From the Department of Molecular Genetics and
Biochemistry, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261 and the § Department of
Pediatrics, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
Received for publication, July 30, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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We have previously demonstrated that adenoviral
gene transfer of the NF- Type 1 diabetes is an autoimmune disease characterized by an
inflammatory response resulting in selective destruction of the insulin
secreting The promoters for a number of cytokine-sensitive genes, including
inducible nitric-oxide synthase, intercellular adhesion molecule 1 (ICAM-1), Fas, and FasL contain binding sites for the NF- In addition to IKK- In our earlier studies we have shown protection of human islets from
the effect of IL-1 Recently it was shown that large protein complexes can be delivered
directly to cells when they are linked to protein transduction domains
(PTDs). PTDs are short, positively charged domains that can freely
cross cell membranes through a receptor and energy independent process
(45-48). Proteins fused to the PTD derived from HIV/Tat or the
Drosophila Antennapedia homeobox protein (Antp) transduce a
variety of different cell types and are even able to cross the
blood/brain barrier when administered to mice by intraperitoneal
injection (49-51). Previously we have identified a series of cationic
peptides that were able to transduce certain cell types as or more
efficiently than the Tat PTD (52). Moreover, we demonstrated that a
specific PTD, termed PTD-5, was able to transduce human islets
efficiently in culture (53).
Given that gene transfer of I Animals--
BALB/c mice were purchased from Taconic
(Germantown, NY). All animals were housed at the University of
Pittsburgh, Center for Biotechnology animal facility in compliance with
the United States Department of Agriculture and National Institutes of
Health regulations. All animal manipulations were conducted and
monitored under protocols reviewed and approved by the Institution
Animal Care and Use Committee.
Peptides--
Peptides PTD-5 (RRQRRTSKLMKR) and PTD-5-NBD
(RRQRRTSKLMKRGGTALDWSWLQTE) were synthesized by the peptide synthesis
facility (University of Pittsburgh) by the solid-phase procedure on an automated peptide synthesizer (PerSeptive Biosystems, Inc., Framingham, MA) using N-(9-fluorenyl)methoxycarbonyl (Fmoc) synthesis
protocols. Peptides were N-terminal conjugated to fluorescent probe
using 6-carboxyfluorescein (6CF, Molecular Probes Inc., Eugene OR) and were subsequently purified and characterized by reversed-phase high
performance liquid chromatography and mass spectrometry. The
construction of the peptide-eGFP fusion protein has been described previously (53).
Islet Isolation and Culture--
Mouse (BALB/c) pancreatic
islets were isolated by intraductal collagenase digestion (Type V, 1.75 mg/ml, Sigma) as described, with modifications (54, 55). For the
in situ peptide transduction, prior to collagenase infusion,
200 µl of a solution of Hanks' balanced salt solution
containing 1 mM peptide or 200 µg of PTD-5-eGFP fusion
protein (6.25 µM) was injected into the common bile duct followed by 2-3 ml of collagenase infusion. The isolated islets were
purified by Ficoll density gradient centrifugation and were handpicked
under a stereomicroscope. In all the experiments, islets of 150-200
µm diameter were used. The purity of the islets was determined by
dithizone staining and was >95% in all isolations. Islets were
maintained in CMRL-1066 supplemented with 2 mM
L-glutamine, 100 µg/ml streptomycin, 100 units/ml
penicillin, and 10% heat-inactivated fetal bovine serum
(Invitrogen, Carlsbad, CA; complete CMRL) in a humidified 5%
CO2 incubator at 37 °C. In addition, isolated islets
were maintained in media containing 200 µM peptide.
Human islets were obtained from the islet isolation core of the
University of Pittsburgh. Pancreas obtained from cadaveric donors were
subjected to digestion, isolation, and purification as described by
Ricordi et al. (56), with modifications (57). The purity of
islets was usually greater than 75% as determined by dithizone
staining. Viability of the cultured islets was usually greater than
80% as assessed by Calcein AM and propidium iodide staining.
The isolated islets were cultured in complete CMRL.
