The Effects of Thrombocytopenia on Vessel Stasis and Macromolecular Leakage after Photodynamic Therapy Using Photofrin

Abstract— Several studies have reported thrombus formation and/or the release of specific vasoactive eicosanoids, suggesting that platelet activation or damage after photodynamic therapy (PDT) may contribute to blood flow stasis. The role of circulating platelets on blood flow stasis and vascular leakage of macromolecules during and after PDT was assessed in an intravital animal model. Sprague-Daw-ley rats bearing chondrosarcoma on the right hind limb were injected intravenously (i.v.) with 25 mg/kg Photofrin 24 h before light treatment of 135 J/cm² at 630 nm. Thrombocytopenia was induced in animals by administration of 3.75 mg/kg of rabbit anti-rat platelet antibody i.v. 30 min before the initiation of the light treatment. This regimen reduced circulating platelet levels from 300000/mm³ to 20000/mm³. Reductions in the luminal diameter of the microvasculature in normal muscle and tumor were observed in control animals given Photofrin and light. Venule leakage of macromolecules was noted shortly after the start of light treatment and continued throughout the period of observation. Animals made thrombocytopenic showed none of these changes after PDT in either normal tissues or tumor. The lack of vessel response correlated with the absence of thromboxane release in blood during PDT. These data suggest that platelets and eicosanoid release are necessary for vessel constriction and blood flow stasis after PDT using Photofrin.     

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder.

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.     

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

QUANTITATIVE ANALYSIS OF INTRACELLULAR BEHAVIOUR OF PORPHYRINS

Abstract Fluorometric analysis performed on L 1210 cells after treatment with Photofrin indicated that the interactions with cellular structures induce a significant modification of the equilibria among the different porphyrin species. This modification turned out to be dependent on the uptake and release processes. Thus, a comparative analysis of the dynamic aspects of the drug accumulation process was performed on cells treated with hematoporphyrin, Photofrin and Photofrin II. The results obtained were interpreted taking into account the different chemical composition of the drugs employed. The porphyrin species mainly released seem to be the monomeric ones and 'unfolded oligomers'. The release process results in further modifications of the aggregation and/or configu-rational state of intracellular porphyrins due to altered internal equilibrium.     

Low molecular weight heparin enhances prostacyclin production by cultured human endothelial cells.

Confluent cultures of vascular endothelial cells derived from the human umbilical vein were incubated in a serum-free medium in the presence of low molecular weight heparin (LMWH) with molecular weights of 4000-6000 dalton (Da), or of unfractionated heparin (UFH) with average molecular weight 12,000 Da, and prostacyclin production was determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. LMWH at 1 U/ml as anti-factor Xa activity significantly increased prostacyclin production after 6h or longer; however, UFH at 1 USP U/ml did not induce such a significant change. The LMWH-induced increase in prostacyclin production occurred at 0.1 U/ml and above after 6 h of treatment. Since prostacyclin is both a potent inhibitor of platelet aggregation and a vasodilator, it was suggested that the increased endothelial cell prostacyclin production induced by LMWH may be a component of the anticoagulant activity of the drug.     

Silkworm-pheophorbide alpha mediated photodynamic therapy against B16F10 pigmented melanoma

In order to apply photodynamic therapy (PDT) to pigmented melanoma, the efficacy of PDT mediated by pheophorbide alpha from silkworm excreta (SPbalpha) and commercial Photofrin against B16F10 melanoma was comparatively studied from the in vivo assay using C57BL/6J mice. From in vitro PDT assay, the proliferation of B16F10 cells treated with SPbalpha (more than 0.5 microg/ml) and light illumination (1.2 J/cm2) were significantly inhibited with the necrotic response. This indicated that the photocytotoxicity of SPbalpha (665 nm) was not influenced by melanin from melanoma. From the assessment of the in vivo photosensitizing activity, the tumor growth was further delayed in groups treated with SPbalpha/PDT compared to that treated with Photofrin /PDT. The survival rate of tumor bearing mice treated with SPbalpha/PDT was closely associated with its photosensitizing effect. In addition, the photosensitizing effect of SPbalpha/PDT showed a dose dependent tendency in light illumination. These results demonstrated that B16F10 melanoma cells were significantly photosensitized by SPbalpha/PDT, regardless of the influence of melanin from melanoma, and SPbalpha/PDT at very low drug dose (1 mg/kg) and light dose (1.2 J/cm2) showed the photosensitizing efficacy surpassing Photofrin against B16F10 melanoma in mice system.     

