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Abstract
Objective—The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the cascade of gene activation that regulates aortic angiogenesis in response to injury.
Methods and Results—Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription–polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNFα gene disruption and recombinant TNFα or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNFα-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNFα and TNFα-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNFα. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNFα gene disruption and promoted by exogenous TNFα. Antibody-mediated inhibition of TNF receptor 1 significantly inhibited angiogenesis. The proangiogenic effect of TNFα was suppressed by blocking VEGF or by ablating aortic macrophages. Exogenous TNFα, however, maintained a limited proangiogenic capacity in the absence of macrophages and macrophage-mediated VEGF production.
Conclusion—Overexpression of TNFα is required for optimal VEGF production and angiogenesis in response to injury. This TNFα/VEGF-mediated angiogenic pathway requires macrophages. The residual capacity of TNFα to stimulate angiogenesis in macrophage-depleted aortic cultures implies the existence of a VEGF-independent alternate pathway of TNFα-induced angiogenesis.
Angiogenesis, the formation of new blood vessels, plays an important role in embryonal development, wound healing, and the ovarian cycle, but it also contributes to the progression of many diseases, including cancer and atherosclerosis. Endothelial cells of preexisting vessels form new vessels in response to angiogenic factors produced by nonendothelial cells.1,2
Among these, vascular endothelial growth factor (VEGF) is recognized as a key regulator of the angiogenic process and an important target of antiangiogenic therapy.3
VEGF is normally expressed in many organs, where it regulates capillary permeability and vessel survival under conditions of angiogenic quiescence.4 Increased expression of VEGF above physiological levels leads to the induction of angiogenesis.5 Anti-VEGF therapy blocks angiogenesis but can also cause side effects, such as hypertension, hemorrhage, thrombosis, and proteinuria, because of inhibition of the physiological functions of VEGF.6 Defining the upstream mechanisms responsible for increased VEGF expression is therefore critical to identifying additional targets of antiangiogenic therapy and possibly reducing its side effects. VEGF is characteristically upregulated in ischemic tissues through hypoxia-inducible transcription factors.7 VEGF levels are also elevated in wounded tissues,8,9 but the underlying mechanisms responsible for this VEGF increase are poorly defined. Understanding how the wounded vessel wall generates angiogenic signals may provide new insights into the mechanisms responsible for the formation of new blood vessels in atherosclerosis and other angiogenesis-related disorders associated with vascular injury.10,11
We previously showed that angiogenesis can be replicated ex vivo by culturing aortic rings in 3-dimensional gels of extracellular matrix.12,13 Angiogenesis in this system is triggered by the injury of the dissection procedure and regulated by paracrine and juxtacrine interactions between endothelial and nonendothelial cells, including macrophages, mural cells, and fibroblasts. Injured explants produce VEGF, which is released into the culture medium before the onset of angiogenesis. Aortic angiogenesis is significantly impaired by blocking VEGF with neutralizing antibodies or VEGF signal transduction inhibitors.14,15 Angiogenic sprouting can also be inhibited by depleting aortic rings of adventitial macrophages, which are required for optimal VEGF production.16 Macrophages promote angiogenesis through their ability to orchestrate the inflammatory response in wounded tissues,17 but it remains unclear how the injury process enables macrophages to promote the production of VEGF required for endothelial sprouting.
