ADAM33 Silencing Inhibits Vascular Smooth Muscle Cell Migration and Regulates Cytokine Secretion in Airway Vascular Remodeling via the PI3K/AKT/mTOR Pathway.

Introduction: Vascular smooth muscle cells (VSMCs) are highly involved in airway vascular remodeling in asthma. Objectives: This study aimed to investigate the mechanisms underlying the effects of a disintegrin and metalloproteinase-33 (ADAM33) gene on the migration capacity and inflammatory cytokine secretion of VSMCs. Methods: Human aortic smooth muscle cells (HASMCs) were transfected with lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 or negative control vectors. The migration capacity of HASMCs was evaluated by a transwell assay. The levels of secreted inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative polymerase chain reaction and Western blot assays were performed to detect mRNA and protein expression levels. Results: Silencing of ADAM33 significantly inhibited the migration of HASMCs. The expression of tumor necrosis factor alpha (TNF-α) in the supernatant of HASMCs was decreased, while that of interferon gamma (IFN-γ) was increased after the transfection of shRNA targeting ADAM33. Insufficient ADAM33 expression also suppressed the expression levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTOR), Rho-associated protein kinases, phospho-forkhead box protein O1 (FOXO1), and cyclin D1, but it did not affect the levels of AKT, mTOR, or Rho. Conclusion: Silencing of the ADAM33 gene inhibited HASMC migration and regulated inflammatory cytokine secretion via targeting the PI3K/AKT/mTOR pathway and its downstream signaling. These data contribute to a better understanding of the regulatory mechanisms of airway vascular remodeling in asthma.

Inhibition of Vascular Smooth Muscle and Cancer Cell Proliferation by New VEGFR Inhibitors and Their Immunomodulator Effect: Design, Synthesis, and Biological Evaluation.

Abnormal vascular smooth muscle cell (VSMC) proliferation has an important role in the pathogenesis of both atherosclerosis restenosis and hypertension. Vascular endothelial growth factor (VEGF) has been shown to stimulate VSMC proliferation. In addition, angiogenesis is one of the hallmarks of cancerous growth. VEGF is the key modulator for the initial stages of angiogenesis that acts through the endothelial-specific receptor tyrosine kinases (VEGFRs). VEGFR-2 blockage is a good approach for suppression of angiogenesis. In order to discover novel VEGFR-2 TK inhibitors, we have designed and synthesized three new series of pyridine-containing compounds. The new compounds were all screened against a panel of three cell lines (HepG-2, HCT-116, and MCF-7). Promising results encouraged us to additionally evaluate the most active members for their in vitro VEGFR-2 inhibitory effect. Compound 7a, which is the most potent candidate, revealed a significant increase in caspase-3 level by 7.80-fold when compared to the control. In addition, Bax and Bcl-2 concentration levels showed an increase in the proapoptotic protein Bax (261.4 Pg/ml) and a decrease of the antiapoptotic protein Bcl-2 (1.25 Pg/ml) compared to the untreated cells. Furthermore, compound 7a arrested the cell cycle in the G2/M phase with induction of apoptosis. The immunomodulatory effect of compound 7a, the most active member, showed a reduction in TNF-α by 87%. Also, compound 7a caused a potent inhibitory effect on smooth muscle proliferation. Docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

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.

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.

Circulating uromodulin inhibits vascular calcification by interfering with pro-inflammatory cytokine signalling.

AIMS: Uromodulin is produced exclusively in the kidney and secreted into both urine and blood. Serum levels of uromodulin are correlated with kidney function and reduced in chronic kidney disease (CKD) patients, but physiological functions of serum uromodulin are still elusive. This study investigated the role of uromodulin in medial vascular calcification, a key factor associated with cardiovascular events and mortality in CKD patients. METHODS AND RESULTS: Experiments were performed in primary human (HAoSMCs) and mouse (MOVAS) aortic smooth muscle cells, cholecalciferol overload and subtotal nephrectomy mouse models and serum from CKD patients. In three independent cohorts of CKD patients, serum uromodulin concentrations were inversely correlated with serum calcification propensity. Uromodulin supplementation reduced phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. In human serum, pro-inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) co-immunoprecipitated with uromodulin. Uromodulin inhibited TNFα and IL-1ß-induced osteo-/chondrogenic signalling and activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated ß cells (NF-kB) as well as phosphate-induced NF-kB-dependent transcriptional activity in HAoSMCs. In vivo, adeno-associated virus (AAV)-mediated overexpression of uromodulin ameliorated vascular calcification in mice with cholecalciferol overload. Conversely, cholecalciferol overload-induced vascular calcification was aggravated in uromodulin-deficient mice. In contrast, uromodulin overexpression failed to reduce vascular calcification during renal failure in mice. Carbamylated uromodulin was detected in serum of CKD patients and uromodulin carbamylation inhibited its anti-calcific properties in vitro. CONCLUSIONS: Uromodulin counteracts vascular osteo-/chondrogenic transdifferentiation and calcification, at least in part, through interference with cytokine-dependent pro-calcific signalling. In CKD, reduction and carbamylation of uromodulin may contribute to vascular pathology.

