Connexin 37, 40, 43 and Pannexin 1 Expression in the Gastric Mucosa of Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis.

Expression of Connexins 37, 40 and 45, Pannexin 1 and Vimentin in Laryngeal Squamous Cell Carcinomas.

Approximately 60% of patients with squamous cell carcinoma (LSCC) have regional occult metastatic disease/distant metastases at the time of diagnosis, putting them at higher risk for disease progression. Therefore, biomarkers are needed for early prognostic purpose. The aim of this study was to analyze the expression pattern of connexins (Cx) 37, 40 and 45, pannexin1 (Panx1) and vimentin in LSCC and correlate with tumor grade (G) and outcome. METHODS: Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. RESULTS: The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. CONCLUSION: Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC.

Connexin37 Regulates Cell Cycle in the Vasculature.

Control of vascular cell growth responses is critical for development and maintenance of a healthy vasculature. Connexins - the proteins comprising gap junction channels - are key regulators of cell growth in diseases such as cancer, but their involvement in controlling cell growth in the vasculature is less well appreciated. Connexin37 (Cx37) is one of four connexin isotypes expressed in the vessel wall. Its primary role in blood vessels relies on its unique ability to transduce flow-sensitive signals into changes in cell cycle status of endothelial (and perhaps, mural) cells. Here, we review available evidence for Cx37's role in the regulation of vascular growth, vessel organization, and vascular tone in healthy and diseased vasculature. We propose a novel mechanism whereby Cx37 accomplishes this with a phosphorylation-dependent transition between closed (growth-suppressive) and multiple open (growth-permissive) channel conformations that result from interactions of the C-terminus with cell-cycle regulators to limit or support cell cycle progression. Lastly, we discuss Cx37 and its downstream signaling as a novel potential target in the treatment of cardiovascular disease, and we address outstanding research questions that still challenge the development of such therapies.

The Expression of Connexin 37, 40, 43, 45 and Pannexin 1 in the Early Human Retina and Choroid Development and Tumorigenesis.

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid. Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma. In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma. Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma. Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye. It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma. Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response. Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism. Additionally, Cx43 might be a potential biomarker of tumor prognosis.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 µg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 µg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

PAR1, a therapeutic target for remote lung injury associated with hind limb ischemia/reperfusion: ERK5/KLF2-dependent lung capillary barrier preservation.

Protease-activated receptor 1 (PAR1) is expressed in pneumocytes and endothelial cells of the alveolar barrier. Its activation by thrombin disrupts the barrier integrity dynamics and induces lung injury in in vitro and in vivo paradigms. Nonetheless, the role of PAR1, as a therapeutic target, in hind limb ischemia/reperfusion (I/R)-mediated remote lung injury has been unclear. Therefore, this study aimed to determine the potential benefit of PAR1 blockade using the selective antagonist SCH79797 in distant lung dysfunction following hind limb I/R injury with special emphasis on the extracellular signal-regulated kinase 5 (ERK5)/Krüppel-like factor 2 (KLF2) axis. Rats were subdivided into control, bilateral hind limb I/R, SCH79797, and SCH79797+BIX02189 (ERK5 inhibitor) groups. PAR1 blockade, ERK5-dependently, alleviated alveolar barrier disruption as evidenced by reductions in both pulmonary systemic leakage of surfactant protein-D and lung fluid accumulation with increase in pulmonary claudin 5, vascular endothelial cadherin, and connexin 37 levels. Such improvements are downstream targets of the ERK5/KLF2-mediated sphingosine-1-phosphate receptor 1 (S1PR1) upregulated expression and pS536-nuclear factor-κB (NF-κB) p65 inhibition. SCH79797 effectively impedes the evoked inflammatory response and oxidative burst by suppressing vascular endothelial growth factor, tumor necrosis factor-α, lipid peroxidation, and neutrophil infiltration while boosting the glutathione antioxidant defense. Accordingly, PAR1 could be a therapeutic target, where its blockade mitigated pulmonary-endothelial barrier disruption via mutual S1PR1 enhancement and NF-κB p65 inhibition following ERK5/KLF2 activation.

Effects of Elevated Downstream Pressure and the Role of Smooth Muscle Cell Coupling through Connexin45 on Lymphatic Pacemaking.

