Effect of Aneurysm and Patient Characteristics on Intracranial Aneurysm Wall Thickness.

Background: The circle of Willis is a network of arteries allowing blood supply to the brain. Bulging of these arteries leads to formation of intracranial aneurysm (IA). Subarachnoid hemorrhage (SAH) due to IA rupture is among the leading causes of disability in the western world. The formation and rupture of IAs is a complex pathological process not completely understood. In the present study, we have precisely measured aneurysmal wall thickness and its uniformity on histological sections and investigated for associations between IA wall thickness/uniformity and commonly admitted risk factors for IA rupture. Methods: Fifty-five aneurysm domes were obtained at the Geneva University Hospitals during microsurgery after clipping of the IA neck. Samples were embedded in paraffin, sectioned and stained with hematoxylin-eosin to measure IA wall thickness. The mean, minimum, and maximum wall thickness as well as thickness uniformity was measured for each IA. Clinical data related to IA characteristics (ruptured or unruptured, vascular location, maximum dome diameter, neck size, bottleneck factor, aspect and morphology), and patient characteristics [age, smoking, hypertension, sex, ethnicity, previous SAH, positive family history for IA/SAH, presence of multiple IAs and diagnosis of polycystic kidney disease (PKD)] were collected. Results: We found positive correlations between maximum dome diameter or neck size and IA wall thickness and thickness uniformity. PKD patients had thinner IA walls. No associations were found between smoking, hypertension, sex, IA multiplicity, rupture status or vascular location, and IA wall thickness. No correlation was found between patient age and IA wall thickness. The group of IAs with non-uniform wall thickness contained more ruptured IAs, women and patients harboring multiple IAs. Finally, PHASES and ELAPSS scores were positively correlated with higher IA wall heterogeneity. Conclusion: Among our patient and aneurysm characteristics of interest, maximum dome diameter, neck size and PKD were the three factors having the most significant impact on IA wall thickness and thickness uniformity. Moreover, wall thickness heterogeneity was more observed in ruptured IAs, in women and in patients with multiple IAs. Advanced medical imaging allowing in vivo measurement of IA wall thickness would certainly improve personalized management of the disease and patient care.

Chronic AICAR treatment prevents metabolic changes in cardiomyocytes exposed to free fatty acids.

The stimulation of glucose transport by metabolic stress is an important determinant of myocardial susceptibility to ischemia and reperfusion injury. Stimulation of glucose transport is markedly impaired in cardiomyocytes chronically exposed to excess free fatty acids (FFA), as occurs in vivo in type 2 diabetes. To determine whether chronic low-grade activation of AMP-activated kinase (AMPK) improves substrate metabolism in cardiomyocytes exposed to FFA, isolated cultured cardiomyocytes were exposed for 7 days to FFA ± the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Glucose transport and glycolysis were then measured during acute metabolic stress provoked by oligomycin. Chronic treatment with AICAR improved basal and oligomycin-stimulated glucose transport in FFA-exposed but not in control cardiomyocytes. Similarly, basal and oligomycin-stimulated glycolysis was reduced in FFA-exposed cardiomyocytes but restored by chronic AICAR treatment. Conversely, fatty acid oxidation was increased in FFA-exposed cardiomyocytes and reduced by chronic AICAR treatment. Chronic AICAR treatment induced in FFA-exposed cardiomyocytes the biogenesis of numerous lipid droplets. Curiously, whereas acute treatment of cardiomyocytes with AICAR increased phosphorylation of the AMPKα subunit on T172, a classical marker of AMPK activation, chronic AICAR treatment almost completely obliterated T172 phosphorylation. However, phosphorylation of the AMPK target protein raptor on S792 was reduced in FFA-exposed cardiomyocytes but restored by AICAR treatment. In conclusion, chronic AICAR treatment induces a metabolic shift in FFA-exposed cardiomyocytes, characterized by improved glucose transport and glycolysis and redirection of fatty acids towards neutral storage. Such metabolic changes in vivo could protect the hearts of patients with type 2 diabetes against ischemia-reperfusion injury.

