Interleukin-8 and de novo mammalian angiogenesis.

In the rat mesenteric-window angiogenesis assay (MWAA), the test tissue is natively vascularized, lacks significant physiological angiogenesis and its homeostasis is unperturbed by surgical intervention. Using the rat MWAA, it is shown here that interleukin-8 (IL-8), administered at approximately physiological doses, is able to induce de novo angiogenesis. Human recombinant IL-8 was administered intraperitoneally at two daily doses of 25 pM, 250 pM and 2.5 nM for 5 consecutive days (days 0-4). Using microscopic, computer-aided techniques including image analysis, the de novo angiogenic response was quantified in groups of animals on days 7, 14 and 21 in terms of the relative vascularized area (VA), a measure of the microvascular spatial extension, and the microvascular length (MVL), a measure of microvascular density or length. The total microvascular length (TMVL) was computed from VA x MVL. A statistically significant angiogenic effect was found in terms of MVL on day 7 and in terms of VA and TMVL on day 14 following the treatment with 2.5 nM, whereas MVL was significantly increased in statistical terms on day 14 following the treatment with IL-8 at the low dose of 25 pM. Notably, IL-8 at the intermediate dose of 250 pM did not induce a statistically significant angiogenic effect in terms of VA, MVL or TMVL on any observation occasion, thereby suggesting that the dose-related angiogenic effect of IL-8 may be nonlinear. This appears to be the first paper showing that IL-8 is able to induce de novo angiogenesis in normally vascularized mammalian tissue.

On the quantitative rat mesenteric-window angiogenesis assay.

The rodent mesenteric-window angiogenesis assay permits the quantitative assessment of angiogenesis in adult, normally-vascularized mammalian tissue in the intact animal. Using appropriate optical magnifications, even very small, newly-formed vessels are recorded. The ultimate value of the model will, of course, depend on how pertinent it is to the features which govern angiogenesis in the tissues that are involved in clinically-relevant, angiogenesis-related diseases.

Nitric oxide suppresses bFGF- and IL-1-alpha-mediated but not VEGF165-mediated angiogenesis in natively vascularized mammalian tissue.

Using the rat mesenteric window angiogenesis assay, we studied the systemic effect of the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester, L-NAME, on angiogenesis induced by basic fibroblast growth factor (bFGF), interleukin-1-alpha (IL-1) or vascular endothelial growth factor (VEGF165). Using technically independent morphometric and image analysis methods, the angiogenic response was quantified in variables related to (i) microvascular spatial extension and (ii) microvascular density. The test tissue, which is natively vascularized and lacks significant angiogenesis physiologically, was unaffected by surgical intervention. Two daily intraperitoneal injections of bFGF (2.20 nmole), IL-1 (1.18 nmole) and VEGF165 (480 pmole) for 5 days elicited significant angiogenesis in the mesenteric windows. L-NAME (0.5 g/L in drinking water) caused further enhancement of the angiogenic response produced by human recombinant bFGF (p<0.001), bovine purified bFGF (p<0.05) or murine recombinant IL-1 (p<0.05). In contrast, the L-NAME treatment did not affect the angiogenic response produced by human and murine recombinant VEGF165. These data suggest that nitric oxide can act as an endogenous suppressor of mammalian de novo angiogenesis, which is a new finding, and, moreover, that angiogenesis induced by VEGF165 on the one hand and by bFGF and IL-1 on the other in the rat mesenteric window depends on different pathways.

Oral administration of a nitric oxide synthase inhibitor enhances de novo mammalian angiogenesis mediated by TNF-alpha, saline and mast-cell secretion.

