Understanding the role of haptoglobin in psoriasis: effects of ultraviolet B.

BACKGROUND: Haptoglobin (Hp) is one of the acute phase proteins, whose main function is to bind free haemoglobin (Hb) and transport it to the liver for degradation and iron recycling. In addition to its role as an Hb scavenger, Hp has been shown to behave as an anti-inflammatory, antioxidant and angiogenic factor. We previously investigated the role of Hp in the pathogenesis of psoriasis, and found that it displays some structural modifications that might be associated with protein function in the disease. Phototherapy is an efficacious treatment for psoriasis, although the biological mechanisms by which phototherapy improves psoriasis are still unclear. AIM: To investigate the effects of ultraviolet (UV)B on Hp to clarify the role of Hp in psoriasis. METHODS: Expression of the genes encoding Hp, interleukin (IL)-6 and IL-10 was assessed in UVB-irradiated and unirradiated HaCaT cells. The biological significance of Hp modulation of UVB treatment was confirmed by ELISA and Western blotting. The Hp gene and protein expression in the skin of patients with psoriasis was also investigated. RESULTS: In vitro results showed that UVB modulated IL-6 and IL-10 gene expression and Hp gene and protein expression in HaCaT cells. The in vivo data also showed that Hp levels were increased in the skin of patients with psoriasis compared with healthy controls. CONCLUSIONS: UVB irradiation was able to modulate Hp production in immortalized keratinocytes. The higher levels of Hp in vivo in both lesional and nonlesional skin suggest that it might have a role in the pathogenesis of the disease.

Haptoglobin from psoriatic patients exhibits decreased activity in binding haemoglobin and inhibiting lecithin-cholesterol acyltransferase activity.

OBJECTIVE: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of haptoglobin (Hpt). BACKGROUND: Hpt is a plasma acute-phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free haemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT). STUDY DESIGN: Hpt was isolated from patients with psoriasis vulgaris, and its activity in haemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. METHODS: Two affinity chromatography steps, the first using resin-coupled haemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by Enzyme-linked immunosorbent assay with stationary haemoglobin or ApoA-I, Hpt in solution and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. RESULTS: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by haemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both haemoglobin binding and LCAT inhibition. CONCLUSIONS: In psoriasis, Hpt displays some structure modification(s), which might be associated with the protein function in the disease.

Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats.

We investigated the role of nitric oxide (NO) in the mitochondrial derangement associated with the functional response to ischemia-reperfusion of hyperthyroid rat hearts. Mitochondria were isolated at 3000 g from hearts subjected to ischemia-reperfusion, with or without N(omega)-nitro-L-arginine (L-NNA, an NO synthase inhibitor). During reperfusion, hyperthyroid hearts displayed tachycardia and low functional recovery. Their mitochondria exhibited O(2) consumption similar to euthyroid controls, while H(2)O(2) production, hydroperoxide, protein-bound carbonyl and nitrotyrosine levels, and susceptibility to swelling were higher. L-NNA blocked the reperfusion tachycardic response and increased inotropic recovery in hyperthyroid hearts. L-NNA decreased mitochondrial H(2)O(2) production and oxidative damage, and increased respiration and tolerance to swelling. Such effects were higher in hyperthyroid preparations. These results confirm the role of mitochondria in ischemia-reperfusion damage, and strongly suggest that NO overproduction is involved in the high mitochondrial dysfunction and the low recovery of hyperthyroid hearts from ischemia-reperfusion. L-NNA also decreased protein content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000 g. This and previous results suggest that the above fraction contains, together with light mitochondria, damaged mitochondria coming from the heaviest fraction, which has the highest cytochrome oxidase activity and capacity to produce H(2)O(2). Therefore, we propose that the high mitochondrial susceptibility to swelling, favoring mitochondrial population purification from H(2)O(2)-overproducing mitochondria, limits hyperthyroid heart oxidative stress.

Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis.

Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pd Delta) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pd Delta and wild-type (wt) ES cells. We found that G6pd Delta ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-X(L). Bcl-X(L) does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Interestingly, ERK activation has opposite outcomes in G6pd Delta ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level.

Evaluation of the antioxidant response in the plasma of healthy or hypertensive subjects after short-term exercise.

Reactive oxygen species are produced during exercise. The antioxidants prevent or limit tissue damages by these species in physiological conditions. In particular, ascorbate and urate scavenge peroxynitrite, which can alter the function of many molecules, including the lecithin-cholesterol acyltransferase (LCAT) enzyme involved in reverse cholesterol transport. The aims of the present study were to compare the plasma antioxidant response to an ergometric test (ET) in hypertensive and healthy subjects, evaluate the exercise-dependent nitrosative stress in plasma, and assess whether the LCAT activity is altered by the exercise. Plasma samples, prepared before and after ET from hypertensive or healthy volunteers, were analysed for their levels of ascorbate, urate, alpha-tocopherol, retinol, nitrotyrosine, and LCAT activity. The alpha-tocopherol and retinol levels did not significantly change in both groups during exercise, while the ascorbate level changed displaying higher increase in controls (+38.8%) than in hypertensives (+17.2%). In these patients, during ET, the urate and nitrotyrosine levels changed more than in normotensives (+13.5 and +40.6% vs -3.1 and +25.2%, respectively). The antioxidants effectively prevented loss or reduction of LCAT activity, as it was similar in hypertensives and normotensives, and did not change after ET. The results demonstrate that exercise is associated with enhanced protein nitrosation, and suggest that the ascorbate or urate levels increase to limit oxidative damage.

The accumulation of alpha-Tocopherol and Retinol in the milk of water buffalo is correlated with the plasma levels of triiodothyronine.

