Functional studies towards the role of a polymorphism in the human connexin37 gene in atherosclerosis

L'athérosclérose et ces conséquences, sont les premières causes de mortalité dans les pays industrialisés. Dans la majorité des cas, ces constatations cliniques sont consécutives à une rupture ou une érosion d'une plaque d'athérome avec formation d'un thrombus obstruant la lumière de l'artère. L'hypertension, le tabac, le diabète, l'hypercholestérolémie, les forces hémodynamiques du flux sanguin ou encore des facteurs génétiques sont parmi les causes les plus importantes favorisant le développement de l'athérosclérose. L'ensemble de ces facteurs induisent une dysfonction endothéliale chronique qui facilite l'adhésion des lymphocytes T, des monocytes et leur transmigration dans les parois artérielles. Suite à cette infiltration, les monocytes se différentient et s'activent en macrophages qui se chargent en lipides pour devenir des cellules spumeuses, formant ainsi la partie grasse de l'athérome. Au niveau du site de la lésion, ces cellules vont sécréter des molécules inflammatoires capables d'induire la migration des cellules musculaires lisses de la média vers l'intima vasculaire où elles vont proliférer et sécréter la matrice extracellulaire pour former la partie fibreuse de l'athérome

Functional differences between human Cx37 polymorphic hemichannels

A polymorphism in the human Cx37 gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus of the Cx37 protein (P319S), has been proposed as a prognostic marker for atherosclerosis. We have recently demonstrated that Cx37 hemichannels control the initiation of atherosclerotic plaque development by regulating ATP-dependent monocyte adhesion in atherosclerosis-susceptible apolipoprotein E-deficient mice. In this study, we have measured the electrical properties of Cx37 hemichannels (HCs) and gap junction channels (GJCs) with voltage-clamp methods. To this end, we have transfected hCx37-P319, hCx37-S319 or empty pIRES-eGFP vector cDNA into communication-deficient HeLa cells. In clones expressing similar levels of Cx37, exposure of single cells to low-Ca(2+) solution induced a voltage-sensitive HC current. The analysis yielded a bell-shaped function g(hc)=f(V(m)) (g(hc): normalized conductance at steady state; V(m): membrane potential) with a maximum around V(m)=-30 mV. The peak g(hc) of Cx37-P319 was 3-fold larger than that of Cx37-S319 HCs. Experiments on cell pairs revealed that Cx37-P319 GJCs exhibited a 1.5-fold larger unitary conductance than Cx37-S319 GJCs. Hence, the larger peak g(hc) of the former may reflect a larger conductance of their HCs. Using the same clones, we found that Cx37-P319 cells released more ATP and were less adhesive than Cx37-S319 cells. The reduction in adhesiveness of Cx37-expressing cells was prevented by extracellular apyrase. We conclude that the differences in biophysical properties between polymorphic HCs may be responsible for inequality in ATP release between Cx37-P319 and Cx37-S319 cells, which results in differential cell adhesion

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

Targeting connexin 43 prevents platelet-derived growth factor-BB-induced phenotypic change in porcine coronary artery smooth muscle cells

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB-induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of alpha-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB-induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis

NADPH oxidase 4 deficiency reduces aquaporin-2 mRNA expression in cultured renal collecting duct principal cells via increased PDE3 and PDE4 activity

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription

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

Real-Time PCR primer sequences (mouse).

<p>Real-Time PCR primer sequences (mouse).</p

phosphodiesterase-3 and phosphodiesterase-4 mediate decreased AQP2 expression by NOX4 depletion.

<p>mRNA expression levels of NOX4, Na,K-ATPase α subunit (NaKα), Na,K-ATPase β subunit (NaKβ) and AQP2 were compared by Q-PCR in mpkCCD_cl4 cells transfected with either scramble siRNA or siNOX4 and challenged or not (Ctl) with either the selective phosphodiesterase-4 inhibitor rolipram (2 µM), selective phosphodiesterase-1 inhibitor vinpocetine (2 µM) or selective phosphodiesterase-3 inhibitor cilostamide (2 µM) for 30 min prior to an additional 24 h incubation in the absence or presence of desmopressin (VP, 10⁻⁹ M). Data is represented as fold induction over untreated cells transfected with scramble siRNA and is expressed as the mean ± SEM of three independent experiments. *P≤0.05; NS: no significant differences.</p

Comparison of Student's t-test values between stimulated groups [cells transfected with siNOX4 and/or challenged with desmopressin (VP)] and control groups (Ctl) of data depicted in Table 2.

<p>Comparison of Student's t-test values between stimulated groups [cells transfected with siNOX4 and/or challenged with desmopressin (VP)] and control groups (Ctl) of data depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087239#pone-0087239-t002" target="_blank">Table 2</a>.</p

TonEBP and NF-κB activity is not affected by NOX4 depletion.

<p>mpkCCD_cl4 cells transfected with either scramble siRNA or siNOX4 were challenged for 24 h with either desmopressin (VP, 10⁻⁹ M) or NaCl-hypertonic medium (500 mOsmol/kg). (A) Q-PCR analysis of aldose reductase (AR), sodium-myo-inositol cotransporter (SMIT) and sodium-chloride-betaine cotransporter (BGT1) was performed for investigation of TonEBP activity. (B) Q-PCR analysis of TNFα and IκBα was performed for investigation of NF-κB activity. Data is represented as fold induction over non-stimulated cells transfected with scramble siRNA and is expressed as the mean ± SEM of three independent experiments.</p