Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1104682 on May 13, 2005

Published online before print May 13, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104682v1
78/2/352    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barcelos, L. S.
Right arrow Articles by Teixeira, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barcelos, L. S.
Right arrow Articles by Teixeira, M. M.
(Journal of Leukocyte Biology. 2005;78:352-358.)
© 2005 by Society for Leukocyte Biology

Impaired inflammatory angiogenesis, but not leukocyte influx, in mice lacking TNFR1

L. S. Barcelos*, A. Talvani*,1, A. S. Teixeira{dagger}, L. Q. Vieira*, G. D. Cassali{ddagger}, S. P. Andrade{dagger} and M. M. Teixeira*,2

* Departments of Bioquímica e Imunologia,
{dagger} Fisiologia e Biofísica, and
{ddagger} Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

2 Correspondence: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627-Pampulha, 31270-901 Belo Horizonte, MG, Brazil. E-mail: mmtex{at}icb.ufmg.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The majority of biological responses classically attributed to tumor necrosis factor {alpha} (TNF-{alpha}) is mediated by p55 receptor (TNFR1). Here, we aimed to clarify the biological role of TNFR1-mediated signals in an in vivo inflammatory angiogenesis model. Polyester-polyurethane sponges, used as a framework for tissue growth, were implanted in C57Bl/6 mice. These implants were collected at days 1, 7, and 14 post-implant for enzyme-linked immunosorbent assay or at days 7 and 14 for hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, used as indexes for angiogenesis, neutrophil, and macrophage accumulation, respectively. In TNFR1-deficient C57Bl/6 mice, there was a significant decrease in sponge vascularization but not in late inflammatory cell influx. It is interesting that levels of vascular endothelial growth factor were significantly lower in TNFR1-deficient than in wild-type mice at days 1 and 7. Levels of angiogenic chemokines, CC chemokine ligand 2/murine homologue of monocyte chemoattractant protein-1 and CXC chemokine ligand 1–3/keratinocyte-derived chemokine, were significantly lower in TNFR1-deficient mice at days 1 and 7 after implantation, respectively. These observations suggest that TNFR1-mediated signals have a critical role in sponge-induced angiogenesis, possibly by influencing the effector state of inflammatory cells and hence, modulating the angiogenic molecular network.

Key Words: TNF-{alpha} • neutrophil • macrophage • p55 receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor {alpha} (TNF-{alpha}) is a pleiotropic cytokine produced by a variety of cell types, including macrophages, neutrophils, lymphocytes, and endothelial cells [1 ]. TNF signals through two distinct cell surface receptors, TNFR1 (TNFRp55–60) and TNFR2 (TNFRp75–80), and the specific intracellular signaling pathways that are activated by TNF determine the specificity of cellular responses in a given cell [2 ]. It has been shown that TNFR1 initiates the majority of the biological activities of TNF-{alpha}, including cell growth and death, oncogenesis, and inflammatory responses [3 ].

TNF-{alpha} has been shown to inhibit growth and induce endothelial cell apoptosis in vitro [4 5 6 ]. However, this cytokine may also induce endothelial cell migration in vitro and angiogenesis in vivo when implanted into corneas, chorioallantoic membranes, or sponge implants [7 8 9 10 ]. These apparent, contrasting effects are likely secondary to the ability of TNF-{alpha} to release proangiogenic and antiangiogenic factors, depending on the local concentration or duration of exposure to TNF-{alpha} [11 , 12 ]. Indeed, TNF-{alpha} may control angiogenesis by regulating the synthesis of secondary mediators such as platelet-activating factor, interleukin-8, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor [13 , 14 ].

Despite the good amount of information concerning TNFR1-mediated inflammatory activities, the involvement of this receptor in angiogenesis is poorly understood. To address the biological role of TNFR1-mediated signals in the in vivo angiogenesis occurring in sites of inflammation, we quantified angiogenesis, inflammation, and mediators of the angiogenic and inflammatory responses after implantation of polyester-polyurethane sponges, used as a framework for tissue growth, in mice genetically engineered to lack the receptor 1 (or p55 receptor) for TNF-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight- to 10-week-old female C57Bl/6J [wild-type (WT)] or TNFR1-deficient mice (TNFR1–/–) were obtained from The Jackson Laboratories (Bar Harbor, ME) and bred and maintained in the animal house of the Department of Biochemistry and Immunology [Universidade Federal de Minas Gerais (UFMG), Brazil]. After sponge implantation, animals were maintained in individual cages with food/water ad libitum and in a controlled environment (temperature and humidity) in the Laboratory of Angiogenesis at the Department of Physiology and Biophysics, UFMG.

Preparation of cannulated sponge discs and implantation
Polyester-polyurethane sponge discs, 5 mm thick and 8 mm diameter (Vitafoam Ltd., Manchester, UK), were used as the matrix for fibrovascular tissue growth. Polyvinyl tubing (12 mm; PE 20, Biovida, Brazil) was secured with silk sutures (Ethicon Ltd., UK) to the center of each disc in such a way that the tube was perpendicular to the disc face. Its open-end cannula were sealed with removable plugs. The cannulated sponge discs were sterilized by soaking overnight in 70% v/v ethanol and by boiling in distilled water for 15 min before the implantation surgery. The animals were anesthetized (tribromoethanol, Sigma Chemical Co., St. Louis, MO, 2.5% v/v, 1 mL/100 g body weight, intraperitoneally), the dorsal hair shaved, and the skin wiped with 70% ethanol. The cannulated sponge discs were aseptically implanted into a subcutaneous (s.c.) pouch that had been made with curved artery forceps through a 1-cm long dorsal midline incision. The cannula was exteriorized through a small incision in a s.c. neck pouch.

Quantification of angiogenesis by hemoglobin measurement
Animals were anesthetized by ether inhalation and killed by cervical dislocation, and the sponge implants were excised carefully, released from the cannula, and weighed. Each implant was homogenized (TR-10, Tekmar, Mason, OH) in 2.0 mL Drabkin reagent (Labtest, São Paulo, Brazil) and centrifuged at 10,000 g for 15 min. The supernatants were filtered through a 0.22-µm filter (Millipore, Bedford, MA). Hemoglobin in the samples was quantified colorimetrically at 540 nm in a spectrophotometer (Emax, Molecular Devices, Sunnyvale, CA). The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel. The results were expressed as µg Hb/mL/mg wet tissue. Previous studies have shown that hemoglobin detection correlated well with other methods for the detection and quantification of angiogenesis in tissues [15 , 16 ].

Quantification of neutrophil tissue accumulation by myeloperoxidase (MPO) activity measurement
Pellets obtained from the centrifugation of sponge homogenates (see above) were suspended in 2.0 mL cooled (4°C) phosphate buffer (0.1 M NaCl, 0.02 M NaPO4, 0.015 M NaEDTA, pH 4.7), vortex-homogenized, and centrifuged at 4°C for 15 min at 10,000 g. Pellets were then resuspended in 2.0 mL (at room temperature) phosphate buffer (0.05 M Na3PO4, 0.5% hexadecyl-trimethylammonium bromide (HETAB), pH 5.4). The suspensions were freeze-thawed three times and centrifuged at 4°C for 15 min at 10,000 g, and supernatants were stored at –20°C until used for MPO assay.

MPO assay reaction
The assay used 25 µL 3,3'-5,5'-tetramethylbenzidine (TMB; Sigma Chemical Co.), dissolved in dimethyl sulfoxide (Merck, Rahway, NJ) in a final concentration of 1.6 mM, 100 µL H2O2, dissolved in phosphate buffer (0.05 M Na3PO4, 0.5% HETAB, pH 5.4) in a final concentration of 0.003% v/v and 25 µL supernatant from tissue sample processing. The reaction was started at 37°C for 5 min in a 96-well microplate by adding the supernatant and the TMB solution. After that, H2O2 was added, and the mixture incubated at 37°C for 5 min. The reaction was stopped by adding 100 µL 4 M H2SO4 and was quantified at 450 nm in a spectrophotometer (Emax, Molecular Devices). The neutrophil content was calculated from a standard curve based on MPO activity expressed as absorbance increase at 450 nm from 5% casein peritoneal-induced neutrophils assayed in parallel. We used casein-elicited neutrophils from WT or TNFR1-deficient mice to construct the standard curves for the relevant strain. The results were expressed in relative number of neutrophils (x104) per mg wet tissue. Under the conditions described here, the MPO assay failed to detect macrophages obtained from the peritoneal cavity of thioglycollate-treated mice and processed the same way (data not shown).

Quantification of macrophage tissue accumulation by N-acetylglucosaminidase (NAG) activity measurement
Pellets obtained from the centrifugation of sponge homogenates were suspended in 2.0 mL cooled (4°C) 0.9% saline containing 0.1% v/v Triton X-100, vortex-homogenized, and centrifuged at 4°C for 10 min at 1500 g. The supernatants were saved and used for NAG assay. If the NAG assay was not carried out immediately, the supernatants were kept frozen until used.

