Adventitial Fibroblasts Release Interleukin 6 After Vascular Injury and Induce Smooth Muscle Cell Proliferation and Neointima Formation

Abstract

Background: Vascular restenosis resulting from neointima formation significantly limits the efficacy of percutaneous interventional therapies compared with bypass surgery. The adventitial layer is involved in neointima formation, but the detailed pathophysiological interplay of the different cell types in this process is still unclear.

Methods: We analyzed the correlation between adventitial and neointimal tissue size in human postmortem restenotic lesions after angioplasty. In porcine and mouse models of vascular injury, we examined early proliferation of fibroblasts and adventitial expansion. Using anti-CD45 antibodies, we identified recruited leukocytes as the source of fibroblast activation following vascular injury in mice. A time-course experiment on neointima formation demonstrated that adventitial activation precedes the proliferation of medial and neointimal smooth muscle cells (SMCs). To further investigate this process, we developed a mouse model enabling the surgical removal and transplantation of adventitial tissue.

Results: We observed that activated adventitial fibroblasts release interleukin 6 and other cytokines, which strongly induce SMC proliferation and migration in vitro. In interleukin 6 knockout mice, supernatants from activated adventitia grafts failed to stimulate SMC proliferation and migration. Furthermore, transplantation of adventitial grafts from interleukin 6 knockout mice did not induce neointima formation. Cell fate tracking experiments using double transgenic reporter mice demonstrated that resident adventitial cells do not directly contribute to the neointimal cellular mass. Instead, medial SMCs were identified as the primary source of neointimal cells.

Conclusions: We show that the release of interleukin 6 by adventitial fibroblasts induces the subsequent proliferation and migration of medial SMC in the process of neointima formation. Thus, we propose a new paradigm for adventitial fibroblasts in this process as a paracrine inflammatory engine. Anti-inflammatory targeting of the vascular adventitia might thus be promising to limit neointima formation.

Keywords: adventitial fibroblasts; interleukin 6; neointima formation; paracrine signaling; smooth muscle cells; vascular restenosis.

Figure 1. Restenotic adventitial and neointimal growth correlate with each other.

A, Patient selection workflow resulting in 24 analyzed peri‐stent samples. B, Both the luminal stenosis and adventitial areas increase over time postangioplasty. C, Representative pictures of adventitial and neointimal thickness assessment in human peri‐stent samples. Inlay 1 presents a representative location of the smallest neointimal size, as compared with inlay 2, showing location of the largest intimal size. Arrows indicate adventitia and neointima in both inlays. Dotted arrow indicates the underlying atherosclerotic lesion not included in the analysis. Scalebars: 1 mm, inlays: 500 μm. D, Adventitial size and (E) adventitial thickness correlate with neointima area at corresponding sites (determined by Pearson correlation analysis). Similarly, (F through H) show representative pictures of adventitial and neointimal thickness assessment in murine wire–induced injury model samples (21 days after vascular injury, scalebars: 200 μm, inlays: 100 μm [1] and 50 μm) and a positive correlation between (G) adventitial size and (H) adventitial thickness, with neointima area at corresponding sites, according to Pearson correlation analysis.

Figure 2. Adventitial proliferation and growth precedes medial proliferation and growth upon vascular injury.

A, Representative pictures of Van Gieson and Ki‐67 staining of uninjured as well as injured femoral arteries, 7, 14, and 21 days after injury. Overlays and inlays are presented. Scalebars: 200 μm, inlays: 100 μm. B, Adventitial thickening was assessed by planimetric analysis after Van Gieson staining of samples from the same experiment. ****P<0.0001 compared with 0 days, presented as mean±SEM, repeated‐measures 2‐way ANOVA. C, Ki‐67⁺ cells are mainly located in the adventitial region 7 days after injury, n=17 (7 days), n=10 (14 days), and n=13 (21 days), *P<0.05, ****P<0.0001 compared with intima and media; repeated‐measures 1‐way ANOVA.

Figure 3. Adventitial activation is critical for neointima formation, but neointimal cells derive from the medial layer and not from the adventitia.

A and B, Morphometric analysis of neointimal lesion formation 21 days following vascular injury and removal of the adventitial layer with or without adventitial tissue removal. n=5, scalebar 200 μm, ****P<0.0001, presented as mean±SEM, 2‐sided unpaired Student t test. C and D, Van Gieson staining and morphometric analysis 21 days following vascular injury in C57BL/6 mice with and without transplantation of the adventitial layer. n=6, scalebar 200 μm, presented as mean±SEM, 2‐sided unpaired Student t test. E, 21 days following vascular injury and transplantation of ubiquitously eGFP‐expressing adventitial layers from C57BL/6‐Tg(CAG‐EGFP)1Osb/J donor mice, immunofluorescence microscopy was performed. Blue: DAPI, green: eGFP, red: α‐smooth muscle actin. n=6, scalebars 200 μm, inlay: 100 μm. None of the mice studied show leakage/trafficking of adventitial (green) cells to the media/neointima. F, 21 days following vascular injury of double transgenic reporter mice, fluorescence microscopy was performed to detect differentiated SMCs before surgery (medially derived, green) and remaining cells (red). n=8, scalebars 200 μm, inlay: 100 μm. AdvTx indicates adventitial layer; eGFP, enhanced green fluorescent protein; SMC, smooth muscle cell; and Tx, transplantation.

Figure 4. Adventitial IL‐6 release is critical for neointima formation.

A, Vascular injury of C57BL/6 mice was followed by adventitia transplantation from donor mice (C57BL/6 or IL‐6^−/− background). Tissue samples were harvested 14 days following transplantation and incubated in serum‐free media for 24 hours. Respective conditioned media were used for functional in vitro experiments on HCASMCs. B, HCASMC proliferation and (C) migration are increased by supplementation of conditioned media derived from adventitial grafts. n=5, *P<0.05, **P<0.01, and ***P<0.001 compared with serum‐free control. ^# P<0.05 and ^## P < 0.01 compared with native aortic adventitia, presented as mean±SEM, repeated‐measures 1‐way ANOVA (B) and 2‐way ANOVA (C). D, Using the same 4a setup, IL‐6^−/− adventitial grafts significantly reduced HCASMC proliferation and (E) migration as compared with transplanted wild‐type adventitial grafts in vitro. n=5, *P<0.05, **P<0.01, compared with wild‐type mice, presented as mean±SEM, 2‐sided unpaired Student t test. F, Representative pictures of Van Gieson staining of neointima formation 21 days after wire‐injured mice, transplanted with wild‐type or IL‐6^−/− infrarenal aorta adventitial grafts, scalebars 200 μm. G through J, Morphometric analysis of neointimal lesion formation parameters 21 days following vascular injury and transplantation of the adventitia of C57BL/6 donor mice or IL‐6^−/− donor mice. n=5, *P<0.05, **P<0.01, presented as mean±SEM, 2‐sided unpaired Student t test. HCASMC indicates human coronary artery smooth muscle cell; IL‐6, interleukin 6; and n.s., not significant.