chloroaluminum-tetrasulfophthalocyanine and Hyperplasia

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.

Topics: Actins; Animals; Apoptosis; Carotid Arteries; DNA; DNA Damage; Electrophoresis, Agar Gel; Fluorescent Antibody Technique, Direct; Hyperplasia; In Situ Nick-End Labeling; Indoles; Male; Nucleosomes; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Sprague-Dawley; Time Factors; Tunica Intima

The multifunctional cytokine, transforming growth factor beta 1 (TGF-beta), plays an important role in the development of injury-associated intimal hyperplasia (IH). Strategies to suppress local TGF-beta activity may have a clinical potential to prevent restenosis caused by IH. Photodynamic therapy (PDT) involves the local generation of cytotoxic free radicals by light activation of photosensitizer dyes and has been shown to inhibit experimental IH. This study investigated whether PDT-generated free radicals can affect TGF-beta activity in a biologic system using vascular smooth muscle cells (SMCs).. The release and activation of TGF-beta by injured SMCs in culture was compared between mechanical injury and PDT. Mechanical injury was induced with a rubber policeman, and PDT was performed with the photosensitizer chloroaluminum sulfonated phthalocyanine (5 micrograms/ml) and 675 nm laser light at subtherapeutic 10 J/cm2 and the in vivo therapeutic dose of 100 J/cm2. Cell viability was assessed by the tetrazolium salt conversion assay, and active and total (active + latent) TGF-beta was determined by enzyme-linked immunosorbent assay in the conditioned media of SMCs 24 hours after treatment. Functional TGF-beta activity was assessed by inhibition of endothelial cell mitogenesis.. Both forms of injury severely reduced (p < 0.0005) SMC viability to less than 15%. In untreated SMC conditioned media, only 14.5% of the total TGF-beta was active (27.7 +/- 8.7 pg per 1 x 10(5) cells). However, after mechanical injury and PDT with 10 J/cm2, there was a significant increase (p < 0.02) in active TGF-beta (60.1 +/- 10.1 pg and 48.6 +/- 21.0 pg, respectively), despite a total reduction of approximately 50%. In contrast to this result, PDT with 100 J/cm2 did not result in increased levels of active TGF-beta (8.1 +/- 3.5 pg), despite having similar levels of total TGF-beta. Consequently, the conditioned media of SMCs that had 100 J/cm2 PDT did not inhibit endothelial cell mitogenesis as compared with the conditioned media of SMCs with mechanical injury and 10 J/cm2 PDT (p < 0.0002).. This report describes two novel findings: (1) injury to SMCs in vitro induces the conversion of biologically latent TGF-beta to active TGF-beta; and (2) the therapeutic PDT dose interferes with this injury activation process. This study substantiates the concept of local cytokine inhibition by PDT in a biologic system and provides new insights into the mechanisms of PDT-mediated inhibition of experimental IH.

Topics: Aluminum; Animals; Cattle; Cell Division; Cells, Cultured; Endothelium, Vascular; Free Radicals; Hyperplasia; In Vitro Techniques; Indoles; Muscle, Smooth, Vascular; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Transforming Growth Factor beta; Tunica Intima

Photodynamic therapy (PDT) has been demonstrated to inhibit experimental intimal hyperplasia and to lead to expedient reendothelialization but negligible repopulation of the vessel media. The mechanism that underlies the differential ingrowth of cells into PDT-treated vessel segments is not understood. Because the extracellular matrix (ECM) is known to modulate specific cell functions, this study was designed to determine whether PDT of isolated ECM affects the function of endothelial cells (ECs) and smooth muscle cells (SMCs).. PDT of bovine aortic EC-ECM was performed with chloroaluminum sulfonated phthalocyanine and 675-nm laser light. Control specimens included untreated ECM, ECM-free plates, and ECM exposed to either light or photosensitizer only. Cell function was characterized by attachment, proliferation, and migration of ECs or SMCs that were plated onto identically treated matrixes.. SMC attachment (86% +/- 0.4% vs 95% +/- 0.4%), proliferation (46% +/- 0.5% vs 100% +/- 1.4%), and migration (40% +/- 1.0% vs 100% +/- 0.9%) were significantly inhibited after PDT of ECM when compared with untreated ECM (all p < 0.001). In contrast, PDT of ECM significantly enhanced EC proliferation (129% +/- 6.2% vs 100% +/- 6.2%; p < 0.03) and migration (118% +/- 2% vs 100% +/- 0.8; p < 0.01), but did not affect attachment.. This report establishes PDT-induced changes in the ECM with a result of inhibition of SMCs and stimulation of EC functions. It provides insight into how PDT-treated arteries can develop favorable EC repopulation without SMC-derived intimal hyperplasia. These findings may help provide a better understanding of the interactions between cells and their immediate environment in vascular remodeling.

