Ana P. Castano

Photodynamic therapy and anti-tumour immunity

Photodynamic therapy (PDT) uses non-toxic photosensitizers and harmless visible light in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis, shut down the tumour microvasculature and stimulate the host immune system. In contrast to surgery, radiotherapy and chemotherapy that are mostly immunosuppressive, PDT causes acute inflammation, expression of heat-shock proteins, invasion and infiltration of the tumour by leukocytes, and might increase the presentation of tumour-derived antigens to T cells.

Mechanisms in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization

The use of non-toxic dyes or photosensitizers (PS) in combination with harmless visible light that is known as photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In a series of three reviews we will discuss the mechanisms that operate in the field of PDT. Part one discusses the recent explosion in discovery and chemical synthesis of new PS. Some guidelines on how to choose an ideal PS for a particular application are presented. The photochemistry and photophysics of PS and the two pathways known as Type I (radicals and reactive oxygen species) and Type II (singlet oxygen) photochemical processes are discussed. To carry out PDT effectively in vivo, it is necessary to ensure sufficient light reaches all the diseased tissue. This involves understanding how light travels within various tissues and the relative effects of absorption and scattering. The fact that most of the PS are also fluorescent allows various optical imaging and monitoring strategies to be combined with PDT. The most important factor governing the outcome of PDT is how the PS interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. Examples of PS that localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes are given. Finally the use of 5-aminolevulinic acid as a natural precursor of the heme biosynthetic pathway, stimulates accumulation of the PS protoporphyrin IX is described.

Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death.

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In the second of a series of three reviews, we will discuss the mechanisms that operate in PDT on a cellular level. In Part I [Castano AP, Demidova TN, Hamblin MR. Mechanism in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagn Photodyn Ther 2004;1:279-93] it was shown that one of the most important factors governing the outcome of PDT, is how the photosensitizer (PS) interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. PS can localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes. An explosion of investigation and explorations in the field of cell biology have elucidated many of the pathways that mammalian cells undergo when PS are delivered in tissue culture and subsequently illuminated. There is an acute stress response leading to changes in calcium and lipid metabolism and production of cytokines and stress proteins. Enzymes particularly, protein kinases, are activated and transcription factors are expressed. Many of the cellular responses are centered on mitochondria. These effects frequently lead to induction of apoptosis either by the mitochondrial pathway involving caspases and release of cytochrome c, or by pathways involving ceramide or death receptors. However, under certain circumstances cells subjected to PDT die by necrosis. Although there have been many reports of DNA damage caused by PDT, this is not thought to be an important cell-death pathway. This mechanistic research is expected to lead to optimization of PDT as a tumor treatment, and to rational selection of combination therapies that include PDT as a component.

