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.

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1(G93A) rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1(G93A) mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1(G93A) rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

The effects of plasminogen activator inhibitor-l (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-i-deficient (PAT-I-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1i--and from PAI-1 wild type (PAI-i 1+) mice underwent limited but significantly different (P < 0.001 ) spontaneous lysis within 3 h (6±1 vs 3±1%, respectively). A 25-A1I '25-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47±5, 66±3, and 87±7% within 8 h in PAI-1 +/+, heterozygous PAT-i-deficient (PA-i +1-), and PAT-i -mice, respectively (P = 0.002 for PAI-i 1+ vs PAI-1-i-mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PA-i +'+ and PAT-I-mice, were 35±5 and 91±3% within 4 h, respectively (P < 0.001). 11 out of 26 PA-i +'+ but only 1 out of 25 PAI-1-i-mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 ,ug endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PA -I -mice after partial amputation of the tail or of the caecum.Thus, disruption of the PAI-i gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis. (J. Clin. Invest. 1993Invest. . 92:2756Invest. -2760

Background —The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg −/− ), in tissue or urokinase plasminogen activator (tPA −/− or uPA −/− ), or in plasminogen activator inhibitor-1 or α 2 -antiplasmin (PAI-1 −/− or α 2 -AP −/− ). Methods and Results —FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6±1.1 mm 3 (mean±SEM), uPA −/− mice had similar infarcts (7.8±1.0 mm 3 , P =NS), tPA −/− mice smaller (2.6±0.80 mm 3 , P <0.0001), PAI-1 −/− mice larger (16±0.52 mm 3 , P <0.0001), and Plg −/− mice larger (12±1.2 mm 3 , P =0.037) infarcts. α 2 -AP −/− mice had smaller infarcts (2.2±1.1 mm 3 , P <0.0001 versus wild-type), which increased to 13±2.5 mm 3 ( P <0.005 versus α 2 -AP −/− ) after intravenous injection of human α 2 -AP. Injection into α 2 -AP −/− mice of Fab fragments of affinospecific rabbit IgG against human α 2 -AP, after injection of 200 μg human α 2 -AP, reduced FCI from 11±1.5 to 5.1±1.1 mm 3 ( P =0.004). Conclusions —Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity ( tPA gene inactivation) reduces whereas its augmentation ( PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity ( Plg gene inactivation or α 2 -AP injection) increases whereas its augmentation (α 2 - AP gene inactivation or α 2 -AP neutralization) reduces infarct size. Inhibition of α 2 -AP may constitute a potential avenue to treatment of FCI.