Recombinant Adenoviral Vector Construction and Gene Transfer to
Isolated Islets--
The construction and propagation of the
adenoviral E1/E3-deleted vectors expressing I Static Glucose-stimulated Insulin Response following in Vitro and
in Situ Peptide Transduction, Adenoviral Infection, and Exposure to
IL-1 NF- Islet Cell Dispersion and Fluorescence-activated Cell Sorting
Analysis of Determination of Islet Viability by Simultaneous Staining of Live
and Dead Cells Using a Two-color Fluorescence Assay--
Viable cells
convert Calcein AM into green fluorescent products and dead cells
incorporate propidium iodide in the nucleus, resulting in an intense
red fluorescence (60). A working phosphate buffer solution containing 1 µg/ml Calcein AM and 10 µg/ml propidium iodide was freshly prepared
according to the manufacturer guidelines (Molecular Probes Inc.).
Islets were incubated in the presence of the dyes for 30 min at
37 °C prior to evaluation under a fluorescence microscope. Pictures
were captured by a two-photon confocal microscope and percent viability
was analyzed using MetaMorphTM software package version
4.6r9 (Universal Imaging Corp., Downingtown, PA). Percentage of viable
cell aggregates over the total was determined by scoring green
versus red fluorescence in at least 25-30 islet cell aggregates.
Statistical Analysis--
All data collected were expressed as
mean ± S.E. and statistics were performed using the SPSS package,
and a p value of less than 0.05 by analysis of variance was
used to indicate statistically significant differences.
Transduction of PTD-5 Peptide into Intact Islets in
Culture--
Earlier studies have demonstrated that PTDs are
capable of mediating internalization of heterologous peptides and
proteins in a receptor- and energy-independent manner into nearly 100% of a variety of cell types (45-49). In particular, we have
demonstrated that human islets can be transduced with a specific PTD,
PTD-5 marker protein complex in culture (53). To examine further the transduction efficiency of PTD-5 into isolated islets, mouse and human
islets were incubated with fluorescently labeled PTD-5. As shown in
Fig. 1, both mouse (B) and
human islets (D) were efficiently transduced with PTD-5
compared with controls (A and C). Analysis of
transduction of human Inhibition of NF- Transduction of PTD-5-NBD to Mouse Islets Prevents
IL-1 Transduction of Islets in Situ--
We have demonstrated above
that PTD-5 is able to efficiently transduce human and mouse islets and
that inhibition of NF- Transduction of PTD-5-NBD Peptide during Isolation of Mouse Islets:
Effect on Cell Viability and Static Glucose-stimulated Insulin
Release--
It has been reported that islets are exposed to osmotic,
mechanical, and ischemic stresses during the isolation procedure, resulting in apoptosis or loss of viability (10-18). To determine whether PTD-5-NBD peptide transduction in situ was able to
reduce the impairment of islet function and viability, the PTD-5-NBD fusion peptide was injected into mouse pancreata prior to collagenase infusion. To determine the viability of the islets following isolation, the isolated islets were stained with Calcein AM and propidium iodide
(60). As shown in Fig. 5, the islets
treated with the PTD-5-NBD peptide showed greater viability compared
with control islets. Analysis of the extent of viable green cells
compared with nonviable red cells demonstrated that treatment with the PTD-5-NBD fusion peptide resulted in ~97% viability as compared with
80-90% in the control.
To confirm that improved cell survival by PTD-5-NBD peptide treatment
in situ resulted in improved islet function, the ability of
the islets to respond to glucose challenge was examined.
