On Connective Tissue Mast Cells as Protectors of Life, Reproduction, and Progeny.

The connective tissue mast cell (MC), a sentinel tissue-residing secretory immune cell, has been preserved in all vertebrate classes since approximately 500 million years. No physiological role of the MC has yet been established. Considering the power of natural selection of cells during evolution, it is likely that the MCs exert essential yet unidentified life-promoting actions. All vertebrates feature a circulatory system, and the MCs interact readily with the vasculature. It is notable that embryonic MC progenitors are generated from endothelial cells. The MC hosts many surface receptors, enabling its activation via a vast variety of potentially harmful exogenous and endogenous molecules and via reproductive hormones in the female sex organs. Activated MCs release a unique composition of preformed and newly synthesized bioactive molecules, like heparin, histamine, serotonin, proteolytic enzymes, cytokines, chemokines, and growth factors. MCs play important roles in immune responses, tissue remodeling, cell proliferation, angiogenesis, inflammation, wound healing, tissue homeostasis, health, and reproduction. As recently suggested, MCs enable perpetuation of the vertebrates because of key effects-spanning generations-in ovulation and pregnancy, as in life-preserving activities in inflammation and wound healing from birth till reproductive age, thus creating a permanent life-sustaining loop. Here, we present recent advances that further indicate that the MC is a specific life-supporting and progeny-safeguarding cell.

Do mast cells contribute to the continued survival of vertebrates?

This study is an attempt to shed light on why the connective tissue mast cell (MC) is preserved in all species with a blood circulatory system, i.e., the vertebrates since >500 million years, which suggests that the MC performs as yet not understood indispensible life-promoting actions. The literature survey focuses on data in published papers on MC functions in immunological and nonimmunological reactions, host protection, pregnancy, inflammation, and wound healing. All data are thus accessible to the reader. The MC is a secretory cell with a unique mediator profile. A distinctive role for MCs is defined not only by their extensive mediator composition but also by their prominent ability to affect the vasculature to expedite selective cell recruitment and permeability changes and to set the stage for an appropriate acquired response. MCs, harboring a wide range of surface membrane receptors, are activated by the major female sex hormones as well as by diverse potentially adverse stimuli. MC activation/degranulation creates a presumably unique triad tissue response in physiological and pathological situations alike: extracellular matrix degradation and tissue remodeling, de novo cell proliferation, and de novo angiogenesis. As shown in the literature, MC-activation is crucial for successful female reproduction in the mouse, implying one of possibly several yet unidentified physiological roles of MCs. Moreover, the activated MC aids newborns to survive to reproductive age owing to its key beneficial actions in inflammation and wound healing. Thus, a not previously described life-perpetuating loop spanning generations are apparently formed, which, hypothetically, could contribute to the continued survival of the vertebrates.

Metronomic chemotherapy and anti-angiogenesis: can upgraded pre-clinical assays improve clinical trials aimed at controlling tumor growth?

Metronomic chemotherapy, which is continuously administered systemically at close to non-toxic doses, targets the endothelial cells (ECs) that are proliferating during tumor angiogenesis. This leads to harmful effects of an even greatly increased number contiguous tumor cells. Although pre-clinical studies of angiogenesis-related EC features in vitro and of the anti-angiogenic and anti-tumor effects in vivo of metronomic chemotherapy have provided valuable insights, clinical trials with this type of therapy have been less successful in inhibiting tumor growth. One possible reason for the apparent disconnect between the pre-clinical and clinical outcomes is that most of the currently used experimental angiogenesis assays and tumor models are incapable of yielding data that can be translated readily into the clinical setting. Many of the assays used suffer from unintentional artifactual effects, e.g., oxidative stress in vitro, and inflammation in vivo, which reduces the sensitivity and discriminatory power of the assays. Co-treatment with an antioxidant or the inclusion of antioxidants in the vehicle often significantly affects the angiogenesis-modulating outcome of metronomic mono-chemotherapy in vivo. This 'metronomic chemotherapy vehicle factor' merits further study, as do the observations of antagonistic effects following metronomic treatment with a combination of standard chemotherapeutic drugs in vivo.