Lymphatic vessels rely on spontaneous lymphatic muscle cell (LMC) contractions and one-way intraluminal valves to efficiently pump lymph and return it into the bloodstream. Intraluminal pressure is known to regulate the contractile function of lymphatics, with pressure elevation leading to increased contraction frequency and decreased amplitude. Contractions are normally initiated by a dominant pacemaker and are highly entrained among strongly coupled LMCs. Previously, we found that connexin45 is the major connexin isoform mediating LMC-LMC electrical coupling. Lymphatics from mice lacking smooth muscle connexin45 display uncoordinated, impaired contractions. Here, we utilized this connexin45-deficient model, pressure myography, and recently developed, novel analytical tools to assess the effects of elevated downstream pressure on the number, location, and frequency of lymphatic pacemakers. Our results show that, in vessels from healthy controls, an increase in downstream pressure resulted in the recruitment/development of new pacemakers and increased contractile frequency while a dominant pacemaker continued to be observed. In contrast, vessels from connexin45-deficient mice displayed significantly more pacemakers, but none were dominant; this worsened with elevated downstream pressure. These results suggest a potential protective mechanism through which the lymphatic vasculature adapts to transient increases in downstream pressure, but which may not be sustained in scenarios with chronic elevated downstream pressure.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Simplified method to quantify valve back-leak uncovers severe mesenteric lymphatic valve dysfunction in mice deficient in connexins 43 and 37.

KEY POINTS: Lymphatic valve defects are one of the major causes of lymph transport dysfunction; however, there are no accessible methods for quantitatively assessing valve function. This report describes a novel technique for quantifying lymphatic valve back-leak. Postnatal endothelial-specific deletion of connexin 43 (Cx43) in connexin 37 null (Cx37-/- ) mice results in rapid regression of valve leaflets and severe valve dysfunction. This method can also be used for assessing the function of venous and lymphatic valves from various species, including humans. ABSTRACT: The lymphatic system relies on robust, spontaneous contractions of collecting lymphatic vessels and one-way secondary lymphatic valves to efficiently move lymph forward. Secondary valves prevent reflux and allow for the generation of propulsive pressure during each contraction cycle. Lymphatic valve defects are one of the major causes of lymph transport dysfunction. Genetic mutations in multiple genes have been associated with the development of primary lymphoedema in humans; and many of the same mutations in mice result in valve defects that subsequently lead to chylous ascites or chylothorax. At present the only experimental technique for the quantitative assessment of lymphatic valve function utilizes the servo-null micropressure system, which is highly accurate and precise, but relatively inaccessible and difficult to use. We developed a novel, simplified alternative method for quantifying valve function and determining the degree of pressure back-leak through an intact valve in pressurized, single-valve segments of isolated lymphatic vessels. With this diameter-based method, the competence of each lymphatic valve is challenged over a physiological range of pressures (e.g. 0.5-10cmH2 O) and pressure back-leak is extrapolated from calibrated, pressure-driven changes in diameter upstream from the valve. Using mesenteric lymphatic vessels from C57BL/6J, Ub-CreERT2 ;Rasa1fx/fx , Foxc2Cre/+ , Lyve1-Cre;Cx43fx/fx , and Prox1-CreERT2 ;Cx43fx/fx ;Cx37-/- mice, we tested our method on lymphatic valves displaying a wide range of dysfunction, from fully competent to completely incompetent. Our results were validated by simultaneous direct measurement of pressure back-leak using a servo-null micropressure system. Our diameter-based technique can be used to quantify valve function in isolated lymphatic valves from a variety of species. This method also revealed that haplodeficiency in Foxc2 (Foxc2Cre/+ ) is not sufficient to cause significant valve dysfunction; however, postnatal endothelial-specific deletion of Cx43 in Cx37-/- mice results in rapid regression of valve leaflets and severe valve dysfunction.

Impaired SMAD1/5 Mechanotransduction and Cx37 (Connexin37) Expression Enable Pathological Vessel Enlargement and Shunting.

OBJECTIVE: Impaired ALK1 (activin receptor-like kinase-1)/Endoglin/BMP9 (bone morphogenetic protein 9) signaling predisposes to arteriovenous malformations (AVMs). Activation of SMAD1/5 signaling can be enhanced by shear stress. In the genetic disease hereditary hemorrhagic telangiectasia, which is characterized by arteriovenous malformations, the affected receptors are those involved in the activation of mechanosensitive SMAD1/5 signaling. To elucidate how genetic and mechanical signals interact in AVM development, we sought to identify targets differentially regulated by BMP9 and shear stress. Approach and Results: We identify Cx37 (Connexin37) as a differentially regulated target of ligand-induced and mechanotransduced SMAD1/5 signaling. We show that stimulation of endothelial cells with BMP9 upregulated Cx37, whereas shear stress inhibited this expression. This signaling was SMAD1/5-dependent, and in the absence of SMAD1/5, there was an inversion of the expression pattern. Ablated SMAD1/5 signaling alone caused AVM-like vascular malformations directly connecting the dorsal aorta to the inlet of the heart. In yolk sacs of mouse embryos with an endothelial-specific compound heterozygosity for SMAD1/5, addition of TNFα (tumor necrosis factor-α), which downregulates Cx37, induced development of these direct connections bypassing the yolk sac capillary bed. In wild-type embryos undergoing vascular remodeling, Cx37 was globally expressed by endothelial cells but was absent in regions of enlarging vessels. TNFα and endothelial-specific compound heterozygosity for SMAD1/5 caused ectopic regions lacking Cx37 expression, which correlated to areas of vascular malformations. Mechanistically, loss of Cx37 impairs correct directional migration under flow conditions. CONCLUSIONS: Our data demonstrate that Cx37 expression is differentially regulated by shear stress and SMAD1/5 signaling, and that reduced Cx37 expression is permissive for capillary enlargement into shunts.

