Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells

Background

Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.

Results

Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N ^G-Nitro-l-arginine methyl ester (l-NAME) treatment or its expression was down-regulated by RNA interference. After long-term l-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O₂ consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.

Conclusion

Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.     

Activation of HIF-1α (hypoxia inducible factor-1α) prevents dry eye-induced acinar cell death in the lacrimal gland

The pathogenesis of immune-mediated lacrimal gland (LG) dysfunction in Sjögren''s syndrome has been thoroughly studied. However, the majority of dry eye (DE) is not related to Sjögren type, and its pathophysiology remains unclear. The purpose of this study was to determine and investigate the protective mechanisms against DE stress in mice. DE induced prominent blood vessel loss without apoptosis or necrosis in the LG. Autophagic vacuoles, distressed mitochondria, and stressed endoplasmic reticulum were observed via electron microscopy. Immunoblotting confirmed the increase in autophagic markers. Glycolytic activities were enhanced with increasing levels of succinate and malate that, in turn, activated hypoxia-inducible factor (HIF)-1α. Interestingly, the areas of stable HIF-1α expression overlapped with COX-2 and MMP-9 upregulation in LGs of DE-induced mice. We generated HIF-1α conditional knockout (CKO) mice in which HIF-1α expression was lost in the LG. Surprisingly, normal LG polarities and morphologies were completely lost with DE induction, and tremendous acinar cell apoptosis was observed. Similar to Sjögren''s syndrome, CD3⁺ and CD11b⁺ cells infiltrated HIF-1α CKO LGs. Our results show that DE induced the expression of HIF-1α that activated autophagy signals to prevent further acinar cell damage and to maintain normal LG function.Dry eye (DE) disease afflicts >15% of the urban population.^{1, 2} It is characterized by discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface because of increases in tear film osmolarity and inflammation. Therefore, it substantially affects the quality of life.^{3, 4} The lacrimal gland (LG), ocular surface, and their interconnecting neural reflex loops work together to produce tears that prevent ocular surface damage from external stimuli. This is defined as the lacrimal functional unit.^{5, 6}LGs can become the target of the immune system and show signs of inflammation that impair its normal function. A progressive loss of exocrine gland function because of glandular damage is induced by lymphocytic infiltration into these target organs. In Sjögren''s syndrome, CD4⁺ T cells infiltrate into accompanying B cells.^{7, 8, 9} Various studies have been conducted on the immune-mediated pathogenesis in LGs of patients with Sjögren''s syndrome. However, most of the DE incidences are non-Sjögren''s syndrome in which nonimmunological injuries that are related to glandular dysfunction are observed.⁸ LG changes and damages in non-Sjogren''s syndrome are less elucidated than those in Sjogren''s syndrome. In Sjögren''s DE disease, the secretory impairment of lacrimal and salivary glands may be related to the extent of lymphocytic infiltration and loss of glandular tissue. However, in non-Sjogren''s disease, inflammatory cell invasion and lymphocytic infiltration are reduced, and other factors may contribute to the impairment in secretory function.^{8, 10, 11}LGs are highly vascularized tissues that secrete water and ions that are transported across the glandular epithelium. Vascular integrity and blood flow are essential for the normal physiological function of LGs. The effects of DE on these factors are not well characterized. The mechanisms by which LG acinar cells protect their secretory function and polarity against DE are also unknown. Recently, we found that prostaglandin (PG) E₂ and cyclooxygenase-2 (COX-2) levels are significantly increased in LGs of humans and mice with DE.¹² However, the mechanisms by which PG synthesis is increased and the exact roles of these PGs in the pathophysiology of DE require further elucidation.The purpose of this study was to investigate the natural protective mechanisms of LGs against DE stress in mice. In addition, we developed a novel hypoxia inducible factor (HIF)-1α conditional knockout (CKO) model to determine the role of HIF-1α in the morphological changes in acinar cells and vascular integrity in DE-induced LGs.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

The effects of delta-9-tetrahydrocannabinol on Krüppel-like factor-4 expression,redox homeostasis,and inflammation in the kidney of diabetic rat