NAG assay reaction
The reaction was started at 37°C for 10 min in a 96-well microplate by the addition of 100 µL p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma Chemical Co.), dissolved in citrate/phosphate buffer (0.1 M citric acid, 0.1 M Na2HPO4, pH 4.5) in a final concentration of 2.24 mM–100 µL supernatant from tissue sample processing, dissolved in citrate/phosphate buffer at appropriate dilutions. The reaction was terminated by the addition of 100 µL 0.2 M glycine buffer (pH 10.6) and was quantified at 405 nm in a spectrophotometer (Emax, Molecular Devices). The macrophage content was calculated from a standard curve based on NAG activity expressed as absorbance increase at 405 nm from 3% thioglycollate peritoneal-induced macrophages assayed in parallel. We used thioglycollate-elicited neutrophils from WT or TNFR1-deficient mice to construct the standard curves for the relevant strain. The results were expressed in relative number of macrophages (x104) per mg wet tissue. Under the conditions described here, the NAG assay failed to detect neutrophils obtained from the peritoneal cavity of casein-treated mice and processed the same way (data not shown).

Enzyme-linked immunosorbent assay (ELISA) for cytokines/chemokines
Animals were anesthetized by ether inhalation and killed by cervical dislocation, and the sponge implants were collected carefully, released from the cannula, and squeezed into microtubes to collect the inflammatory fluid. The squeezed sponge was homogenized in 1.0 mL 1x phosphate-buffered saline (PBS; pH 7.4) containing 0.05% Tween 20 (Difco, Detroit, MI), followed by centrifugation at 4°C for 10 min at 10,000 g. The supernatant was combined with the inflammatory fluid, and ELISA assays were performed using kits from R&D Systems (Minneapolis, MN) for murine VEGF, TNF-{alpha}, CXC chemokine ligand 1–3 (CXCL1–3)/keratinocyte-derived chemokine (KC) and CC chemokine ligand 2 (CCL2)/murine homologue of monocyte chemoattractant protein-1 (JE). Briefly, flat-bottom, 96-well microtiter plates (Nunc, Denmark) were coated with 100 µL/well of the appropriate monoclonal antibodies (mAb) for 18 h at 4°C and then washed with PBS (pH 7.4) containing 0.05% Tween 20 (wash buffer). Nonspecific binding sites were blocked with 300 µL/well 1% bovine serum albumin (BSA) in PBS (blocking buffer). Plates were rinsed with wash buffer, and appropriately diluted samples (in 0.1% BSA in PBS-reagent diluent) were added (100 µL/well), followed by incubation for 18 h at 4°C. Plates were then washed, and 100 µL/well of the appropriate biotinylated detection antibodies diluted in reagent diluent were added for 2 h at room temperature. Plates were then washed, streptavidin-horseadish peroxidase was added (100 µL/well), and the plates were incubated for 20 min at room temperature. Plates were then washed, 100 µL/well of the chromogen substrate o-phenylendiamine (Sigma Chemical Co.) diluted in 0.03 M citrate buffer (pH 5.0), containing 0.02% 30 v/v H2O2, was added, and the plates were incubated in the dark for 30 min at room temperature. The reaction was terminated with 50 µL/well 1 M H2SO4 solution. Plates were read at 492 nm in a spectrophotometer (Emax, Molecular Devices). All samples were assayed in duplicate. The threshold of sensitivity for each cytokine/chemokine was 16 pg/mL.

Histology
At day 14 after implantation, animals were anesthetized by ether inhalation and killed by cervical dislocation. The sponge implants were then collected carefully, released from the cannula, and fixed in formalin (10% in isotonic saline). The implants were embedded in paraffin, and 5 µm-thick sections were obtained. The sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope.

Statistical analysis
Results are presented as the mean ± SEM. Comparisons between two groups were carried out using Student’s t-test for unpaired data. Three or more group comparisons were carried out using one-way ANOVA, and differences between groups were assessed by Student-Newman-Keuls multiple comparisons test. A P value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of inflammatory angiogenesis in sponge implants of C57Bl/6J mice
At fixed times after sponge implantation, neovascularization was measured by evaluating the amount of hemoglobin concentration in the vascularized granulation tissue within the sponge. Similar to our previous work with BALB/c mice [17 ], the hemoglobin content in the C57Bl/6J mice sponges increased progressively after sponge implantation, reaching higher levels on day 14, from which it was kept stable until day 21 (Fig. 1A ).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Time course of vascularization and inflammatory cell influx in C57Bl/6J mice sponge implants. (A) Angiogenesis in implants was measured by hemoglobin concentration. **, P < 0.01, versus day 1; ***, P < 0.001, versus days 1 and 7. (B) Neutrophil and (C) macrophage accumulation in implants was measured by indirect methods based on MPO and NAG activities, respectively. Results represent the relative number of neutrophils (x104) or macrophages (x104) per mg wet tissue and are the mean ± SEM of six to eight animals for each time-point until day 21 after sponge implantation. *, P < 0.05, versus day 1; **, P < 0.01, versus day 1; ***, P < 0.001, versus day 1; #, P < 0.05, versus day 14; ##, P < 0.01, versus day 7.