Low-dosage metronomic chemotherapy and angiogenesis: topoisomerase inhibitors irinotecan and mitoxantrone stimulate VEGF-A-mediated angiogenesis.

Metronomic chemotherapy with cytotoxic agents has been shown to inhibit angiogenesis and, consequently, tumor growth by targeting vascular endothelial cells (ECs). In these regimens, anti-tumor activities additional to anti-angiogenesis may operate. Moreover, chemotherapy typically generates reactive oxygen species in targeted ECs, which can affect angiogenesis. The aim of the present study was to assess the systemic effect of low-dosage metronomic treatment with either irinotecan or mitoxantrone on angiogenesis induced by VEGF-A. Angiogenesis was induced in normal adult rat mesentery by intraperitoneal injection of a low dosage of VEGF-A. Thereafter, irinotecan and mitoxantrone were infused separately continuously at minimally toxic dosages for 14 consecutive days via a subcutaneous osmotic minipump. Angiogenesis was assessed in terms of objective and quantitative variables using morphologic and computerized image analyses. Irinotecan or mitoxantrone significantly stimulated angiogenesis, with ironotecan increasing angiogenesis by 104%, when compared with the vehicle-treated animals. Low-dosage metronomic chemotherapy with irinotecan or mitoxantrone stimulates angiogenesis in the normal mesentery of rats, probably by inducing low-level oxidative stress in the targeted ECs. Whether or not this pertains to tumor angiogenesis may be difficult to confirm, as several anti-tumor modes may operate during low-dosage metronomic chemotherapy.

Rat mesentery angiogenesis assay.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor tissues, as discussed in invited review papers(1,2). Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is translucent and framed by fatty tissue, giving it a window-like appearance. The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in(1); (iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly. Mast-cell-mediated angiogenesis was first demonstrated using this assay(3,4). The model demonstrates a high level of discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., angiogenesis-suppressive, neutral or angiogenesis-stimulating activities(5)); (ii) natural iron-unsaturated human lactoferrin, which stimulates VEGF-A-mediated angiogenesis(6), and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis(7); and (iii) low-molecular-weight heparin fractions produced by various means(8,9). Moreover, the assay is highly suited to studies of the combined effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion. The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated. Review papers (invited) discussing and appraising the assay Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006). Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo.

Tumors are angiogenesis dependent and vascular endothelial growth factor-A (VEGF-A), a heparin-binding protein, is a key angiogenic factor. As chemotherapy and co-treatment with anticoagulant low-molecular-weight heparin (LMWH) are common in cancer patients, we investigated whether angiogenesis in vivo mediated by VEGF-A is modulated by metronomic-type treatment with: (i) the LMWH dalteparin; (ii) low-dosage cytostatic epirubicin; or (iii) a combination of these two drugs. Using the quantitative rat mesentery angiogenesis assay, in which angiogenesis was induced by intraperitoneal injection of very low doses of VEGF, dalteparin sodium (Fragmin(®) ) and epirubicin (Farmorubicin(®) ) were administered separately or in combination by continuous subcutaneous infusion at a constant rate for 14 consecutive days. Dalteparin was administered at 27, 80, or 240 IU/kg/day, i.e., doses that reflect the clinical usage of this drug, while epirubicin was given at the well-tolerated dosage of 0.4 mg/kg/day. While dalteparin significantly stimulated angiogenesis in an inversely dose-dependent manner, epirubicin did not significantly affect angiogenesis. However, concurrent treatment with dalteparin and epirubicin significantly inhibited angiogenesis. The effect of dalteparin is the first demonstration of a proangiogenic effect of any LMWH in vivo. The fact that co-treatment with dalteparin and epirubicin significantly inhibited angiogenesis suggests a complex drug effect.

Low-dose continuous 5-fluorouracil infusion stimulates VEGF-A-mediated angiogenesis.

