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* Faculty of Pharmacy, University of Catanzaro "Magna Graecia", Roccelletta di Borgia, Italy;
MetaPhore Pharmaceuticals, St. Louis, Missouri;
IBAF CNR, Roccelletta di Borgia, Catanzaro, Italy;
Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Italy; and
|| IRCCS "L. Spallanzani", Rome, Italy
Correspondence: Vincenzo Mollace, M.D., Faculty of Pharmacy, University of Catanzaro "Magna Graecia", Roccelletta di Borgia, 88100 Catanzaro, Italy. E-mail: mollace{at}libero.it
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Key Words: SOD-mimetic • monocytes • reservoirs • therapy • AIDS dementia complex • M40401
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Evidence exists suggesting that HIV-1-infected patients are under chronic oxidative stress [19 , 22 ]. Major causes of the increased concentration of free radicals are a depletion of protective systems [glutathione peroxidase, superoxide dismutase (SOD), vitamin E, and selenium] and an increased production of free radicals (superoxide anion and hydroxyl radical) associated with the activation of lymphocytes and M/M; chronic inflammation; increased polyunsaturated fatty acid concentration and lipid peroxidation; and direct or indirect effects of several opportunistic pathogens [19 , 23 ]. In addition, elevated serum levels of hydroperoxides and malondialdehyde (MDA), indicative of oxidative stress, have also been found in asymptomatic HIV-1-infected patients early in the course of the disease [22 ].
Despite the demonstrated role of free radicals in AIDS-dementia complex, a pharmacological approach to neuro-AIDS based on the use of antioxidants is still controversial. Indeed, antiapoptotic/antioxidant strategies should be considered, alongside antiviral approaches, to design the most efficient therapy for AIDS in the near future.
Recently, a class of nonpeptidic, low molecular-weight compounds (M40403) proved to possess comparable catalytic activity with that of the native SOD enzymes. Therefore, the use of these compounds has been suggested for assessing a better therapeutic approach in diseases mediated by superoxide overproduction [24 , 25 ]. These new SOD mimetics represent a breakthrough in chemical design in that they are stable in vivo, possess high activity, and are selective for superoxide with no activity toward H2O2, peroxynitrite, nitric oxide (NO), or hypochlorite. This novel selectivity resides in the nature of the manganese(II) center in these low molecular-weight complexes. The resting oxidation state of the complex is the reduced state, Mn(II). As a consequence, the complex has no reactivity with reducing agents until it is oxidized to Mn(III) by superoxide; therefore, many oxidants will not oxidize these complexes, including NO and oxygen (operating via a simple one-electron oxidation pathway), as well as other two-electron, nonradical oxidants (e.g., OONO-, H2O2, and OCl-).
The unique selectivity of these complexes for superoxide in the presence of other ROS makes it possible then to dissect the role of superoxide in disease models in which ROS are implicated. We have continued our computer-aided design and synthesis program and have recently developed M40401 (the S,S-dimethyl-substituted derivative of the M40403 biscyclohexylpyridyl class of mimetic), which possesses a much higher catalytic activity at pH = 7.4 [25 ]. In fact, its catalytic rate exceeds 1 x 109 M-1 s-1—comparable with the native Cu/Zn SOD enzymes and about 100 times the activity of M40403 at pH = 7.4. As with M40403, M40401 has no catalase activity or reactivity with peroxynitrite. Conversely, M40401 has been shown to produce central effects counteracting peroxidative processes in brain tissues of rats undergoing ischemia/reperfusion brain damage [22 ].
The present experiments have been performed to evaluate the role of superoxide anions in the apoptotic cell death of astroglial cells incubated with supernatants of HIV-1-infected M/M and to ascertain the protective effect of M40401 on HIV-1-related apoptosis of astroglial cells.
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 View larger version (16K): [in a new window] |
Figure 1. Structural formula of M40401, a nonpeptidyl SOD-mimetic compound.
M40401 was used in the present study to antagonize the formation of
superoxide anions in astroglial cells following their incubation with
supernatants from HIV-1-infected M/M.