Glucose-stimulated static insulin release of PTD-5-NBD-treated islets
was ~10-30% higher compared with either untreated or PTD-5 only
(Fig. 6). In addition, the enhanced
secretory response under high glucose stimulation was followed by a
physiologic return to basal levels, when the ambient glucose
concentration was returned to low glucose. The higher level of insulin
release in PTD-NBD islets as compared with PTD-5 control and untreated
islets suggests that inhibition of NF- Transplantation of pancreatic islets promises to be the most
effective approach for treating type 1 diabetes (61, 62). However, the
need for large amounts of islets and the poor survival of the graft
following transplantation represent significant hurdles that need to be
overcome to make the islet replacement theory useful for treating
diabetic patients. Islets undergo enzymatic, osmotic, mechanical, and
ischemic stresses during isolation, resulting in loss of viability,
reduced cell number, and initiation of apoptosis (10-12). These facts
increase the number of islet donors needed to treat a single diabetic
transplant recipient. Loss of viability can also be attributed in part
to detachment of the islets from the surrounding extracellular matrix
that is essential for To improve the viability of islets prior to and following
transplantation, we have been examining gene transfer methods for delivery of agents able to protect islets from dysfunction and death in
culture and to block the immune response to the transplanted islets
in vivo. Previously, we have demonstrated that
adenoviral-mediated gene transfer of the NF- In this report, we demonstrate that both human and mouse islets are
efficiently transduced by cationic peptide transduction domains, in
particular, PTD-5, which functions similarly to the Tat PTD derived
from HIV. The majority of We also have demonstrated the ability to transduce islets in
situ as a way of delivering agents able to preserve islet function during isolation. Importantly, the presence of PTD-5-NBD in the collagenase solution and its intraductal delivery appears to have no
impact on the efficiency of digestion at any step. The yield of islets
was unaffected, demonstrating the safety and feasibility of this
approach. Injection of a fluorescent transduction peptide into the
pancreas prior to islet isolation resulted in almost 40% of the
In the experiments described in this report, we have focused on
inhibiting NF- The use of peptide transduction of protective peptides and fusion
proteins to protect tissue from damage during isolation and culture,
prior to transplant, can be applied to a wide variety of organs in
addition to islets. In preliminary experiments, we have demonstrated
that both cardiac tissue and cartilage can be efficiently transduced,
allowing for peptide-mediated transduction approaches to block ischemic
damage to these and other tissues. Given that NF-B inhibitor I
B to human islets results in
protection from interleukin (IL)-1
-mediated dysfunction and
apoptosis. Here we report that human and mouse islets can be
efficiently transduced by a cationic peptide transduction domain
(PTD-5) without impairment of islet function. PTD mediated delivery of
a peptide inhibitor of the IL-1
-induced I
B kinase (IKK), derived
from IKK
(NBD; Nemo-binding domain), and completely blocked the
detrimental effects of IL-1
on islet function and NF-
B activity,
in a similar manner to Ad-I
B. We also demonstrate that mouse islets
can be transduced in situ by infusion of the transduction
peptide through the bile duct prior to isolation, resulting in 40%
peptide transduction of the
-cells. Delivery of the IKK inhibitor
transduction fusion peptide (PTD-5-NBD) in situ
to mouse islets resulted in improved islet function and viability after
isolation. These results demonstrate the feasibility of using
PTD-mediated delivery to transiently modify islets in situ
to improve their viability and function during isolation, prior to transplantation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells of the pancreas. Proinflammatory cytokines such as
IL-11 and
interferon-
and the free radical, nitric oxide (NO), have been implicated to play key roles in the initial destruction of
-cells leading to the development of the disease (1). In particular, IL-1
is released by resident macrophages in islets in response to a
variety of stimuli that can stimulate NO production by
-cells (1-4). Cytokine-induced NO production by rodent and human
-cells results in islet dysfunction and inhibition of insulin secretion. This
NO-mediated damage has been shown to be attenuated by inducible nitric-oxide synthase inhibitors (5-7). IL-1
exposure also results in up-regulation of Fas on the surface of
-cells, resulting in an
increase in apoptosis (1, 8, 9). Approaches to inhibit the adverse
effects of IL-1
on islets, such as through gene transfer of the IL-1
inhibitor IL-1Ra, have been shown to improve islet function and
viability (9). In addition to the adverse effects of IL-1
, islets
undergo enzymatic, osmotic, mechanical, and ischemic stresses during
isolation, resulting in loss of viability, reduction of cell number,
and initiation of apoptosis (10-12). The loss of viability of isolated
islets has been associated with detachment from the surrounding
extracellular matrix, leading to activation of caspase-3 and NF-
B
(15-18).
B family of
transcription factors (19-22). Proinflammatory cytokines have been
shown to stimulate NF-
B activity in human and rodent islets in
vitro, resulting in
-cell impairment (23). NF-
B binds to a
family of naturally occurring repressors termed I
B (24, 25),
resulting in retention of the transcription factor complex in the
cytoplasm. A variety of inflammatory agents including cytokines,
endotoxin, double stranded RNA, and the viral transactivator Tax
activate the NF-
B by rapid phosphorylation and subsequent
ubiquitin-mediated degradation of the I
B repressor (24, 26-28).
