Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local ...microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
Problem-based learning (PBL) is a pedagogy that has attracted attention for many biomedical engineering curricula. The aim of the current study was to address the research question, 'Does PBL enable ...students to develop desirable professional engineering skills?' The desirable skills identified were communication, teamwork, problem solving and self-directed learning. Forty-seven students enrolled in a biomedical materials course participated in the case study. Students worked in teams to complete a series of problems throughout the semester. The results showed that students made significant improvements in their problem-solving skills, written communication and self-directed learning. Students also demonstrated an ability to work in teams and communicate orally. In conclusion, this case study provides empirical evidence of the efficacy of PBL on student learning. We discuss findings from our study and provide observations of student performance and perceptions that could be useful for faculty and researchers interested in PBL for biomedical engineering education.
Hypertension is a known risk factor for aortic stenosis. The elevated blood pressure increases the transvalvular load and can elicit inflammation and extracellular matrix (ECM) remodeling. Elevated ...cyclic pressure and the vasoactive agent angiotensin II (Ang II) both promote collagen synthesis, an early hallmark of aortic sclerosis. In the current study, it was hypothesized that elevated cyclic pressure and/or angiotensin II decreases extensibility of aortic valve leaflets due to an increase in collagen content and/or interstitial cell stiffness. Porcine aortic valve leaflets were exposed to pressure conditions of increasing magnitude (static atmospheric pressure, 80, and 120 mmHg) with and without 10−6 M Ang II. Biaxial mechanical testing was performed to determine extensibility in the circumferential and radial directions and collagen content was determined using a quantitative dye-binding method at 24 and 48 h. Isolated aortic valve interstitial cells exposed to the same experimental conditions were subjected to atomic force microscopy to assess cellular stiffness at 24 h. Leaflet tissue incubated with Ang II decreased tissue extensibility in the radial direction, but not in the circumferential direction. Elevated cyclic pressure decreased extensibility in both the radial and circumferential directions. Ang II and elevated cyclic pressure both increased the collagen content in leaflet tissue. Interstitial cells incubated with Ang II were stiffer than those incubated without Ang II while elevated cyclic pressure caused a decrease in cell stiffness. The results of the current study demonstrated that both pressure and Ang II play a role in altering the biomechanical properties of aortic valve leaflets. Ang II and elevated cyclic pressure decreased the extensibility of aortic valve leaflet tissue. Ang II induced direction specific changes in extensibility, demonstrating different response mechanisms. These findings help to provide a better understanding of the responses of aortic valves to mechanical and biochemical changes that occur under hypertensive conditions.
Introduction to viral vectors Warnock, James N; Daigre, Claire; Al-Rubeai, Mohamed
Methods in molecular biology (Clifton, N.J.),
01/2011, Letnik:
737
Journal Article
Viral vector is the most effective means of gene transfer to modify specific cell type or tissue and can be manipulated to express therapeutic genes. Several virus types are currently being ...investigated for use to deliver genes to cells to provide either transient or permanent transgene expression. These include adenoviruses (Ads), retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses. The choice of virus for routine clinical use will depend on the efficiency of transgene expression, ease of production, safety, toxicity, and stability. This chapter provides an introductory overview of the general characteristics of viral vectors commonly used in gene transfer and their advantages and disadvantages for gene therapy use.
Current protocols for mechanical preconditioning of tissue engineered heart valves have focused on application of pressure, flexure and fluid flow to stimulate collagen production, ECM remodeling and ...improving mechanical performance. The aim of this study was to determine if mechanical preconditioning with cyclic stretch could promote an intact endothelium that resembled the viability and morphology of a native valve. Confocal laser scanning microscopy was used to image endothelial cells on aortic valve strips subjected to static incubation or physiological strain regimens. An automated image analysis program was designed and implemented to detect and analyze live and dead cells in images captured of a live aortic valve endothelium. The images were preprocessed, segmented, and quantitatively analyzed for live/dead cell ratio, minimum neighbor distance and circularity. Significant differences in live/dead cellular ratio and the minimum distance between cells were observed between static and strained endothelia, indicating that cyclic strain is an important stimulus for maintaining a healthy endothelium. In conclusion,
in vitro
application of physiological levels of cyclic strain to tissue engineered heart valves seeded with autologous endothelial cells would be advantageous.
The demand for biopharmaceutical products is set to see a significant increase over the next few years. As a consequence, the processes used to produce these products must be able to meet market ...requirements. The present paper reviews the current technologies available for animal cell culture and highlights the advantages and disadvantages of each method, while also providing details of recent case studies. Processes are described for both suspension and anchorage‐dependent cell lines.
