The Aryl Hydrocarbon Receptor and Its Ligands Inhibit Myofibroblast Formation and Activation: Implications for Thyroid Eye Disease.

Thyroid eye disease (TED) is a degenerative disease that manifests with detrimental tissue remodeling, myofibroblast accumulation, and scarring in the orbit of affected individuals. Currently, there are no effective therapies for TED that target or prevent the excessive tissue remodeling caused by myofibroblast formation and activation. The canonical cytokine that induces myofibroblast formation is transforming growth factor (TGF)-ß. The TGF-ß signaling pathway is influenced by aryl hydrocarbon receptor (AHR) signaling pathways. We hypothesized that AHR agonists can prevent myofibroblast formation in fibroblasts from patients with TED, and thus AHR ligands are potential therapeutics for the disease. Orbital fibroblasts explanted from patients with TED were treated with TGF-ß to induce myofibroblast formation, contraction, and proliferation. We found that AHR ligands prevent TGF-ß-dependent myofibroblast formation, and this ability is dependent on AHR expression. The AHR and AHR ligands block profibrotic Wnt signaling by inhibiting the phosphorylation of GSK3ß to prevent myofibroblast formation. These results provide new insight into the molecular pathways underlying orbital scarring in TED. These novel studies highlight the potential of the AHR and AHR ligands as future therapeutic options for eye diseases and possibly also for other scarring conditions.

Salinomycin and other polyether ionophores are a new class of antiscarring agent.

Although scarring is a component of wound healing, excessive scar formation is a debilitating condition that results in pain, loss of tissue function, and even death. Many tissues, including the lungs, heart, skin, and eyes, can develop excessive scar tissue as a result of tissue injury, chronic inflammation, or autoimmune disease. Unfortunately, there are few, if any, effective treatments to prevent excess scarring, and new treatment strategies are needed. Using HEK293FT cells stably transfected with a TGFß-dependent luciferase reporter, we performed a small molecule screen to identify novel compounds with antiscarring activity. We discovered that the polyether ionophore salinomycin potently inhibited the formation of scar-forming myofibroblasts. Salinomycin (250 nm) blocked TGFß-dependent expression of the cardinal myofibroblast products α smooth muscle actin, calponin, and collagen in primary human fibroblasts without causing cell death. Salinomycin blocked phosphorylation and activation of TAK1 and p38, two proteins fundamentally involved in signaling myofibroblast and scar formation. Expression of constitutively active mitogen activated kinase kinase 6, which activates p38 MAPK, attenuated the ability of salinomycin to block myofibroblast formation, demonstrating that salinomycin targets the p38 kinase pathway to disrupt TGFß signaling. These data identify salinomycin and other polyether ionophores as novel potential antiscarring therapeutics.

Fibrinolytic activity of endothelial cells from different venous beds.

BACKGROUND: Little is known about the molecular biology of endothelial cells from different venous vascular beds. As a result, our treatment of deep vein thrombosis and pulmonary artery embolism remain identical. As an initial step in understanding venous thromboembolic disease in the trauma and surgical patients, this study sought to investigate the balance between coagulation and fibrinolysis in the pulmonary and deep venous vascular beds and how trauma might influence this balance. MATERIALS AND METHODS: Confluent human iliac vein endothelial cells (HIVECs) and human pulmonary artery endothelial cells (HPAECs), were cultured in the absence or presence of tumor necrosis factor (TNFα; 10 ng/mL) for 24 h. The expression of mediators of coagulation and fibrinolysis were determined by Western blot analysis, and plasminogen activator activity was determined by a fibrin clot degradation assay. RESULTS: After TNFα stimulation, there was decreased expression of endothelial protein C receptor and thrombomodulin in both HIVECs and HPAECs. TNFα stimulation increased urokinase plasminogen activator expression in both HIVECs and HPAECs. There was an increase in the expression of tissue plasminogen activator and plasminogen activator inhibitor-1 in response to TNFα in HPAECs, but not in HIVECs. There was significantly greater clot degradation in the presence of both the conditioned media and cell extracts from HIVECs, when compared with HPAECs. CONCLUSIONS: HPAECs and HIVECs react differently in terms of fibrinolytic potential when challenged with a cytokine associated with inflammation. These findings suggest that endothelial cells from distinct venous vascular beds may differentially regulate the fibrinolytic pathway.

Ascl3 knockout and cell ablation models reveal complexity of salivary gland maintenance and regeneration.

Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3(EGFP-Cre/+) knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.

More than Meets the Eye: The Aryl Hydrocarbon Receptor is an Environmental Sensor, Physiological Regulator and a Therapeutic Target in Ocular Disease.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor originally identified as an environmental sensor of xenobiotic chemicals. However, studies have revealed that the AHR regulates crucial aspects of cell growth and metabolism, development and the immune system. The importance of the AHR and AHR signaling in eye development, toxicology and disease is now being uncovered. The AHR is expressed in many ocular tissues including the retina, choroid, cornea and the orbit. A significant role for the AHR in age-related macular degeneration (AMD), autoimmune uveitis, and other ocular diseases has been identified. Ligands for the AHR are structurally diverse organic molecules from exogenous and endogenous sources. Natural AHR ligands include metabolites of tryptophan and byproducts of the microbiome. Xenobiotic AHR ligands include persistent environmental pollutants such as dioxins, benzo (a) pyrene [B (a) P] and polychlorinated biphenyls (PCBs). Pharmaceutical agents including the proton pump inhibitors, esomeprazole and lansoprazole, and the immunosuppressive drug, leflunomide, activate the AHR. In this review, we highlight the role of the AHR in the eye and discuss how AHR signaling is involved in responding to endogenous and environmental stimuli. We also present the emerging concept that the AHR is a promising therapeutic target for eye disease.

Thinking inside the box: Current insights into targeting orbital tissue remodeling and inflammation in thyroid eye disease.

Thyroid eye disease (TED) is an autoimmune disorder that manifests in the orbit. In TED, the connective tissue behind the eye becomes inflamed and remodels with increased fat accumulation and/or increased muscle and scar tissue. As orbital tissue expands, patients develop edema, exophthalmos, diplopia, and optic neuropathy. In severe cases vision loss may occur secondary to corneal scarring from exposure or optic nerve compression. Currently there is no cure for TED, and treatments are limited. A major breakthrough in TED therapy occurred with the FDA approval of teprotumumab, a monoclonal insulin-like growth factor 1 receptor (IGF1R) blocking antibody. Yet, teprotumumab therapy has limitations, including cost, infusion method of drug delivery, variable response, and relapse. We describe approaches to target orbital fibroblasts and the complex pathophysiology that underlies tissue remodeling and inflammation driving TED. Further advances in the elucidation of the mechanisms of TED may lead to prophylaxis based upon early biomarkers as well as lead to more convenient, less expensive therapies.

TNF-α and NF-κB signaling play a critical role in cigarette smoke-induced epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted.

MicroRNA-130a Is Elevated in Thyroid Eye Disease and Increases Lipid Accumulation in Fibroblasts Through the Suppression of AMPK.

Purpose: Thyroid eye disease (TED) is a condition that causes the tissue behind the eye to become inflamed and can result in excessive fatty tissue accumulation in the orbit. Two subpopulations of fibroblasts reside in the orbit: those that highly express Thy1 (Thy1+) and those with little or no Thy1 (Thy1-). Thy1- orbital fibroblasts (OFs) are more prone to lipid accumulation than Thy1+ OFs. The purpose of this study was to investigate the mechanisms whereby Thy1- OFs more readily accumulate lipid. Methods: We screened Thy1+ and Thy1- OFs for differences in microRNA (miRNA) expression. The effects of increasing miR-130a levels in OFs was investigated by measuring lipid accumulation and visualizing lipid deposits. To determine if adenosine monophosphate-activated protein kinase (AMPK) is important for lipid accumulation, we performed small interfering RNA (siRNA)-mediated knockdown of AMPKß1. We measured AMPK expression and activity using immunoblotting for AMPK and AMPK target proteins. Results: We determined that miR-130a was upregulated in Thy1- OFs and that miR-130a targets two subunits of AMPK. Increasing miR-130a levels enhanced lipid accumulation and reduced expression of AMPKα and AMPKß in OFs. Depletion of AMPK also increased lipid accumulation. Activation of AMPK using AICAR attenuated lipid accumulation and increased phosphorylation of acetyl-CoA carboxylase (ACC) in OFs. Conclusions: These data suggest that when Thy1- OFs accumulate in TED, miR-130a levels increase, leading to a decrease in AMPK activity. Decreased AMPK activity promotes lipid accumulation in TED OFs, leading to excessive fatty tissue accumulation in the orbit.

