Effect of photodynamic therapy on the extracellular matrix and associated components

In many countries, photodynamic therapy (PDT) has been recognized as a standard treatment for malignant conditions (for example, esophageal and lung cancers) and non-malignant ones such as age-related macular degeneration and actinic keratoses. The administration of a non-toxic photosensitizer, its selective retention in highly proliferating cells and the later activation of this molecule by light to form reactive oxygen species that cause cell death is the principle of PDT. Three important mechanisms are responsible for the PDT effectiveness: a) direct tumor cell kill; b) damage of the tumor vasculature; c) post-treatment immunological response associated with the leukocyte stimulation and release of many inflammatory mediators like cytokines, growth factors, components of the complement system, acute phase proteins, and other immunoregulators. Due to the potential applications of this therapy, many studies have been reported regarding the effect of the treatment on cell survival/death, cell proliferation, matrix assembly, proteases and inhibitors, among others. Studies have demonstrated that PDT alters the extracellular matrix profoundly. For example, PDT induces collagen matrix changes, including cross-linking. The extracellular matrix is vital for tissue organization in multicellular organisms. In cooperation with growth factors and cytokines, it provides cells with key signals in a variety of physiological and pathological processes, for example, adhesion/migration and cell proliferation/differentiation/death. Thus, the focus of the present paper is related to the effects of PDT observed on the extracellular matrix and on the molecules associated with it, such as, adhesion molecules, matrix metalloproteinases, growth factors, and immunological mediators. Correspondence


Introduction
Photodynamic therapy (PDT) is a standard treatment for various cancers (lung, esophagus, stomach, cervix, bladder, etc.) as well as for non-malignant conditions such as age-related macular degeneration, actinic keratoses and psoriasis.It is based on the selective retention of a previously administered nontoxic photosensitizer (Figure 1) in the target cells, and irradiation of these cells with visible light at the appropriate wavelength (1).Upon illumination, the photosensitizer generates reactive oxygen species (singlet oxygen and free radicals, such as OH -, HO 2 -and .O 2 -; Figure 2).These reactive species ultimately eliminate highly proliferating cells by damaging membranes, DNA and other cell structures, and also by affecting extracellular matrix (ECM) components.
ECM is a complex network of macromolecules secreted by the cells.It resides between cells as both a barrier and a scaffold on which tissues are built.It is composed of carbohydrates and proteins including adhesion proteins such as fibronectin, vitronectin, laminin, tenascin, and collagen.Cell adhesion and the ECM are involved in signal transduction during a variety of cell functions such as cell motility, morphogenesis, differentiation, and proliferation.In addition to ECM-intracellular signal transduction, cell-cell communication takes place in the extracellular environment when a chemical secreted by a signaling cell interacts with a receptor on the membrane of a second (signal receiving) cell (2).Thus, cell migration comprises cell-cell adhesion and cell-Figure 1. Structure of some photosensitizers used in photodynamic therapy studies.Aminolevulinic acid is not a photosensitizer but a metabolic precursor of protoporphyrin IX.ECM interaction and is orchestrated by cell adhesion molecules and integrin receptors, ECM components, chemoattractant molecules, matrix proteinases, and glycosidases.
In addition to the cytotoxic reaction in the target tissue (direct tumor cell killing), damage to the tumor vasculature, the immunological response associated with leukocyte stimulation, and release of inflammatory mediators like cytokines, growth factors, components of the complement system, acute phase proteins are important mechanisms for the effectiveness of PDT (3).Based on this information, in this paper we present the ECM components and their processes that have been found to be affected by PDT both in vitro and in vivo.

