Proposed Musculoskeletal Examination of Youth and Adolescent Baseball Players.

ABSTRACT: With a wide range of injuries in youth baseball, and more than 12 million amateur baseball players in the United States, a comprehensive list of tests and measures may be helpful to assess strength, mobility, and motor control throughout the kinetic chain to reduce risk of injury in this population. Many studies have looked at youth baseball players using a single test or a small number of tests to determine the prevalence of specific injuries in youth baseball, but to this author's knowledge, there is no comprehensive musculoskeletal screen published at this time specific to youth baseball. The purpose of this article is to review literature published over the last year relative to injury in youth and adolescent baseball players in an effort to update the reader on current concepts, risk factors in this population, and to provide an updated systematic screening process that may be used in reducing injury rates.

Expression of Small Noncoding RNAs in Urinary Exosomes Classifies Prostate Cancer into Indolent and Aggressive Disease.

PURPOSE: This is the first report of the development and performance of a platform that interrogates small noncoding RNAs (sncRNA) isolated from urinary exosomes. The Sentinel™ PCa Test classifies patients with prostate cancer from subjects with no evidence of prostate cancer, the miR Sentinel CS Test stratifies patients with prostate cancer between those with low risk prostate cancer (Grade Group 1) from those with intermediate and high risk disease (Grade Group 2-5), and the miR Sentinel HG Test stratifies patients with prostate cancer between those with low and favorable intermediate risk prostate cancer (Grade Group 1 or 2) and those with high risk (Grade Group 3-5) disease. MATERIALS AND METHODS: sncRNAs were extracted from urinary exosomes of 235 participants and interrogated on miR 4.0 microarrays. Using proprietary selection and classification algorithms, informative sncRNAs were selected to customize an interrogation OpenArray™ platform that forms the basis of the tests. The tests were validated using a case-control sample of 1,436 subjects. RESULTS: The performance of the miR Sentinel PCa Test demonstrated a sensitivity of 94% and specificity of 92%. The Sentinel CS Test demonstrated a sensitivity of 93% and specificity of 90% for prediction of the presence of Grade Group 2 or greater cancer, and the Sentinel HG Test demonstrated a sensitivity of 94% and specificity of 96% for the prediction of the presence of Grade Group 3 or greater cancer. CONCLUSIONS: The Sentinel PCa, CS and HG Tests demonstrated high levels of sensitivity and specificity, highlighting the utility of interrogation of urinary exosomal sncRNAs for noninvasively diagnosing and classifying prostate cancer with high precision.

Collagen biomaterial stimulates the production of extracellular vesicles containing microRNA-21 and enhances the proangiogenic function of CD34+ cells.

CD34+ cells are promising for revascularization therapy, but their clinical use is limited by low cell counts, poor engraftment, and reduced function after transplantation. In this study, a collagen type I biomaterial was used to expand and enhance the function of human peripheral blood CD34+ cells, and potential underlying mechanisms were examined. Compared to the fibronectin control substrate, biomaterial-cultured CD34+ cells from healthy donors had enhanced proliferation, migration toward VEGF, angiogenic potential, and increased secretion of CD63+CD81+ extracellular vesicles (EVs). In the biomaterial-derived EVs, greater levels of the angiogenic microRNAs (miRs), miR-21 and -210, were detected. Notably, biomaterial-cultured CD34+ cells had reduced mRNA and protein levels of Sprouty (Spry)1, which is an miR-21 target and negative regulator of endothelial cell proliferation and angiogenesis. Similar to the results of healthy donor cells, biomaterial culture increased miR-21 and -210 expression in CD34+ cells from patients who underwent coronary artery bypass surgery, which also exhibited improved VEGF-mediated migration and angiogenic capacity. Therefore, collagen biomaterial culture may be useful for expanding the number and enhancing the function of CD34+ cells in patients, possibly mediated through suppression of Spry1 activity by EV-derived miR-21. These results may provide a strategy to enhance the therapeutic potency of CD34+ cells for vascular regeneration.-McNeill, B., Ostojic, A., Rayner, K. J., Ruel, M., Suuronen, E. J. Collagen biomaterial stimulates the production of extracellular vesicles containing microRNA-21 and enhances the proangiogenic function of CD34+ cells.

Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction.

Advanced glycation end-products (AGEs) have been associated with poorer outcomes after myocardial infarction (MI), and linked with heart failure. Methylglyoxal (MG) is considered the most important AGE precursor, but its role in MI is unknown. In this study, we investigated the involvement of MG-derived AGEs (MG-AGEs) in MI using transgenic mice that over-express the MG-metabolizing enzyme glyoxalase-1 (GLO1). MI was induced in GLO1 mice and wild-type (WT) littermates. At 6 h post-MI, mass spectrometry revealed that MG-H1 (a principal MG-AGE) was increased in the hearts of WT mice, and immunohistochemistry demonstrated that this persisted for 4 weeks. GLO1 over-expression reduced MG-AGE levels at 6 h and 4 weeks, and GLO1 mice exhibited superior cardiac function at 4 weeks post-MI compared to WT mice. Immunohistochemistry revealed greater vascular density and reduced cardiomyocyte apoptosis in GLO1 vs. WT mice. The recruitment of c-kit+ cells and their incorporation into the vasculature (c-kit+CD31+ cells) was higher in the infarcted myocardium of GLO1 mice. MG-AGEs appeared to accumulate in type I collagen surrounding arterioles, prompting investigation in vitro. In culture, the interaction of angiogenic bone marrow cells with MG-modified collagen resulted in reduced cell adhesion, increased susceptibility to apoptosis, fewer progenitor cells, and reduced angiogenic potential. This study reveals that MG-AGEs are produced post-MI and identifies a causative role for their accumulation in the cellular changes, adverse remodeling and functional loss of the heart after MI. MG may represent a novel target for preventing damage and improving function of the infarcted heart.

Collagen matrix-induced expression of integrin αVß3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function.

Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold therapeutic promise for treating a variety of cardiovascular diseases. However, further improvements are needed because the effects of CAC therapy remain minimal or transient. The regenerative potential of these cells can be improved by culture on a collagen-based matrix through the up-regulation of key integrin proteins. We found that human CAC function was enhanced by using the matricellular protein CCN1 (CYR61/CTGF/NOV family member 1) to target integrin αV and ß3, which are up-regulated on matrix. Compared to matrix-cultured CACs, CCN1-matrix CACs exhibited a 2.2-fold increase in cell proliferation, 1.8-fold greater migration toward VEGF, and 1.7-fold more incorporation into capillary-like structures in an angiogenesis assay. In vivo, intramuscular injection of CCN1-matrix-cultured CACs into ischemic hind limbs of CD-1 nude mice resulted in blood flow recovery to 80% of baseline, which was greater than matrix-cultured CACs (66%) and PBS (35%) treatment groups. Furthermore, transplanted CCN1-matrix CACs exhibited greater engraftment (11-fold) and stimulated the up-regulation of survival and angiogenic genes (>3-fold). These findings reveal the importance of cell-matrix interactions in regulating CAC function and also reveal a mechanism by which these may be exploited to enhance cell therapies for ischemic disease.

Hedgehog regulates Norrie disease protein to drive neural progenitor self-renewal.

Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.

Comparative genomics identification of a novel set of temporally regulated hedgehog target genes in the retina.

The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development.

Suppressor of fused is required to maintain the multipotency of neural progenitor cells in the retina.

The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.

Surgeon-Therapist Communication Must Be Improved in Rotator Cuff Repair Rehabilitation: An Electronic Survey of Physical Therapists on Postoperative Rehabilitation Protocols and Communication with Treating Surgeons.

