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Division of Experimental Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5
1,
V3, and
V5 integrins
(1, 2, 3, 4). In large vessel-derived endothelium, such as
HUVEC, HIV-1 Tat can act in concert with growth factors like basic
fibroblast growth factor to enhance mitogenesis (3). Under
other conditions, HIV-1 Tat can act as a chemotactic factor for
monocytes that express another VEGF receptor, FLT-1 (1).
In addition to monocyte chemotaxis, HIV-1 Tat increases the expression
of leukocyte adhesion molecules that facilitate the interactions of
inflammatory cells with endothelium (5). HIV-1 Tat also
can trigger apoptosis in PC-12 neuronal cells through the induction of
the apoptotic ligand, TNF- (6). The pleiotropic effects
of this viral protein are believed to be important in the genesis of
Kaposi’s sarcoma and in inflammatory changes seen in the central
nervous system of patients with AIDS dementia. The effects of Tat on small vessel endothelium have not been well characterized. We now report that HIV-1 Tat can induce apoptosis in primary human microvascular endothelial cells (HMVEC) through activation of specific caspases. This effect in small vessel endothelium would enhance transit of virally infected cells and cell-free viral particles from the circulation into the peripheral tissues. Furthermore, HIV-1 Tat as an apoptotic ligand may contribute to endothelial injury syndromes associated with AIDS, such as thrombotic thrombocytopenic purpura.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Abs used were anti-poly(A/DP)-ribose polymerase (anti-PARP) (Boehringer Mannheim, Indianapolis, IN); anti-Bcl-2 (Dako, Glostrup, Denmark, and Transduction Laboratories, Lexington, KY); anti-Bax, anti-Fas, and anti-Fas ligand (anti-Fas-L; Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase-3 and caspase-9 (PharMingen, San Diego, CA); anti-Bcl-xL and anti-Bad (Transduction Laboratories).
Tat protein
HIV-1 Tat protein was purified, lyophilized, and reconstituted in Tat buffer (PBS containing 1 mg of BSA and 0.1 mM/ml DTT) as described (3). The purified Tat protein was endotoxin-free, judging from the timed gel formation assay using the Limulus amebocyte lysate reagent (Sigma, St. Louis, MO). This protein was biologically active, as assessed by its rescue of tat-defective provirus replication in HLM-1 cells (3). Synthetic RGD-containing peptides, HQVSLSKQPTSQPRGD, and basic-rich peptides, SYGRKKRRQRRRPPQ, of Tat were obtained from Intracel (Issaquah, WA).
Cells
HMVEC of lung origin (HMVEC-L) were obtained from Clonetics (San Diego, CA) and cultured in EGM-2 mv medium, containing microvascular endothelial cell growth factors, antimicrobials, and 5% FBS. To avoid phenotypic drift with decreasing expression of various surface receptor molecules, HMVEC-L were not used beyond passage 4.
Stimulation of cells
At 80% confluence, HMVEC-L were starved overnight by placing them in culture medium with the endothelial basal medium, EBM-2 (Clonetics) supplemented with 0.5% FBS. The cells were then stimulated with 10 U/ml heparin with or without 25 ng/ml of HIV-1 Tat protein in fresh EBM-2 medium containing 0.5% FBS for the indicated time periods. It is known that heparin augments the biologic activities of Tat, such as the induction of endothelial cell growth, migration, and invasion in vitro (7). For controls, the cells were stimulated with known apoptotic ligands: LPS (1 µg/ml; Sigma) or with anti-Fas (DX2, 2 µg/ml; PharMingen) as a Fas-L with or without protein G' (2 µg/ml; Sigma) for the indicated time periods. Protein G' is known to enhance anti-Fas-mediated apoptosis according to the manufacturer’s protocol (PharMingen).
Detection of apoptosis
Quantitation of mono- and oligonucleosomes generated from apoptotic cells. Relative amounts of mono- and oligonucleosomes generated from apoptotic HMVEC-L were quantitated with a cell death ELISA kit (Boehringer Mannheim), according to the manufacturer’s protocol. Briefly, Tat-treated, anti-Fas-treated, or control cells were lysed with the manufacturer’s buffer. After centrifugation of the lysates at 200 x g for 10 min, an aliquot of supernatant (cytoplasmic fraction) was transferred onto a streptavidin-coated microtiter plate and incubated for 2 h at room temperature. The plate was then thoroughly washed three times, and 100 µl of substrate solution was added to each plate to develop color, measured at 405 nm.