M2 macrophage-derived exosomes promote the c-KIT phenotype of vascular smooth muscle cells during vascular tissue repair after intravascular stent implantation.

Rationale: For intravascular stent implantation to be successful, the processes of vascular tissue repair and therapy are considered to be critical. However, the mechanisms underlying the eventual fate of vascular smooth muscle cells (VSMCs) during vascular tissue repair remains elusive. In this study, we hypothesized that M2 macrophage-derived exosomes to mediate cell-to-cell crosstalk and induce dedifferentiation phenotypes in VSMCs. Methods:In vivo, 316L bare metal stents (BMS) were implanted from the left iliac artery into the abdominal aorta of 12-week-old male Sprague-Dawley (SD) rats for 7 and 28 days. Hematoxylin and eosin (HE) were used to stain the neointimal lesions. En-face immunofluorescence staining of smooth muscle 22 alpha (SM22α) and CD68 showed the rat aorta smooth muscle cells (RASMCs) and macrophages. Immunohistochemical staining of total galactose-specific lectin 3 (MAC-2) and total chitinase 3-like 3 (YM-1) showed the total macrophages and M2 macrophages. In vitro, exosomes derived from IL-4+IL-13-treated macrophages (M2Es) were isolated by ultracentrifugation and characterized based on their specific morphology. Ki-67 staining was conducted to assess the effects of the M2Es on the proliferation of RASMCs. An atomic force microscope (AFM) was used to detect the stiffness of the VSMCs. GW4869 was used to inhibit exosome release. RNA-seq was performed to determine the mRNA profiles of the RASMCs and M2Es-treated RASMCs. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of the mRNAs. Western blotting was used to detect the candidate protein expression levels. T-5224 was used to inhibit the DNA binding activity of AP-1 in RASMCs. Results: M2Es promote c-KIT expression and softening of nearby VSMCs, hence accelerating the vascular tissue repair process. VSMCs co-cultured in vitro with M2 macrophages presented an increased capacity for de-differentiation and softening, which was exosome dependent. In addition, the isolated M2Es helped to promote VSMC dedifferentiation and softening. Furthermore, the M2Es enhanced vascular tissue repair potency by upregulation of VSMCs c-KIT expression via activation of the c-Jun/activator protein 1 (AP-1) signaling pathway.Conclusions: The findings of this study emphasize the prominent role of M2Es during VSMC dedifferentiation and vascular tissue repair via activation of the c-Jun/AP-1 signaling pathway, which has a profound impact on the therapeutic strategies of coronary stenting techniques.

Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγ-dependent T cells response.

AIMS: Defects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects. METHODS AND RESULTS: Using in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine. CONCLUSION: Altogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing.

Metformin inhibits LPS-induced inflammatory response in VSMCs by regulating TLR4 and PPAR-γ.

