-
[show abstract]
[hide abstract]
ABSTRACT: Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.
Tissue Engineering Part A 06/2010; 16(6):1835-44. · 4.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The association hepatocyte growth factor receptor (Met) tyrosine kinase with prognosis and survival in colon cancer is unclear, due in part to the limitation of detection methods used. In particular, conventional chromagenic immunohistochemistry (IHC) has several limitations including the inability to separate compartmental measurements. Measurement of membrane, cytoplasm, and nuclear levels of Met could offer a superior approach to traditional IHC.
Fluorescent-based IHC for Met was done in 583 colon cancer patients in a tissue microarray format. Using curvature and intensity-based image analysis, the membrane, nuclear, and cytoplasm were segmented. Probability distributions of Met within each compartment were determined, and an automated scoring algorithm was generated. An optimal score cutpoint was calculated using 500-fold crossvalidation of a training and test data set. For comparison with conventional IHC, a second array from the same tissue microarray block was 3,3'-diaminobenzidine immunostained for Met.
In crossvalidated and univariate Cox analysis, the membrane relative to cytoplasm Met score was a significant predictor of survival in stage I (hazard ratio, 0.16; P = 0.006) and in stage II patients (hazard ratio, 0.34; P < or = 0.0005). Similar results were found with multivariate analysis. Met in the membrane alone was not a significant predictor of outcome in all patients or within stage. In the 3,3'-diaminobenzidine-stained array, no associations were found with Met expression and survival.
These data indicate that the relative subcellular distribution of Met, as measured by novel automated image analysis, may be a valuable biomarker for estimating colon cancer prognosis.
Clinical Cancer Research 06/2008; 14(12):3814-22. · 7.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An image based assay has been developed to quantify platelet adhesion on material surfaces. Briefly, citrated platelet rich plasma (PRP) is incubated with materials for 2 h to allow platelet adhesion on the surface, followed by fluorescence labeling of platelets with Celltracker Green. Multiple images are acquired by an automatic fluorescence microscope, IN Cell Analyzer 1000. Platelets are identified and counted by an automatic image analysis algorithm. We have observed that the variance of the counts is considerably greater than expected from simple distribution laws. Statistical analysis of that difference shows that these measurements will often follow a doubly stochastic Poisson process in which the variance is inherently very large. To overcome this, multiple images (n > or = 8 images/well, about 3% of total area) are necessary to achieve accurate counting. This method has been compared to the commonly used enzyme based platelet adhesion assay, lactate dehydrogenase (LDH) assay. It is concluded that the present method is only effective in quantifying adherent platelets when a large number of samples are used. However, this method does provide additional information on platelet morphology and spatial distribution, which is lacking in the LDH assay.
Journal of Biomedical Materials Research Part A 04/2008; 89(1):224-32. · 2.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human umbilical vein endothelial cells (HUVECs) display hydraulic conductivity (L(P)) responses to shear stress that differ markedly from the responses of bovine aortic endothelial cells (BAECs). In HUVECs, 5, 10, and 20 dyn cm(-2) steady shear stress transiently increased L(P) with a return to preshear baseline after a 2-h exposure to shear stress. Pure oscillatory shear stress of 0 +/- 20 dyn cm(-2) (mean+/-amplitude) had no effect on L(P), whereas superposition of oscillatory shear stress on steady shear stress suppressed the effect induced by steady shear stress alone. Shear reversal (amplitude greater than mean) was not necessary for the inhibitory influence of oscillatory shear stress. The transient increase of L(P) by steady shear stress was not affected by incubation with BAPTA-AM (10 microM), suggesting calcium independence of the shear response. Decreasing nitric oxide (NO) concentration with L-NMMA (100 microM), a nitric oxide synthase (NOS) inhibitor, did not inhibit the HUVEC L(P) response to shear stress. At the protein level, 10 dyn cm(-2) shear stress did not affect the total content of occludin, but it did elevate the phosphorylation level transiently. The positive correlation between occludin phosphorylation and hydraulic conductivity parallels observations in BAECs and suggests that occludin phosphorylation may be a general mediator of shear-L(P) responses in diverse endothelial cell types.
Annals of Biomedical Engineering 12/2005; 33(11):1536-45. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/cm(2) for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.
Biomaterials 09/2005; 26(22):4642-53. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The objective of this study was to test whether a glycosaminoglycan component of the surface glycocalyx layer is a fluid shear stress sensor on endothelial cells (ECs). Because enhanced nitric oxide (NO) production in response to fluid shear stress is a characteristic and physiologically important response of ECs, we evaluated NOx (NO2- and NO3-) production in response to fluid shear stress after enzymatic removal of heparan sulfate, the dominant glycosaminoglycan of the EC glycocalyx, from cultured ECs. The significant NOx production induced by steady shear stress (20 dyne/cm2) was inhibited completely by pretreatment with 15 mU/mL heparinase III (E.C.4.2.2.8) for 2 hours. Oscillatory shear stress (10+/-15 dyne/cm2) induced an even greater NOx production than steady shear stress that was completely inhibited by pretreatment with heparinase III. Addition of bradykinin (BK) induced significant NOx production that was not inhibited by heparinase pretreatment, demonstrating that the cells were still able to produce abundant NO after heparinase treatment. Fluorescent imaging with a heparan sulfate antibody revealed that heparinase III treatments removed a substantial fraction of the heparan sulfate bound to the surfaces of ECs. In summary, these experiments demonstrate that a heparan sulfate component of the EC glycocalyx participates in mechanosensing that mediates NO production in response to shear stress. The full text of this article is available online at http://www.circresaha.org.
Circulation Research 12/2003; 93(10):e136-42. · 9.49 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Starling's hypothesis that fluid movement across the microvascular wall is determined by the transmural differences in hydrostatic and osmotic pressures was tested using an in vitro model comprised of bovine aortic endothelial cells grown on a porous support. In all experiments, a 1% bovine serum albumin (BSA) solution was maintained in the abluminal reservoir and the luminal reservoir contained either a 1 or a 5.5% BSA solution. The global osmotic pressure difference across the endothelial layers was thus either 0 or 20.3 cm H2O. When the luminal concentration of BSA was changed from 1 to 5.5% at a hydrostatic pressure differential of 5, 10 or 20 cm H2O, no reverse flow (in the reabsorption direction) was observed even though the hydrostatic pressure differential was far below the global osmotic pressure differential. In another case, the hydrostatic pressure differential was dropped quickly from 20 to 5 cm H2O, while a constant osmotic pressure differential was maintained by 5.5% BSA in the luminal reservoir. A strong transient reabsorption flow was observed over a 30-second period which diminished to undetectable levels within 2.5 min; then a sustained steady-state filtration flow was observed after 20 min. These in vitro experiments support other studies in capillaries showing transient reabsorption that decays to steady-state filtration at longer times.
Journal of Vascular Research 40(4):351-8. · 2.65 Impact Factor
