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.

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)–deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1–deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism.