Objective To compare the effects of hypoxia-inducible drugs using deferoxamine (DFO) and accordion technique (AT) on activating the hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway to promote bone regeneration and remodelling during consolidation phase of distraction osteogenesis (DO). Methods Forty-five specific-pathogen-free adult male Sprague-Dawley (SD) rats were randomly divided into the control group, DFO group, and AT group, with 15 rats in each group. All rats underwent osteotomy to establish a right femur DO model. Then, continuous distraction was started for 10 days after 5 days of latency in each group. During the consolidation phase after distraction, no intervention was performed in the control group; DFO was locally perfused into the distraction area in the DFO group starting at the 3rd week of consolidation phase; cyclic stress stimulation was given in the AT group starting at the 3rd week of consolidation phase. The general condition of rats in each group was observed. X-ray films were conducted at the end of the distraction phase and at the 2nd, 4th, and 6th weeks of the consolidation phase to observe the calcification in the distraction area. At the 4th and 6th weeks of the consolidation phase, peripheral blood was taken for ELISA detection (HIF-1α, VEGF, CD31, and Osterix), femoral specimens were harvested for gross observation, histological staining (HE staining), and immunohistochemical staining [HIF-1α, VEGF, osteopontin (OPN), osteocalcin (OCN)]. At the 6th week of the consolidation phase, Micro-CT was used to observe the new bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the distraction area, and biomechanical test (ultimate load, elastic modulus, energy to failure, and stiffness) to detect bone regeneration in the distraction area. Results The rats in all groups survived until the termination of the experiment. ELISA showed that the contents of HIF-1α, VEGF, CD31, and Osterix in the serum of the AT group were significantly higher than those of the DFO group and control group at the 4th and 6th weeks of the consolidation phase (P<0.05). General observation, X-ray films, Micro-CT, and biomechanical test showed that bone formation in the femoral distraction area was significantly better in the DFO group and AT group than in the control group, and complete recanalization of the medullary cavity was achieved in the AT group, and BMD, BV/TV, Tb.Sp, Tb.N, and Tb.Th, as well as ultimate load, elastic modulus, energy to failure, and stiffness in the distraction area, were better in the AT group than in the DFO group and control group, and the differences were significant (P<0.05). HE staining showed that trabecular bone formation and maturation in the distraction area were better in the AT group than in the DFO group and control group. Immunohistochemical staining showed that at the 4th week of consolidation phase, the expression levels of HIF-1α, VEGF, OCN, and OPN in the distraction area of the AT group were significantly higher than those of the DFO group and control group (P<0.05); however, at 6th week of consolidation phase, the above indicators were lower in the AT group than in the DFO group and control group, but there was no significant difference between groups (P>0.05). Conclusion Both continuous local perfusion of DFO in the distraction area and AT during the consolidation phase can activate the HIF-1α/VEGF signaling pathway. However, AT is more effective than local perfusion of DFO in promoting the process of angiogenesis, osteogenesis, and bone remodelling.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
ObjectiveTo investigate the correlations among the cadual homeobox gene 2 (CDX2), hypoxia inducible factor-1α (HIF-1α) protein expressions, and tumor budding in the colorectal cancer (CRC). MethodsIn this study, 63 CRC specimens surgically removed in the First Affiliated Hospital of Xi’an Jiaotong University from January 2012 to September 2015 were collected. The CDX2 and HIF-1α protein expressions were detected by immunohistochemical staining streptavidin-biotin peroxidase two-step method. The staining and the grade of tumor budding were observed under an optical microscope, and the correlation was analyzed using Spearman test. ResultsThe positive expressions of CDX2 and HIF-1α proteins in the CRC tissues were 35 (55.6%) and 47 (74.6%) cases, respectively, which was a negative correlation in the CRC (rs=–0.302, P=0.017). The positive expressions of CDX2 and HIF-1α proteins in the tumor budding of colorectal cancer were 21 (51.2%) and 26 (63.4%) cases, respectively, which was also a negative correlation in the tumor budding of CRC (rs=–0.336, P=0.031), but there was no statistic correlation between the grade of tumor budding and CDX2 or HIF-1α positive protein expression in the CRC (rs=0.113, P=0.370; rs=–0.026, P=0.838). ConclusionsThe positive expression between CDX2 and HIF-1α has a negative correlation in the same CRC specimen and which has a negative correlation in tumor budding. There is no statistic correlation between grade of tumor budding and CDX2 or HIF-1α protein expression in the CRC. Hypoxia environment may be involved in the downregulation of CDX2 level during the malignant progression of CRC.
