Endothelin-1 promotes osteoprogenitor proliferation and differentiation in fetal rat calvarial cell cultures

Endothelin-1 (ET-1), a peptide produced by vascular endothelial cells, has been suggested to be one of the signaling factors between vascular and osteoblastic cells during bone growth and remodeling. The osteoinductive effects of ET-1 were tested on fetal rat calvaria which have the ability to form bone nodules in culture. ET-1 (10(-10) to 10(-6) M) dose-dependently increased cell proliferation. The effect of ET-1 (10(-8) M) on proliferation was greater than that of dexamethasone (Dex; 10(-8) M). ET-1 also increased the number of bone nodules by 146% over untreated cells, which coincided with a 3.1-fold increase in alkaline phosphatase activity. Limiting dilution assays showed that ET-1 treatment increased the number of osteoprogenitors (CFU-AP and CFU-OB) beyond what would be expected by a proliferative effect alone, indicating that ET-1 also stimulated osteoblast differentiation. Osteocalcin mRNA expression was upregulated as shown by Northern blot analysis. Using cDNA microarray analysis, ET-1 treatment resulted in an expression profile that included an upregulation of 163 genes and expressed sequence tags. Simultaneous addition of ET-1 and Dex to the medium further increased the number of bone nodules and alkaline phosphatase activity over either treatment alone. Our results show that ET-1 promotes both osteoblastic proliferation and differentiation and that the effects of ET-1 and Dex on differentiation are cooperative.     

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFA^fl/fl) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFA^fl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFA^fl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFA^fl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.     

Bone morphogenetic protein 2 induces placental growth factor in mesenchymal stem cells

Bone-forming osteoblasts differentiate from pluripotent mesenchymal stem cells (MSCs) in a multistage process that can be modeled in vitro using MSCs isolated from adult human trabecular bone or bone marrow. To identify new genes involved in osteoblast differentiation, we have performed large-scale gene expression profiling using high-density cDNA microarrays in primary human MSCs treated with the known osteogenic agent bone morphogenetic protein 2 (BMP-2). The vascular endothelial growth factor (VEGF) family member placental growth factor (PlGF) was found as an early regulated gene whose induction was already detected after 2 h treatment with BMP-2. Tissue distribution analysis of PlGF mRNA expression using microarrays revealed a very restricted expression of PlGF only in BMP-2-treated MSCs and in placenta as expected. Ribonuclease protection assay (RPA) confirmed the induction of PlGF and showed preferential expression of the PlGF-1 isoform over PLGF-2 in MSCs and MG63 cells. BMP-2 stimulated PlGF expression in MG63 cells with an EC50 of about 50 ng/ml and mRNA levels peaked between 24 and 32 h after stimulation. Furthermore, induction of PlGF by BMP-2 appeared specific, as other osteogenic agents including vitamin D3, transforming growth factor beta, and basic fibroblast growth factor were inactive. BMP-2 stimulated PlGF secretion from MG63 and MSC cells, but PlGF had no effect on MSC proliferation and osteoblastic differentiation. Based on the known function of PlGF in the recruitment of endothelial and hematopoietic stem cells, these results suggest a paracrine role for MSC-derived PlGF in the angiogenesis and hematopoiesis that accompany BMP-2-induced bone formation.     

Cyclic mechanical strain increases production of regulators of bone healing in cultured murine osteoblasts

