Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

Methods for the Assessment of Active Transforming Growth Factor-ß in Cells and Tissues.

The potent and pluripotent cytokine TGFß has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFß exists in both latent and functionally active forms. Large amounts of latent TGFß are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFß activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFß expression as small changes in TGFß activity levels could be masked by the large amounts of latent TGFß available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFß in cells and tissues. This chapter includes methods for the assessment of TGFß activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

Molecular validation of the precision-cut kidney slice (PCKS) model of renal fibrosis through assessment of TGF-ß1-induced Smad and p38/ERK signaling.

BACKGROUND: The precision-cut kidney slice (PCKS) model appears to be a useful ex vivo model of renal fibrosis. However, little in-depth molecular investigation on the PCKS model has been performed. Therefore, the aim of this study will be to investigate and validate the molecular validity of this model. METHODS: The PCKS model was constructed in male C57BL/6 mice. To induce renal fibrosis, PCKS were incubated in recombinant human TGF-ß1 for 48 h. Protein expression of phosphorylated Smad2 (p-Smad2, cytosolic and nuclear), Smad7, phosphorylated ERK1 (p-ERK1), phosphorylated ERK2 (p-ERK2), and phosphorylated p38 MAPK (p-p38 MAPK) was measured using Western blotting. To assess Smad2/3 heteromeric complex formation and phosphorylated Smad3 (p-Smad3) expression, immunoprecipitation was performed with an anti-Smad2 or an anti-Smad3 antibody, respectively, prior to Western blotting. RESULTS: p-Smad2 and p-Smad3 were significantly upregulated in the PCKS model relative to control (p<0.05). However, we found no significant difference in Smad7 expression between the PCKS model and control (p>0.05). The PCKS model demonstrated significantly greater Smad2/3 complex formation and nuclear translocation relative to control (p<0.05). The PCKS model showed significantly greater expression of p-ERK1, p-ERK2, and p-p38 MAPK relative to control (p<0.05). CONCLUSIONS: The PCKS model displays several well-established molecular markers of renal fibrosis. However, the PCKS model failed to display Smad7 downregulation and appears to display "over-activation" of p-Smad2 and p-Smad3 as well as "under-activation" of ERK1/2 and p38 MAPK signaling vis-à-vis the well-established in vivo unilateral ureteric obstruction model of renal fibrosis.

A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

Assessment of early renal damage in diabetic rhesus monkeys.

The objectives of the study were to improve the model system of diabetic nephropathy in nonhuman primates and assess the early renal damage. Diabetes was induced in monkeys by streptozotocin, and the animals were administered exogenous insulin to control blood glucose (BG). Animals were divided into four groups, including the normal group (N = 3), group A (streptozotocin diabetic model with control of BG < 10 mmol/L, N = 3), group B (streptozotocin diabetic model with control of BG between 15 and 20 mmol/L, N = 4), and group C (streptozotocin diabetic model with control of BG between 15 and 20 mmol/L and high-sodium and high-fat diet, N = 4). The following parameters were evaluated: (1) blood biochemistry and routine urinalysis, (2) color Doppler ultrasound, (3) angiography, (4) renal biopsy, and (5) renal fibrosis-related gene expression levels. Animals in group C developed progressive histologic changes with typical diabetic nephropathy resembling diabetic nephropathy in human patients and exhibited accelerated development of diabetic nephropathy compared with other nonhuman primate models. Significant changes in the expression of the Smad2/3 gene and eNOS in renal tissue were also observed in the early stage of diabetic nephropathy. In conclusion, our model is an excellent model of diabetic nephropathy for understanding the pathogenesis of diabetic nephropathy.