Our data show that NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-beta signalling through a cGMP-dependent pathway. Our findings suggest that NO may play a beneficial role in atherogenesis in part by limiting vascular calcification.

Urinary exosomes, secreted into urine from renal epithelial cells, are known to contain many types of renal functional membrane proteins. Here, we studied whether renal ischemia-reperfusion (I/R) affects urinary exosomal aquaporin-1 (AQP1) excretion in rats subjected to renal I/R and patients who underwent renal transplantation. Immunoblotting studies demonstrated reduction of the urinary exosomal AQP1 level even at 6 h after renal I/R, and the level continued to be low over 96 h after I/R. Renal AQP1 mRNA and protein analyses revealed that the decreased excretion of urinary exosomal AQP1 is associated with renal AQP1 protein retention in the early phase and with a decreased expression level of renal AQP1 in the later phase of renal I/R injury. Decreased abundance of urinary exosomal AQP1 in a recipient patient was also observed at 48 h after renal allograft transplantation. No significant decrease in urinary exosomal AQP1 was observed in a rat model of nephropathy or in patients with proteinuria. Our studies suggest that the renal AQP1 expression level is possibly controlled by its urinary exosomal excretion and indicate that urinary exosomal AQP1 is a novel urinary biomarker for renal I/R injury.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.

a 2 -antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions. J Thromb Haemost 2006; 4: 1602-10.Summary. Background: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. Methods and results:We performed experimental cutaneous wounding in mice with a deficiency of a 2 -antiplasmin (a 2 AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in a 2 AP-deficient (a 2 AP )/) ) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in a 2 AP )/) mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in a 2 AP )/) mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of a 2 AP )/) mice increased dramatically more than in the WT mice.Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of a 2 AP )/) mice. Conclusion:The lack of a 2 AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. Conclusions: This is the first report to describe the crucial role that a 2 AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of a 2 AP on cutaneous wound healing.

Systemic sclerosis results in tissue fibrosis due to the activation of fibroblasts and the ensuing overproduction of the extracellular matrix. We previously reported that the absence of ␣2-antiplasmin (␣2AP) attenuated the process of dermal fibrosis; however, the detailed mechanism of how ␣2AP affects the progression of fibrosis remained unclear. The goal of the present study was to examine the role of ␣2AP in fibrotic change. We observed significantly higher levels of ␣2AP expression in the skin of bleomycininjected systemic sclerosis model mice in comparison with the levels seen in control mice. We also demonstrated that ␣2AP induced myofibroblast differentiation , and the absence of ␣2AP attenuated the induction of myofibroblast differentiation. Moreover, we found that connective tissue growth factor induced the expression of ␣2AP through both the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways in fibroblasts. Interestingly , ␣2AP also induced transforming growth factor-␤ expression through the same pathways, and the inhibition of ERK1/2 and JNK slowed the progression of bleomycin-induced fibrosis. Our findings suggest that ␣2AP is associated with the progression of fibrosis , and regulation of ␣2AP expression by the ERK1/2 and JNK pathways may be an effective antifibrotic therapy for the treatment of systemic sclerosis.

Summary. Background: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)‐β is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. Methods and results: We examined experimental fibrosis in mice with a deficiency of α2‐antiplasmin (α2AP), which is a potent and specific plasmin inhibitor. We found that the lack of α2AP attenuated bleomycin‐induced TGF‐β1 synthesis and fibrosis. In addition, the production of TGF‐β1 from the explanted fibroblasts of α2AP−/− mice decreased dramatically as compared to that in wild‐type mice. Moreover, we found that α2AP specifically induces the production of TGF‐β1 in fibroblasts. Conclusion: The lack of α2AP attenuated TGF‐β1 synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that α2AP plays in TGF‐β1 synthesis during the process of fibrosis. Our results provide new insights into the role of α2AP in fibrosis.

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Objective. Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-␣2-antiplasmin (␣2AP) complex, and we recently implicated ␣2AP in the development of fibrosis through transforming growth factor ␤ (TGF␤) production. This study was undertaken to clarify how ␣2AP induces TGF␤ production and the development of fibrosis.Methods. To clarify the detailed mechanism by which ␣2AP induces TGF␤ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A 2 (iPLA 2 ) and examined whether ATGL/ iPLA 2 is associated with ␣2AP-induced TGF␤ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of ␣2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSclike fibroblasts from the bleomycin-treated mouse skin and examined the production of TGF␤ and prostaglandin F 2␣ (PGF 2␣ ).Results. We found that ␣2AP binding to ATGL promoted PGF 2␣ synthesis through iPLA 2 in fibroblasts, and the PGF 2␣ synthesis that was promoted by ␣2AP induced TGF␤ production in fibroblasts. In addition, the neutralization of ␣2AP attenuated the production of TGF␤ and PGF 2␣ in SSc-like fibroblasts from mice. The ␣2AP deficiency attenuated bleomycininduced fibrosis and PGF 2␣ synthesis, while the administration of PGF 2␣ to ␣2AP-deficient mice facilitated ␣2AP deficiency-attenuated fibrosis.Conclusion. These findings suggest that ␣2AP regulates the development of fibrosis by PGF 2␣ synthesis through ATGL/iPLA 2 . The inhibition of ␣2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.