Impaired Angiogenisis Of Myocardial Microvascular Endothelial Cells In Type 2 Diabetic Rats In Vitro

Vascular endothelial cells are not only for the barrier of blood vessels, but also canrelease various cytokines and active products, which play an important role inpathophysiological function. Therefore, endothelial cell dysfunction is an importantresearch field in vascular complication of type 2 diabetes mellitus (T2DM). Theclinical retrospective studies and animal experiments had found that, the dysfunctionin myocardial angiogenesis, including low capillary density and reduced coronaryartery collateral vessel formation, was the main pathological manifestation in thediabetes-induced ischemic cardiovascular disease. High blood glucose is awell-recognized risk factor for microvascular complication, but indeed, some T2DMpatients with tight blood glucose control still had microvascular complicationoccurrence, which means in addition to the high risk of high blood glucose, other riskfactors were associated with the development and progression of microvascularcomplication. Researchers had found that patients with T2DM accompanied by highserum level of free fatty acids (FFA) most frequently, moreover, the highconcentration of FFA plays an important role in the development of endothelialdysfunction. The complex interaction between metabolic disturbances of T2DM, suchas high blood glucose and lipid metabolic disorder etc., may contribute to thedevelopment and progression of microvascular complication.Goto-Kakizaki (GK) rat is a spontaneously nonobese model of type 2 diabetesmellitus, which was used to isolate myocardial microvascular endothelial cells(MMVEC) and aortic endothelial cells (AEC) for primary culture, by which, theangiogenesis ability changes were compared between microvascular andmacrovascular endothelial cells. Previous studies indicated that the abnormalexpression of proangiogenic factors, their receptors and signal transduction disordersare the leading cause of diabetes-induced angiogenesis impairment. According to thebalance theory between angiogenesis inducible and inhibited factors, we focus on theinducible factors in our studies, comparing the expression changes of angiogenesisinducible factors and their receptors in GK-MMVEC and GK-AEC, furthermore, toinvestigate the possible regulation mechanism of microRNAs in gene expression. This paper is composed of four parts as follows.PartⅠ. GK rats (8～9 weeks of age) with higher fasting and postprandial bloodglucose according with the T2DM diagnosis, were selected for primary cell cultureusing the age-matched normal Wistar rats as control. The MMVEC and AEC werecultured by the method of planting myocardial tissue or aortic rings based on theprevious work of our lab. The molecular markers and functional characteristics wereobserved and identified by immunocytochemistry and DiI-Ac-LDL endocytosis assay.No difference had been found between the endothelial cells derived from GK rats andWistar rats by the characteristics above. To study the changes of angiogenesis abilityof GK-MMVEC and GK-AEC, the parameters of proliferation, migration, adhesionand capillary tube-like formation were used. The results showed that GK-MMVEChad reduced ability of proliferation, migration, adhesion and capillary tube-likeformation comparing with Wis-MMVEC (P＜0.05), whereas, there was no significantdifference between GK-AEC and Wis-AEC by using the same assay above. Theresults suggested that the impaired angiogenesis of myocardial microvascularendothelial cells appeared earlier than that in macrovascular endothelial cells of GKrats.PartⅡ. To analyze why the GK-MMVEC and GK-AEC had different changes ofangiogenesis in vitro, the angiogenesis gene microarrays were used to evaluate theexpression pattern of angiogenic factors, for example growth factors and theirreceptors. Most of the growth factors and their receptors genes had no significantexpression changes in GK-AEC. However, the growth factors and their receptorsgenes were up-regulated in GK-MMVEC. Real-time RT-PCR analysis demonstratedthat there were up-regulated mRNA expression of VEGF (2.14±0.68), Flt-1 (3.48±0.85)and Flk-1 (6.35±1.44) in GK-MMVEC as compared with that in the normalWis-MMVEC (P＜0.01). No significant difference of VEGF, Fit-1, and Flk-1 mRNAexpression had been found between GK-AEC and Wis-AEC. The results of real-timePCR were consistent with the results of gene microarray. However, using Western-blot, protein expressions of VEGF and Flk-1, Flt-li in GK-MMVEC markedlydecreased as compared to the Wis-MMVEC. There was still no significant differenceof VEGF, Flt-1, and Flk-1 protein expression between GK-AEC and Wis-AEC. Theresults of ELISA also showed the VEGF protein secreted to the culture medium inGK-MMVEC was less than that in Wis-MMVEC. Therefore, the "separationphenomenon" of expression between mRNA up-regulation and protein down-regulation of VEGF, Flk-1 and Fit-1, is one of the most important causes ofimpaired angiogenesis in GK-MMVEC. We suggested that the mechanismmentioned-above involves the post-transcription regulation disturbances.PartⅢ. To study the effects of high glucose, high free fatty acids andcombination of