Mouse cytomegalovirus M33 is necessary and sufficient in virus-induced vascular smooth muscle cell migration

Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.     

Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type–Specific Motility

While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX₃C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis.     

Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits Galpha12 and Galpha13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of Galpha12 and Galpha13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominant-negative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through Galpha12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.     

CREG inhibits migration of human vascular smooth muscle cells by mediating IGF-II endocytosis

We previously determined that the cellular repressor of E1A-stimulated genes, (CREG) plays a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). This study aimed to identify the role of CREG in modulating the migration of SMCs. Recombinant virus-mediated CREG expression inhibited the cellular migration of cultured SMCs associated with down-regulated activity of matrix metalloproteinase-9 (MMP-9). In contrast, CREG knockdown via the retroviral transfer of short hairpin RNAs promoted cellular migration. Enzyme-linked immunosorbent assay and endocytosis analysis revealed that CREG knockdown attenuated the internalization and increased secretion of insulin-like growth factor (IGF)-II. Western blot analysis demonstrated that both phosphoinositide 3-kinase (PI3K) and phosphatase Akt were enhanced in CREG knockdown SMCs. Furthermore, the effect of CREG knockdown on SMC migration was abrogated in a dose-dependent manner by the addition of either IGF-II neutralizing antibody or the PI3K inhibitor, LY294002. These results indicate that the CREG knockdown-mediated increase in IGF-II secretion promoted cellular migration in SMCs via the PI3K/Akt signal pathway. Additionally, blockage of IGF-II binding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF2R) by IGF2R antibody or recombinant IGF2R fragment attenuated the endocytosis of IGF-II in cells overexpressing CREG. This indicates that M6P/IGF2R is involved in the regulation of CREG-mediated IGF-II endocytosis. In summary, these data demonstrate for the first time that CREG plays a critical role in the inhibition of SMC migration, as well as maintaining SMCs in a mature phenotype. These results may provide a new therapeutic target for vascular disease associated with neointimal hyperplasia.     

US28 Is a Potent Activator of Phospholipase C during HCMV Infection of Clinically Relevant Target Cells

Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-β in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca⁺⁺ release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca⁺⁺ release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.     

Human cytomegalovirus downregulates expression of receptors for platelet-derived growth factor by smooth muscle cells

Infection by human cytomegalovirus (HCMV) is associated with the development of vascular diseases and may cause severe brain damage in infected fetuses. Platelet-derived growth factor receptors alpha and beta (PDGFR-α and -β) control important cellular processes associated with atherosclerosis and fetal development. In the present investigation, our goal was to determine whether infection by HCMV can influence the expression of PDGFR-α and -β in human smooth muscle cells (SMCs). In connection with HCMV infection in vitro the levels of PDGFR-α and -β at the cell surface and in the total cellular protein of SMCs were reduced in parallel with decreases in the levels of the corresponding mRNAs. These effects were dependent on immediate-early (IE) or early (E) HCMV gene products, since inhibition of late genes did not prevent HCMV from affecting the expression of PDGFR-α and -β. The downregulation of PDGFR caused by HCMV was dose dependent. Furthermore, confocal microscopy revealed that the localization of PDGFR-β was altered in HCMV-infected cells, in which this protein colocalized with proteins associated with endosomes (Rab4 and -5) and lysosomes (Lamp1 and -2), indicating entrance into pathways for protein degradation. Altogether these observations indicate that an IE and/or E HCMV protein(s) downregulates the expression of PDGFR-α and -β in SMCs. This phenomenon may disrupt cellular processes of importance in connection with cellular differentiation, migration, and/or proliferation. These observations may explain why congenital infection with HCMV can cause fetal brain damage.     

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.     

