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Cardiovascular Research 1997 35(2):334-340; doi:10.1016/S0008-6363(97)00120-X
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Copyright © 1997, European Society of Cardiology

Photodynamic therapy inactivates cell-associated basic fibroblast growth factor: a silent way of vascular smooth muscle cell eradication

Randolph G Statius van Eps, Farzin Adili and Glenn M LaMuraglia*

Division of Vascular Surgery and Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA

* Corresponding author. Tel. +1 (617) 726-6997; fax: +1 (617) 726-5780.

Received 21 October 1996; accepted 10 March 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Procedurally related vascular injury results in a smooth muscle cell (SMC) proliferative response which is in part initiated by SMC release of mitogens, including basic fibroblast growth factor (bFGF). This injury-induced proliferative response is believed to be a key event in intimal hyperplasia development. Photodynamic therapy (PDT), a novel approach found to be effective in inhibiting experimental intimal hyperplasia, produces cytotoxic free radicals resulting in localized SMC eradication. However, this form of SMC injury does not induce an inflammatory or proliferative response in the vessel wall. This study investigated whether PDT-generated free radicals could inactivate cell-associated bFGF normally released with cell injury. Methods: PDT of bovine SMC was performed in vitro with the photosensitizer CASPc (5 µg/ml) and 675 nm laser light using three different fluences: 10, 50, and 100 J/cm2. After PDT, SMC viability was determined with the tetrazolium salt (MTT) assay and cell-associated bFGF was quantitated by ELISA. A SMC mitogenesis assay was utilized to detect cell-associated bFGF activity released with SMC injury. Results: In a dose-dependent manner, PDT-generated free radicals reduced cell-associated bFGF levels. After PDT with 100 J/cm2, cell-associated bFGF content was reduced by 88% (P<0.0002). Of special interest was the finding that PDT with 10 J/cm2 significantly (P<0.0002) reduced cell viability to around 50%, without affecting cellular bFGF levels. Consequently, a higher PDT dose (100 J/cm2) was needed to significantly (P<0.001) inhibit the SMC mitogenic response associated with SMC injury. Conclusion: These results provide a mechanism to explain how, unlike mechanical or other forms of SMC injury, optimal doses of PDT can locally eradicate medial vascular SMC without resulting in a bFGF-induced initiation of cell proliferation.

KEYWORDS Photodynamic therapy; Bovine, smooth muscle cell; bFGF; Free radicals; Smooth muscle cell proliferative


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The effectiveness of any vascular intervention for the treatment of occlusive arterial disease may be limited by restenosis due to intimal hyperplasia (IH), an exaggerated vascular healing response associated with injury to the vessel wall [1]. A key event in the initiation of IH formation is the injury-induced stimulation of medial smooth muscle cells (SMC) resulting in proliferation and migration to the intima [2]. The extent of this response is known to correlate with the degree of injury to the arterial wall [3, 4]. The proliferative response of SMC following mechanical injury is thought to be to a large extent mediated by endogenous mitogens, including basic fibroblast growth factor (bFGF), released from damaged cells in the vessel wall [5].

With improved understanding of the IH pathobiology there has been development of novel experimental strategies to deal with this problem [1, 6]. It has been shown that antibodies to bFGF [7, 8], interference with signal-transduction pathways [9], genetic modulation of the cell cycle [10]and low-dose ionizing radiation [11]can decrease the SMC proliferative response and inhibit experimental IH development. In addition, considerable interest has focused on photodynamic therapy (PDT) [12–15]as a means to locally eliminate the SMC population responsible for the hyperplastic process.

PDT involves the combination of light and a photosensitizer dye to produce cytotoxic free radicals which damage cellular membranes and organelles [16]. To perform PDT of the vessel wall, a photosensitizer is administered and the area of interest irradiated with wavelength-specific light. Absorption of light by the photosensitizer leads to the generation of free radicals which result in local eradication of cells in the vessel wall. A remarkable finding after PDT-mediated cell removal to inhibit experimental IH is the lack of an excessive fibroproliferative process in response to injury [12–15]. Despite extensive cytotoxicity, which represents a principal mechanism of endogenous mitogen release [5], there is no increased proliferation or migration of viable SMC at the boundary between PDT and untreated vessel segment [13]. PDT-treated vessels are associated with minimal medial wall SMC repopulation and effective inhibition of experimental IH formation [12–15].