Among the macrophage products identified in aortic cultures is tumor necrosis factor-α (TNFα), an inflammatory cytokine that has the capacity to modulate the angiogenic process.18,19 TNFα is a homotrimeric transmembrane protein that is released into the extracellular space through proteolytic cleavage by the metalloprotease TNFα converting enzyme (TACE or ADAM17).20 TNFα binds to 2 cell membrane receptors, TNFα receptor-1 (TNFR1) and TNFR2. On TNFα binding, TNFRs generate a broad array of downstream signals by variably activating nuclear factor κB, mitogen-activated protein kinase, or caspase-dependent cell death pathways, depending on different contextual cues.21
TNFα has been shown to stimulate VEGF production by isolated cells,22–24 but there is a gap in our understanding of how this cytokine regulates the angiogenic response to injury in complex multicellular environments. In vitro models have investigated the direct effects of TNFα on isolated endothelial cells,25,26 but there are no studies on how endothelial cells respond to TNFα in the presence of macrophages and other vascular cell types. Using the aortic ring model of angiogenesis, we now show that resident macrophage-derived TNFα plays an essential role in the angiogenic response of the vessel wall to injury. Our results demonstrate that TNFα functions as an immediate-response proangiogenic factor in the cascade of gene activation, leading to VEGF production and endothelial sprouting following injury of the vessel wall. Our studies also indicate that TNFα plays an important role in the growth and survival of resident aortic macrophages.
Materials and Methods
For an expanded Materials and Methods section, see the Supplemental Data, available online at http://atvb.ahajournals.org.
Aortic Ring Cultures
Collagen gel cultures of aortic rings from rat and wild-type or TNFα-deficient mice were prepared and measured for angiogenic activity as described.27 Rat or mouse aortic rings were cultured with or without TNFα in the absence or presence of anti-VEGF blocking antibody or nonimmune IgG. Cocultures of aortic rings with aorta-derived macrophages were performed as reported.28 The role of TNFRs in the angiogenic response was studied in cultures of mouse aortic rings treated with anti-TNFR1 or anti-TNFR2 blocking antibodies29–31 or with nonimmune IgG.
Cell Isolation
Aortic macrophages were isolated from colony stimulating factor-1–treated aortic cultures, as described.28 Aortic endothelial cells, aortic smooth muscle cells, and bone marrow macrophages were isolated as reported.32
Macrophage Ablation
Rat aortic rings were depleted of macrophages with liposomal clodronate16,33 and cultured in collagen gels with or without TNFα (5 ng/mL).
Quantitative Reverse Transcription–Polymerase Chain Reaction
The relative expression of TNFα and VEGF in aortic cultures at different time points after injury or TNFα treatment was measured by quantitative reverse transcription–polymerase chain reaction (qRT-PCR).34
Microarray Analysis
The transcriptome of injured and TNFα-treated aortic rings was analyzed with Affymetrix Rat Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA). Microarray data were deposited into the National Center for Biotechnology Information Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under Gene Expression Omnibus series accession numbers GSE23152 and GSE23153.
Western Blot Analysis
Western blotting was used to evaluate TNFα protein expression in rat macrophages, endothelial cells, and smooth muscle cells.
Immunocytochemistry
Macrophages were identified in whole mount preparations of aortic ring cultures by immunoperoxidase or immunofluorescence using antibodies against rat CD68, rat CD163, or mouse F4/80.35 Double immunofluorescence staining with anti-TNFα and anti-CD68 antibodies was used to localize TNFα in aortic macrophages.
ELISA
VEGF produced in aortic cultures was measured with the Quantikine rat and mouse ELISA kits (R&D Systems).
Statistical Analysis
Data were analyzed by the Student t test. The statistical significance of differences between experimental groups was set at P<0.05.
Results
The Angiogenic Response of Aortic Rings to Injury Is Preceded by Upregulated Expression of TNFα and TNFα-Inducible Genes
Aortic rings embedded in collagen gels generate a self-limited angiogenic response that is triggered by the injury of the dissection procedure. Aortic explants start sprouting at day 2 or 3, producing branching neovessels that grow until day 7 or 8.13 Aortic angiogenesis is associated with overexpression of many proangiogenic molecules, including TNFα and VEGF, which work in concert to stimulate vessel growth.16,36 To investigate the temporal sequence of TNFα and VEGF expression before angiogenic sprouting, we performed qRT-PCR time course studies on aortic rings during the first 24 hours of culture. TNFα was upregulated severalfold 10 minutes after the rings were cut, and its expression remained elevated at 24 hours. Upregulated expression of TNFα was followed by a gradual increase in VEGF expression over the following 24 hours (Figure 1). Thus, VEGF expression and angiogenic sprouting were preceded by rapid overexpression of TNFα in response to aortic injury.