These agents increase the activity of the two related I
B kinases,
IKK-
and IKK-
, which phosphorylate I
B. The release and
degradation of I
B following phosphorylation allows NF-
B to
translocate to the nucleus where it binds to its cognate
enhancer/promoter elements upstream of certain proinflammatory genes
(24, 29). In particular, I
B has been shown to inhibit inducible
nitric-oxide synthase gene expression by associating with NF-
B and
preventing its translocation to the nucleus (30). Intracellular
expression of an I
B mutant that is nonphosphorylatable and thus
unable to be degraded prevented the nuclear translocation of the
NF-
B proteins, even in the presence of cytokines (31, 32).
and IKK-
, a regulatory protein known as
IKK-
/NEMO has been shown to be a critical component of IKK complex
(33, 34). IKK-
/NEMO also was identified independently in biochemical
studies as an essential component of the high molecular weight IKK
complex (33, 34). Cells that do not express IKK-
/NEMO are unable to
assemble the high molecular weight IKK complex, preventing stimulation
of IKK activity in response to agents that stimulate the NF-
B
pathway (33, 34). IKK-
/NEMO preferentially associates with IKK-
,
but also binds to IKK-
(34-37). Furthermore, inhibition of the
IKK
-IKK-
/NEMO interaction and subsequent activation of NF-
B
in vivo and in vitro using a cell-permeable
PTD-NEMO-binding domain (NBD) peptide fusion has been reported recently
(38, 39).
by adenoviral gene transfer of several different
genes including IL-1Ra, soluble IL-1 receptors, and insulin-like growth
factor-1 (9, 40). Moreover, we have demonstrated that adenoviral gene
transfer of the nondegradable form of I
B repressor was able to block
IL-1
-mediated dysfunction and apoptosis (41). These results suggest
that specific inhibition of NF-
B is able to inhibit the adverse
effects of IL-1
on
-cells. Although it has been shown that
virus-mediated transfer of anti-apoptotic genes such as Bcl-2 into
islets in culture inhibits apoptosis (42-44), this class of genes is
unable to block the adverse effects of IL-1
on islet function.
Moreover, even though adenoviral vectors do not appear to interfere
with
-cell function in vitro, the inherent immunogenicity
of these viral vectors may be detrimental to islet transplantation.
Although there are promising preclinical results with lentiviral and
adeno-associated virus-mediated gene transfer to islets, there
currently is no clinically appropriate method for improving islet
function and viability by gene transfer.
B was effective in preventing islet
dysfunction and apoptosis, we hypothesized that peptide-mediated transduction of NF-
B inhibitors into islet cells would result in
improved viability and function following IL-1
exposure. Thus, we
have examined the ability of PTD-5-mediated transduction of the
NBD peptide to block islet dysfunction and loss of viability. Here we demonstrate that transduction of mouse islets in culture with
the PTD-5-NBD fusion peptide can prevent IL-1
-dependent suppression of glucose-stimulated insulin release and NF-
B
activation. In addition, we demonstrate the feasibility of transducing
mouse islets in situ by infusion of the transduction peptide
into the mouse pancreas via the common bile duct prior to islet
isolation. Administration of the PTD-5-NBD fusion peptide into mouse
islets in situ before isolation increased their viability
and function following isolation. These results suggest that
peptide-mediated transduction of an NF-
B inhibitor is able to
protect islets from the adverse effects associated with islet
isolation. Clinically, this approach could be used to improve the
viability, function, and mass of human islets following isolation by
in situ delivery of PTD-5-NBD to islets prior to isolation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and LacZ from the
cytomegalovirus promoter has been described previously (41). For
the Ad-I
B infection experiments, groups of 200 islets were washed in
serum-free CMRL-1066 and subsequently infected with adenoviral vectors
encoding I
B and
-galactosidase at 100 multiplicity of infection
for 1 h at room temperature (55). Following infection, islets were washed 2-3 times with fresh medium and then cultured for 48 h in
complete CMRL.
--
To determine the protective effects of NF-
B inhibition
in the presence and absence of IL-1
treatment, static
glucose-stimulated insulin release was used as a functional assay.