In healthy adult rats, the test tissue that is used in the mesenteric-window angiogenesis assay is natively vascularized, lacks physiological angiogenesis, and is unperturbed by surgical intervention. Using the rat MWAA oral treatment with the nitric oxide (NO) synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) enhanced the angiogenic response (compared with controls receiving the inactive enantiomer D-NAME) following i.p. injections of (i) TNF-alpha at an approximate physiological dose, (ii) Compound 48/80, which is a highly selective secretagogue causing mast-cell secretion in situ and a very strong angiogenic response, and (iii) saline of a grade not made for infusion, causing a weak angiogenic response. Angiogenesis was assessed quantitatively using microscopic morphometry and image analysis in terms of objective variables recording the microvascular spatial extension, microvascular density, number and length of microvessel segments (extending between two successive branching points) and the degree of microvessel tortuosity. The data strongly suggest that endogenous NO inhibits all three mammalian angiogenesis reactions, although to a markedly different extent. Notably, the present data are virtually the opposite of those that have been reported from other mammalian angiogenesis models, the test tissues of which display deranged homeostasis, such as surgical intervention and/or ischemia.

Angiogenesis: new aspects relating to its initiation and control.

Angiogenesis, the formation of new microvessels from parent microvessels, involves remodeling the basement membrane and interstitial extracellular matrix (ECM) using degrading proteases produced by the endothelial cells (ECs) and other adjacent cells, and the synthesis of ECM molecules by these cells. Degraded ECM releases previously bound heparin-binding cytokines (and growth factors) which are able to act as ligands to high-affinity receptors on various target cells, including ECs. The EC carries receptors for a number of cytokines which are produced by neighboring cells or released from the ECM and which can either induce or suppress the angiogenic phenotype of the EC. ECs are able to synthesize and secrete cytokines with auto- and paracrine effects. Angiogenesis, which virtually never occurs physiologically in adult tissues (except in the ovary, the endometrium and the placenta), is essential in wound healing and inflammation. Angiogenesis is, in fact, strictly controlled by a redundancy of pro- and anti-angiogenic paracrine peptide molecules, some of which have recently been described. The expression and synthesis of two distinct anti-angiogenic factors is, for example, controlled by the p53 tumor suppressor gene. In certain hypoxic conditions, chronic inflammatory diseases and syndromes, angiogenesis is of pathogenic and prognostic significance. Angiogenesis is, moreover, essential for the growth and metastatic spread of solid tumors. This indicates the potential for developing new therapeutic strategies not only for tumors but also in diseases such as rheumatoid arthritis, psoriasis, liver cirrhosis and diabetic retinopathy. Moreover, the therapeutic induction of angiogenesis in ischemic tissues using recombinant cytokines is also promising for clinical application. In fact, the first successful human gene therapy for stimulating angiogenesis has recently been reported.

Intratumoral microvascular density in malignant lymphomas of B-cell origin.

Sections of surgical lymph-node biopsies of four types of malignant non-Hodgkin's lymphoma of B-cell origin (B-NHL) classified according to the R.E.A.L. terminology or lymphadenitis were immunostained in order to demonstrate endothelial CD34 (QBEnd 10) and to determine the microvascular density and vessel-size distribution using an interactive image-analysis technique. Only microvessels displaying a cross-sectional area corresponding to a diameter of between 3.2 and 34.6 microm were included. The intratumoral microvascular density (iMVD) was found to be significantly higher in chronic lymphatic leukaemia (CLL, n = 13) compared with the clinically more aggressive mantle cell lymphoma (MCL, n = 9) and diffuse large B-cell lymphoma (DLBCL, n = 14). iMVD in CLL was also higher than in the follicular neoplastic parts (FL FOLL) of follicular lymphoma (FL, n = 16). In FL FOLL the microvessel density was, moreover, significantly lower than in the surrounding non-neoplastic FL tissue. In lymphadenitis (LA, n = 10) the iMVD was higher than in DLBCL, FL FOLL and MCL. The data suggest that future studies focusing on the relationship between iMVD and the clinical outcome within each particular NHL group should be carried out in order to verify whether iMVD is a prognostic factor in NHL, as it is in carcinomas.

Hyperplasia of the mesenterial windows precedes that of the small gut in the streptozotocin-diabetic rat.