Neonatal hyperoxia impairs adipogenesis of bone marrow-derived mesenchymal stem cells and fat accumulation in adult mice.

Preterm birth is a risk factor for growth failure and development of respiratory disease in children and young adults. Their early exposure to oxygen may contribute to lung disease because adult mice exposed to hyperoxia as neonates display reduced lung function, changes in the host response to respiratory viral infections, and develop pulmonary hypertension and heart failure that shortens their lifespan. Here, we provide new evidence that neonatal hyperoxia also impairs growth by inhibiting fat accumulation. Failure to accumulate fat may reflect a systemic defect in adipogenic potential of stem cells because bone marrow-derived mesenchymal cells (BMSCs) isolated from the mice grew slower and were more oxidized compared to controls. They also displayed reduced capacity to accumulate lipid and differentiate into adipocytes. BMSCs from adult mice exposed to neonatal hyperoxia express lower levels of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that drives adipocyte differentiation. The defect in adipogenesis was rescued by expressing PPARγ in these cells. These findings reveal early life exposure to high levels of oxygen may suppresses fat accumulation and impair adipogenic differentiation upstream of PPARγ signaling, thus potentially contributing to growth failure seen in people born preterm.

The aryl hydrocarbon receptor pathway controls matrix metalloproteinase-1 and collagen levels in human orbital fibroblasts.

Thyroid eye disease (TED) affects 25-50% of patients with Graves' Disease. In TED, collagen accumulation leads to an expansion of the extracellular matrix (ECM) which causes destructive tissue remodeling. The purpose of this study was to investigate the therapeutic potential of activating the aryl hydrocarbon receptor (AHR) to limit ECM accumulation in vitro. The ability of AHR to control expression of matrix metalloproteinase-1 (MMP1) was analyzed. MMP1 degrades collagen to prevent excessive ECM. Human orbital fibroblasts (OFs) were treated with the pro-scarring cytokine, transforming growth factor beta (TGFß) to induce collagen production. The AHR ligand, 6-formylindolo[3,2b]carbazole (FICZ) was used to activate the AHR pathway in OFs. MMP1 protein and mRNA levels were analyzed by immunosorbent assay, Western blotting and quantitative PCR. MMP1 activity was detected using collagen zymography. AHR and its transcriptional binding partner, ARNT were depleted using siRNA to determine their role in activating expression of MMP1. FICZ induced MMP1 mRNA, protein expression and activity. MMP1 expression led to a reduction in collagen 1A1 levels. Furthermore, FICZ-induced MMP1 expression required both AHR and ARNT, demonstrating that the AHR-ARNT transcriptional complex is necessary for expression of MMP1 in OFs. These data show that activation of the AHR by FICZ increases MMP1 expression while leading to a decrease in collagen levels. Taken together, these studies suggest that AHR activation could be a promising target to block excessive collagen accumulation and destructive tissue remodeling that occurs in fibrotic diseases such as TED.

Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands.

Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism.

BACKGROUND: Cell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF. OBJECTIVE: To determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration. METHODS: Human coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, betaARK(CT) were also used. RESULTS: ATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gbetagamma inhibitor betaARK(CT) inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect. CONCLUSION: ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.

Patterns of gelatinase activation induced by injury in the murine femoral artery.