Integrins, E-cadherin, selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1
Integrins are class of molecules that modulates adhesion to the ECM or the endothelium, and subsequent migration through vessel walls (4) via interaction with ECM ligands, e.g., fibronectins, collagens and laminins.They are ubiquitous transmembrane adhesion molecules that form a link between the extracellular environment and the cytoskeleton.In order to function in signal transduction from one environment to the other, the integrins cluster to form focal adhesion plaques.They constitute a family of heterodimeric cell surface receptors composed of α/ß subunits (5).The identity of the α/ß subunits determines the ECM ligand specificity.Particular integrin/ECM interactions lead to distinct cellular responses, such as cell proliferation and differentiation.Runnels et al. (4) investigated the effects of mild (lethal dose of 15%) benzoporphyrin-derivative monoacid ring A photosensitization on the cell adhesion properties of surviving tumor cells (ovarian carcinoma 3).After pho-tosensitization, ß 1 -containing integrins lost their function, as demonstrated by its diffused pattern on the cell surface, less organized into focal adhesion plaques.It is important to emphasize that this change was not due to the direct binding of the photosensitizer to the integrin receptor at the sites of ECM interaction.This is consistent with the loss of integrin ability to bind to ECM proteins, namely, collagen IV, fibronectin, laminin, and vitronectin, both in vitro and in vivo.Still, the cells retained their capacity to adhere to a collagen IV matrix, suggesting that either alternative ß-subunit integrins or other adhesion molecules were used for this binding.
Another study showed that benzoporphyrin-derivative monoacid ring A PDT interfered with the ability of fibroblasts to adhere to extracellular matrices without altering integrin expression (6).Interestingly, it has been shown that photosensitization with Photofrin ® caused polymorphonuclear leukocytes to adhere to the wall of normal vessel (7) but not to those of tumor capillaries (8).Despite the adherence of neutrophils to the microvascular wall after PDT in vivo, the expression of P-selectin (one of the main adhesion molecules that bind leukocytes) by endothelial cells (EC) was not stimulated (9) but the expression of the adhesion molecule E-selectin (expressed by inflamed endothelium) was increased in HPPH-mediated PDTtreated tumors, facilitating neutrophil migration into the tumor area (10).Intercellular adhesion molecules (ICAM) are members of the Ig superfamily expressed on the surface of EC.Integrins on the surface of leukocytes bind to this molecule in order to form more stable adhesions at sites of tissue inflammation (11).This attachment enables leukocytes to migrate through the EC of capillaries and enter the underlying tissue (Figure 3).The expression levels of the adhesion molecules ICAM-1 and vascular cell adhesion molecule 1 were down-regulated in EC after PDT (12).However, a marked up-regu-lation of the ICAM-1 ligands CD11b and CD11c, which are found on neutrophils, was also associated with PDT-treated tumors (11) (Figure 3).Following in vitro PDT, adhesiveness of malignant cells to EC, as well as their capability to invade the basement membrane (13) decline.Similarly, Vonarx et al. (14) observed that PDT using a hematoporphyrin derivative decreased the adhesiveness of colonic cancer cells to EC monolayers.The effect of PDT was also studied on cadherins, another class of adhesion molecules primarily responsible for the formation of stable junctions between cells in tissues.During cancer progression, however, E-cadherin-mediated adhesion is frequently lost (15).Human adenocarcinoma (WiDr) cells in suspension after aminolevulinic acid (ALA)-PDT were unable to attach to a plastic substratum and showed redistribution of α V ß 3 integrin, with no change in E-cadherin expression (16).Under apoptotic conditions, zinc (II)-phthalocyanine-PDT induced a rapid disorganization of the E-cadherin-mediated cell-cell adhesion, which preceded both the detachment of cells from the substratum via ß 1 integrins.PDT has also been reported to alter cell trypsinization in vitro.Photosensitization with pyridinium zinc (II) phthalocyanine and polyhematoporphyrin significantly decreases the efficiency of trypsinization of RIF-1 (murine fibroblasts), HT29 (human colonic carcinoma), and ECV-304 (human umbilical vein endothelial cells).Also, a correlation seems to exist between increased adhesion and increased tissue transglutaminase (tTGase) activity in these cells (17).This group also proposed that the direct activation of tTGase may play a role in the increased resistance to trypsinization following pyridinium zinc (II) phthalocyanine-PDT.The induction of tTGase has been linked to a suppressive effect on tumor growth (18).The same effect was obtained when sublethal ALA or disulfonated tetraphenylporphyrin-PDT inhibited trypsin-induced detachment of WiDr cells and D54Mg (glioblastoma cells) from a plastic substrate (19).Platelet adhesion to the ECM and fibrinogen can be significantly decreased after PDT of these substrates; however, PDT of collagen resulted in significantly increased platelet adhesion, with large aggregate formation (20).In addition, EC retracted after PDT, enabling neutrophils to adhere to the subendothelial matrix by their ß 2 -integrin adhesion receptors (21).

