Background-Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. Methods and Results-Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. Key Words: atherosclerosis Ⅲ connexins Ⅲ endothelium Ⅲ gap junctions Ⅲ inflammation C ardiovascular diseases currently constitute the major cause of death in developed countries. 1 Atherosclerosis, an inflammatory disease of large and medium-sized arteries, 2 is the most important cause of cardiovascular diseases. The main consequences of atherosclerosis are myocardial infarction, cerebral infarction, and aortic aneurysm. 3 Conclusions-Cx40-mediated Clinical Perspective on p 131Atherosclerosis involves the formation of intimal lesions that are characterized by a dysfunctional endothelium, inflammation, lipid accumulation, cell death, and fibrosis. 2,3 The distribution of atherosclerotic plaques is highly characteristic in humans; the lesions develop predominantly near side branches of arteries where blood flow is disturbed. 4 A variety of substances mediating intercellular communication, including cytokines, chemokines, and growth factors, have been identified to induce, amplify, and modify the atherosclerotic inflammatory process. 5,6 In this context, connexins, a large family of proteins that form hemichannels and gap junction channels enabling transmembrane and intercellular coordination of tissue activity, 7,8 have been involved in atherogenesis. Three connexins are expressed in the vascular wall, namely connexin (Cx)37, Cx40, and Cx43, and important changes in their expression pattern have been reported in Received March 20, 2009; accepted October 28, 2009. From the Division of Cardiology (C.E.C., I.R., B.F., B.R.K.) and Department of Pediatrics (K.E.L.S., M.Z.R.S., M.B., B.F., T.D....

Abstract-We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S-and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with ␣-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S-and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein.In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes. Key Words: 2D-PAGE Ⅲ stent Ⅲ endothelial cells Ⅲ mts1 Ⅲ ␣-smooth muscle actin Ⅲ smoothelin T he concept of smooth muscle cell (SMC) phenotypic heterogeneity has been validated in several species including man (for review see 1 ). We have recently extended this notion to the porcine coronary artery (CA) and isolated from the normal media 2 distinct SMC populations: spindleshaped (S) with the classical "hills-and-valleys" growth pattern and rhomboid (R), which grows as a monolayer. 2 R-SMCs display enhanced proliferative, migratory and proteolytic activities as well as poor level of differentiation compared with S-SMCs. R-SMCs are recovered in higher proportion when SMCs are cultured from the intimal thickening (IT) induced after experimental stent implantation compared with the normal media, indicating that they are crucial for arterial repair, and could represent an atheromaprone phenotype.Our aim was to further characterize the phenotypic features of S-and R-SMCs, to identify them in vivo and possibly to verify their presence in atheromatous plaque and restenotic lesions. We have analyzed protein extracts by means of 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by identification of differentially expressed proteins using tandem mass spectrometry (M...

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-alpha signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-alpha to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-alpha signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-DeltaTRL), TNF-alpha increased the amount of WtCFTR but not CFTR-DeltaTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-DeltaTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

Objectives-Characterize the phenotypic features of media and intima coronary artery smooth muscle cells (SMCs) in mildly stenotic plaques, erosions, stable plaques, and in-stent restenosis. Methods and Results-Expression of ␣-smooth muscle actin (␣-SMA), smooth muscle myosin heavy chains (SMMHCs), and smoothelin was investigated by immunohistochemistry followed by morphometric quantification. The crosssectional area and the expression of cytoskeletal proteins in the media were lower in restenotic lesions and, to a lesser extent, in stable plaques compared with mildly stenotic plaques and erosions. An important expression of ␣-SMA was detected in the intima of the different lesions; moreover, ␣-SMA staining was significantly larger in erosions compared with all other conditions. In the same location, a striking decrease of SMMHCs and a disappearance of smoothelin were observed in all situations. Key Words: ␣-smooth muscle actin Ⅲ erosion Ⅲ smooth muscle myosin heavy chains Ⅲ smoothelin Ⅲ stable plaque T hrombosis after plaque rupture is the most frequent cause of coronary mortality, followed by thrombosis over an erosion, whereas a smaller but significant proportion of deaths corresponds to the presence of stable plaques without evidence of thrombosis. 1 Several factors have been suggested to participate in the mechanisms of plaque rupture such as altered matrix turnover and blood rheology, increased coagulation, and recurrent infections. 2 Macrophages and T lymphocytes are increased at site of plaque rupture, whereas erosions and stable plaques contain relatively few inflammatory cells. Conclusions-Medial

Objectives-Characterize the phenotypic features of media and intima coronary artery smooth muscle cells (SMCs) in mildly stenotic plaques, erosions, stable plaques, and in-stent restenosis. Methods and Results-Expression of ␣-smooth muscle actin (␣-SMA), smooth muscle myosin heavy chains (SMMHCs), and smoothelin was investigated by immunohistochemistry followed by morphometric quantification. The crosssectional area and the expression of cytoskeletal proteins in the media were lower in restenotic lesions and, to a lesser extent, in stable plaques compared with mildly stenotic plaques and erosions. An important expression of ␣-SMA was detected in the intima of the different lesions; moreover, ␣-SMA staining was significantly larger in erosions compared with all other conditions. In the same location, a striking decrease of SMMHCs and a disappearance of smoothelin were observed in all situations. Key Words: ␣-smooth muscle actin Ⅲ erosion Ⅲ smooth muscle myosin heavy chains Ⅲ smoothelin Ⅲ stable plaque T hrombosis after plaque rupture is the most frequent cause of coronary mortality, followed by thrombosis over an erosion, whereas a smaller but significant proportion of deaths corresponds to the presence of stable plaques without evidence of thrombosis. 1 Several factors have been suggested to participate in the mechanisms of plaque rupture such as altered matrix turnover and blood rheology, increased coagulation, and recurrent infections. 2 Macrophages and T lymphocytes are increased at site of plaque rupture, whereas erosions and stable plaques contain relatively few inflammatory cells. Conclusions-Medial

We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation.Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs.We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation.

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation, or NETosis, is a specific and highly efficient process, which is induced by a variety of stimuli leading to expulsion of DNA, proteases and antimicrobial peptides to the extracellular space. However, uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here, we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly, neutrophils isolated from Panx1−/− mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF), a Panx1 channel inhibitor, decreased NETosis in wild-type neutrophils to the extent observed in Panx1−/− neutrophils. Thus, we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target.