Enhancing Endothelial Cell Retention on ePTFE Constructs by siRNA-Mediated SHP-1 Gene Silencing

2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Brandon J. Tefft ◽  
Adrian M. Kopacz ◽  
Wing Kam Liu ◽  
Shu Q. Liu
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Gene Silencing ◽  
Vascular Grafts ◽  
Sh2 Domain ◽  
Great Promise ◽  
Shear Stresses ◽  
Cell Retention ◽  
Stress Dependent

Polymeric vascular grafts hold great promise for vascular reconstruction, but the lack of endothelial cells renders these grafts susceptible to intimal hyperplasia and restenosis, precluding widespread clinical applications. The purpose of this study is to establish a stable endothelium on expanded polytetrafluoroethylene (ePTFE) membrane by small interfering RNA (siRNA)-induced suppression of the cell adhesion inhibitor SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were treated with scrambled siRNA as a control or SHP-1 specific siRNA. Treated cells were seeded onto fibronectin-coated ePTFE scaffolds and exposed to a physiological range of pulsatile fluid shear stresses for 1 h in a variable-width parallel plate flow chamber. Retention of cells was measured and compared between various shear stress levels and between groups treated with scrambled siRNA and SHP-1 specific siRNA. HUVECs seeded on ePTFE membrane exhibited shear stress-dependent retention. Exposure to physiological shear stress (10 dyn/cm2) induced a reduction in the retention of scrambled siRNA treated cells from 100% to 85% at 1 h. Increased shear stress (20 dyn/cm2) further reduced retention of scrambled siRNA treated cells to 55% at 1 h. SHP-1 knockdown mediated by siRNA enhanced endothelial cell retention from approximately 60% to 85% after 1 h of exposure to average shear stresses in the range of 15–30 dyn/cm2. This study demonstrates that siRNA-mediated gene silencing may be an effective strategy for improving the retention of endothelial cells within vascular grafts.

Effects of Fluid Shear Stress on Endothelial Cell Invasion Into Three-Dimensional Matrices

2007 ◽  
Author(s):  
Hojin Kang ◽  
Kayla J. Bayless ◽  
Roland Kaunas
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Cell Invasion ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Shear Stresses ◽  
Collagen Gels ◽  
Fluid Shear

We have previously developed a cell culture model to study the effects of angiogenic factors, such as sphingosine-1-phosphate (S1P), on the invasion of endothelial cells into the underlying extracellular matrix. In addition to biochemical stimuli, vascular endothelial cells are subjected to fluid shear stress due to blood flow. The present study is aimed at determining the effects of fluid shear stress on endothelial cell invasion into collagen gels. A device was constructed to apply well-defined fluid shear stresses to confluent human umbilical vein endothelial cells (HUVECs) seeded on collagen gels. Fluid shear stress induced significant increases in cell invasion with a maximal induction at ∼5 dyn/cm2. These results provide evidence that fluid shear stress is a significant stimulus for endothelial cell invasion and may play a role in regulating angiogenesis.

The Effects of Shear Stress on Endothelial Cells at Hypothermic Temperatures

2002 ◽  
Author(s):  
John H. Slater ◽  
Shailendra Jain ◽  
Robin N. Coger ◽  
Charles Y. Lee
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Low Flow ◽  
Shear Stresses ◽  
Liver Sinusoids ◽  
Endothelial Cell Cultures ◽  
Shear Stress Response

Hypothermic machine perfusion preservation (MPP) has proven to be a successful technique for hypothermic kidney storage, however this technology has not successfully been applied to the liver. Recent research has indicated that the endothelial cells lining the liver sinusoids display rounding phenomena during MPP that is not fully understood. In order to gain a better understanding of endothelial cell shear stress response and the factors that induce rounding, a temperature-controlled micro-shear chamber has been designed and fabricated. The micro-shear chamber has been used to apply shear stresses, corresponding to those imposed during MPP, to rat liver primary endothelial cell cultures in order to form an understanding of how these stresses affect endothelial cell morphology. The chamber allows for the application of shear stresses ranging from 0.2 ± .01 dynes/cm2 to 2.3 ± 0.3 dynes/cm2, corresponding to what occurs during MPP.] Twenty-four hour in vitro experiments with shear stresses ranging from 0 to 1.49 dynes/cm2 at 4 °C were conducted in order to replicate in vivo conditions of the liver during hypothermic MPP. It has been demonstrated that endothelial cell rounding increases with increasing shear and can be prevented by utilizing low flow rates.