Objective To investigate the effect of angiopoietin-like protein 3 (ANGPTL3) on lipid metabolism in patients with obstructive sleep apnea (OSA). Methods A total of 59 OSA patients and 20 healthy controls from the First Affiliated Hospital of Zhengzhou University between May 2023 and February 2024 were included in the study. All participants underwent overnight polysomnography (PSG). Based on the apnea-hypopnea index (AHI), the OSA patients were divided into a mild group and a moderate-to-severe group. Morning blood samples were collected after an 8-hour fast to measure lipid profiles and ANGPTL3 levels. Statistical analyses were performed using SPSS 25.0 software. Results The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and ANGPTL3 were significantly higher, while high-density lipoprotein cholesterol (HDL-C) was significantly lower in the OSA group compared with the control group (P<0.05). ANGPTL3 level was higher in the moderate-to-severe OSA group than that in the mild OSA group and the control group, and higher in the mild OSA group than that in the control group (P<0.05). In the severe hypoxemia group, ANGPTL3 level was significantly higher than that in the mild-to-moderate hypoxemia group (P<0.05). The ANGPTL3 level was also significantly higher in the hyperlipidemia group compared wiht the non-hyperlipidemia group (P<0.05). In the OSA group, ANGPTL3 was positively correlated with TC, TG, percentage of cumulative time with oxygen saturation below 90% in total sleep time (T90) and oxygen desaturation index (ODI), and negatively correlated with lowest arterial oxygen saturation (LSaO2) and mean arterial oxygen saturation (MSaO2). After adjusting for relevant confounding factors, logistic regression analysis indicated that ANGPTL3 might be a potential independent risk factor for OSA, with an odds ratio of 1.021 (95%CI 1.002 - 1.040). Conclusions The level of ANGPTL3 is elevated in OSA patients. The elevation of blood lipid levels in OSA patients may be associated with chronic intermittent hypoxia-induced regulation of ANGPTL3 levels.
Objective To investigate the role of Kv1. 5 in the pathogenesis of pulmonary hypertension simulated by hypobaria and hypoxia, and the effects of dichloroacetate ( DCA) on the Kv1. 5 expression in pulmonary arterial smooth muscle cells ( PASMCs ) and mean pulmonary arterial pressure ( mPAP) . Methods Twenty-four SD rats were randomly divided into a normal group ( N group) , a high altitude group ( HA group) , and a DCA treated group ( DCA group) . The N group were fed in normalconditions, the HA group and DCA group were fed in a hypobaria and hypoxia chamber simulated to an altitude of 5000 meters. In addition, the DCA group rats were gastric gavaged with DCA ( 70 mg · kg - 1 · d - 1 ) .Twenty-one days later, percentage of wall thickness ( WT% ) and percentage of wall area ( WA% ) of the pulmonary arteriole, mPAP, and the ratio of right ventricle / left ventricle and septum ( RV/ LV + S) were evaluated. Real-time PCR, immunohistochemistry and Western blot were carried out to detect the Kv1. 5 expression in PASMCs. Results In the HA group, WT% , and WA% of pulmonary arteriole, mPAP and RV/ ( LV + S) all increased significantly compared with the N group ( P lt;0. 01) . These changes in the DCA group were significantly lower than those in the HA group( P lt; 0. 01) . Furthermore, the protein and mRNA expression of Kv1. 5 in the PASMCs deceased significantly in the HA group compared with the N group( P lt;0. 01) , but recovered in the DCA group ( P lt;0. 01) . Conclusions The expression of Kv1. 5 in PASMCs is tremendously inhibited in rats fed in high altitude, which might be a important role of pulmonaryhypertension. DCA can inhibit the remodeling of pulmonary arterials probably by recovering Kv1. 5 expression.
