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Influence of Class I Interferons on Performance of Vascular Cells on Stent Material in vitro |
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N.
Kipshidze, I. Moussa, V. Chekanov, V. Nikolaychik, A. Khanna, |
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Key
words: Smooth muscle cell, restenosis, stents,interferon. 1.
Introduction Despite intensive investigation, no widely effective treatment modality exists to prevent recurrent stenos is follo wing both vascular surgery or percutaneous interventional procedures [1]. The failed clinical trails using agents that have demonstrable efficacy in experimental animal models probably relates to poor translation from animal models to humans multifactorial nature of human restenosis [2-4]. Thus, new approaches to the problem of restenosis should be aimed at testing agents that target multiple processes involved in the pathogenesis of restenosis [1].
The
main cause of restenosis following stenting procedures is the hyperplastic
response involving migration of smooth muscle cells (SMCs) and fibroblasts
(FBs) from subendothgelial and medial layers of the vessel walls, as well
as expansion of an associated extracellular matrix [2-4]. Most recently,
local antiproliterative strategies including pharmacological stent
coatings (paclitaxel, actinomicin-D, rapamycin, etc.) have demonstrated
inhibition of SMC proliferation in virro, reduced neointimal thickening in
animal models of restenosis and produced promising results in pilot human
studies [4, 5]. However, high local toxicity may after the
andothelialization process after stent implantation and cause late
thrombosis. Therefore, a new strategy to decrease restenosis may be to
arrest SMC growth and decrease collagen production without altering and/or
improving reendothelialization.
Class
I interferons (IFNs), particularly INF-γ, inhibit neointimal
hyperplasia in atherosclerotis vessels and may be useful in the prevention
of post-stents rostenosis [6-8]. INFs may be easily incorporated in stent
coating, we reasoned to study the effective of INFs on viability and
growth characteristics of human aortic endothelial cells (ECs), SMCs and
FBs on stent surface in vitro. 2. Materials and methods. 2.1.
Primary cell procurements and culturing. 2.1.1.
EC isolated and culture
Human
ECs were isolated from coronary vessels (HuCEC) of six anonymous donors by
collagenase digestion according to previously published methods [9-11].
After enzymatic treatment and washing with MCDB-131 (Clonetics, San Diego,
CA), cells were suspended in a MCDB medium, supplemented with 7% fetal
calf serum and 7% human serum epidermal growth factor (10np/ml), having
brain extract (36 μ/ml), sodium heparin (1U/ml), hydrocortisone (1μg/ml),
gentamicin (40μ/ml) and amphotericin B (250 ng/ml). ECs were
identified by positive staining for vWF, uptake of florescent acetylated
LDL and by their histotypic “cobblestone” appearance at confluence.
HuC EC routinely were grown in supplemented medium MCDB-131, and were used
between 3-4 and 6-9. 2.1.2.
SMC isolation and culture
Human
saphenous vein SMCs (HuSV SMC) were obtained from freshly removed
specimens using an explant yechnique [10-12]. Briefly, a segment of vein
was stripped of fat and connective tissue, the endothelium was then
removed by careful stripping, and this segment of vein was chopped into
small fragments of approximately 1x1 mm within a small volume of
DMEN (Hyclone Laboratories, UT). This was supplemented with 10%
feral calf serum (Gibee Life Technology, Grand Island, NY), gentamicin
(40./ml) and amphotericin B (250 ng/ml). The fragment of vascular medial
tissues were transferred to a 25-cm³ culture flask, incubated for 2-3
weeks in a standard cell culture incuator (VWR scientific, Model 5025)
where HuSV SMC migrate out of the explants and become subconfluent.
Subconfluent cultures were passaged by tripsinization. HuSV SMC cells used
for experiments were between passages 6 and 9; their growth was
characterized by typical “hill and valley” configuration. 2.1.3
FB cultures
Normal
human dermal FBs (HUD FB) were obtained from Clonetics and routinely grown
in supplemented medium DMEN. Subconfluent cultures were passaged by
trypsinization and used between passages 4 and 6.