Relationship between SNP rs1764391 and Susceptibility, Risk Factors, Gene-environment Interactions of Acute Myocardial Infarction in Guangxi Han Chinese Population.

BACKGROUND: Large-scale population studies showed that the SNP rs1764391 of Connexin37 gene also known as Cx37 gene may play a pivotal role in the occurrence and development of acute myocardial infarction (AMI). Published results, however, are highly controversial. OBJECTIVE: This study aimed to examine the association between SNP rs1764391 of Cx37 and diseasesusceptibility, several risk factors, and gene-environment interactions of AMI in Guangxi Han Chinese population. METHODS: In this study, 344 healthy controls and 344 AMI patients of Han Chinese population were enrolled. The TaqMan assay was implemented to identify genotypes of Cx37 and allele frequencies of SNP rs1764391 in both the AMI and control groups. RESULTS: Significant differences were detected in TT genotype frequencies of SNP rs1764391 between the AMI and control groups (P < 0.05). In the context of gender stratification, the result was also statistically different in women (P < 0.05). Each variable such as age, BMI, diabetes, high blood pressure, smoking and TC was a risk factor and correlated significantly (P < 0.05) with the development of AMI. HDL-C correlated negatively with the risk of AMI (P < 0.001). BMI, smoking or alcohol consumed interacts significantly (P < 0.017) with the presence of the SNP rs1764391 CC genotype. CONCLUSION: Evidences were presented that Cx37 rs1764391 variation may contribute to the risk for AMI, especially in women and this genetic variant may prove to be a potential biomarker for AMI risk stratification and may prove to be a useful target for therapeutic intervention to further improve prognosis in high-risk patients.

Polymorphism of CONNEXIN37 gene is a risk factor for ischemic stroke in Han Chinese population.

BACKGROUND: Stroke has a high fatality and disability rate, and is one of the main burdens to human health. It is thus very important to identify biomarkers for the development of effective approaches for the prevention and treatment of stroke. Connexin37 is an anti-inflammatory cytokine and is involved in chronic inflammation and atherosclerosis. Recent studies have found that CONNEXIN37 gene variations are associated with atherosclerosis diseases, such as coronary heart disease and stroke, but its association with stroke in distinct human populations remains to be determined. We report here the analysis of the association of the single nucleotide polymorphisms (SNPs) of CONNEXIN37 with ischemic stroke in Han Chinese population. METHODS: Two SNPs of CONNEXIN37 gene were analyzed in 385 ischemic stroke patients and 362 hypertension control patients using ligase detection reaction (LDR) method. RESULTS: Logistic regression analysis demonstrated that, AG and GG genotypes of SNP rs1764390 and CC genotype of rs1764391 of CONNEXIN37 were associated with an increased risk of ischemic stroke, and that G allele of rs1764390 is a risk factor for ischemic stroke. Further, we found that SNP rs1764390 and SNP rs1764391 in CONNEXIN37 were associated with ischemic stroke under additive/dominant model, and recessive/dominant model, respectively. CONCLUSION: Our results indicate that CONNEXIN37 gene polymorphism is an ischemic stroke risk factor in Northern Han Chinese.

GJA4/Connexin 37 Mutations Correlate with Secondary Lymphedema Following Surgery in Breast Cancer Patients.

