The intervention therapy targeting vascular endothelial growth factor (VEGF) has become a specific and effective method for the treatment of diabetic retinopathy (DR). However, some patients did not respond or responded poorly to anti-VEGF therapy, and its effects of eliminating edema and improving vision appear to be unstable in the same patient. Hypoxia-inducible factor-1α (HIF-1α), an important upstream transcriptional regulator of VEGF, is an oxygen concentration-sensitive protein expressed in tissues under hypoxia. It can simultaneously target many downstream target genes except VEGF, such as placental growth factor and angiopoietin-like protein 4, to cause blood-retinal barrier damage and neovascularization, and thus participate in various pathological changes of DR to promote the occurrence and development of DR. Therefore, direct intervention of HIF-1α or targeting one or more downstream target genes regulated by HIF-1α to treat DR may have better efficacy. In the future, the development of effective and safe HIF inhibitors or anti-VEGF with HIF-1α other target gene inhibitors may have broader clinical application prospects.
Objective To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods Eight Sprague Dawley rats were selected to obtain the L1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O2 condition (group A), and under 0.5% O2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Results At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B (t=1.026,P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A (t=22.672,P=0.015;t=18.396,P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B (t=0.594,P=0.781;t=1.251,P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C (t=27.143,P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C (t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C (t=37.648,P=0.006;t=16.729,P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C (t=25.583,P=0.011). Conclusion HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.
ObjectiveTo review the development and applications of hypoxia-inducible factor 1α (HIF-1α) in the strategy of tissue engineered angiogenesis and osteogenesis. MethodThe literature about HIF-1α in tissue engineering technology was reviewed, analyzed, and summarized. ResultsHIF-1α plays a key role in angiogenic-osteogenic coupling, and as an upstream regulator, HIF-1α can regulate the expressions of its target genes related with angiogenesis and osteogenesis. In addition, HIF-1α not only can control and improve the angiogenesis, but also has important significance in proliferation and differentiation of seed cells, especially stem cells, which is the foundation for bone healing. ConclusionsWith the development of tissue engineering technology, the problems in the applications of HIF-1α, such as the effective dose of targeting controlled-release, pro-inflammatory effect, and carcinogenicity, will be explored and solved in the future, so it can be used better in clinical.
Objective To investigate the expression of hypoxia inducible factor 1(HIF1alpha;) in ratsprime; retinae during the embryonic and earlier postnatal period. Methods The retinal expression patterns of HIF-1alpha; protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcriptionpolymerase chain reaction (RT-PCR). Results HIF-1alpha; protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when re tina became more mature. Embryonic rat retina had higher expression of HIF-1alpha; protein and mRNA than postnatal retina, the difference was significant (P＜0.01）. Conclusion The expression of HIF1alpha; in ratsprime;retina e differs from embryonic to earlier postnatal stages.
Objective To investigate the effects and mechanism of 17β-estradiol on the retinal neovasularization in rats with oxygen-induced retinopathy (OIR). MethodsA total of 48 SD rats were randomly divided into control group A, control group B, experimental group A and experimental group B with 12 rats in each group. The rats in control group A and experimental group A received a hypodermic injection of 0.1 ml PBS, and the rats in control group B and experimental group B group received an a hypodermic injection of 0.1 ml 17β-estradiol. At postnatal day 7 (P7) and P14, the mRNA expression of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor (HIF) -1α in the retina were measured by real-time polymerase chain reaction (RTPCR). At P14, endothelial cell nuclei breaking through the internal limiting membrane were counted after staining with hematoxylin and eosin (HE), and the protein expression of VEGF was measured after immunohistochemical staining. The changes of retinal ultrastructure were observed by transmission electron microscopy. ResultsAt P14, the difference of the number of endothelial cell nuclei among four groups was statistically significant(F=10.7, P＜0.05). The number of endothelial cell nuclei in experimental group A was increased greater than that in control group A (P＜0.05) and experimental group B(q=5.16，P＜0.05). But there was no difference between control group A and experimental group B (q=0.25，P＞0.05). The difference of VEGF protein expression among the four groups was statistically significant (P＜0.05). Comparing experimental group A with control group A, B and experimental group B, the difference was statistically significant (P＜0.05). In experimental group A there was ganglion cell swelling, pale staining cytoplasm and mitochondria vacuolizationin, while these were normal in other three groups. At P7 and P14, the differences of VEGF and HIF-1 mRNA expression among four groups were statistically significant(F=14.7,16.1, 13.4, 17.5; P=0.001, 0.005, 0.003, 0.009). At P7, the VEGF mRNA expression in control group B was more than that in control group A (q=5.22, P＜0.05). The VEGF mRNA expression in experimental group B was more than that in experimental group A (q=4.32, P＜0.05). At P14, the VEGF mRNA expression in control group B was more than that in control group A (q=3.72, P＜0.05), but there was no difference of HIF-1 mRNA expression between two groups. The VEGF and HIF-1 mRNA expression in experimental group B were both decreased more than those in experimental group A (q=5.12, 4.08;P＜0.05). Conclusions 17β-estradiol has the effect of two way regulation in VEGF mRNA, which increases VEGF expression in retina under hyperoxic conditions so as to develop the vascular system; which reduces VEGF and HIF-1α expression so as to prevent pathologic neovascularization under hypoxic conditions. It provides some protection from the damage of retinal neovascularization.
