In vitro evidence of cellular adaptation to ozone toxicity in the rat trachea.

Adaptation to prolonged ozone (O3) exposure occurs in the tracheal epithelium of rats and is marked by the presence of ciliated cells with uniform short cilia but is not accompanied by shifts in cell populations, altered characteristics of epithelial secretory cell products, increased cell turnover, or elevated tracheal superoxide dismutase activity. The purpose of this study was to test the hypothesis that adaptation is a result of alterations intrinsic to epithelial cells or to the cells and their matrix, and not due to systemic or neural influences. Rats were preexposed to either filtered air (FA) or 0.96 ppm O3 for 8 hr/night for 60 days, and then their tracheae were removed and exposed to 3 ppm O3 in an explant culture system where behavioral, nasal, exudative, and secretory product influences can be eliminated. After exposure to 3 ppm O3 in vitro, quantitative electron microscopic evaluation demonstrated that the epithelia from the FA preexposure group had significantly more necrotic cells and sloughed cells and fewer ciliated cells than the epithelia from the O3 preexposure group. Thus previous exposure to ozone in vivo induces a change in tracheal epithelium which confers resistance to ozone-induced injury in the explant exposure system

Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways

BACKGROUND: A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. RESULTS: Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. CONCLUSION: These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis

A Case Study in ePortfolio Implementation: A Department-Wide Perspective

This case study documents the trials and tribulations over a 3-year span of one academic department in implementing the ePortfolio as a high-impact practice to its undergraduate students. Failures and successes will be introduced with the resulting lessons learned applied to our current efforts. Pivotal instances that allowed the project partners to gain clarity about the design and implementation of an ePortfolio will be expressed to better understand our journey. The root of our collaborative efforts was based on the product versus process conversation around ePortfolios. Once our mindset shifted, we were able to embrace a more student-centered process ePortfolio that is threaded throughout our curriculum and not sporadically addressed as an add-on assignment

Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity ([Formula: see text]) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells

Quantification of Three-Dimensional Cell-Mediated Collagen Remodeling Using Graph Theory

Background: Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. Methodology/Principal Findings: We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Conclusions/Significance: Definition of early metrics that are able to predict long-term functionality by linking engineere

Perillyl Alcohol Inhibits Breast Cell Migration without Affecting Cell Adhesion

The monoterpene d-limonene exhibits chemotherapeutic and chemopreventive potential in breast cancer patients. D-limonene and its related compounds, perillyl alcohol and perillyl aldehyde, were chosen as candidate drugs for application in a screen for nontoxic inhibitors of cell migration. Using the nontumorigenic human breast cell line MCF-10A, we delineated the toxicity as greatest for the perillyl aldehyde, intermediate for perillyl alcohol, and least for limonene. A noncytotoxic concentration of 0.5 mmol/L perillyl alcohol inhibited the migration, while the same concentration of limonene failed to do so. Adhesion of the MCF-10A cell line and the human breast cancer cell line MDA-MB 435 to fibronectin was unaffected by 1.5 mmol/L perillyl alcohol. 0.4 mmol/L perillyl alcohol inhibited the growth of MDA-MB 435 cells. All migration-inhibiting concentrations of perillyl alcohol for MDA-MB 435 cells proved to be toxic. These results suggest that subtoxic doses of perillyl alcohol may have prophylactic potential in the treatment of breast cancer

Novel Image Analysis Approach Quantifies Morphological Characteristics of 3D Breast Culture Acini with Varying Metastatic Potentials

Prognosis of breast cancer is primarily predicted by the histological grading of the tumor, where pathologists manually evaluate microscopic characteristics of the tissue. This labor intensive process suffers from intra- and inter-observer variations; thus, computer-aided systems that accomplish this assessment automatically are in high demand. We address this by developing an image analysis framework for the automated grading of breast cancer in in vitro three-dimensional breast epithelial acini through the characterization of acinar structure morphology. A set of statistically significant features for the characterization of acini morphology are exploited for the automated grading of six (MCF10 series) cell line cultures mimicking three grades of breast cancer along the metastatic cascade. In addition to capturing both expected and visually differentiable changes, we quantify subtle differences that pose a challenge to assess through microscopic inspection. Our method achieves 89.0% accuracy in grading the acinar structures as nonmalignant, noninvasive carcinoma, and invasive carcinoma grades. We further demonstrate that the proposed methodology can be successfully applied for the grading of in vivo tissue samples albeit with additional constraints. These results indicate that the proposed features can be used to describe the relationship between the acini morphology and cellular function along the metastatic cascade