Chemotaxis Of Smooth Muscle Cells Research Articles

Dyslipidemia regulates thrombospondin-1-induced vascular smooth muscle cell chemotaxis.

Dyslipidemia is a risk factor for intimal hyperplasia (IH). Key to IH is vascular smooth muscle cell (VSMC) migration. Thrombospondin-1 (TSP-1) is a matricellular protein that stimulates VSMC migration. HDL will inhibit and LDL will augment TSP-1-induced VSMC chemotaxis. VSMC chemotaxis will be inhibited by the HDL moiety, S1P, through the S1PR1 receptor, and augmented by the LDL component, LPA, through the LPAR1 receptor. The goal of this study was to determine the effect of HDL and LDL and their receptors on TSP-1-induced VSMC chemotaxis. For VSMC chemotaxis to TSP-1 cells received the following pretreatments: low (25 µg/ml) or optimal (75 µg/ml) concentration of HDL, S1P, optimal (75 µg/ml) or high (175 µg/ml) concentration of LDL, or LPA. For the receptor studies, VSMCs were transfected with siRNA to S1PR1, S1PR3, LPAR1, LPAR2, LPAR3, or a S1PR2 receptor antagonist. The TSP-1-induced chemotaxis results were (1) HDL (25 µg/ml) or LDL (75 µg/ml) exhibited no effect on chemotaxis; (2) HDL (75 µg/ml) inhibited chemotaxis by 50.9 ± 8 % and S1P by 43.4 ± 11.6 %; (3) LDL (175 µg/ml) augmented chemotaxis by 30 ± 10.4 % and LPA by 25.6 ± 12.3 %; (4) S1PR1 and S1PR3 knockdown and S1PR2 antagonist-treated cells augmented chemotaxis; and (5) LPAR1 and LPAR2 knockdown inhibited and LPAR3 knockdown had no effect on chemotaxis. In conclusion, HDL/S1P inhibits, while LDL/LPA stimulates TSP-1-induced VSMC chemotaxis. The HDL/S1P effect is mediated by the S1PR1-3 receptors. The LDL/LPA effects are mediated by the LPAR1 and LPAR2 receptors, but not LPAR3. Therefore, lipids have significant effects on TSP-1-induced VSMC chemotaxis.

Thrombospondin-1-induced smooth muscle cell chemotaxis and proliferation are dependent on transforming growth factor-β2 and hyaluronic acid synthase

Angioplasty causes local vascular injury, leading to the release of thrombospondin-1 (TSP-1), which stimulates vascular smooth muscle cell (VSMC) migration and proliferation, important steps in the development of intimal hyperplasia. Transforming growth factor beta 2 (TGF-β2) and hyaluronic acid synthase (HAS) are two pro-stenotic genes upregulated in VSMCs by TSP-1. We hypothesized that inhibition of TGF-β2 or HAS would inhibit TSP-1-induced VSMC migration, proliferation, and TSP-1 signaling. Our data demonstrate that Inhibition of either TGF-β2 or HAS inhibited TSP-1-induced VSMC migration and proliferation. Activation of ERK 1 was decreased by TGF-β2 inhibition and unaffected by HAS inhibition. TGF-β2 and HAS are not implicated in TSP-1-induced thbs1 expression, while they are each implicated in TSP-1-induced expression of their own gene. In summary, TSP-1-induced VSMC migration and proliferation rely on intact TGF-β2 signaling and HAS function. TSP-1 activation of ERK 1 is dependent on TGF-β2. These data further expand our understanding of the complexity of TSP-1 cellular signaling and the involvement of TGF-β2 and HAS.

A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis). In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

The effects of nicotine on vascular smooth muscle cell chemotaxis induced by thrombospondin-1 and fibronectin

Background Vascular smooth muscle cell (VSMC) migration is an important process in many vascular disorders. Nicotine, thrombospondin-1 (TSP-1) and fibronectin (Fn) separately induce VSMC migration. The hypothesis of this study was that nicotine treatment of vascular cells would augment TSP-1-induced and Fn-induced VSMC migration. Methods VSMCs or endothelial cells (ECs) were treated with serum-free medium or nicotine. Migration of VSMCs was assessed using a modified Boyden chemotaxis chamber to serum-free medium, TSP-1, Fn, EC basal medium, and conditioned EC medium or nicotine-treated conditioned EC medium alone or with supplemented TSP-1 or Fn. Results Nicotine treatment increased VSMC chemotaxis to serum-free medium, but TSP-1 or Fn had no further effect on chemotaxis. Conditioned EC and nicotine-treated conditioned EC enhanced VSMC chemotaxis, which was further augmented by Fn supplementation. Conclusions Nicotine-stimulated EC derived factors induce VSMC migration, which is augmented by the addition of Fn.

Thrombospondin 1, Fibronectin, and Vitronectin are Differentially Dependent Upon RAS, ERK1/2, and p38 for Induction of Vascular Smooth Muscle Cell Chemotaxis

Thrombospondin 1 (TSP-1), fibronectin (Fn), and vitronectin (Vn) promote vascular smooth muscle cell (VSMC) chemotaxis through a variety of second messenger systems, including Ras, ERK1/2, and p38. Ras, ERK1/2, and p38 differentially affect TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Bovine VSMCs were transfected with Ras N17 or treated with the following inhibitors: a farnesyl protein transferase (FPT) inhibitor, PD098059 (ERK1/2 inhibitor), or SB202190 (p38 inhibitor). Thrombospondin 1, Fn, and Vn were used as chemoattractants. Results were analyzed by analysis of variance (ANOVA) with post hoc testing (P < .05). Ras N17 transfection or FPT inhibitor treatment inhibited TSP-1-, Fn-, and Vn-induced chemotaxis. PD098059 or SB202190 resulted in more inhibition of VSMC migration to TSP-1 than to Fn or Vn. Ras appears equally relevant in the signal transduction pathways of TSP-1-, Fn-, and Vn-induced VSMC chemotaxis. Thrombospondin 1-induced migration is more dependent upon ERK1/2 and p38 than Fn- or Vn-included migration.

Leukotriene B 4 mediates vascular smooth muscle cell migration through αvβ3 integrin transactivation

Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B 4 (LTB 4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB 4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB 4 was dependent on the concentration used, being comparable to AngII at 100 nM. Migration induced by LTB 4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB 4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB 4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB 4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases.

Clinical Summaries

Clinical Summaries

Polymorphisms at newly identified lipid-associated loci are associated with blood lipids and cardiovascular disease in an Asian Malay population

We conducted a cross-sectional study of Malay participants aged 40-80 years (n = 2,932) to examine the associations between polymorphisms at newly identified, lipid-associated loci with blood lipid levels and prevalent cardiovascular disease (CVD) in a Malay population in Asia. A polymorphism adjacent to the TRIB1 locus (rs17321515) was associated with elevated total cholesterol and LDL-cholesterol (LDL-C) after adjustment for age and sex (both P values <0.007) and with increased risk of coronary heart disease and CVD [odds ratio (OR) 1.23, 95% confidence interval (95% CI) 1.03-1.46; and OR 1.2, 95% CI 1.02-1.42, respectively] under an additive model of inheritance. In addition, using recessive models of inheritance, polymorphisms on chromosome 19 adjacent to the CILP2 and PBX4 loci (rs16996148) and on chromosome 1 at the GALNT2 locus (rs4846914) were associated with elevated HDL-C (P = 0.005) and lower LDL-C (P = 0.048), respectively. Although novel, the former is consistent with the association between this polymorphism and lower blood triglycerides observed in the initial studies conducted in populations of European ancestry. Neither showed statistically significant association with CVD. These observations should form the basis of further investigation to identify the causative polymorphisms at this locus, and also to understand the mechanistic roles that this protein may play in lipoprotein metabolism in Asians and other populations.

Endothelium-Derived Bone Morphogenic Protein Antagonists May Counteract the Proatherogenic Vascular Effects of Bone Morphogenic Protein 4

