Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE(-/-)) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Toll-like receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE(-/-)TLR4(-/-) mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE(-/-) mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (pro-inflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7-10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE(-/-)TLR4(-/-) mice, and in Eritoran-treated ApoE(-/-) mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.

γδT cells are prevalent in the proximal aorta and drive nascent atherosclerotic lesion progression and neutrophilia in hypercholesterolemic mice.

Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4+ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.

Role of toll-like receptor 4 in intimal foam cell accumulation in apolipoprotein E-deficient mice.

OBJECTIVE: Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. METHODS AND RESULTS: We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by ≈75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by ≈45%. TLR4 deficiency prevented the increased interleukin-1α (IL-1α) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1α levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. CONCLUSIONS: TLR4 contributes to early-stage intimal foam cell accumulation at lesion-prone aortic sites in ApoE KO mice, as does TLR2 to a lesser extent. Intimal SMC surround and penetrate early lesions, where TLR4 signaling within them may influence lesion progression.

Simvastatin inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice: possible role of ERK.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE(-/-)) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. METHODS AND RESULTS: ApoE(-/-) mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. CONCLUSIONS: These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE(-/-) mice at least in part via ERK inhibition.

Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms.

Arterial O(2) levels are thought to modulate vascular smooth muscle cell (VSMC) proliferation and vascular remodeling, but the mechanisms involved are poorly understood. Here, we tested the hypothesis that PHD2, a prolyl hydroxylase domain (PHD)-containing O(2) sensor, modulates growth factor-induced proliferative responses of human pulmonary artery SMC (HPASMC). We found that both PHD1 and PHD2 were robustly expressed by HPASMC, and inhibiting prolyl hydroxylase activity pharmacologically by using the nonselective dioxygenase inhibitor dimethyloxalylglycine (DMOG) inhibited proliferation and cyclin A expression induced by PDGF-AB or FGF-2. Specific knockdown of PHD2 using small interfering RNAs had similar effects. The inhibitory effects of DMOG and PHD2 knockdown on proliferation and cyclin A expression were seen under both normoxic (20% O(2)) and moderately hypoxic (5% O(2)) conditions, and PHD2 expression was not affected by O(2) level nor by stimulation with PDGF or FGF-2, indicating that the proproliferative influence of PHD2 does not involve alterations of its expression. Knockdown of PHD2 increased hypoxia-inducible factor (HIF)-1alpha expression, as expected, but we also found that HIF-1alpha knockdown abolished the inhibitory effect of PHD2 knockdown on PDGF-induced cyclin A expression. Therefore, we conclude that PHD2 promotes growth factor-induced responses of human VSMC, acting by HIF-1alpha-dependent mechanisms. Given the role of PHD2 as an oxygen sensor in mammalian cells, these results raise the possibility that PHD2 links VSMC proliferation to O(2) availability.

Endogenous interleukin-1 alpha promotes a proliferative and proinflammatory phenotype in human vascular smooth muscle cells.

During vascular disease and following injury, vascular smooth muscle cells (VSMC) proliferate and produce inflammation-promoting cytokines and chemokines. Similar phenotypic changes can be elicited in vitro by activation of Toll-like receptors (TLR) within VSMC. TLR-activated VSMC also produce IL-1 alpha, but it is unknown whether endogenous IL-1 alpha stimulates VSMC in an autocrine manner. Here we tested the hypothesis that endogenous IL-1 alpha contributes to TLR-induced proliferation and chemokine release in human VSMC by using RNA interference to knock down IL-1 alpha expression. Knockdown of IL-1 alpha abolished TLR-induced proliferation and suppressed TLR4-induced release of monocyte chemoattractant protein-1 (MCP-1) by VSMC, indicating that endogenous IL-1 alpha plays a crucial role in both responses. Serum, PDGF, FGF-2, and EGF each increased cellular IL-1 alpha concentrations, and IL-1 alpha knockdown inhibited serum- and PDGF-induced DNA synthesis, further indicating that endogenous IL-1 alpha also contributed to VSMC responses to growth factors. IL-1 receptor antagonist, a competitive inhibitor of IL-1 receptor I (IL-1RI), also attenuated TLR-induced proliferation and both basal and TLR-induced MCP-1 expression, indicating at least a partial role of the IL-1RI in mediating these responses. The results support the hypothesis that autocrine actions of endogenous IL-1 alpha, mediated at least in part via IL-1RI signaling, contribute to a proproliferative and proinflammatory phenotypic shift in TLR-activated human VSMC, which might play a pathogenic role in vascular disorders.

Toll-like receptor 3 signaling evokes a proinflammatory and proliferative phenotype in human vascular smooth muscle cells.