Upregulated expression of TNFα precedes VEGF expression and angiogenic sprouting in aortic ring cultures. A, Schematic drawing of the aortic ring model of angiogenesis. New vessels sprout from freshly cut aortic explants embedded in collagen gel. B, qRT-PCR showed rapid upregulation of TNFα gene expression after the aorta had been transected to obtain rings. TNFα mRNA was rapidly overexpressed within minutes after injury, whereas the highest VEGF mRNA levels were detected at 24 hours. C, Venn diagram showing the relationship between the transcriptomes of injured and TNFα-treated rings and identifying many TNFα-inducible genes in the injury transcriptome. D, Representative genes upregulated by both injury and TNFα.
To further evaluate the relationship between injury and TNFα gene induction, we performed microarray studies on freshly cut and TNFα-treated aortic rings and compared the 2 transcriptomes for the presence of commonly upregulated genes. This study showed that 204 genes were upregulated 1.5-fold or more after injury, whereas 245 genes were overexpressed in response to TNFα (Figure 1). Approximately 40% of TNFα-induced genes were identified in the injury transcriptome. Many of these commonly induced genes were chemokines and angioregulatory molecules. Genes induced by both injury and TNFα comprised chemokines that stimulate the migration of monocytes/macrophages (CCL2, CCL7, CCL12) and macrophage-derived chemokines that attract polymorphonuclear cells to sites of inflammation (CCL3, CCL20, CXCL1). Two of these chemokines (CCL2 and CXCL1) have the additional capacity to stimulate angiogenesis.37,38 TNFα induced many soluble angiogenic regulators, including angiopoietin-1, hepatocyte growth factor, EphrinB1, interleukin-6,37,39–41 and bone morphogenetic protein-2.42 Hepatocyte growth factor, interleukin-6, and bone morphogenetic protein-2 were also upregulated in the injury transcriptome. Taken together, these results demonstrate the existence of a TNFα signature of gene expression in the injured aorta before angiogenic sprouting.
Resident Adventitial Macrophages Are the Main Source of TNFα in Aortic Cultures
Aortic outgrowths are composed of a mixed population of cells, including macrophages (Figure 2). To determine the contribution of different cell types to TNFα production, we analyzed TNFα gene expression in isolated macrophages, endothelial cells, and smooth muscle cells. A sufficient number of macrophages was obtained by treating aortic cultures with colony stimulating factor-1, which markedly stimulates macrophage outgrowth.28 qRT-PCR showed abundant TNFα expression by isolated aortic macrophages. No significant expression of TNFα was observed in endothelial or smooth muscle cells by qRT-PCR or Western analysis. Aortic macrophages showed higher mRNA levels of TNFα than bone marrow macrophages. TNFα expression in aorta-derived macrophages was confirmed by double staining isolated aortic macrophages for TNFα and CD68 (Figure 2).
TNFα in aortic cultures is expressed primarily in macrophages. A, Immunoperoxidase staining showed CD68+ macrophages (arrowheads) at the root of an angiogenic outgrowth in a collagen gel culture of rat aorta (asterisk). B, CD68+ macrophages (arrowheads) were closely associated with newly formed microvessels (arrows). C, qRT-PCR demonstrated much higher expression of TNFα in isolated rat aortic macrophages (RAM) compared with rat aortic endothelial cells (RAEC) or smooth muscle cells (RASMC); rat aortic macrophages expressed more TNFα than rat bone marrow–derived macrophages (RBMM). D, Western analysis confirmed lack of TNFα expression in endothelial and smooth muscle cells. E to G, Confocal images of aortic cultures showed coexpression (G, overlay) of CD68 (E, red) and TNFα (F, green) in outgrowing macrophages. Scale bars=200 μm (A), 100 μm (B), 50 μm (E to G).
