BackgroundAccurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition.MethodsHere we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time.ResultsDose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material.ConclusionsThe advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users.

Proliferation and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are critical processes involved in atherosclerosis. Recent studies have revealed that microRNAs (miRNAs) can be detected in circulating blood with a stable form and the expression profiles differ in many cellular processes associated with coronary artery disease (CAD). However, little is known about their role, especially serum-derived miRNAs, in ECs and VSMCs phenotype modulation during atherosclerosis. We compared the miRNA expressions in serum samples from 13 atherosclerotic CAD patients and 5 healthy control subjects and identified 36 differentially expressed miRNAs. The expression of selected miRNAs (miR-135b-5p and miR-499a-3p) was further validated in 137 serum samples. Interestingly, miR-135b-5p and miR-499a-3p directly regulated a common target gene: myocyte enhancer factor 2C (MEF2C) which plays an important role in modulating cell phenotype of cardiovascular systems. Furthermore, our results indicated that the 2 elevated miRNAs could jointly promote ECs and VSMCs proliferation and migration by repressing MEF2C expression. Together, our findings demonstrated a serum-based miRNA expression profile for atherosclerotic CAD patients, potentially revealing a previously undocumented mechanism for cell proliferation and migration mediated by miR-135b-5p and miR-499a-3p, and might provide novel insights into the role of circulating miRNAs in atherosclerosis pathogenesis.

Sma- and Mad-related protein 4 (SMAD4) is the central mediator of the transforming growth factor beta signaling pathway and is closely related to mammalian reproductive ability and the development of ovarian follicles. However, little is currently known about the role of SMAD4 in mammalian follicular granulosa cell (GC) apoptosis or its regulation by miRNAs. Here, we found that the porcine SMAD4 protein was expressed at high levels in GCs and oocytes from primary, preantral, and antral follicles, and only slightly expressed in theca cells; its expression level was down-regulated in apoptotic ovarian GCs, suggesting that SMAD4 may be involved in ovary development and selection. Overexpression and knockdown of SMAD4 increased the proliferation and apoptosis of cultured porcine GCs, respectively. In addition, the use of miRNA mimics and luciferase reporter assays revealed that miRNA-26b (miR-26b) functions as a proapoptotic factor in porcine follicular GCs by targeting the 3'-untranslated region of the SMAD4 gene. Overexpression of miR-26b in follicular GCs suppressed SMAD4 mRNA and protein levels, resulting in down-regulation of the antiapoptotic BCL-2 gene and the promotion of GC apoptosis. Furthermore, transforming growth factor beta 1 (TGF-beta1) down-regulates miR-26b expression in porcine GCs. Taken together, these data suggest that SMAD4 plays a critical role in porcine follicular GC apoptosis and follicular atresia and that miR-26b may have a proapoptotic role in GCs by regulating the expression of SMAD4 in the transforming growth factor beta signaling pathway.