The Role Of Pericytes Apoptosis In Microvascular Dilatation In Rat Experimental Hepatopulmonary Syndrome

Backgroud and objective:Hepatopulmonary syndrome (HPS), defining as liver disease and intrapulmonaryvascular dilatation (IPVD) and hypoxemia, is a serious vascular complication. HPSmarkedly increases the mortality of patients with liver diseases. IPVD is the mainpathological change of HPS. Pericytes with elongated and stellate shaped cytoplasmicprocesses locate at the outer wall of microvessels. They tightly distribute on the wall ofmicrangium along the axis of capillary, and are able to regulate the vascular diameter.Furthermore, pericytes are abundant with constriction proteins and protein kinases, and thusthey possess the function of regulating vascular diameter. Some studies have proved thatpericytes loss was correlated with vascular dilatation in some diseases and caspase-3activation played a key role in the pericytes apoptosis. However, the role of pericytes inIPVD of HPS is unclear. Common bile duct ligation (CBDL) is the establishedexperimental model of human HPS. This study aimed to investigate the role of pericytesapoptosis in microvascular dilatation in rat experimental hepatopulmonarysyndrome. We observed the effects of CBDL serum on pulmonary pericytes apoptosis andcaspase-3activation, detected the pulmonary apoptosis, levels of pericyte markersα-smooth muscle actin (α-SMA), neuron-glial antigen2(NG2) and desmin, and thenumber of these positive cells following CBDL; then assessed the effects of caspase-3inhibition on pulmonary apoptosis, levels of α-SMA，NG2and desmin, the number ofthese positive cells, microvascular dilatation and blood-gas exchange following CBDL.Methods:1Effect of CBDL rats serum on pulmonary pericytes apoptosis.1.1Culture and identification of pulmonary pericytes.1.2Rat experimental HPS was constructed by CBDL, serum was collected in1-wkpoint following CBDL. 1.3Apoptosis and caspase-3level of pulmonary pericytes induced by serum of CBDLrats were detected by TUNEL and western blot.2Pulmonary pericytes variation in CBDL rats.2.1Rat experimental HPS was constructed by CBDL.2.2Pulmonary apoptosis and caspase-3level in CBDL rats were detected by TUNELand western blot.2.3Pulmonary levels of pericyte markers α-SMA, desmin and NG2and the numberof these positive cells in CBDL rats were detected by western blots andimmunohistochemistry.3The effects of caspase-3inhibition on pulmonary pericytes and HPS in CBDLrats.3.1Rat experimental HPS was constructed by CBDL, the caspase-3inhibitor calledZ-DEVD-FMK was administered by intraperitoneal injection daily within2weeks afterCBDL.3.2The effect of caspase-3inhibition on pulmonary apoptosis after CBDL wasdetected by TUNEL.3.3The effects of caspase-3inhibition on pulmonary levels of α-SMA, desmin andNG2and the number of these positive cells after CBDL were assessed by western blots andimmunohistochemistry.3.4The effects of caspase-3inhibition on pulmonary microvascular dilatation andblood-gas exchange after CBDL were evaluated by HE staining and arterial blood gasanalysis.Results:1. Pulmonary pericytes were successfully cultivated. The morphology of culturedcells was spindle or star shaped, single-core, accidentally dual-core, ovoid nuclear, withcytoplasmic processes, vortex or fence grew, and with no contact inhibition.Immunofluorence detection showed that these cells expressed α-SMA, desmin, NG2without CD31.2. TUNEL-positive cells and caspase-3level in pulmonary pericytes were markedlyincreased through the serum of CBDL rats.3. After CBDL, pulmonary TUNEL-positive cells and caspase-3level were significantly increased with a peak at the1-week point, pulmonary levels of pericytemarkers α-SMA, desmin and NG2and the number of these positive cells were markedlydecreased.4. The early administration of caspase-3inhibitor called Z-DEVD-FMK markedlyalleviated the increase in pulmonary TUNEL-positive cells, the reduction in α-SMA,desmin and NG2levels and the number of these positive cells, microvascular dilatation andimproved blood-gas exchange after CBDL.Conclusions:1. We establish a simple and effective method of pulmonary pericytes culture.2. The serum of CBDL rats promoted apoptosis of pulmonary pericytes.3. Caspase-3dependent pericytes apoptosis may be the reason of the reduction inpulmonary levels of α-SMA, desmin and NG2and the number of these positive cells inCBDL animals.4. The early inhibition of caspase-3relieves microvascular dilatation and improvesblood-gas exchange following CBDL may through decreasing pulmonary pericytesapoptosis. The early inhibition of caspase-3maybe a potential strategy to prevent againstexperimental HPS.