Ksenija Bernau

Synergistic Effects of GDNF and VEGF on Lifespan and Disease Progression in a Familial ALS Rat Model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle.

Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotype

Background: Myofibroblasts have heightened expression of contractile genes and drive extracellular matrix formation during pulmonary fibrosis. Results: Enhanced fibronectin assembly by myofibroblasts requires smooth muscle α-actin expression. Conclusion: This study demonstrates a linkage between contractile gene expression and increased assembly of fibronectin fibrils by myofibroblasts. Significance: Targeting contractile gene expression in myofibroblasts may attenuate fibronectin matrix formation during fibrosis. Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Neonatal immune-tolerance in mice does not prevent xenograft rejection.

Assessing the efficacy of human stem cell transplantation in rodent models is complicated by the significant immune rejection that occurs. Two recent reports have shown conflicting results using neonatal tolerance to xenografts in rats. Here we extend this approach to mice and assess whether neonatal tolerance can prevent the rapid rejection of xenografts. In three strains of neonatal immune-intact mice, using two different brain transplant regimes and three independent stem cell types, we conclusively show that there is rapid rejection of the implanted cells. We also address specific challenges associated with the generation of humanized mouse models of disease.

In Vivo Tracking of Human Neural Progenitor Cells in the Rat Brain Using Magnetic Resonance Imaging is Not Enhanced by Ferritin Expression

Rapid growth in the field of stem cell research has generated a lot of interest in their therapeutic use, especially in the treatment of neurodegenerative diseases. Specifically, human neural progenitor cells (hNPCs), unique in their capability to differentiate into cells of the neural lineage, have been widely investigated due to their ability to survive, thrive, and migrate toward injured tissues. Still, one of the major roadblocks for clinical applicability arises from the inability to monitor these cells following transplantation. Molecular imaging techniques, such as magnetic resonance imaging (MRI), have been explored to assess hNPC transplant location, migration, and survival. Here we investigated whether inducing hNPCs to overexpress ferritin (hNPCsFer), an iron storage protein, is sufficient to track these cells long term in the rat striatum using MRI. We found that increased hypointensity on MRI images could establish hNPCFer location. Unexpectedly, however, wild-type hNPC transplants were detected in a similar manner, which is likely due to increased iron accumulation following transplantation-induced damage. Hence, we labeled hNPCs with superparamagnetic iron oxide (SPIO) nanoparticles to further increase iron content in an attempt to enhance cell contrast in MRI. SPIO-labeling of hNPCs (hNPCs-SPIO) achieved increased hypointensity, with significantly greater area of decreased T2* compared to hNPCFer (p < 0.0001) and all other controls used. However, none of the techniques could be used to determine graft rejection in vivo, which is imperative for understanding cell behavior following transplantation. We conclude that in order for cell survival to be monitored in preclinical and clinical settings, another molecular imaging technique must be employed, including perhaps multimodal imaging, which would utilize MRI along with another imaging modality.

In vivo tracking of human neural progenitor cells in the rat brain using bioluminescence imaging.

BACKGROUND Stem cell therapies appear promising for treating certain neurodegenerative disorders and molecular imaging methods that track these cells in vivo could answer some key questions regarding their survival and migration. Bioluminescence imaging (BLI), which relies on luciferase expression in these cells, has been used for this purpose due to its high sensitivity. NEW METHOD In this study, we employ BLI to track luciferase-expressing human neural progenitor cells (hNPC(Luc2)) in the rat striatum long-term. RESULTS We show that hNPC(Luc2) are detectable in the rat striatum. Furthermore, we demonstrate that using this tracking method, surviving grafts can be detected in vivo for up to 12 weeks, while those that were rejected do not produce bioluminescence signal. We also demonstrate the ability to discern hNPC(Luc2) contralateral migration. COMPARISON WITH EXISTING METHODS Some of the advantages of BLI compared to other imaging methods used to track progenitor/stem cells include its sensitivity and specificity, low background signal and ability to distinguish surviving grafts from rejected ones over the long term while the blood-brain barrier remains intact. CONCLUSIONS These new findings may be useful in future preclinical applications developing cell-based treatments for neurodegenerative disorders.

Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF). Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.

Tensin 1 Is Essential for Myofibroblast Differentiation and Extracellular Matrix Formation

&NA; Myofibroblasts, the primary effector cells that mediate matrix remodeling during pulmonary fibrosis, rapidly assemble an extracellular fibronectin matrix. Tensin (TNS) 1 is a key component of specialized cellular adhesions (fibrillar adhesions) that bind to extracellular fibronectin fibrils. We hypothesized that TNS1 may play a role in modulating myofibroblast‐mediated matrix formation. We found that TNS1 expression is increased in fibroblastic foci from lungs with idiopathic pulmonary fibrosis. Transforming growth factor (TGF)‐&bgr; profoundly up‐regulates TNS1 expression with kinetics that parallel the expression of the myofibroblast marker, smooth muscle &agr;‐actin. TGF‐&bgr;‐induced TNS1 expression is dependent on signaling through the TGF‐&bgr; receptor 1 and is Rho coiled‐coiled kinase/actin/megakaryoblastic leukemia‐1/serum response factor dependent. Small interfering RNA‐mediated knockdown of TNS1 disrupted TGF‐&bgr;‐induced myofibroblast differentiation, without affecting TGF‐&bgr;/Smad signaling. In contrast, loss of TNS1 resulted in disruption of focal adhesion kinase phosphorylation, focal adhesion formation, and actin stress fiber development. Finally, TNS1 was essential for the formation of fibrillar adhesions and the assembly of nascent fibronectin and collagen matrix in myofibroblasts. In summary, our data show that TNS1 is a novel megakaryoblastic leukemia‐1‐dependent gene that is induced during pulmonary fibrosis. TNS1 plays an essential role in TGF‐&bgr;‐induced myofibroblast differentiation and myofibroblast‐mediated formation of extracellular fibronectin and collagen matrix. Targeted disruption of TNS1 and associated signaling may provide an avenue to inhibit tissue fibrosis.

The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation

Idiopathic pulmonary fibrosis is a progressive and deadly disorder with very few therapeutic options. Palomid 529 (8‐(1‐hydroxyethyl)‐2‐methoxy‐3‐(4‐methoxybenzyloxy)‐benzo[c]chromen‐6‐one; P529) is a novel dual inhibitor of mechanistic target of rapamycin complex 1/2 (mTORC1/2). In these studies, we investigated the effect of P529 on TGF‐β‐dependent signaling and myofibroblast differentiation. TGF‐β‐induced phosphorylation of the mTORC1 targets, p70 S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E‐BP1), were both dose dependently inhibited by P529 in human lung fibroblasts with maximal inhibition occurring between 10 and 20 μM. mTORC2‐mediated phosphorylation of Akt at the S473 site was partially inhibited with a similar dose dependency, as was TGF‐β‐induced myofibroblast differentiation. Protein levels of TGF‐β‐induced fibronectin and collagen were similarly decreased by P529. At this dose, there was also inhibition of mRNA transcript levels for Col1 and α‐SMA, suggesting inhibition of transcriptional activation. However, there was no effect of P529 on canonical TGF‐β‐induced Smad signaling, as assessed by receptor‐associated Smad2/3 phosphorylation, Smad2/3/4 translocation, or Smad‐driven gene expression, as assessed by Smad‐binding element driven luciferase. Conversely, activation of mTORC1/2 signaling was dependent on TGF‐β type I receptor (ALK5) signaling and on Smad2/3 expression. P529 treatment disrupted TGF‐β‐induced actin stress fiber formation during myofibroblast differentiation, the deposition of new extracellular fibronectin matrix, and linear wound closure by fibroblasts. Likewise, mTOR knockdown inhibited TGF‐β‐induced myofibroblast differentiation. In conclusion, P529 inhibits TGF‐β‐induced myofibroblast differentiation, actin stress fiber formation, and matrix protein expression and deposition. Inhibition of mTORC1/2 by P529 may be a promising approach to inhibit in vivo fibrosis. J. Cell. Biochem. 118: 2241–2249, 2017.

Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response

Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.