Giovanni G. Camici

Genetic deletion of p66Shc adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66Shc (p66Shc−/−) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66Shc−/− mouse. p66Shc−/− and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66Shc−/− and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endothelium-dependent relaxations, increased peroxynitrite (ONOO−) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66Shc−/− diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66Shc−/− but not in WT mice. We report that p66Shc−/− mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.

Final common molecular pathways of aging and cardiovascular disease: Role of the p66Shc protein

Oxidative stress affects the availability of key-regulators of vascular homeostasis and controls a number of signaling pathways relevant to myocardial and vascular disease. Reactive oxygen species are generated by different intracellular molecular pathways principally located in mitochondria. The notion that mice carrying a targeted mutation of the p66(Shc) gene display prolonged lifespan, reduced production of intracellular oxidants, and increased resistance to oxidative stress-induced apoptosis prompted a series of studies aimed at defining the biochemical function of p66(Shc) and its possible implication in cardiovascular diseases. Indeed, p66(Shc-/-) mice are protected against vascular, cardiac, and renal impairment attributable to hypercholesterolemia, aging, diabetes, and ischemia/reperfusion. The present review focuses on the biochemical and physiological function of the p66(Shc) adaptor protein as well as on the mechanisms linking p66(Shc)-associated generation of free radicals to the pathophysiology of aging and cardiovascular disease. On the whole, the evidence so far reported and here discussed supports the concept that pharmacological modulation of p66(Shc) expression and activity may be a novel and effective target for the treatment of atherosclerotic vascular disease as well as myocardial adaptation to hypertrophic, inflammatory and neuro-hormonal stimuli in the overloaded heart.

Paclitaxel Enhances Thrombin-Induced Endothelial Tissue Factor Expression via c-Jun Terminal NH2 Kinase Activation

Paclitaxel is used on drug-eluting stents because it inhibits proliferation of vascular cells. Stent thrombosis remains a concern with this compound, particularly with higher dosages. This study investigates the effect of paclitaxel on tissue factor (TF) expression in human endothelial cells. Paclitaxel enhanced thrombin-induced endothelial TF protein expression in a concentration- and time-dependent manner. A concentration of 10−5 mol/L elicited a 2.1-fold increase in TF protein and a 1.6-fold increase in TF surface activity. The effect was similar after a 1 hour as compared with a 25-hour pretreatment period. Real-time polymerase chain reaction revealed that paclitaxel increased thrombin-induced TF mRNA expression. Paclitaxel potently activated c-Jun terminal NH2 kinase (JNK) as compared with thrombin alone, whereas the thrombin-mediated phosphorylation of p38 and extracellular signal-regulated kinase remained unaffected. Similar to paclitaxel, docetaxel enhanced both TF expression and JNK activation as compared with thrombin alone. The JNK inhibitor SP600125 reduced thrombin-induced TF expression by 35%. Moreover, SP600125 blunted the effect of paclitaxel and docetaxel on thrombin-induced TF expression. Paclitaxel increases endothelial TF expression via its stabilizing effect on microtubules and selective activation of JNK. This observation provides novel insights into the pathogenesis of thrombus formation after paclitaxel-eluting stent deployment and may have an impact on drug-eluting stent design.

Differential Effects of Selective Cyclooxygenase-2 Inhibitors on Endothelial Function in Salt-Induced Hypertension

Background—In view of the ongoing controversy about potential differences in cardiovascular safety of selective cyclooxygenase (COX)-2 inhibitors (coxibs), we compared the effects of 2 different coxibs and a traditional NSAID on endothelial dysfunction, a well-established surrogate of cardiovascular disease, in salt-induced hypertension. Methods and Results—Salt-sensitive (DS) and salt-resistant (DR) Dahl rats were fed a high-sodium diet (4% NaCl) for 56 days. From days 35 to 56, diclofenac (6 mg · kg−1 · d−1; DS-diclofenac), rofecoxib (2 mg · kg−1 · d−1; DS-rofecoxib), celecoxib (25 mg · kg−1 · d−1; DS-celecoxib) or placebo (DS-placebo) was added to the chow. Blood pressure increased with sodium diet in the DS groups, which was more pronounced after diclofenac and rofecoxib treatment (P <0.005 versus DS-placebo) but was slightly decreased by celecoxib (P <0.001 versus DS-placebo). Sodium diet markedly reduced NO-mediated endothelium-dependent relaxations to acetylcholine (10−10-10−5 mol/L) in aortic rings of untreated hypertensive rats (P <0.005 versus DR-placebo). Relaxation to acetylcholine improved after celecoxib (P <0.005 versus DS-placebo and DS-rofecoxib) but remained unchanged after rofecoxib and diclofenac treatment. Vasoconstriction after nitric oxide synthase inhibition, indicating basal NO release, with N&ohgr;-nitro-l-arginine methyl ester (10−4 mol/L) was blunted in DS rats (P <0.05 versus DR-placebo), normalized by celecoxib, but not affected by rofecoxib or diclofenac. Indicators of oxidative stress, 8-isoprostane levels, were elevated in untreated DS rats on 4% NaCl (6.55±0.58 versus 3.65±1.05 ng/mL, P <0.05) and normalized by celecoxib only (4.29±0.58 ng/mL). Conclusions—These data show that celecoxib but not rofecoxib or diclofenac improves endothelial dysfunction and reduces oxidative stress, thus pointing to differential effects of coxibs in salt-induced hypertension.

Dimethyl Sulfoxide Inhibits Tissue Factor Expression, Thrombus Formation, and Vascular Smooth Muscle Cell Activation A Potential Treatment Strategy for Drug-Eluting Stents

Background— Subacute stent thrombosis is a major clinical concern, and the search for new molecules to cover stents remains important. Dimethyl sulfoxide (DMSO) is used for preservation of hematopoietic progenitor cells and is infused into patients undergoing bone marrow transplantation. Despite its intravenous application, the impact of DMSO on vascular cells has not been assessed. Methods and Results— In human endothelial cells, monocytes, and vascular smooth muscle cells (VSMC), DMSO inhibited tissue factor (TF) expression and activity in response to tumor necrosis factor-&agr; or thrombin in a concentration-dependent manner. DMSO did not exert any toxic effects as assessed by phase-contrast microscopy, trypan blue exclusion, and lactate dehydrogenase release. Real-time polymerase chain reaction revealed that inhibition of TF expression occurred at the mRNA level. This effect was mediated by reduced activation of the mitogen-activated protein kinases c-Jun terminal NH2 kinase (51±6%; P=0.0005) and p38 (50±3%; P<0.0001) but not p44/42 (P=NS). In contrast to TF, DMSO did not affect expression of TF pathway inhibitor or plasminogen activator inhibitor-1. In vivo, DMSO treatment suppressed TF activity (41%; P<0.002) and prevented thrombotic occlusion in a mouse carotid artery photochemical injury model. DMSO also inhibited VSMC proliferation (70%; P=0.005) and migration (77%; P=0.0001) in a concentration-dependent manner; moreover, it prevented rapamycin and paclitaxel-induced upregulation of TF expression. Conclusions— DMSO suppresses TF expression and activity, as well as thrombus formation; in addition, it inhibits VSMC proliferation and migration. Given its routine use in modern clinical practice, we propose DMSO as a novel strategy for coating drug-eluting stents and treating acute coronary syndromes.

c-Jun N-Terminal Kinase 2 Deficiency Protects Against Hypercholesterolemia-Induced Endothelial Dysfunction and Oxidative Stress

Background— Hypercholesterolemia-induced endothelial dysfunction due to excessive production of reactive oxygen species is a major trigger of atherogenesis. The c-Jun-N-terminal kinases (JNKs) are activated by oxidative stress and play a key role in atherogenesis and inflammation. We investigated whether JNK2 deletion protects from hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Methods and Results— Male JNK2 knockout (JNK2−/−) and wild-type (WT) mice (8 weeks old) were fed either a high-cholesterol diet (HCD; 1.25% total cholesterol) or a normal diet for 14 weeks. Aortic lysates of WT mice fed a HCD showed an increase in JNK phosphorylation compared with WT mice fed a normal diet (P<0.05). Endothelium-dependent relaxations to acetylcholine were impaired in WT HCD mice (P<0.05 versus WT normal diet). In contrast, JNK2−/− HCD mice did not exhibit endothelial dysfunction (96±5% maximal relaxation in response to acetylcholine; P<0.05 versus WT HCD). Endothelium-independent relaxations were identical in all groups. A hypercholesterolemia-induced decrease in nitric oxide (NO) release of endothelial cells was found in WT but not in JNK2−/− mice. In parallel, endothelial NO synthase expression was upregulated only in JNK2−/− HCD animals, whereas the expression of antioxidant defense systems such as extracellular superoxide dismutase and manganese superoxide dismutase was decreased in WT but not in JNK2−/− HCD mice. In contrast to JNK2−/− mice, WT HCD displayed an increase in O2− and ONOO− concentrations as well as nitrotyrosine staining and peroxidation. Conclusions— JNK2 plays a critical role as a mediator of hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Thus, JNK2 may provide a novel target for prevention of vascular disease and atherosclerosis.

Oxidized Low-Density Lipoprotein Activates p66Shc via Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1, Protein Kinase C-β, and c-Jun N-Terminal Kinase Kinase in Human Endothelial Cells

