Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus.

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke.

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli-induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFß-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFß-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 (col1a1). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFß-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFß-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFß signaling and have implications for our understanding of MSC function in fibrosis.

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3.

The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC's cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC's cytoprotective versus thrombin's proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects.

Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46.

Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas ß-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3ß phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1.

Antibody SPC-54 provides acute in vivo blockage of the murine protein C system.

Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (P<0.001). In summary, monoclonal antibody SPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

In vitro effects of combining Mim8 with factor VIII, FVIIa, and activated prothrombin complex concentrates in thrombin generation assays.

BACKGROUND: Mim8 is a novel antifactor IXa/antifactor X bispecific antibody in clinical development for prophylactic treatment of hemophilia A with and without inhibitors. Patients treated with Mim8 may need supplementary bleed treatment under certain conditions such as surgery or major trauma. OBJECTIVES: This study aimed to better understand the response of Mim8 in thrombin generation assays (TGAs) alone or in combination with other hemostatic proteins. METHODS: We used TGAs with different activators (tissue factor (TF) and activated factor XI) to better understand the similarities and differences between the mode of action of Mim8 and factor VIII (FVIII). Following this, we investigated the effects of mixing Mim8 with the main bleed treatment options for persons with hemophilia A with or without inhibitors: FVIII, activated factor VII (FVIIa), and activated prothrombin complex concentrates (aPCC). RESULTS: The results indicated that for patients without inhibitors, Mim8 does not interfere with FVIII's mode of action. For patients with inhibitors, Mim8 mixed with aPCC results in a strong synergistic effect causing thrombin generation far exceeding the normal levels. Contrary to this, mixing Mim8 with FVIIa results in a more controlled additive effect, visible only when using TF as a trigger, which does not exceed the normal level of thrombin generation. CONCLUSION: These findings support the use of approved clinical doses of FVIIa for bleed treatment of patients with FVIII inhibitors treated with Mim8. Additionally, the findings suggest that concomitant use of FVIII and Mim8 is safe for managing breakthrough bleeds.

Connexin 37 limits thrombus propensity by downregulating platelet reactivity.

BACKGROUND: Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. METHODS AND RESULTS: In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37(-/-) platelets or in murine Cx37(+/+) and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37(-/-) platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37-1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37-1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37-1019C channels for neurobiotin. CONCLUSIONS: We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Gas6 deficiency in recipient mice of allogeneic transplantation alleviates hepatic graft-versus-host disease.

Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6(-/-) mice received allogeneic non-T cell-depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6(-/-) recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6(-/-) recipients' liver. When mice received 0.5 x 10(6) allogeneic T cells with T cell-depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6(-/-) than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6(-/-) T-cell proliferation. We therefore assessed the response of WT or Gas6(-/-) ECs to tumor necrosis factor-alpha. Lymphocyte transmigration was less extensive through Gas6(-/-) than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.

Role of Gas6 in erythropoiesis and anemia in mice.

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.

Unexpected role for the human Cx37 C1019T polymorphism in tumour cell proliferation.

Connexins are a large family of proteins that form gap junction channels allowing exchange of ions and small metabolites between neighboring cells. They have been implicated in pathological processes such as tumourigenesis in which they may act as tumour suppressors. A polymorphism in the human connexin37 (Cx37) gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus (CT) of the Cx37 protein (P319S) has been suggested to be implicated in predisposition to angiosarcomas. In this study, we have used communication-deficient HeLa and SK-HEP-1 cells transfected with Cx37-319S, Cx37-319P or empty vector. We showed that the expression of Cx37-319P limited proliferation of HeLa and SK-HEP-1 cells, whereas Cx37-319S expression was without effect. Using an in vitro kinase assay, we demonstrated phosphorylation of Cx37 CT by glycogen synthase kinase-3 (GSK-3), a kinase known to be implicated in cell proliferation and cancer. GSK-3-induced phosphorylation was associated with reduced gap junctional intercellular communication (GJIC) as measured by microinjection of the tracer neurobiotin. Inhibition of GSK-3 by LiCl or SB415286 reduced phosphorylation of Cx37-319P and increased GJIC. This latter effect on GJIC involved the beta and not the alpha isoform of GSK-3. In contrast, GSK-3 inhibitors were without effect on HeLa cells expressing Cx37-319S. In conclusion, our data indicate functional effects of the Cx37 C1019T polymorphism on GJIC that might contribute to tumour cell growth.

Cell-derived microparticles in haemostasis and vascular medicine.

Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic response and their potential as disease markers, but also their implication in a wide range of physiological and pathological processes. They derive from different cell types including platelets - the main source of microparticles - but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, including ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine.

Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy.

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outside-in" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the beta3 integrin, thereby amplifying outside-in signaling via alphaIIbbeta3. Blocking the Gas6-R-alphaIIbbeta3 integrin cross-talk might be a novel approach to the reduction of thrombosis.

Physiological cerebrovascular remodeling in response to chronic mild hypoxia: A role for activated protein C.

Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5ß1 and αvß3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.

Study of Early Elevated Gas6 Plasma Level as a Predictor of Mortality in a Prospective Cohort of Patients with Sepsis.