In vitro transduced, adenovirus-infected, and control islets
were incubated in the presence of 50 units of recombinant mouse IL-1
(R&D Systems, Minneapolis, MN) for a period of 24-30 h prior to
insulin release studies. The IL-1
containing medium was then removed
and the islets were washed twice in glucose-free Krebs-Ringer
bicarbonate buffer (pH 7.35) containing 10 mM HEPES, 0.5%
bovine serum albumin (Sigma), and a group of 100 islets in triplicate
were conditioned by preincubation for 1 h at 37 °C under low
glucose concentrations (2.8 mM). The preincubation buffer
was discarded and replaced with Krebs-low glucose buffer for a
subsequent 1-h incubation at 37 °C, the Krebs buffer was removed and
stored, and the buffer was replaced with Krebs containing high glucose
(20 mM) and incubated for 1 h at 37 °C. After
1 h, the high glucose buffer was collected and replaced with Krebs
low glucose buffer for a final 1-h incubation. The insulin content of
the buffers collected after each incubation step was determined by a
commercially available enzyme-linked immunosorbent assay kit (ALPCO,
Windham, NH). The islets isolated after in situ
treatment, with or without PTD-5-NBD, were cultured overnight and then
handpicked, washed in Krebs-Ringer bicarbonate buffer, and
glucose-stimulated insulin release was determined as described above.
B p65 Transcription Factor Assay in Mouse Islets--
To
examine the protective effects of NBD peptide from IL-1
exposure on
NF-
B activity in vitro, the isolated islets were transduced with NBD peptide for 2 h, and the islets were
subsequently treated with 50 units of IL-1
for an additional 2 h. As controls, transduced and nontransduced islets with NBD peptide,
with and without IL-1
treatment, were used. All islet groups were
washed twice with cold phosphate-buffered saline and stored at
80 °C until whole cell extracts were prepared. A total of 10 µg
of cellular protein from each group was analyzed for p65 binding
activity according to the manufacturers instructions, using the
enzyme-linked immunosorbent assay-based Trans-AMTM NF-
B
p65 transcription factor assay kit (ActiveMotif, Carlsbad, CA). The
specificity of NF-
B DNA binding activity was confirmed by
competition with a wild type or mutant oligonucleotide with an
immobilized oligonucleotide probe containing the NF-
B consensus site. NF-
B binding activity was measured at 450 nm and the OD reading normalized to protein content was measured using a Bradford protein assay (Bio-Rad) with bovine serum albumin as standard.
-Cells for Peptide Transduction--
Islets isolated
with infusion of PTD-5-6CF peptide in situ were dispersed
into individual cells by treatment with Hank's based enzyme-free cell
dissociation buffer (Invitrogen) at 37 °C for 5-10 min and
incubated in complete CMRL for 2 h at 37 °C prior to cell
sorting.
-Cells were analyzed based on size and endogenous FAD
fluorescence as described (6, 58), using a FACStar flow cytometer (BD
Biosciences), with a laser illuminated at 488 nm and with the
gated cells having forward scatter monitored at 515-535 nm. Under this
selection the majority of the cells gated (>70%) are
-cells (59).
In addition, following uptake of biotinylated PTD-5 coupled to
streptavidin-Alexa Fluor 488 (Molecular Probes Inc.) in
-cells, the
human islets also were dissociated into single cells and peptide
transduction was measured by flow cytometry as described above. The
biotinylated PTD-5 coupled to streptavidin-Alexa Fluor 488 was
prepared as previously described (52).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells by flow cytometry (Fig. 1E)
demonstrated that the majority of the
-cells within the intact
islets, as determined by size and endogenous FAD fluorescence, were
transduced with biotinylated PTD-5 linked to streptavidin-Alexa Fluor
488. To determine whether transduction with PTD-5 affected islet
function, the ability of the treated islets to respond to glucose was
determined. As shown in Fig. 1F, both the PTD-5-treated and
control islets produced similar levels of insulin in response to
increasing concentrations of glucose. These experiments demonstrate
that islets and, in particular,
-cells can be efficiently transduced
with cationic peptides in vitro without any apparent
impairment of glucose signaling and insulin production.
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Fig. 1.
