Simultaneous interference of SP1 and HIF1α retarding the proliferation, migration, and invasion of human microvascular endothelial cells (HMEC‐1) under hypoxia

Abstract

Objective To investigate the regulation of special protein 1 (SP1) and hypoxia‐inducible factor‐1α (HIF1α) on human microvascular endothelial cells (HMEC‐1) under hypoxic conditions. Methods The expression of SP1 and HIF1α under normoxia and hypoxic conditions were assessed by Western blot. SP1 and HIF1α were knocked down by small interfering RNA (siRNA) under hypoxic conditions. The proliferation, migration, and invasion of HMEC‐1 were measured by cell counting kit 8, 5‐ethynyl‐2′‐deoxyuridine and Transwell coculture system. Western blot analysis and Immunofluorescence were carried out to study the mechanisms of simultaneously inhibiting the adenosine triphosphatase (CD39), 5′‐nucleotidase (CD73), adenosine, and vascular endothelial growth factor (VEGF). We compared the inhibitory effects between groups concurrently interfering SP1, HIF‐1α, and ranibizumab under hypoxic conditions. Results Under hypoxic conditions, the protein expression of SP1 and HIF1α was increased in HMEC‐1, contrarily, SP1 siRNA and HIF1α siRNA downregulated the expression. Simultaneous inhibition of SP1 and HIF1α demonstrated a much greater restraint of proliferation, migration, and invasion characteristics on HMEC‐1 than respectively knocking down SP1 or HIF1α and anti‐VEGF drugs (0.5 mg/mL ranibizumab) (siRNA and the VEGF inhibitor were applied separately in different groups). Meanwhile, simultaneous inhibition of SP1 and HIF1α effectively reduced the expression of CD39, CD73, adenosine, and VEGF on HMEC‐1 under hypoxic conditions. Conclusions Our study demonstrated that both SP1 and HIF1α played important roles in HMEC‐1 under hypoxia condition. Simultaneous inhibition of SP1 and HIF1α effectively decreased the activity of HMEC‐1 under hypoxic conditions through the CD39‐CD73‐adenosine and VEGF angiogenesis pathways. Our study may provide a new approach to the treatment of retinal neovascular diseases.

Heme relieves the inhibition of proliferation, migration and angiogenesis of HMEC-1 under hyperoxia by inhibiting BACH1

“…If the retinal blood vessels in Phase I formed normally, the malignant proliferation of blood vessels caused by peripheral retinal ischemia in phase II can be avoided [3], thus ROP children could obtain better visual function. Studies have shown that human retinal angiogenesis is related to the proliferation, migration and angiogenesis of microvascular endothelial cells [2,22], the HMEC-1 is a recognized model of vascular endothelial cells [11,23]. Therefore, we established a model of HMEC-1 induced by 40% hyperoxia in vitro to simulate the relatively hyperoxia environment of phase I ROP, and found that the biological behaviors of HMEC-1 such as cell proliferation, migration and angiogenesis were inhibited in 40% hyperoxia environment.…”

“…HMEC-1 were purchased from American Type Culture Collection (ATCC, Rockefeller of Maryland, the certi cate and STR test result are supplemented in additional le 1,2). According to the previous experimental practice of our research group [11]. The cell culture medium was prepared in MCDB131 medium containing 10% fetal bovine serum (FBS, Gibco, USA), 1% antibiotics (including streptomycin 0.1mg/mL, and penicillin 100U/mL, Gibco), 2mM glutamine(25030, Gibco) and 1μg/ml hydrocortisone (M3451, Abmole, USA) [12].…”

“…Refered to previous method [11], HMEC-1 were seeded in Millicell (1.5×10 4 /well, 200μL) for 12 hours and then adhered to the wall under normoxia condition. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia condition respectively for 48 hours (change the medium every 24 hours).…”

Haem relieves hyperoxia-mediated inhibition of HMEC-1 cell proliferation, migration and angiogenesis by inhibiting BACH1 expression

“…HMEC-1 cells were purchased from American Type Culture Collection (ATCC, Rockefeller of Maryland), and the certificate and STR test results are supplemented in Additional files 1 and 2. According to the previous protocol established by our research group [11], cells were cultured in MCDB131 medium containing 10% foetal bovine serum (FBS, Gibco, USA), 1% antibiotics (including streptomycin 0.1 mg/mL and penicillin 100 U/mL, Gibco), 2 mM glutamine (25,030, Gibco) and 1 μg/mL hydrocortisone (M3451, Abmole, USA) [12]. A working solution of 10 mM haem (51,280, Sigma, USA) was prepared in 20 mM NaOH (diluted in PBS) [9].…”

“…HMEC-1 cells were seeded in Millicell plates (1.5 × 10 4 / well, 200 μL) for 12 h and then cultured under normoxic conditions until the cells adhered to the well bottoms as previously described [11]. The cells were then subjected to the indicated treatments (0 μM haem, 20 μM haem or vehicle) and cultured under normoxic or hyperoxic conditions for 48 h (the medium was changed every 24 h).…”