Lymphedema is a condition resulting from mutations in various genes essential for lymphatic development and function, which leads to obstruction of the lymphatic system. Secondary lymphedema is a progressive and incurable condition, most often manifesting after surgery for breast cancer. Although its causation appears complex, various lines of evidence indicate that genetic predisposition may play a role. Previous studies show that mutations in connexin 47 are associated with secondary lymphedema. We have tested the hypothesis that connexin 37 gene mutations in humans are associated with secondary lymphedema following breast cancer surgery. A total of 2211 breast cancer patients were screened and tested for reference single nucleotide polymorphisms (SNPs) of the GJA4 gene (gap junction protein alpha 4 gene). The results presented in this paper indicate that two SNPs in the 3' UTR (the three prime untranslated region) of the GJA4 gene are associated with an increased risk of secondary lymphedema in patients undergoing breast cancer treatment. Our results provide evidence of a novel genetic biomarker for assessing the predisposition to secondary lymphedema in human breast cancer patients. Testing for the condition-associated alleles described here could assist and inform treatment and post-operative care plans of breast cancer patients, with potentially positive outcomes for the management of disease progression.

Polymorphisms in Gap Junction Proteins and their Role in Predisposition of Acute Myocardial Infarction in Egyptians.

BACKGROUND: Connexin (Cx) proteins are the building blocks of gap junctions. Among these, Cx37 and Cx40 are expressed on vascular system and reported to have cardioprotective role. Linking polymorphisms in genes coding for Cx and coronary artery disease (CAD) risk showed conflicting results in different populations. None has been studied before in Egyptians. Therefore, the aims of this study were to investigate the influence of Cx37 C1019T and Cx40 A71G polymorphisms on the predisposition of acute myocardial infarction (AMI) in Egyptians, to study linkage disequilibrium (LD) and combined effects of single nucleotide polymorphisms (SNPs) and to correlate the genotypes with sVCAM-1 serum levels. METHODS: Total of 201 Egyptian subjects were recruited for the study. They were divided into 104 AMI patients and 97 healthy controls. Genotypes for each participant were determined using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum sVCAM-1was measured by ELISA. RESULTS: Allele frequencies for both Cx37 and Cx40 were not significantly different between AMI and Controls (p=0.93 and p=0.26 respectively). Moreover, studying the dominant and recessive models concluded that none of the genotypes was a risk factor. Both SNPs were not in LD (R2=0.0027). Serum analysis showed higher levels of sVCAM-1 in AMI patients (p<0.0001). sVCAM-1 levels were not significantly different among SNPs (Cx37; p=0.244 and Cx40; p=0.266). CONCLUSION: This study shows that Cx37 C1019T and Cx40 A71G polymorphisms are not associated with cardioprotective role in Egyptians. Moreover, both SNPs are inherited separately and none of the genotypes were associated with higher sVCAM-1 levels.

NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal Spreading: A Novel Role for Cx37 (Connexin 37) and the Protein Tyrosine Phosphatase SHP-2.

OBJECTIVE: Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. APPROACH AND RESULTS: Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). CONCLUSIONS: Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.

C1019T Polymorphism in the Connexin 37 Gene and Myocardial Infarction Risk in Premature Coronary Artery Disease.

Background: The C1019T polymorphism of the connexin-37 (GJA4) gene is a single-nucleotide polymorphisms involved in atherosclerotic plaque rupture and atherosclerosis predisposition. We examined the association between the C1019T polymorphism of the GJA4 gene and the occurrence of myocardial infarction (MI) in patients with premature coronary artery disease (CAD). Methods: Our study recruited 1000 patients with the final diagnosis of premature CAD and classified them into 2 groups: with a history of MI (n = 461) and without it (n = 539). The polymorphism variants were determined via the PCR-RFLP, and then genotyping was conducted through the high-resolution melting method. From a total of 1000 patients, 554 patients, who had been previously followed-up with a median follow-up time of 45.74 months vis-à-vis long-term major adverse cardiac events, were enrolled in this retrospective cohort phase. Results: The frequencies of the wild, heterozygous, and mutant genotypes of the C1019T polymorphism were 54.0%, 40.6%, and 5.4% in the MI group and 49.2%, 43.2%, and 7.6% in the non-MI group (p value = 0.187). After adjustment for the baseline covariates, no difference was found between the MI and non-MI groups apropos the frequency of the heterozygous genotype (p value = 0.625) and the mutant genotype (p value = 0.452). Regarding the level of human connexin-37, the serum level of this marker was not different between the MI and non-MI groups. Conclusion: The C1019T polymorphism of the GJA4 gene may not be useful for predicting the occurrence of MI in patients with premature CAD. The presence of this polymorphism in such patients may also have a low value for predicting long-term CAD complications.

Connexin37 deficiency alters organic bone matrix, cortical bone geometry, and increases Wnt/ß-catenin signaling.