The distinctive vascular healing response after PDT suggest that, besides causing cytotoxicity, PDT-generated free radicals may interfere with important biological mediators that initiate the repair process and thereby profoundly alter the vascular healing process. This may be of particular importance in the vascular system since other means of injury to the vessel wall result in an exuberant healing response with formation of IH [2]. The aim of this study was to investigate a mechanism that could explain why PDT-mediated eradication of vascular cells is not followed by a proliferative response. Since bFGF is considered to be a ‘wound hormone’ that is released from injured and dead cells to activate cell growth [5], this study examined the acute effects of PDT on cell-associated bFGF and pure bFGF. Utilizing an in vitro model, PDT of vascular SMC was performed to test the hypothesis that, besides cytotoxicity, PDT-generated free radicals could target and inactivate cell-associated mitogens, such as bFGF, and thereby inhibit its activity following cell injury.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Primary cultures of bovine smooth muscle cells were obtained from the aortas of freshly slaughtered calves by using the explant technique [17]. Their identity was verified by indirect immunofluorescence using an anti-alpha actin antibody (Biomedical Research Technologies, Inc., Stoughton, MA). Cells were kept in a 37°C, 5% CO2 incubator, refed every 42-72 h with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.6 mol/l L-glutamine (Gibco, Grand Island, NY). Cells were passed at a ratio of 1:5 using 0.05% Trypsin/0.125% EDTA (Gibco) upon reaching confluence and used for experiments between the 2nd and 5th subpassages.

2.2 Photodynamic therapy
To perform PDT of SMC in culture, the cells were seeded at a density of either 4x104/cm2 on 96-well tissue culture plates or 2.5x104/cm2 on 6-well plates (Falcon, Becton Dickinson, Lincoln Park, NJ) and allowed to grow in full medium for 24 h. The photosensitizer, chloroaluminum sulfonated phthalocyanine (CASPc), at a concentration of 5 µg/ml, was subsequently added to the cells in full medium and incubated for another 24-h. After 2 rinses with phosphate-buffered saline (PBS), the confluent cell layer was irradiated with thermoneutral light delivered by an argon-pumped dye laser (Coherent Innova I and Coherent CR 599, Coherent, Palo Alto, CA) tuned at 675 nm for optimal absorption. The end-fiber irradiance was set at 100 mW/cm2 and 3 different fluences (total energies) were applied: 10, 50 and 100 J/cm2. Controls included untreated cells and cells exposed to the photosensitizer or light only. In addition, PDT of SMC was performed in the presence of the free radical quencher, sodium azide (10 and 100 mM, Sigma Chemicals, St. Louis, MO) as a separate control [18].

To perform PDT of pure bFGF, a 1 ml serum-free medium solution containing 0.1% bovine serum albumin (Sigma Chemicals), 5 µg/ml CASPc, and 250 pg/ml bFGF (R&D Systems, Minneapolis, MN) was placed in the wells of 12-well tissue culture plates and irradiated as outlined above.

2.3 Cell viability assay
Smooth muscle cell viability was determined 24 h after PDT treatment using a colorimetric assay based on the uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT, Sigma Chemicals) by viable cells [19]. In brief, the MTT solution (0.5 mg/ml) was added to the cells and incubated at 37°C to allow cleavage of the tetrazolium ring by mitochondrial dehydrogenases and formation of blue formazan crystals. After 3 h, the residual MTT was carefully removed and the crystals were dissolved by incubation with DMSO (Sigma Chemicals) for 30 min. The intensity of the developed color in each well was read by an ELISA reader at 570 nm. The optical density of untreated cells represented 100% viable cells and background color formation of MTT with DMSO added to an empty plate, 0% viable cells. The optical density from the treatment groups was fitted into a linear regression line obtained from the control groups to calculate percent viability.

2.4 Determination of bFGF protein levels
To determine whether PDT could have an acute effect on cellular levels of bFGF, the amount of bFGF protein in SMC lysates was measured using a bFGF immunoassay immediately after treatment (R&D Systems). In this assay, bFGF in the test sample is sandwiched between a monoclonal antibody against human recombinant bFGF coated on the microtiter plate, and a second polyclonal antibody against bFGF conjugated to horseradish peroxidase. A set of known bFGF concentrations are analyzed in parallel to obtain a standard line with the optical densities of the known bFGF concentrations from which the unknown sample concentrations are calculated. Color is developed by addition of hydrogen peroxide and chromogen tetramethylbenzidine and the intensity measured at 450 nm.

