Measurements of CD34+/CD45-dim Stem Cells Predict Healing of Diabetic Neuropathic Wounds.

Management of neuropathic foot ulcers in patients with diabetes (DFUs) has changed little over the past decade, and there is currently no objective method to gauge probability of successful healing. We hypothesized that studies of stem/progenitor cells (SPCs) in the early weeks of standard wound management could predict who will heal within 16 weeks. Blood and debrided wound margins were collected for 8 weeks from 100 patients undergoing weekly evaluations and treatment. SPC number and intracellular content of hypoxia-inducible factors (HIFs) were evaluated by flow cytometry and immunohistochemistry. More SPCs entered the bloodstream in the first 2 weeks of care in patients who healed (n = 37) than in those who did not (n = 63). Logistic regression demonstrated that the number of blood-borne SPCs and the cellular content of HIFs at study entry and the first-week follow-up visit predicted healing. Strong correlations were found among week-to-week assessments of blood-borne SPC HIF factors. We conclude that assays of SPCs during the first weeks of care in patients with DFUs can provide insight into how well wounds will respond and may aid with decisions on the use of adjunctive measures.

Sulforaphane-induced transcription of thioredoxin reductase in lens: possible significance against cataract formation.

PURPOSE: Sulforaphane is a phytochemically derived organic isothiocyanate 1-isothiocyanato-4-methylsulfinyl-butane present naturally in crucifers, including broccoli and cauliflower. Biochemically, it has been reported to induce the transcription of several antioxidant enzymes. Since such enzymes have been implicated in preventing cataract formation triggered by the intraocular generation of oxy-radical species, the purpose of this investigation was to examine whether it could induce the formation of antioxidant enzymes in the eye lens. Thioredoxin reductase (TrxR) was used as the target of such induction. METHODS: Mice lenses were cultured for an overnight period of 17 hours in medium 199 fortified with 10% fetal calf serum. Incubation was conducted in the absence and presence of sulforaphane (5 µM). Subsequently, the lenses were homogenized in phosphate-buffered saline (PBS), followed by centrifugation. TrxR activity was determined in the supernatant by measuring the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-dependent reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Non-specific reduction of DTNB was corrected for by conducting parallel determinations in the presence of aurothiomalate. The reduction of DTNB was followed spectrophotometrically at 410 nm. RESULTS: The activity of TrxR in the lenses incubated with sulforaphane was found to be elevated to 18 times of that observed in lenses incubated without sulforaphane. It was also noticeably higher in the lenses incubated without sulforaphane than in the un-incubated fresh lenses. However, this increase was much lower than that observed for lenses incubated with sulforaphane. CONCLUSION: Sulforaphane has been found to enhance TrxR activity in the mouse lens in culture. In view of the protective effect of the antioxidant enzymes and certain nutrients against cataract formation, the findings suggest that it would, by virtue of its ability to enhance the activity of such enzymes, prevent the tissue against oxidative stress that leads to cataract formation. Additional studies with the activities of other antioxidant enzymes such as quinone oxidoreductase and the levels of Nrf2 are in progress.

Protective effect of caffeine against high sugar-induced transcription of microRNAs and consequent gene silencing: a study using lenses of galactosemic mice.

PURPOSE: Previous studies have shown that caffeine prevents the formation of cataracts induced by a high-galactose diet and consequent oxidative stress. The objective of this study was to investigate if this protective effect is reflected in the attenuation of the transcription of microRNAs (miRNAs) known to induce apoptosis and cell death by gene silencing. METHODS: Young CD-1 mice were fed either a normal laboratory diet or a diet containing 25% galactose with or without 1% caffeine. One week later, the animals were euthanized, and the lenses isolated and promptly processed for RNA isolation and subsequent preparation of cDNAs by reverse transcriptase reaction. Mature miRNA (miR)-specific cDNAs were then quantified with PCR in a 96-well microRNA-specific cassette using an ABI7900HT PCR machine. RESULTS: As expected from previous studies, the lenses were positive for all 84 miRs corresponding to the miRNA probes present in the cassette wells. However, the levels of at least 19 miRs were significantly elevated in galactosemic lenses compared to those in the normal lenses. The majority are proapoptotic. Such elevation was inhibited by caffeine. This has been demonstrated for the first time. CONCLUSIONS: Since aberrant elevation of miRNAs silences various genes and consequently deactivates protein translation, and since caffeine downregulates such aberration, the beneficial effect of caffeine could be attributed to its ability to suppress elevation of toxic miRs and consequent gene silencing.

Effect of high sugar levels on miRNA expression. Studies with galactosemic mice lenses.

PURPOSE: Development of cataract is associated with apoptotic death of the lens epithelial cells. The purpose of this investigation was to examine whether this could be explained by enhancement in the expression of certain pro-apoptotic microRNAs (miRs), known to induce apoptosis by hybridizing with target mRNAs, with the consequence of gene silencing. In addition, it was intended to investigate if such expression could be antagonized by reactive oxygen species (ROS) scavengers. METHODS: CD-1 mice weighing about 20 g were divided into three groups and fed diets, respectively, as follows: Control diet, 25% galactose diet, and 25% galactose diet containing 1% sodium pyruvate. After eight days of such a regimen, the mice were euthanized, their lenses promptly isolated and frozen in liquid nitrogen, and RNAs isolated by extraction with standard methods and converted to cDNAs. The miR-specific cDNAs were then quantified by polymerase chain reaction (PCR) using a 96-well microRNA array cassette using an ABI 7900 HT PCR machine. The results were then analyzed using Bioscience software. RESULTS: The lens samples were positive for all of the 84 miRs expected, on the basis of the specific sequences used for amplification. However, as would be apparent from the microarray plot for the normal and galactosemic lenses, expression of at least 24 apoptotic miRs was upregulated. Six apoptotic miRs were downregulated. In the lenses of animals where the galactose diet was fortified with sodium pyruvate, the expression of 12 miRs was completely prevented. The upregulation observed in the other 14 miRs was also significantly downregulated. A comparison of the galactose and galactose plus pyruvate group clearly indicated that pyruvate inhibits the transcription of apoptotic miRS. CONCLUSIONS: The prevention of galactose-induced enhancement in the expression of apoptotic miRs by pyruvate, a compound well known to scavenge reactive oxygen species as well as to inhibit their formation, strongly suggests that the upregulation of miRs in galactosemic animals is due to generation of reactive oxygen species. This is in conformity with our previous studies showing that pyruvate and other ROS scavengers inhibit apoptosis as well as cataract formation.

Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists.

PURPOSE: Cataract is a significant cause of visual disability with relatively high incidence. It has been proposed that such high incidence is related to oxidative stress induced by continued intraocular penetration of light and consequent photochemical generation of reactive oxygen species, such as superoxide and singlet oxygen and their derivatization to other oxidants, such as hydrogen peroxide and hydroxyl radical. The latter two can also interact to generate singlet oxygen by Haber-Weiss reaction. It has been proposed that in addition to the endogenous enzymatic antioxidant enzymes, the process can be inhibited by many nutritional and metabolic oxyradical scavengers, such as ascorbate, vitamin E, pyruvate, and xanthine alkaloids, such as caffeine. METHODS: Initial verification of the hypothesis has been done primarily by rat and mouse lens organ culture studies under ambient as well as ultraviolet (UV) light irradiation and determining the effect of such irradiation on its physiology in terms of its efficiency of active membrane transport activity and the levels of certain metabolites such as glutathione and adenosine triphosphate as well as in terms of apoptotic cell death. In vivo studies on the possible prevention of oxidative stress and cataract formation have been conducted by administering pyruvate and caffeine orally in drinking water and by their topical application using diabetic and galactosemic animal models. RESULTS: Photosensitized damage to lens caused by exposure to visible light and UVA has been found to be significantly prevented by ascorbate and pyruvate. Caffeine has been found be effective against UVA and UVB. Oral or topical application of pyruvate has been found to inhibit the formation of cataracts induced by diabetes and galactosemia. Caffeine has also been found to inhibit cataract induced by sodium selenite and high levels of galactose. Studies with diabetes are in progress. CONCLUSIONS: Various in vitro and in vivo studies summarized in this review strongly support the hypothesis that light penetration into the eye is a significant contributory factor in the genesis of cataracts. The major effect is through photochemical generation of reactive oxygen species and consequent oxidative stress to the tissue. The results demonstrate that this can be averted by the use of various antioxidants administered preferably by topical route. That they will be so effective is strongly suggested by the effectiveness of pyruvate and caffeine administered topically to diabetic and galactosemic animals.

Effectiveness of topical caffeine in cataract prevention: studies with galactose cataract.

