Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis-and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

The further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Plasminogen is a critical factor of the tissue fibrinolytic system, which mediates tissue repair in the skin and liver. However, the role of the fibrinolytic system in bone regeneration remains unknown. Herein, we investigated bone repair and ectopic bone formation using plasminogen-deficient (Plg -/-) mice. Bone repair of the femur is delayed in Plg -/-mice, unlike that in the wild-type (Plg þ/þ ) mice. The deposition of cartilage matrix and osteoblast formation were both decreased in Plg -/-mice. Vessel formation, macrophage accumulation, and the levels of vascular endothelial growth factor (VEGF) and transforming growth factor-b (TGF-b) were decreased at the site of bone damage in Plg -/-mice. Conversely, heterotopic ossification was not significantly different between Plg þ/þ and Plg -/-mice. Moreover, angiogenesis, macrophage accumulation, and the levels of VEGF and TGF-b were comparable between Plg þ/þ and Plg -/-mice in heterotopic ossification. Our data provide novel evidence that plasminogen is essential for bone repair. The present study indicates that plasminogen contributes to angiogenesis related to macrophage accumulation, TGF-b, and VEGF, thereby leading to the enhancement of bone repair.

Urokinase-type plasminogen activator (uPA) is implicated in the regulation of hepatic regeneration by activating hepatocyte growth factor (HGF). Here, we investigated its role in the hepatic regeneration after Fas-mediated massive hepatocyte death employing mice deficient in either uPA or its inhibitor, plasminogen activator inhibitor-1 (PAI-1). We measured kinetics of hepatic levels of proliferating cell nuclear antigen (PCNA)-labeling index, plasmin activity, mature HGF, and its phosphorylated receptor, c-Met. In the genetically targeted and wild-type mice, hepatocytes fell into the same extent of apoptosis 6 to 12 hours after an intraperitoneal injection with anti-Fas antibody, as judged from histologic analysis and a histon-DNA enzyme-linked immunosorbent assay (ELISA). In the wild-type mice, mature HGF emerged in the liver 6 hours following anti-Fas injection, and hepatic PCNA-labeling index started to increase following 24 hours and peaked at 48 hours. In the uPA ؊/؊ mice, emergence of mature HGF was delayed 12 hours and hepatic regeneration peaked at 96 hours. Supplementation with the uPA gene to the uPA ؊/؊ mice by in vivo lipofection restored hepatic plasmin levels, and improved a delay in the expression of both mature HGF and phosphorylated c-Met, accompanying a normal rate of liver regeneration. In contrast, PAI-1 ؊/؊ mice showed accelerated liver regeneration; mature HGF emerged as early as 3 hours, and PCNA-labeling index increased at 24 hours. This accelerated regeneration was abolished by administration with anti-HGF antibody. These results strongly suggest a physiologic role of uPA in the proteolytic maturation of HGF, and thereby in hepatic regeneration after Fas-mediated massive hepatocyte death. (HEPATOLOGY 2001;33:569-576.)

No abstract

2-antiplasmin (2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine 2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with theneomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated 2-AP allele was observed, and homozygous deficient (2-AP−/−) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of 2-AP mRNA in kidney and liver from 2-AP−/− mice, in contrast to wild-type (2-AP+/+) mice. Immunologic and functional 2-AP levels were undetectable in plasma of 2-AP−/− mice, and were about half of wild-type in heterozygous littermates (2-AP+/−). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for 2-AP+/+, 2-AP+/−, and 2-AP−/− mice. After amputation of tail or toe tips, bleeding stopped spontaneously in 2-AP+/+, as well as in 2-AP+/− and 2-AP−/− mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin–labeled plasma clots was significantly higher in 2-AP−/− than in 2-AP+/+ mice when injecting clots prepared from 2-AP+/+ plasma (78% ± 5% v 46% ± 9%; mean ± SEM, n = 6 to 7; P = .02) or from 2-AP−/−plasma (81% ± 5% v 46% ± 5%; mean ± SEM, n = 5; P = .008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in 2-AP−/− mice, as compared with 2-AP+/+ mice (P ≤ .005). Thus, 2-AP−/− mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.