequently, miR-326-3p was demonstrated to target vesicle-associated membrane protein 3 (VAMP3). Therefore, we hypothesized that circ_0002984 could modulate expression of VAMP3 through sponging miR-326-3p. Furthermore, we confirmed that up-regulation of miR-326-3p rescued the circ_0002984 overexpressing-mediated effects on VMSC viability, migration and inflammation. Additionally, miR-326-3p inhibitor-mediated functions on VSMCs were reversed by knockdown of VAMP3. In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR-326-3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.

Aldehyde dehydrogenase 2 protects against abdominal aortic aneurysm formation by reducing reactive oxygen species, vascular inflammation, and apoptosis of vascular smooth muscle cells.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is an enzyme that detoxifies aldehydes by converting them to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress. Increased oxidative stress plays a pivotal role in abdominal aortic aneurysm (AAA) pathogenesis. Reactive oxygen species (ROS) promote degradation of the extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) apoptosis. Reducing oxidative stress by an ALDH2 activator could have therapeutic potential for limiting AAA development. We hypothesized that ALDH2 deficiency could increase the risk for AAA by decreasing ROS elimination and that an ALDH2 activator could provide an alternative option for AAA treatment. The National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database was used. Human aortic smooth muscle cells (HASMCs) were used for the in vitro experiments. Gene-targeted ALDH2*2 KI knock-in mice on a C57BL/6J background and apolipoprotein E knockout (ApoE KO) mice were obtained. An animal model of AAA was constructed using osmotic minipumps to deliver 1000 ng/kg/min angiotensin II (AngII) for 28 days. Patients with AAA had significantly lower ALDH2 expression levels than normal subjects. ALDH2*2 KI mice were susceptible to AngII administration, exhibiting significantly increased AAA incidence rates and increased aortic diameters. Alda-1, an ALDH2 activator, reduced AngII-induced ROS production, NF-kB activation, and apoptosis in HASMCs. Alda-1 attenuated AngII-induced aneurysm formation and decreased aortic expansion in ApoE KO mice. We concluded that ALDH2 deficiency is associated with the development of AAAs in humans and a murine disease model. ALDH2 deficiency increases susceptibility to AngII-induced AAA formation by attenuating anti-ROS effects and increasing VSMC apoptosis and vascular inflammation. Alda-1 was shown to attenuate the progression of experimental AAA in a murine model.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMß2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Rosmarinic acid inhibits nicotine-induced C-reactive protein generation by inhibiting NLRP3 inflammasome activation in smooth muscle cells.

Atherosclerosis is widely known to be a chronic inflammatory disease. C-reactive protein (CRP), an important inflammatory factor, plays an essential role in the pathogenesis of atherosclerosis. Nicotine, the main addictive component of cigarette, has been shown to induce the production of CRP. The aim of this study was to investigate the effect of rosmarinic acid (RA), a polyphenol with antiinflammatory activity, on nicotine-induced elevation of CRP in vascular smooth muscle cells (VSMCs). We found that pretreatment of VSMCs with RA attenuated nicotine-induced expression of CRP in a time- and dose-dependant manner. In addition, RA also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and reactive oxygen species (ROS) production resulting from nicotine treatment in VSMCs. To confirm these findings in vivo, we constructed a nicotine-induced atherosclerosis rat model. RA did not significantly reduce the serum nicotine level of the rats, whereas it significantly decreased the levels of serum lipids, including concentrations of cholesterol, triglycerides, and low-density lipoprotein cholesterol, and the serum level of CRP. RA also led to diminished nicotine-induced activation of NLRP3 inflammasome and elevation in the CRP level in the aortic tissue of the model rats. The results of this study suggested a protective role of RA in nicotine-induced atherosclerosis by inhibiting the ROS-NLRP3 inflammasome-CRP axial, and RA therefore represented a potential effective therapeutic approach to atherosclerosis, in particular for those who smoke.

Downregulation of the transcriptional co-activator PCAF inhibits the proliferation and migration of vascular smooth muscle cells and attenuates NF-κB-mediated inflammatory responses.

