Role of MiR-126a-3p in Endothelial Injury in Endotoxic Mice

Abstract

Objective: Sepsis poses a serious global health problem with an overall mortality rate of 30%, in which the vascular injury is a major contributor. The study is to determine the expression profile of micro-RNAs in endotoxic vascular walls and their potential roles in sepsis-related vascular injury.

Design: Prospective randomized study.

Setting: Laboratory investigation.

Subjects: Male C57BL/6 mice, average weight 26.5 ± 1.8 g.

Interventions: Endotoxemia was induced in mice via lipopolysaccharide injection (20 mg/kg, intraperitoneal) (Sigma, St. Louis, MO). The control mice were injected with the same amount of saline (500 μL, intraperitoneal). In a subgroup of mice, a high dose of lipopolysaccharide (30 mg/kg, intraperitoneal) was applied to induce endotoxin-related death.

Measurements and main results: The mi-RNA expression profiles in aortas from lipopolysaccharide-induced endotoxic mice were determined. The result demonstrated that some micro-RNAs were aberrantly expressed in endotoxic mouse arteries. Among them, the endothelial cell-enriched/endothelial cell-specific miR-126a-3p was significantly down-regulated in endotoxic mouse arteries, septic human vessels, as well as vascular endothelial cells isolated from endotoxic mice or treated with lipopolysaccharide. The down-regulation of miR-126a-3p occurred at transcriptional level via the decreased expression of Krüppel-like factor 2, which could be inhibited by Krüppel-like factor 2 over-expression via adenovirus expressing Krüppel-like factor 2. The down-regulation of miR-126a-3p in endothelial cells resulted in the increased apoptosis, and decreased proliferation and migration, which were inhibited by miR-126a-3p mimics. In vivo, over-expression of miR-126a-3p via lentivirus attenuated endotoxemia-induced injuries on endothelial function and vascular permeability. We found that SPRED1 and VCAM-1 were two direct target genes of miR-126a-3p related to miR-126a-3p-mediated effects in endotoxemia. Finally, the survival rate of endotoxic mice was significantly increased by the over-expression of miR-126a-3p.

Conclusions: The results suggest that vascular micro-RNAs such as miR-126a-3p may represent novel mechanisms and new therapeutic targets for endotoxemia-induced vascular injury and endotoxic mortality.

Fig.1. Down-regulation of miR-126a-3p in endotoxic mouse arteries, in vascular ECs isolated from endotoxic aortas and lungs, and in septic human vessels

A. miR-126a-3p levels in endotoxic mouse aortas at 6 hours after LPS injection (20 mg/kg, ip) (n=8) and in control aortas isolated from vehicle-treated mice (n=8) (500 μl, ip) determined by qRT-PCR. Note: *p<0.05 compared with that in control aortas. B. miR-126a-3p levels in vascular ECs isolated from endotoxic mouse aortas (macro-vascular ECs) (n=6) and from normal control mouse aortas (n=6) determined by qRT-PCR. Note: *p<0.05 compared with that in ECs from control aortas. C. miR-126a-3p levels in vascular ECs isolated from endotoxic lungs (micro-vascular ECs) (n=6) and from normal control mouse lungs (n=6) determined by qRT-PCR. Note: *p<0.05 compared with that in ECs from control lungs. D. miR-126a-3p levels in vessels of skin biopsy samples from patients with sepsis (n=5) and from control subjects (n=5) determined by qRT-PCR. Note: *p<0.05 compared with that in vessels from control subjects.

Fig. 2. The effects of miR-126a-3p on the proliferation, migration, and apoptosis of cultured mouse aortic ECs

A. Modulation of miR-126a-3p expression in ECs by miR-126a-3p mimics (miR-126 mimics) (30 nM) and AntagomiR-126a-3p (AntagomiR-126a) (30 nM). Note: n=6; *p<0.05 compared with that in oligo control-treated cells. B. Cell proliferation determined by MTT assay. Note: n=6; *p<0.05 compared with that in oligo control-treated cells. C. Cell proliferation determined by BrdU assay. Note: n=6; *p<0.05 compared with that in oligo control-treated cells. D. Cell migration was determined by a modified Boyden chamber assay. Note: n=6; *p<0.05 compared with that in oligo control-treated cells. E. Cell apoptosis determined by TUNEL analysis. Note: n=6; *p<0.05 compared with that in oligo control-treated cells. F. Representative cell images of TUNEL showing the apoptotic ECs with different treatments.

Fig. 3. The effects of miR-126a-3p overexpression on proliferation, migration, and apoptosis of LPS-treated ECs

In this experiment, the mouse ECs were pre-treated with vehicle, control oligo (30 nM) or miR-126a-3p mimics (miR-126 mimics) (30 nM). Twenty-four hours later, the cells were treated with LPS (LPS (1 μg/ml) for 24h. Then, proliferation, migration, and apoptosis of these ECs were determined. One group of vehicle-treated cells without LPS-treatment was used as normal control cells. The levels of miR-126a-3p were determined by qRT-PCR. A. LPS decreased the expression of miR-126a-3p in ECs. Note: n=6; *p<0.05 compared with that in control oligo-treated cells. B. Improved cell proliferation by miR-126a-3p overexpression determined by MTT assay. Note: n=6; *p<0.05 compared with that in control oligo-treated cells. C. Improved cell proliferation by miR-126a-3p overexpression determined by BrdU assay. Note: n=6; *p<0.05 compared with that in control oligo-treated cells. D. Improved cell migration by miR-126a-3p overexpression determined by Boyden chamber assay. Note: n=6; *p<0.05 compared with that in control oligo-treated cells. E. Decreased cell apoptosis by miR-126a-3p overexpression determined by TUNEL assay. Note: n=6; *p<0.05 compared with that in control oligo-treated cells. F. Representative cell images of TUNEL showing the apoptotic ECs with different treatments.