OBJECTIVE: This study aims to explore whether the inhibitory role of metformin could inhibit LPS-induced inflammatory response in vascular smooth muscle cells (VSMCs) and its underlying mechanism. MATERIALS AND METHODS: VSMCs were extracted from aorta of Sprague Dawley rats. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect VSMCs viability after treatment with different concentrations of metformin. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in VSMCs were detected by ELISA (enzyme-linked immunosorbent assay) and qRT-PCR (quantitative Real time-polymerase chain reaction). Protein and mRNA levels of toll like receptor 4 (TLR4) and peroxisome proliferators activated receptor γ (PPAR-γ) in VSMCs were detected by Western blot and qRT-PCR, respectively. Finally, VSMCs were treated with the PPAR-γ antagonist GW9662 and inflammatory indicators in cells were detected. RESULTS: No significant difference in VSMCs viability was found after 0-2 mM metformin treatment or 500 µg/L LPS induction for 24 h. After 500 µg/L LPS induction in VSMCs for 24 h, levels of MCP-1, TNF-α and IL-6 were remarkably elevated. Both mRNA and protein levels of TLR4 in VSMCs were upregulated after 500 µg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. Knockdown of TLR4 remarkably inhibited LPS-induced inflammatory response in VSMCs, manifesting as decreased levels of MCP1, TNF-α and IL-6, which were further downregulated after combination treatment of TLR4 knockdown and 20 mM metformin. Furthermore, both mRNA and protein levels of PPAR-γ in VSMCs were downregulated after 500 µg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. GW9662 treatment resulted in elevated expressions of MCP-1, TNF-α and IL-6, which were reversed by metformin treatment. CONCLUSIONS: Metformin can effectively inhibit the mRNA and protein expressions of IL-6, MCP-1, and TNF-α in LPS-induced VSMCs. The anti-inflammatory effects of metformin inhibit the inflammatory response through downregulating rely on the downregulation of TLR4 expression and upregulation ofng PPAR-γ activity.

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.

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.

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis.

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response. Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology. Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall. Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls. This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

Mechanisms of Trained Innate Immunity in oxLDL Primed Human Coronary Smooth Muscle Cells.

Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNFα and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1α-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.

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.

AIP1 Suppresses Transplant Arteriosclerosis Through Inhibition of Vascular Smooth Muscle Cell Inflammatory Response to IFNγ.

IFNγ-induced vascular smooth muscle cells (VSMCs) inflammatory response plays a key role in transplant arteriosclerosis (TA). However, the mechanisms regulating this process remains poorly defined. Here, we show that ASK1-interacting protein 1 (AIP1) deletion markedly augments the expression of IFNγ-induced chemokines in mouse aortic allografts. Subsequently, donor arterial grafts from AIP1 deficient mice exhibited an accelerated development of TA. Furthermore, AIP1 knockdown significantly increased IFNγ signaling activation in cultured VSMCs and thus enhances chemokines production in response to IFNγ. Together, we conclude that AIP1 functions as an inhibitor of VSMCs inflammation by regulating IFNγ signaling and therefore suppresses TA progression. Our findings suggest that AIP1 might be a potential therapeutic target for chronic transplant rejection. Anat Rec, 302:1587-1593, 2019. © 2018 American Association for Anatomy.

Protein kinase A (PKA) inhibition reduces human aortic smooth muscle cell calcification stimulated by inflammatory response and inorganic phosphate.

AIMS: Smooth muscle cells (SMCs) play a role in medial vascular calcification, which can be stimulated by high levels of serum phosphate and inflammatory mediators. The aim of this study was to investigate whether mitogen-activated protein kinases (MAPKs) (p38 MAPK, ERK1/2, and JNK) and protein kinase A (PKA) can participate in inorganic phosphate (Pi)- and inflammation response-stimulated SMC calcification. MAIN METHODS: We examined the change of Pi- and/or inflammation-stimulated human aortic smooth muscle cell (HASMC) calcification in the presence and absence of inhibitors or small interfering RNAs. KEY FINDINGS: Ca levels were increased in HASMCs incubated with 1.5-3.9 mM Pi, but not with 0.9 mM Pi or compared with non-Pi-treated HASMCs. Furthermore, the addition of interferon-γ (IFN-γ) increased pro-inflammatory cytokines [interleukin (IL)-1α, IL-6, and tumor necrosis factor-α (TNF-α)] in media containing Raw 264.7 cells. Ca levels were significantly increased in HASMCs cultured in IFN-γ-treated medium, compared with non-IFN-γ-treated medium in the presence of Pi (0.9-2.4 mM). The inhibition of p38 MAPK and PKA decreased HASMC calcification stimulated by Pi and IFN-γ-treated medium, though PKA inhibition produced a more significant reduction in calcification than p38 MAPK inhibition. SIGNIFICANCE: These results indicate that PKA inhibition can efficiently reduce Pi- and inflammation-stimulated SMC calcification.

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.

miR-195 suppresses abdominal aortic aneurysm through the TNF-α/NF-κB and VEGF/PI3K/Akt pathway.

