Biomedical subjects

Keith J Gooch

Publications and source records attributed to Keith J Gooch.

11 recordsLinked to original sources

PubMed2006

OxLDL increases endothelial stiffness, force generation, and network formation.

This study investigates the effect of oxidatively modified low density lipoprotein (OxLDL) on the biomechanical properties of human aortic endothelial cells (HAECs). We show that treatment with OxLDL results in a 90% decrease in the membrane deformability of HAECs, as determined by micropipette aspiration. Furthermore, aortic endothelial cells freshly isolated from hypercholesterolemic pigs were significantly stiffer than cells isolated from healthy animals. Interestingly, OxLDL had no effect on membrane cholesterol of HAECs but caused the disappearance of a lipid raft marker, GM1, from the plasma membrane. Both an increase in membrane stiffness and a disappearance of GM1 were also observed in cells that were cholesterol-depleted by methyl-beta-cyclodextrin. Additionally, OxLDL treatment of HAECs embedded within collagen gels resulted in increased gel contraction, indicating an increase in force generation by the cells. This increase in force generation correlated with an increased ability of HAECs to elongate and form networks in a three-dimensional environment. Increased force generation, elongation, and network formation were also observed in cholesterol-depleted cells. We suggest, therefore, that exposure to OxLDL results in the disruption or redistribution of lipid rafts, which in turn induces stiffening of the endothelium, an increase in endothelial force generation, and the potential for network formation.

Animals↗

PubMed2006

Induced cell clustering enhances islet beta cell formation from human cultures enriched for pancreatic ductal epithelial cells.

A better understanding of the culture conditions that stimulate in vitro beta-cell differentiation from islet precursors would be useful for optimizing the production of tissue-engineered islets. In this study, high- and low-adherent substrates and high- and low-serum media were used to control the clustering of human pancreatic ductal epithelial cells and to determine its effect on their transdifferentiation to beta cells. While the initial epithelial cell cultures were devoid of any beta cells as assessed by dithizone staining, dithizone+ cells were generated during the next 3 weeks under all culture conditions. Although the rate of transdifferentiation was low, a approximately 4-fold greater number and percentage of dithizone+ cells were generated following 23-24 days of culture in the least adherent conditions (low-serum medium, low-adherent substrate), which stimulated cell clustering to the highest degree. Insulin immunohistochemistry data correlated well with the dithizone data (r(2) = 0.99), evidence that dithizone is a reliable measure of insulin+ cells. The preferential distribution of the dithizone+ cells to regions of cell aggregation and the increased efficiency of transdifferentiation in conditions that promote cell clustering suggest that cell-cell interactions and/or cell shape changes are important to the transdifferentiation of adult pancreatic ductal epithelial cells to beta cells in vitro.

Cell Aggregation↗

PubMed2006

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress.

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).

Biocompatible Materials↗

PubMed2005

Cholesterol-derivatized polyurethane: characterization and endothelial cell adhesion.

Endothelialization of synthetic surfaces has been challenging with limited success thus far. We investigated the hypothesis that covalent attachment of cholesterol to polyurethane via the urethane nitrogen groups would create a high-affinity surface for attachment and adhesion of endothelial cells. Cholesterol was covalently bound to the polyether polyurethane, Tecothane, by first derivatizing the polyurethane nitrogen groups with bromoalkyl side chains, followed by reacting mercapto-cholesterol to the bromoalkyl sites. Cholesterol-modified polyurethane demonstrated a qualitatively smoother surface per atomic force microscopy than nonmodified and increased surface energy (contact angle measurements) compared with unmodified polyurethane. Cell attachment assays showed a significantly greater number of attached bovine arterial endothelial cells (p = 0.0003) after 45 min of seeding on cholesterol-modified polyurethane versus unmodified polyurethane. Bovine arterial endothelial cells cultivated on cholesterol-modified Tecothane showed significantly greater levels of cell retention compared with unmodified Tecothane when exposed to arterial level shear stress for 2 h (25 dynes/cm2) with 90.0 +/- 6.23% cells remaining adherent compared with unmodified polyurethane, 41.4 +/- 11.7%, p = 0.0070. Furthermore, ovine endothelial precursors, obtained as blood outgrowth endothelial cells, were seeded on cholesterol-modified polyurethane and exposed to 25 dynes/cm2 shear conditions for 2 h, with the retention of 90.30 +/- 3.25% of seeded cells versus unmodified polyurethane, which retained only 4.56 +/- 0.85% (p < 0.001). It is concluded that covalently linking cholesterol to polyurethane results in improved material properties that permit increased endothelial cell retention compared with unmodified polyurethane.

Animals↗

PubMed2005

Shear stress and pressure modulate saphenous vein remodeling ex vivo.

