Praliciguat inhibits progression of diabetic nephropathy in ZSF1 rats and suppresses inflammation and apoptosis in human renal proximal tubular cells.

Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.

Design principles for lymphatic drainage of fluid and solutes from collagen scaffolds.

In vivo, tissues are drained of excess fluid and macromolecules by the lymphatic vascular system. How to engineer artificial lymphatics that can provide equivalent drainage in biomaterials remains an open question. This study elucidates design principles for engineered lymphatics, by comparing the rates of removal of fluid and solute through type I collagen gels that contain lymphatic vessels or unseeded channels, or through gels without channels. Surprisingly, no difference was found between the fluid drainage rates for gels that contained vessels or bare channels. Moreover, solute drainage rates were greater in collagen gels that contained lymphatic vessels than in those that had bare channels. The enhancement of solute drainage by lymphatic endothelium was more pronounced in longer scaffolds and with smaller solutes. Whole-scaffold imaging revealed that endothelialization aided in solute drainage by impeding solute reflux into the gel without hindering solute entry into the vessel lumen. These results were reproduced by computational models of drainage with a flow-dependent endothelial hydraulic conductivity. This study shows that endothelialization of bare channels does not impede the drainage of fluid from collagen gels and can increase the drainage of macromolecules by preventing solute transport back into the scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 106-114, 2018.

Ex vivo gene delivery to hepatocytes: techniques, challenges, and underlying mechanisms.

Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.

Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels.

This work examines how mechanical signals affect the barrier function and stability of engineered human microvessels in microfluidic type I collagen gels. Constructs that were exposed to chronic low flow displayed high permeabilities to bovine serum albumin and 10 kDa dextran, numerous focal leaks, low size selectivity, and short lifespan of less than one week. Higher flows promoted barrier function and increased longevity; at the highest flows, the barrier function rivaled that observed in vivo, and all vessels survived to day 14. By studying the physiology of microvessels of different geometries, we established that shear stress and transmural pressure were the dominant mechanical signals that regulated barrier function and vascular stability, respectively. In microvessels that were exposed to high flow, elevation of intracellular cyclic AMP further increased the selectivity of the barrier and strongly suppressed cell proliferation. Computational models that incorporated stress dependence successfully predicted vascular phenotype. Our results indicate that the mechanical microenvironment plays a major role in the functionality and stability of engineered human microvessels in microfluidic collagen gels.

Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes.

This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of 1.4(-0.6)(+0.9)x10(-6) cm/s and 1.7(-0.5)(+0.8)x10(-6) cm/s (geometric mean+/-95% CI), respectively, and 1.2+/-0.5 (mean+/-95% CI) focal leaks per mm. Exposure to low concentrations (3 microM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 microM) and/or the phosphodiesterase inhibitor Ro-20-1724 (20 microM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.