BACKGROUND: Tumor growth is angiogenesis-dependent. Animal studies have demonstrated that frequent administration of chemotherapeutics may have marked antiangiogenic effects and improved antitumor effects, with less severe toxic side-effects than intermittent maximum tolerated dose chemotherapy. Currently, research focused on low-dose antiangiogenic chemotherapy is increasing. We have recently reported that certain chemotherapeutics, including 5-fluorouracil (5-FU), may in fact stimulate angiogenesis in the tumor-free rat mesenteric window assay. The aim of the present study was to extend the investigation of the angiogenesis-modulating effects of 5-FU by prolonging the continuous infusion treatment time. METHOD: Angiogenesis was induced in the mesenteric test tissue in adult male Sprague-Dawley rats by i.p. injection of VEGF-A, which is a key angiogenic factor in most tumors. During the subsequent angiogenesis, 5-FU was delivered continuously for 14 days by an osmotic pump implanted subcutaneously. The angiogenic response was analyzed by morphometry in the mesenteric windows. RESULTS: The 14-days continuous infusion of 5-FU significantly stimulated angiogenesis. Thus the possibility that the previously reported surprising proangiogenic effect of 5-FU reflected an insufficiently long treatment period can be ruled out. CONCLUSION: The finding that continuously infused 5-FU is able to stimulate angiogenesis in the present rat model of angiogenesis warrants investigation of the mechanisms behind this unexpected finding. It may further have implications for the choice of antiangiogenic chemotherapeutic schedule used for cancer patients.

Dose effects of continuous vinblastine chemotherapy on mammalian angiogenesis mediated by VEGF-A.

Low-dose continuous or metronomic chemotherapy with several agents can exert significant antiangiogenic effects, as shown in preclinical studies. Therapy of this kind is generally well tolerated compared with conventional chemotherapy with high, temporally spaced out bolus doses. A critical point emerges when the effects on angiogenesis of low-toxic metronomic doses of chemotherapeutics in preclinical studies are to be transferred to clinical protocols, as there is a risk that a virtually non-toxic dose might also be ineffective; clearly, dose-effect data are important. We therefore sought to investigate whether a dose-dependent response exists in metronomic vinblastine chemotherapy. The surrogate tumor-free rat mesentery model, allowing the study of antiangiogenic effects per se, was used. Following systemically administered metronomic chemotherapy, it closely reflects the indirectly assessed antiangiogenic and growth-retarding effects in a syngenic cancer model. VEGF-A, which is a central proangiogenic factor in most tumors, was administered i.p. to induce angiogenesis in the mesenteric test tissue and, using morphometry, the angiogenesis-modulating effects of vinblastine were assessed in terms of objective quantitative variables. We report that continuous vinblastine treatment with an apparently non-toxic dose (1.0 mg/kg/week or 0.143 mg/kg/day) for 10 days, and a dose that substantially inhibited the physiologic body-weight gain (2.0 mg/kg/week or 0.286 mg/kg/day) for 6 days, demonstrates a dose-response relationship; the high dose significantly suppresses angiogenesis. To our knowledge, no previous study has reported on a dose-dependent antiangiogenic effect by continuous or metronomic vinblastine treatment in a mammalian in vivo model.

Drug testing with angiogenesis models.

BACKGROUND: The possibility to treat cancers and several angiogenesis- dependent diseases with non-toxic, antiangiogenic agents has revolutionized the therapeutic capabilities in the fields of oncology and ophthalmology, whereas therapeutic angiogenesis, governed by angiogenesis stimulators, is about to enter clinical medicine. OBJECTIVE: To describe and critically evaluate the advantages and limitations of the most important and most frequently used preclinical in vivo angiogenesis assays as well as to appraise the preclinical models that are most widely used for studying antiangiogenic effects in tumors. METHODS: Up-to-date literature survey. RESULTS/CONCLUSION: Only few angiogenesis and tumor models appear to meet realistic standards fully in terms of biological relevance. Improvement of the biological pertinence and sensitivity of such models would apparently facilitate the translatability of preclinical data into clinical practice.

Low-molecular-weight heparins and angiogenesis.