DNA fragmentation assay. DNA generated from Tat-treated, anti-Fas-treated, or control HMVEC-L was analyzed for evidence of fragmentation. Briefly, cells were lysed with lysis buffer containing 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, and 0.2% Triton X-100, and the fragmented DNA in the lysates was separated from the unfragmented chromosomal DNA by precipitation at 12,000 x g for 30 min. The fragmented DNAs in the supernatants were then digested with 100 ng/ml ribonuclease A (Invitrogen, Carlsbad, CA), 20 ng/ml protease K (Invitrogen), and 1% SDS at 37°C for 45 min, purified by the phenol/chloroform extraction method, and precipitated with ethanol/ammonium acetate. The DNA was then electrophoresed on a 1.6% agarose gel containing 0.5 µg/ml ethidium bromide.
Microscopic analysis of apoptosis. HMVEC-L were grown on chamber slides and stained with the fluorescein in situ cell death detection kit (Boehringer Mannheim), based on the TUNEL method. Intracellular fluorescein-labeled fragmented DNA was detected by microscopic analysis, and the percentage of apoptotic cells was derived by averaging the counts from four different fields based on two independent experiments.
Western blot analysis
Cells stimulated with Tat plus heparin or heparin alone were lysed in radioimmunoprecipitation assay buffer (3). Total cell lysates were clarified by centrifugation at 12,000 x g for 20 min. Protein from the clarified supernatants was quantitated by the Lowry method with a Bio-Rad (Hercules, CA) protein assay kit, then separated by SDS-PAGE (30 µg/lane), followed by transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and probed with the indicated primary Abs at 4°C overnight. Immunoreactive bands were visualized using HRP-conjugated secondary Ab and the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).
Quantitation of TNF in culture supernatants
After Tat stimulation, culture supernatants were collected, and cell debris removed by low speed centrifugation at 1500 rpm for 10 min. TNF levels in the clarified supernatants were measured using a commercial TNF Ag capture ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
Measurement of caspase-3 activity
Caspase-3 activity was assayed using a caspase-3 cellular activity assay kit (Biomol, Plymouth Meeting, PA), according to the manufacturer’s protocol. Briefly, 20 µg protein from each cell extract was added to the microtiter wells, and the reaction was initiated by adding 200 µM Ac-DEVD-pNA substrate. In parallel, the samples were reacted with this substrate in the presence of 0.1 µM Ac-DEVD-CHO, a specific caspase-3 inhibitor, to measure the nonspecific hydrolysis of the substrate. Absorbance was read at 37°C at 405 nm in a microtiter plate reader at the indicated time intervals. To examine the effect of the caspase-3 inhibitor on mono- and oligonucleosome release by Tat, HMVEC-L were incubated with serially diluted cell-permeable DEVD-CHO (BioMol) for 1 h, followed by stimulation with 50 ng/ml of Tat plus 10 U/ml heparin for 24 h. The level of apoptotic HMVEC-L was quantitated by measuring mono- and oligonucleosomes with a cell death ELISA kit (Boehringer Mannheim) as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Internucleosomal fragmentation of cellular DNA is a hallmark of
apoptosis. Thus we evaluated the effects of HIV-1 Tat on the
incorporation of fluorescein into the 3'-OH of nicked chromosomal DNA
(TUNEL analysis), on the fragmentation of chromosomal DNA, and on the
release of mono- and oligonucleosomes from chromosomes. Microscopic
analysis of fluorescein-dUTP-labeled cleaved DNA showed that treatment
of HMVEC-L with Tat plus heparin induced apoptosis (Fig. 1
A). When the percentage of
apoptotic cells was derived by averaging the counts from four different
fields based on two independent experiments, the amount of
intracellular fluorescein-labeled fragmented DNA was shown to peak at
25 ng/ml (Fig. 1Ba). The level of cleavage at this
concentration of Tat was at least 7-fold higher than in the cells
treated with heparin alone, indicating that the fragmentation was due
specifically to Tat. This induction of apoptosis was apparent when the
cells were treated for 24 h, but did not appear before 12 h
(Fig. 1Bb). Furthermore, treatment of HMVEC-L with Tat
protein generated a significant amount of fragmented DNA, a hallmark of
apoptosis (Fig. 1C), confirming that stimulation of the
cells with Tat induces apoptosis.