In the present issue of Circulation , Chang et al1 report novel shear stress–sensitive paracrine mechanisms that regulate the activity of bone morphogenetic proteins (BMPs) in the vascular wall. BMP2 and BMP4 are structurally related members of the transforming growth factor-β superfamily. Recent studies demonstrated that vascular endothelial and smooth muscle cells are a significant source of BMPs,2–8 which regulate a host of cellular functions, including cardiovascular development,9 neovascularization in tumors,10 and smooth muscle cell chemotaxis in response to vascular injury,11 and control the balance between proliferation and activation of apoptosis in pulmonary arterial endothelial and smooth muscle cells.12 Article p 1258 Many lines of evidence suggest that BMPs may function as proinflammatory, prohypertensive, and proatherogenic mediators in the vessel wall.13 Recent studies have demonstrated a striking upregulation of BMP2/4 in atheroprone vascular regions and atherosclerotic lesions,5,6,11,14,15 and hypotheses have been put forward that endothelium-derived BMPs contribute to vascular calcification (reviewed elsewhere16,17). In vitro, BMP2 and BMP4 were shown to exert proinflammatory effects. Activation of BMP signaling by either overexpression of BMP2/4 in vascular cells or administration of recombinant BMPs activates NAD(P)H oxidases, which results in cellular oxidative stress and endothelial dysfunction (Figure, A and B).13 Chronic BMP4 infusion in C57Bl/6 and apolipoprotein-null mice also impairs endothelium-dependent vasodilation and induces arterial hypertension in an NAD(P)H oxidase–dependent manner.8 BMP2 and BMP4 also elicit endothelial activation, thus enhancing monocyte adhesiveness.1,3–6 Evidence indicates that in endothelial cells BMP2 and BMP4 activate mitogen-activated protein kinase pathways and nuclear factor-κB,3,6 at least in part, via NAD(P)H oxidase–dependent pathways. Future studies will elucidate in detail the role of BMP receptor subtypes and the interaction between downstream signaling mechanisms induced by BMP2/4 that mediate their proinflammatory effects in the cardiovascular system. A, BMP4 …

Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation☆

Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229+/-9% and 362+/-54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 microM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2+/-0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2+/-0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 microM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 microM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner. VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which-in contrast to Akt and Erk-appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.

Integrin αVβ3, protein kinase C β and Erk activation during hyperglycemia in vascular smooth muscle cells

Cardiovascular disease is responsible for most of the complications associated with diabetes. Atheroma formation involves the growth and migration of vascular smooth muscle cells (SMCs) in neointimal lesions. Hyperglycemia potentiates chemotaxis of SMCs via PI3K and Erk/MAPK signaling pathways, perhaps through activation of protein kinase Cβ (PKCβ). The aim of this study was to determine if integrin αVβ3 modulated PKCβ activation and effects on PI3K and Erk/MAPK signaling pathways under conditions of hyperglycemia. Under elevated glucose conditions (25mmol/L versus 5 mmol/L), activity of the Erk/MAPK and PI3K pathways were significantly increased in cultured mouse aortic SMCs. The association of PKCβ in a complex with the αVβ3 integrin was observed by immunoprecipitation of lysates from the SMCs. A significant 2.4 fold increase in the association of PKCβ with αVβ3 was observed under conditions of high glucose, and scanning electron microscopy demonstrated a correlation with filapodia formation. In a streptozocin-induced diabetes model in mice, elevated Erk/MAPK and PI3K activity was observed in aortas from mice with blood sugars > 450 mg/dl and was associated with recruitment of PKCβby αVβ3 (Mann Whitney U test, P<0.01). In conclusion, our study suggests that PKCβ is central to αVβ3-mediated intracellular signaling inducing Erk1/2 activation by a novel pathway, which regulate events downstream of adhesion and cell migration involved in diabetic vascular responses. Support: NIH R01HL074219

Inhibition of Atherogenesis in BLT1-Deficient Mice Reveals a Role for LTB4 and BLT1 in Smooth Muscle Cell Recruitment

It is known that 5-lipoxygenase and its product, leukotriene B4 (LTB4), are highly expressed in several human pathologies, including atherosclerotic plaque. LTB(4) signals primarily through its high-affinity G protein-coupled receptor BLT1, which is expressed on specific leukocyte subsets. BLT1 receptor expression and function on other atheroma-associated cell types is unknown. To directly assess the role of the LTB4-BLT1 pathway in atherogenesis, we bred BLT1(-/-) mice into the atherosclerosis-susceptible apoE(-/-) strain. Compound-deficient apoE(-/-)/Blt1(-/-) mice fed a Western-type diet had a marked reduction in plaque formation compared with apoE(-/-) controls. Immunohistochemical analysis of atherosclerotic lesions in compound-deficient mice revealed a striking decrease in smooth muscle cells (SMCs) and significant decreases in macrophages and T cells. We report here novel evidence of the expression and function of BLT1 on vascular SMCs. LTB4 triggered SMC chemotaxis, which was pertussis toxin sensitive in Blt1(+/+) SMCs and absent in Blt1(-/-) cells, suggesting that BLT1 was the dominant receptor mediating effector functions through a G protein-coupled signaling pathway. Furthermore, BLT1 colocalized with SMCs in human atherosclerotic lesions. These new findings extend the role of inducible BLT1 to nonleukocyte populations and suggest an important target for intervention to modulate the response to vascular injury.