BACKGROUND: Growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein interacting with anionic phospholipids and TAM tyrosine kinase receptors, is elevated in plasma of septic patients. Previous studies did not find different levels between survivors and non-survivors at admission because either they included a low number of patients (<50) or a low number of non-survivors (5%). OBJECTIVES: To determine, in a larger cohort of septic patients comprising an expected number of non-survivors, the performance of the plasma level of Gas6 and its soluble receptor Axl (sAxl) within 24 hours of admission to predict in-ICU mortality. PATIENTS: Septic adults with or without shock. METHODS: Gas6 and sAxl were prospectively measured by ELISA at day 0, 3, 7, and then weekly until discharge or death. RESULTS: We evaluated 129 septic patients, including 82 with and 47 without shock, with in-ICU mortality rate of 19.4% and in-hospital mortality rate of 26%. Gas6 level was higher in non-survivors than in survivors (238 vs. 167%, P = 0.003); this difference remained constant during the ICU stay. The area under the ROC curve for Gas6 (0.695 [95% CI: 0.58-0.81]) was higher than for sAxl, procalcitonin, CRP, IL-1beta, IL-6 and-alpha, and slightly higher than for IL-8, IL-10, SOFA and APACHEII scores in predicting in-ICU mortality. Considering 249% as a cut-off value, Gas6 measurement had a negative predictive value for mortality of 87%. CONCLUSION: It seems that Gas6 plasma level within 24 hours of ICU admission may predicts in-ICU mortality in patients with sepsis. If our result are confirmed in external validation, Gas6 plasma level measurement could contribute to the identification of patients who may benefit most from more aggressive management.

An engineered factor Va prevents bleeding induced by anticoagulant wt activated protein C.

OBJECTIVE: An increased risk of bleeding is observed in patients receiving activated protein C (APC), which may be a limiting factor for the application of novel APC therapies. Since APC's therapeutic effects often require its cytoprotective activities on cells but not APC's anticoagulant activities, an agent that specifically antagonizes APC's anticoagulant effects but not its cytoprotective effects could provide an effective means to control concerns for risk of bleeding. We hypothesized that superFVa, an engineered activated FVa-variant that restores hemostasis in hemophilia could reduce APC-induced bleeding. APPROACH AND RESULTS: SuperFVa was engineered with mutations of the APC cleavage sites (Arg506/306/679Gln) and a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains, which augment its biological activity and cause high resistance to APC. SuperFVa normalized APC-prolonged clotting times and restored APC-suppressed thrombin generation in human and murine plasma at concentrations where wild-type (wt) FVa did not show effects. Following intravenous injection of APC into BALB/c mice, addition to whole blood ex vivo of superFVa but not wt-FVa significantly normalized whole blood clotting. Blood loss following tail clip or liver laceration was significantly reduced when superFVa was administered intravenously to BALB/c mice prior to intravenous APC-treatment. Furthermore, superFVa abolished mortality (∼50%) associated with excessive bleeding following liver laceration in mice treated with APC. CONCLUSIONS: Our results provide proof of concept that superFVa is effective in preventing APC-induced bleeding and may provide therapeutic benefits as a prohemostatic agent in various situations where bleeding is a serious risk.

Connexins participate in the initiation and progression of atherosclerosis.

Connexins are members of a large family of transmembrane proteins that form hemichannels or gap junctions. These channels allow the exchange of ions and small metabolites between the cytosol and extracellular space or between neighboring cells. Connexins are important in vascular physiology; they support radial and longitudinal cell-to-cell communication in the vascular wall. Four connexins are expressed in the vascular wall: Cx37, Cx40, Cx43, and Cx45. Their expression is not uniform in all blood vessels and varies with vascular territory and species. Significant changes in the expression pattern of vascular connexins have been described during the development of atherosclerosis, a progressive inflammatory disease. In this review, we provide an overview of (1) the tools used to study the involvement of connexins in atherosclerosis, (2) the participation of connexins in atherogenesis, (3) the increasing interest of a polymorphism in the human connexin37 gene as marker of cardiovascular disease, and (4) the possible therapeutic implications of connexins.

Intercellular communication in atherosclerosis.

Cell-to-cell communication is a process necessary for physiological tissue homeostasis and appears often altered during disease. Gap junction channels, formed by connexins, allow the direct intercellular communication between adjacent cells. After a brief review of the pathophysiology of atherosclerosis, we will discuss the role of connexins throughout the different stages of the disease.

Molecular role of Cx37 in advanced atherosclerosis: a micro-array study.

Recently, we showed that connexin37 (Cx37) protects against early atherosclerotic lesion development by regulating monocyte adhesion. The expression of this gap junction protein is altered in mouse and human atherosclerotic lesions; it is increased in macrophages newly recruited to the lesions and disappears from the endothelium of advanced plaques. To obtain more insight into the molecular role of Cx37 in advanced atherosclerosis, we used micro-array analysis for gene expression profiling in aortas of ApoE(-/-) and Cx37(-/-)ApoE(-/-) mice before and after 18 weeks of cholesterol-rich diet. Out of >15,000 genes, 106 genes were significantly differentially expressed in young mice before diet (P-value of <0.05, fold change of >0.7 or <-0.7, and intensity value >2.2 times background). Ingenuity pathway analysis (IPA) revealed differences in genes involved in cell-to-cell signaling and interaction, cellular compromise and nutritional disease. In addition, we identified 100 genes that were significantly perturbed after the cholesterol-rich diet. Similar to the analysis on 10-week-old mice, IPA revealed differences in genes involved in cell-to-cell signaling and interaction as well as to immuno-inflammatory disease. Furthermore, we found important changes in genes involved in vascular calcification and matrix degradation, some of which were confirmed at protein level by (immuno-)histochemistry. In conclusion, we suggest that Cx37 deficiency alters the global differential gene expression profiles in young mice towards a pro-inflammatory phenotype, which are then further influenced in advanced atherosclerosis. The results provide new insights into the significance of Cx37 in plaque calcification.