Deletion of connexin (Cx) 37 in mice leads to increased cancellous bone mass due to defective osteoclast differentiation. Paradoxically; however, Cx37-deficient mice exhibit reduced cortical thickness accompanied by higher bone strength, suggesting a contribution of Cx37 to bone matrix composition. Thus, we investigated whether global deletion of Cx37 alters the composition of organic bone extracellular matrix. Five-month-old Cx37-/- mice exhibited increased marrow cavity area, and periosteal and endocortical bone surface resulting in higher total area in tibia compared to Cx37+/+ control mice. Deletion of Cx37 increased genes involved in collagen maturation (loxl3 and loxl4) and glycosaminoglycans- (chsy1, chpf and has3) proteoglycans- associated genes (biglycan and decorin). In addition, expression of type II collagen assessed by immunostaining was increased by 82% whereas collagen maturity by picrosirius-polarizarion tended to be reduced (p=0.071). Expression of glycosaminoglycans by histochemistry was decreased, whereas immunostaining revealed that biglycan was unchanged and decorin was slightly increased in Cx37-/- bone sections. Consistent with these in vivo findings, MLO-Y4 osteocytic cells silenced for Cx37 gene exhibited increased mRNA levels for collagen synthesis (col1a1 and col3a1) and collagen maturation (lox, loxl1 and loxl2 genes). Furthermore, mechanistic studies showed Wnt/ß-catenin activation in MLO-Y4 osteocytic cells, L5 vertebra, and authentic calvaria-derived osteocytes isolated by fluorescent-activated cell sorter. Our findings demonstrate that altered profile of the bone matrix components in Cx37-deficient mice acts in favor of higher resistance to fracture in long bones.

Protective effects of connexins in atheromatous plaques in patients of carotid artery stenosis.

Fragility of atheromatous plaque in the internal carotid artery can be a risk of brain infarction. The activation of macrophages by oxidative stress and the vulnerability of vascular endothelial cells have been reported to participate in the fragility of atheromatous plaque. Therefore, from the view point of prevention of brain infarction, we investigated the pathological factors which may influence the stabilization of atheromatous plaque. Patients undertaking carotid endoarterectomy (CEA) were continuously screened. Then, 21 samples were obtained from the atheromatous plaques of CEA patients. The expression of connexin (Cx) which composes a gap junction, an intercellular communication organ, was immunohistochemicaly observed. The expression of CD36, an oxidized low-density lipoprotein receptor, was assessed as a marker of oxidative stress. As a result, asymptomatic plaques which were assumed the stable plaques expressed Cx43 along with CD36 expression. In contrast, in the symptomatic plaques, the expression of Cx43 was few and there was almost no coexpression with CD36. The distribution of Cx37 expression was not different between asymptomatic and symptomatic plaques. The expressions of CD36, Cx37 and Cx43 showed no relation to the previous treatment with statins. In conclusion, Cx43 might contribute to the stabilization of atheromatous plaque which is affected by oxidative stress.

Impact of Cx37 on the current density of mononuclear macrophage in atherosclerosis / 医学研究生学报

Objective Little research has been done on how Cx37 changes the current density of mononuclear macrophage in atherosclerosis.The purpose of this study was to detect the effects of Cx37 on the current density of mononuclear macrophage in atherosclerosis.Methods A total of 30 Wistar mice were randomly divided into Cx37+ group and Cx37-group equally.The atherosclerosis model was constructed by a high-fat diet.According to different parts of sample collection, these two groups were subdivided into Cx37+ plaque group, Cx37-plaque group, Cx37+blood group and Cx37-blood group.RT-PCR was applied to detect the expression of Cx37 in different body parts.The mononuclear macrophages were cultured after being separated from blood and plague in both groups.The current density of mononuclear macrophage was detected by the whole cell recording.Results The relative expression of Cx37 in Cx37 + plaque group was higher than that in plaque group ([1.10±0.02] vs [0.60±0.03]).Energy Spectrum CT was used to detect the carotid artery plaque in both Cx37 + and Cx37-groups, which verified the successful model construction.At 80,120 and 160ms, the current density in Cx37 + plaque group([0.61± 0.06], [0.67±0.07], [0.91±0.03]A/cm2) was significantly higher than those in Cx37 + blood group([0.49±0.02], [0.61±0.03], [0.67±0.02]A/cm2) , Cx37-plaque group([0.48±0.02], [0.60±0.02], [0.64±0.02]A/cm2) and Cx37-blood group([0.49±0.02], [0.59±0.02], [0.64±0.02]A/cm2).The same goes for those at 200, 240, 320ms(P<0.05).Conclusion Cx37 has more significant impact on the current density in the plaque of mononuclear macrophage than in the peripheral blood in promoting macrophages activation and atherosclerosis progress.