Fig. 4. The effects of miR-126a-3p over-expression on LPS-induced injuries on vascular endothelial function and vascular permeability, and on LPS-induced mortality

Mice were treated with vehicle (100 μl of saline), lenti-virus vector control (LV-Ctl, 2 × 10⁸ PFU) or LV-miR-126a-3p (LV-miR-126a) (2 × 10⁸ PFU) via external jugular vein. Seven days later, miR-126a-3p expression in aortas was determined. Then, the animals will be treated with vehicle (500 μl of saline, ip) or LPS (20 mg/kg, ip). Six hours later, the vascular permeability in vivo and endothelial function in isolated aortas was determined. A. miR-126a-3p expression in mouse aortas was increased by LV-miR-126a-3p. Note: n=6; *p<0.05 compared with that in aortas from LV-Ctl-treated mice. B. miR-126a-3p overexpression inhibited LPS-induced endothelial dysfunction in endotoxic mice. Note: n=8; *p<0.05 compared with that in LV-Ctl-treated mice. C. miR-126a-3p overexpression inhibited LPS-induced abnormal Evans blue extravasation into the peritoneal cavity in mice. Note: n=8; *p<0.05 compared with that in LV-Ctl-treated mice. C. miR-126a-3p overexpression inhibited LPS-induced abnormal Evans blue leakage in lung in mice. Note: n=6; *p<0.05 compared with that in LV-Ctl-treated mice. For LPS-induced mortality, C57BL/6 mice were pre-treated with vehicle, LV-Ctl or LV-miR-126a-3p. Seven days later, the animals were treated with high dose of LPS (30mg/kg, ip) to induce animal death. The survival rate of these mice was recorded up to 7 days after LPS-injection. Note: n=20; *p<0.05 compared with that in LV-Ctl-treated mice.

Fig. 5. SPRED1 and VCAM-1 are two direct target genes of miR-126a-3p

A. a luciferase reporter construct, containing the putative miR-126a-3p binding sequence from 3′-UTR of the SPRED1 or VCAM-1 mRNA was transfected into HEK293 with vehicle, pDNR-CMV, pmiR-126a-3p . The constructs without the miR-126a-3p binding sequence were used as the truncated controls. pmiR-miR-126a-3p inhibited luciferase activity. In the truncated control group, the inhibitory effect of pmiR-126a-3p disappeared. Note: n=6; *p < 0.05 compared with pDNR-CMV group. B. The effects of miR-126a-3p inhibition or miR-126a-3p overexpression on the protein levels of SPRED1 and VCAM-1 in cultured ECs. Note: n=6; *p < 0.05 compared with oligo control group. C. Representative western blots of SPRED1 and VCAM-1 in ECs with different treatments. D. Increased protein levels of SPRED1 and VCAM-1 in LPS-treated ECs. Note: n=6; *p < 0.05 compared with control group. E. Representative western blots of SPRED1 and VCAM-1 of D. F. Increased protein levels of SPRED1 and VCAM-1 in aortas of LPS-treated mice.Note: n=5; *p < 0.05 compared with control group. G. Representative western blots of SPRED1 and VCAM-1 of F. H. Increased protein levels of SPRED1 and VCAM-1 in skin vessels of patients with sepsis Note: n=5; *p < 0.05 compared with control group. I. Representative western blots of SPRED1 and VCAM-1 of H.

Fig. 6. KLF2 is a key upstream transcription factor involved in the down-regulation of miR-126a-3p in vascular ECs and arteries in endotoxemia

A. Expression of pri-miR-126a-3p in aortas from LPS-injected mice is decreased. n=6; *p<0.05 vs control group. B. Expression of pri-miR-126a-3p in LPS-treated endothelial cells is decreased. n=6; *p<0.05 vs control group. C. Expression of KLF2 protein in aortas from LPS-injected mice is decreased. n=6; *p<0.05 vs control group. D. Expression of KLF2 protein in LPS-treated endothelial cells is decreased. n=6; *p<0.05 vs control group. E. Overexpression of KLF2 via Ad-KLF2 in mouse aortas in vivo. n=6; *p<0.05 vs control group. F. Overexpression of KLF2 via Ad-KLF2 in cultured mouse endothelial cells in vitro. n=6; *p<0.05 vs control group. G. LPS-induced down-regulation of miR-126a-3p in mouse aortas could be efficiently inhibited by KLF2 over-expression via Ad-KLF2 (Fig. 6G and 6H). n=6; *p<0.05 vs Ad-GFP control group. H. LPS-induced down-regulation of miR-126a-3p in mouse ECs could be efficiently inhibited by KLF2 over-expression via Ad-KLF2. =6; *p<0.05 vs Ad-GFP control group.

Fig. 7. Schematic representation shows a miR-126a-3p mechanism of sepsis-related injuries on vascular ECs and vascular integrity

Endotoxemia in sepsis is able to downregulate the expression of EC-enriched miR-126a-3p in the vascular walls via KLF2. The downregulation of miR-126a-3p could induce endothelial dysfunction, and vascular leakage via their target genes such as SPRED1 and VCAM-1 that could induce vascular injury, irreversible multi-organ failure and sepsis-related death.