With the surged prevalence of myopia, the pathogenic mechanism underlying myopia has attracted attention. At present, it is generally believed in the flied that the reduced blood perfusion in the choroid is crucial for myopigenesis. Then, in the process of myopigenesis, how are the blurred visual signals transmitted to the choroidal blood vessels through the retina and retinal pigment epithelium, leading to the reduced choroidal blood perfusion. The cellular and molecular mechanisms underpinning this process remain elusive. In recent years, the theory of scleral hypoxia has attracted much attention. Popular signaling molecules in current research include dopamine, epidermal growth factor, retinoic acid, cholinergic molecules and adenosine, etc. These factors are likely to participate in signal transduction in retina and RPE, thus causing changes in choroidal blood flow and affecting the occurrence and development of myopia. Therefore, these signaling factors and their downstream pathways may provide new ideas for the prevention and control of myopia targets.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 µmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 µmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
ObjectiveTo investigate the effects of hypoxia inducible factor 1α (HIF-1α) overexpression on the differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) into vascular endothelial cells.MethodsSHED was isolated from the retained primary teeth donated by healthy children by using collagenase digestion method. The third generation cells were identified by flow cytometry and alizarin red and alkaline phosphatase (ALP) staining after osteogenic differentiation culture. The SHED were divided into blank control group (SHED without any treatment), empty group (SHED infected with empty lentivirus), HIF-1α overexpression group (SHED infected with HIF-1α overexpression lentivirus), Wnt inhibitor group (SHED interfered by IWR-1), and combination group (HIF-1α overexpressed SHED interfered by IWR-1). Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot were used to analyze the expressions of HIF-1α mRNA and protein in the SHED of blank control group, empty group, and HIF-1α overexpression group. Then the SHED in 5 groups were induced differentiation into vascular endothelial cells for 14 days. The expressions of cell surface marker molecule [von Willebrand factor (vWF) and CD31] were detected by flow cytometry. The mRNA expressions of vascular cell adhesion protein 1 (VCAM-1), KDR (Kinase-inserted domain containing receptor), and VE-cadherin (VE) were analyzed by qRT-PCR. The protein expressions of phosphate-glycogen synthasc kinase 3β (p-GSK3β) and β-catenin were analyzed by Western blot. The tube forming ability of induced cells was detected by Matrigel tube forming experiment. The ability of endothelial cells to phagocytic lipid after differentiation was detected by DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) phagocytosis.ResultsAfter identification, the cells were SHED. After lentivirus transfection, compared with the blank control group and the empty group, the expressions of HIF-1α mRNA and protein in the HIF-1α overexpression group increased significantly (P<0.05). Compared with the blank control group and the empty group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly higher (P<0.05), the relative expression of p-GSK3β protein was significantly lower (P<0.05), the number of tubules formed and the ability to phagocytic lipids significantly increased (P<0.05) in the HIF-1α overexpression group; while the indicators in the Wnt inhibitor group were opposite to those in the HIF-1α overexpression group (P<0.05). Compared with the HIF-1α overexpression group, the expressions of VCAM-1, KDR, and VE mRNA, the percentages of vWF positive cells and CD31 positive cells, and the relative expression of β-catenin protein were significantly lower (P<0.05), the relative expression of p-GSK3β protein was significantly higher, and the number of tubules formed and the ability of phagocytosis of lipids significantly reduced, showing significant differences between groups (P<0.05).ConclusionOverexpression of HIF-1α can promote SHED to differentiate into vascular endothelial cells by activating Wnt/β-catenin signaling pathway.
Objective To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by［3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT］test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P＜0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells. Conclusion Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells. (Chin J Ocul Fundus Dis, 2002, 18: 293-295)
Objective To summarize the advance of bioenergetic metabolic mechanisms of cancer cell. Methods Literatures about the recent studies on the bioenergetic metabolic mechanisms of cancer cell were reviewed.Results Cancer cells required a steady source of metabolic energy in order to continue their uncontrolled growth and proliferation. Accelerated uptake of glucose and glycolysis was one of the biochemical characteristics of hypoxia cancer cells. Glucose transport and metabolism were essential for the survival of tumor cells, leading to poor prognosis. Conclusions The studies on relationships between hypoxia-inducible genes and cancer have come a new understanding of the bioenergetic metabolic mechanisms of cancer cell, become new and important supplementary means of diagnosis and treatment of cancer, and enhanced existing strategies so that the treatment could be more rationally applied and personalized for cancer patients.