ObjectiveTo observe and investigate the effect of HIF-2α in the process of neovascularization of proliferative diabetic retinopathy (PDR).MethodsRetrospective clinical study. From July 2014 to July 2015, 60 eyes of 57 PDR patients diagnosed in Ophthalmology Department of Affiliated Hospital of Qingdao University were included in the study. Twenty-eight eyes of 27 patients received intravitreal injections of 0.5 mg ranibizumab (0.05 ml) at 2-7 days before surgery (ranibizumab group) and other 32 eyes of 30 patients did not (group without ranibizumab). Eighteen eyes of 18 patients with epiretinal membranes were included as controls. Pathological specimens of PDR fibrovascular membrane and premacular membrane were obtained during vitrectomy. The immunohistochemical staining and real-time PCR (RT-PCR) were used to detecting the expression of HIF-2α, Dll4 and VEGF. Kruskal-wallis test was used to compare the expression differences of correlation factors between groups. Spearman correlation analysis was used to analyze the relationship between the two variables.ResultsThe immunohistochemical staining revealed that there were positive expression of HIF-2α, Dll4 and VEGF in all PDR membranes, regardless of the injection of the ranibizumab. The levels of HIF-2α, Dll4 and VEGF protein in the group without ranibizumab were higher than those of the ranibizumab group (t=4.36, 6.01, 4.82; P=0.000, 0.008, 0.016). RT-PCR showed that the differences of the mRNA expression of HIF-2α, Dll4 and VEGF were all statistically significant among the PDR patients and controls (H=18.81,19.60, 20.50; P=0.000, 0.000, 0.000). The expression of HIF-2α, Dll4 and VEGF in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR patients,the expression of HIF-2α, Dll4 and VEGF of the group without ranibizumab was higher than that of the ranibizumab group. The spearman correlation analysis showed that the expression of mRNA between HIF-2α and Dll4, HIF-2α and VEGF were both significantly correlated (r=0.95, 0.87; P＜0.05).ConclusionsThe expression of HIF-2α in the PDR membranes was higher than that of the controls. It is positively correlated with the expression of the DLL4 and VEGF.
