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. 2011 May;31(5):1151-9.
doi: 10.1161/ATVBAHA.111.223917. Epub 2011 Mar 3.

Macrophage-derived tumor necrosis factor-alpha is an early component of the molecular cascade leading to angiogenesis in response to aortic injury

Giovanni Ligresti  1 , Alfred C Aplin, Penelope Zorzi, Ann Morishita, Roberto F Nicosia
Affiliations

Macrophage-derived tumor necrosis factor-alpha is an early component of the molecular cascade leading to angiogenesis in response to aortic injury

Giovanni Ligresti et al. Arterioscler Thromb Vasc Biol. 2011 May.

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.

Conclusions: 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.

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Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Upregulated expression of TNFα precedes VEGF expression and angiogenic sprouting in aortic ring cultures
(A) Schematic drawing of aortic ring model of angiogenesis: New vessels sprout from freshly cut aortic explants embedded in collagen gel. (B) qRT-PCR shows rapid upregulation of TNFα gene expression after the aorta has been transected to obtain rings. TNFα mRNA is rapidly overexpressed within minutes after injury whereas highest VEGF mRNA levels are detected at 24 hr. (C) Venn diagram showing the relationship between the transcriptomes of injured and TNFα-treated rings identifies many TNFα-inducible genes in the injury transcriptome. (D) Table shows representative genes upregulated by both injury and TNFα.
Figure 2
Figure 2. TNFα in aortic cultures is primarily expressed in macrophages
(A) Immuno-peroxidase staining shows CD68+ macrophages (arrowheads) at the root of an angiogenic outgrowth in a collagen gel culture of rat aorta (asterisk). (B) CD68+ macrophages (arrowheads) are closely associated with newly formed microvessels (arrows). (C) qRT-PCR demonstrates much higher expression of TNFα in isolated rat aortic macrophages (RAM) compared to rat aortic endothelial (RAEC) or smooth muscle cells (RASMC); rat aortic macrophages express more TNFα than rat bone marrow derived macrophages (RBMM). (D) Western analysis confirms lack of TNFα expression in endothelial and smooth muscle cells. (E–G) Confocal images of aortic cultures demonstrate co-expression (G, overlay) of CD68 (E, red) and TNFα (F, green) in outgrowing macrophages. Magnification bars = 200 µm (A), 100 µm (B); 50 µm (E,F,G).
Figure 3
Figure 3. Genetic disruption of TNFα impairs angiogenesis and macrophage outgrowth
(A) Collagen gel culture of normal aortic ring contains many newly formed microvessels (arrows). (B) Aortic ring from TNFα knockout mouse shows rare microvessels (arrow) due to marked impairment of angiogenic sprouting. (C) Quantitative analysis demonstrates marked reduction of angiogenesis in cultures of TNFα-deficient aortic rings compared to control (N = 52 aortic rings from 7 animals per group). (D) ELISA of conditioned medium shows that VEGF levels in cultures of TNFα-deficient aortic rings are reduced to 20% of control values (N = 3). (E). TNFα-deficient aortic ring treated with exogenous TNFα produces an angiogenic response (arrows) comparable to control. (F, G). Quantitative analysis of aortic angiogenesis shows that exogenous TNFα (F, N=15) or TNFα producing normal aortic macrophages (G, N=8) restore the angiogenic response of TNFα-deficient aortic rings to normal values. (H) Microvessels sprouted from normal mouse aortic rings are 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 shows no evidence of cells with macrophage features. (J) Immunoperoxidase staining demonstrates 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 is depleted of F4/80+ macrophages. (L) Quantitative evaluation of F4/80-stained cultures demonstrates marked reduction in macrophages in TNFα-deficient aortic rings compared to normal control (N = 10). Magnification bars = 500 µm (A, B, E), 100 µm (H, I, J, K). * p<0.05; ** p<0.01; *** p<0.001.
Figure 4
Figure 4. 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 has concentration-dependent stimulatory or inhibitory effects on growth of microvessels (arrows). (D) Microvessel counts demonstrate >100% stimulation of angiogenesis over time by 5 ng/ml TNFα (N=4). (E) Treatment of aortic cultures with increasing doses of TNFα identifies 5 ng/ml as the optimal pro-angiogenic dose and demonstrates reduced TNFα efficacy at higher doses, with the highest dose having anti-angiogenic activity (N=4). (F, G) Over time, the proangiogenic dose of TNFα induces the formation of a dense periaortic outgrowth which forms a patch (asterisk) on the bottom of the culture dish (G); this outgrowth is less pronounced in the unstimulated control (F). (H) The TNFα-induced aortic patch is composed of a uniform population of CD68+ macrophages. (I) Studies with the thymidine analogue EDU demonstrate occasional labeled nuclei (green) in CD68+ macrophages (red) of untreated control culture. (J) Macrophages exhibit 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). Magnification bars = 500 µm (A, B, C, F, G), 100 µm (H, I, J). * p<0.05, ** p<0.01, *** p<0.001.
Figure 5
Figure 5. The proangiogenic effect of TNFα is significantly inhibited by VEGF blockade
(A) ELISA of aortic culture conditioned medium shows increased production of VEGF in TNFα-treated cultures compared to control (N=3). (B) Antibody-mediated inhibition of VEGF significantly impairs the angiogenic response of aortic rings to TNFα (N=4). (C–F) Micrographs show aortic cultures stimulated with TNFα in the presence of nonimmune IgG (C, E) or anti-VEGF blocking antibody (D, F) and imaged at day 6 (C, D) and 9 (E, F). Note: the anti-VEGF antibody significantly impairs TNFα-stimulated angiogenesis without affecting TNFα-induced macrophage outgrowth and collagen lysis. Representative microvessels in C–F are highlighted by arrows. Magnification bar = 500 µm (C, D, E, F). * p<0.05; ** p<0.01; *** p<0.001.
Figure 6
Figure 6. Macrophage ablation impairs TNFα-induced VEGF production and angiogenesis
(A, B) Whole mount preparations of rat aorta immunostained for CD163 demonstrate depletion of macrophages in clodronate-treated explants (B) compared to PBS control (A); same results are obtained with other macrophages markers (data not shown). (C–F) Macrophage-depleted aortic rings fail to generate an angiogenic response (D) compared to control rings (C), and produce a reduced number of microvessels when treated with TNFα (F) compared to TNFα-treated rings containing macrophages (E). (G, H) Bar graphs show differences in angiogenesis (G, day 6–7, 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 is not affected by antibody-mediated inhibition of VEGF. Magnification bars = 100 µm (A, B), 500 µm (C, D, E, F). * p<0.05; ** p<0.01; *** p<0.001.
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