The Role Of Connexins In The Pathogenesis Of Pulmonary Arterial Hypertension

The role of connexins in the pathogenesis of pulmonary arterial hypertensionObjective: To explore the role of connexins (Cxs) in the pathogenesis of pulmonary arterial hypertension (PAH)induced by monocrotaline (MCT) in rats.Methods: In order to establish the model of PAH, the rats were received an intraperitoneal (IP) injection of MCT (60mg/kg). Two weeks later, they were randomly assigned, this resulted in the following 3 groups: Group A: normal rats with vehide treatment (n=10), the vehicle was gummi arabicum 5% which was gavaged (4ml/kg/day) once dally for a 3-week period. Group B: MCT＋vehide (n=35), the vehicle was gummi arabicum 5% which was gavaged (4ml/kg/day) once daily for a 3-week period. Group C: MCT＋Bosentan (n=22), Bosentan (100mg/kg/day) which was prepared in gummi arabicum 5% (4ml/kg/day) was gavaged once dally for a 3-week period. In every group, we measured the hernodynamic parameters, the ratio of medial wall thickness to external diameter of small pulmonary arteries (%WT) by (ET＋VG) stain (five-micron sections) and light microscopy, the pulmonary tissuse sections were stained by HE (five-micron) and the morphorlogical characteristics of small pulmonary arteries were observed by light microscopy, the pulmonary tissuse sections were stained by uranyl acetate and Pb(NO₃)₂ (100nm) and the ultrastruture of small pulmonary arteries was observed by electromicroscopy. Then we investigated the changes of C×43 and C×37 expression in smooth muscle and endothelial ceils of the small pulmonary arteries using immunofluorescence (sixmicron sections) in every group.Results:â‘ Five-week survival: Five-week survival in group A,B and C was 100%(10/10),31.43% (11/35) and 45.45% (10/22) respectively, with thechi-square test/exact probabilities in 2×2 table, there was no significant diference between group B and C (0.25＜P＜0.50), there was significant diference between group A and B,C (P=0.000).â‘¡Hemodynamic measurements: Mean pulmonary arterial pressure in group A,B and C was 16.96±2.59mmHg,26.55±6.80mmHg and 20.27±6.48mmHg respectively, with one-way ANOVA, there was significant diference between group A and B (P=0.002), there was no significant diference between group B and C (P=0.124), so was between group A and C (P=0.407). Right atrial pressure in group A,B and C was 0.87±0.71mmHg,9.71±5.10mmHg and 6.70±2.26mmHg respectively, with one-way ANOVA, there was significant diference between groupA and B (P=0.001), so was between group A and C (P=0.000), therewas no significant diference between group B and C (P= 0.265). Mean jugular arterial pressure in groupA,B and C was 130.15±22.95mmHg,57.98±6.89mmHg and 80.28±10.01mmHg respectively, with one-way ANOVA, there was significant diference between group A and B (P=0.000),B and C (P=0.000), so was between group A and C (P=0.000).â‘¢% VVT: % WT in group A,B and C was21.10±2.99%,34.36±2.84%and 26.85±3.61% respectively, with one-way ANOVA, therewas significant diference between group A and B (P=0.000),B and C (P=0.000), so was between group A and C (P=0.000).â‘£Changes of morphorlogical characteristics by light microscopy: The wall of small pulmonary arteries was thin and regular in group A; but in group B the intima of small pulmonary arteries proliferated and the media thickened duo to increased smooth muscle cells and disatic fibers, which caused progressive occlusion of the vessel lumen; the changes of small pulmonary arteries in group C were between group A and B.⑤Changes of ultrastruture by electromicroscopy:â… . Intima of small pulmonary arteries: In group A, the endothelial cells were flat; in group B, the endothelial cells which were cubical sticked up, cell nuclei became larger and lobulated, part of intima sticked up into the vessel lumen, basilar membrane thickened; the changes of intima in group C were between group A and B.â…¡. Media of small pulmonary arteries: In group A, there were few layers (0～2) of smooth muscle cells; in group B, the media thickened markedly with 3～4 layers of smooth muscle cells which became larger; the changes of media in group C were between group A and B.â…¢. Gap junctions in the endothelial cells of small pulmonary arteries: In the three groups, gap junctions all were detected in the endothelial cells, but gap junctions were less distinct in group B and C than those in group A.â‘¥C×43 and C×37 expression in the smooth muscle and endothelial cells of small pulmonary arteries using immunofluorescence: C×43 was not detected in the endothelial cells in group A, but C×43 was both detected in group B and C, and the latter was less than the former. In all the three groups, C×37 was detected in the endothelial cells and C×43 were not detected in the smooth muscle cells. In all the three groups, C×37 expression in the smooth muscle cells was not affirmative.Conclusion:â‘ Bosentan is effective in the treatment of MCT-induced PAH.â‘¡In addition to inhibiting the proliferation of pulmonary vascular smooth muscle cells and vasoconstriction of pulmonary vasculature which we have known, inhibiting the expression of C×43 in the endothelial cells of small pulmonary arteries may be one of the mechanisms for favorable effects of Bosentan on PAH.