ObjectiveTo investigate the effects of hypoxia-inducible factor 1α (HIF-1α) small interfere RNA construct pSUPERH1-siHIF1α on the expression of CD18 and ninjurin-1 by K562 (human chronic myelogenous leukemia cell line) cells cultured with serums from patients with early stage of diabetic retinopathy. MethodsK562 cells were cultured in 4 groups as control group (group A), diabetic group (group B), diabetes and pSUPERH1-siHIF1α transfect group (group C) and diabetes and pSUPER-retro transfect group (group D). The cells in group A were cultured in human serum from age-matched healthy control, and in group B, C and D, the cells were cultured in serum from the subjects of early stage of diabetic retinopathy. Twenty-four hours before the cells were cultured by the serum from the subjects of early stage of diabetic retinopathy, the HIF-1α specific siRNA expression vector pSUPERH1-siHIF1α and empty vector pSUPER-retro were transfected into the cells of group C and D, respectively. The percentages of CD18 and ninjurin-1 positive cell on the surface of K562 cells were measured by Flow Cytometry. The adherent rate between K562 and RF/6A was measured by the rose Bengal staining test. ResultsThe percentages of CD18 positive cell in the group A, B, C and D were significantly different (F=14.33, P=0.01). The percentage of group B was significantly higher than that in group A (P=0.001); the percentage of group C was significantly lower than that in group B (P=0.001) and group D (P=0.02); the difference between group C and A was not significant (95%CI=-14.89-2.13, P=0.12). The differences of the percentage of ninjurin-1 positive cell among the group A, B, C and D were significant (F=39.38, P=0.001). The percentage of group B was significantly higher than that in group A (P=0.00); the difference of the percentage between group C and B was not significant (P=0.06), that was also not significant between group C and D (P=0.49). The differences of the adherent rate between K562 and RF/6A (rhesus monkey retinal choroid blood vessel endothelial cell line) among the group A, B, C and D were significant (F=20.62, P=0.00). The adherent rate of group B was significantly higher than that in group A (P=0.00), the adherent rate in group C was significantly lower than that in group B (P=0.01), but it was still significantly higher than that in group A (P=0.002), the difference of adherent rate between group B and D was not significant (P=0.68). ConclusionUnder the early stage of diabetic retinopathy, HIF-1α small interfere RNA pSUPERH1-siHIF1α may significantly suppress the expression of CD18 on the surface of K562 cells, but it may not significantly influence the expression of ninjurin-1 on the surface of K562 cells.
Objective To study the effects of hypoxic preconditioning on the glucose metabol ism of rat BMSCs and its underlying mechanism so as to provide the theoretical basis for the optimization of the stem-cell based therapy. Methods Density gradient centrifugation method was adopted to isolate rat BMSCs from neonatal SD rats (aged 1-3 days). BMSCs were cultured to 4th passage and divided into 4 groups based on different culture conditions: group A in normoxia condition for 24 hours, group B in 1% O2 for 24 hours, group C in 2-methoxyestradiol (20 μmol/L) for 24 hours before hypoxic preconditioning, and group D in hypoxia-inducible factor 1 (HIF-1) specific siRNA (50 μmol/L) for 12 hours before hypoxicpreconditioning. MTT method was appl ied to evaluate the prol iferation of BMSCs. Biochemical analyzer and Real-timefluorescent quantitative PCR were appl ied to detect the glucose uptake, lactate production, and HIF-1α mRNA and Glut-1mRNA levels of BMSCs. Results MTT showed that the absorbance (A) values were 387.67 ± 58.92, 322.50 ± 50.60, 297.00 ± 53.00, and 286.00 ± 41.00 in groups A, B, C, and D, respectively, showing no significant difference among 4 groups (P gt; 0.05). The levels of glucose uptake and lactate production were higher in group B than in groups A, C, and D, showing significant differences (P lt; 0.05); the levels of groups C and D were higher than those of group A, but showing no significant difference (P gt; 0.05). The mRNA expressions of HIF-1α and Glut-1 elevated significantly in group B when compared with those in group A (P lt; 0.05); groups C and D were significantly lower than group B (P lt; 0.05) and were significantly higher than group A (P lt; 0.05). Conclusion Hypoxic preconditioning can stimulate the glucose uptake and metabol ism of rat BMSCs, whose mechanism is probably related to up-regulating the mRNA expressions of HIF-1α and Glut-1.
Objective To investigate the expression pattern of hypoxia-inducible factor 1α (HIF-1α) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. Methods A total of 66 SD rats (female or male) with weight (250 ± 20) g were randomly divided into 3 groups: normal control group (group A, n=6), pseudo injury group (group B, n=6), and spinal cord injury (SCI) group (group C, n=54). In group A, no treatment was given as normal control. In groupB, only laminectomy was appl ied. In group C, laminectomy was appl ied and static compression model of SCI was built at T10 level. The expression of HIF-1α was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with l ight staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of gl ial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-1α was poorly expressed in group A and increased a l ittle in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of gangl ion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then decl ined. At 14 days, it appeared only in a small amount of gangl ion cells of white matter. There was no significant difference in the number of HIF-1α positive cells between groups A and B (t=1.325, P=0.137). The number of HIF-1α positive cells at each time point in group C was more than those in groups A and B (P lt; 0.05), and there were significant differences between all time points in group C (P lt; 0.05). Conclusion The expression of HIF-1α increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.