BACKGROUND: Intimal hyperplasia remains the principal lesion in the development of restenosis after vessel wall injury. Modulation of the extracellular matrix by proteases is a pivotal component of the response to injury. The aim of this study is to characterize the changes in gelatinase (MMP-2/TIMP-2 and MMP-9/TIMP-1) systems in a murine model. METHODS: The murine femoral wire injury model was used in which a microwire is passed through a branch of the femoral and used to denude the common femoral artery. Pluronic gel was used to apply a proteass inhibitor (GM6001) to the exterior of the vessels. Specimens were perfusion-fixed and sections were stained with hematoxylin and eosin and Movat's stain such that morphometry could be performed by using an ImagePro system. Additional specimens of femoral artery were also harvested and snap frozen for Western blotting and zymography to allow for the study of gelatinase expression and activation. Contralateral vessels were used as controls. RESULTS: MMP-2 activity increased significantly at day 1, peaked again at day 7, and then showed a continual decline in activity to day 56. MMP-9 activity peaked early at day 3 and declined thereafter. Protein expression for both MMP-2 and MMP-9 increased significantly after injury and both were maximal at day 14. There was an initial decrease in TIMP-1 and TIMP-2 expression and activity after injury until day 5. Both expression and activation gradually increased thereafter to level out by day 21 and correlated well with the early increases in MMP-2 and MMP-9 activity and their subsequent decline. Local application of protease inhibitor (GM6001) within a pluronic gel decreased cell proliferation, and at 14 d there was a decrease in intimal hyperplasia. CONCLUSIONS: These data demonstrate that femoral wire injury in the mouse is associated with a time-dependent increase in gelatinase activity. Cell proliferation is associated with increased MMP-2/MMP-9 activity and decreased TIMP-2/TIMP-1 activity, whereas migration is associated with increased in MMP-2 activity. Modulation of proteases and their inhibitors control the vessels' response to injury.

TSHR Signaling Stimulates Proliferation Through PI3K/Akt and Induction of miR-146a and miR-155 in Thyroid Eye Disease Orbital Fibroblasts.

Purpose: To investigate the molecular pathways that drive thyroid stimulating hormone receptor (TSHR)-induced cellular proliferation in orbital fibroblasts (OFs) from thyroid eye disease (TED) patients. Methods: Orbital fibroblasts from TED and non-TED patients were treated with TSH and changes in gene expression and proliferation were measured. To determine the role of TSHR, TSHR-specific siRNA was used to deplete TSHR levels. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation. PI3K/Akt activation was analyzed by Western blot. The PI3K inhibitor LY294002 was used to investigate PI3K/Akt signaling in OF proliferation. Expression of TSHR, inflammatory cytokines, proliferation related genes and miR-146a and miR-155 were measured by qPCR. Results: Orbital fibroblasts from TED patients proliferate significantly more than non-TED OFs in response to TSH. TSH-induced proliferation was dependent upon TSHR expression and required the PI3K/Akt signaling cascade. TSHR activation stimulated miR-146a and miR-155 expression. TED OFs produced significantly more miR-146a and miR-155 than non-TED OFs. MiR-146a and miR-155 targets, ZNRF3 and PTEN, which both limit cell proliferation, were decreased in TSH treated OFs. Conclusions: These data reveal that TSHR signaling in TED OFs stimulates proliferation directly through PI3K/Akt signaling and indirectly through induction of miR-146a and miR-155. MiR-146a and miR-155 enhance TED OF proliferation by reducing expression of target genes that normally block cell proliferation. TSHR-dependent expression of miR-146a and miR-155 may explain part of the fibroproliferative pathology observed in TED.

Proton pump inhibitors attenuate myofibroblast formation associated with thyroid eye disease through the aryl hydrocarbon receptor.

Thyroid eye disease (TED) can lead to scar formation and tissue remodeling in the orbital space. In severe cases, the scarring process leads to sight-threatening pathophysiology. There is no known effective way to prevent scar formation in TED patients, or to reverse scarring once it occurs. In this study, we show that the proton pump inhibitors (PPIs), esomeprazole and lansoprazole, can prevent transforming growth factor beta (TGFß)-mediated differentiation of TED orbital fibroblasts to myofibroblasts, a critical step in scar formation. Both PPIs prevent TGFß-induced increases in alpha-smooth muscle actin (αSMA), calponin, and collagen production and reduce TED orbital fibroblast cell proliferation and migration. Esomeprazole and lansoprazole exert these effects through an aryl hydrocarbon receptor (AHR)-dependent pathway that includes reducing ß-catenin/Wnt signaling. We conclude that PPIs are potentially useful therapies for preventing or treating TED by reducing the myofibroblast accumulation that occurs in the disease.

The polyether ionophore salinomycin targets multiple cellular pathways to block proliferative vitreoretinopathy pathology.

