Herein, we describe a method for targeting to and retaining intravenously (IV) injected nanoparticles at the site of acute myocardial infarction (MI) in a rat model. Enzyme-responsive peptide-polymer amphiphiles (PPAs) were prepared and assembled as spherical micellar nanoparticles. The resulting nanoparticles respond to matrix metalloproteineases (MMP-2 and MMP-9) that are upregulated in heart tissue post-myocardial infarction. The nanoparticles undergo a morphological transition from spherical-shaped, discrete materials to network-like assemblies when acted upon by MMPs. We show that 15–20 nm, responsive nanoparticles can be injected IV, undergoing reaction with MMPs in the heart after MI, with the resulting assemblies remaining within the infarct for up to 28 days. The initial studies reported here set the stage for the development of targeting systems for therapeutic delivery for acute MI. Critically, with this development, injection of materials is possible via the IV route immediately following MI, resulting in targeted accumulation and long term retention at the site of MI.

BACKGROUND There is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models. OBJECTIVES We sought to elucidate the tissue level mechanisms underlying the therapeutic benefits of myocardial matrix injection. METHODS Myocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia-reperfusion in Sprague Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks post-injection. Whole transcriptome microarrays were performed on ribonucleic acid (RNA) isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histological quantification confirmed expression of key genes and their activation in altered pathways. RESULTS Principal component analysis of the transcriptomes showed that samples collected from myocardial matrix-injected infarcts are distinct and cluster separately from saline-injected controls. Pathway analysis indicated that these differences are due to changes in several tissue processes that may contribute to improved cardiac healing post-MI. Matrix-injected infarcted myocardium exhibits an altered inflammatory response, reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac hypertrophy and fibrosis, altered metabolic enzyme expression, increased cardiac transcription factor expression, and progenitor cell recruitment, along with improvements in global cardiac function and hemodynamics. CONCLUSIONS These results indicate that the myocardial matrix alters several key pathways post-MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

Transcriptional networks have been shown to evolve very rapidly, prompting questions as to how such changes arise and are tolerated. Recent comparisons of transcriptional networks across species have implicated variations in the cis-acting DNA sequences near genes as the main cause of divergence. What is less clear is how these changes interact with trans-acting changes occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-1 transcriptional network that allows for conserved transcriptional regulation despite continued genetic change. We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single AP-1 family member, distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that, despite their different DNA-binding domains, the AP-1 orthologs regulate a conserved block of genes. This conservation is enabled by concomitant changes in the cisregulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-1 regulatory system in such a way as to compensate for one another. This demonstrates an example of ''coevolution'' between a DNAbinding transcription factor and its cis-regulatory site, reminiscent of the coevolution of protein binding partners.

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Injectable biopolymer hydrogels have gained attention for use as scaffolds to promote cardiac function and prevent negative left ventricular (LV) remodeling post-myocardial infarction (MI). However, most hydrogels tested in preclinical studies are not candidates for minimally invasive catheter delivery due to excess material viscosity, rapid gelation times, and/or concerns regarding hemocompatibility and potential for embolism. We describe a platform technology for progelator materials formulated as sterically constrained cyclic peptides which flow freely for low resistance injection, and rapidly assemble into hydrogels when linearized by disease-associated enzymes. Their utility in vivo is demonstrated by their ability to flow through a syringe and gel at the site of MI in rat models. Additionally, synthetic functionalization enables these materials to flow through a cardiac injection catheter without clogging, without compromising hemocompatibility or cytotoxicity. These studies set the stage for the development of structurally dynamic biomaterials for therapeutic hydrogel delivery to the MI.

Duplication of genes encoding transcription factors plays an essential role in driving phenotypic variation. Because regulation can occur at multiple levels, it is often difficult to discern how each duplicated factor achieves its regulatory specificity. In these cases, a ''systems approach'' may distinguish the role of each factor by integrating complementary large-scale measurements of the regulatory network. To explore such an approach, we integrate growth phenotypes, promoter binding profiles, and gene expression patterns to model the DNA damage response network controlled by the Yeast-specific AP-1 (YAP) family of transcription factors. This analysis reveals that YAP regulatory specificity is achieved by at least three mechanisms: (i) divergence of DNAbinding sequences into two subfamilies; (ii) condition-specific combinatorial regulation by multiple Yap factors; and (iii) interactions of Yap 1, 4, and 6 with chromatin remodeling proteins. Additional microarray experiments establish that Yap 4 and 6 regulate gene expression through interactions with the histone deacetylase, Hda1. The data further highlight differences among Yap paralogs in terms of their regulatory mode of action (activation vs. repression). This study suggests how other large TF families might be disentangled in the future.ChIP-chip ͉ evolution ͉ systems biology

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.

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