Mechanism of oxLDL/β2GPⅠ/aβ2GPⅠ complex promoting the angiogenesis in vascular endothelial cells through TLR4//MyD88/NF-κB signaling pathway

doi:10.11958/20240680

Abstract

Abstract:

Objective To investigate effects of oxidized low density lipoprotein/β2 glycoprotein-Ⅰ/anti-β2 glycoprotein-Ⅰ antibody (oxLDL/β2GPⅠ/aβ2GPⅠ) complex on the proliferation, migration and angiogenesis of vascular endothelial cells and its mechanism. Methods Human umbilical vein endothelial cells (HUVEC) were cultured to logarithmic growth phase and grouped into the control group (normal culture), the oxLDL group (50 mg/L oxLDL), the oxLDL/β2GPⅠ/aβ2GPⅠ group (50 mg/L oxLDL/100 mg/L β2GPⅠ/100 mg/L aβ2GPⅠ) and the VEGF group (100 μg/L VEGF). The gene expressions of VEGF, vascular endothelial cadherin (VE-cadherin), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by real-time quantitative fluorescent PCR (qPCR). Cell counting kit-8 (CCK-8) method was employed to detect cell proliferation. Cell migration and invasion were determined by scratch healing test and Transwell assay. Matrigel tube formation assay was used to observe the angiogenesis of HUVEC. The relative protein expression of TLR4, MyD88 and NF-κB were examined by Western blot assay. Results Compared with the control group, the proliferation activity of cells at 48 h of treatment was increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group (P<0.05). Moreover, compared with the control group, cell migration and angiogenesis were increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group, and the mRNA levels of VEGF, VE-cadherin, MMP-2 and MMP-9 were elevated (P<0.05). Compared with the control group, levels of TLR4 and MyD88 were elevated in the oxLDL/β2GPⅠ/aβ2GPⅠ complex group (P<0.05), as well as levels of p-NF-κB p65/NF-κB p65 (P<0.05). Conclusion oxLDL/β2GPⅠ/aβ2GPⅠ complex may promote the proliferation, migration and tube formation of vascular endothelial cells by regulating TLR4/MyD88/NF-κB signaling pathway.

Key words: atherosclerosis, beta 2-glycoproteinⅠ, neovascularization, pathologic, cell proliferation, cell movement, NF-kappa B, endothelial cells, oxidized low density lipoprotein

CLC Number:

R392.9

Figures/Tables 10

References 19

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基因名称	引物序列（5'→3'）	产物大小/bp
VEGF	上游：AGCTTCGTGTCCTGTATGGC	138
VEGF	下游：TTTCTGGCCACGTCCAGTTT	138
VE-cadherin	上游：CCCACTATCCGATACGAATACC	149
VE-cadherin	下游：ATCCACATCTAGGACGTTGATG	149
MMP-2	上游：TCAAGTTCCCCGGCGATG	225
MMP-2	下游：AGTTGGCCACATCTGGGTTG	225
MMP-9	上游：CAAAGACCTGAAAACCTCCAAC	168
MMP-9	下游：GACTGCTTCTCTCCCATCATC	168
β-actin	上游：GCTTGCGGCATCCACGAGAC	82
β-actin	下游：CGGTGTTGGCATACAGATCCTTACG	82

基因名称	引物序列（5'→3'）	产物大小/bp
VEGF	上游：AGCTTCGTGTCCTGTATGGC	138
VEGF	下游：TTTCTGGCCACGTCCAGTTT	138
VE-cadherin	上游：CCCACTATCCGATACGAATACC	149
VE-cadherin	下游：ATCCACATCTAGGACGTTGATG	149
MMP-2	上游：TCAAGTTCCCCGGCGATG	225
MMP-2	下游：AGTTGGCCACATCTGGGTTG	225
MMP-9	上游：CAAAGACCTGAAAACCTCCAAC	168
MMP-9	下游：GACTGCTTCTCTCCCATCATC	168
β-actin	上游：GCTTGCGGCATCCACGAGAC	82
β-actin	下游：CGGTGTTGGCATACAGATCCTTACG	82

组别	0 h	24 h	48 h	72 h	96 h
对照组	1.00±0.05	1.43±0.14	1.81±0.32	4.15±0.22	5.28±0.34
oxLDL组	0.85±0.42	1.60±0.14	2.70±0.26	4.17±0.16	5.47±0.18
oxLDL/β2GPⅠ/aβ2GPⅠ组	1.05±0.03	1.29±0.18^b	2.40±0.22^a	3.78±0.22^ab	5.54±0.42
F	0.870	4.945^＊	14.040^＊＊	6.025^＊	0.858

组别	0 h	24 h	48 h	72 h	96 h
对照组	1.00±0.05	1.43±0.14	1.81±0.32	4.15±0.22	5.28±0.34
oxLDL组	0.85±0.42	1.60±0.14	2.70±0.26	4.17±0.16	5.47±0.18
oxLDL/β2GPⅠ/aβ2GPⅠ组	1.05±0.03	1.29±0.18^b	2.40±0.22^a	3.78±0.22^ab	5.54±0.42
F	0.870	4.945^＊	14.040^＊＊	6.025^＊	0.858

组别	迁移率/%	侵袭细胞数量/个	管腔节点/%
对照组	100.00±20.78	65.33±18.90	100.00±16.33
oxLDL组	168.70±85.09	173.70±16.50	231.50±55.22
oxLDL/β2GPⅠ/ aβ2GPⅠ组	421.80±83.91^ab	224.70±11.68^ab	369.60±65.47^a
VEGF组	355.00±70.66^ab	192.00±14.73^a	392.90±59.86^ab
F	14.030^＊＊	58.160^＊＊	19.800^＊＊