The Role And Mechanism Of Monocarboxylate Transporter 4 In Diabetic Endothelial Injury

Part 1The morphological observation of endothelium damage in diabetes by scanning ion conductance microscopyObjective To observe endothelium damage in diabetes by scanning ion conductance microscopy (SICM).Methods In this experiment, db/db mice (10-12 weeks old, C57BL/6J background) and wild type mice (C57BL/6J) were used. Conventional immunofluorescence, immunohistochemical staining, and new SICM technique were used to observe aortic endothelium; Acute isolated the aorta of wild type C57BL/6J mice and then cultured in high glucose and/or interleukin-1 (3 (IL-1β), then the aortic endothelium was directly scanned by SICM.Results (1) The conventional immunofluorescence and immunohistochemical staining results indicated that the aortic endothelium completely covered the intima in wild type mice, however aorta endothelium exhibited obvious loss and damage in db/db mice. (2) Three-dimensional and two-dimensional SCIM images showed that the endothelial cells were oval and closely spaced, and the cell size was about 20 um in wild type mice; however aorta endothelium was not smooth and the endothelial cells existed obvious loss and damage in db/db mice, and the proportion of endothelial area decreased significantly. (3) SICM results showed that normal glucose (5.6 mM) did not induce aorta endothelium loss and damage; high glucose (25 mM) induced EC shrinkage, but there was no obvious loss; IL-1β also did not lead to aortic EC loss; but high glucose combined with IL-1β caused obvious damage and loss of aorta endothelium, the proportion of endothelial area decreased significantly.Conclusion SICM is a new technique to observed the morphology of endothelial damage, which has the characteristics of high resolution, simple operation and no damage to tissue.Part 2Isolation, culture and identification of human umbilical vein endothelial cells in vitroObjective To isolate, culture and identify human umbilical vein endothelial cells (umbilical vein endothelial cells, HUVEC) in vitro.Methods 0.1% type I collagenase digestion method was used to isolate endothelial cells, and the growth medium and auxiliary agent (excluding VEGF) were purchased from Promocell Company. Cells were identified according to the cell morphology, tube experiments and CD31 fluorescence staining.Results (1) The perfusion and digestion time was controlled to obtain a large number of cells in 15-18 minutes. (2) The microscope images showed that HUVEC at initial inoculum was round, single or clustered; After 4 hours the cells start to adhere to the dish, and fully adhere after 24 hours; The cell morphology was triangle, short spindle shaped, spindle shaped or irregular in shape, and the nucleus was round or oval shaped, and the nucleoli was prominent; 3-5 days later, the cells gradually grew for monolayer, liking a "cobblestone", which is the typical characteristics of endothelial cells. (3) Cells presented typical of the EC growth characteristics in vitro; CD31 immunofluorescence staining results showed that cells were obvious CD31 positive, and the proportion of CD31 positive cells was more than 95%; tube formation results showed that tubes were formed in about 1 hour. (4) Cells proliferated rapidly after 24 hour, and maintained in logarithmic growth phase from 2-4 days; In the fifth day, cells contacted with each other, which inhibited proliferation, and the cell doubling time was about 24 hour.Conclusion 0.1% type I collagenase digestion method is reasonable and appropriate to isolate HUVEC, and the isolated cells were with high purity and activity.Part 3The role of monocarboxylate transporter 4 in endothelium damage in diabetesObjective To detect the expression of monocarboxylate transporters (MCT), and to explore the effect of high glucose and/or IL-1β on MCT4 expression, and the effect of MCT4 on intracellular lactate, pH level and cell functions in HUVEC.Methods RT-PCR and real-time PCR were used to detect the expression of MCT in HUVEC; the effect of high glucose and/or IL-1β on MCT4 expression were examined by real-time PCR, Western blot and immunofluorescence staining; Specific shRNA target MCT4 was used to silence MCT4, and the effects of MCT4 on intracellular lactate, pH value and cell functions were detected by ELISA, BCECF fluorescence staining, TUNEL, Western blot, CCK-8, scratches and transwell methods.Results (1) In addition to MCT 10 and MCT11, the rest MCT were expressed in HUVEC, and MCT1 and MCT4 mRNA were significantly higher than MCT2 and MCT3. (2) High glucose (25 mM) alone or IL-1β induced apoptosis, and high glucose plus IL-1β increased the proapoptotic effect, and upregulated Bax, while downregulated Bcl-2. (3) High glucose or IL-1β alone did not increase the intracellular lactate, while high glucose combined with IL-1β increased the intracellular lactate dramatically. (4) High glucose did not affect MCT4 mRNA and protein levels, as well as its localization on cell membrane, while high glucose plus IL-1β significantly reduced the mRNA and protein levels of MCT4, as well as its localization on cell membrane. (5) MCT4 silencing significantly increased the intracellular lactate and reduced intracellular pH value in HUVEC. (6) MCT4 silencing induced apoptosis, upregulated BAX and downregulated Bcl-2. (7) MCT4 silencing inhibited the proliferation of HUVEC. (8) MCT4 silencing inhibited the migration of HUVEC.Conclusion High glucose and IL-1β induced HUVEC apoptosis and the inhibition of proliferation and migration, which were related to the downregulation of MCT4.Part 4The study on the mechanism of endothelium damage mediated by monocarboxylate transporter 4 in diabetesObjective To investigate the mechanism of endothelium damage mediated by monocarboxylate transporter 4 in diabetes.Methods Specific shRNA target MCT4 was used to silence MCT4. The effects of MCT4 silencing on proliferation, migration and Akt/Gsk 3β, MEK/Erk, integrin β4/FAK/Src signaling pathways were detected by Western blot, CCK-8, scratches and transwell methods.Results (1) MCT4 Silencing significantly inhibited HUVEC proliferation, so did Akt inhibitor LY294002 and MEK inhibitor AZD6244; whereas MCT4 silencing combined LY294002 or AZD6244 made further efforts on the suppression of proliferation. (2) p-Akt, p-Gsk 3β, p-MEK and p-ERK1/2 were significantly decreased after MCT4 silencing, and LY294002 and AZD6244 also downregulated those phosphate-proteins; MCT4 silencing combined with inhibitors (LY294002 or AZD6244) induced further inhibition of the phosphorylation of Akt, Gsk 3β, MEK and ERK1/2. (3) MCT4 silencing significantly inhibited HUVEC migration, so did the specific FAK inhibitor PF-573228; MCT4 silencing combined with PF-573228 further inhibited HUVEC migration. (4) MCT4 silencing reduced integrin β4, p-FAK and p-Src level; PF-573228 alone did not change integrin β4, but the level of p-FAK and p-Src significantly reduced, whereas MCT4 silencing combined with PF-573228 further decreased p-FAK and p-Src.Conclusion MCT4-mediated endothelium damage in diabetes may be related to the inhibition of Akt/Gsk 3β, MEK/Erk and integrin β4/FAK/Src signaling pathways.