Targeted Disruption of TGF-β–Smad3 Signaling Leads to Enhanced Neointimal Hyperplasia With Diminished Matrix Deposition in Response to Vascular Injury

The role of transforming growth factor (TGF)-beta and its signal in atherogenesis is not fully understood. Here, we examined mice lacking Smad3, a major downstream mediator of TGF-beta, to clarify the precise role of Smad3-dependent signaling in vascular response to injury. Femoral arteries were injured in wild-type and Smad3-null (null) male mice on C57Bl/6 background. Histopathological evaluation of the arteries 1 to 3 weeks after the injury revealed significant enhancement of neointimal hyperplasia in null compared with wild-type mice. Transplantation of null bone marrow to wild-type mice did not enhance neointimal thickening, suggesting that vascular cells in situ play a major role in the response. Null intima contained more proliferating smooth muscle cells (SMC) with less amount of collagen compared with wild-type intima. TGF-beta caused significant inhibition of cellular proliferation in wild-type aortic SMC, whereas the growth of null SMC was only weakly inhibited by TGF-beta in vitro, indicating a crucial role of Smad3 in the growth inhibitory function. On the other hand, Smad3-deficiency did not attenuate chemotaxis of SMC toward TGF-beta. TGF-beta increased transcript level of alpha2 type I collagen and tissue inhibitor of metalloproteinases-1, and suppressed expression and activity of matrix metalloproteinases in wild-type SMC. However, these effects of TGF-beta were diminished in null SMC. Our findings altogether show that the loss of Smad3 pathway causes enhanced neointimal hyperplasia on injury through modulation of growth and matrix regulation in vascular SMC. These results indicate a vasculoprotective role of endogenous Smad3 in response to injury.

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.

Homocysteine promotes p38-dependent chemotaxis in bovine aortic smooth muscle cells

Increased levels of homocysteine in the blood are a risk factor for atherosclerosis. The purpose of this study was to examine the effects of homocysteine on smooth muscle cell (SMC) migration and to determine whether p38 was involved in this process. The effect of 0.5 to 2.0 mmol/L d , l -homocysteine as a chemoattractant for SMCs was assayed with a modified Boyden chamber. To determine the functional role of p38 in SMC chemotaxis induced by d , l -homocysteine, we treated SMCs with a p38 inhibitor, SB203580, before the assay. The number of migrated cells was increased 7.0 +/- 1.2-fold (n = 15; P < .001) by 2.0 mmol/L d , l -homocysteine. SB203580 partially prevented the migration of SMCs toward homocysteine. Preconditioning SMCs with 2.0 mmol/L d , l -homocysteine significantly enhanced chemotaxis toward 10% fetal bovine serum compared with nonconditioned control SMCs (28.9 +/- 3.3-fold vs 15.6 +/- 2.8-fold; P < .05). There was a fourfold p38 activation after exposure of SMCs to 2.0 mmol/L d , l -homocysteine by immunoblot. These results suggest that homocysteine not only is a chemoattractant for SMC but can also enhance SMC chemotactic potential. The mechanism of these effects may involve p38 activation. This study demonstrates that homocysteine can promote chemotaxis of SMCs through a p38-dependent pathway. To our knowledge, this is the first report that homocysteine may influence SMC chemotaxis. It may be an important mechanism for homocysteine-induced atherogenesis, because the migration of SMCs from the media is believed to play a critical role in progressive intimal thickening. Although homocysteine promotes atherogenesis and thrombosis by a variety of mechanisms, the effects of homocysteine on SMC proliferation and migration might be critical elements that may have potential therapeutic implications, because selective blockade of the p38 pathway is feasible.