Inflammation plays a key role in atherogenesis, perhaps promoted by bacterial and viral products present within the artery wall. Vascular smooth muscle cells (VSMC) can express certain bacterially responsive Toll-like receptors (TLR), which promote a proinflammatory and proliferative VSMC phenotype when activated, but it is unknown whether virally activated TLR can regulate VSMC phenotype. Here we tested the role in VSMC of TLR3, which is activated by double-stranded (dsRNA), a molecular signature of viruses. VSMC from multiple vessel types, including human coronary artery (HCoASMC) and mouse aorta (MAoSMC), expressed TLR3 constitutively, and HCoASMC were exquisitely sensitive to dsRNA-stimulated release of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6. dsRNA-induced MCP-1 release was abolished by small interfering RNA-mediated TLR3 knockdown in HCoASMC and was absent in TLR3-/- MAoSMC but was unimpaired in TLR2-/- and in TLR4 signaling-deficient MAoSMC. Exposure to dsRNA also activated ERK1/2 and NF-kappaB in both human and murine SMC, but these effects were absent in SMC from TLR3-deficient mice, demonstrating a crucial role of TLR3 signaling. dsRNA also stimulated proliferation of HCoASMC, indicated by increased DNA synthesis, and induced persistent elevations in the intracellular levels of growth-promoting mediators, including interleukin-1alpha and phospho-ERK1/2. We conclude that exposure of HCoASMC to dsRNA elicits dramatic TLR3-mediated proinflammatory and proproliferative phenotypic changes, responses that could potentially be triggered by viral infection of cells within the arterial wall.

Hypoxia and hypoxia-inducible factor-1alpha promote growth factor-induced proliferation of human vascular smooth muscle cells.

Hypoxia is thought to be a stimulus for the excessive proliferation of vascular smooth muscle cells (VSMC) that contributes to pulmonary hypertension, but the mechanisms involved are unknown. Here we tested whether hypoxia-inducible factor 1-alpha (HIF-1alpha), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O(2)) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), compared with those in normoxia (20% O(2)). Moderate hypoxia elicited increased cellular HIF-1alpha levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1alpha. Knockdown of HIF-1alpha or HIF-1beta levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or 20% O(2) but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1alpha similarly inhibited PDGF-stimulated proliferation, whereas HIF-2alpha knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1alpha expression also inhibited growth factor-induced expression of cyclin A. We conclude that HIF-1alpha promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.

Toll-like receptor 2 mediates persistent chemokine release by Chlamydia pneumoniae-infected vascular smooth muscle cells.

OBJECTIVE: The intracellular bacterium Chlamydia pneumoniae is present in many atherosclerotic lesions, where it could promote inflammation. This study determined whether monocyte chemoattractant protein 1 (MCP-1) release is stimulated in vascular smooth muscle cells (VSMCs) that are exposed to or infected by C pneumoniae and whether toll-like receptor 2 (TLR2) or TLR4 mediate these effects. METHODS AND RESULTS: TLR2 mRNA was expressed constitutively and was upregulated by C pneumoniae exposure in mouse aortic SMC and was inducible by C pneumoniae and TLR3 and TLR4 agonists in human coronary artery SMCs. Exposure to inactivated or viable extracellular C pneumoniae evoked a robust increase in MCP-1 release and activated nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 in wild-type and TLR4 signaling-deficient mouse aortic SMCs but not in TLR2-deficient SMCs, probably because of TLR2-mediated recognition of a chlamydial antigen. Brief exposure to viable C pneumoniae led to active infection of VSMCs, shown by chlamydial protein synthesis, and caused a persistent (>48-hour) MCP-1 release that was also TLR2 dependent. CONCLUSIONS: The results show that VSMCs express functional TLR2 and that TLR2 mediates both a persistent activation of chemokine release in C pneumoniae-infected VSMCs and its acute stimulation by extracellular C pneumoniae. Therefore, TLR2 expressed in VSMCs may promote inflammation within the arterial wall.

Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling.

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. In this study, we tested whether TLR4 signaling promotes a proinflammatory phenotype in human and mouse arterial smooth muscle cells (SMC), characterized by increased cytokine and chemokine synthesis and increased TLR expression. Human arterial SMC were found to express mRNA encoding TLR4 and the TLR4-associated molecules MD-2 and CD14 but not TLR2 mRNA. Mouse aortic SMC, on the other hand, expressed both TLR2 and TLR4 mRNA constitutively. Human SMC derived from the coronary artery, but not those from the pulmonary artery, were found to express cell surface-associated CD14. Low concentrations (ng/ml) of Escherichia coli LPS, the prototypical TLR4 agonist, markedly stimulated extracellular regulated kinase 1/2 (ERK1/2) activity, induced release of monocyte-chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and stimulated IL-1alpha expression in human aortic SMC, and exogenous CD14 enhanced these effects. Expression of a dominant negative form of TLR4 in human SMC attenuated LPS-induced ERK1/2 and MCP-1 release. LPS was a potent inducer of NF-kappaB activity, ERK1/2 phosphorylation, MCP-1 release, and TLR2 mRNA expression in wild-type mice but not in TLR4-signaling deficient mouse aortic SMC. These studies show that TLR4 signaling promotes a proinflammatory phenotype in vascular smooth muscle cells (VSMC) and suggest that VSMC may potentially play an active role in vascular inflammation via the release of chemokines, proinflammatory cytokines, and increased expression of TLR2.