â‘¢There are the grounds for suggesting that the abnormal Cx43 expression in the endothelial cells of small pulmonarv arteries plavs a very important role i n the pathogenesi s of PAH. The role of connexin in the pathogenesis of right ventricular remodelingObjective: To explore the role of connexin43 (Cx43) i n the pathogenesis of right ventricular (RV) remodeling with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats.Methods: In order to establish the model of PAH, the rats were received an intraperitoneal (IP) injection of MCT (60mg/kg). Two weeks later, they were randomly assigned, this resulted in the following 3 groups: Group A: normal ratswith vehicle treatment (n= 10), the vehicle was gummi arabicum 5% which was gavaged (4ml/kg/day) once dally for a 3-week period. Group B: MCT+vehicle (n=35), the vehidewas gummi arabicum 5% which was gavaged (4ml/kg/day) once dally for a 3-week period. Group C: MCT+Bosentan (n=22) , Bosentan (100mg/kg/day) which was prepared in gummi arabicum 5% (4ml/kg/day) was gavaged once dally for a 3-week period. In every group, we measured the hemodynamic parameters, right ventride to left ventricle (RV/LV) thickness ratio, myocardial tissuse sections of RV were stained by HE+PTH (five-micron) and the morphorlogical characteristics were observed by light microscopy, myocardial tissuse sections were stained by uranyl acetate and Pb(NO₃)₂ (100nm) and the ultrastruture was observed by electromicroscopy. Then we investigated the distribution and quantity of Cx43 expression in the RV myocytes using immunoconfocal microscopy (six- micron sections) i n every group.Results:â‘ Hemodynamic measurements: RV systolic pressure in group A, B and C was 25.18±3.31mmHg,34.27±4.21mmHg and 28.62±4.58mmHg respectively, with one-way ANOVA, therewas significant diference between groupA and B ( P=0.000), so was between group B and C( P=0.004), there was no significant diference between group A and C (P=0.069) . The ratios of RV systolic pressure to jugular arterial systolic blood pressure in group A,B and C were 17.78±4.58%,51.68±8.87% and 33.26±6.94% respectively, with one-way ANOVA, there was significant diference between groupA and B (P=0.000),B and C (P=0.000), so was between groupA and C ( P=0.000).â‘¡(RV/LV) thickness ratio : (RV/LV) thickness ratios in group A,B and C were 42.96±5.32%,77.64±16.58% and 68.73±4.20% respectively, with one-way ANOVA, there was significant diference between group A and B ( P=0.000), so was between group A and C( P=0.000), there was no significant diference between group B and C (P=0.300) .â‘¢Changes of morphorlogical characteristics by light microscopy; Compared with group A, group B demonstrated distinct myocardial cells hypertrophy and enlargement of cell nuclei, part of myocardial fibers were loose and interstitial substance were fibrotic; the changes of myocardium in group C were between group A and B.â‘£Changes of ultrastruture by electromicroscopy:â… . Mycardial cells. Mycardial fibers arranged in order and mitochondria were normal in group A; in group B, some of myocardial fibers disorganized and loosened, endoplasmic reticula dilated, and mitochondria swelled; the changes of myocardial cells in group C were between group A and B.â…¡.Interstitial substance: No fiberosis was found in group A; part of interstitial substance appeared clear fiberosis in group B; the changes of interstitial substance in group C were between group A and B.â…¢.Gap junctions: The gap junctions were long and frequent in intercalated disks and rare and short at the sites of side-side cell junctions in group A; but in group B the gap junctions were rare and short in intercalated disks and long and frequent at the sites of side-side cell junctions; the changes of gap junctions in group C were between group A and B.⑤Cx43 expressi on in the RV myocytes using immunoconfocal microscopy:â… .Distribution: In longitudinally sectioned RV myocardi um most of the Cx43 staining was aggregated at the cell termini which are perpendicular to the long axle of myocardium and a little was observed at the sites of side-side cell junctions which are parallel to the long axle of myocardium in group A; but in group B, the Cx43 staining was markly reduced at the cell termini and was significantly increased at the sites of side-side cell junctions, that was to say, the distribution showed varying degree of dispersion over the cell surface; the distribution of Cx43 staining in group C was similar to that in group A.â…¡. Half- quantitative measurement: The area of Cx43 signal per unit area of RV myocardium measured by quantitative immunoconfocal microscopy was not significantly different among group A (2.29±0.69),group B (2.78±1.25) and group C (2.62±1.07) from one-way ANOVA, P=0.149.Conclusion:â‘ Bosentan is effective in reversing partly the RV remodeling.â‘¡In addition to the reduction in pulmonary arterial pressure,pulmonary vascular resistance and interference with the action of endothelin-1(ET-1) on cardiac hypertrophy, affecting the distribution of Cx43 in RV myocardium may be one of the mechanisms of Bosentan's reversing partly the RV remodeling.â‘¢There are the grounds for suggesting that the distribution disturbance of Cx43 in RV myocardium may be one of the mechanisms of RV remodeling.