The Dynamic Response of Vascular Endothelial Cells to Fluid Shear Stress

1981 ◽  
Vol 103 (3) ◽  
pp. 177-185 ◽  
Author(s):  
C. F. Dewey ◽  
S. R. Bussolari ◽  
M. A. Gimbrone ◽  
P. F. Davies
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Dynamic Response ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Shear Stresses ◽  
Vascular Endothelial ◽  
Fluid Shear ◽  
Cell Functions

We have developed an in-vitro system for studying the dynamic response of vascular endothelial cells to controlled levels of fluid shear stress. Cultured monolayers of bovine aortic endothelial cells are placed in a cone-plate apparatus that produces a uniform fluid shear stress on replicate samples. Subconfluent endothelial cultures continuously exposed to 1–5 dynes/cm2 shear proliferate at a rate comparable to that of static cultures and reach the same saturation density (≃ 1.0–1.5 × 105 cells/cm2). When exposed to a laminar shear stress of 5–10 dynes/cm2, confluent monolayers undergo a time-dependent change in cell shape from polygonal to ellipsoidal and become uniformly oriented with flow. Regeneration of linear “wounds” in confluent monolayer appears to be influenced by the direction of the applied force. Preliminary studies indicate that certain endothelial cell functions, including fluid endocytosis, cytoskeletal assembly and nonthrombogenic surface properties, also are sensitive to shear stress. These observations suggest that fluid mechanical forces can directly influence endothelial cell structure and function. Modulation of endothelial behavior by fluid shear stresses may be relevant to normal vessel wall physiology, as well as the pathogenesis of vascular diseases, such as atherosclerosis.

scholarly journals Enhancement of endothelial cell adhesion through treatment with CEACAM6 or TNF-? [Medical College of Wisconsin]

2019 ◽  
Author(s):  
Harsimran Kalsi ◽  
Brandon Tefft
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Statistical Significance ◽  
Lower Percentage ◽  
Cell Retention ◽  
Average Cell ◽  
Coronary Interventions ◽  
Endothelial Cell Adhesion

610,000 Americans die of heart disease every year and over 1 million Americans undergo coronary interventions each year. Risks of these coronary interventions include thrombosis, embolization, infection, and re-narrowing of the artery. However, it is currently believed that improving the adhesion capabilities of endothelial cells may significantly reduce the associated risks of coronary interventions, especially with regards to cardiovascular stent and graft placement procedures. In the present study, we investigated a method to biochemically facilitate endothelial cell adhesion. Using the Reactome database, we identified biochemical methods/targets which could be modified to promote endothelial cell adhesion. Here we describe a potential method of enhancing endothelial cell adhesion through controlled application of two treatment conditions: Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) with Fibronectin-1 (CEACAM6+, FN1+) and Tumor Necrosis Factor-? (TNF-?) with Fibronectin-1 (TNF-?+, FN1+). DAPI stained porcine blood outgrowth endothelial cells (BOECs) were exposed to these treatments or a control treatment (FN1+) for one hour. Each treatment/control condition was then placed into a validated parallel plate flow chamber (PPFC) apparatus (flow rate accuracy and laminar flow were both validated) and exposed to a shear stress of 15 dyn/cm2 (to replicate in vivo shear stress conditions) for 30 minutes using saline solution. Fluorescent images were taken of all replicates before and after shear stress testing in order to determine the effects of treatments on cell adhesion. Statistical analysis of preliminary trials indicated that the treatment groups demonstrated a trend towards higher percentage cell retention (or lower percentage cell loss) with 82.3%, 70.2%, and 50.2% average cell retention for TNF-?, CEACAM6, and the control, respectively; however, statistical significance could not be reached using n=2 and p<0.05. Preliminary results of the TNF-? treatment are promising. In the future, an experiment with a larger sample size and a longer shear stress exposure time (e.g. 1 hour) is needed.

Shear stress with appropriate time-step and amplification enhances endothelial cell retention on vascular grafts

2016 ◽  
Vol 11 (11) ◽  
pp. 2965-2978 ◽  
Author(s):  
Haifeng Liu ◽  
Xianghui Gong ◽  
Xiaohui Jing ◽  
Xili Ding ◽  
Yuan Yao ◽  
...  
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Vascular Grafts ◽  
Time Step ◽  
Cell Retention

Micro-PIV and CFD Studies Show Non-Uniform Wall Shear Stress Distributions Over Endothelial Cells

2010 ◽  
Author(s):  
Sharul S. Dol ◽  
M. Mehdi Salek ◽  
Kayla D. Viegas ◽  
Kristina D. Rinker ◽  
Robert J. Martinuzzi
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Wall Shear Stress ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Shear Stresses ◽  
Stress Distributions ◽  
Micro Piv ◽  
Wall Shear

Wall shear stress acting on arterial walls is an important hemodynamic force determining vessel health. A parallel-plate flow chamber with a 127 μm-thick flow channel is used as an in vitro system to study the fluid mechanics environment. It is essential to know how well this flow chamber performs in emulating physiologic flow regimes especially when cultured cells are present. Hence, the objectives of this work are to computationally and experimentally study the characteristic of the flow chamber in providing a defined flow regime and shear stress to cultured cells and to map wall shear stress distributions in the presence of an endothelial cell layer. Experiments and modeling were performed for the nominal wall shear stresses of 2 and 10 dyn/cm2. Without endothelial cells, the flow field is uniform over 95% of the chamber cross-section and the surfaces are exposed to the target stress level. Using PIV velocity data, the endothelial cell surfaces were re-constructed and flow over these surfaces was then simulated via FLUENT. Once endothelial cells are introduced, local shear variations are large and the velocity profiles are no longer uniform. Due to the velocity distribution between peaks and valleys, the local wall shear stresses range between 47–164% of the nominal values. This study demonstrates the non-uniform shear stress distribution over the cells is non-negligible especially in small vessels or where blockage is important.

Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress

1995 ◽  
Vol 269 (6) ◽  
pp. C1371-C1378 ◽  
Author(s):  
M. Uematsu ◽  
Y. Ohara ◽  
J. P. Navas ◽  
K. Nishida ◽  
T. J. Murphy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Nitric Oxide Synthase ◽  
Mrna Expression ◽  
K Channel ◽  
Shear Stresses ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase

Shear stress enhances expression of Ca(2+)-calmodulin-sensitive endothelial cell nitric oxide synthase (ecNOS) mRNA and protein in bovine aortic endothelial cells (BAEC). The present studies were performed to investigate mechanisms responsible for regulation of ecNOS mRNA expression by shear stress and to determine if this induction of ecNOS mRNA is accompanied by an enhanced nitric oxide (NO) production. Shear stresses of 15 dyn/cm2 for 3-24 h resulted in a two- to threefold increase of ecNOS mRNA content quantified by Northern analysis in BAEC. Shear stresses (1.2-15 dyn/cm2) for 3 h resulted in an induction of ecNOS mRNA in a dose-dependent manner. In human aortic endothelial cells, shear stresses of 15 dyn/cm2 for 3 h also resulted in ecNOS mRNA induction. In BAEC, this induction in ecNOS mRNA was prevented by coincubation with actinomycin D (10 micrograms/ml). The K+ channel antagonist tetraethylammonium chloride (3 mM) prevented increase in ecNOS mRNA in response to shear stress. The ecNOS promotor contains putative binding domains for AP-1 complexes, potentially responsive to activation of protein kinase C (PKC). However, selective PKC inhibitor calphostin C (100 nM) did not inhibit ecNOS induction by shear stress. Finally, production of nitrogen oxides under both basal conditions and in response to the calcium ionophore A-23187 (1 microM) by BAEC exposed to shear stress was increased approximately twofold compared with cells not exposed to shear stress. These data suggest that ecNOS mRNA expression is regulated by K+ channel opening, but not by activation of PKC, and that shear not only enhances ecNOS mRNA expression but increases capacity of endothelial cells to release NO.

Naturally Produced Extracellular Matrix Is an Excellent Substrate for Canine Endothelial Cell Proliferation and Resistance to Shear Stress on PTFE Vascular Grafts

1997 ◽  
Vol 78 (05) ◽  
pp. 1392-1398 ◽  
Author(s):  
A Schneider ◽  
M Chandra ◽  
G Lazarovici ◽  
I Vlodavsky ◽  
G Merin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Cell Attachment ◽  
Thrombus Formation ◽  
Endothelial Cell Proliferation ◽  
Ptfe Graft ◽  
Vascular Prostheses ◽  
Endothelial Cell Attachment

SummaryPurpose: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. Methods: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 X 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (106 cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. Results: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significanly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. Conclusion: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.

scholarly journals Studying dynamic stress effects on the behaviour of THP-1 cells by microfluidic channels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Semra Zuhal Birol ◽  
Rana Fucucuoglu ◽  
Sertac Cadirci ◽  
Ayca Sayi-Yazgan ◽  
Levent Trabzon
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular System ◽  
Circulatory System ◽  
Disease Process ◽  
Microfluidic Channels ◽  
Shear Stresses ◽  
Stress Effects ◽  
Adhesion Behavior ◽  
Cycling System

AbstractAtherosclerosis is a long-term disease process of the vascular system that is characterized by the formation of atherosclerotic plaques, which are inflammatory regions on medium and large-sized arteries. There are many factors contributing to plaque formation, such as changes in shear stress levels, rupture of endothelial cells, accumulation of lipids, and recruitment of leukocytes. Shear stress is one of the main factors that regulates the homeostasis of the circulatory system; therefore, sudden and chronic changes in shear stress may cause severe pathological conditions. In this study, microfluidic channels with cavitations were designed to mimic the shape of the atherosclerotic blood vessel, where the shear stress and pressure difference depend on design of the microchannels. Changes in the inflammatory-related molecules ICAM-1 and IL-8 were investigated in THP-1 cells in response to applied shear stresses in an continuous cycling system through microfluidic channels with periodic cavitations. ICAM-1 mRNA expression and IL-8 release were analyzed by qRT-PCR and ELISA, respectively. Additionally, the adhesion behavior of sheared THP-1 cells to endothelial cells was examined by fluorescence microscopy. The results showed that 15 Pa shear stress significantly increases expression of ICAM-1 gene and IL-8 release in THP-1 cells, whereas it decreases the adhesion between THP-1 cells and endothelial cells.

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