The Anti-angiogenic Activity of rPAI-123 Inhibits Fibroblast Growth Factor-2 Functions

Abstract

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrixbound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1 23 , that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1 23 effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. The vascular endothelium is normally maintained in a differentiated, quiescent state. Pro-angiogenic factors destabilize the quiescent endothelium into migratory, proliferative endothelial cells that are attenuated by anti-angiogenic factors. The pro-and anti-angiogenic molecules have cell survival and death functions that are tightly controlled to maintain a balance (1-4).Many negative regulators of angiogenesis are cleavage products of an existing cellular protein that is not inhibitory in its normal intact conformation (5-7). Induction of endothelial cell apoptosis is one characteristic common to these inhibitors (8). Endothelial cells are more susceptible to apoptosis when they are activated (angiogenic) as compared with quiescent (9, 10). Survival of the latter is dependent upon angiogenic growth factors in the local environment, which can be blocked by antiangiogenic factors. If growth factor functions are blocked, endothelial cells are removed by apoptosis (4, 9, 11).Apoptosis can be induced by diverse stimuli that initiate specific signal transduction pathways. The apoptosis pathways are regulated by pro-and anti-apoptotic molecules that are controlled in part by kinases (12) and proteasomal degradation (13). The c-Jun NH 2 -terminal kinase (JNK) 2 signaling pathway is activated by cellular stress, and its role in apoptosis versus survival remains unclear (14). The role of JNK in cellular proliferation, apoptosis, differentiation, and motility is dependent upon the activity and stability of JNK isoforms and their associated substrates (15-18). The cellular function associated with JNK-substrate complexes is tightly regulated by proteasomal activity (19,20).Plasminogen activator inhibitor-1 (PAI-1) has been shown to have both pro-and anti-angiogenic activity (21-24). It has been suggested that PAI-1 pro-angiogenic versus anti-angiogenic activity is based on the relative amounts of the inhibitor that are in active versus inactive conformations (22,25). Proteolytic molecules plasmin (26) and matrix metalloproteinase 3 (27) cleave and inactivate PAI-1. Potential functions of cleaved PAI-1 have not been studied. We made truncated PAI-1 cDNAs and produced truncated PAI-1 proteins (rPAI-1) to investigate potential PAI-1 functions in the absen...

Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice

“…FGF-2 signaling has been associated with neutrophil-mediated angiogenesis 23 and PAI-1 activity. 22 Immunohistochemical analysis of ischemic muscle tissues demonstrated that the number of F4/80 ϩ cells coexpressing FGF-2 or VEGF-A was not significantly different from vehicle-and PAI-1 inhibitor-treated tissues ( Figure 6B). In contrast, the number of ischemic tissue-resident Gr-1 ϩ cells coexpressing both FGF-2 and VEGF-A was higher in sections derived from PAI-1 inhibitor-treated mice ( Figure 6C).…”

“…22 Therefore, in the present study, we investigated whether Although it is clear that FGF-2-induced angiogenesis requires VEGF signaling, 24 it was unclear whether VEGF-A and FGF-2 signaling are required for PAI-1 inhibitor-mediated tissue neoangiogenesis. Our present data suggest that this might be the case, because VEGF-A and FGF-2 mAb prevented PAI-1 inhibitormediated ischemic tissue recovery and neoangiogenesis in an HL-ischemic model ( Figure 6F-G).…”

“…22 To determine whether PAI-1 inhibition accelerates macrophage recruitment into ischemic tissues, we quantified the number of infiltrating F4/80 ϩ cells 3 days after initiation of HL ischemia. PAI-1 inhibition did not increase the recruitment of macrophages in muscle tissues compared with vehicle treatment (Figure 6A).…”

Antiangiogenic plasma activity in patients with systemic sclerosis

“…Two endothelial cell types were used: human dermal microvascular endothelial cells (HDMECs) and bovine aortic endothelial cells (BAECs). Cell migration was assessed using a standard wounding assay (23)(24)(25)(26)(27)(28)(29)(30). Following the wounding procedure of human dermal blood microvascular endothelial cells (Lonza Biologics, Portsmouth, NH), 100 g of plasma proteins from 20 SSc samples and 10 control samples diluted in 1 ml of serum-free endothelial basal medium (EBM) was added to duplicate wells, and the extent of migration was measured 6 hours later.…”

Small molecule PAI-1 functional inhibitor attenuates vascular smooth muscle cell migration and survival: Implications for the therapy of vascular disease

“…The ability of active PAI-1 to inhibit apoptosis, furthermore, is not due to its urokinase PA (uPA) binding or uPA receptor (uPAR) signaling roles (19) suggesting that LRP-1 may be the more relevant “survival” receptor. Truncated PAI-1 (PAI-1 23 ), a mutant with deletions in much of the heparin-binding domain and the RCL, also stimulated endothelial cell apoptosis (20,21). It appears that an intact RCL is required for the pro-survival function of PAI-1 and disruption of this structure promotes an apoptotic phenotype.…”