The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis

Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.     

miR-4735-3p regulates phenotypic modulation of vascular smooth muscle cells by targeting HIF-1-mediated autophagy in intracranial aneurysm

Intracranial aneurysm (IA) is recognized as a lethal form of cerebrovascular disease mainly featured with a modulated phenotype of vascular smooth muscle cells (SMCs). It is generally believed that enhanced SMC proliferation and migration capabilities are the main characteristics in this process. In this study, we revealed that microRNA-4735 (miR-4735) participates in phenotypic modulation in a hypoxia-inducible factor-1 (HIF-1)-dependent manner of SMCs. miR-4735 targets the 3′-untranslated region of HIF-1. The downregulated expression of miR-4735 in IA tissues leads to elevated expression of HIF-1, which activates autophagy and promotes autophagy-mediated SMC proliferation and migration. Overexpression of miR-4735 suppressed HIF-1 expression and HIF-1-mediated autophagy, which led to impaired SMC proliferation and migration abilities. Forced expression of HIF-1 in miR-4735-overexpressed SMCs rescued the impaired SMC proliferation and migration abilities. In conclusion, miR-4735 plays an important role in phenotypic modulation in IA by regulating autophagy-promoted SMC proliferation and migration.     

Functional properties of smooth muscle cells in ascending aortic aneurysm

Thoracic aortic aneurysm (TAA) develops as a result of complex sequential events that dynamically alter the structure and composition of the aortic vascular extracellular matrix (ECM). The main cellular elements that alter the composition of aortic wall are smooth muscle cells (SMCs). The purpose of the present work was to study alterations of smooth muscle cell functions derived from the patients with TAA and from healthy donors. Since it is believed that TAA associates with bicuspid aortic valve (BAV) and with tricuspid aortic valve (TAV) differed in their pathogenesis, we have compared SMCs and tissue samples from BAV and TAV patients and healthy donors. The comparison was done by several parameters: SMC growth, migration and apoptotic dynamics, metalloproteinase MMP2 and MMP9 activity (zymography), and elastin, collagen, and fibrillin content (Western blot) in both tissue samples and cultured SMCs. Proliferation of BAV and TAV SMCs was decreased and migration ability in scratch tests was increased in TAV-derived SMCs compared to donor cells. BAV-cells migration ability was not changed compared to donor SMCs. Elastin content was decreased in TAA SMCs, whereas the content of fibrillin and collagen was not altered. At the same time, the elastin and collagen protein level was significantly higher in tissue samples of TAA patients than in donorderived samples. SMC proliferation and migration is differently affected in TAV and BAV-associated TAA that supports the idea on different nature of these two TAA groups. Our data also show that SMC functional properties are altered in TAA patients and these alterations could play a significant role in the disease pathogenesis.     

Human cytomegalovirus latent infection of myeloid cells directs monocyte migration by up-regulating monocyte chemotactic protein-1

Following primary infection, human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells from which it reactivates to cause serious disease in immunosuppressed patients such as allograft recipients. HCMV is a common cause of disease in newborns and transplant patients and has also been linked with vascular diseases such as primary and post-transplant arteriosclerosis. A major factor in the pathogenesis of vascular disease is the CC chemokine MCP-1. In this study, we demonstrate that granulocyte macrophage progenitors (GMPs) latently infected with HCMV significantly increased expression of MCP-1 and that this phenotype was dependent on infection with viable virus. Inhibitors of a subset of G(alpha) proteins and PI3K inhibited the up-regulation of MCP-1 in latently infected cultures, suggesting that the mechanism underlying this phenotype involves signaling through a G-protein coupled receptor. In GMPs infected with the low passage viral strain Toledo, up-regulated MCP-1 was restricted to a subset of myeloid progenitor cells expressing CD33, HLA-DR, and CD14 but not CD1a, CD15, or CD16, and the increase in MCP-1 was sufficient to enhance migration of CD14(+) monocytes to latently infected cells. Latent HCMV-mediated up-regulation of MCP-1 provides a mechanism by which HCMV may contribute to vascular disease during the latent phase of infection or facilitate dissemination of virus upon reactivation from latency.     

Increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the alpha/beta hydrolase domain containing 2 gene

Multiple steps, including the migration of vascular smooth muscle cells (SMCs), are involved in the pathogenesis of atherosclerosis. To discover genes which are involved in these steps, we screened mutant mouse lines established by the exchangeable gene trap method utilizing X-gal staining during their embryonic development. One of these lines showed strong reporter gene expression in the vitelline vessels of yolk sacs at embryonic day (E) 12.5. The trap vector was inserted into the fifth intron of alpha/beta hydrolase domain containing 2 (Abhd2) gene which was shown to be expressed in vascular and non-vascular SMCs of adult mice. Although homozygous mutant mice were apparently normal, enhanced SMC migration in the explants SMCs culture and marked intimal hyperplasia after cuff placement were observed in homozygous mice in comparison with wild-type mice. Our results show that Abhd2 is involved in SMC migration and neointimal thickening on vascular SMCs.     

