Fructosyl Amino Oxidase as a Therapeutic Enzyme in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is an age-related disorder that is a global public health problem. The non-enzymatic Maillard reaction results in the formation of advanced glycation end products (AGEs). Accumulation of AGEs in drusen plays a key role in AMD. AGE-reducing drugs may contribute to the prevention and treatment of AGE-related disease. Fructosamine oxidase (FAOD) acts on fructosyl lysine and fructosyl valine. Based upon the published results of fructosamine 3-kinase (FN3K) and FAOD obtained in cataract and presbyopia, we studied ex vivo FAOD treatment as a non-invasive AMD therapy. On glycolaldehyde-treated porcine retinas, FAOD significantly reduced AGE autofluorescence (p = 0.001). FAOD treatment results in a breakdown of AGEs, as evidenced using UV fluorescence, near-infrared microspectroscopy on stained tissue sections of human retina, and gel permeation chromatography. Drusen are accumulations of AGEs that build up between Bruch's membrane and the retinal pigment epithelium. On microscopy slides of human retina affected by AMD, a significant reduction in drusen surface to 45 ± 21% was observed following FAOD treatment. Enzymatic digestion followed by mass spectrometry of fructose- and glucose-based AGEs (produced in vitro) revealed a broader spectrum of substrates for FAOD, as compared to FN3K, including the following: fructosyllysine, carboxymethyllysine, carboxyethyllysine, and imidazolone. In contrast to FN3K digestion, agmatine (4-aminobutyl-guanidine) was formed following FAOD treatment in vitro. The present study highlights the therapeutic potential of FAOD in AMD by repairing glycation-induced damage.

Topical Application of Deglycating Enzymes as an Alternative Non-Invasive Treatment for Presbyopia.

Presbyopia is an age-related vision disorder that is a global public health problem. Up to 85% of people aged ≥40 years develop presbyopia. In 2015, 1.8 billion people globally had presbyopia. Of those with significant near vision disabilities due to uncorrected presbyopia, 94% live in developing countries. Presbyopia is undercorrected in many countries, with reading glasses available for only 6-45% of patients living in developing countries. The high prevalence of uncorrected presbyopia in these parts of the world is due to the lack of adequate diagnosis and affordable treatment. The formation of advanced glycation end products (AGEs) is a non-enzymatic process known as the Maillard reaction. The accumulation of AGEs in the lens contributes to lens aging (leading to presbyopia and cataract formation). Non-enzymatic lens protein glycation induces the gradual accumulation of AGEs in aging lenses. AGE-reducing compounds may be effective at preventing and treating AGE-related processes. Fructosyl-amino acid oxidase (FAOD) is active on both fructosyl lysine and fructosyl valine. As the crosslinks encountered in presbyopia are mainly non-disulfide bridges, and based on the positive results of deglycating enzymes in cataracts (another disease caused by glycation of lens proteins), we studied the ex vivo effects of topical FAOD treatment on the power of human lenses as a new potential non-invasive treatment for presbyopia. This study demonstrated that topical FAOD treatment resulted in an increase in lens power, which is approximately equivalent to the correction obtained by most reading glasses. The best results were obtained for the newer lenses. Simultaneously, a decrease in lens opacity was observed, which improved lens quality. We also demonstrated that topical FAOD treatment results in a breakdown of AGEs, as evidenced by gel permeation chromatography and a marked reduction in autofluorescence. This study demonstrated the therapeutic potential of topical FAOD treatment in presbyopia.

Enzymatic Deglycation of Damaged Skin by Means of Combined Treatment of Fructosamine-3-Kinase and Fructosyl-Amino Acid Oxidase.

