Christopher L. Grigsby

Cardiomyocyte-Specific Deletion of the Vitamin D Receptor Gene Results in Cardiac Hypertrophy

Background— A variety of studies carried out using either human subjects or laboratory animals suggest that vitamin D and its analogues possess important beneficial activity in the cardiovascular system. Using Cre-Lox technology we have selectively deleted the vitamin D receptor (VDR) gene in the cardiac myocyte in an effort to better understand the role of vitamin D in regulating myocyte structure and function. Methods and Results— Targeted deletion of the exon 4 coding sequence in the VDR gene resulted in an increase in myocyte size and left ventricular weight/body weight versus controls both at baseline and following a 7-day infusion of isoproterenol. There was no increase in interstitial fibrosis. These knockout mice demonstrated a reduction in end-diastolic and end-systolic volume by echocardiography, activation of the fetal gene program (ie, increased atrial natriuretic peptide and alpha skeletal actin gene expression), and increased expression of modulatory calcineurin inhibitory protein 1 (MCIP1), a direct downstream target of calcineurin/nuclear factor of activated T cell signaling. Treatment of neonatal cardiomyocytes with 1,25-dihydroxyvitamin D partially reduced isoproterenol-induced MCIP1 mRNA and protein levels and MCIP1 gene promoter activity. Conclusions— Collectively, these studies demonstrate that the vitamin D-VDR signaling system possesses direct, antihypertrophic activity in the heart. This appears to involve, at least in part, suppression of the prohypertrophic calcineurin/NFAT/MCIP1 pathway. These studies identify a potential mechanism to account for the reported beneficial effects of vitamin D in the cardiovascular system.

Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery

Engineering polymeric gene-delivery vectors to release an intact DNA payload at the optimal time and subcellular compartment remains a formidable challenge. An ideal vector would provide total protection of complexed DNA from degradation prior to releasing it efficiently near or within the nucleus of a target cell. While optimization of polymer properties, such as molecular weight and charge density, has proved largely inadequate in addressing this challenge, applying polymeric carriers that respond to temperature, light, pH and redox environment to trigger a switch from a tight, protective complex to a more relaxed interaction favouring release at the appropriate time and place has shown promise. Currently, a paucity of gene carriers able to satisfy the contrary requirements of adequate DNA protection and efficient release contributes to the slow progression of non-viral gene therapy towards clinical translation. This review highlights the promising carrier designs that may achieve an optimal balance of DNA protection and release. It also discusses the imaging techniques and three-dimensional in vitro models that can help study these two barriers in the non-viral gene transfer process. Ultimately, efficacious non-viral gene therapy will depend on the combination of intelligent material design, innovative imaging techniques and sophisticated in vitro model systems to facilitate the rational design of polymeric gene-delivery vectors.

Tonicity-dependent induction of Sgk1 expression has a potential role in dehydration-induced natriuresis in rodents.

In various mammalian species, including humans, water restriction leads to an acute increase in urinary sodium excretion. This process, known as dehydration natriuresis, helps prevent further accentuation of hypernatremia and the accompanying rise in extracellular tonicity. Serum- and glucocorticoid-inducible kinase (Sgk1), which is expressed in the renal medulla, is regulated by extracellular tonicity. However, the mechanism of its regulation and the physiological role of hypertonicity-induced SGK1 gene expression remain unclear. Here, we identified a tonicity-responsive enhancer (TonE) upstream of the rat Sgk1 transcriptional start site. The transcription factor NFAT5 associated with TonE in a tonicity-dependent fashion in cultured rat renal medullary cells, and selective blockade of NFAT5 activity resulted in suppression of the osmotic induction of the Sgk1 promoter. In vivo, water restriction of rats or mice led to increased urine osmolality, increased Sgk1 expression, increased expression of the type A natriuretic peptide receptor (NPR-A), and dehydration natriuresis. In cultured rat renal medullary cells, siRNA-mediated Sgk1 knockdown blocked the osmotic induction of natriuretic peptide receptor 1 (Npr1) gene expression. Furthermore, Npr1-/- mice were resistant to dehydration natriuresis, which suggests that Sgk1-dependent activation of the NPR-A pathway may contribute to this response. Collectively, these findings define a specific mechanistic pathway for the osmotic regulation of Sgk1 gene expression and suggest that Sgk1 may play an important role in promoting the physiological response of the kidney to elevations in extracellular tonicity.

Expression of the vitamin d receptor is increased in the hypertrophic heart.

The liganded vitamin D receptor (VDR) is thought to play an important role in controlling cardiac function. Specifically, this system has been implicated as playing an antihypertrophic role in the heart. Despite this, studies of VDR in the heart have been limited in number and scope. In the present study, we used a combination of real-time polymerase chain reaction, Western blot analysis, immunofluorescence, and transient transfection analysis to document the presence of functional VDR in both the myocytes and fibroblasts of the heart, as well as in the intact ventricular myocardium. We also demonstrated the presence of 1-&agr;-hydroxylase and 24-hydroxylase in the heart, 2 enzymes involved in the synthesis and metabolism of 1,25 dihydroxyvitamin D. VDR is shown to interact directly with the human B-type natriuretic peptide gene promoter, a surrogate marker of the transcriptional response to hypertrophy. Of note, induction of myocyte hypertrophy either in vitro or in vivo leads to an increase in VDR mRNA and protein levels. Collectively, these findings suggest that the key components required for a functional 1,25 dihydroxyvitamin D–dependent signaling system are present in the heart and that this putatively antihypertrophic system is amplified in the setting of cardiac hypertrophy.

Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells.

Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm−2 doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1+ (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP+ cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.

Tuning Physical Properties of Nanocomplexes through Microfluidics-Assisted Confinement

The future of genetic medicine hinges on successful intracellular delivery of nucleic acid-based therapeutics. While significant effort has concentrated on developing nanocarriers to improve the delivery aspects, scant attention has been paid to the synthetic process of poorly controlled nanocomplex formation. Proposed here is a reliable system to better control the complexation process, and thus the physical properties of the nanocomplexes, through microfluidics-assisted confinement (MAC) in picoliter droplets. We show that these homogeneous MAC-synthesized nanocomplexes exhibit narrower size distribution, lower cytotoxicity, and higher transfection efficiency compared to their bulk-synthesized counterparts. MAC represents a physical approach to control the energetic self-assembly of polyelectrolytes, thereby complementing the chemical innovations in nanocarrier design to optimize nucleic acid and peptide delivery.

Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models

Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid-polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3±8.5%) and (37.5±1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA-DNA nanoparticles remained in the intestinal cells even after 24h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA-DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone would not engender a significant therapeutic effect, and this transport modality must be augmented by other means in vivo to render nonviral oral gene delivery practical.

A role for the cell cycle phosphatase Cdc25a in vitamin D-dependent inhibition of adult rat vascular smooth muscle cell proliferation.

We have explored the mechanism(s) underlying 1,25 dihydroxyvitamin Ds (1,25(OH)(2)D) suppression of agonist-induced vascular smooth muscle cell (VSMC) proliferation. Quiescent cultured adult rat VSMC were treated with 1,25(OH)(2)D for 48h and endothelin (ET) or angiotensin II (AII) for the final 24h. We show that VSMC responded to 1,25(OH)(2)D or its less hypercalcemic analogue RO 25-6760 with ∼70% inhibition of ET-dependent (3)H-thymidine incorporation. The inhibition was linked to a comparable reduction in ET-stimulated cyclin-dependent kinase 2 (Cdk2) activity and suppression of an ET-induced Cdk2 activator, cell division cycle 25 homolog A (Cdc25A). Both 1,25(OH)(2)D and RO 25-6760 completely inhibited the ET-dependent increase in Cdc25A mRNA and protein levels, phosphatase and promoter activities. 1,25(OH)(2)D also suppressed AII-induced DNA synthesis, Cdk2 activity and Cdc25A gene transcription. Inhibition of Cdc25A gene expression using a siRNA approach resulted in significant inhibition of ET or AII-dependent Cdk2 activity and (3)H-thymidine incorporation. The Cdc25A siRNA-mediated inhibition of ET or AII-induced Cdk2 activity and DNA synthesis was not additive with that produced by 1,25(OH)(2)D treatment. These data demonstrate that 1,25(OH)(2)D inhibits VSMC proliferation through a Cdc25A-dependent mechanism and suggest that this hormone may prove useful in the management of disorders characterized by aberrant proliferation of VSMC in the vascular wall.

Shape-controlled synthesis of hybrid nanomaterials via three-dimensional hydrodynamic focusing.

Shape-controlled synthesis of nanomaterials through a simple, continuous, and low-cost method is essential to nanomaterials research toward practical applications. Hydrodynamic focusing, with its advantages of simplicity, low-cost, and precise control over reaction conditions, has been used for nanomaterial synthesis. While most studies have focused on improving the uniformity and size control, few have addressed the potential of tuning the shape of the synthesized nanomaterials. Here we demonstrate a facile method to synthesize hybrid materials by three-dimensional hydrodynamic focusing (3D-HF). While keeping the flow rates of the reagents constant and changing only the flow rate of the buffer solution, the molar ratio of two reactants (i.e., tetrathiafulvalene (TTF) and HAuCl4) within the reaction zone varies. The synthesized TTF–Au hybrid materials possess very different and predictable morphologies. The reaction conditions at different buffer flow rates are studied through computational simulation, and the formation mechanisms of different structures are discussed. This simple one-step method to achieve continuous shape-tunable synthesis highlights the potential of 3D-HF in nanomaterials research.

Understanding nonviral nucleic acid delivery with quantum dot-FRET nanosensors.

Nonviral delivery of nucleic acids is a potentially safe and viable therapeutic modality for inherited and acquired diseases. However, current systems have proven too inefficient for widespread clinical translation. The rational design of improved carriers depends on a quantitative, mechanistic understanding of the rate-limiting barriers to efficient intracellular delivery. Separation of the nucleic acid from the carrier is one of the barriers, which may be analyzed by Förster resonance energy transfer (FRET), a mechanism used to detect interactions between fluorescently labeled molecules. When applied to the molecular components of polymer or lipid-based nanocomplexes, FRET provides information on their complexation status, uptake, release and degradation. Recently, the design of FRET systems incorporating quantum dots as energy donors has led to improved signal stability, allowing prolonged measurements, as well as increased sensitivity, enabling direct detection and the potential for multiplexing. The union of quantum dots and FRET is providing new insights into the mechanisms of nonviral nucleic acid delivery through convergent characterization of delivery barriers, and has the potential to accelerate the design of improved carriers to realize the potential of nucleic acid therapeutics and gene medicine.