Ritonavir inhibits HIF-1α-mediated VEGF expression in retinal pigment epithelial cells in vitro.

PURPOSE: Retinal hypoxia-mediated activation of the hypoxia-inducible factor (HIF pathway) leading to angiogenesis is a major signaling mechanism underlying a number of sight-threatening diseases. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising anti-angiogenic role with fewer side effects. Hence, the primary objective of this study was to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial cells treated with ritonavir under hypoxic and normoxic conditions. METHODS: ARPE-19 and D407 cells were cultured in normoxic or hypoxic conditions, alone or in the presence of ritonavir. Quantitative real-time polymerase chain reaction, immunoblot analysis, sandwich ELISA, endothelial cell proliferation, and cytotoxicity were performed. RESULTS: A 12-h hypoxic exposure resulted in elevated mRNA expression levels of both HIF-1α and VEGF in ARPE-19 and D407 cells. Hence, this time point was selected for subsequent experiments. Presence of ritonavir in the culture medium strongly inhibited VEGF expression in a concentration-dependent manner under hypoxic conditions. Immunoblot analysis demonstrated a substantially reduced protein expression of HIF-1α in the presence of ritonavir. Further, hypoxic exposure-induced VEGF secretion was also inhibited by ritonavir, as demonstrated using ELISA. Finally, ritonavir significantly diminished the proliferation of choroid-retinal endothelial (RF/6A) cells demonstrating potential anti-angiogenic activity. Cytotoxicity studies showed that ritonavir is non-toxic to RPE cells. CONCLUSIONS: This study demonstrates for the first time that ritonavir can inhibit HIF-1α and VEGF in ARPE-19 and D407 cells. Such inhibition may form a platform for application of ritonavir in the treatment of various ocular diseases.

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease.

Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.

Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A.

Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step in the mammalian metabolism of beta-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and mature- forms of PAHX were fully active.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Penetration of vascular contrast media into brain tissue and cerebrospinal fluid. An experimental study in rats.

Recent studies suggest that intravenously administered contrast media (CM) penetrate into cerebrospinal fluid (CSF), and the concentration of CM in CSF increases significantly following acetazolamide or probenecid pretreatment. However, corresponding levels of CM in brain tissue and extracellular fluid (ECF) have not been reported. Adult anesthetized rats were injected intravenously with sodium/meglumine diatrizoate and iohexol. The control group received no pretreatment, and the pretreated group received acetazolamide and probenecid before the CM. The amount of each contrast agent was measured in brain tissue and in CSF by high performance liquid chromatography. Pretreated animals attained significantly higher CSF concentrations of diatrizoate and iohexol than control animals. However, tissue ECF concentrations in pretreated animals were not significantly different than in control animals for either agent. The results are consistent with the idea that a flushing action of CSF helps to remove CM from the brain ECF.

Intravascular contrast media and the blood-brain barrier. Testing the new nonionic agent ioxilan.

The effects of diatrizoate, iohexol, and ioxilan on the blood-brain barrier (BBB) were investigated in normal and hypertensive rats. Anesthetized Wistar rats received 14C-inulin as an indicator for BBB disruption. Diatrizoate, iohexol, or ioxilan (350 and 175 mgI/mL) or normal saline was then injected into the carotid artery (2 mL in 30 seconds). Twenty minutes later, the cardiovascular system was flushed, the brain removed, and each hemisphere was digested. BBB disruption, expressed as counts/minute/mg protein, was compared for each hemisphere in each group by analysis of variance. BBB damage in the diatrizoate-350 group was significantly greater than that in all other groups. No significant BBB damage resulted from iohexol or ioxilan relative to normal saline.

Opiate involvement in contrast media-induced blood pressure changes.

The intravenous administration of contrast media (CM) often alters blood pressure (BP). Osmolality plays a role, but the magnitude and even direction of change varies under similar (osmotic) conditions, indicating the involvement of other mechanisms. Male Wistar rats, anesthetized with pentobarbital, received sodium/meglumine diatrizoate, iohexol, or normal saline, 4 ml/kg, via a tail vein, while blood pressure was recorded continuously. Additional groups were pretreated with the opiate antagonist, naloxone (1 mg/kg, IV), or with an equal volume of normal saline 5 minutes prior to the diatrizoate injection. Comparisons of BP change were made with the Student's t-test. Diatrizoate caused a significant (P less than .0002) increase in BP relative to the saline control group, iohexol did not. Thus, the increase with diatrizoate was significantly greater than with iohexol (P less than .00006). Neither the saline nor naloxone pretreatment altered BP significantly. Saline pretreatment did not alter the significant increase in BP produced by the diatrizoate. However, the diatrizoate-induced increase in BP was prevented by the naloxone pretreatment and was significantly less than after the saline pretreatment (P less than .0001). Based on these and previous results, the authors hypothesize that release of endogenous opioids may play a role in BP changes caused by intravenous CM and that significant CM-induced changes may be prevented pharmacologically with the selective opiate blocker, naloxone.

Physiologic effects of contrast media in the rabbit.

The intravenous administration of contrast media (CM) is often associated with alterations in blood pressure (BP) and heart rate (HR). Osmolality is thought to play a role, but the magnitude and even the direction of change may vary under similar osmotic conditions indicating the involvement of other mechanisms. Conscious rabbits received sodium-meglumine diatrizoate (76%, 1 mL/kg, ear vein) every 10 minutes for 1 hour. A similar injection protocol was performed with normal saline and mannitol (36%), equiosmotic to the contrast agent. BP and HR were monitored continuously. Blood samples were collected at the midpoint between each injection for determination of hematocrit, serum osmolality, and iodine concentration. Group parameters at each time point were compared with the Student's t-test. The administration of mannitol caused changes in serum osmolality, hematocrit, and HR as great or greater than the changes caused by equiosmotic diatrizoate. However, BP increased significantly in the diatrizoate group but not in the mannitol group, relative to normal saline. These results suggest that osmolality is important for certain physiologic changes induced by CM, but that BP changes involve mechanisms in addition to osmolality.

Adrenal catecholamines and related changes during different phases of morphine administration--a histochemical study.

(1) Acute morphine treatment of rats reduced the total catecholamine content as well as the noradrenaline content of the adrenal gland. This was accompanied by increased ATPase activity and increased calcium content of the medullary cells. (2) After chronic morphine treatment, the total catecholamine content remained more or less unaltered, but the noradrenaline content was markedly increased together with the noradrenaline-containing areas, in comparison with what was observed in control animals. The ATPase activity of the medullary cells was found to be diminished along with the decreased calcium content. (3) In nalorphine-induced abstinence, a decrease in total catecholamine content together with a reduction of noradrenaline content and of noradrenaline-containing areas were observed. ATPase activity was also found to be increased with increased calcium content.