Shear stress-stimulated endothelial cells induce smooth muscle cell chemotaxis via platelet-derived growth factor-BB and interleukin-1α

Vascular smooth muscle cell (SMC) migration is critical to the development of atherosclerosis and neointimal hyperplasia. Hemodynamic forces such as shear stress and cyclic strain stimulate endothelial cell signal-transduction pathways, resulting in the secretion of several factors, including SMC chemoattractants such as platelet-derived growth factor (PDGF). We hypothesized that mechanical forces stimulate endothelial cells to secrete SMC chemoattractants to induce migration via the mitogen-activated protein kinase (MAPK) pathway. Bovine aortic endothelial cells were exposed to shear stress, cyclic strain, or static conditions for 16 hours. The resulting conditioned medium was used as a SMC chemoattractant in a Boyden chamber. Activation of SMC extracellular signal-regulated protein kinase 1/2 (ERK1/2) was assessed by Western blot analysis. Pathways were inhibited with anti-PDGF-BB or anti-interleukin-1alpha (IL-1alpha) antibodies, or the ERK1/2 upstream pathway inhibitor PD98059. Conditioned medium from endothelial cells exposed to shear stress corresponding to arterial levels of shear stress stimulated SMC migration but lower levels of shear stress or cyclic strain did not. Both PDGF-BB and IL-1alpha were secreted into the conditioned medium by endothelial cells stimulated with shear stress. Both PDGF-BB and IL-1alpha stimulated SMC chemotaxis but were not synergistic, and both stimulated SMC ERK1/2 phosphorylation. Inhibition of PDGF-BB or IL-1alpha inhibited SMC chemotaxis and ERK1/2 phosphorylation. Shear stress stimulates endothelial cells to secrete several SMC chemoattractants, including PDGF-BB and IL-1alpha; both PDGF-BB and IL-1alpha stimulate SMC chemotaxis via the ERK1/2 signal-transduction pathway. These results suggest that the response to vascular injury may have a common pathway amenable to pharmacologic manipulation. One difficulty in the pharmacologic treatment of atherosclerosis or neointimal hyperplasia leading to restenosis is the multiplicity of activated pathways and thus potential treatment targets. This study demonstrates that shear stress, a hemodynamic force that may be a biologically relevant stimulus to induce vascular pathology, stimulates endothelial cells to secrete PDGF-BB and IL-1alpha. Both of these mediators stimulate the SMC ERK1/2 pathway to induce migration, a critical event in the pathogenesis of atherosclerosis and neointimal hyperplasia. Therefore, this study suggests a relevant common target pathway in SMC that is amenable to manipulation for clinical treatment.

Genetic variation in the fibrinogen gene cluster

E levated plasma levels of fibrinogen have been identified as a cardiovascular risk factor in a number of epidemiological studies with a range of endpoints, such as acute myocardial infarction and ischemic stroke. The underlying mechanism for this association is still unclear. One explanation for the association can be found in the acute phase character of fibrinogen. Inflammation is an important mechanism in cardiovascular disease and plasma fibrinogen levels will reflect the level of vascular inflammation. However, the fibrinogen and fibrin that are present in the atherosclerotic plaque can directly contribute locally to the progression of atherosclerosis by increasing the chemotaxis of smooth muscle cells, and affecting the structure and stability of the plaque matrix. . . .

High-mobility group box 1: role in ARDS

Extracellular high-mobility group box 1 (HMGB-1), first isolated approximately 30 years ago from the thymus as a chromosomal protein and recognized as a transcriptional factor, has recently been proposed as one of the late mediators of sepsis and lipopolysaccharide (LPS) endotoxin acute lung injury (ALI) [1,2]. In recent studies, HMGB-1 has been implicated in the development of arthritis, activation of human monocytes, smooth muscle cell chemotaxis, and induction of adhesion molecules in endothelial cells. These observations suggest that HMGB-1 is a key mediator of cell injury, and that its inhibition may be key in improving clinical outcomes. We conducted a translational study to examine the participation of HMGB-1 in the pathogenesis of ALI caused by sepsis. To test the hypothesis that HMGB-1 plays a key role as a late mediator, we first measured its concentrations and those of its related compound, HMGB-2, in the blood and lungs of patients presenting with septic ALI/ARDS, and in a murine model of LPS-induced lung injury. We then studied the localization of the extracellular HMGB-1 protein and examined the anti-inflammatory effects of anti-HMGB-1 antibodies in our LPS-induced lung injury murine model. The direct effects of HMGB-1 and HMGB-2 on the lung were also examined. This study was performed to examine the putative role of HMGB protein in the pathogenesis of ALI. Observations were made (1) in 21 septic patients with ALI and 15 patients with normal lung function, and (2) in a mouse model, 24 hours after intratracheal instillation of lipopolysaccharide. The concentrations of HMGB-1 were increased in plasma and lung epithelial lining fluid of patients with ALI and mice instilled with LPS. LPS-induced ALI was mitigated by anti-HMGB-1 antibody. Although this protein was not detected in the plasma of control humans or mice, the concentrations of HMGB-1 in lung epithelial lining fluid or in bronchoalveolar lavage fluid were unexpectedly high. The nuclear expression of HMGB-1 was apparent in epithelial cells surrounding terminal bronchioles in normal mice, while its nuclear and cytoplasmic expression was observed in alveolar macrophages in LPS-instilled mice. Lung instillation of HMGB-2 did not cause as much inflammation as HMGB-1. Extracellular HMGB-1 may play a key role in the pathogenesis of clinical and experimental ALI. However, its expression in normal airways is noteworthy, and suggests that it also plays a physiologic role in the lung.

