Kem A. Rogers

α5β1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding α5β1 integrin. Whereas α5β1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the α5β1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the α5β1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-β1 integrin antibody, and an anti-α5β1 integrin antibody, but not by an anti-β3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the α5β1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the α5β1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-β1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4.

The adhesion molecules intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function‐blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP‐1 cells interacting with human coronary artery endothelial cells. The number of motile THP‐1 cells was reduced only when VCAM‐1 or both ICAM‐1 and VCAM‐1 were blocked. Blockade of ICAM‐1 and VCAM‐1, either separately or together, reduced to the same degree the distance that THP‐1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM‐1 or VCAM‐1 inhibited pseudopodia formation, but ICAM‐1 blockade induced the formation of filopodia. We suggest that the interactions of endothelial ICAM‐1 and VCAM‐1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.

The p110δ Isoform of PI3K Differentially Regulates β1 and β2 Integrin-Mediated Monocyte Adhesion and Spreading and Modulates Diapedesis

Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA‐1 and VLA‐4 to endothelial ICAM‐1 and VCAM‐1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis.

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

Angiotensin II type 1 receptor blocker inhibits arterial calcification in a pre-clinical model.

AIMS Arterial calcification is a common complication of several disorders and is a strong predictor of mortality. The mechanism underlying arterial calcification is not fully understood and as such, no pharmaceutical therapies are currently available which impede its progression. The aim of this study was to investigate the effects of an angiotensin II (AngII) type 1 receptor blocker (ARB) on arterial calcification. METHODS AND RESULTS Male New Zealand White rabbits were fed an atherogenic diet to induce atherosclerosis and arterial calcification over a period of 12 weeks, with an ARB administered in the final 4 weeks. Using clinically relevant micro-computed tomography, we found that animals fed the atherogenic diet displayed extensive arterial calcification when compared with control. In contrast, administration of the ARB completely inhibited calcification (2.80 ± 1.17 vs. 0.01 ± 0.01% calcified tissue in cholesterol and ARB-treated, respectively; n = 6 and 5; P < 0.05). Calcified regions were characterized by up-regulation of bone morphogenetic protein 2, osteocalcin, and the AngII type 1 receptor and concomitant down-regulation of α-smooth muscle actin, consistent with a phenotypic switch from vascular to osteoblast-like cells. CONCLUSION These data provide the first evidence that angiotensin receptor blockade can inhibit arterial calcification by disrupting vascular osteogenesis and suggest that ARBs may be a novel treatment option for patients suffering from vascular calcification.

Design and implementation of an online systemic human anatomy course with laboratory

Systemic Human Anatomy is a full credit, upper year undergraduate course with a (prosection) laboratory component at Western University Canada. To meet enrollment demands beyond the physical space of the laboratory facility, a fully online section was developed to run concurrently with the traditional face to face (F2F) course. Lectures given to F2F students are simultaneously broadcasted to online students using collaborative software (Blackboard Collaborate). The same collaborative software is used by a teaching assistant to deliver laboratory demonstrations in which three‐dimensional (3D) virtual anatomical models are manipulated. Ten commercial software programs were reviewed to determine their suitability for demonstrating the virtual models, resulting in the selection of Netters 3D Interactive Anatomy. Supplementary online materials for the central nervous system were developed by creating 360° images of plastinated prosected brain specimens and a website through which they could be accessed. This is the first description of a fully online undergraduate anatomy course with a live, interactive laboratory component. Preliminary data comparing the online and F2F student grades suggest that previous student academic performance, and not course delivery format, predicts performance in anatomy. Future qualitative studies will reveal student perceptions about their learning experiences in both of the course delivery formats. Anat Sci Educ 8: 53–62.

The distribution of fibro-fatty atherosclerotic lesions in the aortae of casein- and cholesterol-fed rabbits

Atherosclerotic lesions were induced in the aortas of 50 rabbits by feeding a semi-purified cholesterol-free casein diet or normal rabbit chow with a low level of added cholesterol for 6 or 10 months. Following perfusion fixation, the aortae from these animals were opened along their length, stained with oil red O and photographed en face. Orifice associated lesions were mapped by measuring radial lesion length at 10 degrees intervals circumferentially around ostia. Histology of these lesions revealed abundant smooth muscle cells surrounded by collagen and elastin in the extracellular matrix, typical of fibrous plaques, as well as oil red O staining lipid and some macrophage derived foam cells. These fibro-fatty lesions were found distal and lateral to ostia, at the same locations as fatty streaks seen in rabbits fed a 2% cholesterol diet for 1 week to 2 months in previous studies. The results of this study show that in moderately hypercholesterolemic rabbits fed an atherogenic diet for 6 to 10 months, advanced atherosclerotic plaques develop in the same location as the fatty streaks seen in short term experiments.

Interleukin-1β Reduces Transcellular Monocyte Diapedesis and Compromises Endothelial Adherens Junction Integrity

Objective: Diapedesis occurs through endothelial cell–cell junctions (paracellular) or through individual endothelial cells without disrupting junctions (transcellular). While in vitro studies have provided considerable insight into mechanisms controlling paracellular diapedesis, little is known about what regulates transcellular diapedesis. The authors investigated whether transcellular diapedesis is susceptible to IL‐1β exposure of the endothelium.

The development and assessment of an online microscopic anatomy laboratory course.

Increasing enrollment in post‐secondary institutions across North America, along with an increase in popularity of and demand for distance education is pressuring institutions to offer a greater number and variety of courses online. A fully online laboratory course in microscopic anatomy (histology) which can be taught simultaneously with a face‐to‐face (F2F) version of the same course has been developed. This full year course was offered in the Fall/Winter (FW) terms in both F2F and online formats. To ensure that the online course was of the same quality as the F2F format, a number of performance indicators were evaluated. The same course, offered exclusively online during the summer with a compressed time frame, was also evaluated. Senior undergraduate students self‐selected which version of the course they would enroll in. Course assessment outcomes were compared while incoming grades were used as a predictor for course performance. There were no significant differences between the incoming grades for the F2F FW and Online FW courses; similarly, there were no significant differences between outcomes for these formats. There were significant differences between the incoming grades of the F2F FW and Summer Online students. However, there were no significant differences among any of the outcomes for any of the formats offered. Incoming grades were strong, significant predictors of course performance for both formats. These results indicate that an online laboratory course in microscopic anatomy is an effective format for delivering histology course content, therefore giving students greater options for course selections. Anat Sci Educ 6: 246–256.

Transendothelial Migration of Monocytes in Rat Aorta: Distribution of F-actin, α-Catenin, LFA-1, and PECAM-1

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or α-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent en-dothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by α-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis ...