The involvement of vascular fibroblasts (FBs) and smooth muscle (SM)-like cells in physiological and pathological processes in large vessels (intimal hyperplasia) and microvessels (capillary arterialization), and the realization that these cells are exposed to interstitial flow shear stress (SS), motivate this study of SS on FB migratory activity. Rat adventitial FBs were grown to either 30-50% confluence (subconfluent FBs; SFBs) or full confluence (confluent FBs; CFBs) in culture. Immunofluorescence and Western blotting assays were conducted to evaluate the expression of two phenotype markers: SM alpha-actin and SM myosin heavy chain (MHC). Both assays indicated a significant increase in SM alpha-actin expression in CFBs compared with SFBs, suggesting a phenotype difference between the two cell populations. SFBs and CFBs both expressed minimal SM MHC. Both cell populations were seeded on Matrigel-coated cell culture inserts and exposed to 4 h of either 1 or 20 dyn/cm(2) SS via a rotating disk apparatus in the presence of the chemoattractant platelet-derived growth factor-BB to quantify the effect of SS on SFB and CFB migration. Four hours of 20 dyn/cm(2) SS significantly enhanced SFB migration while it suppressed CFB migratory activity. Four hours of 1 dyn/cm(2) SS did not significantly alter either SFB or CFB migration levels. Because of the distinct migratory responses of SFBs and CFBs in response to SS, phenotype modulation appears to be one way to regulate their involvement in both physiological and pathological remodeling processes.

Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.

To advance the development of point-of-care technology (POCT), the National Institute of Biomedical Imaging and Bioengineering established the POCT Research Network (POCTRN), comprised of Centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. This paper describes the POCTRN and the three currently funded Centers as examples of academic-based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care.

This study describes cocultures of arterial smooth muscle cells (SMC) and endothelial cells (EC) and the influences of their heterotypic interactions on hydraulic conductivity (Lp), an important transport property. A unique feature of these cocultures is that ECs were first grown to confluence and then SMCs were inoculated. Bovine aortic smooth muscle cells (BASMCs) and bovine aortic endothelial cells (BAECs) were cocultured on Transwell Permeable Supports, and then exposed to a pressure-driven transmural flow. Lp across each culture was measured using a bubble tracking apparatus that determined water flux (Jv). Our results indicate that arterial Lp is significantly modulated by EC-SMC proximity, and serum content in culture. The Lp of cocultures was also compared to the predictions of a resistances-in-series model to distinguish the contributions of heterotypic interactions between SMCs and ECs. Conditions that lead to significantly reduced coculture Lp, compared to BAEC monoculture controls, have been uncovered and the lowest Lp in the literature for an in-vitro system are reported. In addition, VE-cadherin immunostaining of intact BAEC monolayers in each culture configuration reveals that EC-SMC proximity on a porous membrane has a dramatic influence on EC morphology patterns. The cocultures with the lowest Lp have ECs with significantly elongated morphology. Confocal imaging indicates that there are no direct EC-SMC contacts in coculture.

The purpose of the study was to examine the effects of arterial coculture conditions on the transport properties of several in vitro endothelial cell (EC) – smooth muscle cell (SMC) – porous filter constructs in which SMC were grown to confluence first and then EC were inoculated. This order of culturing simulates the environment of a blood vessel wall after endothelial layer damage due to stenting, vascular grafting or other vascular wall insult. For all coculture configurations examined, we observed that hydraulic conductivity (Lp) values were significantly higher than predicted by a resistances-in-series (RIS) model accounting for the Lp of EC and SMC measured separately. The greatest increases were observed when EC were plated directly on top of a confluent SMC layer without an intervening filter, presumably mediated by direct EC – SMC contacts that were observed under confocal microscopy. The results are the opposite of a previous study that showed Lp was significantly reduced compared to an RIS model when EC were grown to confluency first. The physiological, pathophysiological and tissue engineering implications of these results are discussed.

Rat aortic fibroblasts were cultured either subconfluent (<20%) or confluent (>80%). Western blotting was conducted to evaluate the amount of two phenotype markers of smooth muscle cells (SMCs), SM α-actin (α-SMA) and SM myosin heavy chain (SM-MHC). The results showed that subconfluent fibroblasts were native fibroblasts and confluent fibroblasts were myofibroblasts. These two types of cell were then plated on slides. 8 dyn/cm 2 of shear stress was applied to fibroblasts for 6h and myofibroblasts for 24h, respectively. Using immunostaining we found that expressions of both α-SMA in fibroblasts and SM-MHC in myofibroblasts were significantly upregulated under shear by 2.3-fold and 2.2-fold, respectively, based on the increase of fluorescent intensity compared to static controls. These results indicate that shear stress can promote fibroblast differentiation into myofibroblast, and myofibroblast differentiation into SMC. In another experiment, SMCs, fibroblasts, and myofibroblasts were plated in Boyden chambers, and 13 dyn/cm 2 of shear was applied to the cells for 4h. We found the migration of fibroblasts was enhanced by shear, whereas, the migration of both SMCs and myofibroblasts were suppressed. Taken together, we speculate that the interstitial flow shear stress might play an important role in phenotype modulation and migration of fibroblast and myofibroblast during neointima formation and vascular remodeling.