Hydrolysis of plasma arginine to citrulline by arginine deiminase (ADI) was recently shown to suppress lipopolysaccharide-induced nitric oxide (NO) synthesis. Since arginine is the precursor of NO, and the latter modulates angiogenesis, we explored whether ADI treatment significantly affected tube-like (capillary) formation of human umbilical vein endothelial cells. Inhibition occurred in a dosedependent manner, both in the chorioallantoic membrane and the murine Matrigel plug assay. Inhibition of angiogenesis by ADI was reversed when a surplus of exogenous arginine was provided, indicating that its antiangiogenic effect is primarily due to arginine depletion, although other pathways of interference are not entirely excluded. Arginine deiminase is also shown to be as a potent inhibitor of tumour growth in vitro as in vivo, being effective at nanogram quantities per millilitre in CHO and HeLa cells. Thus, it could be highly beneficial in cancer therapy because of its two-pronged attack as both an antiproliferative and an antiangiogenic agent.
Low-intensity ultrasound (LIUS) has recently been considered to be an effective method to induce cartilage repair and/or regeneration after injury. Nevertheless, there is no study to provide a cellular mechanism or signal pathways of LIUS stimulation. The current study is designed to investigate the effects of LIUS on the mechanotransduction pathways in C-28/I2, an immortalized human chondrocyte cell line. C-28/I2 cells were treated with LIUS at an intensity of 200 mW/cm2 using Noblelife from Duplogen. The role of stretch-activated channels (SAC) and integrins that are most well-known mechanoreceptors on the chondrocyte cell surface was first examined in mediating the LIUS effects on the expression of type II collagen and aggrecan. When analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, gadolinium (a specific inhibitor of SACs) or GRGDSP (a peptide inhibitor of integrins) specifically reduced the LIUS-induced elevation of type II collagen and aggrecan expressions depending on the incubation time. In addition, the LIUS treatment of C-28/I2 cells induced the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) but not p38 kinase among the members of the mitogen-activated protein kinases (MAPKs). The phosphorylation of ERK by LIUS was repressed by a specific inhibitor of the ERK pathway and integrin function. These results suggest that the LIUS signal might be mediated via canonical mechanoreceptors of SACs and integrins and subsequently through JNK and ERK pathways. The present study provides the first evidence for the activation of the mechanotransduction pathways by LIUS in human chondrocytes.
Mesenchymal stem cells (MSCs) have a capacity to differentiate into the chondrogenic lineage and are a valuable allogenic source for cartilage tissue engineering. However, they still have critical limitations of relatively inefficient chondrogenic differentiation in vitro and of dedifferentiation and/or hypertrophic changes at late stages of differentiation. Numerous approaches using biochemical and mechanical factors have been tried but have so far failed to overcome these problems. Recent studies by other groups and ours have shown that low-intensity ultrasound (LIUS) is an efficient tool for promoting the chondrogenic differentiation of MSCs both in vitro and in vivo. A series of our experiments suggests that LIUS not only induces chondrogenic differentiation of MSCs but also has diverse additional activities that enhance the viability of MSCs, increase possibly the integrity of the differentiated tissues and delays hypertrophic changes during differentiation. Therefore, LIUS could be an innovative and versatile tool for chondrogenic differentiation of MSCs and for cartilage tissue engineering.
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