Sylvie Kossodo

Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.

Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.

Photodynamic Therapy Induces Apoptosis in Intimal Hyperplastic Arteries

Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.

Clinical Potential of Photodynamic Therapy in Cardiovascular Disorders

Atherosclerosis and intimal hyperplasia remain obstacles for surgeons to overcome following vascular reconstructions. Even with all of the technical improvements that have occurred in the past several decades, long term patency following intervention is hindered by these inherent adverse developments. Today, the use of light is seen as a potential treatment modality in vascular surgery. Photodynamic therapy (PDT) has been used in the treatment of cancer, and because of its continued success in vascular experimental models it is now being tested in clinical trials for vascular diseases. PDT offers the surgeon many advantages, and it may have unlimited uses in the clinical setting. Is PDT the ultimate treatment modality for the cardiovascular surgeon and will it help to overcome the inherent failures associated with vascular reconstructions? It may be too early to answer these questions, but with the current successes demonstrated by PDT, there is a need for further testing in clinical trials. In the near future, PDT may be used clinically as a treatment modality to inhibit restenosis and intimal hyperplasia following surgical intervention.

Effects of UVR and UVR-induced Cytokines on Production of Extracellular Matrix Proteins and Proteases by Dermal Fibroblasts Cultured in Collagen Gels¶

Synthesis of extracellular matrix (ECM) proteins and their degradation by matrix metalloproteinases (MMP) are part of the dermal remodeling resulting from chronic exposure of skin to ultraviolet radiation (UVR). We have compared two alternative mechanisms for these responses, namely, a direct mechanism in which UV‐B or UV‐A is absorbed by fibroblasts and an indirect mechanism in which cytokines, produced in skin in response to UVR, stimulate production of the ECM proteins and MMP. These studies were carried out on human dermal fibroblasts grown in contracted, free‐floating 9 day old collagen gels as a dermal equivalent. Synthesis of tropoelastin, collagen, fibrillin, MMP‐1, ‐2, ‐3 and ‐9 and tissue inhibitors of metalloproteinases (TIMP)‐1 and ‐2 were measured. Tropoelastin, collagen and fibrillin levels were stable between days 4 and 10, and MMP and TIMP decreased by day 10. Neither UV‐B (2.5–50 mJ/cm2) nor UV‐A (2–12 J/cm2) altered synthesis of ECM proteins, but UV‐A increased MMP‐1 and ‐3 production. Tropoelastin synthesis increased in response to transforming growth factor‐β1 (5 ng/mL) treatment. Both interleukin‐1β and tumor necrosis factor‐α (10 ng/mL) decreased fibrillin messenger RNA levels but increased MMP‐1, ‐3 and ‐9 synthesis markedly. Collagen synthesis was not modulated by UV‐B, UV‐A or cytokine treatment. These results indicate that certain cytokines may have greater effects on production of ECM proteins and MMP than absorption of UV‐B and UV‐A by fibroblasts grown in dermal equivalents and suggest that the former pathway may play a role in the dermal remodeling in photoaged skin.

Mechanisms of reduced human vascular cell migration after photodynamic therapy

Restenosis results from intimal hyperplasia and constrictive remodeling following cardiovascular interventions. Photodynamic therapy (PDT) has been shown to inhibit intimal hyperplasia in vivo by preventing neointimal repopulation of the treated vessel. This study was undertaken in an attempt to further dissect the mechanisms by which PDT acts on secreted and extracellular matrix proteins to inhibit migration of cultured human vascular cells. PDT of three‐dimensional collagen gels inhibited invasive human smooth muscle cell (SMC) migration, whereas cell‐derived matrix metalloproteinase production remained unaltered. Additionally, PDT generated cross‐links in the collagen gels, a result substantiated in an ex vivo model whereby PDT rendered the treated vessels resistant to pepsin digestion and inhibited invasive migration of SMC and fibroblasts. These data support the premise that by inducing matrix protein cross‐links, rendering the vessel resistant to degradation, in vivo PDT inhibits repopulation of the vessel and therefore intimal hyperplasia.

Vaskuläre photodynamische Therapie hemmt die Migration von Fibroblasten durch die Modulation extrazellulärer Matrix: Implikationen zur postinterventionellen Restenosehemmung

Hintergrund: Postinterventionelle Gefasrestenosen stellen ein wesentliches Hindernis fur befriedigende Funktionsraten dar. Experimentelle Studien belegen eine Restenosehem-mung durch photodynamische Therapie (PDT). Die zugrundeliegenden Mechanismen sind jedoch nur unvollstandig bekannt.