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Human astrocytoma cell line
The astroglial cell line Lipari was derived from a 51-year-old
male patient who presented a large, right front-temporal mass
(astrocytoma) [23
]; these cells are not permissive to
HIV-1 infection [14
]. Cells were expanded and cultured
by seeding them in 25 cm2 plastic flasks at a density of
0.7 x 106 cells/flask in complete medium and
incubated at 37°C in humidified air containing 5% CO2.
HIV-1 strain and M/M infection
A monocytotropic strain of HIV-1, HIV-1Ba-L, was
used in all experiments. Characteristics and genomic sequence of this
strain have been described previously [26
,
27
]. The virus was expanded in M/M, collected, filtered,
and stored at -80°C before use [28
]. Its
concentration was 2.1 x 108 genomes, corresponding to
35 ng p24 gag antigen (Ag) and 5000 tissue culture-infectious doses,
50% per ml (TCID50/ml), as assessed by virus titration in
M/M. Macrophages were challenged for 2 h with 300
TCID50/ml virus, then extensively washed with warm medium
to remove the excess of virus, and finally cultured in complete medium
at the same conditions as before. Macrophages were washed and fed every
7 days with fresh complete medium. Supernatants of HIV-1- and
mock-infected M/M were collected at day 14 after virus challenge, spun
to remove cells and cellular debris, and stored at -80°C until use.
Virus production was determined by the antigen-capture assay using a
commercially available p24 gag Ag kit (Abbott Pomezia, Italy).
Challenge of astrocytes
Astrocytes were incubated with supernatants from HIV-1-infected
M/M or mock-infected M/M for 2 h at 37°C in a humidified
incubator. When required, M40401 (5, 10, and 30 µM),
L-NAME (100 µM), and AZT (0.1 µM) were added
immediately before exposure to supernatants of HIV-1- or mock-infected
M/M. Cells were then carefully and repeatedly washed to remove M/M
supernatants and cultured in complete medium with and without a daily
treatment with all the compounds used in this study.
Trypan blue exclusion test of cell viability
The dye exclusion test was used to determine the number of
viable cells after exposure of astrocytes, treated or not treated with
supernatants of HIV- or mock-infected M/M. At different timepoints
after treatment, astrocytes were trypsinized, exposed to dye, and then
visually examined to determine whether cells take up or exclude dye.
The live cells that possess intact cell membranes exclude trypan blue,
whereas dead cells do not.
Evaluation of programmed cell deaths in astrocytes
FACS
Astroglial cells exposed or not exposed to supernatants of
HIV-1-infected M/M were detached gently from plastic 6–8 days after
exposure. Aliquots of 5 x 105 cells were centrifuged
at 300 g for 5 min, and pellets were washed with PBS, placed
on ice, and overlaid with 0.5 ml hypotonic fluorocrome solution
containing 50 µg/ml propidium iodide (PI), 0.1% sodium citrate, and
0.1% Triton X-100. After gentle resuspension in this solution, cells
were left at 4°C for 30 min in the absence of light before analysis.
PI-stained cells were analyzed with a FACScan flow cytometer, and
fluorescence was measured between 565 and 605 nm. The data were
acquired and analyzed by the Lysis II program.
Immunocytochemical studies
Astroglial apoptotic nuclei were assessed by in situ terminal
deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate
(dUTP)-biotin nick-end labeling (TUNEL) of DNA strand breaks. Briefly,
astroglial cells were permeabilized by a 20-min incubation at room
temperature in 0.15 Triton X/0.15 sodium citrate; washed in 10 mM PBS,
pH 7.2; reacted for 1 h at 37°C with TdT and biotin-labeled dUTP
in 30 mM Tris-HCl, pH 7.2, 140 mM sodium cacodylate, and 1 mM cobalt
chloride; and visualized using streptavidin-alcalin phosphatase complex
with natol-fast red. Cells were coverslipped under DPX
mounting, and TUNEL-positive cells were assessed. Negative controls
included sections incubated with biotin-labeled dUTP in the absence of
TdT.