Proteoglycans
Proteoglycans are a class of glycoconjugates consisting of a core protein covalently linked to one or more linear polymers of repeating disaccharide units named glycosaminoglycans.They reside on the cell surface membrane (e.g., syndecan, decorin, glypican) or within the ECM including cartilage, basement membranes and connective tissue (e.g., versican, aggrecan, perlecan).They form a cover of negative charge, which coats virtually all animal cells, playing a role in the integrity of the ECM (22).The glycosaminoglycans found in the ECM include chondroitin/dermatan sulfates, heparan sulfates, and hyaluronan (23).Information regarding PDT and proteoglycan is scarce in the literature.In the repopulating cells of the adventitia of balloon-injured carotid arteries of rats after methylene blue-PDT there was a significant decrease in versican mRNA (24), a proteoglycan which seems to be involved in cell proliferation and is often found in tissues exhibiting elevated proliferation, as is the case during development and in a variety of tumors (25).Despite the essential role of glycosaminoglycans, research involving the effect of PDT on the metabolism of this molecule remains open.

Cytokines
Cytokines are small-secreted proteins that regulate the development and differentiation of blood cells and control the activities of lymphocytes during the immune response.If targeted cells are not destroyed, photooxidative stress leads to transcription and translation of various stress response and cytokine genes.Release of various inflammatory cytokines has been reported to be modulated by PDT (Figure 3) and factors such as tumor necrosis factor-alpha (TNFα) and interleukins (IL-1ß, IL-2, IL-6, IL-8, IL-10) may also have a potential meaning for PDT effectiveness (26).Up-regulation of IL-6 was observed in Photofrin ® photosensitization of epithelial HeLa (cervix carcinoma) cells (27) and EMT6 cells (mam-mary carcinoma) (28).In another study, hypericin photoactivation enhanced IL-6 mRNA production in poorly differentiated but not in well-differentiated nasopharyngeal cancer cells (29).Photofrin-mediated PDT of solid tumors induces host cell infiltration associated with an intense inflammatory response involving the expression of IL-1ß, TNF-α, and IL-6 (10).Also, the decrease of the PDT-mediated tumor cure rate after blocking the functions of some cytokines proves the significance of the immunological response correlated with photosensitization (30).Diamino acid derivatives of protoporphyrin IX evoked an immunological response when the light was applied, stimulating an increase of IL-1ß and IL-6 in sera of treated mice.An interesting finding is that untreated tumor metastases localized at distant sites from the origin of the tumor, underwent regression after PDT treatment (3).This modulation shows a secondary mechanism mediated by PDT.It has been shown that PDT is able to enhance the inherent immunogenicity of at least some tumor cells, as demonstrated by its protection against subsequent tumor inoculation and induction of tumoricidal activity in the spleen and of increasing numbers of interferon (IFN)-γ-secreting splenic cells.Thus, in situ PDT amplifies the host immune response.Incubation of immature dendritic cells with PDT-generated tumor cell lysates in vitro stimulated IL-12 production, whereas incubation with UV and ionizing irradiation-generated tumor cell lysates did not.These results may indicate enhancement of PDT effectiveness in generating host antitumor immune response and potential protection against metastases outside the treatment field (31).A PDT-generated vaccine was also obtained by treating squamous cell carcinoma with a benzoporphyrin derivative and later with a lethal X-ray dose.When injected peritumorally into mice with established squamous cell carcinoma, these treated cells produced a significant therapeutic effect, including growth retardation, tumor regression and cure.Importantly, vaccine cells retrieved from the treatment site 1 h after injection were intermixed with dendritic cells (32).On the other hand, Jee et al. (33) reported that over-expression of IL-6 in ALA-PDT-treated human basal cell carcinoma increased the anti-apoptotic activity.A number of studies have suggested that the cytokines (34) found in untreated tumors are more likely to contribute to tumor growth and progression and to immunosuppression than to promote an effective host antitumor response.For example, IL-6 is also believed to function as a growth factor for various tumors (e.g., colorectal carcinomas) (35).On the other hand, PDT-mediated reduction in function of IL-6 and other cytokine receptors prevents the occurrence of the predicted cytokine effects on cells.The altered cytokine responsiveness is predicted to affect functions of both normal and tumor cells in the post-PDT tissue environment and may determine the treatment outcome in patients undergoing PDT (36).Nevertheless, the real benefit of their up-or down-regulation for the outcome of PDT treatment of tumors remains to be investigated.Moreover, cancer incidence and progression may be subjected to functional polymorphisms of inflammatory cytokine genes and to altered expression of inflammatory cytokines (34).