MicroRNA 21 promotes fibrosis of the kidney by silencing metabolic pathways

MicroRNA-21 contributes to fibrosis in the kidney by posttranscriptionally regulating lipid metabolism genes. Defeating Fibrosis Although small—just 22 nucleotides in length—microRNA-21 (miR-21) packs a mighty punch, posttranscriptionally regulating the expression of many genes. Furthermore, miR-21 dysregulation has been linked to cardiac disease and cancer. Now, Chau et al. show that dysregulated miR-21 also contributes to kidney fibrosis, an inappropriate wound-healing response that promotes organ failure. The authors first identified miRNAs that were up-regulated in two mouse models of kidney injury. On the basis of preliminary analyses, Chau et al. focused on miR-21. In mice, miR-21 is up-regulated in the kidney soon after injury, before fibrosis appears. Moreover, miR-21 is up-regulated in human kidneys from patients with problems such as acute kidney injury. Although mice that lack miR-21 are healthy and display relatively normal gene expression in the kidney, after injury, a derepressed set of miR-21 target mRNAs becomes apparent, and they develop much less fibrosis than their littermates that express miR-21. In normal mice, inhibition of miR-21 with complementary oligonucleotides likewise reduces kidney fibrosis after injury. To understand how miR-21 amplifies kidney fibrosis, the authors examined kidney gene expression profiles in mice with and without miR-21 after kidney injury. About 700 genes were derepressed in kidneys from mice without miR-21; surprisingly, genes involved in metabolic pathways—particularly involving fatty acid and lipid oxidation—were among the up-regulated genes, whereas those involved in immune or cell proliferation pathways were not. One derepressed gene, encoding peroxisome proliferator–activated receptor α (PPARα), a regulator of lipid metabolism, is a direct target of miR-21. Overexpression of PPARα in the kidney during injury inhibited fibrosis in mice; conversely, in mice that lacked PPARα, inhibition of miR-21 no longer protected against kidney fibrosis. The finding that miR-21 is a major player in kidney fibrosis suggests that drugs that inhibit miR-21, like the complementary oligonucleotides used in this study, might prove to be useful therapies in humans. Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. To understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models and identified 24 commonly up-regulated miRNAs. Among them, miR-21 was highly elevated in both animal models and in human transplanted kidneys with nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21−/− mice suffered far less interstitial fibrosis in response to kidney injury, a phenotype duplicated in wild-type mice treated with anti–miR-21 oligonucleotides. Global derepression of miR-21 target mRNAs was readily detectable in miR-21−/− kidneys after injury. Analysis of gene expression profiles up-regulated in the absence of miR-21 identified groups of genes involved in metabolic pathways, including the lipid metabolism pathway regulated by peroxisome proliferator–activated receptor-α (Pparα), a direct miR-21 target. Overexpression of Pparα prevented ureteral obstruction–induced injury and fibrosis. Pparα deficiency abrogated the antifibrotic effect of anti–miR-21 oligonucleotides. miR-21 also regulated the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation Mpv17l was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for antifibrotic therapies.

Mechanisms in photodynamic therapy: Part three—Photosensitizer pharmacokinetics, biodistribution, tumor localization and modes of tumor destruction

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as cancer therapy, some of its most successful applications are for non-malignant disease. The majority of mechanistic research into PDT, however, is still directed towards anti-cancer applications. In the final part of series of three reviews, we will cover the possible reasons for the well-known tumor localizing properties of photosensitizers (PS). When PS are injected into the bloodstream they bind to various serum proteins and this can affect their phamacokinetics and biodistribution. Different PS can have very different pharmacokinetics and this can directly affect the illumination parameters. Intravenously injected PS undergo a transition from being bound to serum proteins, then bound to endothelial cells, then bound to the adventitia of the vessels, then bound either to the extracellular matrix or to the cells within the tumor, and finally to being cleared from the tumor by lymphatics or blood vessels, and excreted either by the kidneys or the liver. The effect of PDT on the tumor largely depends at which stage of this continuous process light is delivered. The anti-tumor effects of PDT are divided into three main mechanisms. Powerful anti-vascular effects can lead to thrombosis and hemorrhage in tumor blood vessels that subsequently lead to tumor death via deprivation of oxygen and nutrients. Direct tumor cell death by apoptosis or necrosis can occur if the PS has been allowed to be taken up by tumor cells. Finally the acute inflammation and release of cytokines and stress response proteins induced in the tumor by PDT can lead to an influx of leukocytes that can both contribute to tumor destruction as well as to stimulate the immune system to recognize and destroy tumor cells even at distant locations.

Bone Marrow Ly6Chigh Monocytes Are Selectively Recruited to Injured Kidney and Differentiate into Functionally Distinct Populations

Roles for monocyte/macrophages (Mφ) in directing the development of tissue fibrosis are increasingly recognized. Macrophages form a heterogeneous group of inflammatory leukocytes, and the mechanisms by which they acquire heterogeneity and its functional significance are unclear. We used the unilateral ureteral obstruction model of progressive kidney fibrosis to explore macrophage heterogeneity and function further. Unilateral ureteral obstruction kidney Mφs form three distinct subpopulations defined by the marker Ly6C, all of which are derived from a single Ly6Chigh bone marrow monocyte population selectively recruited to the kidney. Conditional ablation of these Mφs in vivo in CD11b-DTR mice is potently antifibrotic. The mRNA transcription profile of these populations is consistent with differential functional roles for each subpopulation, with Ly6Clow macrophages transcribing genes consistent with selective profibrotic or M2-type function. Furthermore, bone marrow chimerism studies indicate that although resident kidney macrophages proliferate markedly to comprise up to 40% of the inflammatory macrophage population, they do not contribute to fibrosis. Our data identify Ly6C as a marker of functionally discrete tissue macrophage subsets and support a model of selective recruitment of Ly6Chigh bone marrow monocytes to the kidney that differentiate into three populations of kidney macrophages, including a profibrotic Ly6Clow population.