The consensus in aging is that inflammation, cellular senescence, free radicals, and epigenetics are contributing factors. Skin glycation through advanced glycation end products (AGEs) has a crucial role in aging. Additionally, it has been suggested that their presence in scars leads to elasticity loss. This manuscript reports fructosamine-3-kinase (FN3K) and fructosyl-amino acid oxidase (FAOD) in counteracting skin glycation by AGEs. Skin specimens were obtained (n = 19) and incubated with glycolaldehyde (GA) for AGE induction. FN3K and FAOD were used as monotherapy or combination therapy. Negative and positive controls were treated with phosphate-buffered saline and aminoguanidine, respectively. Autofluorescence (AF) was used to measure deglycation. An excised hypertrophic scar tissue (HTS) (n = 1) was treated. Changes in chemical bonds and elasticity were evaluated using mid-infrared spectroscopy (MIR) and skin elongation, respectively. Specimens treated with FN3K and FAOD in monotherapy achieved an average decrease of 31% and 33% in AF values, respectively. When treatments were combined, a decrease of 43% was achieved. The positive control decreased by 28%, whilst the negative control showed no difference. Elongation testing of HTS showed a significant elasticity improvement after FN3K treatment. ATR-IR spectra demonstrated differences in chemical bounds pre- versus post-treatment. FN3K and FAOD can achieve deglycation and the effects are most optimal when combined in one treatment.

Biomarkers of disease in human nails: a comprehensive review.

Diagnostic, monitoring, response, predictive, risk, and prognostic biomarkers of disease are all widely studied, for the most part in biological fluids or tissues, but there is steadily growing interest in alternative matrices such as nails. Here we comprehensively review studies dealing with molecular or elemental biomarkers of disease, as opposed to semiological, pharmacological, toxicological, or biomonitoring studies. Nails have a long history of use in medicine as indicators of pathological processes and have also been used extensively as a matrix for monitoring exposure to environmental pollution. Nail clippings are simple to collect noninvasively as well as to transport and store, and the matrix itself is relatively stable. Nails incorporate, and are influenced by, circulating molecules and elements over their several months of growth, and it is widely held that markers of biological processes will remain in the nail, even when their levels in blood have declined. Nails thus offer the possibility to not only look back into a subject's metabolic history but also to study biomarkers of processes that operate over a longer time scale such as the post-translational modification of proteins. Reports on ungual biomarkers of metabolic and endocrine diseases, cancer, and psychological and neurological disorders will be presented, and an overview of the sampling and analytical techniques provided.

A Potential Role for Fructosamine-3-Kinase in Cataract Treatment.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Analysis of protein glycation in human fingernail clippings with near-infrared (NIR) spectroscopy as an alternative technique for the diagnosis of diabetes mellitus.

BACKGROUND: Glycated keratin allows the monitoring of average tissue glucose exposure over previous weeks. In the present study, we wanted to explore if near-infrared (NIR) spectroscopy could be used as a non-invasive diagnostic tool for assessing glycation in diabetes mellitus. METHODS: A total of 52 patients with diabetes mellitus and 107 healthy subjects were enrolled in this study. A limited number (n=21) of nails of healthy subjects were glycated in vitro with 0.278 mol/L, 0.556 mol/L and 0.833 mol/L glucose solution to study the effect of glucose on the nail spectrum. Consequently, the nail clippings of the patients were analyzed using a Thermo Fisher Antaris II Near-IR Analyzer Spectrometer and near infrared (NIR) chemical imaging. Spectral classification (patients with diabetes mellitus vs. healthy subjects) was performed using partial least square discriminant analysis (PLS-DA). RESULTS: In vitro glycation resulted in peak sharpening between 4300 and 4400 cm-1 and spectral variations at 5270 cm-1 and between 6600 and 7500 cm-1. Similar regions encountered spectral deviations during analysis of the patients' nails. Optimization of the spectral collection parameters was necessary in order to distinguish a large dataset. Spectra had to be collected at 16 cm-1, 128 scans, region 4000-7500 cm-1. Using standard normal variate, Savitsky-Golay smoothing (7 points) and first derivative preprocessing allowed for the prediction of the test set with 100% correct assignments utilizing a PLS-DA model. CONCLUSIONS: Analysis of protein glycation in human fingernail clippings with NIR spectroscopy could be an alternative affordable technique for the diagnosis of diabetes mellitus.

Glycated nail proteins: a new approach for detecting diabetes in developing countries.

OBJECTIVE: To assess glycation of nail proteins as a tool in the diagnosis of diabetes. METHODS: Glycation of nail proteins was assessed using a modified photometric nitroblue tetrazolium-based assay, which provides information about average glucose values of the last 6-9 months. Analysis is possible on 10 mg of nail clippings with a within-run coefficient of variation (CV) of 11%. The analyte is extremely stable. The reference range for glycated nail protein (0.55-3.60 µmol/g nail) increases upon ageing. RESULTS: In diabetics (n = 112), values for glycated nail protein are significantly higher (median: 4.07 µmol/g nail, IQR: 2.37-6.89 µmol/g nail, P < 0.0001) than in non-diabetics (n = 116). ROC analysis shows an AUC of 0.848 (specificity 93.1%; sensitivity 68.9%). CONCLUSION: This affordable method is a simple alternative for diagnosing diabetes in remote areas as the pre-analytical phase (including all processes from the time a laboratory request is made by a physician until the sample is ready for testing) is extremely robust.