The involvement of the vascular system in malignancy encompasses not only angiogenesis, but also systemic hypercoagulability and a pro-thrombotic state, and there is increasing evidence that pathways of blood coagulation and angiogenesis are reciprocally linked. In fact, cancer atients often display hypercoagulability resulting in markedly increased thromboembolism, which requires anti-coagulant treatment using heparins, for example. Clinical trials reveal that treatment with various low-molecular-weight heparins (LMWHs) improves the survival time in cancer patients receiving chemotherapy compared with those receiving unfractionated standard heparin (UFH) or no heparin treatment, as well as in cancer patients receiving LMWH as thrombosis prophylaxis during primary surgery. This anti-tumor effect of the heparins appears to be unrelated to their anti-coagulant activity, but the mechanisms involved are not fully understood. Tumor growth and spread are dependent on angiogenesis and it is noteworthy that the most potent endogenous pro- and anti-angiogenic factors are heparin-binding proteins that may be affected by systemic treatment with heparins. Heparin and other glycosaminoglycans play a role in vascular endothelial cell function, as they are able to modulate the activities of angiogenic growth factors by facilitating the interaction with their receptor and promoting receptor activation. To date, preclinical studies have demonstrated that only LMWH fragments produced by the heparinase digestion of UFH, i.e. tinzaparin, exert anti-angiogenic effects in any type of tissue in vivo. These effects are fragment-mass-specific and angiogenesis-type-specific. Data on the effect of various LMWHs and UFH on endothelial cell capillary tube formation and proliferation in vitro are also presented. We hope that this paper will stimulate and facilitate future research designed to elucidate whether the anti-angiogenic or anti-tumor effects of commercial LMWHs in their own right are agent specific and whether anti-angiogenic properties increase the anti-tumor properties of the LMWHs in the clinic.

On metronomic chemotherapy: modulation of angiogenesis mediated by VEGE-A.

Tumors are angiogenesis dependent. Preclinical studies have shown that well-tolerated continuous low dose, i.e. metronomic, chemotherapy can exert significant antiangiogenic effects per se and thereby a greater antitumor influence than conventional chemotherapy with high, spaced-out bolus doses. There are however, no means of quantitatively assessing the antiangiogenic effect of chemotherapy in tumors. We therefore used a surrogate tumor-free, non-surgical rat mesentery model and quantitatively studied the dose effect of metronomic treatment with cisplatin, cyclophosphamide, doxorubicin, fluorouracil and paclitaxel on VEGF-A-mediated angiogenesis, a characteristic of tumors. Cyclophosphamide and paclitaxel treatment exerted significant dose-dependent antiangiogenic effects, whereas doxorubicin treatment produced insignificant effects. By contrast, metronomic cisplatin and fluorouracil treatment occasionally significantly stimulated angiogenesis in a dose-dependent, non-linear manner. To our knowledge, this is the first report of metronomic chemotherapy stimulating angiogenesis in vivo. The data suggest that the angiogenic response to cisplatin, cyclophosphamide, fluorouracil and paclitaxel was significantly influenced by the presence of antioxidants in the vehicles or when co-treated with N-acetylcystein, a widely used free-radical scavenger. The data relating to the metronomic scheduling were compared with bolus treatment data for the identical agent formulations in the same experimental model. Cisplatin, cyclophosphamide and paclitaxel caused approximately the same overall, agent-specific angiogenesis-modulating effects following metronomic and bolus treatments. Moreover, apparently secondary delayed effects of chemotherapy affected capillary sprouting.

The anti-tumour effect of low-dose continuous chemotherapy may partly be mediated by thrombospondin.