PURPOSE: The primary objective of the study was to investigate the possible inhibition of cataract formation by topical administration of caffeine using the galactosemic rat model. It was hypothesized that caffeine will do so by acting as scavenger of reactive oxygen species known to be generated under hyperglycemic conditions. METHODS: Cataract was induced by feeding young rats a diet containing 24% galactose for a period of 25 days. A control group of such rats was treated with a placebo eye drop preparation containing hydroxy propyl methyl cellulose as a wetting agent. In the experimental group, the rats were treated with the above preparation mixed with 72 mM caffeine. RESULTS: Administration of caffeine eye drops was found to significantly inhibit the onset as well as the progress of cataract formation. By day 25 on the galactose diet, all the animals in the control group developed advanced white opacity spread over the entire area of the lens. In the caffeine group, the formation of such opacity remained strikingly inhibited. The lenses remained largely transparent. The transparency data paralleled the higher concentration of glutathione maintained by caffeine treatment. Its levels in the placebo group were 0.8, 0.5, and 0.4 µmoles/g lens wt. on days 5, 15, and 25 against a consistent basal control value of ~3 µmoles/g over the entire period. In the caffeine group, the corresponding values were nearly 3 µmoles/g till day 15, but decreasing to ~2 µmoles/g on day 25. The levels were hence significantly higher than in the caffeine untreated group, remaining relatively closer to the basal controls. In addition, the compound was found effective in inhibiting morphological changes induced by galactose. CONCLUSIONS: Micromolar amounts of topical caffeine have been found to be significantly effective in inhibiting the formation of galactose cataract, strongly suggesting its possible usefulness against diabetic cataracts. The effects are attributed to its ability to prevent oxidative stress and consequent maintenance of tissue metabolic and transport functions, in addition to preventing the induction of apoptosis.

Inhibition of selenite-induced cataract by caffeine.

PURPOSE: The objective of the investigation was to study possible inhibition of oxidative stress and cataract formation by caffeine in vivo. METHODS: Oxidative stress and consequent cataract formation was induced by intraperitoneal administration of a single dose of sodium selenite (1.16 µmol) to Sprague-Dawley rat pups on day 9 postnatally. In experiments designed to inhibit such cataract formation, the pups were pretreated intraperitoneally with caffeine (5.15 µmol), starting 2 days prior to the administration of selenite and continuing such treatment till day 21, when the experiments were terminated. The extent of tissue damage caused by the selenite was assessed biochemically by measurements of the levels of GSH and ATP in the isolated lenses. Cataract formation and its prevention were monitored by examining the eye with pen light illumination and subsequent photography of the isolated lenses. RESULTS: Injection of selenite led to a significant loss of lens clarity because of cataract formation. In the group treated with caffeine, the formation of cataract was significantly prevented. In the caffeine-untreated group, the levels of lens GSH and ATP were substantially lower than in the caffeine-treated group. The levels of GSH decreased from a value of ∼8.2 µmol to ∼2 µmol/g wet weight of the lens. The content of ATP decreased from ∼2.5 µmol to about ∼ 1 µmol. In the case of caffeine-treated group, these decreases were significantly prevented from taking place, the corresponding values of GSH and ATP being ∼5.8 and ∼1.6 µmol/g, respectively. CONCLUSION: Over all, the results suggest that caffeine can exert a significant preventive effect against cataract formation induced by agents generating reactive oxygen species such as sodium selenite.

UV-B-induced damage to the lens in vitro: prevention by caffeine.

Ultraviolet (UV) irradiation is one of the significant risk factors in the genesis of cataracts. Pathogenetically, the process can be triggered by the intraocular generation of various reactive species of oxygen that are well known to be initiated by the penetration of light, especially of the UV frequencies. The contribution of UV exposure in the etiology of this disease is likely to increase further due to ozone depletion in the upper atmosphere. The present studies were undertaken to examine if the UV effects can be attenuated with the xanthine-based alkaloids primarily present in tea and coffee. We have examined this possibility by in vitro lens culture studies with caffeine. As expected, mice lenses incubated in Tyrode solution exposed to UV at 302 nm are physiologically damaged, as evidenced by the inhibition of the active transport of (86)Rb(+), an ion acting as a surrogate of the K(+). There was a simultaneous decrease in the levels of adenosine triphosphate and glutathione. The addition of caffeine to the medium prevented such deleterious effects. That caffeine and perhaps other xanthinoids have a protective effect against cataract formation induced by UV has hence been demonstrated for the first time.

Induction of ultraviolet cataracts in vitro: prevention by pyruvate.

Ultraviolet (UV) radiation is one of the important cataract risk factors. The present studies examined the hypothesis that this effect is due to the UV penetration through the cornea and subsequent induction of a photochemical generation of reactive species of oxygen (ROS) in the aqueous and lens. The hypothesis was ascertained by rat lens organ culture studies conducted under UV (365 nm), with media containing micromolar levels of riboflavin, with and without pyruvate, the latter acting as an ROS scavenger. The implication of ROS in the UV-induced damage was confirmed by measurements of peroxide generation. Damage to the lens was assessed physiologically by measuring the decrease in its active transport of rubidium ions. Biochemically, it was assessed by measuring the lowering of adenosine triphosphate and glutathione. The incorporation of pyruvate in the medium protected the lens against these deleterious effects. That the beneficial effect of pyruvate is attributable to its ROS-scavenging property was proven by the peroxide depletion in its presence, commensurate with its own utilization in parallel. A protective effect of this keto acid against UV-induced tissue damage has been shown for the first time, suggesting its clinical usefulness against UV irradiation-induced pathologies. Hence, further studies on the possible protective effects of such alpha-keto acids against UV damage are in progress.