Chandan Alam

Effects of low dose ionizing radiation on murine chronic granulomatous tissue

PurposeSubstantial clinical evidence shows the efficacy of low-dose radiotherapy in the treatment of a wide variety of benign conditions. However, experimental investigations into these empirically clinical observations remain scarce. We investigated in vivo low-dose radiation effects on chronic granulomatous tissue by using the air pouch model in mice.Material and MethodsChronic granulomatous air pouches were induced in mice and dosed according to 4 protocols: group I: sham control; group II: 2 Gy on day 2; group III: 2 Gy on day 6; group IV: 5 daily doses of 0.5 Gy from day 2 to 6. On day 7 after granuloma induction the granuloma wet and dry weight was estimated, the vascular content was assessed by the formation of vascular casts incorporating carmine, the inducible nitric oxide synthase (iNOS)- and heme oxygenase 1 (HO-1)-expression in tissue homogenates was assessed by Western blot analysis, and the immunohistochemical localization of iNOS was carried out in cryostat sections of the granulomatous tissue.ResultsWe did not observe any significant reduction in granulomatous tissue wet weight or dry weight following the different radiation treatments, which indicates that anti-proliferative effects in response to the low radiation doses used, are probably not involved in the effects of anti-inflammatory radiotherapy. A single dose of 2 Gy on day 2, as well as fractionated treatment with 5×0.5 Gy lead to an increase in vascularity. iNOS-expression in the homogenized granulomatous tissue was decreased, being most pronounced after single-dose irradiation with 2 Gy on day 2, early on in the acute phase of inflammation. In contrast, the HO-1-expression was increased in all irradiated groups.ConclusionLow doses of radiation interfere with the NO- and the HO-1 pathway. Since NO contributes to several aspects of inflammation such as oedema formation and inflammatory pain, we put forward the hypothesis, that the inhibitory effect of low doses of ionizing radiation on the NO pathway is one radiobiological mechanism underlying the clinically observed efficacy of anti-inflammatory radiotherapy and might result in the reduction of swelling as well as relief of pain. Furthermore, the suppression of iNOS activity could be due to the increase in the stress protein HO-1 by low dose radiotherapy.ZusammenfassungZielEs gibt zahlreiche klinische Beweise dafür, daß die niedrig dosierte Strahlentherapie in der Behandlung verschiedener gutartiger Erkrankungen erfolgreich ist. Experimentelle Untersuchungen dieser klinisch-empirischen Beobachtungen liegen jedoch nahezu nicht vor. Wir untersuchten in vivo die Effekte der niedrig dosierten Bestrahlung auf chronisch granulomatöses Gewebe unter Verwendung des Air-Pouch-Modells in der Maus.Material und MethodenChronisch granulomatöses Gewebe wurde induziert und bestrahlt: Gruppe I: scheinbestrahlte Kontrolle; Gruppe II: 2 Gy am Tag 2; Gruppe III: 2 Gy am Tag 6; Gruppe IV: 5×0,5 Gy an den Tagen 2 bis 6. Am Tag 7 nach Induktion des Granuloms wurden das Feucht- und Trockengewicht des Granuloms bestimmt, dessen Vaskularität wurde mit Hilfe des Carmin-Assays beurteilt, die Expression der induzierbaren Stickoxydsynthase (iNOS) und der Häm-Oxygenase 1 (HO-1) wurden in homogenisiertem granulomatösem Gewebe (Western-Blot-Analyse) sowie die iNOS-Immunlokalisation in Cryostat-Schnitten untersucht.ErgebnisseWir beobachteten unter den verschiedenen Behandlungsregimen keine signifikante Verminderung des Feucht- sowie des Trockengewichts des Granuloms, was darauf schließen läßt, daß antiproliferative Effekte in Reaktion auf die applizierten niedrigen Strahlendosen für die Effektivität der Entzündungsbestrahlung keine Rolle spielen. Einzeitbestrahlung mit 2 Gy am Tag 2 sowie fraktionierte Bestrahlung mit 5×0,5 Gy führten zu einer Erhöhung der Vaskularität. Die Western-Blot-Analyse der iNOS-Expression demonstrierte in allen bestrahlten Gruppen im Durchschnitt eine verminderte iNOS-Konzentration, die am ausgeprägtesten nach Einzeitbestrahlung mit 2 Gy am Tag 2 war, das heißt in der akuten Entzündungsphase. Die immunhistochemisch bestimmte Anzahl an iNOS-positiv markierten Zellen innerhalb der verschiedenen Bestrahlungsgruppen zeigte im wesentlichen ein vergleichbares Muster. Im Gegensatz dazu demonstrierte die Western-Blot-Analyse der HO-1-Expression erhöhte HO-1-Konzentrationen in allen bestrahlten Gruppen.SchlußfolgerungNiedrig dosierte ionisierende Strahlung interferiert mit dem NO- sowie dem HO-System. Da NO zu verschiedenen Aspekten der Entzündung wie Ödem und Schmerz beiträgt, postulieren wir, daß der inhibitorische Effekt niedriger Strahlendosen auf das NO-System einer der strahlenbiologischen Mechanismen der klinisch beobachteten Effektivität der Entzündungsbestrahlung ist und zu einem Rückgang des Entzündungsödems sowie einer Verminderung des Entzündungsschmerzes führen könnte. Des weiteren könnte die Suppression der iNOS-Aktivität auf eine strahleninduzierte Expression des Streßproteins HO-1 zurückzuführen sein.

