Pascal J. Goldschmidt-Clermont

A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis

BACKGROUND Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction. METHODS To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion. RESULTS The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4). CONCLUSIONS We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.

Aging, Progenitor Cell Exhaustion, and Atherosclerosis

Background—Atherosclerosis is largely attributed to chronic vascular injury, as occurs with excess cholesterol; however, the effect of concomitant vascular aging remains unexplained. We hypothesize that the effect of time in atherosclerosis progression is related to obsolescence of endogenous progenitor cells that normally repair and rejuvenate the arteries. Methods and Results—Here we show that chronic treatment with bone marrow–derived progenitor cells from young nonatherosclerotic ApoE−/− mice prevents atherosclerosis progression in ApoE−/− recipients despite persistent hypercholesterolemia. In contrast, treatment with bone marrow cells from older ApoE−/− mice with atherosclerosis is much less effective. Cells with vascular progenitor potential are decreased in the bone marrow of aging ApoE−/− mice, but cells injected from donor mice engraft on recipient arteries in areas at risk for atherosclerotic injury. Conclusions—Our data indicate that progressive progenitor cell deficits may contribute to the development of atherosclerosis.

Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system

OBJECTIVE Methylation of the promoter region of the estrogen receptor gene alpha (ER alpha) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. METHODS We used Southern blot analysis to determine the methylation state of the ER alpha gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. RESULTS An age related increase in ER alpha gene methylation occurs in the right atrium (range 6 to 19%, R = 0.36, P < 0.05). Significant levels of ER alpha methylation were detected in both veins and arteries. In addition, ER alpha gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10 +/- 2% versus 4 +/- 1%, P < 0.01). In endothelial cells explanted from human aorta and grown in vitro, ER alpha gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER alpha gene methylation (19-99%). CONCLUSIONS Methylation associated inactivation of the ER alpha gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation.

The signal transduction pathway leading to the activation of the transcription factor NF-kappaB remains incompletely characterized. We demonstrate that in HeLa cells, transient expression of a constitutively active mutant of the small GTP-binding protein rac1 (V12rac1) leads to a significant increase in NF-kappaB transcriptional activity. In addition, expression of a dominant-negative rac1 mutant (N17rac1) inhibits basal and interleukin 1beta-stimulated NF-kappaB activity. Gel shift analysis using nuclear extract prepared from HeLa cells infected with a recombinant adenovirus encoding N17rac1 (Ad.N17racl) showed reduced levels of cytokine-stimulated DNA binding to a consensus NF-kappaB binding site. We demonstrate that rac proteins function downstream of ras proteins in the activation of NF-kappaB. In addition, V12rac1 stimulation of NF-kappaB activity is shown to be independent of the ability of rac proteins to activate the family of c-jun amino-terminal kinases. In an effort to further explore how rac proteins might regulate NF-kappaB activity, we demonstrate that expression of V12rac1 in HeLa cells or stimulation with cytokine results in a significant increase in intracellular reactive oxygen species (ROS). Treatment of cells with either of two chemically unrelated antioxidants inhibits the rise in ROS that occurs following V12rac1 expression as well as the ability of V12rac1 to stimulate NF-kappaB activity. These results suggest that in HeLa cells, rac1 regulates intracellular ROS production and that rac proteins function as part of a redox-dependent signal transduction pathway leading to NF-kappaB activation.

Platelet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists

BACKGROUND Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.

Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts

Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.

Microtubule Binding to Smads May Regulate TGFβ Activity

Abstract Smad proteins are intracellular signaling effectors of the TGFβ superfamily. We show that endogenous Smad2, 3, and 4 bind microtubules (MTs) in several cell lines. Binding of Smads to MTs does not require TGFβ stimulation. TGFβ triggers dissociation from MTs, phosphorylation, and nuclear translocation of Smad2 and 3, with consequent activation of transcription in CCL64 cells. Destabilization of the MT network by nocodazole, colchicine, or a tubulin mutant disrupts the complex between Smads and MTs and increases TGFβ-induced Smad2 phosphorylation and transcriptional response in CCL64 cells. These data demonstrate that MTs may serve as a cytoplasmic sequestering network for Smads, controlling Smad2 association with and phosphorylation by activated TGFβ receptor I, and suggest a novel mechanism for the MT network to negatively regulate TGFβ function.

Deficient Smad7 expression: A putative molecular defect in scleroderma

Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) β has been implicated in the pathogenesis of scleroderma. Smad proteins are signaling transducers downstream from TGF-β receptors. Three families of Smads have been identified: (i) receptor-regulated Smad2 and -3 (R-Smads); (ii) common partner Smad4 (Co-Smad); and (iii) inhibitory Smad6 and -7 (I-Smads, part of a negative feedback loop). We have investigated the signaling components for the TGF-β pathway and TGF-β activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro. Basal level and TGF-β-inducible expression of Smad7 are selectively decreased, whereas Smad3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-β signaling events, including phosphorylation of Smad2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with Smad7 restores normal TGF-β signaling in scleroderma fibroblasts. These results suggest that alterations in the Smad pathway, including marked Smad7 deficiency and Smad3 up-regulation, may be responsible for TGF-β hyperresponsiveness observed in scleroderma.

Contribution of Monocytes/Macrophages to Compensatory Neovascularization: The Drilling of Metalloelastase-Positive Tunnels in Ischemic Myocardium

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1–negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2′-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with &bgr;-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.