Smooth Muscle Cell-Mineralocorticoid Receptor as a Mediator of Cardiovascular Stiffness With Aging.

Stiffening of the vasculature with aging is a strong predictor of adverse cardiovascular events, independent of all other risk factors including blood pressure, yet no therapies target this process. MRs (mineralocorticoid receptors) in smooth muscle cells (SMCs) have been implicated in the regulation of vascular fibrosis but have not been explored in vascular aging. Comparing SMC-MR-deleted male mice to MR-intact littermates at 3, 12, and 18 months of age, we demonstrated that aging-associated vascular stiffening and fibrosis are mitigated by MR deletion in SMCs. Progression of cardiac stiffness and fibrosis and the decline in exercise capacity with aging were also mitigated by MR deletion in SMC. Vascular gene expression profiling analysis revealed that MR deletion in SMC is associated with recruitment of a distinct antifibrotic vascular gene expression program with aging. Moreover, long-term pharmacological inhibition of MR in aged mice prevented the progression of vascular fibrosis and stiffness and induced a similar antifibrotic vascular gene program. Finally, in a small trial in elderly male humans, short-term MR antagonism produced an antifibrotic signature of circulating biomarkers similar to that observed in the vasculature of SMC-MR-deleted mice. These findings suggest that SMC-MR contributes to vascular stiffening with aging and is a potential therapeutic target to prevent the progression of aging-associated vascular fibrosis and stiffness.

Direct contribution of vascular mineralocorticoid receptors to blood pressure regulation.

Hypertension is an extremely prevalent cardiovascular risk factor and current antihypertensive therapies do not adequately treat hypertension in many affected individuals. Thus, a better understanding of mechanisms of hypertension could lead to novel therapies. Mineralocorticoid receptors (MR) are known to regulate blood pressure by responding to aldosterone in the kidney to regulate sodium retention. Recent evidence supports a direct contribution of the vasculature to control of BP and suggests the possibility that MR antagonists may also lower blood pressure by acting on extrarenal MR. This review summarizes existing research considering the role of the vascular MR in regulating vasoreactivity and blood pressure. Multiple studies indicate a role for vascular MR in modulating vasoconstriction and vasorelaxation. Activation of MR in vascular endothelial and smooth muscle cells leads to increased reactive oxygen species production and decreased availability of nitric oxide, important regulators of vascular reactivity. Transgenic mouse models, including an endothelial MR overexpressing mouse and a smooth muscle cell-specific MR-knockout mouse, support a direct role for vascular MR in control of blood pressure. This new evidence demonstrating that vascular MR directly contribute to control of vasoreactivity and blood pressure supports vascular MR and the pathways they control as novel therapeutic targets to treat hypertension.

Time Course Analysis of Gene Expression Patterns in Zebrafish Eye During Optic Nerve Regeneration.

It is well-established that neurons in the adult mammalian central nervous system (CNS) are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in approximately 21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after ONX can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. To define different phases of regeneration after ONX, alpha tubulin 1 (tuba1) and growth-associated protein 43 (gap43), markers previously shown to correspond to morphophological events, were measured by real time quantitative PCR (qPCR). Microarray analysis was then performed at defined intervals (6 hours, 1, 4, 12, and 21 days) post-ONX and compared to SHAM. Results show that optic nerve damage induces multiple, phase-related transcriptional programs, with the maximum number of genes changed and highest fold-change occurring at 4 days. Several functional groups affected by optic nerve regeneration, including cell adhesion, apoptosis, cell cycle, energy metabolism, ion channel activity, and calcium signaling, were identified. Utilizing the whole eye allowed us to identify signaling contributions from the vitreous, immune and glial cells as well as the neural cells of the retina. Comparisons between our dataset and transcriptional profiles from other models of regeneration in zebrafish retina, heart and fin revealed a subset of commonly regulated transcripts, indicating shared mechanisms in different regenerating tissues. Knowledge of gene expression patterns in all components of the eye in a model of successful regeneration provides an entry point for functional analyses, and will help in devising hypotheses for testing normal and toxic regulatory factors.

Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.

BACKGROUND: Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. RESULTS: QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 - 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 - 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. CONCLUSION: This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.