The
number of cells in subconfluent cultures was determined by Alamar Blue
(AB) assay (Alamar BioSciences, Sacramento, CA), based on the bioreduction
of a fluorogenic substrate. The resulting values were converted to cell
numbers using standard calibration curves [12]. Individual calibration
curves were constructed for each cell type and for each cell medium.
HuSV
SMC and HuD FB were rendered quiescent by replacing their medium with DMEN
containing 2% fetal calf serum (serum starved medium) 24h before
IFN treatment. AB assay has shown that this level of serum is sufficient
to maintain viabiulity and constant cell density but not simulate
proliferation [12].
All
cultures were ascertained to e free of micoplasma/acholeplasma
contamination by evaluation of cultures supernatants from each cell type
grown from two passages in corresponding media, without antibiotics, using
a commercial micoplasma detection kit (Genprobe, San Diego, CA). 2.2
Biomaterials
Assay
were conducted in 24 well plates (Costar, Cambridge, MA). The cells were
conjured enema uncanny on tissue culture (TC) surface and placed on the
bottom of the wells or on substrates made of 316-1 stainless steel. The
metal substrates were 1-1.2 mm thick and 1.88 mm². They were cut from
metal sheets and sterilized by incubation in 0.5 M NaOH for 30 min.
Following repeated rising in distilled water, the disks were affixed to
the bottom of the wells by gravity. To mimic a stent, stainless-steel
disks were coated with fibrin meshwork impregnated wit different doses of
IFN-γ or IFN-α in our laboratory (VN). Atop of such layer three
types of human vascular cells-ECs, SMCs and FBs-were cultured, whereas
control cells were seeded onto fibrin-coated surfaces. Concentration of
recombinant IFN varied from 5, 10, 15 and 20 ng/cm².
Cell
growth on the TC plastic surface was assessed every day for up to 7 days
of culturing by the AB assay. Additional morphometric data were obtained
by phase-contrast light microscopy. 2.2.1.
Cell quantification.
The number of cells plated in each experiment was determined
directly by using a standard hemocytometer (Haussek Scientific, Norshem,
PA). After attachment, quantitation of viable cells attached to the
substrates was performed by AB assay [12]. AB was added to cell cultures
at 1:10 v/v ratio and incubated for 3 h at 37 C in the incubator. The
fluorescence yield was obtained by measuring in a spectrofluorometer
(Model 650-10S, Hitachi, Japan) set at excitation and emission wavelengths
of 560 and 590 nm. The resulting values were converted to cell numbers
using a standard calibration curve.
The
viable cells were enumerated by AB assay. Individual experiments were
repeated at least three times. Normalized viability index was calculated. 2.2.2.
Cell growth evaluation
EC were seeded onto a
96-well Dynatech pigmented plate at an initial density of 10,000 cell/cm²
for evaluation of growth rate. After 24 h, the number of cells was
indicated by AB assay and EC monolayers were treated with various doses.
Each test-dose was performed in six wells. Quantitation by AB assay was
performed for 7 days, and refreshment of culture medium was done after
each assay. PECAM-1, ICAM-1 (R&D Systems, Minneapolis, MN, USA),
elastase and lactate dehydrogenase (Sigma, USA) were used in these
experiments. 2.2.3.
Phase-contrast light microscopic examination
This system allowed us to
directly visualize the spreading and adhesion of the, cells to the metal
wire surfaces in culture. 2.2.4.
Statistical analysis
All experiments were
carried out in sextuplicate and repeated at least three times. Where
appropriate, the data are presented as mean standard deviation. The
significance of variability between the various results was determined by
the ANOVA test. 3.
Results 3.1.
Impact on cell viability and growth
Experiments in TC
demonstrated that IFN treatment affected growth rates for all three types
of cells. We observed, however, that growth inhibition effect was maximal
with SMC and was minimal with FB and EC.