Lose the Nintendo and thou shall be healed! Restoring vision in malingering.

Non-organic visual loss can be hard to prove or explain to the parents of affected children at times. Here, we describe a simple yet effective approach that may help solve both issues by ensuring that the patient refrains from visual stimuli.

MITOGEN-ACTIVATED PROTEIN INHIBITORS: FULL-FIELD ELECTRORETINOGRAM DEMONSTRATING GENERALIZED RETINAL DYSFUNCTION.

PURPOSE: To report a patient with generalized retinal toxicity to mitogen-activated protein inhibitors. METHODS: Retrospective case report. RESULTS: Full-field electroretinogram findings indicate a generalized toxicity to the use of the mitogen-activated protein inhibitor trametinib. There was an improved response and resolution of serous detachments after decreasing the dose. CONCLUSION: Mitogen-activated protein inhibitors may affect global retinal function, as opposed to the serous detachments that are concentrated in the posterior pole. This may be of importance in further understanding the underlying pathologic mechanisms.

Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation Spectroscopy.

Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K.

Real-World Outcomes in Patients with Diabetic Macular Edema Treated Long Term with Ranibizumab (VISION Study).

AIM: Evaluate long-term real-world treatment patterns and associated effectiveness and safety outcomes in patients with diabetic macular edema (DME) treated ≥36 months with 0.5mg ranibizumab. METHODS: Open-label observational effectiveness study in 9 Belgian clinics. Included were primary treated eyes of 55 DME patients between August 2014 and March 2015 and followed for 3.5±1.8 years. Eyes were 21.8% treatment (TX)-naïve, 9.1% non-naïve with exclusive prior anti-VEGF treatment (PRIOR-anti-VEGF), and 63.6% non-naïve with other prior treatments (PRIOR-other). Intravitreal injections with ranibizumab were administered per ophthalmologists' best clinical judgment. Trend testing of changes in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) over time occurred using mixed regression analysis. RESULTS: The mean±SD number of treatments in the first year was 5.1±3.0 (TX-naïve), 4.5±2.7 (PRIOR-anti-VEGF) and 5.6±3.1 (PRIOR-other). At 12 months, BCVA increased by 8.9±16.4 letters from 59.7±9.3 at baseline in TX-naïve (p<0.0001), by 11.8±9.9 from 61.6±8.5 in PRIOR-anti-VEGF (p=0.03), and by 4.2±10.6 from 58.2±14.6 in PRIOR-other groups (p=0.0002). BCVA remained stable for the remainder of follow-up in all groups. CRT decreased over the first 2 months by monthly rates of -43.8µm in TX-naïve (p=0.04), -75.7µm in PRIOR-anti-VEGF (p=0.02), and -65.8µm in PRIOR-other eyes (p=0.0003), showing stability afterwards. No unknown adverse events were recorded; a painful eye following injection was registered with a possible relationship to the treatment. CONCLUSION: This real-world study confirms the effectiveness of ranibizumab in preventing a decline in BCVA and demonstrated initial improvement and subsequent retention of BCVA in DME patients ≥36 months. Ranibizumab initially reduced and then maintained CRT. However, these data reveal that treatment intensity and BCVA and CRT outcomes are lower than those found in early efficacy trials. Under-treatment likely accounts for this efficacy-effectiveness gap. Yet, intravitreal ranibizumab is an effective and safe long-term treatment for DME under conditions of significant heterogeneity in patients and treatment patterns.

UV Fluorescence-Based Determination of Urinary Advanced Glycation End Products in Patients with Chronic Kidney Disease.