Serum Amyloid P Inhibits Fibrosis Through FcγR-Dependent Monocyte-Macrophage Regulation in Vivo

The fibrosis that occus in kidney after tissue injury can be inhibited by serum amyloid protein, which stimulates the cytokine interleukin 10. Suppressing Fibrosis When it comes to repairing damaged tissues or organs, there can be too much of a good thing. Although sometimes the body’s natural repair processes lead to complete restoration of normal tissue after an injury, at other times these processes continue unrestrained, leading to the replacement of normal tissue with fibrous scars—potentially causing organ failure and death. Such fibrosis is a common underlying feature of a wide variety of diseases, including cirrhosis of the liver, cardiovascular disease, rheumatoid arthritis, and progressive kidney disease. Even though fibroproliferative diseases are a leading cause of death in Western societies, there are currently no therapies that target fibrosis directly. Now, Duffield and colleagues describe a potential antifibrotic therapy for fibrosis of the kidney. The body’s repair processes are triggered by a range of harmful events, such as physical trauma, infections, toxin-induced damage, or an inadequate supply of blood. Tissue repair progresses through several stages, including an inflammatory response with an influx of white blood cells like monocytes, apoptosis and necrosis, the activation of wound-healing myofibroblasts, and the deposition and remodeling of extracellular matrix. When this matrix expands in an apparently uncontrolled manner, fibrous scars—consisting primarily of collagen—are formed. Compounds that inhibit the formation of these scars would be welcome tools for treating fibroproliferative diseases. Duffield’s group sought to determine whether serum amyloid P (SAP)—a protein previously shown to inhibit fibrosis in the lung and heart—might also suppress fibrosis in the kidney. SAP can bind ligands on the surface of apoptotic cells as well as Fcγ receptors on certain immune cells, and binding of SAP to the apoptotic cell ligands induces Fcγ receptor–dependent phagocytosis. These observations suggest that SAP might localize to sites of injury, aiding in the removal of wounded tissue. Duffield’s team showed that administration of human SAP to mice with experimentally induced kidney injury markedly decreased fibrosis. The SAP was recruited to the injured kidney where it was associated with dead cells. In the lung and heart, SAP appears to act by inhibiting fibrocytes, which directly produce collagen matrix. In the kidney, however, fibrocytes do not participate in fibrosis. Instead, the researchers determined, kidney fibrosis in mice depends on inflammatory monocytes and macrophages. SAP binds to and suppresses these cells, an effect that depends on expression of the anti-inflammatory cytokine IL-10 and regulated binding to Fcγ receptors, which are expressed on monocytes and macrophages. Given that a form of human SAP is now being tested in a phase 1 clinical trial in healthy volunteers, we may soon know whether it suppresses fibrotic disease in humans. New therapies that target chronic inflammation with fibrosis are urgently required. Increasing evidence points to innate activation of inflammatory cells in driving chronic organ fibrosis. Serum amyloid P is a naturally circulating soluble pattern recognition receptor, a member of the family of pentraxin proteins. It links danger-associated molecular pattern recognition to Fcγ receptor–mediated phagocytosis. Here we show that fibrosis progression in the mouse kidney is significantly inhibited by therapeutic administration of human serum amyloid P, regulated by activating Fcγ receptors, and dependent on inflammatory monocytes and macrophages, but not fibrocytes. Human serum amyloid P–mediated inhibition of mouse kidney fibrosis correlated with specific binding of human serum amyloid P to cell debris and with subsequent suppression of inflammatory monocytes and kidney macrophages in vitro and in vivo, and was dependent on regulated binding to activating Fcγ receptors and interleukin-10 expression. These studies uncover previously unidentified roles for Fcγ receptors in sterile inflammation and highlight serum amyloid P as a potential antifibrotic therapy through local generation of interleukin-10.

