Timothy E. O'Toole

The inner world of cell adhesion: integrin cytoplasmic domains

Many of the interactions between cells and their environment are mediated by the integrin family of heterodimeric transmembrane receptors. The past decade has been a broad-based effort to decipher the rules by which integrins function. Integrins bind both intracellular and extracellular ligands and thus transfer signals across the membrane in both directions. The cytoplasmic domains of these receptors play a key role in this bidirectional flow of information and in the formation of direct physical linkages between protein structures on the inside and outside of the cell.

Mutation of the Cytoplasmic Domain of the Integrin Subunit DIFFERENTIAL EFFECTS ON CELL SPREADING, RECRUITMENT TO ADHESION PLAQUES, ENDOCYTOSIS, AND PHAGOCYTOSIS

The cytoplasmic domain of the β subunit of the αβ3integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the β3(ITYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ilepartially preserved these functions. We further found that substitution of Tyrwith Ala also abolished αβ3-mediated cell spreading. The effects of these and other mutations on additional functions of αβ3were also studied. Progressive truncations of β3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of αβ3to preestablished adhesion plaques and a gradual decrease in the ability of αβ3to mediate internalization of fibrinogen-coated particles. The Tyr Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr, and at a conserved area close to the transmembrane domain of β3, decreased integrin recruitment to preestablished adhesion plaques but allowed αβ3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of β3did not affect the constitutive endocytosis of αβ3.

Involvement of tumor cell integrin αvβ3 in hematogenous metastasis of human melanoma cells

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin αvβ3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin αvβ3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either αvβ3, αIIbβ3 or no β3 integrin, we found that only αvβ3 strongly supported metastasis. Inhibition of tumor cell αvβ3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow αvβ3, but not αIIbβ3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing αvβ3, but not those expressing αIIbβ3 or no β3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between αvβ3 expression and protease processing. Furthermore, αvβ3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin αvβ3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin αIIbβ3 cannot substitute for αvβ3 in this respect.

Episodic Exposure to Fine Particulate Air Pollution Decreases Circulating Levels of Endothelial Progenitor Cells

Rationale: Acute and chronic exposures to airborne particulate matter (PM) have been linked in epidemiological studies to a wide spectrum of cardiovascular disorders that are characterized by a dysfunctional endothelium. The pathophysiological mechanisms underlying these associations are unclear. Objective: To examine whether exposure to fine PM with an aerodynamic diameter of <2.5 &mgr;m (PM2.5) affects the circulating levels of endothelial progenitor cell (EPC) populations, systemic inflammation and coagulation. Methods and Results: Phenotypically distinct EPC populations were quantified by flow cytometry in young (18 to 25 years) adult humans exposed to episodic increases in PM2.5 along the Wasatch Mountain Front in Utah. In addition, Sca-1+/Flk-1+ cells were measured in the peripheral blood of mice exposed to concentrated particles from ambient air in Louisville, Ky. In both studies, PM exposure was negatively correlated with circulating EPC levels. In humans, statistically significant associations between PM2.5 exposure and the plasma levels of platelet–monocyte aggregates, high-density lipoprotein, and nonalbumin protein were also observed. Episodic increases in PM2.5 did not change plasma levels of C-reactive protein, interleukin-1&bgr;, interleukin-6, fibrinogen, or serum amyloid A. Conclusions: Episodic exposure to PM2.5 induces reversible vascular injury, reflected in part by depletion of circulating EPC levels, and increases in platelet activation and the plasma level of high-density lipoprotein. These changes were also accompanied by an increase in nonalbumin protein and may be related to mechanisms by which exposure to particulate air pollution increases the risk of cardiovascular disease and adverse cardiovascular events.

Reductive Metabolism of AGE Precursors: A Metabolic Route for Preventing AGE Accumulation in Cardiovascular Tissue

OBJECTIVE To examine the role of aldo-keto reductases (AKRs) in the cardiovascular metabolism of the precursors of advanced glycation end products (AGEs). RESEARCH DESIGN AND METHODS Steady-state kinetic parameters of AKRs with AGE precursors were determined using recombinant proteins expressed in bacteria. Metabolism of methylglyoxal and AGE accumulation were studied in human umbilical vein endothelial cells (HUVECs) and C57 wild-type, akr1b3 (aldose reductase)-null, cardiospecific-akr1b4 (rat aldose reductase), and akr1b8 (FR-1)-transgenic mice. AGE accumulation and atherosclerotic lesions were studied 12 weeks after streptozotocin treatment of C57, akr1b3-null, and apoE- and akr1b3-apoE–null mice. RESULTS Higher levels of AGEs were generated in the cytosol than at the external surface of HUVECs cultured in high glucose, indicating that intracellular metabolism may be an important regulator of AGE accumulation and toxicity. In vitro, AKR 1A and 1B catalyzed the reduction of AGE precursors, whereas AKR1C, AKR6, and AKR7 were relatively ineffective. Highest catalytic efficiency was observed with AKR1B1. Acetol formation in methylglyoxal-treated HUVECs was prevented by the aldose reductase inhibitor sorbinil. Acetol was generated in hearts perfused with methylglyoxal, and its formation was increased in akr1b4- or akr1b8-transgenic mice. Reduction of AGE precursors was diminished in hearts from akr1b3-null mice. Diabetic akr1b3-null mice accumulated more AGEs in the plasma and the heart than wild-type mice, and deletion of akr1b3 increased AGE accumulation and atherosclerotic lesion formation in apoE-null mice. CONCLUSIONS Aldose reductase–catalyzed reduction is an important pathway in the endothelial and cardiac metabolism of AGE precursors, and it prevents AGE accumulation and atherosclerotic lesion formation.

Lipid Peroxidation Product 4-Hydroxy-trans-2-nonenal Causes Endothelial Activation by Inducing Endoplasmic Reticulum Stress

Background: Oxidized lipids cause endothelial activation. Results: Endothelial activation by the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, was associated with ER stress and was prevented by chaperones of protein folding. Conclusion: ER stress regulates endothelial activation by oxidized lipids. Significance: Vascular inflammation caused by oxidized lipids could be attenuated by decreasing ER stress. Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.

Environmental risk factors for heart disease.

In this review, we discuss current evidence linking environmental pollutants to cardiovascular disease (CVD). Extensive evidence indicates that environmental factors contribute to CVD risk, incidence, and severity. Migrant studies show that changes in the environment could substantially alter CVD risk in a genetically stable population. Additionally, CVD risk is affected by changes in nutritional and lifestyle choices. Recent studies in the field of environmental cardiology suggest that environmental toxins also influence CVD. Exposure to tobacco smoke is paradigmatic of such environmental risk and is strongly and positively associated with increased cardiovascular morbidity and mortality. In animal models of exposure, tobacco smoke induces endothelial dysfunction and prothrombotic responses and exacerbates atherogenesis and myocardial ischemic injury. Similar mechanism may be engaged by other pollutants or food constituents. Several large population-based studies indicate that exposure to fine or ultrafine particulate air pollution increases CVD morbidity and mortality, and the plausibility of this association is supported by data from animal studies. Exposure to other chemicals such as polyaromatic hydrocarbons, aldehydes, and metals has also been reported to elevate CVD risk by affecting atherogenesis, thrombosis, or blood pressure regulation. Maternal exposure to drugs, toxins, and infection has been linked with cardiac birth defects and premature CVD in later life. Collectively, the data support the notion that chronic environmental stress is an important determinant of CVD risk. Further work is required to assess the magnitude of this risk fully and to delineate specific mechanisms by which environmental toxins affect CVD.

Acrolein activates matrix metalloproteinases by increasing reactive oxygen species in macrophages

Acrolein is a ubiquitous component of environmental pollutants such as automobile exhaust, cigarette, wood, and coal smoke. It is also a natural constituent of several foods and is generated endogenously during inflammation or oxidation of unsaturated lipids. Because increased inflammation and episodic exposure to acrolein-rich pollutants such as traffic emissions or cigarette smoke have been linked to acute myocardial infarction, we examined the effects of acrolein on matrix metalloproteinases (MMPs), which destabilize atherosclerotic plaques. Our studies show that exposure to acrolein resulted in the secretion of MMP-9 from differentiated THP-1 macrophages. Acrolein-treatment of macrophages also led to an increase in reactive oxygen species (ROS), free intracellular calcium ([Ca2+](i)), and xanthine oxidase (XO) activity. ROS production was prevented by allopurinol, but not by rotenone or apocynin and by buffering changes in [Ca2+](I) with BAPTA-AM. The increase in MMP production was abolished by pre-treatment with the antioxidants Tiron and N-acetyl cysteine (NAC) or with the xanthine oxidase inhibitors allopurinol or oxypurinol. Finally, MMP activity was significantly stimulated in aortic sections from apoE-null mice containing advanced atherosclerotic lesions after exposure to acrolein ex vivo. These observations suggest that acrolein exposure results in MMP secretion from macrophages via a mechanism that involves an increase in [Ca2+](I), leading to xanthine oxidase activation and an increase in ROS production. ROS-dependent activation of MMPs by acrolein could destabilize atherosclerotic lesions during brief episodes of inflammation or pollutant exposure.

Acrolein exposure is associated with increased cardiovascular disease risk.

Background Acrolein is a reactive aldehyde present in high amounts in coal, wood, paper, and tobacco smoke. It is also generated endogenously by lipid peroxidation and the oxidation of amino acids by myeloperoxidase. In animals, acrolein exposure is associated with the suppression of circulating progenitor cells and increases in thrombosis and atherogenesis. The purpose of this study was to determine whether acrolein exposure in humans is also associated with increased cardiovascular disease (CVD) risk. Methods and Results Acrolein exposure was assessed in 211 participants of the Louisville Healthy Heart Study with moderate to high (CVD) risk by measuring the urinary levels of the major acrolein metabolite—3‐hydroxypropylmercapturic acid (3‐HPMA). Generalized linear models were used to assess the association between acrolein exposure and parameters of CVD risk, and adjusted for potential demographic confounders. Urinary 3‐HPMA levels were higher in smokers than nonsmokers and were positively correlated with urinary cotinine levels. Urinary 3‐HPMA levels were inversely related to levels of both early (AC133+) and late (AC133−) circulating angiogenic cells. In smokers as well as nonsmokers, 3‐HPMA levels were positively associated with both increased levels of platelet–leukocyte aggregates and the Framingham Risk Score. No association was observed between 3‐HPMA and plasma fibrinogen. Levels of C‐reactive protein were associated with 3‐HPMA levels in nonsmokers only. Conclusions Regardless of its source, acrolein exposure is associated with platelet activation and suppression of circulating angiogenic cell levels, as well as increased CVD risk.

Exposure to acrolein by inhalation causes platelet activation.

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5ppm for 6h) or sub-chronic (1ppm, 6h/day for 4days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.