Vein graft failure remains an important clinical challenge, but factors contributing to vein graft failure have not clearly been defined. We investigated the role of the mechanical environment in vein remodeling in an ex vivo perfusion system. Porcine saphenous veins were subjected to five different ex vivo hemodynamic environments, including one mimicking an arterial bypass graft, for one week in order to independently assess the effects of shear stress and pressure on vein remodeling. The extent of intimal hyperplasia decreased with culture under increasing shear stress, with veins cultured under the lowest levels of shear stress exhibiting the greatest ratio of intimal/medial area, 0.15+/-0.03, which was greater than that of fresh veins (0.06+/-0.01, p<0.05). All perfused veins displayed characteristics of both medial hypertrophy and eutrophic remodeling, with those veins cultured under elevated pressures showing greater increases in mass and area than those cultured under venous pressures. Medial area correlated with the average pressure under which veins were cultured (R2=0.95, p<0.001), with veins cultured under bypass graft conditions, which were exposed to the greatest pressure during the one week culture, exhibiting the largest medial area (1.69+/-0.15 mm2), which was significantly greater than that of fresh veins (1.08+/-0.05 mm2, p<0.05). However, pulsatility was not a necessary stimulus for medial growth, as increases in medial area were observed in culture conditions in which steady flow and pressure were present. Our results suggest that pressure and shear stress act independently to regulate vein remodeling, influencing changes in vessel size as well as the nature of the remodeling.

Animals↗

PubMed2005

Hemodynamic conditions alter axial and circumferential remodeling of arteries engineered ex vivo.

We previously demonstrated that growth and remodeling was stimulated in arteries elongated ex vivo using step increases in axial strain. Viability and vasoactivity were similar to fresh arteries, however there was a substantial decrease in the ultimate circumferential stress. To test the hypothesis that the subphysiological perfusion conditions (i.e., low pressure and flow) previously used caused the reduction, arteries were subjected to the identical elongation protocol (50% increase over 9 days) while being perfused with physiological levels of flow, viscosity and pulsatile pressure. A significant increase in unloaded length was achieved by elongation under both perfusion conditions, although the increase was less under physiological (7 +/- 1%) than under subphysiological conditions (19 +/- 2%, p < 0.005). When length at physiological stress was estimated using mechanical testing data the values were similar. The ultimate circumferential stress of arteries elongated under physiological conditions was increased (33%), whereas the ultimate axial stress was decreased (50%) as compared with arteries elongated under subphysiological conditions. Elongated arteries under both perfusion conditions showed significant increases in proliferation and collagen mass, and similar viability and appearance to fresh arteries. These data suggest that there is substantial cross-talk between perfusion conditions and axial strain that modulates arterial remodeling and length.

Animals↗

PubMed2005

Mechanical properties of native and ex vivo remodeled porcine saphenous veins.

When grafted into an arterial environment in vivo, veins remodel in response to the new mechanical environment, thereby changing their mechanical properties and potentially impacting their patency as bypass grafts. Porcine saphenous veins were subjected for one week to four different ex vivo hemodynamic environments in which pressure and shear stress were varied independently, as well as an environment that mimicked that of an arterial bypass graft. After one week of ex vivo culture, the mechanical properties of intact saphenous veins were evaluated to relate specific aspects of the mechanical environment to vein remodeling and corresponding changes in mechanics. The compliance of all cultured veins tended to be less than that of fresh veins; however, this trend was more due to changes in medial and luminal areas than changes in the intrinsic properties of the vein wall. A combination of medial hypertrophy and eutrophic remodeling leads to significantly smaller (p<0.05) wall stresses measured in all cultured veins except those subjected to bypass graft conditions relative to stresses measured in fresh veins at corresponding pressures. Our results suggest that the mechanical environment effects changes in vessel size, as well as the nature of the remodeling, which contribute to altering vein mechanical properties.

Animals↗

PubMed2004

Successful growth and characterization of mouse pancreatic ductal cells: functional properties of the Ki-RAS(G12V) oncogene.

