Aravind Athiviraham

Wnt/β-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance

Wnt signaling transduces evolutionarily conserved pathways which play important roles in initiating and regulating a diverse range of cellular activities, including cell proliferation, calcium homeostasis, and cell polarity. The role of Wnt signaling in controlling cell proliferation and stem cell self-renewal is primarily carried out through the canonical pathway, which is the best-characterized the multiple Wnt signaling branches. The past 10 years has seen a rapid expansion in our understanding of the complexity of this pathway, as many new components of Wnt signaling have been identified and linked to signaling regulation, stem cell functions, and adult tissue homeostasis. Additionally, a substantial body of evidence links Wnt signaling to tumorigenesis of cancer types and implicates it in the development of cancer drug resistance. Thus, a better understanding of the mechanisms by which dysregulation of Wnt signaling precedes the development and progression of human cancer may hasten the development of pathway inhibitors to augment current therapy. This review summarizes and synthesizes our current knowledge of the canonical Wnt pathway in development and disease. We begin with an overview of the components of the canonical Wnt signaling pathway and delve into the role this pathway has been shown to play in stemness, tumorigenesis, and cancer drug resistance. Ultimately, we hope to present an organized collection of evidence implicating Wnt signaling in tumorigenesis and chemoresistance to facilitate the pursuit of Wnt pathway modulators that may improve outcomes of cancers in which Wnt signaling contributes to aggressive disease and/or treatment resistance.

Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine

With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

Multifaceted signaling regulators of chondrogenesis: Implications in cartilage regeneration and tissue engineering

Defects of articular cartilage present a unique clinical challenge due to its poor self-healing capacity and avascular nature. Current surgical treatment options do not ensure consistent regeneration of hyaline cartilage in favor of fibrous tissue. Here, we review the current understanding of the most important biological regulators of chondrogenesis and their interactions, to provide insight into potential applications for cartilage tissue engineering. These include various signaling pathways, including fibroblast growth factors (FGFs), transforming growth factor β (TGF-β)/bone morphogenic proteins (BMPs), Wnt/β-catenin, Hedgehog, Notch, hypoxia, and angiogenic signaling pathways. Transcriptional and epigenetic regulation of chondrogenesis will also be discussed. Advances in our understanding of these signaling pathways have led to promising advances in cartilage regeneration and tissue engineering.

The Prodomain-Containing BMP9 Produced from a Stable Line Effectively Regulates the Differentiation of Mesenchymal Stem Cells

Background: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. Methods: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. Results: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80ºC, repeated thawing cycles, and extended storage at 4ºC. Conclusions: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.

Neural EGF-like protein 1 (NELL-1): Signaling crosstalk in mesenchymal stem cells and applications in regenerative medicine

Bone tissue regeneration holds the potential to solve both osteoporosis and large skeletal defects, two problems associated with significant morbidity. The differentiation of mesenchymal stem cells into the osteogenic lineage requires a specific microenvironment and certain osteogenic growth factors. Neural EGF Like-Like molecule 1 (NELL-1) is a secreted glycoprotein that has proven, both in vitro and in vivo, to be a potent osteo-inductive factor. Furthermore, it has been shown to repress adipogenic differentiation and inflammation. NELL-1 can work synergistically with other osteogenic factors such as Bone Morphogenic Protein (BMP) −2 and −9, and has shown promise for use in tissue engineering and as a systemically administered drug for the treatment of osteoporosis. Here we provide a comprehensive up-to-date review on the molecular signaling cascade of NELL-1 in mesenchymal stem cells and potential applications in bone regenerative engineering.

Sox9 augments BMP2-induced chondrogenic differentiation by downregulating Smad7 in mesenchymal stem cells (MSCs)

