Effect Of Cell Adhesion Molecule 3(CADM3) On Specific Adhesion Of Inflammmatory Cells To Hypoxic Pulmonary Vascular

Objective Hypoxia is a pathogenic factor of many diseases and is a common pathological process.In recent years,the study found that when the gas inhaled partial pressure of oxygen or oxygen concentration drops(also known as alveolar hypoxia,for example,plain entering plateau),Monocyte from peripheral blood(CD45+/CD11b+)was significantly increased infiltration in the lungs,especially aggregation in pulmonary perivascular extensive;at the same time,the synthesis and secretion of a large number of inflammatory mediators can be detected in lung tissue.More and more studies have shown that these inflammatory cells and inflammatory cytokines are important for the development of hypoxic pulmonary hypertension.It is interesting that,there is no similar inflammatory reaction around the systemic circulation under the same experimental conditions.Cell adhesion molecular 3,is a kind of immunoglobin adhesion molecule and belongs to the Nectin family protein,which is mainly located on the cell membrane.Previous studies have shown that CADM3 is a cell adhesion molecule specifically expressed on nerve tissue,which plays an important role in the formation of neurons.Our previous studies found that CADM3 was increased in hypoxic human umbilical vein endothelial cells and required for monocyte adhesion.However,it is still unclear whether CADM3 mediates the infiltration of mononuclear cells in pulmonary circulation.The purpose of this study was to investigate the role of CADM3 in the specific adhesion of inflammatory cells to hypoxic pulmonary vascular.Methods According to the experimental design,rat pulmonary arterial endothelial cell(RPAEC),rat aortic endothelial cells(RAEC),human peripheral blood mononuclear cell(U937,THP-1)and peripheral blood leukocytes were divided into different groups.U937: normoxic RPAEC + normoxic U937(CPE+CU),hypoxic RPAEC + normoxic U937(HPE+CU),hypoxic RPAEC + hypooxic U937(HPE+HU),normoxic RAEC + normoxic U937(CAE+CU),hypoxic RAEC + normoxic U937(HAE+CU)and hypoxic RAEC + hypoxic U937(HAE+HU);THP-1: normoxic RPAEC + normoxic THP-1(CPE+CT),hypoxic RPAEC + normoxic THP-1(HPE+CT),hypoxic RPAEC + hypooxic THP-1(HPE+HT),normoxic RAEC + normoxic THP-1(CAE+CT),hypoxic RAEC + normoxic THP-1(HAE+CT)and hypoxic RAEC + hypoxic THP-1(HAE+HT).Hypoxia group(1%O2)were placed in the three gas incubator cultured 6h and the control group(21%O2)was cultured in the CO2 incubator for the same time.1.Precent of Di I+ cell was detected by flow cytometry;fluorescence quantitative was used to detect cell lysates fluorescence density;fluorescence microscopy was used to analysis the adhesion of endothelial cell and U937.2.Western blot was performed to tested the protein levels of CADM3 and HIF-1?;the fluorescence intensity of CADM3 and p65 were observed by immunofluorescence assay.3.Adhesion of endothelial cell and U937 changed by anti CADM3 antibody was detect by flow cytometry.Results 1.Effects of hypoxia on the adhesion between endothelial cells and U937 cell: flow cytometry analysis and fluorescence density of cell lysates results showed that Di I+ cell precent and cell lysates density in HPE+CU group was much higher than that of CPE+CU group.The difference was significant(P<0.05);There had no obvious difference of Di I+ cell precent and cell lysates density between HPE+HU group and HPE+CU group;there was no significant difference was observed in RAEC and U937 cell adhesion under hypoxic or normoxic conditions.The results of fluorescence microscope showed monocyte adhesion rate and endothelial cell adhesion ability were significantly increased in HPE+CU group.The difference was significant when compared with CPE+CU(P<0.05).However,no difference was detected between HPE+HU and HPE+CU.2.Hypoxia promoted the adhesion of THP-1 cells and peripheral blood leukocytes to RPAEC: flow cytometry analysis showed that the percent of Di I+ cell in HPE+CT group was much higher than that of CPE+CT.The difference was significant(P<0.05).There had no obvious difference of Di I+ cell percent between HPE+HT and HPE+CT.Interestingly,no significant difference was observed in RAEC and THP-1 cell adhesion under hypoxic or normoxic conditions;Fluorescence density CD11b+ of cell lysates from the group of hypoxic RPAEC was much higher than that from normoxic RPAEC(P<0.05).The other peripheral blood leukocytes had no difference.In addition,there was no significant difference in adhesion between peripheral blood leukocytes and RAEC under hypoxic or normoxic conditions. 3.Western-Blot and immunofluorescence assay were performed to detect the protein levels of CADM3,the protein levels of CADM3 in hypoxia RPAEC group was higher than that in normoxic RPAEC group,the difference was significant(P<0.05).There was no significant difference in normoxia RPAEC+LPS group and normoxia RPAEC group,hypoxia RPAEC+LPS group and hypoxia RPAEC group.In addition,the expression of CADM3 was no significant difference in the groups of RAEC.4.The location of p65 was detected by immunofluorescence assay,the p65 protein in normoxia RPAEC and hypoxia RPAEC group was located in the cytoplasm;normoxia RPAEC +LPS group and hypoxia RPAEC+LPS group,the p65 protein was located in the cell nucleus.5.Flow cytometry was used to detect the adhesion of U937 to RPAEC after testing anti CADM3 antibody.Results showed that the cell adhesion of testing anti CADM3 antibody group was significantly inhibited compared with the hypoxia group,the difference was significant(P<0.05).Conclusion 1.Hypoxia induced the adhesion of monocytes /CD11b+ cells in peripheral blood to RPAEC increased,which is mainly related to activation of RPAEC by hypoxia.2.Expression of CADM3 increased in hypoxia RPAEC which is not associated with the inflammation by LPS stimulation.3.CADM3 was involved in the adhesion between monocytes and RPAEC.