For cell lysate preparation, the medium was removed and the cells lysed immediately after treatment using a cell lysing reagent (Proteins International, Rochester Hills, MI) suspended in the assay diluent (R&D Systems). The cell lysate was then clarified at 2000 rpm for 15 min and assayed within 1 h. For the purpose of normalizing bFGF protein levels, a group of untreated SMC were seeded in separate wells in parallel and counted at the time of PDT treatment. The cell numbers were analyzed after trypsinization using an electronic coulter counter (Multisizer, Coulter Electronics Ltd., Luton, England) and represented the amount of cells present at the time of PDT treatment. To exclude the possibility that immediately after PDT treatment there was detachment and loss of cells, pilot experiments were performed to determine the number of detached cells in the supernatant after PDT treatment. The results demonstrated that there was no difference in the number of detached cells between PDT and control cells (data not shown).

2.5 Preparation of conditioned medium
To induce the release of cellular bFGF, PDT-treated and control SMC were injured by vigorously scraping them from the plastic substratum with a rubber policeman in the presence of low serum (1%) medium [20]. The cells were allowed to condition the medium for 30 min at 37°C after which the suspension was centrifuged at 2000 rpm for 15 min. The supernatant was assayed for SMC growth-promoting activity and the conditioned media of SMC left undisturbed in their dishes served as control. To determine whether the SMC growth-promoting activity of the injured SMC-conditioned media was mediated by bFGF, a neutralizing antibody against bFGF (R and D Systems) or a non-immune control antibody (normal rabbit IgG, R&D Systems) was added to the conditioned media of mechanically disrupted SMC.

2.6 Mitogenesis assay
To functionally evaluate cellular bFGF release associated with SMC injury, [3H]thymidine incorporation in SMC was used as an indicator of DNA replication [21]. Cells were seeded in low serum medium at a density of 6x103/cm2 and allowed to attach for 24 h. The mechanically disrupted SMC-conditioned medium was subsequently added and incubated with the cells for 48-h and 2.5 µCi of [3H]-thymidine (New England Nuclear, Boston, MA) was included in the medium for the last 5 h. The cells were then washed 3 times with PBS, dissolved in 0.1 N NaOH and placed in Ready Gel scintillation fluid (Beckman Instruments, Inc., Fullerton, CA). Cell-incorporated radioactivity was counted with a scintillation counter (Beckman Instruments, Inc.).

To directly examine the effect of PDT on the mitogenic activity of pure bFGF, PDT was performed of bFGF in solution as described above. The PDT-treated bFGF solution was then supplemented with calf serum to make a 1% calf serum solution and added to SMC to evaluate SMC mitogenesis. Baseline control for these experiments included 1% calf serum medium containing 0.1% BSA and 5 µg/ml CASPc, and the positive control was a non-irradiated bFGF solution containing the photosensitizer.

2.7 Statistical analysis
All data are expressed as mean±standard deviation (s.d.). For comparison of means between multiple groups, a one-way analysis of variance and Tukey's HSD post-hoc test for multiple comparisons was applied (Statistica, Statsoft, Tulsa, OK). P-values of less than 0.05 were considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 PDT-mediated SMC cytotoxicity
To study the relationship between PDT-mediated cytotoxicity and its effects on cell-associated bFGF, SMC viability was assessed after in vivo therapeutic (100 J/cm2) and subtherapeutic (50 and 10 J/cm2) doses of PDT [12]. Immediately after PDT treatment with all doses, the cells remained attached to the culture plates and there were no gross changes in cell morphology as assessed by phase-contrast microscopy. Approximately 1 h after PDT treatment, changes in the cell shape with deterioration of the normal cell membrane contour was first observed. Although most cells remained attached to the tissue culture plate, there was no evidence of SMC survival with PDT doses of 50 and 100 J/cm2 (P<0.0005) as determined by the tetrazolium salt (MTT) conversion assay (Fig. 1). After PDT with 10 J/cm2, SMC viability decreased to around 50% (P<0.0005), whereas exposure of SMC to either light (100 J/cm2) or photosensitizer only had no effect on cell viability (Fig. 1).


Figure 1
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  		Fig. 1 Effect of PDT on smooth muscle cell viability. Viability, as determined by the tetrazolium salt conversion assay, of PDT-treated SMC with total fluences of 10, 50 and 100 J/cm2 and SMC exposed to either the photosensitizer (DO) or light (LO) only is compared to untreated (NT) SMC viability, which represents 100% viable cells. Values are mean±s.d. *P<0.0005 versus NT, DO and LO; #P<0.0005 versus PDT with 10 J/cm2 (ANOVA, n = 6).

 
3.2 PDT effects on cell-associated bFGF
To determine whether PDT-mediated cytotoxicity is accompanied by specific effects on cell-associated bFGF, the concentration of bFGF in SMC lysates was quantitated by ELISA in untreated controls and immediately after PDT treatment. PDT of SMC resulted in a dose-dependent decrease in cellular levels of bFGF (Fig. 2). Whereas 10 J/cm2 failed to affect cellular bFGF levels, there was a significant decrease (P<0.0005) after PDT with 50 and 100 J/cm2. The effect of PDT on cell-associated bFGF required the production of free radicals since the interaction of light or the photosensitizer alone, did not affect bFGF levels (Fig. 2). To exclude the possibility that cellular bFGF leaked out of the cells during laser irradiation, bFGF was measured in the medium immediately after PDT treatment. No detectable levels of bFGF could be measured in the medium of either PDT-treated SMC, or untreated cells (data not shown).


Figure 2
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  		Fig. 2 Effect of PDT on cell-associated levels of bFGF. The concentration of bFGF, as measured by ELISA, in SMC lysates is plotted for PDT of SMC with 10, 50 and 100 J/cm2 and the different controls: non-treated (NT) and exposure of SMC with the photosensitizer drug (DO) or light (LO) only. Values are mean±s.d. *P<0.0002 versus NT, DO, LO and PDT 10 J/cm2; #P<0.0005 versus PDT 50 J/cm2 (ANOVA, n = 8 for NT, PDT 10, 50 and 100 J/cm2, n = 6 for LO and DO).

 
To further examine whether the PDT effect on cell-associated bFGF was mediated through the generation of free radicals, a free radical quencher, sodium azide, was added to SMC immediately prior to irradiation. The addition of sodium azide to non-treated SMC did not affect the cellular levels of bFGF (data not shown). However, sodium azide protected cellular bFGF levels from the PDT effect in a dose-dependent manner (Fig. 3).


Figure 3
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  		Fig. 3 Reversal of the PDT effect on cell-associated levels of bFGF by the free radical quencher, sodium azide. The concentration of bFGF in SMC lysates is plotted for non-treated SMC (NT), PDT-treated SMC with 100 J/cm2 (PDT) and PDT-treated SMC that received sodium azide (PDT + 10 mM and 100 mM NaN3). Values are mean±s.d. *P<0.0005 versus NT and PDT + 100 mM NaN3,; #P<0.0005 versus PDT + 10 mM NaN3 (ANOVA, n = 8 for NT, n = 3 for PDT + 10 and 100 mM NaN3).

 
3.3 Functional consequence of PDT effects on cell-associated bFGF
To assess whether the reduced measurable levels of cell-associated bFGF after PDT had a functional significance, a SMC mitogenesis assay was used to determine bFGF growth-promoting activity. For this purpose, mechanical injury of cells was utilized as a means to release cellular bFGF [21]. As expected, the conditioned media of mechanically injured SMC significantly increased SMC mitogenesis (P<0.0005) as compared to the conditioned media of untreated SMC (Fig. 4). Correlating with the PDT effects on cellular levels of bFGF, PDT of SMC prior to mechanical injury decreased the growth-promoting activity of the conditioned media in a dose-dependent manner (Fig. 4).


Figure 4
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  		Fig. 4 Effect of PDT on growth-promoting activity of injured SMC-conditioned media. The conditioned media of control non-treated (NT) SMC, mechanically injured (INJ) SMC and injured SMC that were PDT-treated with 10, 50 and 100 J/cm2 were used to assess its growth-promoting activity ([3H]thymidine incorporation) on SMC. Values are mean±s.d. *P<0.0002 versus NT; #P<0.01 versus NT; ##P<0.001 versus INJ (ANOVA, n = 25 for NT, n = 15 for INJ, and n = 9 for PDT 10, 50 and 100 J/cm2).

 
To assess the importance of bFGF in the increase of SMC mitogenesis after mechanical injury, an anti-bFGF neutralizing antibody was used to inhibit its activity. Addition of the antibody to the conditioned media of mechanically wounded SMC completely removed its growth-promoting activity (Fig. 5). In fact, the presence of the antibody in the conditioned media of injured SMC decreased SMC mitogenesis below the level of untreated SMC control. Addition of the antibody to control medium also significantly (P<0.001) reduced SMC mitogenesis (58.5±23.5%, n = 6) below the level of untreated SMC control. These findings may be explained by the inhibition of bFGF present in 1% calf serum medium used to prepare the conditioned media (see Section 2). Alternatively, the presence of the antibody during the incubation time could inhibit autocrine bFGF function in SMC [22].