Milk is the most important source of Retinol and alpha-Tocopherol for calves. These antioxidants save the food quality and prevent lipid oxidation in the mammary gland and the calf growing tissues. In Bubalus bubalis, seasonal changes for the plasma levels of both antioxidants were not found. The levels of Retinol and alpha-Tocopherol in the milk were 2 and 1.7 times higher in winter than in summer, respectively. These levels were correlated with the plasma level of triiodothyronine, and markedly increased in cows injected with triiodothyronine in summer. The cytosol from alveolar epithelial cells of mammary glands was incubated with alpha-Tocopherol and 3H-Retinol and, after gel filtration chromatography, both antioxidants were found associated with proteins migrating as a single peak of 33 kD. The amount of alpha-Tocopherol and Retinol binding proteins was 1.5 and 2.3 times higher in winter than in summer respectively. The Retinol binding proteins migrated as two bands (33 and 16 kD) by electrophoresis in denaturing and reducing conditions. Our data suggest that triiodothyronine enhances the transport of both liposoluble antioxidants through the blood-mammary barrier, and demonstrate that proteins of the mammary epithelial cells are involved in such a transport.

Lecithin-cholesterol acyltransferase activity during maturation of human preovulatory follicles with different concentrations of ascorbate, alpha-tocopherol and nitrotyrosine.

The enzyme lecithin-cholesterol acyltransferase (LCAT) transfers an acyl chain from lecithin to cholesterol or oestradiol, thus playing a crucial role in reverse cholesterol transport and follicular synthesis of potent long-lived oestrogens. The mechanism of catalysis is biphasic, as it is based on a phospholipase and an esterifying activity. Sulfhydryl groups were previously reported to be required for the esterification step. Lecithin-cholesterol acyltransferase has previously been shown to be inhibited by thiol oxidants such as peroxynitrite. Peroxynitrite also converts tyrosine to nitrotyrosines. In the present study, high levels of nitrotyrosine associated with low LCAT activity, and vice versa, were found in human preovulatory follicular fluids. Follicular fluids were also analysed for oestradiol (E) and progesterone (P) concentrations. The E/P ratio, which decreases as ovulation approaches, was used to evaluate the maturation status of each follicle. Enzyme activity was negatively correlated with the E/P ratio. Ascorbate (Asc) and alpha-tocopherol (Toc) were titrated in follicular fluid and plasma to evaluate their accumulation or consumption in the follicle. High LCAT activity was found in follicular fluids where Asc and Toc had accumulated, whereas lower activity was associated with Asc and Toc consumption. The consumption of both antioxidants was positively correlated with the E/P ratio. The results suggest that as follicle maturation progresses, Toc and Asc concentrations increase in follicular fluid, thus protecting LCAT from oxidative damage and loss of activity.

Synthesis of ascorbate and urate in the ovary of water buffalo.

Blood flow interruption is associated with oxygen depletion and loss of factors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We studied the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were aspirated from each antral follicle, before and during incubation of the ovaries at 39 degrees C. Urate, ascorbate, retinol and alpha-tocopherol in the fluid were, titrated by High Performance Liquid Chromatography (HPLC) with spectrophotometric or spectrofluorimetric detection. The total antioxidant capacity of follicular fluid was determined as absorbance decrease, following addition of a source of radical chromophores. The more the incubation progressed, the higher levels of urate, ascorbate and total antioxidant capacity were found. Conversely, changes in concentration of the liposoluble antioxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-gamma-lactone oxidase in microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD-p-nitroanilide cleaving activity, was found related with the immunoreactivity to anti-CPP32 antibodies. Negative correlation between the enzyme activity (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of nitrotyrosine, a product of peroxynitrite attack on proteins, was measured in follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amount was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concentrations of ascorbate or urate may be associated with oxidative stress during follicular atresia.

Estradiol esterification in the human preovulatory follicle.

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K(m) and V(max) values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.

Haptoglobin inhibits lecithin-cholesterol acyltransferase in human ovarian follicular fluid.

The activity of the enzyme lecithin-cholesterol acyltransferase (LCAT; E.C. 2.3.1.43) is involved in the removal of cholesterol excess from peripheral cells. This activity is stimulated by the HDL (high density lipoprotein) apolipoprotein A1 (ApoA1). Haptoglobin (Hpt) was previously found to be associated with ApoA1 in ovarian follicular fluid. LCAT activity was analyzed in follicular fluids, collected from an IVF program, containing different amounts of Hpt or Hpt/ApoA1 ratio. Addition of purified Hpt to follicular fluid caused a decrease in the enzyme activity, which was measured as the rate of synthesis of cholesteryl esters. In the fractions of fluid proteins, as obtained by gel filtration chromatography, Hpt and HDL were titrated by ELISA while the LCAT activity was assayed by using radioactive cholesterol and purified HDL. When isolated LCAT was incubated with fractions containing different Hpt/ApoA1 ratios, the enzyme activity was found negatively correlated with the Hpt/ApoA1 ratio (P < 0.01). LCAT kinetic parameters were measured in two fractions with the same amount of ApoA1 (5 microg/ml) but different amounts of Hpt (0.69 or 3.77 microg/ml): the V(max) did not change while the K(m) values were 24.1 or 78.6 microM in the presence of the low or high Hpt level, respectively. The analysis of fluids associated with cytoplasmically mature MII oocytes, in a cross-sectional study, confirmed that a negative correlation exists between the Hpt/ApoA1 ratio and the LCAT activity (P < 0.01). The results suggest that Hpt inhibits the reverse transport of cholesterol by preventing ApoA1 stimulation of the LCAT activity.