Iatrogenic acute limb ischemia with complete traumatic rupture of the popliteal artery associated with total knee arthroplasty.

Acute limb ischemia (ALI) related to total knee arthroplasty (TKA) is rare. Most occlusions are caused by thrombus formation in the popliteal artery (PA). Currently such cases are revascularized using less invasive approaches such as endovascular therapy or Fogarty thrombectomy. We report a case of ALI in a 65-year-old woman with complete rupture of the PA due to a TKA procedure. She had resting pain and motor paralysis in her right lower extremity after TKA. Contrast-enhanced computed tomography showed occlusion of the right femoropopliteal artery. Subsequently, she was referred to our hospital with a diagnosis of ALI. Initially, a less invasive revascularization procedure was unsuccessfully attempted. Therefore, we performed an emergency distal bypass and succeeded in revascularization. Intraoperative examination revealed a complete rupture of the PA. Postoperatively, the patient exhibited no signs of myonephropathic metabolic syndrome. Although there was significant motor impairment, the affected limbs were successfully salvaged. ALI with complete rupture of the PA associated with TKA has not been reported previously. In cases of iatrogenic ALI after TKA, it would be essential to consider diagnostic and revascularization methods that account for the possibility of severe injury to the PA. Learning objective: Acute limb ischemia after total knee arthroplasty is a rare and life- and limb-threatening condition. The underlying pathological mechanism is often thrombus occlusion due to mechanical stimuli of the popliteal artery (PA). There are no established treatments for this condition, and less invasive approaches such as endovascular procedures and Fogarty thrombectomy are often used. However, in cases involving severe damage to the PA, bypass surgery may be necessary, and revascularization procedures should be considered accordingly.

TGF-ß signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells.

Histological distinction between enchondroma and chondrosarcoma is difficult because of a lack of definitive biomarkers. Here, we found highly active transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signalling in human chondrosarcomas compared with enchondromas by immunohistochemistry of phosphorylated SMAD3 and SMAD1/5. In contrast, the chondrogenic master regulator SOX9 was dramatically down-regulated in grade 1 chondrosarcoma. Paternally expressed gene 10 (PEG10) was identified by microarray analysis as a gene overexpressed in chondrosarcoma SW1353 and Hs 819.T cells compared with C28/I2 normal chondrocytes, while TGF-ß1 treatment, mimicking higher grade tumour conditions, suppressed PEG10 expression. Enchondroma samples exhibited stronger expression of PEG10 compared with chondrosarcomas, suggesting a negative association of PEG10 with malignant cartilage tumours. In chondrosarcoma cell lines, application of the TGF-ß signalling inhibitor, SB431542, increased the protein level of PEG10. Reporter assays revealed that PEG10 repressed TGF-ß and BMP signalling, which are both SMAD pathways, whereas PEG10 knockdown increased the level of phosphorylated SMAD3 and SMAD1/5/9. Our results indicate that mutually exclusive expression of PEG10 and phosphorylated SMADs in combination with differentially expressed SOX9 is an index to distinguish between enchondroma and chondrosarcoma, while PEG10 and TGF-ß signalling are mutually inhibitory in chondrosarcoma cells.

Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

Bone morphogenic protein (BMP) signaling up-regulates neutral sphingomyelinase 2 to suppress chondrocyte maturation via the Akt protein signaling pathway as a negative feedback mechanism.

Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.

SnoN suppresses maturation of chondrocytes by mediating signal cross-talk between transforming growth factor-ß and bone morphogenetic protein pathways.

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-ß (TGF-ß) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-ß type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-ß pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-ß-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-ß to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.