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Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise, Fgf2 -/- mice transplanted with retroviral BCR-ABL leukemia survive significantly longer than their +/+ counterparts when treated with TKI. Thus, inhibition of FGFR can modulate stromal function, reduce exosome secretion, and may be a therapeutic option to overcome resistance to TKIs. This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Latently infected CD4+ T cells are the main barrier to complete clearance of HIV infection, but it is unclear what mechanisms govern latent HIV infection in vivo. To address this question, we developed a new panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV transcripts that define distinct blocks to transcription. We applied this panel of assays to CD4+ T cells freshly isolated from HIV-infected patients on suppressive antiretroviral therapy (ART) to quantify the degree to which different mechanisms inhibit HIV transcription. In addition, we measured the degree to which these transcriptional blocks could be reversed ex vivo by T cell activation (using anti-CD3/CD28 antibodies) or latency-reversing agents. We found that the main reversible block to HIV RNA transcription was not inhibition of transcriptional initiation but rather a series of blocks to proximal elongation, distal transcription/polyadenylation (completion), and multiple splicing. Cell dilution experiments suggested that these mechanisms operated in most of the HIV-infected CD4+ T cells examined. Latency-reversing agents exerted differential effects on the three blocks to HIV transcription, suggesting that these blocks may be governed by different mechanisms.

Despite intensive study, it is unclear which mechanisms are responsible for latent HIV infection in vivo. One potential mechanism is inhibition of HIV transcriptional elongation, which results in short abortive transcripts containing the trans-activation response (TAR) region. Because the relative levels of total (including short) and processive transcripts provide measures of HIV transcriptional initiation and elongation, there is a compelling need for techniques that accurately measure both. Nonetheless, prior assays for total transcripts have been semi-quantitative and have seen limited application to patient samples. This manuscript reports the validation of quantitative reverse transcription (RT) droplet digital PCR assays for measurement of total (TAR) and processive (R-U5/gag) HIV transcripts. Traditional RT priming strategies can efficiently detect the TAR region on long HIV transcripts but detect 10-fold higher than elongated transcripts, implying a substantial block to transcriptional elongation in vivo. This approach may be applied to other difficult-to-prime RNA targets.

BackgroundIn HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels.MethodsThirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition.ResultsThe addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling.ConclusionsTreatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels.

AIMS:We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(–/–) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(–/–) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS:HypoE/SR-BI(–/–) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS:Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

BackgroundMuscle atrophy, fatty infiltration, and fibrosis of the muscle have been described as important factors governing outcome after rotator cuff injury and repair. Muscle fibrosis is also thought to have a role in determining muscle compliance at the time of surgery. The transforming growth factor-β (TGF-β) pathways are highly conserved pathways that exert a potent level of control over muscle gene expression and are critical regulators of fibrosis in multiple organ systems. It has been shown that TGF-β can regulate important pathways of muscle atrophy, including the Akt/mammalian target of rapamycin pathway. The purpose of this study was to evaluate the expression of TGF-β and its downstream effectors of fibrosis after a massive rotator cuff tear (RCT) in a previously established rat model.MethodsTo simulate a massive RCT, infraspinatus and supraspinatus tenotomy and suprascapular nerve transection were performed on Sprague-Dawley rats with use of a validated model. Two and 6 weeks after surgery, supraspinatus muscles were harvested to study alterations in TGF-β signaling by Western blotting, quantitative polymerase chain reaction, and histologic analysis.ResultsThere was a significant increase in fibrosis in the rotator cuff muscle after RCT in our animal model. There was a concomitant increase in TGF-β gene and protein expression at both 2 and 6 weeks after RCT. Evaluation of the TGF-β signaling pathway revealed an increase in SMAD2 activation but not in SMAD3. There was an increase in profibrotic markers collagen I, collagen III, and α-smooth muscle actin.ConclusionsTGF-β signaling is significantly upregulated in rat supraspinatus muscles after RCTs.

Backgroundextracellular matrix (ECM) components are instrumental in maintaining homeostasis and muscle fiber functional integrity. Skeletal muscle hypertrophy is associated with ECM remodeling. Specifically, recent studies have reported the involvement of matrix metalloproteinases (MMPs) in muscle ECM remodeling. However, the functional role of MMPs in muscle hypertrophy remains largely unknown.Methodsin this study, we examined the role of MMP-2 in skeletal muscle hypertrophy using a previously validated method where the plantaris muscle of mice were subjected to mechanical overload due to the surgical removal of synergist muscles (gastrocnemius and soleus).Resultsfollowing two weeks of overload, we observed a significant increase in MMP-2 activity and up-regulation of ECM components and remodeling enzymes in the plantaris muscles of wild-type mice. However, MMP-2 knockout mice developed significantly less hypertrophy and ECM remodeling in response to overload compared to their wild-type littermates. Investigation of protein synthesis rate and Akt/mTOR signaling revealed no difference between wild-type and MMP-2 knockout mice, suggesting that a difference in hypertrophy was independent of protein synthesis.Conclusiontaken together, our results suggest that MMP-2 is a key mediator of ECM remodeling in the setting of skeletal muscle hypertrophy.