Disruption of the TNFα Gene Causes Reduced Angiogenesis and Macrophage Outgrowth
To define the role of endogenous TNFα in the angiogenic response to injury, aortic rings from TNFα knockout mice were compared with normal aortic rings for their capacity to sprout in collagen gel culture. Disruption of the TNFα gene caused a marked reduction in angiogenic sprouting. Impairment of angiogenesis in the absence of TNFα correlated with a significant downregulation in VEGF mRNA expression (data not shown) and reduction in VEGF protein levels (Figure 3). The angiogenic response of TNFα-deficient aortic rings was restored to normal values by adding exogenous TNFα to the culture medium or by coculturing the TNFα-deficient rings with TNFα producing normal aortic macrophages (Figure 3). These observations indicate that TNFα production is required for optimal angiogenesis in response to injury of the aortic wall.
Genetic disruption of TNFα impairs angiogenesis and macrophage outgrowth. A, Collagen gel culture of normal aortic ring contained many newly formed microvessels (arrows). B, Aortic ring from TNFα knockout mouse contained rare microvessels (arrow) because of marked impairment of angiogenic sprouting. C, Quantitative analysis demonstrated marked reduction of angiogenesis in cultures of TNFα-deficient aortic rings compared with control (n=52 aortic rings from 7 animals per group). D, ELISA of conditioned medium showed that VEGF levels in cultures of TNFα-deficient aortic rings were reduced to 20% of control values (n=3). E, TNFα-deficient aortic ring treated with exogenous TNFα produced an angiogenic response (arrows) comparable to control. F and G, Quantitative analysis of aortic angiogenesis showed that exogenous TNFα (F, n=15) or TNFα producing normal aortic macrophages (G, n=8) restored the angiogenic response of TNFα-deficient aortic rings to normal values. H, Microvessels sprouted from normal mouse aortic rings were surrounded by clusters of single cells with features (rounded morphology, granular cytoplasm) characteristic of macrophages (arrowheads). I, The angiogenic outgrowth in TNFα-deficient aortic rings showed no evidence of cells with macrophage features. J, Immunoperoxidase staining demonstrated numerous F4/80+ macrophages in the adventitia of a normal mouse aortic ring in collagen gel culture. K, The adventitia of TNFα-deficient aortic ring was depleted of F4/80+ macrophages. L, Quantitative evaluation of F4/80-stained cultures demonstrated marked reduction in macrophages in TNFα-deficient aortic rings compared with normal control (n=10). Scale bars=500 μm (A, B, and E), 100 μm (H to K). *P<0.05, **P<0.01, ***P<0.001.
Close examination of TNFα-deficient aortic ring cultures showed a reduced number of single cells with morphological features of macrophages compared with control. Immunocytochemical staining for the mouse macrophage marker F4/80 confirmed this observation and demonstrated that aortic macrophages in the absence of endogenous TNFα were reduced to 34% of control (Figure 3).
To identify the TNFα receptor involved in the angiogenic response, aortic cultures were treated with blocking antibodies against TNFR1 or TNFR2. Angiogenic sprouting was markedly impaired in cultures treated with anti-TNFR1 antibody. This inhibitory effect was associated with a marked reduction in the outgrowth of single cells, including macrophages (data not shown). The anti-TNFR2 antibody caused a modest and not statistically significant reduction in angiogenesis (Supplemental Figure I).