Simultaneous two-photon excitation of photofrin in relation to photodynamic therapy

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-gamma) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-gamma) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-gamma excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-gamma PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-gamma killing of vascular endothelial cells in vitro. The 2-gamma absorption properties of Photofrin were investigated in the 750-900 nm excitation wavelength range. It was shown that 2-gamma excitation dominates over 1-gamma excitation above 800 nm. The 2-gamma absorption spectrum of Photofrin in the 800-900 nm excitation wavelength range was measured. The 2-gamma cross section decreased from about 10 GM (1 GM = 10(-50) cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-gamma contribution was negligible. Cell death was monitored with the use of 2-gamma scanning laser microscopy. The light doses required for killing were high (6300 J cm(-2) for approximately 50% killing), but 2-gamma cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-gamma PDT due to its low 2-gamma cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-gamma cross sections (>100 GM), which will be required for 2-gamma PDT of AMD (and other conditions) to be clinically practical.     

Platelet adhesion to photodynamic therapy-treated extracellular matrix proteins

Photodynamic therapy (PDT) produces reactive species that alter vascular wall biology and vessel wall proteins. In this study, we examined platelet adhesion to PDT-treated (photosensitizer = Photofrin; fluence 100 J/cm2; lambda = 630 nm) extracellular matrix (ECM), fibrinogen, von Willebrand factor (vWF) and collagen Types I and III, under flow conditions in a recirculating perfusion chamber. Platelet adhesion was quantified by image analysis. The effect of PDT on vWF was assessed by measuring the binding of domain-specific antibodies to treated vWF. PDT significantly decreased platelet adhesion to the ECM, fibrinogen and vWF. However, PDT of collagen resulted in significantly increased platelet adhesion, with large aggregate formation. PDT affected mostly the A1 (glycoprotein [GP]-Ib-binding site), A2 and A3 (collagen-binding site) domains of vWF but not the D'-D3 (factor VIII-binding site) and B-C1 (GP-IIb/IIIa-binding site) domains. In conclusion, PDT can alter the ECM, resulting in decreased platelet adhesion. However, vessels with high collagen content, such as veins and small arteries, may become increasingly prone to thrombosis. The results of this study may thus play a role in understanding the thrombogenic properties and mechanisms of vascular PDT.     

Magnetic Resonance Imaging Evaluation of Photodynamic Therapy-Induced Hemorrhagic Necrosis in the Murine M1 Tumor Model

Abstract— Proton magnetic resonance imaging (MRI) and histological methods were used to evaluate photodynamic therapy (PDT)-induced hemorrhagic necrosis in the murine Ml tumor within 72 h of treatment of male DBA/2 mice. The effects of three photosensitizing drugs were investigated: Photofrin (n = 4), Zn (II) phthalocyanine (n = 7) and benzoporphyrin derivative monoacid ring A (n = 11). As noted in previous studies of PDT using MRI, MRI makes possible serial, noninvasive, in vivo observation of tissue response to PDT. Our serial study of MRI and histological data confirms that tumors responded in the same way to PDT treatment using the three photosensitizing drugs: vascular damage followed by hemorrhagic necrosis. Most importantly and unlike previous MRI studies of PDT, we used a very high field magnet that enhanced the effect of magnetic susceptibility on image signal when blood is processed by the body after PDT-induced hemorrhagic necrosis. This last finding demonstrates the utility of high field magnets and the importance of localized, serial experiments in future magnetic resonance studies of PDT.     