Topics: Aluminum; Animals; Aorta; Cattle; Cell Adhesion; Cell Division; Cell Movement; Cells, Cultured; Endothelium, Vascular; Extracellular Matrix; Free Radicals; Hyperplasia; Indoles; Lasers; Muscle, Smooth, Vascular; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Tunica Intima; Tunica Media

To study the cytotoxic effect of photodynamic therapy (PDT) on myointimal hyperplasia (MIH) in 120 New Zealand white rabbits using the chromophore chloroaluminum phthalocyanine tetrasulfonate (APtS).. A common carotid artery (CCA) injury model was used to initiate MIH. Photodynamic therapy was administered 1 week after injury (inhibition arm) or 6 weeks after injury (treatment arm). The inhibition arm CCAs were harvested 6 weeks after therapy. The treatment arm CCAs were harvested 1 week or 6 weeks after therapy. Each evaluation included four subgroups (n = 10 each): control, drug only, laser only, and drug plus laser.. An established CCA balloon injury model was used. Photodynamic therapy was administered by exposing CCAs to continuous external laser irradiation 30 minutes after treatment with a 2.5-mg/kg intravenous dose of APtS (fluence = 25 J/cm2, lambda = 672 nm). The control and drug-only subgroups received sham reoperations without laser exposure.. Following harvest, the CCAs were evaluated for area of stenosis and cell density.. In the inhibition arm, no PDT effect was seen on intimal cell density or area stenosis. In the treatment arm, intimal cell density was markedly diminished (P < .05) in the rabbits in the drug-laser group that were killed 1 week but not 6 weeks after PDT compared with rabbits in the control, drug-only, and laser-only groups. Area stenosis was not significantly affected by PDT.. Marked acute cytotoxicity of PDT on MIH was verified in vivo in the treatment arm. No sustained benefit of PDT was seen in the inhibition or the treatment arms. Refinements in dosimetry will be necessary to achieve long-term benefit of PDT for MIH.

Topics: Animals; Carotid Artery Injuries; Carotid Artery, Common; Collagen; Elastin; Hyperplasia; Indoles; Microscopy, Electron; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Rabbits; Tunica Intima

There is no clinically useful therapy for the suppression of vein bypass graft intimal hyperplasia (IH). Photodynamic therapy (PDT), a technique that uses light to activate otherwise biologically inert photosensitizers to produce cytotoxic effects, has been demonstrated to successfully inhibit experimental IH in balloon-injured arteries. The purpose of this study was to investigate the efficacy of PDT as a method to reduce vein graft IH.. Reversed external jugular vein bypass grafts of the common carotid artery were performed in 28 male Sprague-Dawley rats. The animals received either chloroaluminum sulfonated phthalocyanine (2.5 mg/kg intravenously) 24 hours before the ex vivo irradiation of the vein grafts (VG) with 100 joule/cm2 at 675 nm (PDT VG) or saline solution as control (CON VG). Preharvest bromodeoxyuridine was administered to label proliferating cells. All vein grafts were perfusion fixed within 96 hours for a pilot study or at 2 and 4 weeks for the main study. Histology, immunohistochemistry, and morphometric analysis were performed.. There was no acute thrombus formation in the hypocellular PDT VG with occasional platelets but no leukocytes adherent to the luminal surface. Intimal areas of the PDT VG were 18% at 2 weeks and 53% at 4 weeks of the CON VGs (p < 0.05). Medial areas and percent of stenoses were also significantly less in PDT than in CON VG. However, intimal hyperplasia noted in the longitudinal sections within 2 mm of the anastomoses did not demonstrate a difference between PDT and CON VG. Intimal hyperplasia of both PDT and CON VG consisted of smooth muscle cells, verified by immunohistochemistry. Bromodeoxyuridine-labeled cells were more abundant in 2-week than in 4-week specimens, were found most frequently in the intimal areas of the CON VG body, and were equivalent in the anastomoses of PDT VG and CON VG.. These data suggest that PDT of vein grafts suppresses the development of IH in the body of the vein graft but does not affect IH adjacent to the anastomoses. The artery may be the source of proliferating smooth muscle cells that contribute to the anastomotic vein graft IH.

Topics: Anastomosis, Surgical; Animals; Blood Platelets; Bromodeoxyuridine; Carotid Artery, Common; Cell Division; Hyperplasia; Indoles; Jugular Veins; Male; Organometallic Compounds; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Sprague-Dawley; Tunica Intima; Veins

Intimal hyperplasia (IH) is a focal arterial problem that still eludes successful therapy. We have previously demonstrated the feasibility of use of photodynamic therapy (PDT) for the acute treatment of experimental IH with light to activate an otherwise biologically inert photosensitizer. The purpose of this study was to determine the acute and long-term effects of PDT inhibition of IH on the artery wall.. Segmental IH was induced by balloon injury localized to the cervical common carotid artery of 33 rats. The photosensitizer chloroaluminum sulfonated phthalocyanine (5 mg/kg) for the experimental group or saline solution for the control group was administered intravenously. Twenty-four hours later, all instrumented portions of arteries were irradiated at 675 nm to induce cytotoxic injury in the PDT-treated arteries as compared with laser only-treated arteries for controls. Animals were killed at 1, 2, 4, and 16 weeks.. There were no untoward side effects in either group. All PDT-treated arteries were devoid of smooth muscle or inflammatory cells in the treated media. There was no evidence of arterial degeneration of PDT-treated arteries. Only three arteries in the PDT group developed IH, whereas it was universal in all controls. In control arteries, immunocytochemistry with bromodeoxyuridine revealed maximal intimal and medial cell proliferation at 1 week, and morphometric analysis demonstrated a maximal IH at 2 weeks. Immunocytochemistry staining for smooth muscle cell actin was positive for the IH in control and when present in PDT-treated arteries, whereas the adventitia of PDT-treated arteries were positive after 2 weeks. Electron microscopy demonstrated early myofibroblast migration to the adventitia, and at 16 weeks occasional myofibroblasts were noted in the media of PDT-treated arteries. There was complete reendothelial cell covering of the intima by 4 weeks.. These in vivo data demonstrate that PDT is an effective local method for the treatment of experimental IH. There is no evidence of significant recurrence of IH or arterial degeneration. Further studies with PDT may provide novel approaches to the understanding and treatment of arterial IH.

Topics: Animals; Carotid Artery Diseases; Hyperplasia; Immunohistochemistry; Indoles; Male; Microscopy, Electron; Organometallic Compounds; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Sprague-Dawley; Time Factors; Tunica Intima

Photodynamic therapy, the light activation of photosensitizers into cytotoxic mediators, has been a successful treatment for experimental intimal hyperplasia (IH). To understand the basis of the photosensitizer chloroaluminum sulfonated phthalocyanine (CASPc)-mediated photoinhibition of intimal hyperplasia in the rat common carotid artery model, we studied photosensitizer partitioning in hyperplastic as compared to normal arterial tissue. Serum clearance of CASPc is exponential with, a half-life of 300 minutes. Laser-induced fluorescence and spectrofluorimetric analyses of artery tissue demonstrated an approximately 60% lower uptake and retention of CASPc by normal arterial tissue as compared to arteries with IH; the differences become more pronounced at 24 h. Fluorescent microscopy of arterial tissue demonstrated increased uptake of the CASPc by the artery with IH. However, by 24 h it is primarily the IH tissue that has retained the CASPc, with clearance of the dye from the media of normal or hyperplastic arteries. These data demonstrate that IH, like neoplastic tissue, has an increased accumulation of CASPc compared to normal artery. The preferential partitioning into hyperplastic tissue has implications for therapeutic targeting of this cellular population with photodynamic therapy.

Topics: Animals; Arteries; Carotid Artery, Common; DNA; Half-Life; Hyperplasia; Indoles; Lasers; Male; Microscopy, Fluorescence; Organometallic Compounds; Phototherapy; Radiation-Sensitizing Agents; Rats; Rats, Sprague-Dawley; Tunica Intima