Hyperplastic growth of the mesenterial windows abutting the small gut occurs in lactating rats (Bergström and Norrby 1988) and chronically diabetic rats (Norrby et al. 1983). In the present study, early events in the mesenterial windows and the small gut in streptozotocin-diabetic rats were examined. The area of the mesenterial windows had already increased significantly on day 1 and hyperplasia in terms of increased DNA content, as well as an increase in histamine content (a mast-cell marker), was established from day 2 of diabetes. The increase in total mesenterial window content of DNA, histamine and protein was roughly linear and parallel from day 2 to day 19. The small-gut circumference increased transiently on day 1, but the small-gut mucosal volume was unaffected on days 1 and 2. The small-gut wet weight increased significantly from day 5, whereas elongation was not observed until day 19. The difference in time between the appearance of hyperplasia and the growth of the mesenterial windows and their adjoining gut and the rate with which the hyperplasia proceeds in the two tissues indicate that the regulatory mechanisms of early hyperplastic growth in these tissues are not identical. The factor(s) causing mesenterial window growth and hyperplasia is/are as yet unknown.

NO and de novo mammalian angiogenesis: further evidence that NO inhibits bFGF-induced angiogenesis while not influencing VEGF165-induced angiogenesis.

Using the non-surgical rat mesenteric window angiogenesis assay, we studied the systemic effect of (i) the nitric oxide (NO)-releasing vasodilator isosorbide-5-mononitrate (ISMN) and (ii) the NO-synthase inhibitor L-NAME on angiogenesis induced by the intraperitoneal injection of bFGF and VEGF165. The response was assessed objectively and quantitatively by microscopic morphometry and image analysis in terms of the vascularized area (VA; a measurement of microvessel spatial extension), the microvascular length (MVL; a composite measurement of microvessel density), the total microvascular length (TMVL=VA x MVL), the number of microvessel segments per unit tissue volume (No. MS), the length of the microvessel segments (Le. MS) and the degree of microvessel tortuosity (MVT). Additional architectural features of the network were assessed in terms of variables introduced here: the number of microvessel branching points per unit tissue volume (No. BP), the index of interconnecting microvessel loop formation (In. LF), the index of microvessel intersection (In. IS), the number of microvessel sprouts per unit tissue volume (No. SP) and their length (Le. SP). In bFGF-mediated angiogenesis, L-NAME significantly, augmented angiogenesis, whereas ISMN significantly inhibited angiogenesis. By contrast, neither L-NAME nor ISMN affected the angiogenic response to VEGF165.

Mesenterial-window hyperplasia in the lactating rat.

By examining the true mesentery adjacent to the small gut before and after weaning, as well as in age-matched controls, we found that the number of mesenteric windows, their total area, and their total DNA content increased significantly during lactation. Simultaneously, Feulgen-DNA absorption analysis in individual mesenteric cells showed that these had a normal DNA stemline, and a normal distribution within the G1-G2 range. The hyperplasia and growth developed and declined temporally somewhat parallel in the mesenterial windows and their adjacent small gut, suggesting that an as yet unknown common factor(s) governs the hyperplastic growth in both these tissues. The novel physiological, mesenterial-window hyperplasia in the lactating rat described in the present study may prove useful for studies of the mesenteric function and the regulation of cell proliferation.

Mitogenesis in wound-healing cells in diabetic rats.

The healing of perforated mesenterial window is delayed in insulin-deficient rats after 4 weeks of streptozoticin-induced diabetes. In the present study we investigated the mitogenic capacity of the two predominating cell types in the healing mesenterial window in such rats. The labelling index (LI) in fibroblasts and mesothelial cells, normally constituting approximately 96% of all tissue-bound cells, was estimated in early and later phases of healing within (a) a 1 mm-wide zone surrounding the perforation and (b) centrally in wounds after healing by closure. The mitotic index (MI) of these cells was estimated at various distances from the perforation. Adjacent unperforated control mesenteric windows served as internal controls. Proliferation increased on days 1 and 2 post-perforation, whereafter it gradually diminished, fibroblasts showing a higher LI than mesothelial cells days 3-7 after closure. On day 1 post-perforation the relative increase of LI was greatest in diabetic mesenteries. During the period just preceding healing by closure. LI of both fibroblasts and mesothelial cells was, however, significantly reduced in diabetic animals. The impaired mitogenesis in these wound-healing cells in diabetic rats may thus be of pathogenic significance in the delayed healing in such animals.

On mast cell-mediated mitogenesis in normal and hyperplastic mesenterial windows in female rats.

The outcome of the mast cell-mediated mitogenesis in hyperplastic membranous mesenterial windows of lactating rats as well as in normal mesenterial windows of age-matched and young virgin female rats was studied quantitatively in vivo and in organ culture. Besides elucidating the effect of age and tissue hyperplasia on mitogenic responsiveness, this approach should provide some insight into the pathogenic mechanics of the previously reported supranormal mast cell-mediated mitogenic reaction that emerges in similarly hyperplastic mesenterial windows of diabetic rats. Mast cell secretion was elicited by Compound 48/80 and the histamine release, which was quantified fluorometrically, was unaffected by lactation. The young female rats showed a statistically significant mast cell-dependent mitogenesis taken as the mitotic index and the fraction of the predominating fibroblasts and mesothelial cells in the (S+G2) cell cycle phases after Feulgen-DNA absorption analysis of the cells in situ. Although there was an age-dependent decrease in mitogenesis, the older lactating and non-lactating virgin control rats also showed mast cell-mediated mitogenesis measured as the specific DNA activity. The hyperplastic mesenterial tissue of the lactating animals showed a virtually normal mitogenic reactivity following local mast cell secretion, but at a lower level than in the age-matched controls. This finding suggests that the supranormal mast cell-mediated mitogenesis previously found in the hyperplastic mesenterial windows of diabetic animals is causally related to the diabetic condition rather than to the hyperplastic state of the test tissue.

Raised 5-hydroxytryptamine concentrations in enterochromaffin cells in adult coeliac disease.

We measured cytofluorometrically the concentration of 5-hydroxytryptamine (5-HT, serotonin) of individual enterochromaffin (EC) cells in adult coeliac and non-coeliac small intestinal mucosa. The distributions of 5-HT concentration within populations of EC cells in control and coeliac mucosae were log normal and thus contained one single population of EC cells. The median 5-HT concentration per EC cell, and the number of EC cells both increased in coeliac disease, but showed signs of normalisation when gluten was withdrawn from the diet. The results indicate that, besides inducing EC cell hyperplasia, gluten is capable of producing reversible changes in functions of EC cells in adult coeliac disease.

In vivo models of angiogenesis.

The process of building new blood vessels (angiogenesis) and controlling the propagation of blood vessels (anti-angiogenesis) are fundamental to human health, as they play key roles in wound healing and tissue growth. More than 500 million people may stand to benefit from anti- or pro-angiogenic treatments in the coming decades [National Cancer Institute (USA), Cancer Bulletin, volume 3, no. 9, 2006]. The use of animal models to assay angiogenesis is crucial to the search for therapeutic agents that inhibit angiogenesis in the clinical setting. Examples of persons that would benefit from these therapies are cancer patients, as cancer growth and spread is angiogenesis-dependent, and patients with aberrant angiogenesis in the eye, which may lead to blindness or defective sight. Recently, anti-angiogenesis therapies have been introduced successfully in the clinic, representing a turning point in tumor therapy and the treatment of macular degeneration and heralding a new era for the treatment of several commonly occurring angiogenesis-related diseases. On the other hand, pro-angiogenic therapies that promote compensatory angiogenesis in hypoxic tissues, such as those subjected to ischemia in myocardial or cerebral hypoxia due to occluding lesions in the coronary or cerebral arteries, respectively, and in cases of poor wound healing, are also being developed. In this review, the current major and newly introduced preclinical angiogenesis assays are described and discussed in terms of their specific advantages and disadvantages from the biological, technical, economical and ethical perspectives. These assays include the corneal micropocket, chick chorioallantoic membrane, rodent mesentery, subcutaneous (s.c.) sponge/matrix/alginate microbead, s.c. Matrigel plug, s.c. disc, and s.c. directed in vivo angiogenesis assays, as well as, the zebrafish system and several additional assays. A note on quantitative techniques for assessing angiogenesis in patients is also included. The currently utilized preclinical assays are not equivalent in terms of efficacy or relevance to human disease. Some of these assays have significance for screening, while others are used primarily in studies of dosage-effects, molecular structure activities, and the combined effects of two or more agents on angiogenesis. When invited to write this review, I was asked to describe in some detail the rodent mesenteric-window angiogenesis assay, which has not received extensive coverage in previous reviews.