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C) and fluorescein incorporation into the nicked
chromosomal DNA (Fig. 1A). Quantitation of
fluorescein-labeled cells by microscopy indicated that 
40% of the
cells were stained within 6 h of anti-Fas treatment, whereas
16% of the cells were stained within 24 h of Tat treatment (Fig. 1B). Taken together, the assays indicate that HIV-1 Tat
protein specifically induces apoptotic cell death in HMVEC-L. Tat does not induce Fas or TNF in HMVEC-L
Activation of Fas- or TNF-receptors by Fas-L and TNF,
respectively, is a well characterized trigger of apoptosis
(8, 9, 10, 11). We examined the possible role of Fas or TNF in
mediating Tat-induced apoptosis. Western blot analysis indicated that
HMVEC-L express Fas but not Fas-L, and that Fas expression was not
significantly altered by Tat treatment (Fig. 2A). Similarly, secretion of
TNF into the culture supernatants was not induced by Tat treatment
of HMVEC-L, using a highly specific ELISA (R&D Systems) (data not
shown). These data indicate that Tat-induced apoptosis was not mediated
by Fas or TNF.
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Changes in the concentration-dependent homo- or heterodimer
formation of Bcl family molecules play a key role in apoptosis. Thus we
examined Bcl expression after Tat treatment, by using Western blot
analysis. Bcl-2 protein was not detected by this method using two
different anti-Bcl-2 Abs (Fig. 2B), although these Abs
were proficient in Bcl-2 detection in other tested cell lines
(12, 13, 14). Interestingly, expression of
Bcl-xL, another known apoptotic suppressor, was
detected in HMVEC-L, but was not altered after Tat treatment (Fig. 2B). This suggested that removal of the
Bcl-xL suppressor is not essential for
Tat-induced apoptosis. Similarly, expression of Bax, a pro-apoptotic
molecule, was unaltered by Tat. Bad, which can replace Bax in the
Bax-Bcl-xL complex (thereby enhancing apoptosis),
was also not detected in HMVEC-L by Western blotting (Fig. 2B). Because some of the Bcl family molecules were not
detected in the Tat-treated cells but were apparent in HMVEC-L treated
for 1 h with anti-Fas, we repeated this experiment by
stripping and reprobing the same membrane with the indicated Abs to
confirm the absence of these molecules. Consistent with the results in
Fig. 2, A and B, Fas-L, Bcl-2, and Bad proteins
were not detected in HMVEC-L treated with Tat for 1 h, whereas
those proteins were detected in anti-Fas-treated HMVEC-L and in
untreated Jurkat cells (Fig. 2C). Stimulation of the cells
with either Tat or anti-Fas did not significantly change the amount
of Bcl-xL. These data confirm the results shown
in Fig. 2B, in which Bcl and Bax family members do not
participate in Tat-induced apoptosis in HMVEC-L.
Effect of Fas stimulation on the expression of Bcl family molecules
To further confirm the specific lack of Bcl family expression and
induction by Tat, the expression of Bcl family members was examined
after stimulation of HMVEC-L with anti-Fas plus protein G'.
Interestingly, expression of Bcl-2 and Bad, which was not detected upon
Tat stimulation, was found to be induced by anti-Fas plus protein
G'. Expression of each protein was detectable after 1 h of
anti-Fas stimulation (Fig. 3). It is
noteworthy that the observed apoptosis by Fas ligation occurred despite
appreciable amounts of induced Bcl-2, an anti-apoptotic molecule.
It is possible that Fas ligation gives rise to an excess of the
pro-apoptotic molecule, Bax, so that Bax exhausts the available Bcl-2
(by heterodimerizing with the latter), thus resulting in a molar excess
of Bax-Bax homodimers. To test this possibility, Bax expression in
response to Fas stimulation was investigated. Activation of HMVEC-L
with anti-Fas caused augmented Bax expression, peaking at 3 h
of treatment (Fig. 3). Expression of Bcl-xL was
not changed upon Fas stimulation. Taken together, these data show that
Bcl-2 and Bad were not expressed in Tat-treated HMVEC-L (Fig. 2B) but were induced by anti-Fas treatment (Fig. 3), and
that the relative molar concentrations of homo- and heterodimers of the
Bcl family may regulate Fas-mediated apoptosis.
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Several different pathways can cause apoptosis; some involve
activation of specific caspases, whereas others do not. To test whether
the caspase pathway participates in Tat-induced apoptosis in HMVEC-L,
we measured apoptosis using a PARP cleavage assay. We observed basal
level cleavage of the PARP precursor protein (116 kDa) into a smaller
23-kDa fragment in the unstimulated cells (Fig. 4, A and B), which
might be due to the starvation of HMVEC-L in low serum culture. The
amount of full-length PARP was strongly increased following Tat
treatment, and the subsequent cleavage of the precursor protein was
dependent in part upon the duration of Tat stimulation (Fig. 4A). Similar results were obtained when cells were treated
with LPS, a known inducer of PARP degradation and apoptosis in
endothelial cells (Fig. 4B). Thus, Tat-induced HMVEC-L
apoptosis correlated with the extent of biochemical cleavage of PARP, a
known substrate of caspases.
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Because PARP cleavage is activated by caspases, particularly
caspase-9 and -3, we further investigated specific caspase involvement
in this process. Caspase-9 expression was evaluated upon Tat treatment
by immunoblot analysis. No expression of this enzyme was detected (data
not shown). However, Western analysis showed that caspase-3 (32 kDa)
was expressed and cleaved into 17- to 19-kDa molecules upon treatment
with Tat (Fig. 5A). These data
indicate that PARP cleavage resulted from activated caspase-3 but not
activated caspase-9. Thus, we directly quantitated caspase-3 activity
in the Tat-treated cell lysates using a caspase-3 specific activity
assay kit. We found that caspase-3 activity increased in response to
Tat treatment, peaking at 3 h (Fig. 5B), a
corroboration of the Western analysis (Fig. 5A). However, it
is unclear why a second peak of caspase-3 activity appeared at 24-h
treatment, a result that did not parallel with the Western analysis of
procaspase-3 cleavage (compare Fig. 5, A to B).
Taken together, these data suggest that Tat-induced apoptosis of
HMVEC-L occurs through caspase-3, followed by PARP cleavage.
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Because Tat treatment increased caspase-3 activity, we examined
whether the cell-permeable caspase-3 specific inhibitor, DEVD-CHO,
abrogated Tat-induced apoptosis. To this end, endothelial cells were
exposed to the indicated concentrations of this inhibitor before
treatment with Tat plus heparin. The amount of mono- and
oligonucleosomes generated from each sample was then quantitated with a
cell death ELISA kit. Nucleosomal cleavage was significantly reduced in
a dose-dependent manner upon pretreatment of HMVEC-L with
cell-permeable DEVD-CHO (Fig. 6). These
results indicate that caspase-3 plays a major role in Tat-mediated
apoptosis in HMVEC-L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The basic domain of Tat has homology with heparin-binding growth
factors such as VEGF (15, 16). Tat can co-opt VEGF
signaling pathways by its specific binding to cognate VEGF receptors.
In addition, a classical RGD integrin-binding domain is found in the
amino terminus of HIV-1 Tat. This moiety can activate integrins,
which normally bind to extracellular matrix proteins like fibronectin
(3, 17). Therefore, cell adhesion properties can be
altered by these receptor interactions with HIV-1 Tat.