PTD-5-mediated delivery of peptide into mouse
and human islets. Fluorescently labeled PTD-5 in media was
added to the isolated islets and cultured at 37 °C. Following
incubation for 1-2 h, the islets and cells were extensively washed.
Cellular localization was visualized by the multiphoton laser scanning
confocal microscope system. A, control isolated mouse
islets; B, mouse islets treated with PTD-5-6CF;
C, control isolated human islets; D, human islets
treated with PTD-5-6CF; E, transduction of -cells as
evaluated by flow cytometry of biotinylated PTD-5 coupled to
streptavidin-Alexa Fluor 488 (SA-488); and F, static
glucose-stimulated insulin release by peptide-transduced islets as
compared with control. L1, initial low (2.8 mM)
glucose; H, high glucose (20 mM); and
L2, second low glucose concentrations. The data represents
the mean ± S.E. of four independent experiments in triplicate.
The bars are presented as percentage of insulin secretion
above the control group of untreated islets exposed to the first 2.8 mM glucose treatment given a relative value of 100%.
B in Mouse Islets by Peptide-mediated
Transduction Prevents IL-1
-induced Impairment--
We have
previously demonstrated that adenoviral-mediated gene transfer of I
B
was able to block IL-1
-mediated islet dysfunction in cultured islets
(41). This result suggests that islet integrity and function, at least
in culture, can be preserved by inhibition of NF-
B activity in
-cells. To determine whether peptide-mediated transduction of an
NF-
B inhibitor into islets is able to inhibit NF-
B activity,
blocking IL-1
-mediated islet dysfunction, a peptide containing PTD-5
fused to the IKK
peptide inhibitor of IKK kinase (NBD) through a
diglycine spacer was synthesized (PTD-5-NBD) and used for islet
transduction experiments (39). As a positive control for the effects of
NF-
B inhibition, the Ad-I
B vector was also used for gene
transfer. As shown in Fig. 2,
transduction of mouse islets with the PTD-5-NBD fusion peptide was able
to prevent IL-1
-induced impairment of glucose-stimulated insulin release by cultured islets. Moreover, the inhibition of
IL-1
-mediated islet dysfunction by the fusion peptide was similar to
that conferred by adenoviral-mediated gene transfer of I
B. Thus,
PTD-mediated delivery of an inhibitor of the I
B kinase is effective
in blocking the detrimental effects of IL-1
on islet function.
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Fig. 2.
Transduction with PTD-5-NBD and adenoviral
gene transfer of I B
to mouse islets inhibits the
IL-1
-induced impairment of static glucose
stimulation insulin release. Two-hundred islets were transduced
with PTD-5-NBD and control PTD-5 alone or infected with Ad-lacZ and
Ad-I
B. Half of each group of islets was exposed to 50 units of mouse
IL-1
for 30-36 h. Then the islets were subjected to
glucose-stimulated insulin release and assayed by enzyme-linked
immunosorbent assay. The data represents the mean ± S.E. of four
independent experiments in triplicate. The bars are
presented as percentage above control with insulin secretion by
untreated islets exposed to the first 2.8 mM glucose
treatment shown as a value of 100%.
-stimulated NF-
B Activation--
To demonstrate that
transduction of the NBD peptide inhibited NF-
B activation, analysis
of the NF-
B binding activity was performed using a NF-
B p65
transcription factor assay on PTD-5-NBD-transduced islets. As shown in
Fig. 3, NF-
B activity was
significantly increased in nontransduced islets following treatment
with IL-1
. However, in the NBD peptide-treated islets exposed to
IL-1
, the NF-
B level was similar to control nontransduced islets.
These results demonstrate that transduction of islets with the NBD
peptide blocked IL-1
-mediated induction of NF-
B binding
activity.
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Fig. 3.
Inhibition of NF- B
activation in PTD-5-NBD-transduced islets treated with IL-1
in
vitro. Isolated islets were transduced with an IKK inhibitor, NBD
peptide, and subsequently treated with 50 units of IL-1
.
Nontransduced islets were used as controls. Results are shown as
percentage of control where NF-
B activation in the lysates of
nontransduced islets was taken to be 100%. The data are expressed as
mean ± S.E. of four independent experiments in duplicate. **,
p < 0.01.
B activity by transduction with the PTD-5-NBD
fusion peptide prevents IL-1
-mediated islet dysfunction in culture.