 
By using MPO- and NAG-based assays, we evaluated the time-course of neutrophil and macrophage accumulation in the sponges, respectively. There was a progressive increase in neutrophil accumulation into sponges, which was prominent on the 7th day after sponge implantation (Fig. 1B) . Similarly, macrophage accumulation increased progressively and appeared to peak on day 14 after sponge implantation (Fig. 1C) . At day 21 after implantation, we observed a fall in macrophage content (Fig. 1C) . This correlation, observed between angiogenesis and inflammatory cell infiltration, was also evident in histopathological sections of sponge tissues (data not shown).

Neovascularization and inflammatory cell accumulation in TNFR1-deficient mice
Angiogenesis and leukocyte influx were evaluated at days 7 and 14 after sponge implantation. There was a significant decrease in sponge vascularization in TNFR1-deficient mice when compared with WT control mice at both time-points (Fig. 2A ). The sponge vascularization was restored to WT control levels in TNFR1-deficient mice at day 21 post-implantation (WT=2.59±0.26 vs. TNFR1–/–=3.76±0.55 µg/mL hemoglobin/mg sponge). It is interesting that at day 7, but not at day 14 after sponge implantation, we found a significant decrease in the tissue neutrophil and macrophage content in TNFR1-deficient mice, as assessed by the tissue MPO and NAG activities (Fig. 3 ). There was no significant difference in the weight of the sponges at all times evaluated (day 7: WT=105±4 mg vs. TNFR1–/–=94±3 mg; day 14: WT=126±6 mg vs. TNFR1–/–=114±5 mg; day 21: WT=104±4 mg vs. TNFR1–/–=93±8 mg sponge; n=6 in each group).



View larger version (61K):
[in this window]
[in a new window]
  		Figure 2. TNFR1-deficient mice exhibit decreased angiogenic response. (A) Angiogenesis in implants was measured by hemoglobin concentration and was evaluated at days 7 and 14 after sponge implantation. Results represent the mean ± SEM of six to eight animals for each group. **, P < 0.01, and ****, P < 0.0001, when compared with C57Bl/6J WT sponges. The H&E-stained histological sections from (B) WT control animal show the pattern of the fibrovascular tissue infiltration at day 14 post-implantation. Angiogenesis in TNFR1–/– sponges (C) is decreased when compared with WT control implants. Symbols indicate blood vessels (BV) and sponge matrix (SM). Photos are x66 from original magnification. KO, Knockout..

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Neutrophil and macrophage accumulation in sponges of WT and TNFR1-deficient mice. (A) Neutrophil and (B) macrophage influx was evaluated at days 7 and 14 after sponge implantation. The recruitment was evaluated by measuring MPO and NAG activities, respectively. Results represent the relative number of neutrophils (x104) or macrophages (x104) per mg wet tissue and are the mean ± SEM of six to eight animals for each group. **, P < 0.01, when compared with C57Bl/6J WT sponges. KO, Knockout.

 
In agreement with the above findings, histological examination of 14-day sponge sections showed the presence of an extensive neutrophil infiltrate in both groups. There was greater growth of new blood vessels in the WT control group (Fig. 2B) as compared with TNFR1-deficient mice (Fig. 2C) . Indeed, in the latter group, there were relatively few newly formed vessels. The surrounding granulation tissue was apparently more cellular and with more collagen deposition in TNFR1-deficient than in WT mice.

VEGF, TNF-{alpha}, CXCL1–3/KC and CCL2/JE protein levels in WT and TNFR1-deficient mice
The production of cytokines and chemokines within sponge tissue drives the angiogenic and inflammatory processes. Thus, we measured the concentrations of these mediators on days 1, 7, and 14 after implantation. VEGF was already detectable on day 1 after sponge implantation and peaked on day 7, and levels diminished significantly at day 14 (Fig. 4A ). In TNFR1-deficient mice, VEGF levels were markedly lower on days 1 and 7 but were similar to those of WT mice on day 14 (Fig. 4A) . Levels of TNF-{alpha} were detectable on day 1, peaked at day 7, and maintained at high levels thereafter (Fig. 4B) . In TNFR1-deficient mice, the concentration of this cytokine was lower on days 1 and 7 but increased to levels similar to those found in WT mice on day 14 (Fig. 4B) .



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Altered cytokine and chemokine profiles in sponges of TNFR1–/– mice. ELISA was carried out with specific mAb for (A) VEGF, (B) TNF-{alpha}, (C) CXCL1–3/KC, and (D) CCL2/JE. Results are expressed as the mean ± SEM of six to eight animals for each group and for each time-point until day 14 after sponge implantation. **, P < 0.01; ****, P < 0.0001, when compared with respective WT control sponges. KO, Knockout.