Endothelial Cx40 limits myocardial ischaemia/reperfusion injury in mice.

AIMS: Gap junctions are indispensable for the function of heart and blood vessels by providing electrical coupling and direct cell-to-cell transfer of small signalling molecules. Gap junction channels between neighbouring cells are composed of 12 connexins (Cx). Changes in Cx43 expression, localization, and channel properties in cardiomyocytes contribute to infarction and reperfusion injury of the heart. It is increasingly recognized that deleterious consequences of ischaemia/reperfusion (IR) are modulated by the inflammatory response and endothelial function. The role of the endothelial connexins, i.e. Cx40 and Cx37, in cardiac IR injury is, however, not known. METHODS AND RESULTS: Following 30 min ischaemia and 24 h reperfusion, we found a significant increase in myocardial infarct size in mice with endothelial-specific deletion of Cx40 (Cx40del), but not in Cx37-deficient mice. The cardioprotective effect of endothelial Cx40 was associated with a decrease in neutrophil infiltration. Moreover, beneficial effects of endothelial Cx40 were not observed in isolated Langendorff-perfused hearts, suggesting direct involvement of endothelial-leucocyte interactions in the cardiac injury. Single-dose administration of methotrexate, a CD73 activator, reduced infarct size and neutrophil infiltration into the infarcted myocardium in Cx40del but not in control mice. Similar to Cx40del mice, CD73-deficient mice showed increased sensitivity to cardiac IR injury, which could not be conversed by methotrexate. CONCLUSION: Endothelial Cx40, but not Cx37, is implicated in resistance of the heart to IR injury by activation of the CD73 pathway. Thus, the Cx40-CD73 axis may represent an interesting target for controlling reperfusion damage associated with revascularization in coronary disease.

Lack of association between connexin40 polymorphisms and coronary artery disease.

OBJECTIVE: Cx40 is a gap junction protein important for cell-cell communication in the endothelium. Polymorphisms in the promoter region of the human Cx40 gene, -44G>A and +71A>G, were shown to reduce Cx40 transcription by half. As mice with an endothelial-specific deletion of Cx40 are more susceptible to atherosclerosis, this study was designed to discover a correlation between these polymorphisms and atherosclerosis in European populations. METHODS AND RESULTS: 803 patients referred to the Geneva University Hospitals for elective coronary angiography were divided according to the number of significantly stenosed vessels (from 0 to 3) and were genotyped for the Cx40 polymorphisms. Genotype distribution in the control group was -44GG/+71AA=59.8%, -44AG/+71AG=35.1% and -44AA/+71GG=5.2%. Surprisingly, this distribution was similar in the CAD group, with -44GG/+71AA=58.5%, -44AG/+71AG=37.6% and -44AA/+71GG=3.8% (p=0.67). Moreover, no significant association between histological carotid plaque composition of culprit lesions and Cx40 polymorphisms could be detected in 583 Dutch patients of the Athero-Express study. CONCLUSIONS: Despite a clear antiatherogenic role of Cx40 in mice, our study could not detect an association of Cx40 promoter polymorphisms and CAD in human. Moreover, a correlation with atherosclerotic plaque stability or hypertension could not be demonstrated either. Connexin polymorphisms affecting channel function may be of greater importance for cardiovascular disease than polymorphisms affecting the expression level of the protein.

Gap junction protein Cx37 interacts with endothelial nitric oxide synthase in endothelial cells.

OBJECTIVE: The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. A C1019T Cx37 gene polymorphism, encoding a P319S substitution in the regulatory C terminus of Cx37 (Cx37CT), correlates with arterial stenosis and myocardial infarction in humans. This study was designed to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction. METHODS AND RESULTS: Using a high-throughput phage display, we retrieved 2 binding motifs for Cx37CT: WHK ... [K,R]XP ... and FHK ... [K,R]XXP ... , the first being more common for Cx37CT-319P and the second more common for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed 77.7% homology with residues 843 to 854 of endothelial nitric oxide synthase (eNOS). In vitro binding of this peptide or of the homologous eNOS sequence to both Cx37CT isoforms was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like and eNOS(843-854) increased the frequency of events with conductances higher than 300 pS. We demonstrated that eNOS coimmunoprecipitated with Cx37 in a mouse endothelial cell (EC) line (bEnd.3), human primary ECs, and a human EC line transfected with Cx37-319P or Cx37-319S. Cx37 and eNOS colocalized at EC membranes. Moreover, a dose-dependent increase in nitric oxide production was observed in ECs treated with Cx37 antisense. CONCLUSIONS: Overall, our data show for the first time a functional and specific interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease.