TNFα Stimulates Aortic Angiogenesis and Macrophage Outgrowth
To further characterize the angioregulatory function of TNFα, the effect of this cytokine was tested in collagen gel cultures of rat aorta. TNFα potently stimulated angiogenesis in a dose-dependent manner, causing a marked increase in vessel number and length. The proangiogenic effect of TNFα was most pronounced at 5 ng/mL. TNFα was less stimulatory at higher doses, exhibiting antiangiogenic activity at 100 ng/mL (Figure 4). Proangiogenic doses of TNFα markedly stimulated macrophage outgrowth over time. This effect was accompanied by enhanced collagen lysis around the explants. TNFα treatment caused formation of cellular patches composed of CD68+ macrophages on the bottom of the culture dish, beneath the aortic rings. DNA synthesis studies of cultures incubated with a thymidine analog showed a 285% increase in macrophage proliferation in response to TNFα treatment compared with control (Figure 4). The effect of TNFα on macrophage outgrowth switched from stimulatory to inhibitory at higher doses. Maximum inhibition of macrophages was obtained with 100 ng/mL (data not shown). Taken together with the TNFα knockout experiments, studies with exogenous TNFα establish a link between the capacity of this cytokine to regulate macrophage outgrowth and its angioregulatory properties.
TNFα has dose-dependent effects on angiogenesis and macrophage outgrowth. A, 7-day-old control aortic ring culture. B, Culture treated with 5 ng/mL TNFα. C, Culture treated with 100 ng/mL TNFα. TNFα had concentration-dependent stimulatory or inhibitory effects on growth of microvessels (arrows). D, Microvessel counts demonstrated >100% stimulation of angiogenesis over time by 5 ng/mL TNFα (n=4). E, Treatment of aortic cultures with increasing doses of TNFα identified 5 ng/mL as the optimal proangiogenic dose and demonstrated reduced TNFα efficacy at higher doses, with the highest dose having antiangiogenic activity (n=4). F and G, Over time, the proangiogenic dose of TNFα induced the formation of a dense periaortic outgrowth that formed a patch (asterisk) on the bottom of the culture dish (G); this outgrowth was less pronounced in the unstimulated control (F). H, The TNFα-induced aortic patch was composed of a uniform population of CD68+ macrophages. I, Studies with the thymidine analog ethynyl deoxyuridine (EdU) demonstrated occasional labeled nuclei (green) in CD68+ macrophages (red) of untreated control culture. J, Macrophages exhibited a higher rate of proliferation in TNFα-treated cultures. K, Bar graph shows quantitative analysis of macrophage (MΦ) proliferation in CD68-stained control and TNFα-treated aortic cultures (n=10). Scale bars=500 μm (A to C, F, and G), 100 μm (H to J). *P<0.05, **P<0.01, ***P<0.001.
TNFα Requires VEGF for Optimal Stimulation of Angiogenesis
To evaluate the effect of TNFα on the expression of VEGF, conditioned media of aortic cultures were analyzed by VEGF ELISA. VEGF levels at day 3 increased from 100 pg/mL in the untreated control to 320 pg/mL in TNFα-treated cultures (Figure 5). VEGF concentration remained elevated (220 pg/mL) at day 6 following TNFα treatment but decreased to 30 pg/mL in control cultures. To define the role of VEGF in TNFα-induced angiogenesis, TNFα-treated aortic cultures were incubated with an anti-VEGF blocking antibody. Inhibition of VEGF almost completely abrogated the stimulatory effect of TNFα on angiogenesis, indicating that VEGF is an important mediator of TNFα-induced angiogenesis. The anti-VEGF antibody had no significant effect on macrophage outgrowth or collagen lysis (Figure 5).
The proangiogenic effect of TNFα is significantly inhibited by VEGF blockade. A, ELISA of aortic culture conditioned medium showed increased production of VEGF in TNFα-treated cultures compared with control (n=3). B, Antibody (Ab)–mediated inhibition of VEGF significantly impaired the angiogenic response of aortic rings to TNFα (n=4). C to F, Micrographs show aortic cultures stimulated with TNFα in the presence of nonimmune IgG (C and E) or anti-VEGF blocking antibody (D and F) and imaged at day 6 (C and D) and day 9 (E and F). Note: the anti-VEGF antibody significantly impaired TNFα-stimulated angiogenesis without affecting TNFα-induced macrophage outgrowth and collagen lysis. Representative microvessels in C to F are highlighted by arrows. Scale bar=500 μm (C to F). *P<0.05, **P<0.01, ***P<0.001.