 
Chemokines may affect angiogenesis by directly activating endothelial cells or by inducing leukocyte infiltration. The chemokines CXCL1–3/KC and CCL2/JE were detectable throughout the observation period in sponge tissues implanted into the s.c. tissue of WT mice (Fig. 4C and 4D) . CXCL1–3/KC was detectable on day 1 after sponge implantation and peaked on day 7. Thereafter, there was a drop in CXCL1–3/KC production (Fig. 4C) . In gene-deficient mice, CXCL1–3/KC was produced at low levels on day 7 but detectable at levels similar to those of WT mice at day 14 (Fig. 4C) . CCL2/JE peaked on day 1 after implantation, and levels dropped rapidly but remained above the detection limit of the assay. In the gene-deficient mice, the early production of CCL2/JE was lost, and low levels of the protein were detected throughout the observation period (Fig. 4D) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TNF-{alpha} is a relevant angiogenic candidate in sponge-induced angiogenesis, as it elicits a broad spectrum of cellular responses including activation of leukocytes and endothelial cells [10 , 18 19 20 21 ]. Moreover, TNF-{alpha} is a potent modulator of the expression of inflammatory and proangiogenic molecules [13 , 22 23 24 25 26 27 ]. Nevertheless, little is known about receptor-mediated signals that are involved in the biological activity of TNF during inflammatory angiogenesis and especially, in the model of sponge-induced angiogenesis. Once TNFR1 signals are important for the actions of TNF-{alpha} on endothelial cells, we hypothesized that angiogenesis development would be reduced in sponges from mice lacking TNFR1 function. Indeed, we now demonstrate a significant decrease in sponge vascularization in these mice, which was accompanied by strongly reduced levels of the angiogenic molecule VEGF. Vascularization was assessed by evaluating the hemoglobin content, and this method bears a good correlation with other quantitative techniques to evaluate angiogenesis in sponges [15 , 16 ]. There was also a significant inhibition of TNF-{alpha} production in TNFR1-deficient mice, suggesting that TNF production is auto-regulated by TNFR1 activation. Moreover, as TNF-{alpha} levels are decreased in our gene-deficient mice, it is likely that any activation of the TNFR2 would also be diminished. These observations demonstrate a critical role for TNFR1-mediated signals in promoting angiogenesis and suggest that production and action of VEGF and TNF-{alpha} at the site of sponge implantation may be relevant for the effects observed. It is interesting that angiogenesis was restored 21 days after sponge implantation in TNFR1-deficient mice, raising the possibility that the late (day 14) increase in TNF-{alpha} and action on TNFR2 levels could be compensating for the absence of TNFR1. This possibility clearly deserves further investigation.

Activated macrophages appear to be major cellular protagonists capable of promoting angiogenesis by virtue of their secretory products [23 , 28 , 29 ]. Neutrophils can also synthesize angiogenic molecules, suggesting that these cells are directly angiogenic [30 31 32 33 ]. In the present model, which tries to mimic chronic inflammatory and wound-healing processes, a cascade of inflammatory events is triggered after implantation and culminates in new blood vessel formation [34 , 35 ]. Here, similar to our previous study [17 ], we show that angiogenesis development was progressive and parallel with the inflammatory cell accumulation in C57Bl/6J mice implants.

Angiogenesis was prevented in TNFR1-deficient mice, and there was also inhibition of the accumulation of neutrophils or macrophages in sponges at day 7 but no difference at day 14 after implantation. These results are in contrast with those of Mori and colleagues [36 ], who showed an enhancement in angiogenesis and continuous reduction in leukocyte infiltration in a model of excisional skin wounds in TNFR1-deficient mice. Although there were differences in background of strains (C57Bl/6 vs. BALB/c) and gender (female vs. male), skin wounds and sponge models are models of wound-healing and inflammatory angiogenesis. There are differences, however, in the nature and in the duration of stimulus and in the blood supply to these tissues. The implanted sponge is an artificial matrix, which is initially nonvascularized and noncellularized (i.e., contains no biochemical activity). In contrast, the skin is a highly vascular organ, and skin wounds are well-supplied with resident cells that can deliver the stimuli to start the repair process. In addition, in the sponge model, the injuring stimulus (the own sponge implant) is continuous, whereas the skin model follows a characteristic time-course until complete tissue repair and new tissue generation are attained. For example, whereas Mori et al. [36 ] measured only a transient TNF-{alpha} up-regulation, TNF-{alpha} levels in the sponge implant system are sustained. Another interesting difference that could be related to the type of stimulation is a much shorter duration of leukocyte accumulation observed after skin wound when compared with the sponge model [36 ]. Altogether, the differences mentioned above could account for the different needs of TNF-{alpha} during inflammatory angiogenesis. Regardless of the explanation, it is clear that TNFR1 has relevant, proangiogenic activity in sponge implants, which is separable, at least in the later stages, from its effects on leukocyte recruitment.