Aortic valve ectopic calcification occurs exclusively on the fibrosa surface. This may be due to the distinct mechanical environments on either side of the valve, or to the existence of unique, ...side-specific endothelial sub-phenotypes. The study aim was to determine if side-specific endothelial cells (ECs) would differentially express cell-cell and cell-matrix adhesion molecules in response to elevated levels of equibiaxial tensile strain.
Side-specific porcine aortic valve ECs were isolated and strained at 10% or 20% using a Flexcell 4000T for 24 h, and compared to static controls. The quantity and pattern of distribution of adhesion proteins was then assessed using ELISA and fluorescence microscopy, respectively. The adhesion proteins of interest were platelet endothelial cell adhesion molecule-1 (PECAM-1), beta1-integrin, VE-cadherin, and vinculin.
Overall, ventricular ECs were more reactive to changes in cyclic strain, with significant increases in VE-cadherin and vinculin at 20% strain. However, the expression of beta1-integrin was significantly increased at 20% strain in fibrosa ECs. Expression of PECAM-1 was not significantly changed at all strain levels for both sub-populations of ECs.
Endothelial cells isolated from the fibrosa and ventricularis surfaces of porcine aortic valves showed significantly different expression profiles of cell-cell and cell-extracellular matrix adhesion molecules under elevated tensile strain. These differences in response to cyclic strain suggest that different endothelial sub-phenotypes exist on the fibrosa and ventricularis surfaces of the aortic valve.
An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. Previous studies by this group have shown that ...exposure to constant static pressure leads to enhanced collagen synthesis in porcine aortic valve leaflets. In this study, the effect of cyclic pressure was evaluated using a custom-designed pressure system. Different pressure magnitudes (100, 140, and 170 mmHg) as well as pulse frequencies (0.5, 1.167, and 2 Hz) were studied. Collagen synthesis, cell proliferation, sGAG synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were chosen as markers for valvular biological responses. Results showed that aortic valve leaflets responded to cyclic pressure in a magnitude and frequency-dependent manner. Increases in pressure magnitude (with the frequency fixed at 1.167 Hz) resulted in significant increases in both collagen and sGAG synthesis, while DNA synthesis remained unchanged. Responses to pulse frequency (with the magnitude fixed at 100 mmHg) were more complex. Collagen and sGAG synthesis were increased by 25 and 14% respectively at 0.5 Hz; but were not affected at 1.167 and 2 Hz. In contrast, DNA synthesis increased by 72% at 2 Hz, but not at 0.5 and 1.167 Hz. Under extreme pressure conditions (170 mmHg, 2 Hz), collagen and sGAG synthesis were increased but to a lesser degree than at 170 mmHg, and 1.167 Hz. Cell proliferation was not affected. A notable decline in a-SMC actin was observed over the course of the experiments, although no significant difference was observed between the cyclic pressure and control groups. It was concluded that cyclic pressure affected biosynthetic activity of aortic valve leaflets in a magnitude and frequency dependent manner. Collagen and sGAG synthesis were positively correlated and more responsive to pressure magnitude than pulse frequency. DNA synthesis was more responsive to pulse frequency than pressure magnitude. However, when combined, pressure magnitude and pulse frequency appeared to have an attenuating effect on each other. The number of alpha-SMC actin positive cells did not vary with cyclic pressure, regardless of pulse frequency and pressure magnitude.
Mechanical in vitro preconditioning of tissue engineered heart valves is viewed as an essential process for tissue development prior to in vivo implantation. However, a number of pro-inflammatory ...genes are mechanosensitive and their elaboration could elicit an adverse response in the host. We hypothesized that the application of normal physiological levels of strain to isolated valve interstitial cells would inhibit the expression of pro-inflammatory genes. Cells were subjected to 0, 5, 10, 15 and 20% strain. Expression of VCAM-1, MCP-1, GM-CSF and OPN was then measured using qRT-PCR. With the exception of OPN, all genes were significantly up regulated when no strain was applied. MCP-1 expression was significantly lower in the presence of strain, although strain magnitude did not affect the expression level. VCAM-1 and GM-CSF had the lowest expression levels at 15% strain, which represent normal physiological conditions. These findings were confirmed using confocal microscopy. Additionally, pSMAD 2/3 and IκBα expression were imaged to elucidate potential mechanisms of gene expression. Data showed that 15% strain increased pSMAD 2/3 expression and prevented phosphorylation of IκBα. In conclusion, cyclic strain reduces expression of pro-inflammatory genes, which may be beneficial for the in vitro pre-conditioning of tissue engineered heart valves.
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