Cardiac hypertrophy is a major predictor of heart failure and a prevalent disorder with high mortality. Little is known, however, regarding mechanisms governing the transition from stable cardiac hypertrophy to decompensated heart failure. Here, we tested the role of autophagy, a conserved pathway mediating bulk degradation of long-lived proteins and cellular organelles that can lead to cell death. To quantify autophagic activity, we engineered a line of "autophagy reporter" mice and confirmed that cardiomyocyte autophagy can be induced by short-term nutrient deprivation in vivo. Pressure overload induced by aortic banding induced heart failure and greatly increased cardiac autophagy. Load-induced autophagic activity peaked at 48 hours and remained significantly elevated for at least 3 weeks. In addition, autophagic activity was not spatially homogeneous but rather was seen at particularly high levels in basal septum. Heterozygous disruption of the gene coding for Beclin 1, a protein required for early autophagosome formation, decreased cardiomyocyte autophagy and diminished pathological remodeling induced by severe pressure stress. Conversely, Beclin 1 overexpression heightened autophagic activity and accentuated pathological remodeling. Taken together, these findings implicate autophagy in the pathogenesis of load-induced heart failure and suggest it may be a target for novel therapeutic intervention.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM-LDL) on osteoblastic differentiation of murine marrow stromal cells, M2-10B4. MM-LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogenactivated protein kinase-dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM-LDL to regulate adipogenesis was also assessed. Treatment of M2-10B4 as well as 3T3-L1 preadipocytes with MM-LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator-activated receptor (PPAR) ␥ agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter-reporter construct experiments, MM-LDL may be acting in part through activating PPAR␣. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the "lipid hypothesis of osteoporosis." (J Bone Miner Res

IntroductionAdipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency.MethodsADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage.ResultsPRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs.ConclusionPretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.

The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism.The osteoblast-specific factor-2/core binding factor ␣-1 (Osf2 1 /Cbfa1),hereafter called Cbfa1, is a transcription factor recently shown to be a master regulator of osteoblastic differentiation. Komori et al. (1) and Otto et al. (2) observed that targeted disruption of osteoblast-specific Cbfa1 in vivo blocks skeletogenesis, and that heterozygous mutations of Cbfa1 in both human and mice lead to cleidocranial dysplasia, an autosomal dominant disorder (2-4). As further evidence of its role in differentiation, Ducy and colleagues (5) showed that forced expression of Cbfa1 induced expression of principal osteoblastspecific genes such as bone sialoprotein, osteopontin, and osteocalcin in non-osteoblastic cells. Furthermore, Ducy et al. (5) and Banerjee et al. (6) demonstrated that disruption of Cbfa1 by antisense oligonucleotides in osteoblast cultures inhibited expression of osteoblastic differentiation markers and formation of mineralized nodules.The cAMP pathway, the major mediator of PTH signaling in osteoblasts (7), can have either anabolic or catabolic effects (8, 9) depending on the cell line used, the stage of differentiation, culture conditions, and treatment duration (10 -12). PTH/ cAMP have been shown to regulate expression of several osteoblastic differentiation genes including Cbfa1-regulated osteoblast-specific genes such as osteopontin (13), type I collagen (14, 15), bone sialoprotein (16), and osteocalcin (11). Although sequences conferring cAMP responsiveness have been found in the promoters of osteocalcin (8) and bone sialoprotein (16), it remains largely unknown w...

Osteoarthritis is one of the most common diseases, and it affects 12% of the population around the world. Although the disease is chronic, it significantly reduces the patient’s quality of life. At present, stem cell therapy is considered to be an efficient approach for treating this condition. Mesenchymal stem cells (MSCs) show the most potential for stem cell therapy of osteoarthritis. In fact, MSCs can differentiate into certain mesodermal tissues such as cartilage and bone. Therefore, in the present study, we applied adipose tissue-derived MSCs to osteoarthritis treatment. This study aimed to evaluate the clinical efficiency of autologous adipose tissue-derived MSC transplantation in patients with confirmed osteoarthritis at grade II and III. Adipose tissue was isolated from the belly, and used for extraction of the stromal vascular fraction (SVF). The SVF was mixed with activated platelet-rich plasma before injection. The clinical efficiencies were evaluated by the pain score (VAS), Lysholm score, and MRI findings. We performed the procedure in 21 cases from 2012 to 2013. All 21 patients showed improved joint function after 8.5 months. The pain score decreased from 7.6±0.5 before injection to 3.5±0.7 at 3 months and 1.5±0.5 at 6 months after injection. The Lysholm score increased from 61±11 before injection to 82±8.1 after injection. Significant improvements were noted in MRI findings, with increased thickness of the cartilage layer. Moreover, there were no side-effects or complications related to microorganism infection, graft rejection, or tumorigenesis. These results provide a new opportunity for osteoarthritis treatment. Level of evidence: IV.