BACKGROUND: The adaptive response of bone to mechanical strain, for which angiogenesis is required, is underscored during fracture healing. Vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-beta1) are critical regulators of angiogenesis. The purpose of this study was to examine the effect of strain on the production of VEGF and TGF-beta1. STUDY DESIGN: MC3T3-E1 mouse osteoblasts underwent cyclic strain (low, 0.1 Hz, or high, 0.2 Hz) for 24 or 48 hours. VEGF and TGF-beta1 protein levels were determined by ELISA, and Northern blot analysis was performed for VEGF mRNA. Alkaline phosphatase (an osteoblast differentiation marker) activity was determined by functional enzymatic assay. All measurements were standardized for cell number by crystal violet colorimetric assay. Statistical significance was determined by t-test, ANOVA, and the Tukey-Kramer test. RESULTS: Protein production of VEGF and TGF-beta1 was dose-dependently elevated by strain (p < 0.05); alkaline phosphatase did not rise significantly. Northern blot analysis of strained osteoblast cells demonstrated increased VEGF mRNA. Cyclic strain was found to be progressively destructive in a dose-dependent manner, causing 51% and 70% decreases in cell number under low and high strain, respectively (p < 0.01). CONCLUSIONS: We demonstrated simultaneous, dose-dependent increases in VEGF and TGF-beta1 protein production by osteoblastic cells in response to increasing strain. VEGF mRNA also increased in response to strain. This strain-induced increase in angiogenic cytokines suggests a potential mechanism by which injured bone may recruit a new blood supply. But we also found increasing strain to increase cellular toxicity, suggesting that cyclic mechanical strain may select for a subpopulation of osteoblasts.     

rhBMP-2, rhVEGF(165), rhPTN and thrombin-related peptide, TP508 induce chemotaxis of human osteoblasts and microvascular endothelial cells.

Osteogenesis and angiogenesis are inter-linked and tightly regulated processes involved in growth, repair, and bone remodeling. Bone morphogenic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), pleiotrophin (PTN) and thrombin-related peptide, TP508 have all been found to have the ability to promote bone fracture healing by enhancing both the osteogenesis and angiogenesis processes. One of the underlying mechanisms proposed is that mediators for osteogenesis may also be involved in mediating angiogenesis and vice versa. The aim of this study was to examine the chemotactic effects of rhBMP-2, rhVEGF(165), rhPTN and TP508 on human osteoblasts and endothelial cells. Using a direct-viewing chemotaxis assay system, we report for the first time, the direct quantitative observation of chemotaxis of both human osteoblastc cells and microvascular endothelial cells towards sources of rhBMP-2, rhVEGF(165), rhPTN and TP508. This study confirmed that rhBMP-2, rhVEGF(165), rhPTN and TP508 have chemotactic effects on both human osteoblastic and endothelial cells, indicating that these factors are directly involved in promoting angiogenesis and osteogenesis by recruiting osteoblasts and endothelial cells via chemotaxis.     

血管内皮生长因子在甲状腺癌的表达及意义

血管内皮生长因子特异性作用于血管内皮细胞,通过促进内皮细胞的增生,增加血管通透性,以诱导肿瘤血管及淋巴管生成,在肿瘤的发生发展中起到关键作用.研究表明,VEGF及其受体的表达与甲状腺癌的发生,淋巴结转移,预后密切相关,抑制VEGF及其受体的表达在甲状腺未分化癌及髓样癌的治疗中有一定的应用前景.     

Angiogenesis induced by the implantation of self-bone marrow cells: a new material for therapeutic angiogenesis

Bone marrow, contains various primitive cells that are thought to secrete several angiogenic growth factors and may also differentiate into endothelial cells. The present study was conducted to investigate the possibility that bone marrow cells could be a novel material to induce angiogenesis. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rat bone marrow cells was examined by immunohistochemistry. The production of VEGF was compared in tissue culture supernatant under the conditions of normoxia and hypoxia. The process of angiogenesis that occurred following the implantation of bone marrow cells was determined using a rat cornea model. VEGF- and bFGF-positive cells were found in rat bone marrow. The production of VEGF from bone marrow cells was significantly more enhanced by hypoxic conditions than by normoxic conditions. The rat cornea model showed that bone marrow cell implantation created new vessels. The implantation of self-bone marrow cells is a novel and simple method of inducing angiogenesis.     