Macrophages Are Mediators of TNFα-Induced VEGF Production and Angiogenesis
To investigate the role of macrophages in TNFα-induced angiogenesis, aortic rings were depleted of resident adventitial macrophages by overnight treatment with liposomal clodronate before collagen embedding. Macrophage ablation markedly impaired the spontaneous angiogenic response of the aortic rings. Addition of exogenous TNFα restored angiogenesis in macrophage-depleted cultures to control values. In the absence of aortic macrophages, however, the TNFα effect was blunted and insufficient to further potentiate the angiogenic response. The reduced efficacy of TNFα in macrophage-depleted cultures correlated with markedly reduced VEGF levels (Figure 6). This finding suggested that TNFα maintains a limited capacity to stimulate angiogenesis in the absence of macrophages, independently of VEGF production. To evaluate this hypothesis, macrophage-depleted aortic cultures treated with TNFα were supplemented with anti-VEGF blocking antibody or nonimmune IgG control. Inhibition of VEGF had no effect on the residual proangiogenic activity of TNFα in macrophage-depleted cultures. The efficacy of the anti-VEGF antibody was confirmed in VEGF-treated control cultures in which the stimulatory effect of recombinant VEGF was completely abolished by the blocking antibody (data not shown). These results indicate that aortic macrophages, which are the primary source of TNFα, also function as one of the targets and mediators of the TNFα proangiogenic activity during the early stages of the angiogenic response. They also demonstrate the existence of a VEGF-independent pathway of angiogenic stimulation by TNFα that becomes manifest following ablation of resident aortic macrophages.
Macrophage ablation impairs TNFα-induced VEGF production and angiogenesis. A and B, Whole mount preparations of rat aorta immunostained for CD163 demonstrated depletion of macrophages in clodronate-treated explants (B) compared with phosphate-buffered saline control (A). The same results were obtained with other macrophages markers (data not shown). C to F, Macrophage-depleted aortic rings failed to generate an angiogenic response (D) compared with control rings (C) and produced a reduced number of microvessels when treated with TNFα (F) compared with TNFα-treated rings containing macrophages (E). G and H, Bar graphs show differences in angiogenesis (G, days 6 to 7, n=4) and VEGF production (H, n=3) between macrophage-depleted rings and control rings treated with TNFα or left untreated. I, The residual proangiogenic effect of TNFα in clodronate treated cultures was not affected by antibody-mediated inhibition of VEGF. Scale bars=100 μm (A and B), 500 μm (C to F). *P<0.05, **P<0.01, ***P<0.001.
Discussion
Angiogenesis in response to tissue injury is a complex process regulated by a cascade of inflammatory stimuli that enable preexisting blood vessels to switch from proliferative quiescence to an angiogenic phenotype.1,43 Although many studies have investigated the role of angiogenic factors, inflammatory cytokines, and chemokines in this process, the molecular sequence of events that orchestrates the initial stages of the angiogenic cascade is incompletely understood and not fully characterized.
The angiogenic process can be accurately replicated ex vivo by culturing rat or mouse vessel explants in collagen gels under serum-free conditions.12,44 Angiogenesis in this system is triggered by injury and mediated by adventitial macrophages, which are essential for optimal VEGF production and angiogenic sprouting.16 The angiogenic response of the aortic explants is a self-limited process that begins 2 to 3 days after injury, lasts 5 to 6 days, and is followed by vascular regression during the second week of culture. We used this model to define the sequence of molecular events responsible for the initial sprouting of neovessels from the injured aortic wall. Aortic angiogenesis is preceded by an upregulated expression of many inflammatory cytokines and chemokines, which work in concert with VEGF to induce sprouting of new vessels.16,36 Among these is TNFα, a macrophage-derived cytokine that has been implicated in the regulation of reactive and pathological angiogenesis.45 Macrophages in aortic cultures act as injury sensors and transducers of the angiogenic response, but the molecular mechanisms by which these cells translate injury signals into VEGF production and angiogenesis are poorly understood. Because TNFα has the capacity to induce VEGF in different vascular cell types,22–24 we hypothesized that macrophages used this cytokine as an early regulator of the angiogenic response of the wounded aortic wall.