P300/CBP-associated factor (PCAF) regulates vascular inflammation. This study was to explore the effect of PCAF on the proliferation and migrationof vascular smooth muscle cells (VSMCs) and neointimal hyperplasia in balloon-injured rat carotid artery. Downregulation of PCAF remarkably suppressed VSMCs proliferation and migration induced by lipopolysaccharide, and also significantly inhibit the nuclear translocation of nuclear factor-kappaB p65. Meanwhile, downregulation of PCAF inhibited the mRNA expression of tumor necrosis factor-α and interleukin-6, and also the levels in culture supernatants. Moreover, downregulation of PCAF profoundly reduced the intima area and the ratio of intima area to media area in balloon-injured rat carotid artery. In addition, the expression of PCNA and NF-κB p65 in intima were decreased by downregulation of PCAF. These results highlight that PCAF may be a potential target for prevention and treatment of neointimal hyperplasia and restenosis after angioplasty.

MFG-E8 mediates arterial aging by promoting the proinflammatory phenotype of vascular smooth muscle cells.

BACKGROUND: Among older adults, arterial aging is the major factor contributing to increased risk for cardiovascular disease-related morbidity and mortality. The chronic vascular inflammation that accompanies aging causes diffuse intimal-medial thickening of the arterial wall, thus increasing the vulnerability of aged vessels to vascular insults. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a biomarker for aging arteries. This integrin-binding glycoprotein, induced by angiotensin II, facilitates vascular smooth muscle cell (VSMC) proliferation and invasion in aging vasculatures. This study investigated whether MFG-E8 directly mediates the initial inflammatory responses in aged arteries or VSMCs. METHODS: A model of neointimal hyperplasia was induced in the common carotid artery (CCA) of aged mice to exacerbate age-associated vascular remodeling. Recombinant MFG-E8 (rMFG-E8) was administered to the injured artery using Pluronic gel to accentuate the effect on age-related vascular pathophysiology. The MFG-E8 level, leukocyte infiltration, and proinflammatory cell adhesion molecule (CAM) expression in the arterial wall were evaluated through immunohistochemistry. By using immunofluorescence and immunoblotting, the activation of the critical proinflammatory transcription factor nuclear factor (NF)-κB in the injured CCAs was analyzed. Immunofluorescence, immunoblotting, and quantitative real-time polymerase chain reaction were conducted using VSMCs isolated from the aortas of young and aged mice to assess NF-κB nuclear translocation, NF-κB-dependent gene expression, and cell proliferation. The extent of intimal-medial thickening in the injured vessels was analyzed morphometrically. Finally, Transwell migration assay was used to examine VSMC migration. RESULTS: Endogenous MFG-E8 expression in aged CCAs was significantly induced by ligation injury. Aged CCAs treated with rMFG-E8 exhibited increased leukocyte extravasation, CAM expression, and considerably increased NF-κB activation induced by rMFG-E8 in the ligated vessels. Exposure of early passage VSMCs from aged aortas to rMFG-E8 substantially increased NF-κB activation, proinflammatory gene expression, and cell proliferation. However, rMFG-E8 attenuated VSMC migration. CONCLUSIONS: MFG-E8 promoted the proinflammatory phenotypic shift of aged VSMCs and arteries, rendering the vasculature prone to vascular diseases. MFG-E8 may constitute a novel therapeutic target for retarding the aging processes in such vessels.

Inhibitory effect of recombinant human CXCL8(3-72)K11R/G31P on atherosclerotic plaques in a mouse model of atherosclerosis.

Context: Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration. Objective: This study is to investigate the therapeutic effect of G31P on atherosclerosis through a mouse model. Materials and methods: A mouse model of atherosclerosis was generated through feeding the ApoE-/- mice with high fat diet for 12 weeks. G31P was injected subcutaneously into the mice. The levels of keratinocyte chemoattractant (KC), CXCR2, TNF-α, and IFN-γ were analyzed through ELISA. The expressions of MMP-2, MMP-9, PCNA, and Mef2a in aortic tissues were detected through RT-qPCR. In A7r5 cells, the levels of p-ERK, ROCK1, and ROCK2 were analyzed by western blot. Intracellular calcium levels were measured through Fluo-3 AM assay. Results and disccussion: G31P suppressed the abnormal lipid profile and decreased the levels of KC, MMP-2, MMP-9, PCNA, and Mef2a in a mouse model of atherosclerosis. In addition, G31P also inhibited the expressions of p-ERK, ROCK1, ROCK2, and decreased the calcium concentrations in A7r5 cells. Conclusions: These findings indicate the potential therapeutic effects of G31P in suppressing the development of atherosclerosis by antagonizing the IL-8 receptor. G31P inhibits the proliferation and migration of VSMCs through regulating the Rho-kinase, ERK, and calcium-dependent pathways.