Pattern formation of vascular smooth muscle cells subject to nonuniform fluid shear stress: mediation by gradient of cell density

Smooth muscle cells (SMCs) are organized in various patterns in blood vessels. Whereas straight blood vessels mainly contain circumferentially aligned SMCs, curved blood vessels are composed of axially aligned SMCs in regions with vortex blood flow. The vortex flow-dependent feature of SMC alignment suggests a role for nonuniform fluid shear stress in regulating the pattern formation of SMCs. Here, we demonstrate that, in experimental models with vascular polymer implants designed for the observation of neointima formation and SMC migration under defined fluid shear stress, nonuniform shear stress possibly plays a role in regulating the direction of SMC migration and alignment in the neointima of the vascular implant. It was found that fluid shear stress inhibited cell growth, and the presence of nonuniform shear stress influenced the distribution of total cell density and induced the formation of cell density gradients, which in turn directed SMC migration and alignment. In contrast, uniform fluid shear stress in a control model influenced neither the distribution of total cell density nor the direction of SMC migration and alignment. In both the uniform and nonuniform shear models, the gradient of total cell density was consistent with the alignment of SMCs. These observations suggest that nonuniform shear stress may regulate the pattern formation of SMCs, possibly via mediating the gradient of cell density in the neointima of vascular polymer implants.     

Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to protein-tyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK.Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK.Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2.FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH2-terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration.     

Anti-HCMV and KSHV effect of a trapping ligand antagonist for herpesvirus-encoded GPCR

We have found and synthesized a trapping ligand peptide H22-LP (the conservative sequence is NAHCALL) from a random phage library according to the broad-spectrum trapping receptor H22, which derived from the residue 14-35 near the N-terminal region of receptor US28 on HCMV. In this study, we will evaluate its potential as an efficient antagonist of US28 and the anti-virus activity, acting as a broad spectrum chemokine receptors antagonist. Stable expression of US28 and ORF74 in NIH/3T3 cells were successfully constructed in vitro. Flow cytomety was used to determine the concentration of Ca2+ induced by H22-LP, and the binding of H22-LP and US28 was confirmed by enzyme-linked immunosorbent assay (ELISA). Antivirus activity of H22-LP on HCMV and KSHV was evaluated by anti-virus experiments. Our data suggest that H22-LP is an effectual antagonist of receptor US28 of HCMV and ORF74 of KSHV in the transfection assay, and it has potential to inhibit infection of HCMV and KSHV. These results provide support for the development of anti-virus strategies based on targeted inhibiting the infection of herpesvirus.     

Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells

The focal adhesion (FAK) non-receptor protein-tyrosine kinase (PTK) links both extracellular matrix/integrin and growth factor stimulation to intracellular signals promoting cell migration. Here we show that both transient and stable overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) inhibits serum and platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (SMC) migration in wound healing and in vitro Boyden Chamber chemotaxis assays, respectively. Expression of FRNK, but not a point mutant of FRNK (FRNK L1034S), disrupted the formation of a complex containing both FAK and the activated PDGF-beta receptor and resulted in reduced tyrosine phosphorylation of endogenous FAK at the Tyr-397 binding site for Src family PTKs. As demonstrated using FAK-deficient and FAK-reconstituted fibroblasts, FAK positively contributed to PDGF-BB-stimulated ERK2/MAP kinase activity, and in SMCs, ERK2/MAP kinase activity was required for PDGF-BB-stimulated chemotaxis. Stable expression of FRNK but not FRNK L1034S expression in SMCs lowered the extent and duration of stimulated ERK2/MAP kinase activation at low but not at high PDGF-BB concentrations. Importantly, stable expression of FRNK in SMCs did not affect SMC morphology or proliferation in culture. Because the increased migration of vascular SMCs in response to extracellular matrix proteins and growth factors contributes to neointima formation, our results show that FAK inhibition by FRNK expression may provide a novel approach to regulate abnormal vascular SMC migration in vivo.     

