Luis M. Alvarez

An engineered bivalent neuregulin protects against doxorubicin-induced cardiotoxicity with reduced proneoplastic potential.

Background— Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1&bgr; is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1&bgr; (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor–like domain of neuregulin-1&bgr; (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1&bgr;. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. Methods and Results— NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). Conclusions— NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels.

The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues.

Continuous Desulfurization of Dibenzothiophene with Rhodococcus rhodochrous IGTS8 (ATCC 53968)

Desulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrousIGTS8 was demonstrated in a 2‐L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L−1 h−1) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first‐order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h−1. Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off‐gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.

Engineered Bivalent Ligands to Bias ErbB Receptor-mediated Signaling and Phenotypes

The ErbB receptor family is dysregulated in many cancers, and its therapeutic manipulation by targeted antibodies and kinase inhibitors has resulted in effective chemotherapies. However, many malignancies remain refractory to current interventions. We describe a new approach that directs ErbB receptor interactions, resulting in biased signaling and phenotypes. Due to known receptor-ligand affinities and the necessity of ErbB receptors to dimerize to signal, bivalent ligands, formed by the synthetic linkage of two neuregulin-1β (NRG) moieties, two epidermal growth factor (EGF) moieties, or an EGF and a NRG moiety, can potentially drive homotypic receptor interactions and diminish formation of HER2-containing heterodimers, which are implicated in many malignancies and are a prevalent outcome of stimulation by native, monovalent EGF, or NRG. We demonstrate the therapeutic potential of this approach by showing that bivalent NRG (NN) can bias signaling in HER3-expressing cancer cells, resulting in some cases in decreased migration, inhibited proliferation, and increased apoptosis, whereas native NRG stimulation increased the malignant potential of the same cells. Hence, this new approach may have therapeutic relevance in ovarian, breast, lung, and other cancers in which HER3 has been implicated.

Multilayer Thin Film Coatings Capable of Extended Programmable Drug Release: Application to Human Mesenchymal Stem Cell Differentiation

The promise of cellular therapy lies in healing damaged tissues and organs in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Postnatal stem cells are ideally suited for cellular therapies due to their pluripotency and the ease with which they can be cultured on functionalized substrates. Creating environments to control and successfully drive their differentiation toward a lineage of choice is one of the most important challenges of current cell-based engineering strategies. In recent years, a variety of biomaterials platforms have been prepared for stem cell cultures, primarily to provide efficient delivery of growth or survival factors to cells and a conductive microenvironment for their growth. Here, we demonstrate that repeating tetralayer structures composed of biocompatible poly(methacrylic acid), poly(acrylamide), and poly(ethylene oxide)-block-poly(ε-caprolactone) micelles arrayed in layer-by-layer films can serve as a payload region for dexamethasone delivery to human mesenchymal stem cells (MSCs). This architecture can induce MSC differentiation into osteoblasts in a dose-dependent manner. The amount of Dex loaded in the films is controlled by varying the deposition conditions and the film thickness. Release of Dex is tuned by changing the amount of covalent cross-linking of multilayers via thermal treatments. The multilayer architecture including payload and cell-adhesion region introduced here are well suited for extended cell culture thus affording the important and protective effect of both Dex release and immobilization. These films may find applications in the local delivery of immobilized therapeutics for biomedical applications, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.