Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia

Abstract

Individuals with diabetes mellitus suffer from impaired angiogenesis and this contributes to poorer peripheral arterial disease outcomes. In experimental peripheral arterial disease, angiogenesis and perfusion recovery are impaired in mice with diabetes. We recently showed that a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) is upregulated in ischemic endothelial cells and plays a key role in post-ischemic angiogenesis and perfusion recovery following experimental peripheral arterial disease. Here we investigated the role of miR29a in the regulation of endothelial cell ADAM12 expression in ischemia and how hyperglycemia negatively affects this regulation. We also explored whether modulating miR29a can improve impaired post-ischemic angiogenesis associated with hyperglycemia. Additionally, we tested whether miR29a modulation could improve post ischemic angiogenesis in the setting of impaired vascular endothelial growth factor signaling. We forced miR29a expression in ischemic endothelial cells and assessed ADAM12 expression. We also evaluated whether hyperglycemia in vivo and in vitro impair ischemia-induced ADAM12 upregulation and miR29a downregulation. Lastly, we determined whether modulating endothelial cell miR29a expression in ischemia and hyperglycemia could improve impaired endothelial cell functions. We found under ischemic conditions where ADAM12 is upregulated in endothelial cells, miR29a is downregulated. Forced expression of miR29a in ischemic endothelial cell prevented ADAM12 upregulation . In ischemic hind limbs of mice with type 1 diabetes and in endothelial cells exposed to simulated ischemia plus hyperglycemia, ADAM12 upregulation and miR29a downregulation were blunted while angiogenesis was impaired. Knocking down miR29a with an miR29a inhibitor was sufficient to improve ADAM12 upregulation and angiogenesis in simulated ischemia plus hyperglycemia. It was also sufficient to improve perfusion recovery in type 1 diabetes mellitus mice in vivo and angiogenesis in vitro even when vascular endothelial growth factor signaling was impaired with blocking antibodies. In conclusion, MiR29a regulates endothelial cell ADAM12 upregulation in ischemia and this is impaired in hyperglycemia. Modulating miR29a improves impaired post-ischemic angiogenesis associated with hyperglycemia. Impact statement Individuals with diabetes are more likely to develop peripheral arterial disease (PAD), and when PAD is present, in those with diabetes, it is more severe and there is currently no effective medical treatment for impaired blood flow which occurs in diabetics with PAD. The current work advances the field by providing an understanding of a molecular mechanism involved in impaired post ischemic angiogenesis in diabetes. It shows for the first time that failure to downregulate miR29a in ischemic diabetic tissues is a major contributing factor to poor perfusion recovery in experimental PAD, and miR29a is elevated in skeletal muscle samples from human diabetics compared with levels in those without diabetes. Knocking down the elevated miR29a in ischemic diabetic mouse hind limbs improved perfusion recovery following experimental PAD. This shows miR29a modulation as a novel therapeutic target for improving blood flow in diabetics with PAD.

miR-548j-5p Regulates Angiogenesis in Peripheral Artery Disease

“…improved impaired post-ischemic angiogenesis in diabetes [17]. In the present study, miR-548j-5p was observed to facilitate migration and tube formation in EPCs, indicating that miR-548j-5p may contribute to angiogenesis in PAD by regulating the angiogenic activities of EPCs.…”

MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice

“…Male C57BL6 mice (8 weeks of age) were given multiple low-dose STZ (MLD-STZ), intraperitoneal doses of 40 mg/kg of STZ in citrate buffer (pH 4.5) for 5 days consecutively. The level of hyperglycemia in the experimental and control groups of mice were determined by measuring hemoglobin A1C (PTS Diagnostics, IN, U.S.A) and fast blood glucose as previously described [ 12 ], and the groups were found to have identical levels of hemoglobin A1C and fast blood glucose. Three weeks after diabetes induction, all nondiabetic and diabetic mice received unilateral hindlimb artery ligation and excision as described previously [ 12 , 13 ].…”

“…The level of hyperglycemia in the experimental and control groups of mice were determined by measuring hemoglobin A1C (PTS Diagnostics, IN, U.S.A) and fast blood glucose as previously described [ 12 ], and the groups were found to have identical levels of hemoglobin A1C and fast blood glucose. Three weeks after diabetes induction, all nondiabetic and diabetic mice received unilateral hindlimb artery ligation and excision as described previously [ 12 , 13 ]. To understand the function of miR-133a on perfusion recovery, miR-133a was knocked down using cholesterol-conjugated miR-133a antagomir retro-orbitally at a dose of 8 mg/kg body weight in STZ-induced diabetic mice day 1 before HLI; on the other hand, miR-133a was overexpressed in the ischemic limb by using an intramuscular injection of pCMV-miR-133a followed by electroporation day 3 before HLI.…”

“…Mouse muscle tissue and cells were homogenized in RIPA lysis buffer containing 1% protease inhibitor cocktail (Sigma–Aldrich, Shanghai, China) as described previously [ 12 ]. Equal amounts of protein in homogenate samples were separated in SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, U.S.A) and blotted with primary antibodies and corresponding peroxidase-conjugated secondary antibodies.…”

Hsa‐miRNA‐29a protects against high glucose‐induced damage in human umbilical vein endothelial cells

“…Moreover, the increased expression of HBP1, a target gene of miRNA-29a, mediates resisting hypoxic damage by miR-29a. Chen et al 14 demonstrated that miRNA-29a upregulates ADAM metallopeptidase domain 12 (ADAM12) in ischemic endothelial cells, which is impaired in hyperglycemia. 12 Studies have shown that the expression of miRNA-29a decreased significantly in human amniotic mesenchymal stem cells (hAMSCs) exposed to serum-free and hypoxi, and miRNA-29a knockdown resulted in decreased apoptosis of hAMSCs by increasing myeloid cell leukemia-1 (MCL-1).…”