Selective inhibition of endothelin receptor A as an anti-angiogenic and anti-proliferative strategy for human pancreatic cancer

Endothelin-1 (ET-1) plays a major role in tumor proliferation and angiogenesis of various types of cancer acting through endothelin receptors A and B (ETRA and ETRB). The aim of this study was to analyze theET-1/ETRsystem inhumanpancreatic cancer cell linesandto evaluate the effect of a selective endothelin A inhibitor in vitro and in vivo in an orthotopic mouse model. Three different human pancreatic cancer cell lines, MiaPaCa-2, AsPC-1, and Panc-1, were studied. We found that proliferation of human pancreatic carcinoma cells expressing ETRA was significantly reduced with a selective antagonist. Hypoxic conditions led to improved results compared to a normoxic environment (MiaPaCa-2: -53% vs. -18%; AsPC-1: -54% vs. -46%). Proliferation of ETRA negative Panc-1 cells was not decreased. In vivo, the selective ETRA inhibition resulted in reduced angiogenesis as measured by lower microvessel densities (MiaPaCa-2: -47%; AsPC-1: -55%). The blockade of ETRA decreased the volume (MiaPaCa-2: -87%; AsPC-1: -28%) and metastatic spread (MiaPaCa-2: -95.5%; AsPC-1: -27%) of receptorpositive tumors, thereby increasing survival in experimental pancreatic cancer. ETRA blockade did not show an effect on ETRA negative Panc-1 tumors. Therefore, targeting ETRA with a selective antagonist might provide a new approach to reducing proliferation and angiogenesis in human pancreatic cancer. Presented in part at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida, May 18–21, 2003. This work was supported by the R.S. Hirshberg Foundation and the Deutsche Forschungsgemeinschaft (Grant HO 1843/2-1).     

血管内皮生长因子重组腺病毒转染鼠骨髓基质细胞及其产物促内皮细胞增殖的研究

目的探讨腺病毒介导血管内皮生长因子165(VEGF165)基因转染骨髓基质细胞(BMSCs)后对VEGF基因的转录、表达及其产物生物活性的影响。方法体外培养4只大鼠骨髓基质细胞,用携带VEGF165基因的重组腺病毒转染培养细胞;通过逆转录聚合酶链反应、免疫印记法和酶联免疫吸附法检测VEGF在转染细胞内的转录、表达和细胞外分泌情况;通过血管内皮细胞增殖实验测定转染VEGF165基因后的BMSCs培养上清中VEGF蛋白的生物活性。结果腺病毒介导VEGF165基因转染BMSCs后可以获得有效的转录及表达,其分泌于培养上清中的表达产物可明显促进大鼠主动脉血管内皮细胞的增殖(P<001),具有很强的生物学活性。结论腺病毒可安全、有效地转染BMSCs,VEGF165基因转染的鼠BMSCs可有效表达具有生物活性的VEGF蛋白。     

Enhanced cytoprotection and angiogenesis by bone marrow cell transplantation may contribute to improved ischemic myocardial function.

OBJECTIVES: Heat shock proteins (HSPs) are cytoprotective proteins. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor. This study aimed to elucidate the possible role of cytoprotection and angiogenesis on cardiac function after bone marrow cell transplantation (BMT). METHODS: Myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation. A total of 5 x 10(6) bone marrow-mononuclear cells were transplanted into the ischemic zone by direct injection. At 1, 3, 7, 14 and 28 days post-transplantation, cardiac function was evaluated by echocardiography. The expressions of HSP32, HSP70 and VEGF were assessed by immunofluorescence and RT-PCR. The number of vessels was examined by immunohistochemistry. The differentiation of the transplanted cells was determined by immunofluorescence. RESULTS: Echocardiography showed BMT led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction and fraction of shortening. Immunofluorescence revealed that the expressions of HSP32, HSP70 and VEGF were promoted in both transplanted bone marrow cells and recipient cardiomyocytes. RT-PCR showed that the mRNA expression levels of HSP32, HSP70 and VEGF in the BMT group were markedly higher in comparison with injection of peripheral blood cells or saline (P<0.01) by day 7. Seven days later, the vessel count showed that angiogenesis had been induced to a significantly greater degree in the BMT groups. Fourteen days later, specific markers for myocardial or vascular endothelial cells were detected in the transplanted bone marrow cells. CONCLUSIONS: BMT upregulated the expressions of HSP32, HSP70 and VEGF in both transplanted bone marrow cells and recipient endogenous cardiomyocytes in the early phase post-transplantation. This enhanced cytoprotection and angiogenesis, and contributed to the functional recovery following cardiac infarction. In the late phase, the transplanted bone marrow cells might differentiate into both myocardial and vascular endothelial cells that enhanced the ischemic cardiac function further.