The chemokine network tightly controls cell recruitment and activation at inflammatory sites [37 , 38 ]. We observed that the production of the neutrophil-active chemokine CXCL1–3/KC was significantly decreased at day 7 but not at day 14 after sponge implantation. These data are coincident with the decreased neutrophil accumulation into sponges at day 7 but not at day 14 post-implantation. Similar to the neutrophils, macrophage influx was decreased early but not at day 14 after implantation. The macrophage-active chemokine CCL2/JE was significantly decreased at day 1. Nevertheless, there was no difference of CCL2/JE levels between WT controls and TNFR1-deficient mice at later stages (days 7 and 14). It is not clear whether this initial fall in CCL2/JE would be accountable for the decrease of macrophage numbers at day 7. Preliminary experiments in our laboratory suggest that macrophage recruitment is similar in WT and CCL2/JE-deficient mice (our own unpublished results), suggesting that this chemokine is not relevant for macrophage accumulation and that other functionally redundant chemokines, such as CCL3/macrophage-inflammatory protein-1{alpha} or CCL5/regulated on activation, normal T expressed and secreted, may be produced concomitantly and compensate for the loss of CCL2/JE. In conclusion, we found a good correlation between neutrophil influx and CXCL1–3/KC production but not between macrophage influx and CCL2/JE production. In addition to their ability to promote leukocyte recruitment, chemokines may affect and regulate angiogenesis via activation of leukocytes and induction of proangiogenic molecules [13 , 25 ]. Thus, although the chemokines modulated by TNFR1 may be or not (in the case of CCL2/JE) necessary for the recruitment and accumulation of leukocytes, these chemokines may participate in the cascade of events leading to leukocyte and endothelial cell activation and release of proangiogenic molecules such as VEGF.

Overall, this report suggests that the functional activity of TNFR1 favors an angiogenic phenotype in a model of inflammatory angiogenesis. TNFR1 activation is associated with greater production of VEGF and proangiogenic chemokines, CXCL1–3/KC and CCL2/JE, but is not crucial for the recruitment of leukocytes. TNFR1 signals may be important for the activation of the recruited inflammatory cells, hence, facilitating release of proangiogenic factors and angiogenesis.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brasil) and Fundação do Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG).


   FOOTNOTES
 
1 Current address: Instituto de Ciências Biológicas e Saúde, Pontifícia Universidade Católica de Minas Gerais, Belo Horizonte, MG, Brazil. Back

