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.
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 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.
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 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 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.
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 observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
ObjectiveTo observe the expression and mechanism of hypoxia-inducible factor-1α (HIF-1α) and p53 protein at the altitude of 5000 meter plateau hypoxia environment in rats, as well as the effect of Astragalus injection. MethodsSixty Sprague Dawley rats were randomly divided into the Astragalus injection intervention group and normal saline control group, 30 rats in each group. Astragalus injection group rats were intraperitoneal injected of Astragalus injection (15 ml/kg) before 30 minutes into the plateau environment simulation cabin, normal saline group rats were intraperitoneal injected with the same volume of saline. 30 minutes after injection, rats in each group were reared in the plateau experiment cabin which simulated altitude of 5000 m (oxygen partial pressure 11.3 kPa) for 2, 6, 8, 12, 24 hours, each time period of 6 rats. When get out, the rats were executed immediately and eyes were harvested. Retinal sections were studied by hematoxylin eosin stain, and immunohistochemical method for HIF-1α and p53 expression. ResultsFor control rats, after 2 hours in the cabin, there was edema in retinal layers. HIF-1α and p53 were expressed mainly in the cytoplasm of retinal layers. When the periods in cabin extended, there was atrophy of retinal nerve fiber layer, swelling and degeneration of ganglion cells. The expression of HIF-1α and p53 was increased. Compared with the control group, the intervention group rat had similar but less severe retinal changes, and the expression of HIF-1α and p53 was significantly decreased (P<0.05). ConclusionAstragalus injection can reduce pathological retinal damage in rats at high altitude environment, and its mechanism may be associated with reduced HIF-1α, p53 expression.