Ultrastructural studies
Cells for electron microscopy were fixed in 2.5% glutaraldehyde
in PBS, pH 7.4, at 4°C and then washed for 2 h in PBS and
post-fixed in osmium tetroxide, 1.33% for 2 h at 4°C. After
several washes in PBS, the cells were dehydrated in graded alcohol,
transferred into toluene, and embedded in Epon 812 resin. The resin was
allowed to polymerize in a dry oven at 60°C for 24 h. Thin
sections were cut with a glass knife Reichert microtome, tained with
toluidine blue, and examined on an Axioscope microscope. Ultrathin
sections were cut on a Reichert microtome using a diamond knife,
stained with uranyl-acetate-lead-hydroxide, and evaluated and
photographed on a Philips electron microscope CM 10 (Philips Electronic
Instruments, Mt. Vernon, NY).
MDA determinations
MDA has been used as a biochemical marker for lipid peroxidation
and was measured by a method described previously [25
].
In particular, levels of MDA were measured in astroglial cell
homogenates 8 days after cell exposure to supernatants of HIV-infected
M/M as indicated above. In particular, astroglial cells were frozen in
liquid nitrogen and homogenized in potassium chloride (1.15%).
Chloroform (2 ml) was then added to each homogenate and spun for 30
min. The organic layer of the sample was removed and dried under
nitrogen gas and reconstituted with 100 µl saline. MDA generation was
evaluated by the assay of thiobarbituric acid (TBA)-reacting compounds.
In particular, the addition of a solution of 20 µl sodium dodecyl
sulphate (SDS; 8.1%), 150 µl 20% acetic acid solution (pH 3.5), 150
µl 0.8% TBA, and 400 µl distilled water produced a chromogenic
product that was extracted in n-butanol and pyridine. The
organic layer was removed, and MDA was read at 532 nm and expressed as
nmol MDA/mg protein of cell homogenate.
Statistical analysis
Statistical significance and standard deviations were assessed
using the Student’s t-test.
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When astrocytes were incubated with supernatants of such HIV-infected M/M, a dramatic reduction of cell viability was seen by optical microscopy and trypan blue exclusion. This phenomenon was time-dependent starting at day 6, reaching its maximum at day 10 after incubation with HIV-1-infected M/M supernatants, and only 11% of astrocytes were viable compared with control (astrocytes not exposed to HIV-1-infected M/M supernatants; Fig. 2 ). In sharp contrast, supernatants of mock-infected M/M only marginally affected astrocyte viability.
 View larger version (20K): [in a new window] |
Figure 2. HIV-1-infected M/M induce astrocytic cell death. Incubation of
astroglial cells with supernatants from HIV-1-infected M/M ( )
time-dependently (3, 6, 8, and 10 days after supernatant exposure)
reduced cell viability as assessed by the trypan blue exclusion method.
The viability of astroglial cells was only affected marginally by
incubation with supernatants from mock-infected M/M ( ) compared with
untreated controls (•).Values are the mean out of four independent
experiments. Error bars represent standard deviations. *,
P < 0.05 astroglial cells treated with supernatants of
HIV-infected M/M versus astrocytes incubated with supernatants of
mock-infected M/M or control.
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Figure 3. M40401 prevents apoptosis in astroglial cells induced by HIV-1-infected
M/M. Supernatants from HIV-1-infected M/M (M/M+HIV) produce apoptotic
cell death of astroglial cells greater than supernatants from
mock-infected M/M (M/M) as evaluated by FACS analysis 8 days after
exposure to supernatants. M40401, but not L-NAME (100 µM)
or AZT (0.1 µM), antagonized this effect. Values are the mean out of
four independent experiments. Error bars represent standard deviations.
*, P < 0.05 astroglial cells treated with supernatants
of HIV-infected M/M versus M40401-treated cells.
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Figure 4. MDA increases within astroglial cells incubated with supernatants of
HIV-infected M/M (M/M+HIV) but not after exposure to supernatants from
mock-infected M/M (M/M). M40401 (30 µM) antagonized MDA
overproduction, and L-NAME (100 µM) and AZT (0.1 µM)
failed to antagonize lipid peroxydation. Values are the mean out of
four independent experiments. Error bars represent standard deviations.
*, P < 0.05 astroglial cells treated with supernatants
of HIV-infected M/M versus astrocytes incubated with supernatants of
mock-infected M/M or control.
