Vascular homeostasis depends on the balance of at least two main systems: VEGF/VEGF receptor (VEGFR) and the Ang-Tie ligand-receptor system. Numerous other factors are involved, including endostatin, angiostatin, PDGF and fibroblast growth factor-2.

VEGF, one of the main transcriptional targets of HIF-1α, is the primary cytokine associated with angiogenesis. In addition to HIF-1α and chronic hypoxia, other inducers of VEGF in SSc are interleukin-1β, PDGF and transforming growth factor (TGF)-β (16). The VEGF family includes VEGF-A, -B, -C and -D and placenta growth factor. VEGF-A (usually referred to as VEGF) is released by fibroblasts, macrophages, endothelial cells and T cells and is involved in angiogenesis at many levels (17). The effects of VEGF are regulated by its three tyrosine kinase receptors (VEGFR-1/Flt-1, VEGFR-2/Flk-1 and VEGFR-3). VEGFR-2/Flk-1 is required for the mitotic response of endothelial cells to VEGF (18). Endostatin is the most potent inhibitor of VEGF-induced angiogenesis (17).

AO in SSc is associated with increased osteoclastogenesis and higher VEGF levels (19,20). In patients with SSc the proangiogenic VEGF-A and its receptors are paradoxically overexpressed despite insufficient angiogenesis, correlating with disease progression and fingertip ulcer development (19,20). An explanation could be the overexpression of VEGF 165b, an inhibitory splice variant of VEGF leading to insufficient angiogenesis; in patients with SSc, VEGF165b correlates with the extent of videocapillaroscopic damage and loss (21). The typical capillaroscopic changes in SSc have been interpreted as failed angiogenic attempts following VEGF stimulation, resulting in a disturbed capillary network (19).

The Ang-Tie ligand-receptor system is important in the regulation of vascular integrity and quiescence. The constitutive Ang-1/Tie2 interaction is the default vascular homeostasis control pathway, while Ang-2 acts as a dynamically regulated vessel-destabilizing cytokine (22).

The Ang-Tie ligand-receptor comprises two receptor kinases, Tie1 and Tie2, and four corresponding ligands, Ang-1, −2, −3 and −4 (23). Ang-1 inhibits VEGF-induced formation of blood vessels, the expression of adhesion molecules and TNF-α-stimulated leukocyte transmigration (22). Ang-2, a Tie2 antagonist, which is almost undetectable in endothelium at rest, is rapidly induced during activation, resulting in endothelial destabilization required for angiogenesis and/or inflammation in the presence of VEGF, while in the absence of VEGF Ang-2 causes vascular regression (24). Ang-1 is significantly decreased while serum Ang-2 is substantially elevated in SSc, particularly in patients with a ‘late’ nailfold videocapillaroscopy pattern, correlating with erythrocyte sedimentation rate and C-reactive protein levels, and lung involvement (25). The Ang-1/Ang-2 imbalance in patients with SSc suggests a shift toward vascular regression and angiostasis (26).

Bone remodeling is a dynamic process that requires functional coordination between osteoclasts, osteoblasts and osteocytes. In SSc the generalized prevalence of osteoporosis appears to be increased, involving several humoral and cellular players (4,9).

Angiopoietin-like proteins (ANGPTLs) are a family of proteins with structural similarities to the angiopoietins, which do not bind to Tie receptors, and have roles in lipid and glucose metabolism, inflammation, hematopoiesis and cancer (27). Of these, ANGPTL4, regulated by HIF-1α, is able to stimulate osteoclasts even when HIF-1α is deficient (28). Its N-terminal fragment inhibits angiogenesis, while the C-terminal fragment modulates cell adhesion. ANGTL4 is overexpressed in the osteoclasts, synovial cells, synovial fluid and serum of patients with rheumatoid arthritis (RA), suggesting its involvement in RA erosions (29). Although arthritis is an independent predictive factor for disease progression in patients with early SSc. it seems unlikely that SSc-associated AO is due to synovial ANGPTL4 over-expression driving osteoclast-mediated bone resorption, as AO is associated with vascular involvement rather than synovitis (2,29). To date, there are no studies in which the ANGPTL4 level in SSc has been assessed. However, ANGPTL4 and ANGPTL3 share numerous features, and the level of ANGPTL3 has been found to be increased in patients with SSc, and associated with the prevalence of digital ulcers, suggesting the involvement of ANGPTL3 in the pathogenesis of SSc-associated microangiopathy (30). In this regard, clarifying the role of ANGPTLs in SSc may lead to further understanding of the complex SSc pathogenesis.