Apoptosis Induction and Inhibition of Colon-26 Tumour Growth and Angiogenesis: Findings On COX-1 and COX-2 Inhibitors in Vitro & in Vivo and Topical Diclofenac in Hyaluronan

The chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs) results in reduced colonic adenocarcinoma incidence1. When administered to existing tumours, NSAIDs induce tumour regression2. Other states susceptible to NSAIDs include basal cell carcinoma (BCC) and the precancerous lesion actinic keratosis (AK)3 using a topical delivery system, HYAL CT1101 (3% diclofenac in 2.5% hyaluronan). The advent of inducible cyclooxygenase (COX-2) raises the question as to its role in tumour growth and development. The role of COX of any isoform remains speculative. Tumour derived prostaglandin-E2 (PGE2) may result in inhibition of macrophage and NK cell function or there may be direct effects on tumour cells. COX-2 has been reported in many human colonic adenocarcinomas but not all4, 5. Studies with the dual COX-1/2 inhibitor sulindac have shown that familial intestinal polyposis is reduced in the clinic6, whilst the murine equivalent is modified by selective COX-2 inhibition or gene deletion7.

The depletion of substance P by diclofenac in the mouse

Diclofenac in hyaluronan is analgesic and angiostatic. The depletion of substance P may be a common mechanism. Mice received diclofenac, diclofenac in hyaluronan, or saline i.v. for 5 days and snout substance P assessed: saline 2.80 +/- 0.23; 0.5 mg/kg diclofenac 2.03 +/- 0.20 (P < 0.05); diclofenac in hyaluronan 1.88 +/- 0.21 (P < 0.02); capsaicin 1.45 +/- 0.26 fmol/mg tissue (P < 0.005). Substance P recovered by 5 days (diclofenac in hyaluronan, capsaicin) and 24 h (diclofenac). Diclofenac may deplete substance P in analgesia, and hyaluronan prolong the depletion.

Modelling angiogenesis in inflammation

Angiogenesis is an integral component of chronic inflammatory lesions and is essential for tissue development and repair. The inhibition of this process is a target for the development of novel therapeutics against chronic inflammation, especially those diseases where angiogenic blood vessels feature prominently, such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and psoriasis. The development of these vessels is stimulated by factors produced within the inflammatory milieu and are derived from inflammatory cells, not least macrophages, which produce angiogenic factors under the hypoxic conditions found within these tissues. Macrophages for example have the extraordinary capacity to produce just about every angiogenic growth factor and cytokine known [1, 2, 3], such as tumour necrosis factor (TNF)-α, basic fibroblast growth factor (FGF-2), transforming growth factor (TGF)-β, angiotropin, prostaglandin (PG) E2, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, platelet-derived growth factor (PDGF), interleukin (IL)-6, vascular endothelial growth factor (VEGF), nitric oxide (NO), and angiotensin converting enzyme (ACE), but not all, angiogenin and platelet factor (PF) 4 being absent from their armamentarium. They do synthesise thrombospondin-1 (TP-1), which may be angiogenic or angiostatic depending on whether it is matrix bound or in the soluble or truncated form [4, 5, 6]. Thus, angiostatic factors may also be synthesised or elaborated, e.g. macrophage-derived enzymes such as metallo-elastase may mediate angiostatin release [7].

Tetrahydro-derivatives of cortisone promote granulomatous tissue angiogenesis in vivo on topical application in hyaluronan.

Hyaluronan is an essential component of the extracellular matrix and alters the quality of wound healing as well as modulating angiogenesis. It is also used as a topical and i.v. drug delivery system. We have previously reported that cortisone in hyaluronan gel inhibits granulomatous tissue angiogenesis, as does tetrahydrocortisol (THF) administered s.c. We have investigated the effects of tetrahydrocortisone (THE) and THF administered s.c. on granulomatous tissue angiogenesis, and compared this with their topical administration in hyaluronan. Carmine/gelatin vascular casts of murine chronic granulomatous air pouches were formed and the vascularity index calculated as μg carmine/mg granuloma dry mass. THF inhibited angiogenesis as previously reported; however, THE stimulated angiogenesis significantly. On topical application in 2.5% hyaluronan both steroids dramatically stimulated granulomatous tissue angiogenesis, THE by 100% and THF by 300% at 1mg/kg. Previous work has shown that topical hyaluronan alone has little effect on granulomatous tissue angiogenesis. Thus the topical application of the tetrahydro-steroid derivatives results in the stimulation of angiogenesis and converts the angiostatic properties of THF to an angiogenic profile. These formulations may therefore have potential as topical therapies, e.g. for the acceleration of wound healing.