Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-β (TGF-β) modulation. Using an adult fmod-null (fmod-/-) ...mouse model, this study further elucidates the interplay between FMOD and TGF-β expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod-/- and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod-/- mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod-/- wounds exhibited early elevation (within 24hours post-wounding) of type I and type II TGF-β receptors as well as unexpectedly high fibroblast expression of TGF-β3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-β3, fmod-/- fibroblasts were significantly less motile than WT fibroblasts. fmod-/- fibroblasts were also more susceptible to migration inhibition by TGF-β3, leading to profound delays in dermal cell migration. Increased scarring in fmod-/- mice indicates that TGF-β3's antimotility effects predominate over its antifibrotic effects when high TGF-β3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-β bioactivity.
The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal ...repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.
Abstract Pluripotent and/or multipotent stem cell-based therapeutics are a vital component of tissue engineering and regenerative medicine. The generation or isolation of safer and readily available ...stem cell sources will significantly aid clinical applications. We report here a technique using a single molecule, recombinant human fibromodulin protein (FMOD), to reprogram human fibroblasts into multipotent cells. Like virally-induced pluripotent stem (iPS) cells, FMOD reprogrammed (FReP) cells express pluripotency markers, form embryoid bodies (EBs), and differentiate into ectoderm, mesoderm, and endoderm derivatives in vitro . Notably, FReP cells regenerate muscle and bone tissues but do not generate teratomas in vivo . Unlike iPS cells, undifferentiated FReP cells proliferate slowly and express low proto-oncogene c-MYC and unexpectedly high levels of cyclin-dependent kinase inhibitors p15 Ink4B and p21 WAF1/Cip1 . Remarkably, in a fashion reminiscent of quiescent stem cells, the slow replicative phenotype of undifferentiated FReP cells reverses after differentiation induction, with differentiating FReP cells proliferating faster and expressing less p15 Ink4B and p21 WAF1/Cip1 than differentiating iPS cells. Overall, single protein, FMOD-based, cell reprograming bypasses the risks of mutation, gene instability, and malignancy associated with genetically-modified iPS cells, and provides an alternative strategy for engineering patient-specific multipotent cells for basic research and therapeutic application.
Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., an endangered ancient legume species, endemic to the Gobi desert in north-western China. As the only evergreen broadleaf shrub in this area, A. ...mongolicus plays an important role in the region's ecological-environmental stability. Despite the strong potential of A. mongolicus in providing new insights on drought tolerance, sequence information on the species in public databases remains scarce. To both learn about the role of gene expression in drought stress tolerance in A. mongolicus and to expand genomic resources for the species, transcriptome sequencing of stress-treated A. mongolicus plants was performed.
Using 454 pyrosequencing technology, 8,480 and 7,474 contigs were generated after de novo assembly of RNA sequences from leaves of untreated and drought-treated plants, respectively. After clustering using TGICL and CAP3 programs, a combined assembly of all reads produced a total of 11,357 putative unique transcripts (PUTs). Functional annotation and classification of the transcripts were conducted by aligning the 11,357 PUTs against the public protein databases and nucleotide database (Nt). Between control and drought-treated plants, 1,620 differentially expressed genes (DEGs) were identified, of which 1,106 were up-regulated and 514 were down-regulated. The differential expression of twenty candidate genes in metabolic pathways and transcription factors families related to stress-response were confirmed by quantitative real-time PCR. Representatives of several large gene families, such as WRKY and P5CS, were identified and verified in A. mongolicus for the first time.
The additional transcriptome resources, gene expression profiles, functional annotations, and candidate genes provide a more comprehensive understanding of the stress response pathways in xeric-adapted plant species such as A. mongolicus.
► We constructed a cDNA library of Saos2 cells to identify NELL-1 interacting proteins. ► APR3 was identified as a direct binding protein of NELL-1 through phage display. ► The inhibitory effect of ...APR3 on osteoblast proliferation is enhanced by NELL-1. ► NELL-1 and APR3 binding can promote osteoblast differentiation and mineralization.
Nel-like protein 1 (NELL-1) is an osteoinductive molecule associated with premature calvarial suture closure. Here we identified apoptosis related protein 3 (APR3), a membrane protein known as a proliferation suppressor, as a binding protein of NELL-1 by biopanning. NELL-1 and APR3 colocalized on the nuclear envelope of human osteoblasts. NELL-1 significantly inhibited proliferation of osteoblasts co-transfected with APR3 through further down-regulation of Cyclin D1. The co-expression of NELL-1 and APR3 enhanced Ocn and Bsp expression and mineralization. RNAi of APR3 significantly reduced the differentiation effect of NELL-1. These findings suggest that the effects of NELL-1 on osteoblastic differentiation and proliferation are partly through binding to APR3.
APR3physically interacts with NELL1 by pull down(View interaction)
APR3 and NELL1colocalize by fluorescence microscopy(View interaction)
NELL1physically interacts with SMEK1 by phage display(View interaction)
NELL1physically interacts with EIF4B by phage display(View interaction)
NELL1physically interacts with APR3 by anti bait coimmunoprecipitation(View interaction)
NELL1physically interacts with EIF5B by phage display(View interaction)
NELL1physically interacts with DNTTIP2 by phage display(View interaction)
NELL1physically interacts with EEF1A1 by phage display(View interaction)
NELL1physically interacts with Nexilin by phage display(View interaction)
NELL1physically interacts with PLCG1 by phage display(View interaction)
NELL1physically interacts with APR3 by phage display(View interaction)
NELL1physically interacts with SLTM by phage display(View interaction)
NELL1physically interacts with SSRP1 by phage display(View interaction)
NELL1physically interacts with NOL10 by phage display(View interaction)
The Food and Drug Administration–approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like ...adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, in vitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible.
Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue ...regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.
Using the CHA
DS
-VASc score to recognize the risk of stroke in patients with atrial fibrillation has been well-established. However, few studies have assessed whether the CHA
DS
-VASc score has a ...similar predictive value in recurrence after myocardial infarction (MI).
We conducted a retrospective observational cohort study of adult inpatients with MI. The CHA
DS
-VASc and modified CHA
DS
-VASc (MCHA
DS
-VASc) scores of all patients were calculated. The associations of both scores with recurrent MI were analyzed.
A total of 6,700 patients with MI (60.0 ± 11.1 years, 77.2% men) were enrolled, and 759 (11.3%) presented a definite recurrence during hospitalization. After multivariable adjustment by logistic regression in patients with MI, the CHA
DS
-VASc and MCHA
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-VASc scores were independently associated with recurrence. The MCHA
DS
-VASc score showed a better predictive value for risk of recurrence than that of CHA
DS
-VASc in overall area under the receiver operating characteristic curve (AUC) 0.757 vs. 0.676 or male patients (AUC 0.759 vs. 0.708). MCHA
DS
-VASc was superior to CHA
DS
-VASc for identifying "truly high-risk" patients with MI, regardless of overall patients or sex-specific subgroups. The two scores had a similar focus on the identification of "low-risk" patients in overall or women, but not in men.
The CHA
DS
-VASc and MCHA
DS
-VASc scores for predicting recurrence are validated in patients with MI. However, MCHA
DS
-VASc could be more helpful to secondary prevention than CHA
DS
-VASc after MI, especially in men. The superiority of MCHA
DS
-VASc compared with CHA
DS
-VASc in women is just more discriminatory for "truly high-risk" patients.
The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective ...protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.
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