BACKGROUND: Tumour growth is dependent on angiogenesis. Antiangiogenic chemotherapy, i.e. continuous or metronomic low-dose chemotherapy, is a method for administrating cytostatics at a low and well-tolerated concentration without prolonged breaks. The target is the genetically stable endothelial cells playing a pivotal role in angiogenesis within the tumour. Different mediators could mediate the antiangiogenic effect of metronomic chemotherapy. One of these mediators could be thrombospondin (TSP). TSP is a potent inhibitor of angiogenesis and might therefore be important in controlling tumour growth. This study was designed to evaluate the effects of low-dose continuous or moderate-dose bolus chemotherapy on tumour growth and on tumour expression of TSP. MATERIALS AND METHODS: Rats bearing a malignant prostate tumour (Dunning AT-1) not expressing TSP were treated systemically with cyclophosphamide, doxorubicin or paclitaxel and the combination of cyclophosphamide and doxorubicin. Tumour growth and body weight were measured during the treatment. CD36, one of TSP's main receptors, was also analysed. The expression pattern of TSP-1, TSP-2 and CD36 was investigated using immunohistochemistry and Western blot analyses. Q-PCR was used to analyse TSP-1 mRNA expression. RESULTS: Low-dose cyclophosphamide and paclitaxel re-induced the expression of TSP in the tumours. However, following a bolus dose of doxorubicin, tumours showed no expression of TSP. Both cyclophosphamide and doxorubicin treatments decreased the tumour weight by more than 60% compared with vehicle controls. When cyclophosphamide and doxorubicin were combined the tumour weight was reduced by 47%, while paclitaxel reduced the tumour weight by 18% compared to the vehicle controls. CONCLUSIONS: Systemic low-dose continuous treatment of a rat prostate cancer model with cyclophosphamide and paclitaxel induced the expression of TSP in tumour tissue and inhibited tumour growth. These findings support the hypothesis that the anti-tumour effect of low-dose metronomic chemotherapy, at least with certain chemotherapeutics, is partly mediated by induction of endogenous antiangiogenic factors.

Is tumor vascularity in prostate core biopsies a predictor of PSA recurrence after radical prostatectomy?

The purposes of this study were to evaluate if tumour vascularity by Chalkley counting (TVC) in prostate core biopsies can be a predictor of PSA recurrence after radical prostatectomy in prostate cancer and to estimate the concordance between the TVC in core biopsies and the subsequently examined prostatectomy specimen. All patients, with Gleason score < or =7 in core biopsy, clinical stage T1 or T2 who had a radical prostatectomy during 1990-1997 at Sahlgrenska University Hospital, were selected as a primary group. Patients with neoadjuvant hormonal therapy were excluded. The patients were divided into two groups, one with PSA recurrence and one group without PSA recurrence. 25 patients had PSA recurrence during the follow up period and 25 patients from non-recurrence group were randomly selected. TVC was assessed from the prostate tissue by immunostaining against CD34. TVC was statistically significant predictor of PSA relapse. The PSA-free survival rate was only 17% in patients within the highest TVC quartile compared to 67% in patients within the lowest TVC quartile.

Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo.

BACKGROUND: Lactoferrin, LF, a multifunctional iron- and heparin-binding protein, present in exocrine body secretions and leukocytes, is remarkably resistant to proteolysis. Ingested bovine iron-unsaturated LF, apo-bLF, suppresses VEGF-A-mediated angiogenesis in a previously described rat mesentery angiogenesis assay, possibly explaining, at least in part, its established anticancer effect in rats and mice. METHODS: Using the same experimental system, we have now studied the effect of (i) ingested human apo-LF, apo-hLF, on angiogenesis mediated by VEGF-A and bFGF, (ii) ingested human iron-saturated LF, holo-hLF, on VEGF-A-mediated angiogenesis and (iii) subcutaneous continuously infused apo-hLF on VEGF-A-mediated angiogenesis. RESULTS: Ingested holo-hLF did not affect VEGF-A-mediated angiogenesis. Ingested apo-hLF (from one and the same batch) significantly enhanced VEGF-A-mediated angiogenesis but did not affect bFGF-mediated angiogenesis. Moreover, subcutaneously infused apo-hLF also significantly stimulated VEGF-A-mediated angiogenesis. CONCLUSION: Taken together, the data suggest that apo-hLF exerts a specific proangiogenic effect in VEGF-A-mediated angiogenesis. Clearly, human and bovine apo-LF exert opposite effects on VEGF-A-induced angiogenesis. Differences in molecular features between human and bovine LFs of possible significance for the outcome are discussed. In hypoxia, compensatory collateral circulation is mediated primarily by VEGF-A. We hypothesize that systemically administered apo-hLF may promote collateral blood vessel formation at hypoxic sites in normal tissue, thus counteracting ischemia and infarction.

