Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angio-genesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.Tumor cell invasion and metastatic processes require the coordinated and temporal regulation of a series of adhesive, proteolytic and migratory events 1 . The plasminogen activator (PA)-plasmin proteolytic system has been implicated in these processes. Urokinase-type (uPA) and tissue-type (tPA) plasminogen activators are serine proteases that catalyze the conversion of inactive plasminogen into plasmin, a broadly acting enzyme able to degrade a variety of extracellular matrix proteins and to activate metalloproteinases and growth factors 2,3 . Plasminogen and uPA bind to their specific receptors directing plasmin activity to the migrating tumor cell surface. The activities of PA are directly controlled by specific inhibitors, the PA inhibitors 1 and 2 (PAI1 and PAI2) (ref. 4).Many studies have focused on the role of uPA in cellular invasion and metastasis. Much of the data supporting the role of uPA in these events derives from in vitro and in vivo experiments demonstrating a correlation between uPA expression and cell invasion and metastasis as well as reduction of metastatic potential by using natural or synthetic serine protease inhibitors, neutralizing antibodies to uPA or antisense oligonucleotides 5,6 . PAI1 may also be directly involved in cancer progression. Both tumor cells and capillary endothelial cells express higher levels of PAI1 than other cell types [7][8][9] . Surprisingly, this inhibitor is necessary for optimal invasion of cultured lung cancer cells 10 , and an increasing number of clinical studies have demonstrated that high PAI1 levels indicate a poor prognosis for the survival of patients suffering from a variety of cancers [11][12][13] . However, as PAI1 is an acute-phase reactant 14 , it remains undetermined whether the increased PAI1 levels causally contribute to, or rather are the consequence of, the malignancy.Various observations indicate that the PA system may provide both surface-associated protease activity and an adhesion mechanism for cells through interaction with vitronectin. Deng et al. suggested that the balance between cell adhesion and cell detachment is governed by PAI1 (ref. 15). The PAI1-mediated release of cells attached to vitronectin seems to occur independently of the abili...

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.