However, significant damage to islets occurs during the isolation
period, reducing islet viability and function in culture. Thus, it was
of significant interest to determine whether islets can be transduced
in situ prior to isolation to deliver agents, such as the
NBD peptide, that are able to prevent islet dysfunction and apoptosis.
To determine whether islets can be transduced in situ,
pancreas from BALB/c mice were injected with a PTD-5-6CF peptide as
well as with a PTD-5-eGFP fusion protein and an eGFP control protein.
Islets isolated from PTD-5-6CF-treated pancreata showed extensive
transduction by confocal microscopy (Fig.
4B) compared with controls
(Fig. 4A). Analysis of
-cells from the isolated islets by
fluorescence-activated cell sorting showed that at least 30-40% of
the
-cells were transduced (Fig. 4E). Islets treated with
recombinant PTD-5-eGFP fusion protein in situ (Fig.
4D) also showed evidence of transduction compared with the
eGFP control (Fig. 4C), albeit at reduced levels compared with the 12-amino acid peptide, PTD-5-6CF. However, these results clearly demonstrate that islets and in particular,
-cells, can be
modified by peptide-mediated transduction in situ prior to isolation.
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Fig. 4.
In situ transduction of pancreatic
islets with peptides. A, 200 µl of fluorescently
labeled PTD-5 (1 mM) and PTD-5-eGFP (6.25 µM)
fusion protein were infused into the pancreas before collagenase
treatment. Immediately after purification, the islets were examined by
confocal microscopy. A, control untreated islets;
B, PTD-5-6CF; C, control eGFP; and D,
PTD-5-eGFP. The efficiency of PTD-5, peptide transduction was
determined by dissociation of islets into single cells and analyzed by
flow cytometry. The -cells were gated based on size and endogenous
FAD fluorescence. E, the percentage of
-cells positive
for fluorescence in PTD-5-6CF-treated islets. Results are
representative of three independent experiments (A-D), or
the mean ± S.E. of four independent experiments (E).
*, p < 0.05.
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Fig. 5.
Enhanced viability of pancreatic islets
transduced with anti-apoptotic peptide PTD-5-NBD, an inhibitor of
NF- B. Mouse islets were transduced
in situ with PTD-5-NBD and control PTD-5 alone before islet
isolation and subsequently cultured with the peptide for 12-18 h. The
viability of the islets was determined by staining with Calcein AM and
propidium iodide and visualized by fluorescence/confocal microscopy.
Viable cells in the islets are stained green (green
fluorescence), whereas dead cells appear red (red
fluorescence). A, control untreated islets; B,
islets transduced with control peptide PTD-5; and C, islets
transduced with PTD-5-NBD. Percentage of viable and dead cells
aggregates over the total was determined by scoring green
versus red fluorescence using the MetaMorphTM
software package as described under "Experimental Procedures"
(D). Results are representative of four independent
experiments (A-C) and mean ± S.E. of four
independent experiments (D). *, p < 0.05;
**, p < 0.01.
B during the islet isolation
procedure is able to improve islet viability and therefore function in
culture following isolation.
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Fig. 6.
In situ pancreatic transduction of
PTD-5-NBD before islet isolation improves glucose-stimulated insulin
release. Nontransduced islets were isolated along with islets
isolated after transduction with either PTD-5 alone or PTD-5-NBD and
were subjected to static glucose-stimulated insulin release.
L1, initial low (2.8 mM) glucose; H,
high glucose (20 mM); and L2, second low glucose
concentrations. The data represents the mean ± S.E. of three
independent experiments performed in triplicate. **, p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell viability and function (13-18). In
addition, release of inflammatory cytokines such as IL-1
from the
pancreatic tissue during the isolation procedure contributes to islet
dysfunction and reduced mass. The reduction in islet viability and
increased
-cell death prior to transplant most likely leads to
stimulation of a stronger immune response toward the allogeneic islets.
B inhibitor, I
B, to
islets results in complete inhibition of IL-1
-mediated islet
dysfunction (41). However, there currently are no clinically applicable
gene transfer approaches to modify islets in culture. In addition,
significant damage to islets occurs during the isolation procedure,
necessitating the delivery of protective agents to islets in
situ. To date, gene transfer to islets in situ has been
extremely inefficient.