Our study demonstrates the following points. (1) Aortic injury induces overexpression of both TNFα and VEGF. (2) TNFα is upregulated within minutes after injury. (3) The increase in TNFα is followed by overexpression of VEGF over the following 24 hours. (4) The transcriptome of injured aortic rings contains a TNFα signature of gene expression. (5) TNFα in aortic cultures is produced primarily by resident macrophages. (6) Disruption of the TNFα gene causes reduced VEGF production, angiogenesis, and macrophage outgrowth. (7) Angiogenesis is markedly impaired by antibody-mediated inhibition of TNFR1. (8) Exogenous TNFα promotes both angiogenesis and macrophage proliferation. (9) The angiogenic response to TNFα is significantly inhibited by neutralizing VEGF with a blocking antibody. (10) Pharmacological ablation of macrophages markedly inhibits VEGF production and significantly impairs the angiogenic response to exogenous TNFα. (11) Exogenous TNFα retains a limited capacity to stimulate angiogenesis through VEGF-independent mechanisms in the absence of macrophages.
Taken together, these findings demonstrate that VEGF production in a wounded vasculature is the result of upregulated expression of TNFα, which acts as an immediate proangiogenic response gene in macrophages activated by the injury process. We reported previously that the angiogenic response of cultured aortic rings is associated with an increased production of VEGF, which peaks during the first 24 hours after injury.14 VEGF plays a central role in the angiogenic response of the aortic wall because endothelial sprouting can be markedly impaired by treating aortic cultures with anti-VEGF neutralizing antibodies or VEGF signal transduction inhibitors.14,15 In the present study, we identified TNFα as an upstream inducer of VEGF expression in the cascade of angiogenic gene activation triggered by the aortic injury. TNFα was upregulated within minutes after wounding of the aortic wall, and disruption of the TNFα gene caused marked reduction in VEGF mRNA expression, VEGF protein levels, and angiogenesis. Our additional finding that aortic angiogenesis could be significantly inhibited by blocking TNFα signaling with an anti-TNFR1 neutralizing antibody supports this conclusion and is in keeping with an earlier study by others in which genetic disruption of TNFR1 was shown to cause impaired VEGF production and neovascularization in a subcutaneous vivo model of inflammatory angiogenesis.46
Our results confirm previous observations that TNFα is rapidly overexpressed in wounded tissues47–49 and has the capacity to induce VEGF expression in different cell types.22–24 To our knowledge, however, no studies have defined the relationship between the rapid rise in TNFα expression and the induction of VEGF in a model in which the mRNA and protein levels of these molecules can be directly correlated with the angiogenic response. We were able to fill this gap by studying molecular events occurring before angiogenic sprouting in the isolated aortic wall. For this particular application, the ex vivo aortic ring model offered considerable advantages over in vitro and in vivo models of angiogenesis. In vitro models of endothelial wound healing are limited by the lack of nonendothelial cell types, such as macrophages, which are involved in the angiogenic response to injury. Angiogenesis in aortic ring cultures is instead the result of paracrine interactions between endothelial and nonendothelial cells types.13 Compared with the more complex in vivo models, aortic ring cultures can be easily monitored over time and pharmacologically modulated under chemically defined culture conditions.
Microarray analysis revealed a distinct TNFα signature of gene expression in aortic rings 24 hours after injury. Upregulated genes included macrophage stimulatory chemokines, such as CCL2, CCL7, and CCL12, and macrophage-derived chemokines that attract polymorphonuclear cells to sites of inflammation (CCL3, CCL20, CXCL1).50 The increase in macrophage outgrowth was associated with enhanced proteolysis resulting in periaortic rarefaction of the collagen gel. Exogenous TNFα induced expression of the interstitial collagenase matrix metalloproteinase (MMP) 13, which most likely contributed to the TNFα-stimulated angiogenic sprouting and collagen lysis.51 TNFα also induced MMP3 and MMP9, which, together with MMP13, were also upregulated by injury in the absence of exogenous TNFα.