Advanced glycation end products (AGEs) are a class of proteins or lipids that are non-enzymatically glycated and oxidized after contact with aldose sugars. The accumulation of AGEs results in carbonyl stress, which is characteristic for diabetes mellitus, uremia, atherosclerosis and vascular dysfunction. In recent decades, several innovative methods have been developed to measure the concentration of AGEs in blood or urine. In the present study, we evaluated the use of UV fluorescence as an alternative tool to detect urinary AGEs in four groups of well characterized chronic kidney disease (CKD) patients over a wide range of kidney insufficiency and in a group of healthy subjects. Using an excitation wavelength of 365 nm, the fluorescence spectra of urinary AGEs were recorded in the 400-620 nm emission range. When considering the emission peaks at 440 nm and 490 nm, a significantly higher AGE-specific fluorescence intensity was detected in CKD patients compared to healthy subjects (p < 0.0001 and p = 0.0001, respectively). The urinary creatinine adjusted fluorescence emission spectra in the group of CKD patients with diabetes mellitus were comparable with those of CKD patients without diabetes mellitus. Creatinine-adjusted fluorescence emission spectra were highest in CKD patients with proteinuria, moderate in CKD patients without proteinuria and lowest in healthy controls (p < 0.0001 at both emission wavelengths). In a multiple regression analysis, age, CRP and insulin treatment were predictors of fluorescence intensity at the emission wavelength of 440 nm. Age and insulin treatment were predictors at 490 nm. The presented method is a simple, cheap, alternative method to monitor the AGE-load in the CKD population.

Fructosamine-3-Kinase as a Potential Treatment Option for Age-Related Macular Degeneration.

Age-related macular degeneration is the leading cause of blindness in the developed world. Since advanced glycation end products (AGEs) are implicated in the pathogenesis of AMD through various lines of evidence, we investigated the potential of fructosamine-3-kinase (FN3K) in the disruption of retinal AGEs, drusenoid material and drusenoid lesions in patients with AMD. AGE-type autofluorescence was measured to evaluate the effects of FN3K on glycolaldehyde-induced AGE-modified neural porcine retinas and unmodified human neural retinas. Eye pairs from cigarette-smoke- and air-exposed mice were treated and evaluated histologically. Automated optical image analysis of human tissue sections was performed to compare control- and FN3K-treated drusen and near-infrared (NIR) microspectroscopy was performed to examine biochemical differences. Optical coherence tomography (OCT) was used to evaluate the effect of FN3K on drusenoid deposits after treatment of post-mortem human eyes. FN3K treatment provoked a significant decrease (41%) of AGE-related autofluorescence in the AGE-modified porcine retinas. Furthermore, treatment of human neural retinas resulted in significant decreases of autofluorescence (-24%). FN3K-treated murine eyes showed less drusenoid material. Pairwise comparison of drusen on tissue sections revealed significant changes in color intensity after FN3K treatment. NIR microspectroscopy uncovered clear spectral differences in drusenoid material (Bruch's membrane) and drusen after FN3K treatment. Ex vivo treatment strongly reduced size of subretinal drusenoid lesions on OCT imaging (up to 83%). In conclusion, our study demonstrated for the first time a potential role of FN3K in the disruption of AGE-related retinal autofluorescence, drusenoid material and drusenoid lesions in patients with AMD.

A rare case of concomitant sicca keratopathy and ipsilateral central facial palsy in Wallenberg's dorsolateral medullary syndrome.

Objective: To describe a patient with a right-sided supranuclear facial palsy and concomitant sicca keratopathy of the right eye following right-sided dorsolateral medullary infarction. Methods: Our patient underwent a complete ophthalmologic and neurologic examination including biomicroscopy, fundus examination, cranial nerve examination, Shirmer I test, and magnetic resonance imaging of the brain. Results: A 61-year-old woman presented in emergency with a central facial nerve palsy on the right side and truncal ataxia. Neurologic assessment revealed a concurrent dysphagia, dysarthria, hypoesthesia of the right face, and weakness of the right upper limb. Magnetic resonance imaging of the brain showed an old left-sided cerebellar infarction, but a recent ischemic infarction at the level of the right dorsolateral medulla oblongata was the cause of our patient's current problems. One month after diagnosis of the right-sided dorsolateral medullary syndrome, there were complaints of ocular irritation and a diminished visual acuity in the right eye. Biomicroscopy showed a sicca keratopathy with nearly complete absence of tear secretion on the Shirmer I test, but with normal eye closure and preserved corneal reflexes and sensitivity. Conclusion: A dorsolateral medullary syndrome can have a variable expression in symptomatology. Our case is special because of the combination of an ipsilateral supranuclear facial palsy with normal upper facial muscle function together with an ipsilateral sicca keratopathy as a result of a nearly absent tear secretion. We hypothesized that the mechanism underlying the patient's sicca keratopathy ipsilateral to the supranuclear facial palsy involved the superior salivatory nucleus, which is situated in the caudal pons inferiorly of the motor facial nucleus and is most probably affected by a superior extension of the infarcted area in the right medulla oblongata.