IFN- γ abrogated the
mitogenic response of SMC and FB to FGF stimulation by not EC, and t did
not inhibit EC growth VEGF stimulation. IFN-α was able to inhibit EC
growth and did not influence growth rates of SMC and FB. Biochemical
analysis of lactate dehydrogenase demonstrated no difference in the
relative activity at day seven between controls and IFN (α and γ)-treated
cells. We
also studied the effect of different doses of IFN on the expression of
cell adhesive molecules P and E-selections and PECAM and ICAM–1. These
molecules up regulated by IFN in EC. Media derived from quiescent human
SMC displayed low immunoreactivity elastase activity with conditioned
media after IFN-γ treatment, but not after IFN-a treatment, which had
approximately threefold greater activity. 4.
Disscusion 4.1.
IFN for prevention of restenosis
IFNs are a family of
naturally occurring proteins that are produced by cells of the immune
system. Three classes of IFNs have been identified: α, β and
γ. Each class has different effects though their activities overlap.
Together, the IFNs direct the immune system’s attack on viruses,
bacteria and other foreign substances. Once IFNs have detected and
attacked a foreign substances, they alter it by slowing, blocking or
changing it’s growth or function.
IFN is considered
analogous to tumor suppression protein.
It inhibits the proliferation and migration of cells, and blocks the
effects of many oncogones and growth factors [13].
IFNs induce and regulate
tumor suppressior protein RB and p53 [13, 14]. Both regulate the cell
cycle [15]. It is believed that IFN may, in corcert with the tumor
suppressor proteins inside the cell, mediate tumor suppressor function and
that the protein inside the cell cannot be totally effective without
adequate IFN outside the cell.
Despire
intensive investigation, no widely effective treatment modality exists to
prevent recurrent stenosis following procedural revascularization. It was
shown that the cytokine-induced activation and proliferation of medial
vascular SMCs leading to neointimal hyperplasia is one of the most
critical cellular events in the formation of transplant arteriopathy and
balloon angioplasty-induced restenosis [16, 17]. Balloon injury of the
arterial wall and stent implantation not only induced increased SMC
proliferation, enhances elastic recoil and abnormalities in thrombosis,
both of which contributes to regrowth of the intima and to the production
of restenosis lesions[18].
Accumulation of T
lymphocytes and monocyte activation of SMCs (proliferation, migration and
matrix deposition) are among the factors leading to inflammation and to
onset of vascular deseases including restenosis [19, 20]. Vessel injury
helps us to realize that proiflammatory cytokines and growth factors
influence SMC proliferation and migration. Produced by T lymphocytes. IFN-γ
have immunoregulatory functions and act on similar receptors, designated
Class II cytokines receptors [2].
Accordinglyof the
regulation of genes involved in SMC proliferation, particularly by
naturally occurring inhibitors, is important. It was previously
demonstrated that IFNs inhibits cholesterol accumulation and foam cell
formation cell formation by downregulating the scavenger receptor on
microphages [21].
Castronuovo
et al. [23] showed that
IFN-γ reduced the development of neointimal hyperplasia following
arterial injury. They showed also that SMC restores its proliferative
activity within 7 days after discontinuation of IFN-γ treatment. It
was proposed that efficacy of IFN- γcn be enhanced by lengthening the
period of treatment.
Interaction of FB with the
F-derived extracellular matrix is an important modulator of IFN-γ
responsiveness and this interaction may play a role in the low
immunogenicity of allogenic FB growth [24], which have an important role
in restenosis.
In
cultured SMC, IFN-γ inhibits apoptosis. Genetic disruptions of IFN-γ
signaling in a mouse model of restenosis significantly reduced the
vascular proliferative response and neointimal proliferation [25]. Ribault
et al. [26] studied IFN-γ
expression inhibited SMC growth. They demonstrated that neointimal
formation in the rat carotid balloon injury model was reduced to the same
extent by adenoviral gene transfer of IFN-γ and driven by the
SM-myosin heavy chain enhanced.
However, other studied
showed a different influence of IFN-γ and IFN-β on endothelial
and SMCs. Various inflammatory cytokines, primarily IFN-γ, increase
the expression of allograft inflammatory factor-1 (AIF-1), which plays an
important role in SMC activation subsequent to arterial injury [27].