Photodynamic therapy plus low-dose cyclophosphamide generates antitumor immunity in a mouse model

Photodynamic therapy (PDT) is a modality for the treatment of cancer involving excitation of nontoxic photosensitizers with harmless visible light-producing cytotoxic reactive oxygen species. PDT causes apoptosis and necrosis of tumor cells, destruction of the tumor blood supply, and activation of the immune system. The objective of this study was to compare in an animal model of metastatic cancer PDT alone and PDT combined with low-dose cyclophosphamide (CY) a treatment that has been proposed to deplete regulatory T cells (T-regs) and increase the immune response to some tumors. We used J774 tumors (a highly metastatic reticulum cell sarcoma line) and PDT with benzoporphyrin derivative monoacid ring A, verteporfin for injection (BPD; 1-mg/kg injected i.v. followed after 15 min by 150 J/cm2 of 690-nm light). CY (50 or 150 mg/kg i.p.) was injected 48 h before light delivery. PDT alone led to tumor regressions and a survival advantage but no permanent cures were obtained. BPD–PDT in combination with low-dose CY (but not high-dose CY) led to 70% permanent cures. Low-dose CY alone gave no permanent cures but did provide a survival advantage and was shown to reduce CD4+FoxP3+ T-regs in lymph nodes, whereas high-dose CY reduced other lymphocyte classes as well. Cured animals were rechallenged with J774 cells, and the tumors were rejected in 71% of mice. Cured mice had tumor-specific T cells in spleens as determined by a 51Cr release assay. We conclude that low-dose CY depletes T-regs and potentiates BPD–PDT, leading to tumor cures and memory immunity.

Chitosan acetate bandage as a topical antimicrobial dressing for infected burns.

ABSTRACT An engineered chitosan acetate bandage preparation (HemCon) is used as a hemostatic dressing, and its chemical structure suggests that it should also be antimicrobial. We previously showed that when a chitosan acetate bandage was applied to full-thickness excisional wounds in mice that had been infected with pathogenic bioluminescent bacteria (Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus aureus), it was able to rapidly kill the bacteria and save the mice from developing fatal infections. Wound healing was also stimulated. In the present study, we asked whether a chitosan acetate bandage could act as a topical antimicrobial dressing when it was applied to third-degree burns in mice contaminated with two of these bacterial species (P. aeruginosa and P. mirabilis). Preliminary experiments established the length of burn time and the number of bacteria needed to produce fatal infections in untreated mice and established that the chitosan acetate bandage could adhere to the infected burn for up to 21 days. In the case of P. aeruginosa infections, the survival rate of mice treated with the chitosan acetate bandage was 73.3% (whereas the survival rate of mice treated with a nanocrystalline silver dressing was 27.3% [P = 0.0055] and that of untreated mice was 13.3% [P < 0.0002]). For P. mirabilis infections, the comparable survival rates were 66.7%, 62.5%, and 23.1% respectively. Quantitative bioluminescent signals showed that the chitosan acetate bandage effectively controlled the growth of bacteria in the burn and prevented the development of systemic sepsis, as shown by blood culture. These data suggest that chitosan acetate bandage is efficacious in preventing fatal burn infections.

Effect of chitosan acetate bandage on wound healing in infected and noninfected wounds in mice

HemCon® bandage is an engineered chitosan acetate preparation designed as a hemostatic dressing, and is under investigation as a topical antimicrobial dressing. We studied its effects on healing of excisional wounds that were or were not infected with Staphylococcus aureus, in normal mice or mice previously pretreated with cyclophosphamide (CY). CY significantly suppressed wound healing in both the early and later stages, while S. aureus alone significantly stimulated wound healing in the early stages by preventing the initial wound expansion. CY plus S. aureus showed an advantage in early stages by preventing expansion, but a significant slowing of wound healing in later stages. In order to study the conflicting clamping and stimulating effects of chitosan acetate bandage on normal wounds, we removed the bandage from wounds at times after application ranging from 1 hour to 9 days. Three days application gave the earliest wound closure, and all application times gave a faster healing slope after removal compared with control wounds. Chitosan acetate bandage reduced the number of inflammatory cells in the wound at days 2 and 4, and had an overall beneficial effect on wound healing especially during the early period where its antimicrobial effect is most important.