The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival

Human cytomegalovirus (HCMV) is a widespread pathogen that can lay dormant in healthy individuals and establish lifelong latent infection. This successful co-existence is facilitated by a number of viral gene products that manipulate host cellular functions and immune responses. Among these immunomodulatory genes are four G-protein coupled receptors (GPCRs) encoded by HCMV, designated US27, US28, UL33, and UL78. Studies have shown the US28 gene product to be a functional chemokine receptor that signals both constitutively and in a ligand-dependent manner, resulting in a wide range of cellular effects. In previous work, we have found that US27 expression results in at least two biological effects: enhanced CXCR4 signaling and increased in cellular proliferation in HEK293 cells. Here, we examined the involvement of two protein domains, the DRY box and the C-terminal intracellular domain (CTD) of US27, in mediating both cell proliferation and survival. While both domains were required for a proliferative effect, loss of either domain only moderately impacted cell survival, suggesting that US27 may interact with cell survival pathways through protein regions other than the DRY box and CTD. Quantitative RT-PCR arrays were used to profile changes in cellular gene expression in the HEK293-US27 cell line, and down-regulation of cell cycle regulators CDKN1A/p21/CIP1 (cyclin dependent kinase inhibitor 1A) and SESN (Sestrin2 or Hi95) was observed. These results indicate that increased cell proliferation due to US27 may be linked to suppression of negative growth regulators, and that these interactions require the DRY box and CTD.     

Xenobiotic pregnane X receptor promotes neointimal formation in balloon-injured rat carotid arteries

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Recent studies demonstrated that PXR was also expressed in the vasculature and protected the vessels from endogenous and exogenous insults, thus representing a novel gatekeeper in vascular defense. In this study, we examined the potential function of PXR in the neointimal formation following vascular injury. In the rat carotid artery after balloon injury, overexpression of a constitutively active PXR increased the intima-to-media ratio in the injured region. PXR increased cell proliferation and migration in cultured rat aortic smooth muscle cells (SMCs) by inducing the expressions of cyclins (cyclin A, D1, and E) and cyclin-dependent kinase 2. In addition, PXR increased the phosphorylation and activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Inactivation of ERK1/2 and p38 MAPK pathways using selective inhibitors (U0126 and SB203580) abrogated PXR-induced SMC proliferation and migration. Furthermore, cigarette smoke particles (CSP) activated PXR in SMCs. Knockdown of PXR by small interfering RNA suppressed the cell proliferation, migration, and activation of the MAPK pathways by CSP. These findings suggested a novel role for PXR in promoting SMC proliferation and migration, and neointimal hyperplasia. Therefore, PXR may be a potential therapeutic target for vascular disease related to xenobiotics such as cigarette smoking and other environmental pollutants.     

Study of gastric fluid induced cytokine and chemokine expression in airway smooth muscle cells and airway remodeling

Asthma is a chronic airway inflammatory disease. Chronic aspiration by gastric fluid in gastroesophageal reflux disease (GERD) is considered a primary inflammatory factor exacerbating or predisposing patients to asthma. Airway smooth muscle cells (SMCs) are considered an important component in airway remodeling. To investigate the role of gastric fluid in airway SMC inflammation and airway remodeling, we examined gastric fluid-induced cytokine and chemokine profiles, airway SMC migration and matrix metalloproteinase expression in rat primary rat airway SMCs. The T helper cell type 2 (Th2) cytokines interleukin 4, interleukin 6 and tumor necrosis factor 2 (TNF-α) and the chemokines, lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), cytokine-induced neutrophil chemoattractant 2 (CINC-2), CINC-3, fractalkine, ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor were induced by gastric fluid in primary cultured rat airway SMCs. Migration of rat airway SMCs was enhanced by gastric fluid and conditioned medium. The migration of rat airway SMCs enhanced by gastric fluid was associated with actin polymerization and activation of focal adhesion kinase. Matrix metalloproteinase 2 expressions in airway SMCs was enhanced by gastric fluid and conditioned medium. The results suggest potential mechanisms by which gastric fluid aspiration might influence SMC-mediated airway remodeling.