The hypothesis that the stimulatory effect of TNFα on angiogenesis is mediated by leukocytes was originally proposed on the basis of the observation that TNFα inhibits the proliferation of isolated endothelial cell in vitro52,53 but stimulates inflammatory angiogenesis in vivo.18,54–56 Ablation of macrophages has been shown to reduce angiogenesis during wound healing,57 but there are no in vivo studies on the effect of macrophage depletion on the proangiogenic activity of TNFα. To evaluate the role of macrophages in TNFα-induced angiogenesis, we pretreated aortic rings with liposomal clodronate, which selectively kills phagocytic cells.16 Macrophage ablation significantly impaired the proangiogenic effect of TNFα. The reduced proangiogenic activity of TNFα was most likely caused by the marked reduction in VEGF production in these cultures. This result establishes the macrophage as a key mediator of the TNFα proangiogenic activity.
TNFα exhibited a limited capacity to induce vessel sprouting in macrophage-depleted cultures. This residual proangiogenic activity was not due to VEGF because VEGF levels were markedly decreased and antibody-mediated inhibition of VEGF had no effect in the absence of macrophages. This VEGF-independent proangiogenic activity may be due to a direct stimulatory effect of TNFα on endothelial cell migration56,58 or the ability of this cytokine to induce expression in mural cells of other angiogenic factors. Our microarray studies showed that TNFα induces expression of hepatocyte growth factor, angiopoietin-1, EphrinB1, bone morphogenetic protein-2, and the angiogenic chemokines CCL2 and CXCL1, which could all function as stimulators of angiogenesis in the absence of VEGF.37,38,40–42,59
A previous study with a subcutaneous in vivo model showed that low doses of TNFα stimulated angiogenesis, whereas high doses were inhibitory.19 To evaluate whether this bimodal effect of TNFα angiogenesis was related to macrophages, we tested increasing doses of TNFα in the aortic ring model. Our studies confirmed this observation and showed stimulation of angiogenesis with 5 to 10 ng/mL TNFα and marked inhibition with 100 ng/mL. Whereas the proangiogenic low doses of TNFα concurrently stimulated angiogenesis and macrophage outgrowth, the antiangiogenic high doses were potently inhibitory for both. The inhibitory effect of TNFα was limited to macrophages and microvessels, as mesenchymal cell outgrowths were not significantly affected.
In summary, our study identifies TNFα as an immediate response gene in the proangiogenic cascade of gene activation triggered by injury of the aortic wall. Our results indicate that TNFα is required for optimal VEGF production and angiogenesis in aortic cultures. This TNFα/VEGF-mediated pathway of angiogenic induction is critically dependent on macrophages, which produce TNFα and mediate its proangiogenic effect. The residual angiogenic activity observed in macrophage-deficient aortic cultures treated with exogenous TNFα implies the existence of a VEGF-independent alternate pathway of TNFα-induced angiogenesis (Supplemental Figure II). More studies are, however, needed to further characterize the TNFα-mediated angiogenic cascade and to define the mechanisms by which TNFα blocking agents currently used in the clinic affect TNFα-induced angiogenic pathways.60 A greater knowledge of how TNFα regulates macrophage-induced angiogenesis may provide new insights into the molecular pathways responsible for the progression of atherosclerosis, cancer, and other pathological conditions associated with vascular injury and inflammatory angiogenesis.
Sources of Funding
Work presented in this article was supported by National Heart, Lung, and Blood Institute Grant HL52585 and a Merit Review Grant from US Department of Veterans Affairs, Office of Research and Development, Biomedical Laboratory Research Program.
Disclosures
None.
- Received October 15, 2010.
- Accepted February 15, 2011.
- © 2011 American Heart Association, Inc.
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