Growth factors and growth factor receptors
Growth factors are polypeptides that control the growth and differentiation of animal cells.The predicted functions of growth factors and cytokines are critically dependent on the receptor status of the target cells (37).Epidermal growth factor receptor (EGFR) is over-expressed in a wide variety of solid human tumors and is considered to enhance cell proliferation, motility, adhesion, and invasion, and angiogenesis (38).As a consequence, inhibition of EGFR activity has been examined as an approach to the management Photodynamic therapy and the extracellular matrix www.bjournal.com.br of solid tumors (39).An inhibition of the EGF response by down-regulation of EGFR tyrosine kinase has been noted in PDT-treated cells in vitro and in vivo (40).Treatment of lung fibroblasts with ALA-PDT and Photofrin-PDT caused a marked loss (>90%) of EGFR within the period of light treatment (36).These investigators also observed that this reduction of EGFR paralleled the loss of EGF signaling toward the ERK pathway.The same protocol applied to FaDu cells (squamous cell carcinoma) showed that the EGFR level decreased even further during a subsequent 1-h culture period, besides the loss of fully processed leukemia inhibitory factor receptor α.On the other hand, only minor reduction of oncostatin M receptor ß was detected in both HeLa and FaDu cells.Two other growth factors play an important role in the pathogenesis of neovascular agerelated macular degeneration.Schmidt-Erfurth et al. (41) observed that vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were up-regulated in the vascular endothelium of the choroid of treated areas, whereas they were absent in adjacent PDT-unexposed regions and in control tissue.PEDF is an antagonist of VEGF and inhibits VEGF-induced EC growth and migration, and the development of retinal neovascularization (42).PDT has a stimulating effect on the release of VEGF in murine tumors treated with Photofrin ® -mediated PDT (43).On the other hand, synthesis of PEDF mRNA was detected in ganglion cells, cells of the inner nuclear layer, and retinal pigment epithelial cells of normal rat eyes (44).Presumably, angiogenic inhibitors such as PEDF are responsible for the described reduction in human vascular cell migration after PDT (45), and the simultaneous release of PEDF may balance the effects of VEGF (41).Transforming growth factor (TGF) inhibits proteases involved in matrix breakdown or matrix destruction and increases the expression of a number of matrix-associated structural genes such as collagen and fibronectin (46).It was shown that chloroaluminum-sulfonated phthalocyanine-PDT inactivated the functional activity of matrix-associated growth factors such as TGF-ß and basic fibroblast growth factor (bFGF), and decreased cellular bFGF levels of PDT-treated bovine smooth muscle cells (47).In addition, it was demonstrated that PDT of collagen matrix, that mimics the cell-free vascular matrix after PDT in vivo, significantly decreased TGF-ß and bFGF mRNA levels in fibroblasts not treated with PDT (48).

Prostanoids
Prostanoids are the cyclooxygenase (COX) metabolites of arachidonic acids and exert a range of actions in the body.Treatment of tumor cells with Photofrin ® -based PDT releases prostanoids, including prostaglandin E 2 (PGE 2 ), prostacyclin and thromboxane as well as von Willebrand factor, a protein involved in platelet adhesion and aggregation (43,49).Expression of the enzyme prostaglandin endoperoxide synthase 2, also known as COX-2, is regulated by nuclear factor kappa B and produces the inflammatory mediators known as eicosanoids (including PGE 2 and leukotrienes).PDT induces increased expression of COX-2 along with increased PGE 2 synthesis (50), and suppresses the expression of tissue inhibitor of metalloproteinases (51).An increase in the number of cures was obtained after systemic administration of the COX-2 inhibitor [N-(2-cyclohexyloxy-4-nitrophenyl)-methane sulfonamide] (NS-398) which decreased the PDT-induced expression of both PGE 2 and VEGF in BA (mouse mammary carcinoma) tumors.COX-2 inhibitors act as potentiators of the anti-tumor effectiveness of PDT when they are given after illumination.This antitumor effect is probably caused by the inhibition of angiogenesis, which is necessary for tumor regrowth (52).On the other hand, the release of thromboxane from endothelial cells after PDT is partly responsible for the vascular shutdown (49).