Connexin43 modulates neutrophil recruitment to the lung.

Transmigration of neutrophils through the microvascular endothelium is a cardinal event of acute inflammation. It has been suggested that gap junctions made of connexin43 (Cx43) may serve as a conducting pathway to spread inflammatory signals within the lung capillary network. To determine whether Cx43 contributes to neutrophil transmigration in vivo, the number of transmigrated neutrophils was monitored in lungs of Cx43 mouse models subjected to inflammation by intratracheal instillations of Pseudomonas aeruginosa lipopolysaccharide (LPS). Cx43 was detected in inflamed lungs independently of neutrophil recruitment, whereas Cx43 up-regulation was not detected in mice genetically protected from inflammation. Mice heterozygous for the Cx43 gene (gja1) showed a 56% (P < 0.01) reduction in airway neutrophil count. In contrast, increased (P < 0.05) neutrophil recruitment in response to LPS was observed in a mouse model expressing a mutant Cx43 with enhanced channel conductivity. In vitro adhesion assays showed that reduced conductivity of Cx43 channels with (43)Gap26, a Cx43 blocking peptide, decreased adhesion of neutrophils to endothelial cells. Finally, we found that instillation of (43)Gap26 in inflamed lungs reduced neutrophil transmigration by 65% (P < 0.05). These results indicate that inflammatory mediators up-regulate alveolar Cx43 to promote neutrophil recruitment to the airspace. Cx43 may therefore represent a pharmacological target in lung diseases characterized by excessive neutrophil recruitment to the airways.

Connexin37 protects against atherosclerosis by regulating monocyte adhesion.

A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.

Gap junctional communication does not contribute to the interaction between neutrophils and airway epithelial cells.

Cystic fibrosis (CF) is characterized by intense neutrophil migration into the airways. Increasing evidence indicates that interaction between neutrophils and airway epithelial cells contributes to the modulation of the inflammatory response. Blood neutrophils were reported to express connexins and form gap junctions with endothelial cells, thereby establishing gap junctional communication. We tested whether altered communication between human neutrophils and airway epithelial cells may contribute to the exaggerated inflammatory response observed in CF patients. Microinjections did not reveal dye coupling between activated blood neutrophils. By contrast, diffusion of calcein between neutrophils and airway epithelial cells of CF or non-CF origin was observed in transmigration and adhesion assays. This diffusion was prevented with probenicid, an inhibitor of ATP-dependent organic anion pumps, but not with gap junction blockers. Finally, RT-PCR failed to detect mRNAs for six connexins in blood neutrophils. These results suggest that gap junctional communication does not contribute to neutrophil-airway epithelial cell interaction.

Gap junctional communication in tissue inflammation and repair.

Local injury induces a complex orchestrated response to stimulate healing of injured tissues, cellular regeneration and phagocytosis. Practically, inflammation is defined as a defense process whereby fluid and white blood cells accumulate at a site of injury. The balance of cytokines, chemokines, and growth factors is likely to play a key role in regulating important cell functions such as migration, proliferation, and matrix synthesis during the process of inflammation. Hence, the initiation, maintenance, and resolution of innate responses depend upon cellular communication. A process similar to tissue repair and subsequent scarring is found in a variety of fibrotic diseases. This may occur in a single organ such as liver, kidneys, pancreas, lung, skin, and heart, but fibrosis may also have a more generalized distribution such as in atherosclerosis. The purpose of this review is to summarize recent advances on the contribution of gap junction-mediated intercellular communication in the modulation of the inflammatory response and tissue repair.