Change Of Barrier Function In Pulmonary Microvascular Endothelial Cells From Bleomincy-injuried Rats And Its Correlation With Pulmonary Fibrosis

AIM:As the basic structural and functional unit of the blood vessels, the important physiological function of endothelial cells is to act as a barrier to regulate the substance exchange of the blood vessels; to maintain the stabilization of the inner cellular environment; and to prevent proximal and distal organs and tissues from damage. Endothelial cells adhere to extracellular matrix to form intact endothelium barrier. Upon PF, both increasing of the blood vessel permeability and continuous opening of the endothelial cells junction are observed in the origination phase of the alveolus inflammation and the phase of fibrosis formation. This malfunction of the endothelial barrier provides a paracellular pathway to exchange macro-molecule crossing endothelium. It plays a significant role for the invasion of inflammation cells to alveolus and pulmonary interstitium and activation of fibroblasts proliferation during injury of lung. Together with cystoskeleton,endothelial cell gap junction connexin linked with actin and influenced by intracellular [Ca2+]i. This study utilized electronic microscopy, cell culture, immunohistochemistry, and confocal laser scanning microscopy to observe morphological changes of the rat PMVECs during pneumonia and PF; to compare the difference of the intracellular [Ca2+]i of PMVECs and analyze the expression of its positive feedback factor—intermediate conductance Ca2+-activated potassium channel protein 1(Kca3.1), in order to explore the change and its mechanism of barrier function of PMVECs in BLM-injured rats and the gain the knowledge of its contribution in the PF process.METHODS:20 SD rats, female and male, weight 150±20 g(provided by experimental animal center of the Fourth Military Medical University), were divided randomly into control group and BLM group, 10 ones each group. BLMA5(Tianjin Taihe pharmaceuticals, Inc. 8 mg, batch number 041103) was diluted into 4 g/L by physiological saline solution. The BLM group received BLM (5 mg/kg) treatment intratracheally, whereas the control group received same-volume physiological saline solution instead. Primary PMVECs were obtained and cultured from the peripheral lung tissues and loaded with Fluo-3/AM (America, Molecular Probe, Inc.), respectively. Confocal laser scanning microscope was used to measure the [Ca2+]i and expression of intermediate conductance Ca2+-activated potassium channel protein 1 (Kca3.1) in the cells. The expression of Kca3.1 was also analyzed by immunocytochemistry and image quantitative analysis methods. Transmission electron microscope was used to observe the morphology of PMVECs in pneumonia group, BLM group and control group (electron microscope samples provided by Dr.Yin Qian, Department of Radiology, Tangdu Hospital, Fourth Military Medical University). RESULTS:1. Observation of PMVECs by TEM:â‘ . Tight junctions: The luminal surface of ECs in control group was slick and without lanthanum particles adhering, and the tight junction between ECs was closed. In pneumonia 7 d group, little lanthanum particles were found at the luminal surface of ECs; tight junctions were open., and ECs were in the proliferation state. In pneumonia 14 d group, the luminal surface of ECs regained slick and no lanthanum particle was found at the suface; ECs regained normal; and there was a small quantity of collagen deposit surrounding ECs. In BLM groups, tight junctions kept open, and lanthanum particles and platelets adhered to the luminal surface of ECs. ECs were in the proliferation state and had digitations at the luminal surface, especially in 7 d , 14 d and 21 d groups.â‘¡. Micropinocytotic vesicles: Micropinocytotic vesicles were significantly decreased in pneumonia 7 day group but increased in BLM groups, especially in BLM 7 d group. Large quantity of collagen fibers deposited surrouding ECs. In some place, ECs direct contacted with fibroblasts.2. Intracellular Ca2+ of PMVECs mainly located in cytoplasm. The [Ca2+]i in BLM group (166.56±24.17) was significantly higher than that in control group(95.79±13.51, P<0.01).3. Kca3.1 of PMVECs mainly located at the cellular membrane. Expression of Kca3.1 in BLM group was much lower than that in control group by indirect immunofluorescence assay(679.29±206.98 vs 958.75±188.84, P<0.01)and immunocytochemical method (169.08±14.81 vs 189.78±13.73, P<0.01). CONCLUSION:1. In BLM groups, the abnormal barrier function and increased vasopermeability of PMVECs, together with high adhesiveness and transportation function were the important factors for inflammatory cells and vasoactive substances get into interstitium.2. The main reason of barrier functional abnormality found in PMVECs was [Ca²⁺]i overloading,which might have close correlation with phenotype transforming and function changing of EC.3. Decreasing of Kca3.1 activity in PMVECs might be correlated with [Ca²⁺]i overloading during PF.