Antiangiogenic effect of metronomic paclitaxel treatment in prostate cancer and non-tumor tissue in the same animals: a quantitative study.

Well-tolerated continuous or metronomic chemotherapy can exert a marked antiangiogenic and thus superior antitumor effect compared with conventional high-dose, temporarily spaced-out chemotherapy, as shown in preclinical studies. There is, however, no means of directly assessing the antiangiogenic effect in a tumor, a serious impediment to assessing the effects of putative antiangiogenic chemotherapeutics or treatments. In an attempt to circumvent or minimize this impediment we studied the antiangiogenic effect of well-tolerated metronomic paclitaxel therapy in a surrogate tumor-free tissue that allows true quantitative analysis as well as in syngeneic At-1 prostate cancer in the same rat. This novel model allows an accurate comparison of the angiogenesis-modulating effect of chemotherapy in the two tissues to be made. The effect of chemotherapy on VEGF-A-mediated angiogenesis, a characteristic of most tumors, was assessed truly quantitatively by microscopic morphometry and image analysis in the tumor-free mesentery. The chemotherapy significantly suppressed VEGF-A-mediated angiogenesis in the mesentery to an extent that closely mirrored the significant increase in tumor necrosis measured morphometrically and the significant decrease in tumor growth rate. This finding opens an avenue to study quantitatively and systematically the antiangiogenic effect of chemotherapeutic modalities and treatments that approximately mirror their antiangiogenic effect in the At-1 tumor.

Testicular lymphoma--a retrospective, population-based, clinical and immunohistochemical study.

From a population-based registry, 35 patients with histologically verified testicular lymphomas were identified: diffuse large B-cell lymphomas (DLBCL) in 33 and peripheral T-cell lymphomas in two cases. Twenty-two patients had localized disease (Pe stage I and II). Twenty-eight patients received systemic chemotherapy, 17 of whom also received intrathecal prophylaxis, and 12 out of these 17 also received radiotherapy to the contralateral testis. In the Pe stage I/II group, 7 out of 21 patients in complete remission (CR) relapsed. In 5 of them the CNS was involved (isolated CNS relapse in three). Remarkably late relapses occurred (up to 127 months). Intrathecal prophylaxis seemed to reduce the frequency of relapses involving the CNS, but the relatively short follow-up (median 45 months, range 34-88, for censored patients) prevents firm conclusions regarding efficacy. The outcome for the stage IV patients was poor, with only 1 out of 11 patients in continuous CR. Immunohistochemical analysis of the DLBCL tumours revealed that 31% had the germinal centre B-cell-like phenotype. CD44 was expressed in all the tumours of stage IV patients but in less than half of the Pe stage I/II patients. A high intratumoural microvessel density was correlated with a high degree of Ki-67 positive tumour cells and an inferior overall survival.

Chemotherapy and antiangiogenesis: drug-specific effects on microvessel sprouting.

Tumors are angiogenesis dependent. Some chemotherapeutics have been shown to be able to suppress angiogenesis and thus tumor growth in vivo at low, well-tolerated doses. Not much is known about the angiogenesis-modulating effects of chemotherapeutics in vivo, however. Microvessel sprouting is inherent to angiogenesis. Using the rat mesentery assay, we studied the effect of cyclophosphamide, doxorubicin and paclitaxel at a low, atoxic dose on the number of sprouts per unit tissue volume (No. SP) and their length (Le. SP) at the edge of the expanding network in VEGF165-mediated angiogenesis. A single dose of each cytotoxic drug was administered i.v. 7 days before the animals were sacrificed. Cyclophosphamide significantly lengthened the shortest Le. SP and shortened the longest Le. SP, doxorubicin did not significantly affect Le. SP, whereas paclitaxel significantly shortened both the shortest and the longest Le. SP. No correlation was found between the present results and the distinctly drug-specific results of microvessel segment number and length analyzed within central parts of the same expanding network. To our knowledge, this is the first quantitative report on the effect of chemotherapy on angiogenesis sprouting in vivo. Collectively, the data suggest that cyclophosphamide, doxorubicin and paclitaxel at a non-toxic dose primarily target different intrinsic components of the angiogenic cascade, leading to distinctly drug-specific effects.