Fibrinopeptide A Induces Expression of C-Reactive Protein via the ROS-ERK1/2/ P38-NF-κB Signal Pathway in Vascular Smooth Muscle Cells.

BACKGROUND/AIMS: Atherosclerosis is a chronic inflammatory disease in the artery walls. Fibrinopeptide A (FPA) is a biomarker of the activation of coagulation system, and a high concentration of FPA in blood occurs in patients with ischemic heart disease etc. However, there exist few studies on the pathological effects of FPA in cardiovascular system. Therefore, the present study examined the effect of FPA on CRP expression in VSMCs and the molecular mechanisms. METHODS: mRNA and protein expression was identified by quantitative real-time PCR and Western blot, respectively. Reactive oxygen species (ROS) and the immunofluorescence staining were observed by a fluorescence microscope. Plasma FPA and CRP level was determined by ELISA. RESULTS: FPA induced the expressions of CRP, IL-1ß and IL-6 in VSMCs, and anti-IL-1ß and anti-IL-6 neutralizing antibodies partially reduced FPA-induced CRP expression in VSMCs. The subchronic administration of FPA to rats increased FPA level in plasma and CRP expression in the aortic artery walls. The further studies showed that FPA promoted superoxide anion generation in VSMCs. Antioxidant NAC antagonized FPA-stimulated superoxide anion generation and inhibited FPA-induced CRP expression in VSMCs. FPA activated ERK1/2 and p38 phosphorylation, and PD98059 and SB203580 reduced FPA-induced CRP expression. Moreover, NAC inhibited the activation of ERK1/2 and p38. In addition, FPA enhanced NF-κB level in the nuclei of VSMCs, and PDTC reduced FPA-induced expression of CRP. CONCLUSIONS: FPA induces CRP expression in VSMCs via ROS-ERK1/2/p38-NF-κB signal pathway. This finding for the first time provides an experimental evidence for pro-inflammatory effect of FPA.

Osteopontin may be a driver of abdominal aortic aneurysm formation.

OBJECTIVE: Previous in vitro and animal studies have suggested that osteopontin (OPN), an inflammatory extracellular matrix protein, is involved in the formation and growth of abdominal aortic aneurysms (AAAs). However, the mechanism by which this occurs continues to be nebulous. The relationship between OPN and inflammation-suppressing lymphocytes present in the human AAA condition was investigated and presented herein. METHODS: Serum OPN concentrations were measured in healthy, risk factor-matched non-AAA and AAA patients by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to determine the source of OPN secretion using aortic tissue collected from multiorgan donors and AAA patients undergoing open surgical repair. Vascular smooth muscle cells (VSMCs) were exposed to various inflammatory mediators, and OPN expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction and ELISA. The inflammatory nature of OPN and the aortic wall was determined using a TR1 suppressor cell induction assay as a surrogate and characterized by ELISA and fluorescence-activated cell sorting. RESULTS: OPN was found to be elevated in both the plasma and aortic homogenate of AAA patients compared with controls. On immunohistochemistry, OPN localized to the tunica media of the diseased aorta but was minimally expressed in healthy aorta. In vitro, cigarette smoke extract was the most potent stimulator of OPN secretion by VSMCs and increased both messenger RNA and supernatant concentrations. OPN demonstrated an ability to inhibit the induction of interleukin 10-secreting TR1 lymphocytes, a depleted population in the AAA patient, from naive precursors. Last, neutralizing receptor targets of OPN in the setting of AAA homogenate coincubation abrogated the inhibition of TR1 induction. CONCLUSIONS: OPN, secreted by the VSMCs of the tunica media, is elevated in the circulating plasma and aortic wall of patients with AAA. It can inhibit the induction of the TR1 suppressor cell, leading to an overall proinflammatory state contributing to progressive aortic wall breakdown and dilation.

LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway.