HIV-1 Tat activation of Flk-1/KDR (1), FLT-1 (1, 3), and 51 and
V3 integrins
(4, 16, 17) may cause cells to be "confused" by the
multiplicity of pathways simultaneously activated, whereas these
phenomena do not normally occur in the presence of physiological
ligands. Alterations in normal growth factor receptor and adhesion
receptor signaling can lead to apoptosis by such confused cell
signaling (18). To test whether a profusion of
inappropriate signals by the RGD motif or basic peptide of Tat causes
apoptosis, the levels of apoptosis of HMVEC-L treated with these
peptides were measured by quantitating mono- and oligonucleosomes
released from the treated cells. Our data showed that significant
chromosomal cleavage occurred when the cells were treated with high
concentrations of the RGD-containing peptide (data not shown). However,
at the corresponding concentrations of Tat (picomolar unit),
these peptides did not induce a significant level of apoptosis (data
not shown). These data suggest that the contributions of the peptide to
the observed Tat-mediated apoptosis are nominal. This result implies
that the signaling cascades leading to Tat-mediated apoptosis might be
distinct from those leading to RGD-mediated angiogenesis and
inflammation in vascular cells (1, 3, 7, 15, 16, 17, 18).
Recent reports demonstrate that several members of the caspase family
play important roles as effector molecules in endothelial apoptosis
(19). For instance, TL-1, a novel tumor necrosis
factor-like cytokine, induces a pathway activating caspase-3. Caspase-3
is a central component of the proteolytic cascade, which culminates in
apoptosis in large vessel aortic endothelial cells
(20, 21). Participation of caspases has also been
demonstrated in the LPS-induced apoptosis of HMVEC (22).
Our study demonstrates that HIV-1 Tat induces apoptosis in HMVEC-L
through PARP cleavage upon activation of caspase-3, and not through the
modulation in expression of several Bcl family molecules. However, it
is unclear why a second peak of caspase-3 activity appeared at 24-h
treatment, a result that did not parallel with the Western analysis of
procaspase-3 cleavage (Fig. 5). It is possible that if the caspase
blocker lacks specificity for an individual caspase and therefore
titrates other caspases, the concentration of the caspase-3 inhibitor
would not be sufficient to maintain the observed inhibitory effect at
24 h. Additional study is required to confirm this
possibility.
Interestingly, Bcl-2, Bad, and Bax proteins were detected in
anti-Fas-treated, but not untreated, HMVEC-L cultures (Fig. 3). The
levels of precursor PARP were also significantly higher in HMVEC-L
cultures stimulated for 1 h with Tat or LPS. However, these
findings must be considered in light of 1) the brevity of the
stimulation period relative to the magnitude of the results, and 2)
earlier reports in which the solubility (and therefore the recovery) of
the relevant factors may have been affected by apoptotic subcellular
redistribution (23, 24, 25, 26), a phenomenon that could account
for the increases observed in the present study.
Endothelial cell injury, teleologically, would act in the interests of the virus by disturbing a component of innate immunity, that of the endothelial barrier, thus allowing the passage of pathogens between the circulation and tissue compartments. In the particular type of microvascular endothelium that we examined (derived from the lung), this Tat-mediated apoptosis could facilitate the egress of HIV virions and HIV-infected cells as they take residence in lung parenchyma. Because different forms of endothelium vary with regard to expression of important cell surface receptors as well as growth properties, it will be necessary to examine microvascular cells derived from other tissue sources, particularly brain, to elucidate mechanisms whereby HIV may transit into other tissue sites. Data (from T. Kim and S. Avraham, unpublished observations) indicate that brain endothelium, but not bone marrow endothelium, respectively, is susceptible to Tat-mediated apoptosis. Elucidating the molecular mechanisms of apoptosis upon HIV-endothelial interaction may lead to novel strategies that will limit dissemination of virus in the host, and thereby contribute to the goal of eradicating HIV-1 with antiretroviral drugs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerome E. Groopman, Division of Experimental Medicine, Harvard Institutes of Medicine/Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Room 309, Boston, MA 02115. E-mail address: jgroopma{at}caregroup.harvard.edu
3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; HMVEC, primary human microvascular endothelial cells; HMVEC-L, HMVEC of lung origin; PARP, poly(A/DP)-ribose polymerase; Fas-L, Fas ligand.
Received for publication May 18, 2001. Accepted for publication July 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V5 integrin to the basic domain of the HIV Tat protein and vitronectin. J. Cell Biol. 121:461. and activation of non-N-methyl-D-aspartate receptors by a NF
B-independent mechanism. J. Biol. Chem. 273:17582.
-chain: defective Fas ligand expression in -chain-deficient mice. J. Immunol. 159:4737.[Abstract]
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