Diabetes mellitus is a complex, multifactorial disorder that is attributed to pancreatic β cell dysfunction. Pancreatic β cell dysfunction results in declining utilization of glucose by peripheral tissues as kidney and it leads to nephropathy. Excessive production and accumulation of free radicals and incapable antioxidant defense system lead to impaired redox status. Macromolecular damage may occur due to impaired redox status and also immune imbalance. Δ9-Tetrahydrocannabinol (THC) is the main active ingredient in cannabis. THC acts as an immunomodulator and an antioxidant agent. Our aim was to evaluate the effects of THC in the diabetic kidney. We analyzed macromolecular damage biomarkers as protein carbonyl (PCO), lipid hydroperoxide (LHP), malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and antioxidant defense system biomarkers as thiol fractions (T-SH, NP-SH, P-SH) and Cu/Zn-superoxide dismutase activity for the antioxidative effects of THC. Furthermore, mRNA expression of Krüppel-like factor-4, secreted immunopositive cell number changes of interleukin-6, nuclear factor κβ (NF-κβ), and peroxisome proliferator-activated receptor-γ and tumor necrosis factor α (TNF-α) levels were analyzed for the immunomodulatory activity of THC. Diabetic rats showed significantly increased levels of PCO, LHP, MDA, and 8-OHdG when compared with controls (P < 0.05 for each parameter). THC significantly reduced the elevated levels of PCO and 8-OHdG (P < 0.05 for both parameters) and also LHP and MDA levels were insignificantly reduced by THC. Also, thiol fractions insignificantly increased in THC administered diabetic kidney when compared with diabetic rats. The NF-κβ cell number significantly decreased in the diabetic rats treated with THC compared with the diabetic group. According to our data, THC has ameliorative effects on the impaired redox status of diabetic kidney and also it acts as an immunomodulator. Therefore, THC might be used as a therapeutic agent for diabetic kidneys but its usage in the healthy kidney may show adverse effects.     

The flavonoid quercetin induces hypoxia-inducible factor-1α (HIF-1α) and inhibits cell proliferation by depleting intracellular iron

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1α (HIF-1α) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1α and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1α expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein–chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1α by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1α and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells

The adult human anterior cruciate ligament (ACL) has a poor functional healing response, whereas the medial collateral ligament (MCL) does not. The difference in intrinsic properties of these ligament cells can be due to their different response to their located microenvironment. Hypoxia is a key environmental regulator after ligament injury. In this study, we investigated the differential response of ACL and MCL fibroblasts to hypoxia on hypoxia-inducible factor-1α, vascular endothelial growth factor, and matrix metalloproteinase-2 (MMP-2) expression. Our results show that ACL cells responded to hypoxia by up-regulating the HIF-1α expression significantly as compared to MCL cells. We also observed that in MCL fibroblasts response to hypoxia resulted in increase in expression of VEGF as compared to ACL fibroblasts. After hypoxia treatment, mRNA and protein levels of MMP-2 increased in both ACL and MCL. Furthermore we found in ACL pro-MMP-2 was converted more into active form. However, hypoxia decreased the percentage of wound closure for both ligament cells and had a greater effect on ACL fibroblasts. These results demonstrate that ACL and MCL fibroblasts respond differently under the hypoxic conditions suggesting that these differences in intrinsic properties may contribute to their different healing responses and abilities.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4°C

This study evaluated the protective effects of supplementation with three different sugars on the motility, morphology and DNA integrity of rat epididymal sperm chilled and stored at 4°C Epididymides were obtained from each donor. Rat epididymal sperm was diluted in Ham's F10 plus raffinose, Ham's F10 plus trehalose, Ham's F10 plus fructose, and Ham's F10 medium for control purposes. Thereafter, the extended sperm were chilled and stored in liquid form at 4°C. Sperm motility, morphological abnormalities and DNA damage were determined at 0 and 12h after chilling. No significant difference was observed in any of the parameters evaluated at 0h, before storage (P>0.05). After 12h of storage, all sugar additives led to statistically higher motility, normal sperm morphology and DNA integrity in comparison to the control group. Raffinose gave the best motility percentages (32.86±1.84%) after 12h of storage at 4°C, compared to the other groups (P<0.001). In conclusion, Raffinose, trehalose and fructose provided a better protection of sperm functional parameters against chilling injury, in comparison to the control group.     

The expression patterns of β1,4 galactosyltransferase I and V mRNAs, and Galβ1-4GlcNAc group in rat gastrocnemius muscles post sciatic nerve injury