Objective— Deletion of the mitochondrial gene p66Shc protects from endothelial dysfunction and atherosclerotic plaque formation in mice fed a high-fat diet. However, the molecular mechanisms underlying this beneficial effect have not yet been delineated. The present study was designed to elucidate the proatherogenic mechanisms by which p66Shc mediates oxidized low-density lipoprotein (oxLDL) uptake by the endothelium, a critical step in plaque formation. Methods and Results— Incubation of human aortic endothelial cells with oxLDL led to phosphorylation of p66Shc at Ser36. Inhibition of lectin-like oxLDL receptor-1 prevented p66Shc phosphorylation, confirming that this effect is mediated by lectin-like oxLDL receptor-1. OxLDL also increased phosphorylation of protein kinase C &bgr;2 (PKC&bgr;2) at both Thr641 and Ser660, as well as c-Jun N-terminal kinase (JNK). Furthermore, inhibition of PKC&bgr;2 prevented the activation of JNK, suggesting that PKC&bgr;2 is upstream of JNK. Finally, p66Shc silencing blunted oxLDL-induced O2 −. production, underscoring the critical role of p66Shc in oxLDL-induced oxidative stress in endothelial cells. Conclusion— In this study we provide the molecular mechanisms mediating the previously observed atherogenic properties of p66Shc. Taken together, our data set the stage for the design of novel therapeutic tools to retard atherogenesis through the inhibition of p66Shc.

Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells

AIMS The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. METHODS AND RESULTS Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65(-/-) mouse embryonic fibroblasts reconstituted with non-acetylatable Lys(310)-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH(2)-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. CONCLUSION We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosis.

Deletion of the Activated Protein-1 Transcription Factor JunD Induces Oxidative Stress and Accelerates Age-Related Endothelial Dysfunction

Background— Reactive oxygen species are major determinants of vascular aging. JunD, a member of the activated protein-1 family of transcription factors, is emerging as a major gatekeeper against oxidative stress. However, its contribution to reactive oxygen species homeostasis in the vasculature remains unknown. Methods and Results— Endothelium-dependent vasorelaxation was impaired in young and old JunD−/− mice (6 and 22 months old) compared with age-matched wild-type mice. JunD−/− mice displayed an age-independent decline in endothelial nitric oxide release and endothelial nitric oxide synthase activity and increased mitochondrial superoxide formation and peroxynitrite levels. Furthermore, vascular expression and activity of the free radical scavengers manganese and extracellular superoxide dismutase and aldehyde dehydrogenase 2 were reduced, whereas the NADPH oxidase subunits p47phox, Nox2, and Nox4 were upregulated. These redox changes were associated with premature vascular aging, as shown by reduced telomerase activity, increased &bgr;-galactosidase–positive cells, upregulation of the senescence markers p16INK4a and p53, and mitochondrial disruption. Interestingly, old wild-type mice showed a reduction in JunD expression and transcriptional activity resulting from promoter hypermethylation and binding with tumor suppressor menin, respectively. In contrast, JunD overexpression blunted age-induced endothelial dysfunction. In human endothelial cells, JunD knockdown exerted a similar impairment of the O2−/nitric oxide balance that was prevented by concomitant NADPH inhibition. In parallel, JunD expression was reduced in monocytes from old versus young healthy subjects and correlated with mRNA levels of scavenging and oxidant enzymes. Conclusions— JunD provides protection in aging-induced endothelial dysfunction and may represent a novel target to prevent reactive oxygen species–driven vascular aging.

Selective COX-2 Inhibitors and Renal Injury in Salt-Sensitive Hypertension

In view of the ongoing controversy of cardiorenal safety of selective COX-2 inhibitors (coxibs), the present study was designed to examine the effects of 2 different coxibs, celecoxib and rofecoxib, compared with a traditional NSAID, diclofenac, and placebo on renal morphology and function in salt-sensitive hypertension. Salt-sensitive (DS) and salt-resistant (DR) Dahl rats were fed with NaCl-enriched diet (4% NaCl) for 8 weeks. Diclofenac (DS-diclofenac), rofecoxib (DS-rofecoxib), celecoxib (DS-celecoxib), or placebo was added to chow from weeks 6 to 8. Immunostaining for monocytes/macrophages (ED1) and cytotoxic T lymphocytes (CD8) was performed. In addition, renal morphology and proteinuria were assessed. Renal cortex mRNA was isolated for determination of COX-2, eNOS, and CRP mRNA by real-time reverse-transcriptase polymerase chain reaction. Untreated hypertensive animals showed glomerular injury including collapsing glomerulopathy, mesangial sclerosis, mesangiolysis, extracapillary proliferation, protein drops, and an especially high grade of glomerulosclerosis (P<0.05 versus DR-placebo) and CD8-positive and ED1-positive cells (P<0.01 versus DR-placebo), which was improved by celecoxib but not by diclofenac and rofecoxib. C-reactive protein mRNA in renal cortex was increased in DS-placebo animals (P<0.05 versus DR-placebo) and normalized by celecoxib (P<0.05 versus DS-placebo), whereas eNOS mRNA was decreased in the DS-rofecoxib group (P<0.05 versus DR-placebo, DS-celecoxib, and DS-diclofenac). Proteinuria was observed in hypertensive animals (P<0.0001 versus DR-placebo), increased by rofecoxib (P<0.05 versus DS-placebo), and normalized by celecoxib (P=0.0015 versus DS-placebo). This head-to-head comparison of selective and nonselective COX inhibitors demonstrates differential effects of coxibs on renal morphology and function in salt-dependent hypertension.