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Figure 5. Immunocytochemical studies on HIV-1-infected M/M-related apoptosis in
astrocytes. (a) Incubation of astroglial cells with HIV-1-infected M/M
supernatants leads to DNA fragmentation as shown by appearance in
immunocytochemical preparation of TUNEL-positive cells (red spots, see
arrowheads). (b) M40401 (30 µM) antagonized the generation of
apoptosis in astrocytes subsequent to incubation with supernatants from
HIV-1-infected M/M; no evidence of apoptotic cells was shown under
these conditions. These are representative photomicrographs (original
optical microscopy, 40x) out of four independent
experiments.
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 View larger version (174K): [in a new window] |
Figure 6. Ultrastructural analysis of astroglial cells. (a) Control cell line.
The cells are large with irregular nuclei composed mainly by
euchromatin with a few peripheric heterochromatin. Numerous dense
mitochondria, dilated endoplasmic reticulum, and cytoscheleton
filaments are shown in the cytoplasm (original magnification, x4900).
(b) Incubation (2 h) of astroglial cells with supernatants from
mock-infected M/M did not modify ultrastructural images of astroglial
cells as displayed 8 days later (original magnification, x4900).
(c–e) Astroglial cells exposed to supernatants of HIV-1-infected M/M
undergo apoptotic cell death 6–8 days after exposure. In fact, the
cells displayed 8 days after 2 h exposure to supernatants show an
increase of plasma-membrane protrusions; in many cells, a developed
cytoplasmic blebbing, large vacuoles as a result of cytoplasmic loss,
and cells in which cytoplasm is almost completely absent can be
observed. The chromatin is condensed and marginalized, expressing DNA
fragmentation (original magnification, x1900, x2750, and x3800).
(f) The effect of HIV-1-infected M/M on astroglial
cells is strongly antagonized by coincubation with M40401 (30 µM). In
particular, it is shown that cells maintain the normal architecture and
the normal ratio between cytoplasm and nuclei, which appear almost
completely normal. (g and h) The coincubation with AZT (0.1 µM), an
antiviral compound acting on HIV replication, failed to inhibit the
proapoptotic effect of supernatants of HIV-1-infected M/M, thus
confirming that apoptosis is not a result of the direct infection of
astroglial cells by HIV-1. Ultrastructural analysis was performed 8
days after exposure to M/M supernatants in the presence or absence of
compounds.
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The mechanisms underlying cell death of CNS cells in HIV-related brain disorders are to be clarified still. However, for a long time, the release of neurotoxins by HIV-infected M/M has been believed to play a crucial role in neuro-AIDS [31 ]. In particular, evidence exists that M/M are well-recognized as a major target of HIV-1 in the body, playing a role as reservoir of the virus [31 , 32 ]. They are poorly sensitive to the cytopathic effect of HIV-1, can survive for long time after infection, and thus, produce large quantities of virus particles during their lifespan. Their pathogenic relevance in HIV-1 infection is particular in the CNS, a sequestered compartment where the majority of cells infected by HIV-1 is indeed M/M [33 , 34 ]. The alteration of the homeostasis induced by HIV-1 infection, with consequent production of toxic factors, is claimed to be the main cause of neuronal damage during AIDS. In particular, the release of some coating component of HIV-1, such as gp120 glycoprotein, by HIV-1-infected M/M produces direct and indirect effects in the CNS [35 , 36 ]. Indeed, direct microinfusion of gp120 into several areas of rat CNS is capable of producing apoptotic cell death involving an enhanced cytokine release [9 , 10 , 37 ]. In vitro incubation of human astroglial cells and neurons with gp120 leads to exaggerated release of free radicals and, in turn, to neurodegenerative effects by activation of cytokine network and excitatory amino acid N-methyl-D-aspartate (NMDA)-receptor sensitization [29 , 38 , 39 ]. Previous studies have shown that HIV-1-infected M/M are able to induce apoptosis of T-lymphocytes or astroglial cells via overproduction of several factors, including prostaglandins, CD95-ligand, and free-radicals [14 , 40 ]. Furthermore, arachidonic acid produced by infected M/M has been shown to be able to cause neuronal damage [29 ]. Finally, it has been demonstrated that HIV-1 infection induces a heavy perturbation of oxidative status of M/M, including increased production of MDA and decreased synthesis of endogenous glutathione [41 ], thus indicating that the interaction of HIV-1 with macrophages/microglial cells and the release of HIV-1 components into the CNS represent apoptotic cell death of brain cells in neuro-AIDS via ROS overproduction.
