Debbie Beasley

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-1&agr; (IL-1&agr;) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1&agr; 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. Conclusion—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.

Hypoxia stimulates proliferation and interleukin-1α production in human vascular smooth muscle cells

Several lines of evidence indicate that hypoxia is a stimulus to vascular smooth muscle cell (VSMC) proliferation that occurs in pulmonary hypertension. The present study tested the hypothesis that low O(2) tension directly stimulates human VSMC proliferation by inducing them to produce interleukin (IL)-1, a potent autocrine growth factor for human VSMC. Human VSMC derived from pulmonary artery, aorta, or saphenous vein were incubated in either a normal in vitro O(2) environment (20% O(2)) or in chambers containing low (approximately 1%) or moderate (5%) O(2). Levels of IL-1alpha and IL-1beta mRNA increased in human VSMC after 24-48 h of incubation in low O(2) compared with levels in normoxic cells and then decreased upon subsequent reoxygenation. Levels of cell-associated IL-1alpha also increased progressively after 24-48 h in low O(2); however, detectable IL-1alpha was not released from the cells in the media. IL-1beta was detectable in cell lysates and supernatants; however, the levels were not affected by exposure to low O(2). mRNA encoding for tumor necrosis factor-alpha (TNF-alpha), a related cytokine and VSMC mitogen, was not detectable in human VSMC exposed to either low or 20% O(2). Proliferation of human VSMC was not stimulated during exposure to low O(2), despite the fact that cells remained responsive to the mitogenic effect of exogenous IL-1. Interestingly, however, exposure to 5% O(2) enhanced proliferation of human VSMC but did not induce IL-1alpha production. Inhibition of IL-1 binding to the type I IL-1 receptor by exogenous addition of IL-1-receptor antagonist (10 microgram/ml) did not attenuate the proliferation rates of human VSMC incubated in 20%, 5%, or low O(2) or in human VSMC that were reoxygenated after exposure to low O(2). These results demonstrate two direct and distinct effects of hypoxia on VSMC. Exposure to moderately low O(2) tension induces VSMC proliferation, independent of IL-1, whereas exposure to very low O(2) tension induces production of IL-1alpha.

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.

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-&kgr;B 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.

Constitutive expression of interleukin-1α precursor promotes human vascular smooth muscle cell proliferation

Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the failure of vascular surgeries and contributes to the development of atherosclerotic lesions. Evidence that interleukin-1 (IL-1) is a mitogen for cultured VSMC has implicated its release by activated macrophages in the development of atherosclerosis. VSMC also produce IL-1, including the precursor form of IL-1α. However, it is not known whether IL-1α precursor is processed to mature IL-1α or released from VSMC, nor is it known whether either precursor or mature IL-1α functions as an autocrine growth factor. The goals of the present study were to establish whether proliferation is enhanced in human VSMC transfectants producing IL-1α constitutively at levels comparable to those produced after activation, and to determine which domains of IL-1α are important for its activity. Human VSMC were stably transfected with expression vectors directing constitutive expression of either full-length IL-1α precursor [IL-1α-(1-271)], its NH2-terminal domain [IL-1α-(1-112)], or mature IL-1α [IL-1α-(113-271)]. Both IL-1α-(1-271) and IL-1α-(113-271) stable transfectants produced moderate levels of IL-1α (0.2-1.0 ng/106cells) and released low levels of IL-1α into the supernatant (<20 pg/ml). VSMC stably transfected with either IL-1α-(1-271) or IL-1α-(113-271) expression plasmids proliferated rapidly compared with nontransfected or vector-transfected VSMC and displayed a distinct morphology characterized by elongated, spindle-shaped cells. Stable transfection with IL-1α-(1-271) was somewhat more effective than transfection with IL-1α-(113-271). Interestingly, VSMC transfected with IL-1α-(113-271) expression plasmids also expressed IL-1α-(1-271) mRNA, suggesting that IL-1α-(113-271) activates an IL-1-induced IL-1 autocrine loop. In contrast, neither proliferation rates nor morphology was affected by stable transfection with IL-1α-(1-112) expression plasmids. Exogenous IL-1 receptor antagonist partially reversed the enhanced DNA synthesis in VSMC transfected with either IL-1α-(1-271) or IL-1α-(113-271) expression plasmids, suggesting that the pro-proliferative effect of VSMC-derived IL-1α is at least partially mediated by signaling via the type I IL-1 receptor. These results demonstrate that IL-1α precursor is an autocrine growth factor for human VSMC and further indicate that amino acids 113-271 play a crucial role in its actions.

COX-2 and cytosolic PLA2mediate IL-1β-induced cAMP production in human vascular smooth muscle cells