BACKGROUND: There is no consensus on postoperative rotator cuff repair protocols in orthopedic or physical therapy literature. Despite surgical management, the frequency of rotator cuff retears continues to be high. OBJECTIVES: This study is designed to investigate the current concepts of postoperative rehabilitation and to evaluate the state of communication between referring surgeons and treating physical therapists. METHODS: A survey was conducted over a 2-year period, performed by an online survey company. RESULTS: Six hundred responses were obtained from physical therapists. Most rehab protocols were based on size of tear, tissue quality, and open versus arthroscopic repair. Current intervention concepts and professional experience guided protocol development. Thirty-three percent of therapists receive operative notes ≤ 25% of the time. Sixteen percent reported not receiving operative notes and not having access to the physician >50% of the time. Most patients were seen within 2 weeks, with passive range of motion started in 83% of cases. Sixty percent started active-assist range of motion at ≤ 4 weeks. Sixty-four percent of therapy was continued for 12 to 16 weeks. Patient compliance, poor tissue quality, and rapid rehab progression were reported as common causes of failure. CONCLUSION: Most rehabilitation programs follow protocols developed by surgeons and physical therapists. Tissue quality, size of tear, and repair type are usually documented in the operative report, and are rarely conveyed to the therapist. This study highlights the lack of communication between the physician and the therapist. Improving communication regarding the findings at surgery, opening lines of communication, and making alterations to the protocol may improve patient outcomes.

Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction.

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium's mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.

Electroconductive nanoengineered biomimetic hybrid fibers for cardiac tissue engineering.

We report for the first time the preparation of a fibrous material composed of surface grafted spherical nanosilver and collagen using one-step electrospinning. The resulting composite showed comparable morphology to the control without nanosilver, but had improved electrical conductivity. Under electrical stimulation, fibrous materials containing nanosilver increased connexin-43 expression and proliferation of neonatal cardiomyocytes. Furthermore, composites containing nanosilver prevented biofilm formation but did not activate macrophages.

Epigenetic Activation of Pro-angiogenic Signaling Pathways in Human Endothelial Progenitors Increases Vasculogenesis.

Human endothelial colony-forming cells (ECFCs) represent a promising source of adult stem cells for vascular repair, yet their regenerative capacity is limited. Here, we set out to understand the molecular mechanism restricting the repair function of ECFCs. We found that key pro-angiogenic pathways are repressed in ECFCs due to the presence of bivalent (H3K27me3/H3K4me3) epigenetic marks, which decreases the cells' regenerative potential. Importantly, ex vivo treatment with a combination of epigenetic drugs that resolves bivalent marks toward the transcriptionally active H3K4me3 state leads to the simultaneous activation of multiple pro-angiogenic signaling pathways (VEGFR, CXCR4, WNT, NOTCH, SHH). This in turn results in improved capacity of ECFCs to form capillary-like networks in vitro and in vivo. Furthermore, restoration of perfusion is accelerated upon transplantation of drug-treated ECFCs in a model of hindlimb ischemia. Thus, ex vivo treatment with epigenetic drugs increases the vascular repair properties of ECFCs through transient activation of pro-angiogenic signaling pathways.

Methylglyoxal-Induced Endothelial Cell Loss and Inflammation Contribute to the Development of Diabetic Cardiomyopathy.

