A two-dimensional finite element model of intercellular cAMP signaling through gap junction channels.

While cyclic adenosine monophosphate (cAMP) is typically considered an intracellular signal, it has been shown to spread between adjacent cells through connexin-based gap junction channels, promoting gap junctional intercellular communication (GJIC). Gap junction-mediated signaling is critical for the coordinated function of many tissues, and have been linked with cardiovascular disease, neurogenerative disease, and cancers. In particular, it plays a complex role in tumor suppression or promotion. This work introduces a two-dimensional finite element model that can describe intercellular cAMP signaling in the presence of gap junctions on membrane interfaces. The model was utilized to simulate cAMP transfer through one and two gap junction channels on the interface of a cluster of two pulmonary microvascular endothelial cells. The simulation results were found to generally agree with what has been observed in the literature in terms of GJIC. The research outcomes suggest that the proposed model can be employed to evaluate the permeability properties of a gap junction channel if its cAMP volumetric flow rate can be experimentally measured.

Hyperspectral imaging and adaptive thresholding to identify agonist-induced cAMP signals in pulmonary microvascular endothelial cells.

Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Förster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest - ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE1) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.

A three-dimensional finite element model of cAMP signals.

This paper presents a three-dimensional finite element model for cyclic adenosine monophosphate (cAMP) signaling. Governing equations for the synthesis, diffusion, and degradation of cAMP were numerically implemented using the finite element method. Simulated results were displayed as time course plots of cAMP concentrations at selected nodes within the discretized geometry. The validity of the finite element model was assessed by comparing simulated results against analytical or other numerical solutions of cAMP concentration distribution for a spherical cellular volume. An endothelial cell was also simulated using its discretized geometry obtained from microscopic cellular cross-sectional images. Simulated solutions using the spherical cellular volume produced near identical cAMP concentration plots to the analytical solutions and were in good agreements with numerical results obtained from VCell, an existing software package for modeling cell biological systems. The validated 3-D finite element model was then employed to simulate the cAMP signaling pathway within a pulmonary microvascular endothelial cell geometry.

Milestones in the development and implementation of FRET-based sensors of intracellular signals: A biological perspective of the history of FRET.

FÓ§rster resonance energy transfer (FRET) has been described for more than a century. FRET has become a mainstay for the study of protein localization in living cells and tissues. It has also become widely used in the fields that comprise cellular signaling. FRET-based probes have been developed to monitor second messenger signals, the phosphorylation state of peptides and proteins, and subsequent cellular responses. Here, we discuss the milestones that led to FRET becoming a widely used tool for the study of biological systems: the theoretical description of FRET, the insight to use FRET as a molecular ruler, and the isolation and genetic modification of green fluorescent protein (GFP). Each of these milestones were critical to the development of a myriad of FRET-based probes and reporters in common use today. FRET-probes offer a unique opportunity to interrogate second messenger signals and subsequent protein phosphorylation - and perhaps the most effective approach for study of cAMP/PKA pathways. As such, FRET probes are widely used in the study of intracellular signaling pathways. Yet, somehow, the potential of FRET-based probes to provide windows through which we can visualize complex cellular signaling systems has not been fully reached. Hence we conclude by discussing the technical challenges to be overcome if FRET-based probes are to live up to their potential for the study of complex signaling networks.

A two-dimensional finite element model of cyclic adenosine monophosphate (cAMP) intracellular signaling.

In this work, we present a two-dimensional finite element analysis (FEA) model that describes fundamental intracellular signals of cyclic adenosine monophosphate (cAMP) in a general fashion. The model was subsequently solved numerically and the results were displayed in forms of time-course plots of cAMP concentration at a cellular location or color-filled contour maps of cAMP signal distribution within the cell at specific time points. Basic intracellular cAMP signaling was described in this model so it can be numerically validated by verifying its numerical results against available analytical solutions and against results obtained from other numerical techniques reported in the literature. This is the first important step before the model can be expanded in future work. Model simulations demonstrate that under certain conditions, sustained cAMP concentrations can be formed within endothelial cells (ECs), similar to those observed in rat pulmonary microvascular ECs. Spatial and temporal cAMP dynamic simulations indicated that the proposed FEA model is an effective tool for the study of the kinetics and spatial spread of second messenger signaling and can be expanded to simulate second messenger signals in the pulmonary vasculature.

MicroRNAs as potential clinical biomarkers: emerging approaches for their detection.

MicroRNAs (miRNAs) have emerged as novel post-transcriptional regulators of gene expression. These short non-coding RNAs are involved in diverse biological processes and their dysregulation is often observed under diseased conditions. Therefore, miRNAs hold great potential as clinical biomarkers of physiological and pathological states and extensive efforts are underway to develop efficient approaches for their detection. We review recent advances and discuss the promises and pitfalls of emerging methods of miRNA profiling and detection.