Cartilage injuries caused by arthritis or trauma pose formidable challenges for effective clinical management due to the limited intrinsic proliferative capability of chondrocytes. Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries. Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) and can thus be explored as a potential therapeutic agent for cartilage defect repair. However, BMP2 also induces robust endochondral ossification. Although the precise mechanisms through which BMP2 governs the divergence of chondrogenesis and osteogenesis remain to be fully understood, blocking endochondral ossification during BMP2-induced cartilage formation may have practical significance for cartilage tissue engineering. Here, we investigate the role of Sox9-donwregulated Smad7 in BMP2-induced chondrogenic differentiation of MSCs. We find that overexpression of Sox9 leads to a decrease in BMP2-induced Smad7 expression in MSCs. Sox9 inhibits BMP2-induced expression of osteopontin while enhancing the expression of chondrogenic marker Col2a1 in MSCs. Forced expression of Sox9 in MSCs promotes BMP2-induced chondrogenesis and suppresses BMP2-induced endochondral ossification. Constitutive Smad7 expression inhibits BMP2-induced chondrogenesis in stem cell implantation assay. Mouse limb explant assay reveals that Sox9 expands BMP2-stimulated chondrocyte proliferating zone while Smad7 promotes BMP2-intitated hypertrophic zone of the growth plate. Cell cycle analysis indicates that Smad7 induces significant early apoptosis in BMP2-stimulated MSCs. Taken together, our results strongly suggest that Sox9 may facilitate BMP2-induced chondrogenesis by downregulating Smad7, which can be exploited for effective cartilage tissue engineering.

Damage control: Harnessing prostaglandin E2 as a potential healing factor of tissue injuries

Increasing prostaglandin E2 by knocking out its inhibitor 15-hydroxyprostaglandin dehydrogenase (15-PDGH) or administering a compound that inhibits 15-PDGH was recently found to improve healing in hematopoietic stem cell transplants, colitis recovery, and hepatogenesis after transection in mice. These results are suggestive of pharmacologic therapies or even genetic therapy that could improve patient outcomes, especially since the excess PGE2 and the 15-PDGH inhibitor have proven to be non-toxic. However, elevated levels of PGE2 are associated with increased risk of cancer and blood clotting problems. It would be unacceptable to treat a cancer patient with chemotherapy and replenish the hematopoietic stem cells with the help of PGE2, only to have increased expression of PGE2 and induce another cancer. Therefore, to assess the most therapeutic aspects of PGE2, it is important to consider effects that could induce disease.

Efficacy of an Arthroscopic Virtual Based Simulator for Orthopedic Surgery Residents by Year in Training

Objectives: Arthroscopic virtual-reality stimulators are an attractive option for resident training and are increasing across training programs. However, no study has analyzed the utility of the simulator for trainees based on their post-graduate level of training (PGY-level). The primary aim of this study was to determine the utility of the ArthroSTM arthroscopic simulator for orthopedic trainees based on their level of training (to determine at what point in training the simulator offers the most benefit for trainees). We hypothesized that all levels would show similar improvements in performance following completion of the training modules. Methods: Eighteen orthopedic surgery residents performed pre-training diagnostic knee and shoulder tasks on the ArthoSTM simulator. Subjects completed a series of training modules, then repeated the diagnostic tasks. Mean composite scores were calculated. Group differences for discrete variables were evaluated using ANOVA (analysis of variance). Correlation coefficients (R2) were calculated for improvements in mean composite score as a function of subject year-in-training. Results: Average improvement in composite score for participants as a whole was 11.2 points [SD 10; p-value 0.0003] for the knee simulator and 14.9 points [SD 10.9; p-value 0.0352] for the shoulder simulator. When broken down by PGY-level, all groups showed improvement, with greater improvements seen for junior-level residents in the knee simulator and greater improvements seen for senior-level residents in the shoulder simulator. ANOVA for the score improvement variable in the knee simulator data amongst the different PGY-groups yielded an f-value of 1.640 (p-value 0.2258) and that for the shoulder simulator data amongst the different PGY-groups yielded an f-value of 0.2292 (p-value 0.917). The correlation coefficient (r2) was -0.866 for the knee score improvement and 0.887 for the shoulder score improvement (Image 1). Conclusion: We found that residents training on a virtual arthroscopic simulator made significant improvements in both knee and shoulder arthroscopic skills. Most importantly, we report for the first time that the knee simulator appears to be more beneficial for junior residents, while the shoulder simulator appears to be more beneficial for senior residents. Our study adds to the mounting evidence supporting virtual arthroscopic simulator-based training for orthopedic surgery residents.