Inhibition
of rabbit IFN-γ by secreted myxoma viral immunomodularity
glycoprotein (M-T7) inhibits intimal hyperplasia after antiplasty injury
and may be critical for prevention of plaque growth after vascular injury
[28]. Dlmolino and Castellot [29] showed that haparin, by suppressing
serum and glucocorticoid-regulated kinase (sgk), inhibits SMC
proliferation in animals and in culture,
but sgk expression is not suppressed by IFN-β.
On the other hand, it was
demonstrated [18] that expression of recombinant porcine IFN-β in SMC
reduced cell proliferation significantly in vitro and that supernatants
derived from β IFN vector inhibited SMC proliferation related to
controls.
Mintzer et al. [30] showed that
β IFN inhibited vascular smooth muscle growth while having no
growth-inhibitory effect on ECs obtained from the same blood vessels,
making it a potential candidate for treating pathologic condition where
abnormal SMC proliferation is implicated, such as restenosis follwing
balloon angioplasty or smooth muscle proliferation following vascular
stenting. Autieri and Agrawal [16] showed that IFN-γ inhibits
proliferation of SMC in culture and reduces arterial restenosis
balloon-injured arteries by decreasing proliferation and intimal
formation. Stephan et al. [18] showed that IFN-γ activated
macrophages inhibit fibrogenesis of FBs by releasing antifibrogenic or
fibrotic factors. Gaydos [31] also showed that when IFN-γ is used
there is inhibition of a productive growth cycle in a doserelated
response.
This study demonstrated that
IFN is capable of affecting vascular EC and SMC growth characteristics. In
our experiments, SMC were more sensitive to IFN treatment than EC. The
effect appears to be cytostatic, because there was no statistically
significant increase in lactate dehydrogenase activity that suggested that
both IFN-α and IFN-γ were not toxic to the vascular cells. 4.2.
Local delivery
Chemoprevention of restenosis
after PCI may be one application of IFN the problems with toxicity and
route of administration can be solved.
Systemic administration of a
large number pharmaceuticals, which have shown promise in in-vitro and
animal trials, has proven to be largely ineffective in reducing the
occurrence of restenosis in patients following angioplasty, perhaps due to
insufficient local concentrations and limitation on maximal doses.
Given the increased frequency
(up to 80-90%) with which stents [1, 32] are being deployed for the
treatment of cardiovascular disease, it is not surprising that interest
has turned from catheter-based drug delivery toward stentbased
approaches. Stents provide an excellent scaffold for delivery at
high local concentrations over relatively long time period.
We developed a therapeutic
nontoxic dose (5-10 ng/cm) of IFN that does not effect EC but reduces SMC
proliferation. These data correlate with other reports. In these studies,
it was demonstrated that IFN reduces SMC growth [22, 23, 33-35].
Other investigators also showed
the varying influence of different types of IFN on SMC activity [27, 28,
33].
We did not observe elastase of
the SMC media with IFN-α; however, experiments demonstrated that
there was a statistically significant difference when IFN-γ was used
that may represent an indirectly decreased production of collagen.
Its
favorable influence on both SMC growth and decreased collagen production
in the absence of endothelial damage may an advantage over more cytotoxic
agents (taxol, actinomicin D, etc.) [4,32].
IFN may have role in the
treatment of vulnerable plaques due to its anti-inflammatory effect.
However, decreased production of callogen, increased expression of the
cell adhesive molecules P and E-selections, and PECAM and ICAM-1 by IFN-γ
may represent the potential problem [36] of increasing thrombogenicity,
and therefore vectorial delivery (coating of outer surface of the stent)
may be necessary to overcome up-regulation of cell adhesive, molecules in
EC. 5.
Conclusion
Our studies demonstrate that
IFN-γ arrests SMC growth on metal surfaces in the absence of
significant EC toxicity. These effects are dose dependant. In vivo
experiments are planned to study the effects of an IFN-γ-eluting
stent implantation on restenosis. Acknowledgment
The research was supported in
part by a Leon Hess Research Fund at Lenox Hill Hospital (New York, NY)
and A. Ward Ford Foundation (Wausau, WI). The authors owe much gratitude
to John R. Peterson, MD for his review of the manuscript and to Cathy
Kennedy for editorial assistance.
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