R G Statius van Eps1, G M LaMuraglia. 1. Division of Vascular Surgery, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
Abstract
PURPOSE: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
PURPOSE: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
Authors: G M LaMuraglia; J Schiereck; J Heckenkamp; G Nigri; P Waterman; D Leszczynski; S Kossodo Journal: Am J Pathol Date: 2000-09 Impact factor: 4.307