Thermoresponsive Citrate-Based Graphene Oxide Scaffold Enhances Bone Regeneration from BMP9-Stimulated Adipose-Derived Mesenchymal Stem Cells

Effective bone tissue engineering is important to overcome the unmet clinical challenges as more than 1.6 million bone grafts are done annually in the United States. Successful bone tissue engineering needs minimally three critical constituents: osteoprogenitor cells, osteogenic factors, and osteoinductive/osteoconductive scaffolds. Osteogenic progenitors are derived from multipotent mesenchymal stem cells (MSCs), which can be prepared from numerous tissue sources, including adipose tissue. We previously showed that BMP9 is the most osteogenic BMP and induces robust bone formation of immortalized mouse adipose-derived MSCs entrapped in a citrate-based thermoresponsive hydrogel referred to as PPCNg. As graphene and its derivatives emerge as promising biomaterials, here we develop a novel thermosensitive and injectable hybrid material by combining graphene oxide (GO) with PPCNg (designated as GO-P) and characterize its ability to promote bone formation. We demonstrate that the thermoresponsive behavior of the hybrid material is maintained while effectively supporting MSC survival and proliferation. Furthermore, GO-P induces early bone-forming marker alkaline phosphatase (ALP) and potentiates BMP9-induced expression of osteogenic regulators and bone markers as well as angiogenic factor VEGF in MSCs. In vivo studies show BMP9-transduced MSCs entrapped in the GO-P scaffold form well-mineralized and highly vascularized trabecular bone. Thus, these results indicate that GO-P hybrid material may function as a new biocompatible, injectable scaffold with osteoinductive and osteoconductive activities for bone regeneration.

Early Rates of Revision of Knee Cartilage Restoration Surgery and Conversion to Arthroplasty within Five Years

Objectives: To examine the early rates of revision surgery for common knee cartilage restoration procedures performed in the United States, and subsequent conversion to total or unicondylar knee arthroplasty within five years. Methods: Using the MarketScan Research Databases (Truven Health Analytics), we examined all patients who underwent knee microfracture (CPT 29879), autologous chondrocyte implantation (ACI) (CPT 27412) or osteochondral grafting (CPT 27415, 27416, 29866, 29867), within the encompassed timeframe (2003-2014), and who remained tracked within the system for two contiguous years post-operatively to assess rate of revision surgery for complications and five contiguous years to assess revision to either total or unicondylar knee arthroplasty. Results: Although microfracture remains the most common articular cartilage restorative procedure, its rates have been slowly declining since 2009, where as rates of ACI and osteochondral grafting have remained relatively stable (Image). 76,304 microfracture patients, average age 48.3 (SD=13.6), with minimum follow-up of 2 years were examined. 6,366 patients (8.3%) underwent at least one revision cartilage procedure within two years. 93% of these patients had either repeat microfracture or chondroplasty, 4.7% underwent osteochondral grafting, and 2.3% underwent ACI. 1,030 patients (1.3%) required repeat surgery secondary to stiffness or infection: 94% of these patients required manipulation under anesthesia (MUA) and the remaining 6% required open or arthroscopic incision and drainage (I&D). 24,892 patients who underwent microfracture were enrolled for at least 5 years. 428 (1.7%) of these patients subsequently underwent knee arthroplasty within 5 years. Of the 868 patients who underwent ACI as the index procedure, 139 patients (16.0%) underwent a revision cartilage procedure within two years; most commonly chondroplasty (77.8%). Average age of ACI patients was 34.5 (SD=13.5). 46 ACI patients (2.1%) returned to the OR for complications; most commonly MUA for stiffness (98.3%). Of the 233 ACI patients who were enrolled for 5 years, 16 (6.9%) of patients underwent knee arthroplasty. 2,151 patients underwent osteochondral grafting as the index procedure, with the average age being 34.5 (SD=14.1). 331 (15.4%) patients underwent revision cartilage surgery, most commonly arthroscopic chondroplasty (52.6%). 107 (5%) of patients underwent surgery for complications, most commonly MUA (80.3%). Of the 741 patients who underwent osteochondral grafting that were enrolled for 5 years, 16 (2.2%) subsequently underwent conversion to knee arthroplasty. Conclusion: While the incidence of arthroscopic microfracture has been slowly decreasing since 2009, it remains the most common cartilage restorative procedure performed in the USA. Of the procedures examined, those undergoing ACI were more likely to require subsequent chondroplasty, where as patients undergoing osteochondral grafting were more likely to require MUA for stiffness. Conversion to arthroplasty within five years was lowest in the microfracture group and highest in the ACI group. The most common revision cartilage procedure was chondroplasty and most common complication requiring surgery was stiffness in all groups. Data from this study may help providers better inform patients on the rates of revision surgery and efficacy of these procedures in delaying total joint arthroplasty.