Glycation in human fingernail clippings using ATR-FTIR spectrometry, a new marker for the diagnosis and monitoring of diabetes mellitus.

OBJECTIVES: Although HbA1c is a good diagnostic tool for diabetes, the precarity of the health system and the costs limit the use of this biomarker in developing countries. Fingernail clippings contain ±85% of keratins, which are prone to glycation. Nail keratin glycation may reflect the average glycemia over the last months. We explored if attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) can be used as a non-invasive tool for assessing glycation in diabetes. DESIGN AND METHODS: Using ATR-FTIR spectroscopy, glycation and deglycation experiments with fructosamine 3-kinase allowed to identify the spectrum that corresponds with keratin glycation in fingernail clippings. Clippings of 105 healthy subjects and 127 diabetics were subjected to the standardized ATR-FTIR spectroscopy method. RESULTS: In vitro glycation resulted in an increased absorption at 1047cm-1. Following enzymatic deglycation, this peak diminished significantly, proving that the AUC between 970 and 1140cm-1 corresponded with glycated proteins. Within-run CV of the assay was 3%. Storage of nail clippings at 37°C for 2weeks did not significantly change results. In diabetics, glycated nail protein concentrations (median: 1.51µmol/g protein, IQR: 1.37-1.85µmol/g protein) were significantly higher than in the controls (median: 1.19µmol/g protein, IQR: 1.09-1.26µmol/g protein) (p<0.0001). ROC analysis yielded an AUC of 0.92 at a cut-off point of 1.28µmol/g nail (specificity: 82%; sensitivity: 90%). No correlation was observed between the glycated nail protein concentrations and HbA1c. CONCLUSIONS: Protein glycation analysis in fingernails with ATR-FTIR spectroscopy could be an alternative affordable technique for diagnosing and monitoring diabetes. As the test does not consume reagents, and the preanalytical phase is extremely robust, the test could be particularly useful in developing countries.

Complicated spastic paraplegia in patients with AP5Z1 mutations (SPG48).

OBJECTIVE: Biallelic mutations in the AP5Z1 gene encoding the AP-5 ζ subunit have been described in a small number of patients with hereditary spastic paraplegia (HSP) (SPG48); we sought to define genotype-phenotype correlations in patients with homozygous or compound heterozygous sequence variants predicted to be deleterious. METHODS: We performed clinical, radiologic, and pathologic studies in 6 patients with biallelic mutations in AP5Z1. RESULTS: In 4 of the 6 patients, there was complete loss of AP-5 ζ protein. Clinical features encompassed not only prominent spastic paraparesis but also sensory and motor neuropathy, ataxia, dystonia, myoclonus, and parkinsonism. Skin fibroblasts from affected patients tested positive for periodic acid Schiff and autofluorescent storage material, while electron microscopic analysis demonstrated lamellar storage material consistent with abnormal storage of lysosomal material. CONCLUSIONS: Our findings expand the spectrum of AP5Z1-associated neurodegenerative disorders and point to clinical and pathophysiologic overlap between autosomal recessive forms of HSP and lysosomal storage disorders.

Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling.

We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube-like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor-alpha. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX-2 (NS-398) and epidermal growth factor receptor (EGF-R) tyrosine kinase (ZD1839), but is independent of KDR/Flk-1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX-2 and EGF-R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.

Glycation of nail proteins: from basic biochemical findings to a representative marker for diabetic glycation-associated target organ damage.