them on expression of VEGF, its receptors and changes ofangiogenesis ability in cultured MMVEC. The regulation of VEGF, Flt-1, Flk-1mRNA and protein expression in MMVEC after incubation with high glucose, highfree fatty acids and combination of them were investigated by Real-time RT-PCR andWestern blot. The results indicated that D-glucose and FFA could regulate theexpression of VEGF and receptors in a time and dose-dependent manner. The resultsof Real-time RT-PCR showed that after 4 h incubation with 33.3mmol/L D-glucose,the expression of VEGF mRNA reached the peak, but Flt-1, Flk-1 mRNA decreasedto the minimum(P＜0.01 vs. control), then restored in some degree, but did not returnto the basal levels within 24h. After treatment with FFA (PA 0.125mmol/L+OA0.25mmol/L), VEGF and Flk-1 mRNA were inhibited significantly and reached theminimum at 4h, while Fit-1 at 12h(P＜0.01 vs. control). The lowest level of VEGF,Flt-1 and Flk-lmRNA reached at 12h, 2h, 8h, respectively after treatment with thecombination of D-glucose and FFA (D-glucose33.3mmol/L+PA 0.125mmol/L+ OA0.25mmol/L). The results of Western blot indicated that after 8 h incubation with33.3mmol/L D-glucose, the expression of VEGF protein arrived to the maximum(P＜0.05 vs. control), while Flt-1,Flk-1 protein reached the minimum at 24h (P＜0.01vs. control). When it was incubated with FFA (PA 0.125mmol/L+OA 0.25mmol/L),the inhibition of VEGF, Flt-1, Flk-1 protein reached to the minimum at 24 h, 8 h, 24hrespectively, (P＜0.01 vs. control). While MMVEC was treated with the combinationof D-glucose and FFA (D-glucose33.3mmol/L+PA 0.125mmol/L+OA 0.25mmol/L),the lowest level of VEGF, Flt-l, Flk-1 protein reached at 24h, 12h and 24 h (P＜0.01vs. control). In the experiment, the most effective concentration of D-glucose and FFAwere 33.3mmol/L, PA 0.25mmol/L+OA 0.5mmol/L (P＜0.01 vs. control) respectively.In the angiogenesis experiment, pretreated with high glucose(33.3 mmol/L) orFFA (PA 0.125mmol/L+OA 0.25mmol/L) for 16h, no influence had been found onmigration, adhesion and capillary tube-like formation of MMVEC after incubationwith high glucose(33.3 mmol/L, P＞0.05). While the FFA could inhibited theangiogenesis ability obviously (P＜0.05) comparing with the control. The combinationof the high glucose and FFA had significant inhibition on MMVEC migration, adhesion comparing to the group treated with FFA only (P＜0.05), and there wasalmost no capillary tube-like formation. The results demonstrated that incubation withhigh D-glucose in short time could inhibit the expression of Flt-1 and Flk-1, butinduced the up-regulation of VEGF, therefore, had no effects on angiogenesis ofMMVEC in vitro. However, both the VEGF and their receptors were inhibited by FFAafter short time treatment, which contributed the decreased angiogenesis ability. Morelikely, the high glucose in short time was the coordinated risk factor in FFA-inducedangiogenesis impairment.PartⅣ. In order to investigate the mechanism of impaired angiogenesis ofGK-MMVEC, microRNAs (miRNAs) microarray was used to assess the miRNAsexpression. We found that the 2 times up-regulated miRNAs included let-7e,miR-129, miR-291-5p, miR-320, miR-327, mir-333, miR-363-5p, miR-370,miR-494, miR-503, miR-664. Through four published prediction algorithms(TargetScan, miranda, PicTar, TargetScanS) analysis, 9 miRNAs were predicted totarget factors and their receptors, including VEGF, Flk-1, IGF-1, IGF-1R and so on,which related closely with angiogenesis. The miR-320, which had most target genes,were confirmed by miRNAs real-time PCR. The predicted target genes of miR-320included Flk-1, IGF-1 and IGF-1 receptor. We only analyzed the expression of IGF-1,IGF-1RmRNA and protein by real-time PCR and Western Blot in the study. Theresults showed that the protein expression of IGF-1 and IGF-1R were decreasedsignificantly, meanwhile, mRNA of IGF-1 was up-regulated. There was no changeof IGF-1R mRNA expression in GK-MMVEC. Therefore, we suggested that thedown-regulation of IGF-1 and IGF-1R protein without mRNA down-regulationprobably were induced by the abnormal high expression of miR-320, and theup-regulated of miR-320 was related to the impaired angiogenesis in GK-MMVEC.In summary, the present study demonstrated that the impaired angiogenesis ofmyocardial microvascular endothelial cells appeared earlier than that inmacrovascular endothelial cells of GK rats. We suggested that the "separationphenomenon" of expression between mRNA up-regulation and proteindown-regulation of VEGF, Flk-1 and Flt-1, is one of the most important causes ofimpaired angiogenesis in GK-MMVEC. The down-regulation of IGF-1 and IGF-1Rprotein without mRNA down-regulation probably were induced by the abnormal highexpression of miR-320, and the up-regulated of miR-320 was related to the impairedangiogenesis in GK-MMVEC. D-glucose and FFA could regulate the expression of VEGF and its receptors in a time and dose-dependent manner. Incubation with highD-glucose in short time had no effects on angiogenesis of MMVEC in vitro. However,FFA induced decrease of angiogenesis ability in short time treatment. The highglucose in short time was the coordinated risk factor in FFA-induced angiogenesisimpairment.