The present experiments show that incubation of human-cultured astroglial cells with supernatants from HIV-1-infected M/M leads to apoptotic cell death of astrocytes, an effect that is driven by overproduction of superoxide anions. This is expressed by sustained generation of thiobarbituric-reactive products (showing the occurrence of lipid peroxidation) in astroglial cells undergoing HIV-1-related apoptosis, with both effects being dose-dependently antagonized by the nonpeptidic SOD mimic, M40401.
For a long time, it has been known that oxidative stress contributes to many aspects of HIV-1 disease pathogenesis, including viral replication, inflammatory response, decreased immune-cell proliferation, loss of immune function, chronic weight loss, and increased sensitivity to drug toxicity [42 ]. On the basis of this evidence, it is likely that the enhancement of endogenous antioxidant moieties and the exogenous supply of free radical scavengers may reduce the brain damage accompanying HIV-1 infection. Evidence exists that the enhancement of antioxidant levels in infected cells leads to reduced HIV-1 replication and probably, HIV-1-related cell damage. In fact, lecithinized SOD (PC-SOD) added exogenously inhibits HIV types 1 and 2 replication in MT-4 cells [43 ]. In addition, PC-SOD shows synergistic interaction with anti-HIV compounds such as AZT, ddI, ddC, KNI-272, and dextran sulfate. PC-SOD also inhibited the oxidative stress-induced depletion of sulfhydryls, which are the cause of diminished antioxidant defenses in HIV-1-infected patients. Conversely, the use of antioxidants other than SOD or catalase has also been investigated to obtain a protective effect in HIV-related cell damage. Indeed, ferulic acid (FA) and ethyl ferulate (EF) are able to induce a marked decrease of HIV-1 p24 gag Ag release [44 ]. Furthermore, SOD and the hydroxyl radical scavengers dimethylthiourea and thiourea inhibited acute HIV-1 replication in M/M and dose-dependently inhibited N-acetyl-cystine (NAC)-mediated enhancement of HIV-1 replication, thus suggesting that oxygen radicals play an important role in self-sustained HIV-1 replication in M/M and that oxygen-radical scavengers other than NAC should be considered as therapeutic agents for AIDS patients [45 ]. This has also been confirmed by the coadministration of the antiviral compound AZT with antioxidants, which has been demonstrated to increase its therapeutic potential [42 ].
Despite this experimental evidence, the use of peptidic enzymes, such as SOD, catalase, or antioxidants other than peptidic scavengers, failed to produce relevant effects when used in the treatment of neurodegenerative disorders [46 ]. This is likely a result of the difficulty of these molecules to gain access to brain tissues and to their limited stability and half-life. Recently, a new class of nonpeptidic SOD mimetic has been developed that possesses the same activity of native SOD and a more reliable pharmacological profile for their use during in vivo experimental procedures. In particular, M40401, the nonpeptidyl SOD mimic used in our experiments, is a stable, low molecular-weight, manganese-containing, nonpeptidic molecule possessing the function and catalytic rate of native SOD enzymes but having the advantage of being a much smaller molecule [24 , 25 ]. Because M40401 has been shown to reduce MDA formation in astroglial cells incubated with supernatants of HIV-infected M/M and because this effect is accompanied by relevant antiapoptotic activity, it is likely that this compound may be a useful pharmacological tool in HIV-related apoptotic cell death of human astroglial cells in vitro. In addition, because M40401 is able to produce neuroprotective effects in vivo when injected peripherally, it is likely to suggest a potential role of these compounds in brain disorders including neuro-AIDS [22 , 47 ].
In conclusion, our results suggest that overproduction of superoxide anions (and possibly other free-radical species), possibly via the release of proinflammatory substances by HIV-infected macrophages/microglial cells, may contribute in astroglial apoptotic cell death in neuro-AIDS. In addition, because of their innovative pharmacological profile, the use on novel, nonpeptidyl SOD mimetics may represent the basis for alternative and efficient strategies in the treatment of neuro-AIDS.
Received August 7, 2001; accepted August 13, 2001.
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