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β1,4-galactosylation is performed by a family of β1,4-galactosyltransferases (β1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides and play roles in sciatic nerve regeneration after sciatic nerve injury. In the present study, the expression of β1,4-galactosyltransferase (β1,4-GalT) I, V mRNAs and Galβ1-4GlcNAc group were examined in rat gastrocnemius muscles after sciatic nerve crush and transection. Real time PCR revealed that β1,4-GalT I and V mRNAs expressed at a high level in normal gastrocnemius muscles and decreased gradually from 6 h, reached the lowest level at 2 weeks, then restored gradually to relatively normal level at 4 weeks after sciatic nerve crush. In contrast, in sciatic nerve transection model, β1,4-GalT I and V mRNAs decreased gradually from 6 h, and remained on a low level at 4 weeks in gastrocnemius muscles after sciatic nerve transection. In situ hybridization indicated that β1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellite cells under normal conditions and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 4 weeks after sciatic nerve transection. Furthermore, lectin blotting showed that the expression level of the Galβ1–4GlcNAc group decreased from 6 h, reached the lowest level at 2 weeks, and restored to relatively normal level at 4 weeks after sciatic nerve crush. RCA-I lectin histochemistry demonstrated that Galβ1–4GlcNAc group localized in numerous membranes of myocytes and muscle satellite cells in normal and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 2 and 4 weeks after sciatic nerve transection. These results indicated that the expressions of β1,4-GalT I, V mRNAs and Galβ1–4GlcNAc group were involved in the process of denervation and reinnervation, which suggests that β1,4-GalT I, V mRNAs and Galβ1-4GlcNAc group may play an important role in the muscle regeneration.     

In vitro effects of sodium selenite on nuclear 3,5,3’-triiodothyronine (T3) receptor gene expression in rat pituitary GH4C1 cells

The present study was undertaken in order to investigate the effects of sodium selenite on:

1. The growth of rat pituitary GH₄C₁ cells;
2. The nuclear T₃ receptor gene expression;
3. The cytoplasmic protein phosphorylation; and
4. The prolactin secretion in rat pituitary GH₄C₁ cell line.

Sodium selenite (up to 2.5 μM) has no inhibitory effect on GH₄C₁ cell proliferation as well as the prolactin secretion. On the other hand, 0.5 μM sodium selenite significantly decreases the rate of mRNA synthesis and/or degradation of both, the α1 form of the T₃ receptor (TRα1) and the α2 isoform of the T₃ receptor. At 1 μM of sodium selenine, significant changes in the electrophoretic profile of low molecular mass cytoplasmic proteins were found, moreover, sodium selenite (1 μM) also considerably affects phosphorylation of a higher molecular mass proteins. The results based on the in vitro experiments suggest that sodium selenite may affect specific processes at the pretranslational level as well as it may also take part in processes of posttranslational modification of protein(s), the cell vitality and the cell growth remaining unchanged.     

Effect of Astragalus polysaccharides on expression of TNF-α, IL-1β and NFATc4 in a rat model of experimental colitis

AimAstragalus membranaceus is a Chinese medicinal herb and has been shown to improve hapten-induced experimental colitis. One of its major components is polysaccharides. We investigated the effect of Astragalus polysaccharides (APS) on expression of TNF-α, IL-1β and NFATc4 in a rat model of experimental colitis.MethodsThe experimental colitis model was induced by TNBS. Forty five rats were divided into five groups (n = 9): Normal control group, receiving ethanol vehicle with no TNBS during induction and IP saline injection during treatment; TNBS colitis model group (TNBS + IP saline), receiving only IP saline vehicle treatment; APS low dose group (TNBS + L-APS), receiving APS 100 mg/kg; APS high dose group (TNBS + H-APS), receiving APS 200 mg/kg; and positive control group (TNBS + Dexm), receiving dexamethasone 0.3 mg/kg. The clinical features, macroscopic and microscopic scores were assessed. The expressions of TNF-α, IL-1β and NFATc4 were measured by real-time PCR and ELISA assays.ResultsCompared to normal control rats, TNBS + IP saline had significant weight loss, increased macroscopic and microscopic scores, higher disease activity index (DAI) up-regulation of TNF-α, IL-1β and NFATc4 mRNA expression and up-regulation of TNF-α and IL-1β protein expression. Compared to TNBS + IP saline, treatment with APS or dexamethasone significantly reduced DAI, partially but significantly prevented TNBS colitis-induced weight loss and improved both macroscopic and microscopic scores; high dose APS or dexamethasone significantly down-regulated TNF-α and IL-1β expressions (both mRNA and protein) and up-regulated NFATc4 mRNA and protein expression. The effect of high dose APS and dexamethasone is comparable.ConclusionsAPS significantly improved experimental TNBS-induced colitis in rats through regulation of TNF-α, IL-1β and NFATc4 expression.     

The effect of N-Acetyl cysteine on the expression of Fxr (Nr1h4), LXRα (Nr1h3) and Sirt1 genes,oxidative stress,and apoptosis in the liver of rats exposed to different doses of cadmium

Molecular Biology Reports - The aim of this study was to consider the expression of farnesoid X receptor (Fxr), liver X receptor (LXRα) and sirtuin 1 (Sirt1), oxidative stress, inflammation,...