Received November 22, 2004; revised April 5, 2005; accepted April 6, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baud, V., Karin, M. (2001) Signal transduction by tumor necrosis factor and its relatives Trends Cell Biol. 11,372-377[CrossRef][Medline]
  2. MacEwan, D. J. (2002) TNF receptor subtype signaling: differences and cellular consequences Cell. Signal. 14,477-492[CrossRef][Medline]
  3. Chen, G., Goeddel, D. V. (2002) TNF-R1 signaling: a beautiful pathway Science 296,1634-1635[Abstract/Free Full Text]
  4. Frater-Schroder, M., Risau, W., Hallmann, R., Gautschi, P., Bohlen, P. (1987) Tumor necrosis factor type {alpha}, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo Proc. Natl. Acad. Sci. USA 84,5277-5281[Abstract/Free Full Text]
  5. Sato, N., Nariuchi, H., Tsuruoka, N., Nishihara, T., Beitz, J. G., Calabresi, P., Frackelton, A. R., Jr (1990) Actions of TNF and IFN-{gamma} on angiogenesis in vitro J. Invest. Dermatol. 95,85S-89S[CrossRef][Medline]
  6. Clermont, F., Adam, E., Dumont, J. E., Robaye, B. (2003) Survival pathways regulating the apoptosis induced by tumour necrosis factor-{alpha} in primary cultured bovine endothelial cells Cell. Signal. 15,539-546[CrossRef][Medline]
  7. Leibovich, S. J., Polverini, P. J., Shepard, H. M., Wiseman, D. M., Shively, V., Nuseir, N. (1987) Macrophage-induced angiogenesis is mediated by tumour necrosis factor-{alpha} Nature 329,630-632[CrossRef][Medline]
  8. Mahadevan, V., Hart, I. R., Lewis, G. P. (1989) Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique Cancer Res. 49,415-419[Abstract/Free Full Text]
  9. Vanderslice, P., Munsch, C. L., Rachal, E., Erichsen, D., Sughrue, K. M., Truong, A. N., Wygant, J. N., McIntyre, B. W., Eskin, S. G., Tilton, R. G., Polverini, P. J. (1998) Angiogenesis induced by tumor necrosis factor-{alpha}; is mediated by {alpha}4 integrins Angiogenesis 2,265-275[CrossRef][Medline]
  10. Gao, B., Saba, T. M., Tsan, M. F. (2002) Role of {alpha}(v)ß(3)-integrin in TNF-{alpha}-induced endothelial cell migration Am. J. Physiol. Cell Physiol. 283,C1196-C1205[Abstract/Free Full Text]
  11. Fajardo, L. F., Kwan, H. H., Kowalski, J., Prionas, S. D., Allison, A. C. (1992) Dual role of tumor necrosis factor-{alpha} in angiogenesis Am. J. Pathol. 140,539-544[Abstract]
  12. Heba, G., Krzeminski, T., Porc, M., Grzyb, J., Ratajska, A., Dembinska-Kiec, A. (2001) The time course of tumor necrosis factor-{alpha}, inducible nitric oxide synthase and vascular endothelial growth factor expression in an experimental model of chronic myocardial infarction in rats J. Vasc. Res. 38,288-300[CrossRef][Medline]
  13. Montrucchio, G., Lupia, E., Battaglia, E., Passerini, G., Bussolino, F., Emanuelli, G., Camussi, G. (1994) Tumor necrosis factor {alpha}-induced angiogenesis depends on in situ platelet-activating factor biosynthesis J. Exp. Med. 180,377-382[Abstract/Free Full Text]
  14. Yoshida, S., Ono, M., Shono, T., Izumi, H., Ishibashi, T., Suzuki, H., Kuwano, M. (1997) Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor {alpha}-dependent angiogenesis Mol. Cell. Biol. 17,4015-4023[Abstract]
  15. Hu, D. E., Hiley, C. R., Smither, R. L., Gresham, G. A., Fan, T. P. (1995) Correlation of 133Xe clearance, blood flow and histology in the rat sponge model for angiogenesis. Further studies with angiogenic modifiers Lab. Invest. 72,601-610[Medline]
  16. Machado, R. D., Santos, R. A., Andrade, S. P. (2000) Opposing actions of angiotensins on angiogenesis Life Sci. 66,67-76[CrossRef][Medline]
  17. Barcelos, L. S., Talvani, A., Teixeira, A. S., Cassali, G. D., Andrade, S. P., Teixeira, M. M. (2004) Production and in vivo effects of chemokines CXCL1–3/KC and CCL2/JE in a model of inflammatory angiogenesis in mice Inflamm. Res. 53,576-584[CrossRef][Medline]
  18. Mason, M. J., Van Epps, D. E. (1989) In vivo neutrophil emigration in response to interleukin-1 and tumor necrosis factor-{alpha} J. Leukoc. Biol. 45,62-68[Abstract]
  19. Vallien, G., Langley, R., Jennings, S., Specian, R., Granger, D. N. (2000) Expression of endothelial cell adhesion molecules in neovascularized tissue Microcirculation 7,249-258[CrossRef][Medline]
  20. Robertson, M., Liversidge, J., Forrester, J. V., Dick, A. D. (2003) Neutralizing tumor necrosis factor-{alpha} activity suppresses activation of infiltrating macrophages in experimental autoimmune uveoretinitis Invest. Ophthalmol. Vis. Sci. 44,3034-3041[Abstract/Free Full Text]
  21. Viemann, D., Goebeler, M., Schmid, S., Klimmek, K., Sorg, C., Ludwig, S., Roth, J. (2004) Transcriptional profiling of IKK2/NF-{kappa} B- and p38 MAP kinase-dependent gene expression in TNF-{alpha}-stimulated primary human endothelial cells Blood 103,3365-3373[Abstract/Free Full Text]