On the 48/80-induced secretion of tissue mast cells and its mitogenic effect on nearby cells in the intact rat.

Histamine release from tissue-bound mast cells and cell proliferation in the proper mesentery in the intact rat was quantitated following in intraperitoneal injection of graded doses of compound 48/80. The dose-response curves were sigmoid-like in linear-log plots. ED50 for histamine release was 0.035-0.040 and for increased cell proliferation 0.040-0.048 microgram per g BW. The proliferative response following mast-cell secretion ceased after a period of between 48-72 h, irrespective of whether a high or a low dose of 48/80 was used. Basal on the net rate of histamine synthesis (ca. 0.45 microgram/g mesentery wet weight/h) after an initial injection of 48/80, on the extent of histamine release and the proliferative response after a repeated injection of 48/80, it is concluded that there is a lag period of at least 3 days before proliferation can be re-stimulated by renewed 48/80-induced mast-cell secretion.

Local mitogenic effect of tissue mast cell secretion.

The effect of drug-induced mast cell secretion on proliferation was studied in fibroblast-like and mesothelial-like cells in organ-cultured rat mesentery. Mast cell degranulation achieved by Compound 48/80 was followed by a marked mitogenic reaciton in the surrounding tissue cells. The drug itself lacked mitogenic effect on cultured guinea-pig mesentery, the mast cells of which are unresponsive to the drug, and on a human normal fibroblast-like cell line. In contrast, histamine at about 10-(10) M, a major mast cell component, induced marked mitogenesis in guinea-pig mesentery without causing degranulation of mast cells. It is concluded that secreting rat-tissue mast cells release a mitogenic factor or factors acting locally on nearby tissue cells.

Intradermal mast-cell secretion causing cutaneous mitogenesis.

This study demonstrated that mast-cell secretion elicited by intradermal injection of compound 48/80 initiated cell proliferation in rat skin. The mast-cell secretion was confirmed by microscopical demonstration of degranulation of mast cells, and by quantifying histamine release, whereas the proliferation was assessed by measuring DNA-synthesis and the frequency of mitosis. The results, which fully support our previous findings in the true mesentery, indicate that the common type of mast cell, when appropriately activated, is capable of playing a role in governing cell proliferation in diverse tissues.

Mast cell histamine, a local mitogen acting via H2-receptors in nearby tissue cells.

The tissue mast cell is the major storage site of histamine in the body. The present study is concerned with the effect of one histamine H1- and one histamine H2-receptor antagonist on proliferation in the rat mesentery following drug-induced mast-cell secretion. The H2-receptor antagonist, but not the H1-receptor antagonist, significantly suppressed mast-cell-mediated proliferation in vivo and in organ-cultured mesentery. This finding indicates that mast-cell-histamine is a mitogen acting directly on histamine H2-receptors in surrounding tissue cells.

Basic fibroblast growth factor and de novo mammalian angiogenesis.

Using the rat mesenteric-window assay, the de novo angiogenic effect of basic fibroblast growth factor (bFGF) in the intact, adult, normally vascularized test tissue was analyzed using quantitative microscopy including image analysis. Basic FGF was injected ip at doses of 10, 100, and 1000 ng twice daily for 4.5 days and the animals, which were not subjected to surgery, were sacrificed in groups every week for 6 consecutive weeks. The scale and dynamics of the angiogenic response were measured in terms of variables related to microvascular spatial expansion and density. The growth factor-induced angiogenesis in a dose-dependent, nonlinear manner and the angiogenesis displayed distinctly dose-dependent kinetics. The middle dose thus caused angiogenesis that peaked already at Day 7, thereby suggesting a direct effect. The high dose caused angiogenesis that peaked at Day 14, which also appears to be compatible with a direct effect. The low dose caused a more prolonged angiogenic response which did not peak until Day 21 or 28, depending on which variable was measured, which is indicative of an indirect effect. Clearly, the findings suggest that the molecular and cellular mechanisms of bFGF-mediated angiogenesis are dose-dependent.