Patients with SSc and pAO exhibit increased osteoclastogenesis, associated with elevated VEGF plasma levels (20). Osteoclasts are terminally differentiated cells derived from cells with a monocyte/macrophage lineage. Hypoxia acts as a major stimulator of osteoclast formation and bone resorption. The secretion of VEGF-A by hypoxic osteoclasts, regulated by RANKL-mediated activation of HIF-1α, is dependent on osteoclast size (31). However, osteoclasts express VEGFR1 (Flt-1) and, to some extent, VEGFR2 (Flk-1). Thus, local hypoxia could indirectly influence osteoclastogenesis via autocrine and paracrine secretion of VEGF under the control of HIF-lα (31). The direct role of the VEGF-VEGFR system in osteoclastogenesis and activity was demonstrated in a study of osteopetrotic mice, characterized by the absence of functional macrophage colony-stimulating factor (M-CSF) resulting in severe osteoclast deficiency (32). The results revealed that M-CSF and VEGF play almost overlapping roles in osteoclastic bone resorption Moreover, endostatin, a potent angiogenic inhibitor, also inhibits VEGF-stimulated osteoclastic bone resorption (33). In a study of patients with SSc, endostatin was found to be increased at all stages, while angiostatin, a platelet-derived angiogenesis inhibitor, was increased only later in the disease, and was associated with osteoarticular and lung involvement (34).

Osteocytes, embedded in bone matrix and isolated from vessels, act as mechanical sensors, mediated by hypoxia. Mechanical unloading, which may occur during advanced SSc, results in increased numbers of HIF-1α-expressing osteocytes (35). Hypoxic osteocytes positively regulate osteoclastic differentiation through the secretion of growth differentiation factor 15, regulated by HIF-1α (36). Hypoxia also decreases sclerostin secretion by osteocytes during bone remodeling (22). It may be hypothesized that osteocytic hypoxia and mechanical unloading may both be present in patients with SSc and severe hand involvement, contributing to AO.

The osteoblast has a central role in the control of VEGF-mediated angiogenesis in bone. Osteoblasts express VEGF and both VEGF-A receptors, which are upregulated by hypoxia. The overexpression of HIF-1α by osteoblasts leads to increased angiogenesis and osteogenesis, coupled by osteoblast-generated VEGF (37). Wnt signaling is osteogenic by promoting mesenchymal stem cell differentiation, and its inactivation leads to osteoporosis (37). Also, the endothelial-myofibroblast transition involves the canonical Wnt and Notch signaling pathways, and dysregulated Wnt signaling is involved in the pathogenesis of SSc-associated fibrosis (37). The Wnt canonical pathway is activated by TGF-β (38). The involvement of Wnt in SSc-related AO is currently unclear, but HIF-1α and the osteoblast-specific transcription factor Osterix in osteoblasts synergistically inhibit the Wnt pathway (39). Bone morphogenic proteins (BMPs) are modulators of the Wnt pathway, while sclerostin and dickkopf-1 are endogenous Wnt pathway antagonists (37,40). Crosstalk exists between the Wnt pathway and other signaling pathways, including the Notch signaling pathway. Wnt and Notch are overexpressed in SSc (37,38). Notch pathway activation inhibits Wnt-induced osteogenesis (37). Notch signaling is activated in SSc, playing an important role in fibrosis (40), but its contribution to AO is not known. However, in Hajdu-Cheney syndrome, a rare disease evolving with AO due to Notch2 gain-of-function mutation, osteoclast hyperactivation along with endothelial impairment are involved (41).

TGF-β/BMP signaling has critical regulatory functions in osteoblast differentiation and bone formation, in addition to angiogenesis and endothelial cell-vascular smooth muscle cell interactions, and TGF-β/BMP signaling is the master regulator of fibrosis in SSc (42). Connective tissue growth factor (CTGF) negatively regulates BMP-2-induced signaling and osteoblast differentiation, and in SSc CTGF is profibrotic, along with TGF-β (43).

The main hypoxia-activated participants that are possibly involved in SSc-related AO and their effects on the endothelial and bone cells are summarized in Table I.