Hindlimb Ischemia Impairs Endothelial Recovery and Increases Neointimal Proliferation in the Carotid Artery

Abstract

Peripheral ischemia is associated with higher degree of endothelial dysfunction and a worse prognosis after percutaneous coronary interventions (PCI). However, the role of peripheral ischemia on vascular remodeling in remote districts remains poorly understood. Here we show that the presence of hindlimb ischemia significantly enhances neointima formation and impairs endothelial recovery in balloon-injured carotid arteries. Endothelial-derived microRNAs are involved in the modulation of these processes. Indeed, endothelial miR-16 is remarkably upregulated after vascular injury in the presences of hindlimb ischemia and exerts a negative effect on endothelial repair through the inhibition of RhoGDIα and nitric oxide (NO) production. We showed that the repression of RhoGDIα by means of miR-16 induces RhoA, with consequent reduction of NO bioavailability. Thus, hindlimb ischemia affects negative carotid remodeling increasing neointima formation after injury, while systemic antagonizzation of miR-16 is able to prevent these negative effects.

Figure 1

Effects of hindlimb ischemia on vascular carotid remodeling. (a) Left: Representative images of ki67 staining (green) in balloon-injured carotid arteries at 2 days after injury. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Right: Quantitative data derived from arterial sections stained with Ki67. Carotid arteries were explanted from BI and FL-BI groups at 2 days after balloon injury. NS: not significant. (b) Left: Representative images of haematoxylin and eosin staining in balloon-injured carotid arteries at 14 days in BI and FL-BI groups. Scale bars, 100 µm. Right: Bar graphs represent the morphometric analysis of arterial sections. Neointima /media ratio of arteries in differently treated groups is shown. Quantitative data derived from arterial sections at different levels from each animal in each group. *P < 0.05 versus BI group; n = 10 for group. (c) Left: Representative sections of carotid arteries stained for CD68⁺ macrophages (brown). Carotid arteries were explanted from BI and FL-BI groups at 14 days after balloon injury. Right: Quantitative analysis from arterial sections stained with CD68⁺cells. *P < 0.05 versus BI group; n = 7. (d) Relative expression of MYOCD, MMP9, VCAM and ICAM mRNA transcripts in balloon-injured arteries, evaluated at 14 days after balloon injury. *P < 0.01 versus BI group; n = 7.

Figure 2

Effects of hindlimb ischemia on carotid artery endothelium. (a) Left: Representative sections of carotid arteries immunostained for the specific endothelial cell marker CD31. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in carotid arteries sections explanted from BI and FL-BI groups at 14 days after balloon injury. Scale bars = 50 µm. Right: Bar graphs represents the percentage of re-endothelializated circumference of the common carotid artery. *P < 0.05 versus BI group, n = 6 for group. (b) Schematic model of the experimental setup. (c) Expression levels of selected miRNAs in the carotid artery endothelium 14 days from injury. *P < 0.05 versus BI group; n = 6. (d) Relative expression of eNOS, VCAM and ICAM mRNA transcripts in carotid artery endothelium 14 days after injury. *P < 0.05 versus BI group; n = 6.

Figure 3

miR‐16 inhibits eNOS expression and activation (a) Cultured ECs were transfected with miR-16 Mimic, miR-16 Inhibitor, Mimic-NC, or Inhibitor-NC. The levels of eNOS mRNA were analyzed at 48 hours after transfection by real-time RT-PCR. *P < 0.01 versus cells transfected with Mimic-NC; n = 5. (b) eNOS expression and activation were determined using western blot analysis. Representative immunoblotting (upper) and quantification (lower) of eNOS and p-eNOS in HUVEC transfected with miR-16 Mimic. *P < 0,01 versus cells transfected with Mimic-NC; n = 4 (c) Measurement of Nitric Oxide (NO) production in ECs transfected with miR-16 mimic or mimic-NC. (*P < 0.05 versus cells transfected with mimic-NC). (d) Left: Representative images of EdU incorporation Assay in ECs in response to TNF-α. Right: Percentage of proliferating ECs (green) after transfection with miR-16 Inhibitor or Inhibitor-NC. *P < 0.05 vs. inhibitor NC.

Figure 4

miR-16 upregulation leads to increased activation of RhoA by targeting RhoGDIα in endothelial cell. (a) Expression levels of RhoGDIα in ECs from rat carotid artery of BI and FL-BI groups at 14 days after balloon injury. *P < 0.01 versus control group; n = 5. (b) Cultured ECs were transfected with miR-16 Mimic, miR-16 Inhibitor, Mimic-NC, or Inhibitor-NC, showing modulation in RhoGDIα levels 48 hours after manipulation of miR-16 levels (real-time RT-PCR). *P < 0.05 versus cells transfected with Mimic-NC; n = 4. (c) Representative immunoblotting (upper) and quantification (lower) of RhoGDIα protein levels in HUVEC transfected with miR-16 Mimic, miR-16 Inhibitor, Mimic-NC or Inhibitor-NC. *P < 0,01 versus cells transfected with Mimic-NC; n = 4 (d) Representative immunoblotting (upper) and quantification (lower) of p-AKT in ECs transfected with miR-16 Inhibitor. *P < 0,01 versus cells transfected with Inhibitor-NC; n = 5. (e) Assay of RhoA activation in ECs transfected with miR-16 Inhibitor and stimulated with TNF. *P < 0.05 versus cells tranfected with Inhibitor-NC and treated with TNFα; n = 4.

Figure 5

Systemic delivery of antagomiR-16 promotes endothelial recovery and inhibits neointima formation in the carotid artery of rats with hindlimb ischemia. (a) Schematic model of the experimental setup. (b) Expression levels of miR-16 in vascular endothelium. Total RNAs were obtained from vascular endothelium of rat carotid artery 14 days after injury. *P < 0.01 versus control group; n = 6. (c) Relative expression of RhoGDIα and eNOS mRNA transcripts in vascular endothelium of rat carotid artery 14 days after injury. *P < 0.05 versus control group; n = 6. (d) Left: Representative images of Haematoxylin and eosin staining in balloon-injured carotid arteries at 14 days in rats treated with or without Antago-16. Scale bars, 100 µm. Right: Bar graphs represent the morphometric analysis of arterial sections. Neointima /media ratio of arteries in differently treated groups is shown. *P < 0.05 versus NC group; n = 7. (e) Left: Representative sections of carotid arteries immunostained for the specific endothelial cell marker CD31. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Carotid arteries were explanted from experimental groups at 14 days after balloon injury. Scale bars = 50 µm. Right: Bar graphs represents the percentage of re-endothelializated circumference of the common carotid artery. *P < 0.05 versus rat treated with antagomir scrambled; n = 6 for group. (f) Left: Representative sections of carotid arteries stained for the macrophage (brown) marker CD68. Carotid arteries were explanted from rats at 14 days after balloon injury. Right: Quantitative data derived from arterial sections stained with CD68 positive cells. *P < 0.05 versus control; n = 5.