-cells can be transduced in culture
without a significant effect on islet function. Moreover, we have
demonstrated that PTD-5-mediated delivery of a peptide inhibitor of the
I
B kinase (IKK) is able to block IL-1
-mediated induction of
NF-
B as well as islet dysfunction in culture. The inhibitory effect
of the PTD-5-NBD peptide in blocking IL-1
was similar to the
protective effect conferred by adenoviral-mediated gene transfer of
I
B. The use of the peptide to protect islets in culture, however,
has significant advantages over gene transfer in regard to simplicity
and cost as well as risk. Our results using the PTD-5-NBD peptide are
similar to those previously reported for Tat PTD-mediated delivery of
Bcl-2 to islets to block apoptosis (63). However, in contrast to the
Bcl-2 fusion, the peptide inhibitor of NF-
B is also able to block
induction of NO, and thus preserve islet dysfunction, in addition to
blocking cell apoptosis. Thus the inhibition of NF-
B activation by
PTD-5-NBD is able to prevent loss of islet function as well as improve
islet viability.
-cells being transduced, with an additional percentage most likely
being weakly transduced. Transduction of islets with the PTD-5-eGFP
fusion protein appeared to be less efficient, however, the difference
in transduction most likely is because of the 160-fold difference in
the concentration used between PTD-5-6CF and PTD-5-eGFP. Furthermore,
these results may be because of the differences between the eGFP and
6CF cargos with respect to efficiency of internalization and the
stability and function of the markers following internalization. The
delivery of the PTD-5-NBD peptide to islets in situ resulted in an increase in the percent of viable cells within the islets as well
as improved islet function. Thus, this approach could be used to
improve the quality of islets during isolation, resulting in recovery
of greater islet mass and improved islet function. Indeed, in
preliminary experiments with human islets, injection of the PTD-5
peptide into the pancreas prior to isolation also resulted in efficient
islet transduction.2
Moreover, the injection of the PTD-5-NBD peptide into the human pancreas resulted in islet isolation with greater
-cell mass and
viability. Taken together, these results suggest that the PTD-5-NBD
peptide could be used to modify islets in situ prior to
isolation to improve the integrity of islets for transplantation.
B by peptide-mediated transduction to improve islet
function. Although we demonstrate that inhibition of NF-
B activity
in response to IL-1
protects the islet from dysfunction and death,
it is highly likely that additional pathways may have to be targeted.
For instance, inhibiting the effects of interferon-
through delivery
of signal transducers and activators of transcription inhibitory
peptides or proteins could improve islet function. Moreover, delivery
of inhibitors of apoptosis such as Bcl-2, dnCaspase 9, or agents that
block free radical damage such as manganese-superoxide dismutase could
also be able to improve islet mass and function following isolation.
Regardless of the therapeutic agent to be delivered, the use of peptide
transduction has the advantage over gene transfer in that it can be
applied in situ. Moreover, peptide transduction results in
only transient modification of the cells, eliminating any risks
associated with long term gene expression of anti-apoptotic gene products.
B activation plays
a major role in mediating ischemic tissue damage, the PTD-5-NBD fusion
peptide could be used to protect other tissues from damage during
isolation. Taken together, our results demonstrate the feasibility of
using peptide transduction domains to modify tissues in situ
to improve their viability prior to transplantation. Moreover, we have
demonstrated that inhibition of NF-
B activity is clearly protective
to islets in culture and in situ.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Nick Giannoukakis and XiaoLi Lu for assistance and helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a grants from the Juvenile Diabetes Research Foundation, the Muscular Dystrophy Association, and National Institutes of Health Contract AR-6-2225.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom all correspondence should be addressed: Dept. of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, W1246, Biomedical Science Tower, Pittsburgh, PA 15261. Tel.: 412-648-9268; Fax: 412-383-8837; E-mail: probb@pitt.edu.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M207700200
2 R. Bottino, K. Rehman, P. Robbins, and M. Trucco, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
IL-1, interleukin-1
;
6CF, 6-carboxyfluorescein;
IKK, I
B kinase;
NBD, NEMO-binding domain;
NF-
B, nuclear factor
B;
NO, nitric oxide;
PTD, peptide transduction domain.
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