Previous studies have evaluated role of Akt/mTOR signaling in rotator cuff muscle atrophy and determined that there was differential in signaling following tendon transection (TT) and suprascapular nerve (SSN) denervation (DN), suggesting that atrophy following TT and DN was modulated by different protein degradation pathways. In this study, two muscle proteolytic systems that have been shown to be potent regulators of muscle atrophy in other injury models, the ubiquitin-proteasome pathway and autophagy, were evaluated following TT and DN. In addition to examining protein degradation, this study assessed protein synthesis rate following these two surgical models to understand how the balance between protein degradation and synthesis results in atrophy following rotator cuff injury. In contrast to the traditional theory that protein synthesis is decreased during muscle atrophy, this study suggests that protein synthesis is up-regulated in rotator cuff muscle atrophy following both surgical models. While the ubiquitin-proteasome pathway was a major contributor to the atrophy seen following DN, autophagy was a major contributor following TT. The findings of this study suggest that protein degradation is the primary factor contributing to atrophy following rotator cuff injury. However, different proteolytic pathways are activated if SSN injury is involved.

Repair of peripheral nerve defects with current synthetic, tubular nerve conduits generally shows inferior recovery when compared with using nerve autografts, the current gold standard. We tested the ability of composite collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to stimulate neurite extension on a promising aligned, nanofiber poly-L-lactide-co-caprolactone (PLCL) scaffold. In vitro, the hydrogels significantly increased neurite extension from dorsal root ganglia explants. Consistent with these results, the addition of hydrogels as luminal fillers within aligned, nanofiber tubular PLCL conduits led to improved sensory function compared to autograft repair in a critical-size defect in the sciatic nerve in a rat model. Sensory recovery was assessed 3 and 12 weeks after repair using a withdrawal assay from thermal stimulation. The addition of hydrogel did not enhance recovery of motor function in the rat model. The NGF led to dose-dependent improvements in neurite out-growth in vitro, but did not have a significant effect in vivo. In summary, composite collagen/hyaluronan hydrogels enhanced sensory neurite outgrowth in vitro and sensory recovery in vivo. The use of such hydrogels as luminal fillers for tubular nerve conduits may therefore be useful in assisting restoration of protective sensation following peripheral nerve injury.

Rotator cuff tears (RCTs) are among the most common injuries seen in orthopedic patients. Chronic tears can result in the development of muscular atrophy and fatty infiltration. Despite the prevalence of RCTs, little is known about the underlying molecular pathways that produce these changes. Recently, we have shown that mammalian target of rapamycin (mTOR) signaling plays an important role in muscle atrophy that results from massive RCTs in a rat model. The purpose of this study was therefore to extend our understanding of mTOR signaling and evaluate its role in fatty infiltration after a combined tendon transection and suprascapular nerve denervation surgery. Akt/mTOR signaling was significantly increased and resulted in the up-regulation of two transcription factors: SREBP-1 and PPARγ. We also saw an increase in expression of adipogenic markers: C/EBP-α and FASN. Upon treatment with rapamycin, an inhibitor of mTOR, we observed a decrease in mTOR signaling, activity of transcription factors, and reduction in fatty infiltration. Therefore, our study suggests that mTOR signaling mediates rotator cuff fatty infiltration via SREBP-1 and PPARγ. Clinically, our finding may alter current treatment methods to address rotator cuff fatty infiltration.

Muscle extracellular matrix (ECM) plays an important role in maintaining muscular integrity and force transduction. However, the role of ECM in skeletal muscle atrophy remains unknown. In this study, we employed two clinically relevant mouse models of Achillotenotomy and hindlimb suspension to simulate Achilles tendon rupture and hindlimb disuse. The gastrocnemius was harvested following two weeks of treatment. We hypothesized that degradation of muscle ECM basement membrane lead to dysfunction of muscle contractility. Our results demonstrated a significant reduction of gastrocnemius single twitch force, isometric tetanic force, and contraction velocity following tendon rupture (p

Proteogenomics refers to the integration of comprehensive genomic, transcriptomic, and proteomic measurements from the same samples with the goal of fully understanding the regulatory processes converting genotypes to phenotypes, often with an emphasis on gaining a deeper understanding of disease processes. Although specific genetic mutations have long been known to drive the development of multiple cancers, gene mutations alone do not always predict prognosis or response to targeted therapy. The benefit of proteogenomics research is that information obtained from proteins and their corresponding pathways provides insight into therapeutic targets that can complement genomic information by providing an additional dimension regarding the underlying mechanisms and pathophysiology of tumors. This review describes the novel insights into tumor biology and drug resistance derived from proteogenomic analysis while highlighting the clinical potential of proteogenomic observations and advances in technique and analysis tools. [ABSTRACT FROM AUTHOR]