Background Studies to dissect the role of calcineurin in pathological cardiac remodeling have relied heavily on murine models, where genetic gain- and loss-of-function manipulations are initiated at or before birth. However, the great majority of clinical cardiac pathology occurs in adults. Yet, nothing is known about the effects of calcineurin when its activation commences in adulthood. Further, despite the fact that ventricular hypertrophy is a well established risk factor for heart failure, the relative pace and progression of these two major phenotypic features of heart disease are unknown. Methods and Results We engineered mice harboring in cardiomyocytes a constitutively active calcineurin transgene driven by a tetracycline-responsive promoter element. Expression of the mutant calcineurin transgene was initiated for variable lengths of time to determine the natural history of disease pathogenesis, and to determine when, if ever, these events are reversible. Activation of the calcineurin transgene in adult mice triggered rapid and robust cardiac growth with features characteristic of pathological hypertrophy. Concentric hypertrophy preceded the development of systolic dysfunction, fetal gene activation, fibrosis, and clinical heart failure. Further, cardiac hypertrophy reversed spontaneously when calcineurin activity was turned off, and expression of fetal genes reverted to baseline. Fibrosis, a prominent feature of pathological cardiac remodeling, manifested partial reversibility. Conclusions Together, these data establish and define the deleterious effects of calcineurin signaling in adult heart and reveal that calcineurin-dependent hypertrophy with concentric geometry precedes systolic dysfunction and heart failure. Furthermore, these findings demonstrate that during much of the disease process, calcineurin-dependent remodeling remains reversible.

Prostate embryonic development, pubertal and adult growth, maintenance, and regeneration are regulated through androgen signaling-mediated mesenchymal-epithelial interactions. Specifically, the essential role of mesenchymal androgen signaling in the development of prostate epithelium has been observed for over 30 years. However, the identity of the mesenchymal cells responsible for this paracrine regulation and related mechanisms are still unknown. Here, we provide the first demonstration of an indispensable role of the androgen receptor (AR) in sonic hedgehog (SHH) responsive Gli1-expressing cells, in regulating prostate development, growth, and regeneration. Selective deletion of AR expression in Gli1-expressing cells during embryogenesis disrupts prostatic budding and impairs prostate development and formation. Tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wildtype urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. RNA-sequencing analysis showed significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis in AR-deficient Gli1-expressing cells. Among these altered pathways, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. We further demonstrated the activation of TGFβ1 signaling in AR-deleted prostatic Gli1-expressing cells, which inhibits prostate epithelium growth through paracrine regulation. These data demonstrate a novel role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, morphogenesis, and renewal, and elucidate the mechanism by which mesenchymal

Edited by Xiao-Fan WangEmerging evidence has shown that the hepatocyte growth factor (HGF) and its receptor, MET proto-oncogene, receptor tyrosine kinase (MET), promote cell proliferation, motility, morphogenesis, and angiogenesis. Whereas up-regulation of MET expression has been observed in aggressive and metastatic prostate cancer, a clear understanding of MET function in prostate tumorigenesis remains elusive. Here, we developed a conditional Met transgenic mouse strain, H11 Met/؉ : PB-Cre4, to mimic human prostate cancer cells with increased MET expression in the prostatic luminal epithelium. We found that these mice develop prostatic intraepithelial neoplasia after HGF administration. To further assess the biological role of MET in prostate cancer progression, we bred H11 Met/؉ /Pten LoxP/LoxP :PBCre4 compound mice, in which transgenic Met expression and deletion of the tumor suppressor gene Pten occurred simultaneously only in prostatic epithelial cells. These compound mice exhibited accelerated prostate tumor formation and invasion as well as increased metastasis compared with Pten LoxP/LoxP :PB-Cre4 mice. Moreover, prostatic sarcomatoid carcinomas and lesions resembling the epithelial-to-mesenchymal transition developed in tumor lesions of the compound mice. RNA-Seq and qRT-PCR analyses revealed a robust enrichment of known tumor progression and metastasis-promoting genes in samples isolated from H11 Met/؉ /Pten LoxP/LoxP :PB-Cre4 compound mice compared with those from Pten LoxP/LoxP :PB-Cre4 littermate controls. HGF-induced cell proliferation and migration also increased in mouse embryonic fibroblasts (MEFs) from animals with both Met transgene expression and Pten deletion compared with Pten-null MEFs. The results from these newly developed mouse models indicate a role for MET in hastening tumorigenesis and metastasis when combined with the loss of tumor suppressors.