Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences

Abstract

Objective-The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood. Methods and Results-Monolayers of porcine aortic endothelial cells (PAECs) or porcine aortic valve endothelial cells (PAVECs) were exposed to 20 dyne/cm 2 steady laminar shear stress for 48 hours, with static cultures serving as controls. Multiple microarray comparisons were made using RNA from sheared and control batches of both cell types. More than 400 genes were significantly differentially expressed in each comparison group. The resulting profiles were validated at the transcription and protein level and expression patterns confirmed in vivo by immunohistochemistry. PAVECs were found to be less intrinsically inflammatory than PAECs, but both cell types expressed similar antioxidant and antiinflammatory genes in response to shear stress. PAVECs expressed more genes associated with chondrogenesis, whereas PAECs expressed osteogenic genes, and shear stress had a protective effect against calcification. Conclusions-Transcriptional differences between PAVECs and PAECs highlight the valvular endothelial cell as a distinct organ system and suggest more attention needs to be given to valvular cells to further our understanding of similarities and differences between valvular and vascular pathology. Key Words: aortic valve Ⅲ shear stress Ⅲ inflammation Ⅲ calcification Ⅲ endothelial cell A ortic valve disease is associated with significant mortality and morbidity and is a strong risk factor for additional cardiovascular events. 1,2 Valvular degeneration is characterized by the development of stenosis or insufficiency, and by the time it is clinically manifested, it is usually only treatable by prosthetic valve replacement. 3 Explants of diseased valves reveal a wide spectrum of pathology, including sclerotic and calcific lesions, thrombus formations, bacterial vegetations, and fractured matrix fibers. 4,5 Aortic valve disease was originally thought to be the result of the continuous barrage of hemodynamic and mechanical forces over time, but recent evidence suggests a much more active biological progression involving inflammation, oxidation, angiogenesis, calcification, and osteogenesis. 6 -8 The vascular endothelium is a critical mediator of hemodynamic and humoral stimuli, and that endothelial inflammation and atherosclerosis occur preferentially at sites of disturbed or oscillatory flow. 9 Valvular endothelial dysfunction is also a hallmark of leaflet degeneration, and similarly characterized by the expression of proinflammatory adhesion receptors. 10,11 Interestingly, much of the aforementioned valvular pathology seems to occur preferentially on the aortic surface of the leaflet, which experiences a complex circulating flow that is different from the unidirectional flow on the ventricular side of the leaflet. This suggests that disturbed flow may play a causal role in the initiation of valvular pathology...

Isolation of Valvular Endothelial Cells

“…In the presence of shear flow, the endothelial cells down regulated calcific genes such as BMP-4 and POSTN to retain their quiescent phenotype. Shear flow seems to act as a protective measure for endothelial cell types from chondro/osteogenic differentiation, which is a possible explanation of the ventricular endothelial protection 8 .…”

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

“…EC isolated from vascular and valvular sources display differences in gene expression and other properties, which is a major indication of endothelial cell heterogeneity [37]. Heart valve endothelial cells are different from those in the aorta [38], [39]; for example, in response to mechanical stress, porcine aortic It is hypothesized and scientifically becoming more significant that biomechanical and biochemical factors play an important and potentially synergistic role in determining the local homeostatic environment of the aortic VIC. As demon-strated in in vitro studies, application of mechanical stimulation such as tension and addition of cytokine stimulation such as TGF-1 were found to synergistically alter the contractile and biosynthetic proteins of aortic VICs to a greater extent than each factor alone [36].…”

“…This could be readily achieved for example by determining the positive expression of nucleoside phosphorylase and an absence of Interleukin-1 alpha which are distinct characteristics of valve ECs (as opposed to vascular ECs) [39] . Finally, the combined effects of low dose (10 -6 M) Nicotine and OSS regulation of BMMSCs may have synergistic benefits; however this remains to be determined.…”

“…Four diff t outcomes, such as collagen assay to measure ECM production (2), QRT PCR to quantify gene expression [39], [96], [137], immunostaining to locate protein distribution [29], [36] and histology to observe the valve tri layer ECM structure [9] were evaluated. Each outcome has a sample size of 3, which limit the power analysis of the experiment.…”

Cellular Events Under Flow States Pertinent to Heart Valve Function

“…Mechanical stimuli are sensed by the cell membrane receptors which are later transferred to the cytoskeleton; consequently this initiates a biochemical signaling cascade 19 . In BMSCs, the cytoskeletal structure has shown to be altered after exposure to fluid-induced shear stress 46 .…”