Angiogenesis is the process of new capillary blood vessels growth and is instrumental under both physiologic and pathologic conditions. Physiologic conditions include embryogenesis, growth, tissue repair after injury and the female reproductive cycle whereas pathologic angiogenesis can occur in chronic inflammatory and fibroproliferative disorders and tumorigenesis of cancer. Angiogenesis is similar to but distinct from vasculogenesis which describes the de novo formation of blood vessels from angioblasts or endothelial progenitor cells, process that mostly occurs during embryogenesis 11. On the other hand, angiogenesis describes the sprouting of new vessels from pre-existing vasculature which can occur both in embryonic and adult life. The regulation of angiogenesis is determined by a dual, yet opposing balance of angiogenic and angiostatic factors that promote or inhibit neovascularization, respectively.

Molecules that originally promote angiogenesis include members of the CXC chemokine family, characteristically heparin binding proteins which on structural level have four highly conserved cysteine amino acid residues, with the first two cysteines separated by one nonconserved amino acid residue. CXC chemokines display unique diverse roles in the regulation of angiogenesis resulting from dissimilarity in structure. Therefore, members that contain in the NH₂-terminus a three amino-acid motif (ELR) such as IL-8/CXCL8, epithelial neutrophil activating protein (ENA)-78/CXCL5, growth-related genes (GROs, a, β, γ/CXCL1, 2, 3), granulocyte chemotactic protein (GCP)-2/CXCL6 and neutrophil activating protein (NAP)-2/CXCL7, originally promote angiogenesis 1112. There are two candidate CXC chemokine receptors that mediate this effect: CXCR1 and CXCR2 1112. Another crucial promoter of angiogenesis is vascular endothelial growth factor (VEGF) a dimorphic glycoprotein with multifunctional roles in both the development of vasculature and the maintenance of vascular structure and function 1314. Its expression is induced when most cell types are subjected to hypoxia 15. Finally, another positive regulator of aberrant vascular remodeling in pulmonary fibrosis is basic fibroblast growth factor (bFGF) which has been shown originally to stimulate the proliferation of cells of mesodermal origin, including fibroblasts 16. In addition, it has been shown that inappropriate expression of bFGF can result in tumor production through promotion of uncontrolled cell proliferation and aberrant angiogenesis 171819.

By contrast, other members of the CXC chemokine family that do not contain the angiogenic ELR motif (ELR-) behave as potent inhibitors of angiogenesis. Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit aberrant angiogenesis. Furthermore, the angiostatic ELR- members of the CXC chemokine family include the interferon (IFN)-γ inducible protein (IP)-10/CXCL10, monokine induced by IFN-γ (MIG)-2 and IFN-γ-inducible T-cell a chemoattractant (ITAC)/CXCL11 1112. The latter inhibit angiogenesis via interaction with the specific CXC chemokine receptor CXCR3 which is expressed in Th1 and natural killer (NK) cells. Additionally, pigment epithelium-derived factor (PEDF) is an inhibitor of new vessel formation, first described in retinal pigmented epithelial cells during diabetic retinopathy and then in young proliferating fibroblasts 20. Its expression in retinal cell lines has been documented to be directly regulated by VEGF 21. PEDF angiostatic activities are specific for new developing vessels and its expression has been detected in kidney, pancreas, prostate, pleura, testes, bone, within peripheral blood cells and recently in lung 21.

Several transcription factors play instrumental role in promoting angiogenesis and sensing the environmental cues that drive this process. Strieter et al. 22 identified two transcription factors that stand out and appreciated the "master switches" that control aberrant angiogenesis. These are nuclear factor-κB (NF-κB) and hypoxia inducible factor-1a (HIF-1a). Both factors are under strict regulation. NF-κB plays an essential role as a "master switch" in the transactivation of angiogenic CXC chemokines as shown in detail for CXCL8 (Figure 1). Generation of reactive oxygen species activates NF-κB and sets in motion a process that releases NF-κB in the cytoplasm and leads to its translocation into the nucleus where it binds with the promoters of angiogenic CXC chemokines resulting to the activation of target genes 23. In addition, it has been shown that VEGF promotes the expression of angiogenic chemokines (i.e CXCL8) from endothelial cells in an autocrine and paracrine way 13 (Figure 1). On the other hand, HIF-1a serves as a critical transcription factor for cellular and systemic oxygen homeostasis. Under hypoxic conditions HIF-1a is subsequent to activation and translocation into the nucleus. There it dimerizes with HIF-1b and the heterodimer recognizes the hypoxia response element found in the promoter region of several target genes (i.e VEGF) resulting to gene expression (Figure 1) 2425.

The past ten years parallels have been drawn between the biology of cancer and pulmonary fibrosis. The unremitting recruitment and maintenance of the altered fibroblast phenotype with generation and proliferation of immortal myofibroblasts is reminiscent with the transformation of cancer cells 262728293031323334353637. A hallmark of tumorigenesis is the production of new blood vessels to facilitate tumor growth. A number of novel treatments targeting angiogenesis are in varying stages of clinical development for cancer 38. On the other hand several chronic fibroproliferative disorders including IIPs are associated with aberrant angiogenesis 39. In parallel with the biology of the fibroblast proliferation and deposition of ECM in IIPs, a considerable number of studies have examined the role of angiogenesis/vascular remodeling in wound healing and its contribution to the fibroproliferation and ECM deposition characterizing these disorders 39.