The mechanisms for the development of diabetic cardiomyopathy remain largely unknown. Methylglyoxal (MG) can accumulate and promote inflammation and vascular damage in diabetes. We examined if overexpression of the MG-metabolizing enzyme glyoxalase 1 (GLO1) in macrophages and the vasculature could reduce MG-induced inflammation and prevent ventricular dysfunction in diabetes. Hyperglycemia increased circulating inflammatory markers in wild-type (WT) but not in GLO1-overexpressing mice. Endothelial cell number was reduced in WT-diabetic hearts compared with nondiabetic controls, whereas GLO1 overexpression preserved capillary density. Neuregulin production, endothelial nitric oxide synthase dimerization, and Bcl-2 expression in endothelial cells was maintained in the hearts of GLO1-diabetic mice and corresponded to less myocardial cell death compared with the WT-diabetic group. Lower receptor for advanced glycation end products and tumor necrosis factor-α (TNF-α) levels were also observed in GLO1-diabetic versus WT-diabetic mice. Over a period of 8 weeks of hyperglycemia, GLO1 overexpression delayed and limited the loss of cardiac function. In vitro, MG and TNF-α were shown to synergize in promoting endothelial cell death, which was associated with increased angiopoietin 2 expression and reduced Bcl-2 expression. These results suggest that MG in diabetes increases inflammation, leading to endothelial cell loss. This contributes to the development of diabetic cardiomyopathy and identifies MG-induced endothelial inflammation as a target for therapy.

Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems.

Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation.

The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.

Timing underpins the benefits associated with injectable collagen biomaterial therapy for the treatment of myocardial infarction.

Injectable hydrogel biomaterials are promising therapies to promote repair and regeneration post-myocardial infarction (MI). However, the timing of delivery and the mechanisms through which biomaterial treatments confer their benefits are translational issues that remain to be addressed. We assessed the efficacy of an injectable collagen matrix at 3 different delivery time points post-MI. Infarcted mice received the matrix or control (saline) treatment at 3 h, 1 week or 2 weeks after MI. The earlier treatment delivery better prevented negative ventricular remodeling and long-term deterioration of cardiac function (up to 3 months), whereas waiting longer to administer the matrix (1 and 2 weeks post-MI) reduced the therapeutic effects. Collagen matrix delivery did not stimulate an inflammatory response acutely and favorably modulated inflammation in the myocardium long-term. We found that the matrix interacts with the host tissue to alter the myocardial cytokine profile, promote angiogenesis, and reduce fibrosis and cell death. This work highlights that the timing of delivery can significantly affect the ability of an injectable hydrogel to protect the post-MI environment, which will be an important consideration in the clinical translation of cardiac biomaterial therapy.

Preparation and characterization of circulating angiogenic cells for tissue engineering applications.

Circulating angiogenic cells (CACs) are a heterogeneous cell population of bone marrow (BM) origin. These cells are most commonly derived from the peripheral blood, bone marrow, and cord blood, and are one of the leading candidates for promoting vascularization in tissue engineering therapies. CACs can be isolated by culturing peripheral blood mononuclear cells (PBMCs) on fibronectin or by flow cytometry to obtain more specific subpopulations. Here we will describe how to generate a population of CACs, and how to characterize the cells and confirm their phenotype. Also, we will provide select methods that can be used to assess the angiogenic and endothelial cell-like properties of the CACs.

Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice.

AIMS: Methylglyoxal (MG) accumulates in diabetes and impairs neovascularization. This study assessed whether overexpressing the MG-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischaemic tissue of streptozotocin-induced diabetic mice. METHODS AND RESULTS: After 24 h of hyperglycaemic and hypoxic culture, BMCs from GLO1 overexpressing and wild-type (WT) diabetic mice were compared for migratory potential, viability, and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (eGFP) mice that overexpress GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischaemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells and also arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared with WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+), and Flk(+)) was enhanced post-ischaemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into the hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. CONCLUSION: This study demonstrates that protection from MG uniquely in BM is sufficient to restore BMC function and neovascularization of ischaemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.

The effect of encapsulation of cardiac stem cells within matrix-enriched hydrogel capsules on cell survival, post-ischemic cell retention and cardiac function.

Transplantation of ex vivo proliferated cardiac stem cells (CSCs) is an emerging therapy for ischemic cardiomyopathy but outcomes are limited by modest engraftment and poor long-term survival. As such, we explored the effect of single cell microencapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVß3 and α5ß1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability under hypoxic stress conditions and, when compared to standard suspended CSC, media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, cardiac structure and function. Single cell encapsulation prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.