  22. Camussi, G., Bussolino, F., Salvidio, G., Baglioni, C. (1987) Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor J. Exp. Med. 166,1390-1404[Abstract/Free Full Text]
  23. Koch, A. E., Polverini, P. J., Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner, V. M., Elner, S. G., Strieter, R. M. (1992) Interleukin-8 as a macrophage-derived mediator of angiogenesis Science 258,1798-1801[Abstract/Free Full Text]
  24. Mackay, F., Loetscher, H., Stueber, D., Gehr, G., Lesslauer, W. (1993) Tumor necrosis factor {alpha} (TNF-{alpha})-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55 J. Exp. Med. 177,1277-1286[Abstract/Free Full Text]
  25. Yoshida, S., Ono, M., Shono, T., Izumi, H., Ishibashi, T., Suzuki, H., Kuwano, M. (1997) Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor {alpha}-dependent angiogenesis Mol. Cell. Biol. 17,4015-4023
  26. McCourt, M., Wang, J. H., Sookhai, S., Redmond, H. P. (1999) Proinflammatory mediators stimulate neutrophil-directed angiogenesis Arch. Surg. 134,1325-1331[Abstract/Free Full Text]
  27. Yamashiro, S., Kamohara, H., Yoshimura, T. (2000) Alteration in the responsiveness to tumor necrosis factor-{alpha} is crucial for maximal expression of monocyte chemoattractant protein-1 in human neutrophils Immunology 101,97-103[CrossRef][Medline]
  28. Polverini, P. J., Cotran, P. S., Gimbrone, M. A., Jr, Unanue, E. R. (1977) Activated macrophages induce vascular proliferation Nature 269,804-806[CrossRef][Medline]
  29. Sunderkotter, C., Steinbrink, K., Goebeler, M., Bhardwaj, R., Sorg, C. (1994) Macrophages and angiogenesis J. Leukoc. Biol. 55,410-422[Abstract]
  30. Gaudry, M., Bregerie, O., Andrieu, V., El Benna, J., Pocidalo, M. A., Hakim, J. (1997) Intracellular pool of vascular endothelial growth factor in human neutrophils Blood 90,4153-4161[Abstract/Free Full Text]
  31. Scapini, P., Calzetti, F., Cassatella, M. A. (1999) On the detection of neutrophil-derived vascular endothelial growth factor (VEGF) J. Immunol. Methods 232,121-129[CrossRef][Medline]
  32. Scapini, P., Lapinet-Vera, J. A., Gasperini, S., Calzetti, F., Bazzoni, F., Cassatella, M. A. (2000) The neutrophil as a cellular source of chemokines Immunol. Rev. 177,195-203[CrossRef][Medline]
  33. Scapini, P., Morini, M., Tecchio, C., Minghelli, S., Di Carlo, E., Tanghetti, E., Albini, A., Lowell, C., Berton, G., Noonan, D. M., Cassatella, M. A. (2004) CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A J. Immunol. 172,5034-5040[Abstract/Free Full Text]
  34. Andrade, S. P., Vieira, L. B., Bakhle, Y. S., Piper, P. J. (1992) Effects of platelet activating factor (PAF) and other vasoconstrictors on a model of angiogenesis in the mouse Int. J. Exp. Pathol. 73,503-513[Medline]
  35. Andrade, S. P., Machado, R. D., Teixeira, A. S., Belo, A. V., Tarso, A. M., Beraldo, W. T. (1997) Sponge-induced angiogenesis in mice and the pharmacological reactivity of the neovasculature quantitated by a fluorimetric method Microvasc. Res. 54,253-261[CrossRef][Medline]
  36. Mori, R., Kondo, T., Ohshima, T., Ishida, Y., Mukaida, N. (2002) Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration FASEB J. 16,963-974[Abstract/Free Full Text]
  37. Bernardini, G., Ribatti, D., Spinetti, G., Morbidelli, L., Ziche, M., Santoni, A., Capogrossi, M. C., Napolitano, M. (2003) Analysis of the role of chemokines in angiogenesis J. Immunol. Methods 273,83-101[CrossRef][Medline]
  38. Rot, A., von Andrian, U. H. (2004) Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells Annu. Rev. Immunol. 22,891-928[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Dent. Res.Home page
I. Andrade Jr., T.A. Silva, G.A.B. Silva, A.L. Teixeira, and M.M. Teixeira
The Role of Tumor Necrosis Factor Receptor Type 1 in Orthodontic Tooth Movement
J. Dent. Res., November 1, 2007; 86(11): 1089 - 1094.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. N. Athanasopoulos, D. Schneider, T. Keiper, V. Alt, U. R. Pendurthi, U. M. Liegibel, U. Sommer, P. P. Nawroth, C. Kasperk, and T. Chavakis
Vascular Endothelial Growth Factor (VEGF)-induced Up-regulation of CCN1 in Osteoblasts Mediates Proangiogenic Activities in Endothelial Cells and Promotes Fracture Healing
J. Biol. Chem., September 14, 2007; 282(37): 26746 - 26753.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. N. Athanasopoulos, M. Economopoulou, V. V. Orlova, A. Sobke, D. Schneider, H. Weber, H. G. Augustin, S. A. Eming, U. Schubert, T. Linn, et al.
The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms
Blood, April 1, 2006; 107(7): 2720 - 2727.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104682v1
78/2/352    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Barcelos, L. S.
Right arrow Articles by Teixeira, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barcelos, L. S.
Right arrow Articles by Teixeira, M. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS