Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

Transcutaneous Flexible Sensor for In Vivo Photonic Detection of pH and Lactate.

Clinical research shows that frequent measurements of both pH and lactate can help guide therapy and improve patient outcome. However, current methods of sampling blood pH and lactate make it impractical to take readings frequently (due to the heightened risk of blood infection and anemia). As a solution, we have engineered a subcutaneous pH and lactate sensor (PALS) that can provide continuous, physiologically relevant measurements. To measure pH, a sheet containing a pH-sensitive fluorescent dye is placed over 400 and 465 nm light-emitting diodes (LEDs) and a filter-coated photodetector. The filter-coated photodetector collects an emitted signal from the dye for each LED excitation, and the ratio of the emitted signals is used to monitor pH. To measure lactate, two sensing sheets comprising an oxygen-sensitive phosphorescent dye are each mounted to a 625 nm LED. One sheet additionally comprises the enzyme lactate oxidase. The LEDs are sequentially modulated to excite the sensing sheets, and their phase shift at the LED drive frequency is used to monitor lactate. In vitro results indicate that PALS successfully records pH changes from 6.92 to 7.70, allowing for discrimination between acidosis and alkalosis, and can track lactate levels up to 9 mM. Both sensing strategies exhibit fast rise times (< 5 min) and stable measurements. Multianalyte in vitro models of physiological disorders show that the sensor measurements consistently quantify the expected pathophysiological trends without cross talk; in vivo rabbit testing further indicates usefulness in the clinical setting.

Clinical evaluation of a novel subcutaneous lactate monitor.

Lactate levels are commonly used as an indirect measure to assess metabolic stress in clinical conditions like sepsis. Dynamic lactate measurements are recommended to assess and guide treatment in patients with shock and other critical care conditions. A minimally invasive, continuous lactate monitor has potential to improve clinical decisions and patient care. The purpose of the study was to evaluate continuous lactate measurements of a novel enzymatic Continuous Lactate Monitor (CLM) developed in our laboratory. Lactate levels were monitored during incremental cycling exercise challenges as a tool for hyperlactatemia. Six healthy individuals 18-45 y/o (4 males, 2 females) participated in the study. CLM devices were inserted subcutaneously in the postero-lateral trunk below the renal angle, one hour before the exercise challenge. Each exercise challenge consisted of a 3 to 12-min warm up period, followed by up to 7, 4-min incremental workload bouts separated by rest intervals. Continuous lactate measurements obtained from CLM were compared with commercial lactate analyzer (Abbott iSTAT) measurements of venous blood (plasma) drawn from the antecubital vein. Blood was drawn at up to 25 time points spanning the duration of before exercise, during exercise, and up to 120 min post exercise. Area under the curve (AUC), and delay time were calculated to compare the CLM readings with plasma lactate concentration. Average plasma lactate concentration increased from 1.02 to 16.21 mM. Ratio of AUC derived from CLM to plasma lactate was 1.025 (0.990-1.058). Average dynamic delay time of CLM to venous plasma lactate was 5.22 min (2.87-10.35). Insertion sites examined 48 h after CLM removal did not show signs of side effects and none required medical attention upon examination. The newly developed CLM has shown to be a promising tool to continuously measure lactate concentration in a minimally invasive fashion. Results indicate the CLM can provide needed trends in lactate over time. Such a device may be used in the future to improve treatment in clinical conditions such as sepsis.

Cell contact guidance via sensing anisotropy of network mechanical resistance.

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*| -1 - |X*| -1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.

Matrix crosslinking enhances macrophage adhesion, migration, and inflammatory activation.

Macrophages are versatile cells of the innate immune system that can adopt a variety of functional phenotypes depending on signals in their environment. In previous work, we found that culture of macrophages on fibrin, the provisional extracellular matrix protein, inhibits their inflammatory activation when compared to cells cultured on polystyrene surfaces. Here, we sought to investigate the role of matrix stiffness in the regulation of macrophage activity by manipulating the mechanical properties of fibrin. We utilize a photo-initiated crosslinking method to introduce dityrosine crosslinks to a fibrin gel and confirm an increase in gel stiffness through active microrheology. We observe that matrix crosslinking elicits distinct changes in macrophage morphology, integrin expression, migration, and inflammatory activation. Macrophages cultured on a stiffer substrate exhibit greater cell spreading and expression of αM integrin. Furthermore, macrophages cultured on crosslinked fibrin exhibit increased motility. Finally, culture of macrophages on photo-crosslinked fibrin enhances their inflammatory activation compared to unmodified fibrin, suggesting that matrix crosslinking regulates the functional activation of macrophages. These findings provide insight into how the physical properties of the extracellular matrix might control macrophage behavior during inflammation and wound healing.

Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way. STATEMENT OF SIGNIFICANCE: Fibrin hydrogels are used as a naturally derived model to study interactions between cells and their surrounding extracellular matrix (ECM). ECM stiffness influences cell state. Cells in 3D culture considerably modify the stiffness of their pericellular space, which can be quite heterogeneous at the micron-scale. Here we present and evaluate a method to pattern stiffness within fibrin hydrogels using a laser scanning confocal microscope and selective photo crosslinking. We believe that this technique can aid future studies of cell-ECM interactions by enabling researchers to modify the pericellular distribution of stiffness.

Sprouting angiogenesis induces significant mechanical heterogeneities and ECM stiffening across length scales in fibrin hydrogels.

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment, have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.

Vascularization and innervation of slits within polydimethylsiloxane sheets in the subcutaneous space of athymic nude mice.

Success of cell therapy in avascular sites will depend on providing sufficient blood supply to transplanted tissues. A popular strategy of providing blood supply is to embed cells within a functionalized hydrogel implanted within the host to stimulate neovascularization. However, hydrogel systems are not always amenable for removal post-transplantation; thus, it may be advantageous to implant a device that contains cells while also providing access to the circulation so retrieval is possible. Here we investigate one instance of providing access to a vessel network, a thin sheet with through-cut slits, and determine if it can be vascularized from autologous materials. We compared the effect of slit width on vascularization of a thin sheet following subcutaneous implantation into an animal model. Polydimethylsiloxane sheets with varying slit widths (approximately 150, 300, 500, or 1500 µm) were fabricated from three-dimensional printed molds. Subcutaneous implantation of sheets in immunodeficient mice revealed that smaller slit widths have evidence of angiogenesis and new tissue growth, while larger slit widths contain native mature tissue squeezing into the space. Our results show that engineered slit sheets may provide a simple approach to cell transplantation by providing a prevascularized and innervated environment.

Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.

Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging Microscopy as a surrogate for the metabolic state of the cells. We observed that cells seeded in tumor ECM had higher relative levels of free NADH, consistent with a higher glycolytic rate, than those seeded in normal ECM. These results demonstrate that the ECM plays an important role in the growth of cancer cells and their associated vasculature.

Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering.

UNLABELLED: Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVß1 and α5ß1, and several laminin binding integrins (α7ß1, α6ß1, α3ß1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs. STATEMENT OF SIGNIFICANCE: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury.

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells.

Vibrational spectroscopy, both infrared absorption and Raman spectroscopy, have attracted increasing attention for biomedical applications, from in vivo and ex vivo disease diagnostics and screening, to in vitro screening of therapeutics. There remain, however, many challenges related to the accuracy of analysis of physically and chemically inhomogeneous samples, across heterogeneous sample sets. Data preprocessing is required to deal with variations in instrumental responses and intrinsic spectral backgrounds and distortions in order to extract reliable spectral data. Data postprocessing is required to extract the most reliable information from the sample sets, based on often very subtle changes in spectra associated with the targeted pathology or biochemical process. This review presents the current understanding of the factors influencing the quality of spectra recorded and the pre-processing steps commonly employed to improve on spectral quality. It further explores some of the most common techniques which have emerged for classification and analysis of the spectral data for biomedical applications. The importance of sample presentation and measurement conditions to yield the highest quality spectra in the first place is emphasised, as is the potential of model simulated datasets to validate both pre- and post-processing protocols.

Novel insights from 3D models: the pivotal role of physical symmetry in epithelial organization.

3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties.

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.

Multivariate statistical methodologies applied in biomedical Raman spectroscopy: assessing the validity of partial least squares regression using simulated model datasets.

Raman spectroscopy is fast becoming a valuable analytical tool in a number of biomedical scenarios, most notably disease diagnostics. Importantly, the technique has also shown increasing promise in the assessment of drug interactions on cellular and subcellular levels, particularly when coupled with multivariate statistical analysis. However, with respect to both Raman spectroscopy and the associated statistical methodologies, an important consideration is the accuracy of these techniques and more specifically, the sensitivities which can be achieved, and ultimately the limits of detection of the various methods. The purpose of this study is thus the construction of a model simulated dataset with the aim of testing the accuracy and sensitivity of the partial least squares regression (PLSR) approach to spectral analysis. The basis of the dataset is the experimental spectral profiles of a previously reported Raman spectroscopic analysis of the interaction of the cancer chemotherapeutic agent cisplatin in an adenocarcinomic human alveolar basal epithelial cell-line, in vitro, and is thus reflective of actual experimental data. The simulated spectroscopic data are constructed by adding known perturbations which are independently linear in drug doses as well as cytological responses experimentally determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. It is demonstrated that, through appropriate choice of dose range, PLSR against the respective targets can differentiate between the spectroscopic signatures of the direct chemical effect of the drug dose and the indirect cytological effect it produces.

Raman spectroscopy in nanomedicine: current status and future perspective.

Raman spectroscopy is a branch of vibration spectroscopy that is capable of probing the chemical composition of materials. Recent advances in Raman microscopy have significantly added to the range of applications, which now extend from medical diagnostics to exploring the interfaces between biological organisms and nanomaterials. In this review, Raman is introduced in a general context, highlighting some of the areas in which the technique has been successful in the past, as well as some of the potential benefits it offers over other analytical modalities. The subset of Raman techniques that specifically probe the nanoscale, namely surface- and tip-enhanced Raman spectroscopy, will be described and specific applications relevant to nanomedical applications will be reviewed. Progress in the use of traditional label-free Raman for investigation of nanoscale interactions will be described, and recent developments in coherent anti-Stokes Raman scattering will be explored, particularly its applications to biomedical and nanomedical fields.

Spectral cross-correlation as a supervised approach for the analysis of complex Raman datasets: the case of nanoparticles in biological cells.

Spectral cross-correlation is introduced as a methodology to identify the presence and subcellular distribution of nanoparticles in cells. Raman microscopy is employed to spectroscopically image biological cells previously exposed to polystyrene nanoparticles, as a model for the study of nano-bio interactions. The limitations of previously deployed strategies of K-means clustering analysis and principal component analysis are discussed and a novel methodology of spectral cross-correlation analysis is introduced and compared with the performance of classical least squares analysis, in both unsupervised and supervised modes. The previous study demonstrated the feasibility of using Raman spectroscopy to map cells and identify polystyrene nanoparticles in a lipid rich environment, which is suggestive of the membrane rich endoplasmic reticulum. However, short comings in identification of all nanoparticle signatures in the cell using K-means clustering are apparent, as highlighted by principal component analysis of the identified clusters which demonstrates that K-means clustering does not identify all regions where spectral signatures of the nanoparticles are evident. Thus, two more sophisticated analytical approaches to the extraction of the nanoparticle signatures from the Raman spectral datasets, namely classical least squares analysis and cross-correlation analysis, were employed and are demonstrated to improve the identification of spectroscopic signatures characteristic of polystyrene nanoparticles in a cellular environment. Additionally, to investigate the local biochemical environment in which the nanoparticles are trafficked, a pure spectrum of 3-sn-phosphatidyl ethanolamine was cross-correlated against the Raman dataset, further suggesting the particles are indeed localized in a lipid rich environment. Furthermore, to demonstrate the robustness and versatility of the analysis method, a spectrum of pure RNA was used to demonstrate that a differentiation could be made between DNA of the nucleus and RNA of the nucleolus using the supervised spectral cross-correlation technique.

Activin-betaA signaling is required for zebrafish fin regeneration.

Vertebrate limb regeneration occurs in anamniotes such as newts, salamanders, and zebrafish. After appendage amputation, the resection site is covered by a wound epidermis capping the underlying mature tissues of the stump from which the blastema emerges. The blastema is a mass of progenitor cells that constitute an apical growth zone. During outgrowth formation, the proximal blastemal cells progressively leave the zone and undergo the differentiation that results in the replacement of the amputated structures. Little is known about the mechanisms triggering regenerative events after injury. The zebrafish caudal fin provides a valuable model to study the mechanisms of regeneration. Zebrafish blastemal cells express specific genes, such as the homeobox-containing transcription factors msxB and msxC, and secreted signal FGF20a. In this study, we set out to identify signals that are transcriptionally upregulated after fin amputation and before blastema formation. Accordingly, a gene encoding a TGFbeta-related ligand, activin-betaA (actbetaA), was found to be strongly induced within 6 hr after fin amputation at the wound margin, and later in the blastema. Inhibition of Activin signaling through two specific chemical inhibitors, SB431542 and SB505124, lead to the early and complete block of regeneration. The morpholino knockdown of actbetaA and its receptor alk4 impaired the progression of regeneration. Closer examination of the phenotype revealed that Activin signaling is necessary for cell migration during wound healing and blastemal proliferation. These findings reveal a role of Activin-betaA signaling in the tissue repair after injury and subsequent outgrowth formation during epigenetic regeneration of the vertebrate appendage.

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair.

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.