In the present study, the function of microRNA (miR)­195 on abdominal aortic aneurysm (AAA) and its possible mechanism were investigated. Reverse transcription­quantitative polymerase chain reaction analysis was used to detect the expression of miR­195 in patients with AAA. The expression levels of miR­195 in patients with AAA were effectively increased. The present study also used miR­195 mimics to increase the expression of miR­195, and ELISA kits and western blot analysis were used to analyze the levels of interleukin (IL)­1ß and IL­6, and the protein expression levels of matrix metalloproteinase (MMP)­2, MMP­9, tumor necrosis factor (TNF)­α, nuclear factor (NF)­κB, vascular endothelial growth factor (VEGF), phosphoinositide 3­kinase (PI3K) and phosphorylated (p­)Akt. The overexpression of miR­195 promoted the levels of IL­1ß and IL­6, induced the protein expression of MMP­2 and MMP­9, upregulated the protein expression of TNF­α and NF­κB, and suppressed the protein expression levels of VEGF, PI3K and p­Akt in angiotensin II­vascular smooth muscle cells. In addition, TNF­α promoted the pre­inflammatory effect of miR­195 on the protein expression levels of TNF­α and NF­κB, levels of IL­1ß and IL­6, and protein expression levels of MMP­2 and MMP­9 in the angiotensin II­vascular smooth muscle cells. Suppression of PI3K promoted the pre­inflammatory effect of miR­195 on the protein expression of PI3K, p­Akt and VEGF, the levels of IL­1ß and IL­6, and the protein expression of MMP­2 and MMP­9 in angiotensin II­vascular smooth muscle cells. Combined, these results suggested that miR­195 suppressed AAA inflammation through the TNF­α/NF­κB and VEGF/PI3K/Akt pathways.

Is there a role for exosomes in foetoplacental endothelial dysfunction in gestational diabetes mellitus?

Gestational diabetes mellitus (GDM) is a disease of pregnancy associated with endothelial dysfunction in the foetoplacental vasculature. Foetoplacental endothelial dysfunction is characterized by changes in the l-arginine-adenosine signalling pathway and inflammation. The mechanisms involved in these alterations are suggested to be hyperglycaemia, hyperinsulinemia, and oxidative stress. These conditions increase the release of exosomes, nanovesicles that are generated from diverse cell types, including endothelial cells. Since exosomes can modulate vascular function, they may play an important role in foetoplacental endothelial dysfunction seen in GDM pregnancies. In this review, we summarized current knowledge on the potential role of exosomes in foetoplacental endothelial dysfunction seen in this disease of pregnancy.

Human Cytomegalovirus-Encoded Receptor US28 Is Expressed in Renal Allografts and Facilitates Viral Spreading In Vitro.

BACKGROUND: Renal transplantation is the preferred treatment for patients with end-stage renal disease. Human cytomegalovirus (HCMV) activation is associated with decreased renal graft function and survival. Human cytomegalovirus encodes several immune modulatory proteins, including the G protein-coupled receptor US28, which scavenges human chemokines and modulates intracellular signaling. METHODS: Our aim was to identify the expression and localization of US28 in renal allograft biopsies by immunohistochemistry and determine its role in viral spreading in vitro. RESULTS: Immunohistochemistry revealed US28 in 31 of 34 renal transplant biopsies from HCMV-seropositive donors. Expression was independent of HCMV viremia or IgG serostatus. US28 was predominantly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs) and tubular epithelial cells, with a median positivity of 20% and 40%, respectively. Also, US28-positive cells were present within arterial neointima. In contrast to US28, HCMV-encoded immediate early antigen was detected in less than 5% of VSMCs, tubular epithelial cells, interstitial endothelium, interstitial inflammatory infiltrates, and glomerular cells.Primary VSMCs were infected with green fluorescent protein-tagged wild type or US28-deficient HCMV. The viral spreading of US28-deficient HCMV, via culture medium or cell-to-cell transmission, was significantly impeded as shown by green fluorescent protein (ie, infected) cell quantification and quantitative real-time polymerase chain reaction. Additionally, the number and size of foci was smaller. CONCLUSIONS: In summary, HCMV-encoded US28 was detected in renal allografts from HCMV-positive donors independent of viremia and serostatus. Also, US28 facilitates HCMV spreading in VSMCs in vitro. Because the vasculature is affected in chronic renal transplant dysfunction, US28 may provide a potential target for therapeutic intervention.

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.