Figure 5
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  		Fig. 5 Neutralizing antibody against bFGF inhibits growth-promoting activity of conditioned media of injured SMC. The conditioned media of control non-treated (NT) SMC, mechanically injured (INJ) SMC and injured SMC that were pre-treated with an anti-bFGF neutralizing antibody (20 µg/ml) or a normal rabbit IgG control (20 µg/ml), were used to assess its growth-promoting activity ([3H]thymidine incorporation) on SMC. Values are mean±s.d. *P<0.0005 versus NT, INJ and INJ + control IgG; #P<0.0002 versus NT (ANOVA, n = 25 for NT, n = 15 for INJ, n = 6 for INJ + bFGF Ab, and n = 3 for INJ + control IgG).

 
3.4 PDT effects on bFGF in solution
To confirm that PDT can inactivate the SMC mitogenic function of bFGF, PDT was performed of pure bFGF in solution. The addition of 250 pg/ml of bFGF to SMC led to a significant increase in SMC mitogenesis (Fig. 6). In a light-dose dependent way, PDT of bFGF resulted in a decrease in its mitogenic activity on SMC (Fig. 6). These results confirm that bFGF is sensitive to the photochemical reaction induced by PDT, which leads to inactivation of its SMC mitogenic function.


Figure 6
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  		Fig. 6 PDT-mediated inhibition of bFGF activity. The functional activity of pure bFGF in solution was determined with a SMC mitogenesis assay ([3H]thymidine incorporation). PDT of bFGF in solution (250 pg/ml) was performed with fluences of 10 and 100 J/cm2. Negative control included a 5 µg/ml CASPc medium solution (CASPc), which was compared with SMC mitogenesis exposed to an untreated bFGF-CASPc solution (bFGF+CASPc), as a positive control, and light-irradiated bFGF-CASPc solutions (bFGF + PDT 10 and 100 J). Values are mean±s.d. *P<0.0005 versus CASPc, and bFGF + PDT 100 J; #P<0.001 versus bFGF+CASPc (ANOVA, n = 3).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The principal finding of this study is that PDT-generated free radicals inactivate cell-associated bFGF. Consequently, the bFGF-induced stimulation of SMC mitogenesis after cellular injury in vitro is inhibited by PDT. In this regard, PDT represents a unique way of cell eradication, since other means of either sublethal or lethal cell injury have been associated with bFGF release. It has been demonstrated that mechanical injury and disruption of the plasma membrane of endothelial cells resulted in release of a bFGF-like molecule [20]. In accordance with these findings, a recent study showed that mechanical injury to SMC in culture caused release of bFGF that activated replication of neighboring SMCs [23]. Furthermore, other forms of cell injury such as hypoxia [24], endotoxin [25]and gamma-irradiation [26]have been shown to cause cellular bFGF release. These experimental data and the fact that bFGF does not possess a signal peptide to direct its release via the ‘classic’ secretory pathway have led to the proposal that cell death or injury are the most likely mechanisms for cellular bFGF release [22]. The importance of bFGF in stimulating SMC mitogenesis after SMC injury was substantiated by the present study, which demonstrated complete inhibition of these effects with a neutralizing bFGF antibody.

In support of the concept that bFGF acts as a ‘wound hormone’ to initiate tissue repair after injury, there is strong evidence to indicate that this mitogen plays an important role in the initial proliferative response of SMC after vascular injury [27]. Balloon injury and other forms of injury to the vessel wall such as thermal injury [28], stent implantation [29]and gamma irradiation [30], which are all associated with widespread cell death, results in an increased SMC proliferative response. The extent of SMC proliferation and subsequent neointimal formation has been found to be proportional to the degree of vascular injury [3, 4]. The role of bFGF in these initial injury responses has been confirmed by the finding that anti-bFGF antibodies can inhibit SMC proliferation [7]and suppress neointimal lesions after experimental balloon injury [8].

PDT of the vessel wall is a form of injury that results in local eradication of vascular cells, and yet, unlike other forms of injury, there is an absence of a proliferative or inflammatory response. This consistent histologic finding after therapeutic doses of PDT in experimental models of IH formed the basis of this study. It brings up the consideration that PDT-mediated cell injury not only targets cellular membranes but may also affect cell-associated bFGF and possibly other important cellular cytokines. The present study focused on the effects of PDT on cell-associated bFGF because of its well-established biological importance in initiating SMC proliferation after cell injury [5, 27]. It has been demonstrated that proteins can undergo free-radical-induced modification [17, 18]. Free radicals react with sensitive amino acids such as histidine, methionine, tyrosine and tryptophan, and the susceptibility of proteins to free radical damage depends mainly on their amino acid composition, the importance and location of susceptible amino acids that mediate protein conformation and activity, and the cellular location [18]. Whether PDT-generated free radicals can target and affect cell-associated growth factors, such as bFGF, has not been studied before.

The present study demonstrated that free radicals generated in SMC can destroy cell-associated bFGF. The sensitivity of bFGF to PDT was confirmed by the finding that PDT of pure bFGF in solution inhibited its SMC mitogenic function. The photosensitizer, CASPc, used in this study is known to bind to protein molecules [31], which may help explain the photochemical reaction with important proteins, such as bFGF, upon light activation. Whether photosensitizers with different chemical characteristics or cellular distribution could elicit the same effect is not known and requires further study. The requirement of PDT-produced free radicals to inactivate bFGF was demonstrated by the fact that neither the photosensitizer nor the light only had any effects on bFGF. In addition, the free radical quencher, sodium azide, protected bFGF from the PDT-induced reaction. Sodium azide primarily quenches singlet oxygen, but can also react with other excited states [18]. Therefore, no conclusion can be drawn as to which specific free radical pathways are involved in the inactivation of bFGF.

The effects of PDT on cell-associated bFGF was studied as a model to test the hypothesis that free radicals can react with cellular mediators and thereby modify the biological response associated with cell injury. Because of potential free radical reactions with proteins, lipids and other chemical structures, it should be emphasized that PDT does not specifically affect a biological factor. In fact, recent in vitro studies from this laboratory have demonstrated that PDT-produced free radicals can profoundly alter the biological characteristics of the extracellular matrix [17]and inactivate matrix-associated transforming growth factor-β [32]. Thus, the distinct vascular healing response after PDT-induced SMC eradication is likely of a multifactorial nature, mediated by free radical cytotoxicity and potential chemical reactions with a host of cellular mediators.

Of special interest in the present study was the finding that the threshold dose for PDT-mediated cytotoxicity was lower than for cellular bFGF inactivation. Although PDT with 10 J/cm2 caused significant SMC death, it had no effect on cellular bFGF levels. Likewise, PDT with 50 and 100 J/cm2 equally eradicated cell viability, but there was significantly less effect on cell-associated bFGF after doses of 50 J/cm2 than PDT with 100 J/cm2. This observation, which correlated with the mitogenic response of SMC after different doses of PDT (Figs. 4 and 6Go), may have important implications for the dosimetry of PDT for clinical applications. Considering the importance of bFGF and other growth factors in determining the outcome of the vascular response to injury, it can be conceived that mere eradication of the SMC population may not be sufficient for effective inhibition of IH. Moreover, histologic examination of PDT arteries with subtherapeutic doses of PDT have shown complete local eradication of SMC but with subsequent delayed IH development [12]. It can therefore be speculated that, besides cytotoxicity, PDT-mediated inactivation of bFGF and potentially other cellular biological mediators may be imperative for its successful inhibition of experimental neointima formation.

If this can be achieved in the clinical setting, PDT may prove to be a silent and effective way of eradicating the SMC population involved in neointima formation and the problem of restenosis. It is hoped that identifying the key PDT parameters to achieve this goal will allow refinement of vascular PDT for human use.

Time for primary review 21 days.


   Acknowledgements
 
Supported in part by NIH grants HL02583 and ONR Contract N00014-91-C-0084. Dr Statius van Eps is recipient of a Netherlands Heart Foundation grant (R94138) and a grant from the US Department of Energy (DE-FG02-91-ER61228). Dr Adili was recipient of a grant from the Deutsche Forschungsgemeinschaft (Ad 106/2-1). The authors express their gratitude to Hassan Albadawi for excellent technical assistance, Drs. Michael T. Watkins and R. Rox Anderson for helpful discussions, and acknowledge Ciba-Geigy for kindly providing the chloro-aluminum sulfonated phthalocyanine.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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 PERSPECT VASC SURG ENDOVASC THERHome page
J. Heckenkamp and G. M. Lamuraglia
Intimal Hyperplasia, Arterial Remodeling, and Restenosis: An Overview
Perspectives in Vascular Surgery and Endovascular Therapy, January 1, 1999; 11(2): 71 - 94.
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