Matrix metalloproteinase
Matrix metalloproteinases (MMP) are extracellular proteolytic enzymes (endopeptidases) capable of digesting various structural components of the ECM.Growth, invasion and metastatic potential of solid tumors depend on the formation and development of new blood vessels concomitant with the degradation of the ECM.It was shown that ALA and light induced MMP-1 and MMP-3 expression in normal and scleroderma fibroblasts, besides reducing collagen type I mRNA expression (53).Also, after repeated PDT, lesional skin biopsies of patients with localized scleroderma showed a marked induction of MMP-1 in the dermis (54); a paracrine mechanism seemed to occur, as evidenced by the induction of MMP-1 and MMP-3 protein levels of fibroblasts stimulated with previously PDT-treated keratinocyte medium.Thus, induction of collagen-degrading enzymes together with a reduction of collagen production was thought to be responsible for the antisclerotic effects of ALA-PDT observed in vivo (55).PDT can also increase both the expression and the enzymatic activity of MMP-9 in BA tumors (56).The inflammatory response and host cell infiltration, described previously, suggest that the increased expression of MMP-9 observed in tumor tissue after PDT involves the influx of MMP-9-expressing inflammatory host cells, as opposed to direct PDTinduced expression of MMP-9 in inflammatory cells present within tumor tissue at the time of treatment.These results demonstrate that EC and infiltrating host cells are sources of MMP-9 in PDT-treated BA tumors (56).

Protein cross-linking
As presented previously, PDT is able to induce expression of MMP.Yet, the singlet oxygen generated during PDT interacts with amino acid residues in proteins to generate reactive species.These newly generated free radicals interact with other molecules to form cross-links (57), inactivating matrix-residing growth factors.Cross-linked collagen shows an increased resistance to protease degradation.Indeed, PDT of cell-free matrix using phthalocyanine-PDT induced matrixprotein cross-linking, which resulted in resistance to metalloproteinase digestion (58).Thus, PDT might generate matrix protein cross-links, hindering invasive cellular migration (45), which was also evidenced by cell detachment from the collagen gel surface after PDT, possibly because of induced structural alterations of matrix binding sites (58).Heckenkamp et al. (48) showed that PDT had an inhibitory effect on smooth muscle cell and fibroblast migration, and no significant change in the secretion of MMP was observed.It has been theorized that ECM cross-linking is a major contributor to the inhibition of cell migration following PDT.

Final considerations
It is widely recognized that the interaction of cells with the ECM has profound effects on a number of biological processes, as can be observed in the intertwined relation between angiogenesis, tumorigenesis and ECM metabolism.For this reason, post-PDT responses involving ECM components help to modulate the fate of cells, especially the surviving cells.This occurs because the effects of PDT are not limited to the site where photosensitization takes place, but is rather propagated in a chain reaction.For example, PDT can induce a complex immune response that may enhance anti-tumor immunity.ECM components will be affected, even when they are not the primary targets, and thus modulation of this interaction as a result of photosensitization is of great importance.As Castano and colleagues (59) stated in a recent review, the ideal cancer therapy, besides destroying the primary tumor, should also trigger the immune system to recognize, pursue and destroy any remaining tumor cells, located either at or near the site of the primary tumor, or distant micrometastases.We should also keep in mind that the action of PDT on specific molecular pathways depends on cell line, fluence rate and photosensitizer used.Table 1 summarizes the information discussed in the preceding sections regarding the investigation of various aspects of PDT modulation of the ECM and related components.Other important molecules involved in the regulation of cellular functions, such as proteoglycans, deserve more attention for a better understanding of the effects of photosensitization on the remaining surviving cells and on the tissue bed as a whole.Finally, besides the dual selectivity of PDT, that is the photosensitizer localization in the target tissue and

Table 1 .
Summary of the effect of photodynamic therapy (PDT) on some molecules of the extracellular matrix and related components.