Chemotherapy and antiangiogenesis--drug-specific, dose-related effects.

Dose-response effects of fluorouracil, paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide and etoposide on VEGF165/164-mediated angiogenesis using the rat mesenteric-window angiogenesis assay are reported. VEGF is a pivotal pro-angiogenic factor in most tumors. Microvessel spatial extension, density, pattern formation and segment length were assessed quantitatively and objectively. A single i.v. injection of each drug was given at a low, intermediate or high dose, 7 days before sacrifice. All the drugs elicited significant responses in terms of one or more measured variables. Only paclitaxel, doxorubicin and cyclophosphamide significantly suppressed the overall angiogenic response (p < or = 0.0001, p < or = 0.0002 and p < or = 0.05, respectively), however. Taking toxicity into account, paclitaxel was more potent in inhibition of angiogenesis than the other agents. No clear correlation was found between drug half-life, the degree of toxic effects (in terms ofbody weight changes) and the antiangiogenic effect. The antiangiogenic effects were distinctly drug specific.

Tumour-type-specific capillary endothelial cell stainability in malignant B-cell lymphomas using antibodies against CD31, CD34 and Factor VIII.

The microvessel density (MVD) was assessed in lymph nodes infiltrated by diffuse large B-cell lymphomas, mantle cell lymphomas, chronic lymphatic leukemia and follicular lymphomas, and in lymphadenitis. Serial sections of formalin-fixed and paraffin-embedded tissue were stained with antibodies against CD31, CD34 or Factor VIII. Using light microscopy and computerised image analysis, the number and size of individual immunostained vessel profiles within a preselected area size range corresponding to capillaries, postcapillary venules, small collecting venules and small arterioles were determined. A significantly larger number of vessels were registered following staining with anti-CD34 than with anti-CD31 or anti-Factor VIII. Moreover, among the smallest capillary-sized vessel profiles in all lesion types, there was a selective relative loss of stainability of anti-CD31 and anti-Factor VIII, resulting in a substantial total loss of visualised capillary-sized vessels compared with anti-CD34. In fact, the number of non-detected capillaries following staining with anti-CD31 and anti-Factor VIII was significantly tumour type specific. These findings influence how we evaluate MVD data in B-cell lymphomas and possibly also other tumour types, as well as data relating to capillary endothelium-related functional variables of proliferation, apoptosis and maturation when different double-labelling immunohistochemical techniques are used and different tumour types are analysed.

Mast cells and angiogenesis.

Angiogenesis is tightly regulated by pro- and anti-angiogenic factors. Secreting mast cells are able to induce and enhance angiogenesis via multiple in part interacting pathways. They include mast cell-derived (i) potent pro-angiogenic factors such as VEGF, bFGF, TGF-beta, TNF-alpha and IL-8, (ii) proteinases and heparin, that release heparin-binding pro-angiogenic factors lodged on cell surfaces and in the extracellular matrix (ECM), (iii) histamine, VEGF, and certain lipid-derived mediators that induce microvascular hyperpermeability having pro-angiogenic effects, (iv) chemotactic recruitment of monocytes/macrophages and lymphocytes that are able to contribute with angiogenesis-modulating molecules, (v) activation of platelets that release pro-angiogenic factors, (vi) activation of neighboring stationary non-mast cells, which secrete pro-angiogenic factors, ECM-degrading proteinases and stem cell factor which attracts, mitogenically stimulates and activates mast cells, (vii) auto- and paracrine stimulation of mast cells by stem cell factor, (viii) recruitment of mast cells by pro-angiogenic factors such as VEGF, bFGF and TGF-beta. As a result of ECM-degradation and changes in the microenvironment following initial mast cell secretion, the mast cell populations may change significantly in number, phenotype and function. In tumor models, mast cells have been shown to play a decisive role in inducing the angiogenic switch which precedes malignant transformation. There is, moreover, strong evidence that mast cells significantly influence angiogenesis and thus growth and progression in human cancers.