Interleukin (IL)-1 is a potent vasodilator that causes prolonged induction of prostacyclin (PGI2) and cAMP synthesis in human vascular smooth muscle cells (HVSMC). The present study investigated IL-1 induction of PG synthetic enzymes in HVSMC and tested their respective roles in PGI2 and cAMP production. Cyclooxygenase (COX)-1 mRNA was not detectable in either control or IL-1-treated HVSMC, as assessed by RT-PCR. In contrast, COX-2 mRNA was detectable in control HVSMC, increased markedly (16-fold) after 1 h of IL-1 exposure, and increased further (52-fold) after 24 h. COX-2 protein levels, assessed by Western analysis, were increased concomitantly. HVSMC contained mRNA encoding both the secreted and cytosolic forms of phospholipase A2(sPLA2 and cPLA2, respectively). IL-1 stimulation did not affect sPLA2mRNA levels, but cPLA2 mRNA levels increased at 8 h, after the initial induction of PG synthesis. HVSMC constitutively expressed PGI2synthase mRNA, and its levels were not affected by IL-1. A selective COX-2 inhibitor, NS-398, reversed IL-1-induced PGI2 and cAMP production, supporting a role of COX-2 in mediating increased PG synthesis. IL-1-induced cAMP was also reversed by a selective cPLA2 inhibitor, AACOCF3, but not by thioetheramide phosphorylcholine, which inhibits sPLA2 preferentially over cPLA2, supporting a requirement for cPLA2-derived arachidonic acid in IL-1-induced PG synthesis. The delayed induction of cPLA2 mRNA was also attenuated by NS-398, suggesting that it was secondary to the initial COX-2-induced PG synthesis. Together, the results support the hypothesis that IL-1 induces intracellular PG synthesis in HVSMC via rapid upregulation of COX-2, which utilizes cPLA2-derived arachidonic acid to generate PG metabolites that regulate adenylate cyclase.

Phorbol ester and interleukin-1 induce interleukin-6 gene expression in vascular smooth muscle cells via independent pathways.

Previous studies using bioassays suggest that interleukin 6 (IL-6) is a major secretory product of vascular smooth muscle cells (VSMC), which is induced by proinflammatory cytokines. This study investigated whether activation of the protein kinase C (PKC) pathway induces IL-6 gene expression and release in VSMC, by using both bioassay and specific immunoassay methods to measure IL-6 release. Activation of PKC with a phorbol ester, PMA (phorbol myristate acetate), induced a rapid and transient (1-4 h) increase in the levels of both 1.2- and 2.4-kb IL-6 transcripts in rat aortic SMCs (RASMC), as determined by Northern analysis, which was followed by increased release of bioactive IL-6, as determined by a B9 cell-proliferation assay. IL-1, a physiological activator of PKC, induced a rapid increase in IL-6 messenger RNA (mRNA) levels, which was sustained at 24 h. PMA-induced IL-6 mRNA levels in RASMC were markedly attenuated after downregulation of PKC with PMA and by the selective PKC inhibitor, bisindolylmaleimide. In contrast, IL-1-induced increases in IL-6 mRNA were not affected by either PKC downregulation or bisindolylmaleimide. Angiotensin II (Ang II), also known to activate PKC, likewise induced a rapid increase in IL-6 mRNA levels and IL-6 release in RASMC, but the effect was not blocked by PKC downregulation. VSMC derived from human saphenous vein (HSVSMC) released substantial amounts of immunoreactive IL-6 in the absence of stimulation by exogenous growth factors, and both PMA and IL-1 markedly increased IL-6 release. Furthermore, downregulation of PKC and bisindolylmaleimide blocked the effect of PMA but not that of IL-1 in HSVSMC. These results suggest that activation of phorbol ester-responsive PKC induces IL-6 gene expression in both rat and human VSMC. In contrast, IL-1 and Ang II activate IL-6 gene expression by a pathway distinct from that of phorbol ester-responsive PKC.

Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1α-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.

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.

DOUBLE-STRANDED RNA AND BACTERIAL LPS-INDUCED ACTIVATION OF VASCULAR SMOOTH MUSCLE CELLS: ROLE OF TOLL-LIKE RECEPTOR 3 AND TOLL-LIKE RECEPTOR 4

A growing body of evidence supports a central role of inflammation in the pathogenesis of atherosclerosis, however the triggers that initiate the inflammatory process have not been definitively identified. Infectious agents, including both bacteria and viruses, are among the proposed initiators of inflammation, yet the molecular mechanisms by which microbes activate cells in the vessel wall are unknown. Recent evidence supports a central role of transmembrane Toll-like receptors (TLRs) in cellular activation by microbial products. Ten different TLRs are activated by distinct microbial ligands, and in turn elicit differential cellular responses. To test the possible role of TLRs in vascular smooth muscle cell (VSMC) activation, we characterized the expression of TLR mRNAs in VSMC, and characterized the activation of VSMC by their respective microbial ligands. VSMC isolated from either human pulmonary artery or human aorta were found to express mRNA encoding TLR3, TLR4 and TLR6. TLR3 has recently emerged as an important receptor for poly ( IC), a mimic of double-stranded RNA that is produced by many viruses, whereas TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Poly ( IC) was a potent stimulator of human VSMC, markedly stimulating the cellular release of monocyte-chemoattractant protein-1 (MCP-1), and the levels of cell-associated interleukin-1alpha ( IL-1alpha ), a potent SMC mitogen. LPS likewise stimulated MCP-1 release and IL-1alpha synthesis. Both poly ( IC) and LPS markedly induced NFkappaB activation in human VSMC, and in aortic SMC derived from wild-type mice that express functional TLR4. Poly ( IC) also activated NFkappaB in VSMC derived from mice that express a non-functional form of TLR4, whereas LPS was ineffective. Expression of a dominant negative form of TLR3 attenuated poly ( IC)-, but not LPSor IL-1alpha-induced NFkappaB activation in mouse aortic SMC. Poly ( IC ) also markedly stimulated proliferation in human VSMC and in mouse aortic VSMC derived from either wild-type or TLR4 mutant mice. In contrast, bacterial LPS did not affect proliferation either when added alone or in the presence of the LPS transfer factor CD14. These studies support the hypothesis that double-stranded RNA activates VSMC via TLR3, whereas bacterial LPS activates VSMC via TLR4, and that TLR3 and TLR4 appear to elicit differential effects on VSMC proliferation.