Proliferative vitreoretinopathy (PVR) is characterized by membranes that form in the vitreous cavity and on both surfaces of the retina, which results in the formation of tractional membranes that can cause retinal detachment and intrinsic fibrosis of the retina, leading to retina foreshortening. Currently, there are no pharmacologic therapies that are effective in inhibiting or preventing PVR formation. One of the key aspects of PVR pathogenesis is retinal pigment epithelial (RPE) cell epithelial mesenchymal transition (EMT). Here we show that the polyether ionophore compound salinomycin (SNC) effectively inhibits TGFß-induced EMT of RPE cells. SNC blocks the activation of TGFß-induced downstream targets alpha smooth muscle actin (αSMA) and collagen 1 (Col1A1). Additionally, SNC inhibits TGFß-induced RPE cell migration and contraction. We show that SNC functions to inhibit RPE EMT by targeting both the pTAK1/p38 and Smad2 signaling pathways upon TGFß stimulation. Additionally, SNC is able to inhibit αSMA and Col1A1 expression in RPE cells that have already undergone TGFß-induced EMT. Together, these results suggest that SNC could be an effective therapeutic compound in both the prevention and treatment of PVR.

Plasminogen activator system and vascular disease.

Vascular diseases, such as atherosclerosis, thromboembolic disorders and stroke, in addition to surgical procedures such as restenosis, all share the plasminogen activator system as a central component in the pathogenesis of vascular injury. Since the development of plasminogen deficient mice our knowledge of the effects of this proteolytic system in cardiovascular disease has vastly increased. The plasminogen activator system plays a key role in vascular homeostasis and constitutes a critical response mechanism to cardiovascular injury. The central components of the PA system are the proteolytic activators, urokinase-plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), plasminogen (plg) and its degradation product, plasmin, together with the major inhibitors of this system, plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2). An extensive network of additional proteases, inhibitors, receptors and modulators directly associate and are influenced by the PA system, the largest group being the Matrix Metalloproteinases (MMPs) and their respective inhibitors the Tissue inhibitors of MMPs (TIMPS).

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway.

BACKGROUND: Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). METHODS: Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. CONCLUSIONS: Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.

Mechanisms of sphingosine-1-phosphate-induced akt-dependent smooth muscle cell migration.

BACKGROUND: Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets that stimulates migration of vascular smooth muscle cells (VSMC) in vitro. S-1-P will activate akt, which can regulate multiple cellular functions including cell migration. Akt activation is downstream of phosphatidylinositol 3'-kinase (PI3-K) and phosphoinositide-dependent protein kinase-1 (PDK1). The objective of this study was to examine the regulation of akt signaling during smooth muscle cell (SMC) migration in response to S-1-P. METHODS: Murine arterial SMC were cultured in vitro. Linear wound and microchemotaxis assays of migration in Boyden chambers were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Assays were performed for PI3-K, PDK1, akt, and GSK3beta in the presence of various inhibitors and after transfection with the G beta gamma inhibitor beta ARK(CT). RESULTS: S-1-P induced time-dependent PI3-K, PDK1, and akt activation. The migratory responses in both assays to S-1-P were blocked by aktI. Activation of akt and dephosphorylation of its downstream kinase, GSK3 beta, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly decreased activation of both PI3-K and akt. Inhibition of G beta gamma inhibited akt activation through a decrease in activation of both PI3-K and PDK1. Although inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited activation of both PI3K and akt. PDK1 responses were unchanged by inhibition of GTPases. Inhibiting the generation of reactive oxygen species with N-acetylcysteine and of epidermal growth factor receptor with AG1478 inhibited PDK1 activation in response to S-1-P. CONCLUSION: S-1-P-mediated migration is akt-dependent. S-1-P-mediated akt phosphorylation is controlled by a G beta gamma-dependent PI3-K activation, which requires the GTPase rho and G beta gamma. PDK1 activation requires G beta gamma-dependent generation of reactive oxygen species and epidermal growth factor receptor activation.

Insulin-induced epidermal growth factor activation in vascular smooth muscle cells is ADAM-dependent.

BACKGROUND: With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. This study examines the role of insulin actions on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor-linked tyrosine kinases and is key to many of their responses. OBJECTIVE: To determine the pathway of EGFR transactivation by insulin in human vascular smooth muscle cells (VSMC). METHODS: VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the A disintegrin and metalloproteinase domain (ADAM) inhibitors tumor necrosis factor-alpha protease inhibitor (TAPI)-0 and TAPI-1, heparin-binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. RESULTS: Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration-dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. CONCLUSION: Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. This is the first description of cross-talk via ADAM between insulin and EGFR in VSMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target.