There is increasing evidence supporting the notion that vascular remodeling in fibroproliferative disorders appears to be regulated by an imbalance between angiogenic and angiostatic factors. Seminal observation implicating angiogenic activity as an important aspect of progressive fibrosis was originally made by Turner-Warwick in 1963, when she demonstrated the presence of anastomoses between the systemic and pulmonary microvasculature in lungs of patients with IPF 40. Despite these data suggesting a potential role of neovascularization in fibrogenesis, the exact contribution of aberrant vascular remodeling to the progression of fibrosis has been, so far, largely ignored. On the other hand, the pathology of IPF demonstrates temporal and regional heterogeneity and presents with distinct pathogenetic components compared to other IIPs that may explain major discrepancies in terms of clinical course, prognosis and responsiveness to treatment. On the basis of this conception, the last decade, a number of reports addressed intriguing questions arising from the above data. These include the following: 1) Is the primary vascular abnormality a lack or an excess of neovascularization and consequently what is the role of angiogenesis in the fibrotic process? 2) Is there any association of vascular remodeling with the histopathologic pattern of the IIP? or 3) any correlation with parameters of disease severity?

The vascular remodeling phenomenon has been also described in the experimental model of bleomycin-induced pulmonary fibrosis. The role of neovascularization during the pathogenesis of experimental pulmonary fibrosis was originally raised by Peao and coworkers 60. In line with human data 40 investigators reported aberrant vascular remodeling in the peribronchial areas of the lungs proximal to fibrotic regions and accompanied by architectural distortions of the alveolar capillaries. While these eloquent studies implicated the presence of angiogenesis in the pathogenetic cascade of IPF, so far, there have been no investigations to delineate factors that regulate neovascularization and subsequent fibrosis. To demonstrate proof of the principle that CXC chemokines regulate angiogenic and angiostatic activity in IPF, Keane et al. effectively assessed the relevance of macrophage inflammatory protein 61 and CXCL10 62 with the augmented net angiogenic activity in the in vivo model of pulmonary fibrosis. Neutralization of (MIP)-2 attenuated both angiogenic activity and the fibrotic response to bleomycin, whereas a relative deficiency of IFN-γ inducible angiostatic regulator CXCL10 was also noted. In addition, systemic administration of CXCL10 inhibited fibroplasia and angiogenesis, supporting the premise that aberrant angiogenesis enhances fibroblast proliferation and ECM deposition. In agreement with these findings, Burdick et al. 63 stated that instillation of the angiostatic CXCL11 produced a marked decrease of fibrotic areas and an attenuation of the dysregulated vascular remodeling.

Fueled by the prospect that anti-angiogenic treatment could be beneficial for pulmonary fibrosis, Hamada et al. 64 tested the efficacy of anti-VEGF gene therapy in the bleomycin model of pulmonary fibrosis. Administration of a specific VEGF receptor that blocks its activity produced a significant anti-fibrotic, anti-inflammatory and anti-angiogenic effect, suggesting an important role for VEGF through its versatile properties. In addition, Jiang et al. 65 used CXCR3 deficient mice and delineated potential mechanisms through which the CXCR3/CXCR3-ligands biological axis exerts a protective role by shifting the Th equilibrium toward resolution of the injurious response. Moreover, Belperio et al. 52 by using a murine model of bronchiolitis obliterans syndrome (BOS) conducted a proof-of-concept analysis and demonstrated that multiple angiogenic CXC chemokines and their receptors (CXCR2) are involved in a dual fashion in the pathogenetic pathway of experimental BOS.

The latter results have clear therapeutic implications since inhibition of angiogenic mediators or administration of angiostatic chemokines reduced lung collagen deposition and attenuated the exaggerated matrix remodeling. On the basis of this concept, neutralization of proangiogenic environment should be pursued. However, the latter statement should be treated with caution for the following reasons: 1) Findings derived from the bleomycin model of pulmonary fibrosis may not be applicable to human disease since pathogenetic components seen in bleomycin-induced pulmonary fibrosis do not demonstrate areas compatible with fibroblastic foci, the leading edge of human fibrosis. In addition, there are clear limitations to this model in terms of its self-limiting nature, the rapidity of its development and the close association with inflammation that accompanies the lung injury 66. Regarding the experimental model of BOS, it also presents with substantial weaknesses due to its heterotopic positioning, discounting the influence of adjacent airway mucosa 52. 2) Moreover, the aforementioned studies were unable to investigate the complete angiogenic and angiostatic network involved in the pathogenesis of the disease. Therefore, results could be misleading due to the lack of knowledge of a variety of mediators that may have a direct effect on fibroblast proliferation and collagen gene expression. Therefore, their potential contribution to vascular and matrix remodeling can not be excluded. Maybe an investigation of several angiogenic pathways in a single experiment could help us circumvent this problem. However, the above limitations are not to diminish the scientific value and accuracy of these studies but to underline the necessity for further analyses using more representative experimental models in combination with human studies.