CXCL6 is an important paracrine factor in the pro-angiogenic human cardiac progenitor-like cell secretome.

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.

Tumor-conditioned Gr-1(+)CD11b(+) myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro.

Gr-1(+)CD11b(+) cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1(+)CD11(+) cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1(+)CD11b(+) cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1(+)CD11b(+) cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1(+)CD11b(+) cells. Incubation of Gr-1(+)CD11b(+) cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1(+)CD11b(+) cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1(+)CD11b(+) myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1(+)CD11b(+) cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.

CXCL6 antibody neutralization prevents lung inflammation and fibrosis in mice in the bleomycin model.

IPF is a chronic, progressive pulmonary disease, leading to respiratory failure. In search of mechanisms of IPF, we used the bleomycin-induced lung-injury model in mice, which causes acute inflammation that may progress to chronic lung inflammation and fibrosis. Here, we asked whether CXCL6/GCP-2, a member of the CXC chemokine superfamily, may be involved in IPF development. First, we reported an increase of CXCL6 levels in BALF from patients with IPF, as well as in the lung of mice, 24 h after bleomycin administration. To investigate whether CXCL6 played a role in experimental bleomycin-induced pulmonary fibrosis, we treated mice with an anti-mCXCL6 mAb that has been shown to inhibit neutrophil chemotaxis in vitro. CXCL6 antibody blockade attenuated acute inflammation with a reduced pulmonary neutrophil influx, IL-1ß, CXCL1, and TIMP-1 production. In the later phase (14 days after bleomycin exposure), lymphocyte recruitment and fibrosis markers, such as collagen and TIMP-1, were diminished, as well as collagen deposition and fibrotic lesion the lung. Therefore, the data suggest that CXCL6 contributes to experimental pulmonary fibrosis, and CXCL6 inhibition might be used to reduce lung toxicity associated with bleomycin treatment.

Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells.

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1beta and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1beta was enhanced synergistically by TNF-alpha but inhibited by IFN-gamma, which synergized with IL-1beta to produce the angiostatic CXCL10/IFN-gamma-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.