CCL11 (Eotaxin) induces CCR3-dependent smooth muscle cell migration.

CCL11 (Eotaxin) is a potent eosinophil chemoattractant that is abundant in atheromatous plaques. The major receptor for CCL11 is CCR3, which is found on leukocytes and on some nonleukocytic cells. We sought to determine whether vascular smooth muscle cells (SMCs) possessed functional CCR3. CCR3 mRNA (by RT-PCR) and protein (by Western blot analysis and flow cytometry) were present in mouse aortic SMCs. CCL11 induced concentration-dependent SMC chemotaxis in a modified Boyden chamber, with maximum effect seen at 100 ng/mL. SMC migration was markedly inhibited by antibody to CCR3, but not to CCR2. CCL11 also induced CCR3-dependent SMC migration in a scrape-wound assay. CCL11 had no effect on SMC proliferation. CCR3 and CCL11 staining were minimal in the normal arterial wall, but were abundant in medial SMC and intimal SMC 5 days and 28 days after mouse femoral arterial injury, respectively, times at which SMCs possess a more migratory phenotype. These data demonstrate that SMCs possess CCR3 under conditions associated with migration and that CCL11 is a potent chemotactic factor for SMCs. Because CCL11 is expressed abundantly in SMC-rich areas of the atherosclerotic plaque and in injured arteries, it may play an important role in regulating SMC migration.

Chemokine receptor–8 (CCR8) mediates human vascular smooth muscle cell chemotaxis and metalloproteinase-2 secretion

AbstractThe response of the arterial vascular wall to injury is characterized by vascular smooth muscle cell (VSMC) migration, a process requiring metalloproteinase production. This migration is induced by cytokines, however the agonists involved are not fully defined. The CC chemokine receptor 8 (CCR8) is expressed on monocytes and T lymphocytes and is the sole receptor for the human CC chemokine 1 (CCL1, I-309) and for the viral chemokine, vCCL1 (viral macrophage inflammatory protein 1 [vMIP-1]). We have reported that CCR8 is expressed on human umbilical vein endothelial cells (HUVECs) and mediates chemotaxis induced by CCL1. The conditioned medium from incubation mixtures of lipoprotein(a) (Lp(a)) and HUVECs (LCM) contained CCL1 and stimulated both monocyte and HUVEC chemotaxis, providing novel mechanisms for the atherogenicity of Lp(a). We now report that CCL1, vCCL1, and LCM stimulate chemotaxis of human VSMCs that is blocked by murine monoclonal antibody against CCR8 and by the G-protein inhibitor pertussis toxin. The effect of anti-CCR8 was specific, as this antibody failed to effect the chemotaxis of VSMCs in response to CCL3 or by platelet-derived growth factor BB (PDGF-BB). VSMCs contained CCR8 mRNA and CCR8 antigen coprecipitated with VSMC membranes. Antibodies against metalloproteinase-2 (MMP-2) inhibited the CCL1-induced chemotaxis of VSMCs, whereas anti–MMP-9 was less effective. CCL1 induced VSMC pro–MMP-2 mRNA and protein secretion. Poxvirus MC148 inhibited the increase in MMP-2 induced by CCL1, documenting that CCR8 was the receptor responsible. In mouse femoral arteries, CCR8 and TCA3 antigen colocalized with VSMCs and were up-regulated after injury. The induction of CCR8 and CCL1/TCA3 under conditions associated with VSMC proliferation and migration raises the possibility that CCR8 may play an important role in vessel wall pathology.