BACKGROUND/AIMS: Lipopolysaccharide (LPS) is a potent activator of vascular smooth muscle cells (VSMCs) proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) and Ras-related C3 botulinum toxin substrate 1 (Rac1) expression using small interfering RNA (siRNA) in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation. METHODS: VSMCs proliferation was monitored by 5-ethynyl-2'-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA), smooth muscle 22α (SM22α), myosin heavy chain (MYH) and transient receptor potential channel 1 (TRPC1) were detected by qRT-PCR. The expression of total Akt, p-Akt (308), p-Akt (473), SM22α, MYH and TRPC1 protein was analysed by Western blot. RESULTS: Treatment with TLR4 siRNA (siTLR4) or Rac1 siRNA (siRac1) significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1. CONCLUSION: This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect.

PCSK9 regulates the chemokine receptor CCR2 on monocytes.

Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. METHODS AND RESULTS: PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-ß or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. CONCLUSION: PCSK9 is regulated in VSMCs by TLR-4 - SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.

Inflammatory and immune responses in the arterial media.

Inflammatory arterial diseases differentially affect the compartments of the vessel wall. The intima and adventitia are commonly involved by the disease process, with luminal and microvascular endothelial cells playing a critical role in the recruitment and activation of leukocytes. In contrast, the avascular media is often spared by immune-mediated disorders. Surprisingly, vascular smooth muscle cells (VSMCs), the predominant and often exclusive cell type of the media, are capable of robust proinflammatory responses to diverse stressors. The multiple cytokines and chemokines produced within the media can profoundly affect macrophage and T cell function, thus amplifying and shaping innate and adaptive immune responses. On the other hand, VSMCs and the extracellular matrix that they produce also display significant anti-inflammatory properties. The balance between the pro- and anti-inflammatory effects of VSMCs and their extracellular matrix versus the strength of the inciting immunologic events determines the pattern of medial pathology. Limitations on the extent of medial infiltration and injury, defined as medial immunoprivilege, are typically seen in arteriosclerotic diseases, such as atherosclerosis and transplant vasculopathy. Conversely, breakdown of medial immunoprivilege that manifests as more intense leukocytic infiltrates, loss of VSMCs, and destruction of the extracellular matrix architecture is a general feature of certain aneurysmal diseases and vasculitides. In this review, we consider the inflammatory and immune functions of VSMCs and how they may lead to medial immunoprivilege or medial inflammation in arterial diseases.

Synergistic effect of atorvastatin and Cyanidin-3-glucoside on angiotensin II-induced inflammation in vascular smooth muscle cells.

Statins have often been used in atherosclerosis treatment because of its pleiotropic effects on inflammation. However, some adverse effects of high doses of statin show reverse effects after withdrawal. Cyanidin-3-glucoside (C3G) is a powerful anti-inflammation and antioxidant that has been of interest for use in combination with low doses of statin, which may be alternative treatment for atherosclerosis. The objective is to investigate the synergistic effect of atorvastatin and C3G in angiotensin II (Ang II)-induced inflammation in vascular smooth muscle cells. Human aortic smooth muscle cells (HASMCs) were exposed to Ang II with or without atorvastatin and C3G alone, or in combination. The results revealed that the combination of atorvastatin and C3G produces synergism against inflammation and oxidative stress. The mechanism of the combination of atorvastatin and C3G suppressed the translocation of the p65 subunit of NF-κB from cytosol to nucleus, and attenuated the expression of proteins including inducible nitric oxide synthase, intracellular adhesion molecule 1(ICAM-1), and vascular cell adhesion molecule 1(VCAM-1), in addition to nitric oxide (NO) production. Moreover, C3G exerts the antioxidative properties of atorvastatin through down-regulating NOX1 and promoting the activity of the Nrf2(-)ARE signaling pathway and downstream proteins including heme oxygenase (HO-1), NAD(P)H:quinoneoxidoreductase 1 (NQO-1), and glutamate-cysteine ligase catalytic subunit (γ-GCLC), besides increasing the activity of superoxide dismutase (SOD) enzymes. Taken together, these results suggest that a combination of low dose statins and C3G might serve as a potential regulator of the atherosclerosis process which is mediated by attenuating oxidative stress, thereby inhibiting NF-κB and activating Nrf2 signaling pathways induced by Ang II.