BACKGROUND: Although assessment of glycated nail proteins may be a useful marker for monitoring of diabetes, their nature and formation are still poorly understood. Besides a detailed anatomical analysis of keratin glycation, the usefulness of glycated nail protein assessment for monitoring diabetic complications was investigated. METHODS: 216 patients (94 males, 122 females; mean age ± standard deviation: 75.0 ± 8.7 years) were enrolled. Glycation of nail and eye lens proteins was assessed using a photometric nitroblue tetrazolium-based assay. Following chromatographic separation of extracted nail proteins, binding and nonbinding fractions were analyzed using one-dimensional gel electrophoresis. Using a hand piece containing a latch-type-bur, a meticulous cutting of the nail plate into superficial and deep layers was performed, followed by a differential analysis of fructosamine. RESULTS: Using SDS PAGE, four and two bands were identified among the nonglycated and glycated nail fraction respectively. Significantly lower fructosamine concentrations were found in the superficial nail layer (mean: 2.16 ± 1.37 µmol/g nails) in comparison with the deep layer (mean: 4.36 ± 2.55 µmol/g nails) (P<0.05). A significant higher amount of glycated eye lens proteins was found in diabetes mellitus patients (mean: 3.80 ± 1.57 µmol/g eye lens) in comparison with nondiabetics (mean: 3.35 ± 1.34 µmol/g eye lens) (P<0.05). A marked correlation was found between glycated nail and glycated eye lens proteins [y (glycated nail proteins) = 0.39 + 0.99 x (eye lens glycated proteins); r2 = 0.58, P<0.001]. The concentration of glycated eye lens proteins and the HbA1c level were found to be predictors of the concentration of glycated nail proteins. CONCLUSIONS: Glycation of nail proteins takes place in the deep layer of finger nails, which is in close contact with blood vessels and interstitial fluid. Glycation of nail proteins can be regarded as a representative marker for diabetic glycation-associated target organ damage.

Invasion of retinal pigment epithelial cells: N-cadherin, hepatocyte growth factor, and focal adhesion kinase.

PURPOSE: To investigate the role of N-cadherin and hepatocyte growth factor (HGF) in the invasion of collagen type I by human retinal pigment epithelial (RPE) cells. METHODS: RPE sheets from eight human eyes were used for characterization through Western blot analysis of the expression of cadherin in total lysates or after immunoprecipitation with anti beta-catenin antibody. First-passage primary cultures of RPE sheets were successfully established from 28 of 56 human eyes. First-passage primary RPE cell cultures on glass substrate, consisting of patches of cells, were used for immunocytochemistry. Fifteen first-passage primary RPE cell cultures in culture vessels were grown to confluence. Four of the 15 first-passage primary RPE cell cultures were investigated for cadherin expression by immunocytochemistry, and the other 11 were further subcultured for two to six passages. These 11 cultures were used for functional assays and investigated for expression of cadherin at regular time intervals. Cells from passage-3 to -4 primary RPE cell cultures were tested for invasion into collagen type I gels, with or without neutralizing antibodies for HGF and N-cadherin, respectively. Activation of the c-Met receptor for HGF and of focal adhesion kinase (FAK) was investigated by immunoprecipitation with anti-phosphotyrosine antibody, gel electrophoresis, and immunostaining on Western blot. Levels of HGF in conditioned medium (CM) of RPE cells were determined by ELISA. RESULTS: RPE cells in culture displayed two phenotypes: Both fibroblast-like and epithelioid cells were present in all 15 first-passage primary cultures and in further passaged cultures derived therefrom. When seeded on collagen, all RPE cells acquired a fibroblast-like phenotype and invaded the collagen type I gel. RPE cells also stimulated branching morphogenesis of MDCK/AZ epithelial canine kidney cell colonies inside collagen. HGF was found in RPE CM, suggesting an autocrine loop for invasion through its c-Met receptor. HGF-neutralizing antibody inhibited invasion of collagen. The major cadherin expressed by RPE cells in culture was N(euronal)-cadherin. Invasion of collagen by RPE cells was also inhibited by an N-cadherin-neutralizing antibody. N-cadherin and c-Met coimmunoprecipitated in RPE cells. FAK and c-Met were both phosphorylated in RPE cells in culture, and phosphorylation was inhibited by antibodies neutralizing either N-cadherin or HGF. CONCLUSIONS: The present investigation provides evidence for an autocrine HGF/c-Met loop that stimulates RPE cell invasion into collagen through FAK. The invasion-stimulatory molecule N-cadherin also activates FAK in invasive RPE cells.

Structure and function of the N-cadherin/catenin complex in retinoblastoma.

PURPOSE: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.