Original articleInterleukin-19 increases angiogenesis in ischemic hind limbs by direct effects on both endothelial cells and macrophage polarization
Introduction
Peripheral artery disease (PAD) is often associated with diabetes and coronary artery disease, leading to significant morbidity (amputation) and mortality (myocardial infarction) in patients. Identification and characterization of molecules which not only limit tissue inflammation but also increase capillary density, collateral formation and perfusion have the potential to salvage ischemic tissue and can lead to new therapies for tissue repair and neovascularization. Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion, and accordingly ischemic revascularization is a complex process involving multiple processes and cell types. While neovascularization and inflammation are independent biological processes, they are linked in response to injury and ischemia, and both inflammatory and anti-inflammatory cytokines participate in these processes. Endothelial cell (EC) paracrine and autocrine stimulation can result in migration and proliferation, and are an essential component of normal and pathophysiological processes including wound healing and angiogenesis [1], [2], [3]. In addition to well characterized angiogenic cytokines like VEGF, FGF, and CXCL1, it is accepted that many pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and IL-18 increase EC migration, proliferation, tube formation, and increased vascularity in vivo [4], [5], [6]. One exception is Interleukin-12, which is both pro-inflammatory and potently anti-angiogenic [7]. On the other hand, the role of and direct effects of anti-inflammatory interleukins on EC in initiation of angiogenesis are less clear. The prototypical anti-inflammatory cytokine, IL-10, has anti-angiogenic activity and is associated with VEGF down regulation, reduction of FGF and VEGF induced proliferation of microvascular EC [8]. Similarly, IL-4 can inhibit VEGF production and reduce vascularization, but can also induce migration and tube like structure formation in EC, activities consistent with angiogenesis [9], [10], [11]. IL-13 attenuates EC tube formation, and IL-20 has both pro- and anti-angiogenic effects [12], [13], [14], [15]. Macrophages also participate in angiogenesis as the M2, or alternatively activated macrophages express several pro-angiogenic cytokines and thus must be included in any discussion of angiogenesis in vivo [16], [17]. In summary, direct pro-angiogenic effects on EC, polarization of macrophage M2 phenotype, and inhibition of anti-angiogenic cytokines are all recognized pathways leading to angiogenesis; a modality which could reduce inflammation but not impair revascularization and would have obvious clinical benefits.
Interleukin-19 (IL-19) was discovered in 2001 [18], and is considered to be part of the IL-10 sub-family which includes IL-20, IL-22 and IL-24 [19], [20]. IL-19 promotes an anti-inflammatory Th2 rather than the Th1 response in T-lymphocytes [21], [22]. Unlike IL-10, IL-19 expression and activity are not restricted to leukocytes and is rather unique among interleukins. For example, neither IL-10, IL-4, nor IL-33 is expressed by EC or vascular smooth muscle cells (VSMC), precluding potential autocrine effects of these interleukins on the vasculature [23]. Little is reported regarding IL-19 effects on macrophage.
We recently reported that IL-19 was expressed in angiogenic tissue, and has potent pro-angiogenic effects on multiple human EC types, (umbilical vein, coronary artery, and microvascular) [24]. This manuscript reported that IL-19 is chemotactic and mitogenic for EC, promotes tube-like structure formation on Matrigel, and microvessel formation in the mouse aortic ring assay. These inaugural studies, though novel, were all cell culture or ex vivo based, and lacked validation in a relevant in vivo model of angiogenesis. Rodent hind limb ligation and ischemia is a well-established model for induction of neovascularization in vivo [25], [26]. In the present study, multiple but complementary approaches were used to determine if IL-19 regulated neovascularization in the hind-limb ischemia model. In this manuscript we determined that in contrast to IL-10, IL-19 can increase perfusion in ischemic hind limbs and exerts its angiogenic effects by at least three mechanisms: direct effects on EC gene expression; local and systemic M2 macrophage polarization and VEGF-A expression, and suppression of IL-12 expression in macrophage. Together, this implicates IL-19 as a link for two major processes; anti-inflammation and angiogenesis, and could identify IL-19 as a previously unrecognized pro-angiogenic modality in treatment of PAD.
Section snippets
Animals
Wild type C57BL/6 mice were purchased from Jackson Labs. IL-19 knockout mice were generated using the VelociGene method and IL-19−/− mice identified by genotyping of tail DNA by PCR using specific primers as we described [27]. Age and sex-matched male and female littermates were used for these studies. The hind limb ischemia model was performed as described [28]. Briefly, mice were anesthetized by injection of ketamine and xylazine, the femoral artery is dissected from the femoral vein, the
IL-19 expression is induced in murine ischemic hind limb
IL-19 expression is induced in angiogenic tissue [24], and initial experiments validated IL-19 expression in ischemic murine hind limb. Figs. 1A and B show that IL-19 expression is very low, but detectible in non-ischemic hind limb control, but is significantly increased by 505.8% 3 days post-ligation surgery. IL-19 expression declines by 7 days post-ligation surgery but remains elevated above baseline levels. Macrophages are detected 3 days post-ligation, and decrease, but remain detectable 7 days
Discussion
In a previous report using cell culture and ex vivo approaches, we determined that IL-19 had angiogenic potential. The present study extends those data to show that IL-19 can increase perfusion of ligated murine hind-limbs by multiple mechanisms and effects on multiple target cells. A major point of novelty is that unlike other Th2 interleukins, IL-19 appears to have pro-angiogenic effects in ligated murine hind limbs.
IL-19 expression is rapidly induced 3 days post ligation surgery, consistent
Funding
This work was supported by grants HL115575 and HL117724 from the National Heart Lung, and Blood Institute of the National Institutes of Health, and Grant 13GRNT1685003 from the American Heart Association to MVA. K.G. was supported by the American Heart Association Post-doctoral Fellowship 11POST7530001.
Disclosures
The author(s) declare no competing financial interests or any other conflict of interest.
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