BACKGROUND & AIMS: The Ki-RAS oncogene is altered in pancreatic ductal neoplasms. Pancreatic ductal cells (PDCs) were purified from cytokeratin 19 (K19)-Ki-RAS(G12V) transgenic mice and control littermates to identify properties of Ki-Ras activation in a cell-type-specific context. Because Ki-RAS mutation has prognostic significance in patients treated with radiation, we studied the influence of Ki-RAS status on radiation survival. METHODS: Pancreatic ductal fragments from mice with Ki-RAS(G12V) mutation or wild-type (WT)-Ki-RAS were cultured. Growth curves, electron microscopy, flow cytometry, and analysis of signaling and cell-cycle proteins were established. Farnesyltransferase inhibitor (FTI) treatment with R115777 before and after irradiation was used to determine the effect of Ki-Ras farnesylation on cell survival. RESULTS: PDCs from WT and K19-Ki-RAS(G12V) mice had features of ductal cells with formation of 3-dimensional structures on collagen without differences in morphology, growth, and cell-cycle distribution. This may result from up-regulation of p16INK4 and p27(Kip1) and lack of hyperstimulation of the mitogen-activated protein kinase pathway in Ki-RAS(G12V) PDCs. No differences in radiation survival between Ki-RAS(G12V) PDCs and WT PDCs were observed. However, Ki-RAS(G12V) PDCs expressing mutant p53(V143A) had enhanced survival compared with WT PDCs transduced with p53(V143A). R115777 treatment sensitized Ki-RAS(G12V) PDCs and Ki-RAS(G12V)/p53(V143A) PDCs, but not WT PDCs. CONCLUSIONS: Novel characterization of murine WT PDCs and Ki-RAS(G12V) PDCs is described. Induction of cell-cycle regulators and lack of mitogen-activated protein kinase hyperstimulation likely are responsible for constraining activated Ki-RAS(G12V)-mediated proliferation. Because its activation was required for sensitization by an FTI, R115777 may be useful against pancreatic tumors expressing oncogenic Ki-Ras.

Animals↗

PubMed2004

Salmon fibrin supports an increased number of sprouts and decreased degradation while maintaining sprout length relative to human fibrin in an in vitro angiogenesis model.

Salmon-derived fibrin has been proposed as a preferred alternative to human or bovine fibrin because of its reduced potential for disease transmission. Here we evaluate salmon fibrin as an alternative ECM support for therapeutic angiogenesis applications, such as vascularizing engineered tissues. Human umbilical vein endothelial cells (HUVEC) seeded on gelatin beads and suspended in either salmon or human fibrin sprouted and formed capillary-like structures. Sprout length was generally increased with the addition of bFGF and VEGF and further increased with the addition of phorbol myristate acetate (PMA). The number of sprouts per bead was increased 61-188% in salmon fibrin relative to human fibrin (alpha < 0.0005) in cultures receiving growth factors and PMA, while average sprout lengths were similar for HUVEC within human or salmon fibrin. Additionally, under these conditions in the absence of a protease inhibitor, HUVEC appeared to degrade human, but not salmon, fibrin. These results support the idea that salmon fibrin may be an attractive alternative ECM able to support microvascular network formation.

Animals↗

PubMed2003

Tissue engineering of arteries by directed remodeling of intact arterial segments.

Traditional approaches to generating tissue-engineered arteries in vitro rely on expansion of cells in culture to seed appropriate scaffolds. In most envisioned applications, small autologous blood vessels would be harvested and used as a source for these cells. We propose that small autologous arteries, not the cells derived from them, may be an attractive starting point for engineered arteries. This approach capitalizes on the ability of intact arteries to grow and remodel in response to chronic changes in their mechanical environment. Carotid arteries from juvenile (approximately 30-kg) pigs were stretched longitudinally in an ex vivo perfusion system over 9 days. This resulted in a 40% increase in artery length at physiological longitudinal stress and a 20 +/- 3% increase when unstressed. Control arteries were perfused for 9 days ex vivo at their physiological loaded length. Control and elongated arteries displayed native appearance (macroscopic and histological), excellent viability (cellularity and mitochondrial activity), normal vasoactivity, and similar mechanical properties (ultimate stress and ultimate strain) as compared with freshly harvested arteries. Growth, as opposed to just redistribution of existing mass, contributed to elongation as evidenced by an increase in artery weight. Results on elongation of arteries from neonatal and adolescent pigs are also presented and discussed.

Animals↗

PubMed2002

Differential effects of growth factors on tissue-engineered cartilage.

The effects of four regulatory factors on tissue-engineered cartilage were examined with specific focus on the ability to increase construct growth rate and concentrations of glycosaminoglycans (GAG) and collagen, the major extracellular matrix (ECM) components. Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured in medium with or without supplemental insulin-like growth factor (IGF-I), interleukin-4 (IL-4), transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor (PDGF). IGF-I, IL-4, and TGF-beta1 increased construct wet weights by 1.5-2.9-fold over 4 weeks of culture and increased amounts of cartilaginous ECM components. IGF-I (10-300 ng/mL) maintained wet weight fractions of GAG in constructs seeded at high cell density and increased by up to fivefold GAG fractions in constructs seeded at lower cell density. TGF-beta1 (30 ng/mL) increased wet weight fractions of total collagen by up to 1.4-fold while maintaining a high fraction of type II collagen (79 plus minus 11% of the total collagen). IL-4 (1-100 ng/mL) minimized the thickness of the GAG-depleted region at the construct surfaces. PDGF (1-100 ng/mL) decreased construct growth rate and ECM fractions. Different regulatory factors thus elicit significantly different chondrogenic responses and can be used to selectively control the growth rate and improve the composition of engineered cartilage.

Animals↗