Effects of photodynamic therapy on adhesion molecules and metastasis

Photodynamic therapy (PDT) induces among numerous cell targets membrane damage and alteration in cancer cell adhesiveness, an important parameter in cancer metastasis. We have previously shown that hematoporphyrin derivative (HPD)-PDT decreases cancer cell adhesiveness to endothelial cells in vitro and that it reduces the metastatic potential of cells injected into rats. The present study analyzes the influence of PDT in vivo on the metastatic potential of cancers cells and in vitro on the expression of molecules involved in adhesion and in the metastatic process. Photofrin and benzoporphyrin derivative monoacid ring A (BPD) have been evaluated on two colon cancer cell lines obtained from the same cancer [progressive (PROb) and regressive (REGb)] with different metastatic properties. Studies of BPD and Photofrin toxicity and phototoxicity are performed by colorimetric MTT assay on PROb and REGb cells to determine the PDT doses inducing around 25% cell death. Flow cytometry is then used to determine adhesion-molecule expression at the cell surface. ICAM-I, MHC-I, CD44V6 and its lectins (àHt1.3, PNA, SNA and UEA) are studied using cells treated either with BPD (50 ng/ml, 457 nm light, 10 J/cm2) or Photofrin (0.5 microgram/ml, 514 nm light, 25 J/cm2). Changes of metastatic patterns of PROb cells have been assessed by the subcutaneous injection of non-lethally treated BPD or Photofrin cells and counting lung metastases. First, we confirm the metastatic potential reduction induced by PDT with respectively a 71 or 96% decrease of the mean number of metastases (as compared with controls) for PROb cells treated with 50 ng/ml BPD and 10 or 20 J/cm2 irradiation. Concerning Photofrin-PDT-treated cells, we find respectively a 90 or 97% decrease (as compared with controls) of the mean number of metastases for PROb cells treated with 0.5 microgram/ml Photofrin and 25 or 50 J/cm2 irradiation. Then, we observe that CD44V6, its lectins (àHt1.3, PNA, SNA) and MHC-I are significantly decreased (compared with the other molecules tested) in PROb and REGb cells after both BPD and Photofrin PDT treatment. These modifications in adhesion-molecule expression, particularly of CD44V6, can thus account only for part of the decrease in the metastatic potential of PDT-treated cancer cells. Changes in adhesion-molecule expression induced by PDT are only transient, implying that the rate of metastatic reduction is probably not linked simply to these changes.     

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.     

IN VIVO 31P NMR STUDY OF COMBINED HYPERTHERMIA and PHOTODYNAMIC THERAPIES OF MAMMARY CARCINOMA IN THE MOUSE

Although the sequence and time interval effects of combined photodynamic therapy (PDT) and hyperthermia tumor treatments have been studied using survival curves, tumor regrowth, and cloning assays, the metabolic response to combined treatment measured by nuclear magnetic resonance (NMR) spectroscopy has not yet been clarified. In this study, mammary carcinoma in the flank of C3H mice was subjected to PDT (12.5 mg/kg Photofrin II, 632 +/- 1 nm at 200 J/cm2) and water bath hyperthermia (43.5 degrees C, 30 min) with no delay or 4 h delay between treatments. In vivo 31P-NMR spectroscopy was employed to measure energy metabolism and pH of the tumors before and serially after treatment for up to 1 week. The data revealed significant differences in the time course of high energy phosphate levels between treatment combinations, which may reflect the biological effectiveness of the combined treatments. Our observations indicate that 31P-NMR spectroscopy can be used to evaluate the metabolic response of tumors to treatment with combined PDT and hyperthermia.     

PHARMACOKINETICS OF FLUORESCENT POLYPORPHYRIN PHOTOFRIN II IN NORMAL RAT TISSUE AND RAT BLADDER TUMOR

The transient behavior of the molecular components responsible for fluorescence emission of the photosensitizing polyporphyrin Photofrin II has been studied quantitatively in the liver, small intestines, bladder and muscles of rats. Relative concentrations of the substance were determined fluorometrically in vivo using a Kr(+)-laser (wavelength = 406.7 nm) and a mercury arc lamp (wavelength = 405 or 550 nm) for fluorescence excitation of Photofrin II. Fluorescence was detected at the maxima of the emission bands, at 630 or 690 nm. The results of the experiments show that Photofrin II can be clearly detected by its fluorescence in all the organs investigated from 3 h up to at least 28 days after systemic application of the substance. Within this investigational period the fluorescing components of Photofrin II are released continuously from the organs. In all the tissues examined, an initial decrease with time constants between 2 and 42 h followed by a slow decay with time constants between about 300 and 600 h can be observed. In addition the pharmacokinetics of the fluorescent components of Photofrin II in chemically induced rat bladder tumors with different stages of malignancy were compared to healthy rat bladder tissue. In a time range of 2-10 days after intravenous injection Photofrin II shows a fluorescence 2-5 times brighter in rat bladder tumors than in healthy bladder tissue.     

CRITICAL IMPORTANCE OF THE TRIPLET LIFETIME OF PHOTOSENSITIZER IN PHOTODYNAMIC THERAPY OF TUMOR

The relation between the lifetimes of the triplet states of various porphyrins and their photosensitizing effects on the photodynamic therapy (PDT) of tumor has been examined. Diethylene-triamine pentaacetic acid ester of 4-[1-(2-hydroxy-ethyloxy)ethyl]-2-vinyl deuteroporphyrin-IX gallium (III) complex (Ga-DP), zinc (II) complex (Zn-DP), and manganese (III) complex (Mn-DP) and Photofrin II (PII) are used as the photosensitizer. The triplet lifetimes have been measured for the samples adsorbed on filter paper (FP) and found to be 57 ms (Ga-DP), 26 ms (Zn-DP), less than or equal to 10 microseconds (Mn-DP) and 9 ms (PII). The phosphorescence of Ga-DP in tumor-bearing golden hamsters are measured both in tumor tissue and in liver. They show bi-exponential decay with the lifetimes of about 5 and 20 ms. From the values, the generation rate, kct[3O2], of singlet molecular oxygen in living animal tissue may be estimated to be an order of 10(2) s-1. The PDT effects have been quantitatively investigated for in vitro experiments; upon irradiation the growth inhibitions of mouse p388 leukemia cells are obtained as a function of concentration of Ga-DP, Zn-DP, Mn-DP and PII. The experimental results indicate that the PDT effects depend essentially on the triplet lifetimes of the photosensitizers.     

含悬挂羧基手臂聚乳酸材料的内皮细胞黏附及其细胞活性

为了考察内皮化材料表面的细胞活性, 在前期工作的基础上, 分别在聚乳酸(PLA)、乳酸-苹果酸共聚物(PLMA), 以及含悬挂羟基或羧基的乳酸-苹果酸共聚物膜(PLMAHE,PLMACA)表面种植人脐静脉内皮细胞(HUVEC), 成功地制备了内皮化表面. 通过测定内皮化材料表面内皮细胞释放的内皮型一氧化氮合酶(eNOS)以及一氧化氮的释放量, 间接考察了内皮细胞的抗凝血活性; 另外, 通过内皮化表面的血小板黏附实验, 直接观察了血小板在内皮细胞上的黏附情况. 实验结果表明, 含羧基材料表面的内皮细胞活性比PLA和PLMAHE的高; 相对其它材料PLMACA能更有效地保留黏附于其表面内皮细胞的活性, 其单位内皮细胞的eNOS以及NO的释放量分别为(41.8±8.1) μmol/104 cells和(0.76±0.16) U/104 cells. 电镜照片(SEM)显示, 各种材料表面的内皮细胞均能有效地减少血小板的黏附与聚集; 在内皮细胞脱落的区域, PLMACA仍能较好地实现其抑制血小板黏附的功能, 有望成为新型血管修复(替代)材料.     

Quantitative in vitro demonstration of two-photon photodynamic therapy using photofrin and visudyne

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.     

A COMPARISON OF SERUM KINETICS AND TISSUE DISTRIBUTION OF PHOTOFRIN II FOLLOWING INTRAVENOUS AND INTRAPERITONEAL INJECTION IN THE MOUSE

Abstract Although hematoporphyrin derivative (HPD) and its 'purers' variety Photofrin II are the most widely used tissue sensitizers in both clinical and experimental photodynamic therapy (PDT), quantitative studies of tissue distribution have been few. We have extracted and measured Photofrin II in several organs of the normal mouse including those of relevance to urological practice. In view of the reported heterogeneities in the distribution within tissues of various cytotoxics when administered intraperitoneally. we have compared results for Photofrin II given by this route with those for intravenous injection. Although both routes of administration gave equally consistent results, differences in absolute tissue concentration as a function of time after injection were found for several but not all tissues. Furthermore, the porphyrin accumulated following intravenous administration seemed to contain more of the non-polar photodynamically active component than that accumulated following the intraperitoneal route. We attempt to explain these differences by reference to published data on porphyrin binding to serum proteins.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

Photochemically modulated endothelial cell thrombogenicity via the thrombomodulin-tissue factor pathways

Photodynamic therapy (PDT) is based on a photochemical reaction using a photosensitizer and light to produce reactive oxygen species that have biological effects. Although its application in some fields is largely based on thrombosis, in the vascular setting thrombosis must be prevented. In this study we examined the effects of PDT on the changes in activity of thrombomodulin (TM) and tissue factor (TF) as important regulators of the coagulation process of endothelial cells. Human umbilical vein endothelial cells were treated with PDT (chloro-aluminum-sulfonated phthalocyanine, lambda = 630 nm) at different light-energy doses, and TM and TF levels were measured using fluorescence spectroscopy. Microparticles (MP) were analyzed using flow cytometry analysis. PDT alters the thrombogenic state of endothelial cells by causing decreased expression of TM and increased expression of functional TF in a light-energy dose-dependent way. PDT-treated endothelial cells shed large numbers of MP containing high levels of TF. TF functionality of PDT-treated cells, measured by a Factor Xa-generating assay, was high. TF was located mostly intracellularly and in MP. The disturbed anticoagulant balance described in this study may explain the occurrence of thrombosis induced by PDT and, if not contained, dispute the suitability of PDT as an adjuvant modality to treat vascular restenosis.