The aortic valve (AV) experiences a complex mechanical environment, which includes tension, flexure, pressure, and shear stress forces due to blood flow during each cardiac cycle. This mechanical environment regulates AV tissue structure by constantly renewing and remodeling the phenotype. In vitro, ex vivo and in vivo studies have shown that pathological states such as hypertension and congenital defect like bicuspid AV (BAV) can potentially alter the AV's mechanical environment, triggering a cascade of remodeling, inflammation, and calcification activities in AV tissue. Alteration in mechanical environment is first sensed by the endothelium, which in turn induces changes in the extracellular matrix, and triggers cell differentiation and activation. However, the molecular mechanism of this process is not understood very well. Understanding these mechanisms is critical for advancing the development of effective medical based therapies. Recently, there have been some interesting studies on characterizing the hemodynamics associated with AV, especially in pathologies like BAV, using different experimental and numerical methods. Here, we review the current knowledge of the local AV mechanical environment and its effect on valve biology, focusing on in vitro and ex vivo approaches. © 2013 Biomedical Engineering Society.

Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) affect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this effect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of inflammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL.

Background: Aortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation of bone morphogenic protein (BMP) pathway in the endothelium of the ventricularis and fibrosa is associated with human AV calcification. Methods and Results: Human calcified AVs obtained from AV replacement surgeries and non-calcified AVs from heart transplantations were used for immunohistochemical studies. We found SMAD-1/5/8 phosphorylation (a canonical BMP pathway) was higher in the calcified fibrosa than the non-calcified fibrosa while SMAD-2/3 phosphorylation (a canonical TGFβ pathway) did not show any difference. Interestingly, we found that BMP-2/4/6 expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both calcified and non-calcified AV cusps; however, BMP antagonists (crossvienless-2/BMPER and noggin) expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both disease states. Moreover, significant expression of inhibitory SMAD-6 expression was found only in the non-calcified ventricularis endothelium. Conclusions: SMAD-1/5/8 is preferentially activated in the calcified fibrosa endothelium of human AVs and it correlates with low expression of BMP antagonists and inhibitory SMAD6. These results suggest a dominant role of BMP antagonists in the side-dependent calcification of human AVs.

Musculoskeletal pathologies associated with decreased bone mass, including osteoporosis and disuse-induced bone loss, affect millions of Americans annually. Microgravity-induced bone loss presents a similar concern for astronauts during space missions. Many pharmaceutical treatments have slowed osteoporosis, and recent data shows promise for countermeasures for bone loss observed in astronauts. Additionally, high magnitude and low frequency impact such as running has been recognized to increase bone and muscle mass under normal but not microgravity conditions. However, a low magnitude and high frequency (LMHF) mechanical load experienced in activities such as postural control, has also been shown to be anabolic to bone. While several clinical trials have demonstrated that LMHF mechanical loading normalizes bone loss in vivo, the target tissues and cells of the mechanical load and underlying mechanisms mediating the responses are unknown. In this review, we provide an overview of bone adaptation under a variety of loading profiles and the potential for a low magnitude loading as a way to counteract bone loss as experienced by astronauts. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), in part, due to alterations in gene expression in the endothelium. While numerous in vitro studies have shown how anti-atherogenic flow and pro-atherogenic flow differently regulate gene expression of cultured endothelial cells, similar in vivo studies have been scarce. Recently, we developed a mouse model of atherosclerosis that rapidly develops robust atherosclerosis by partially ligating the left carotid artery (LCA) branches, while using the contralateral right carotid (RCA) as control. We also developed a novel method to collect endothelial-enriched RNAs from the carotids of these animals, which enabled us to perform genome-wide expression analyses of mRNAs and miRNAs in the arterial endothelium exposed to either d-flow or s-flow. These microarray results were used to identify novel mechanosensitive genes such as DNA methyltransferase-1 and miR-712 that play key roles in atherosclerosis. Here, we report these endothelial mRNA and miRNA expression profiles with in-depth information on experimental procedures along with an example of usage of these data.

MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same 'seed sequence' as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive 'athero-miRs' may provide a new treatment paradigm in atherosclerosis.

Disturbed blood flow induces apoptosis of vascular endothelial cells, which causes atherosclerosis. In this issue, Heo et al. (2011. J. Cell Biol. doi:10.1083/jcb.201010051) sheds light on p53’s role in this phenomenon. Disturbed flow induces peroxynitrite production, which activates protein kinase C ζ and it’s binding to the E3 SUMO (small ubiquitin-like modifier) ligase PIASy (protein inhibitor of activated STATy). This leads to p53 SUMOylation and its export to the cytosol, where it binds to the antiapoptotic protein Bcl-2 to induce apoptosis.

Background: The pathomechanisms of atherosclerosis and vascular remodelling are under intense research. Only a few in vivo tools to study these processes longitudinally in animal experiments are available. Here, we evaluated the potential of micro-CT technology. Methods: Lumen areas of the common carotid arteries (CCA) in the ApoE^-/- partial carotid artery ligation mouse model were compared between in vivo and ex vivo micro-CT technique and serial histology in a total of 28 animals. AuroVist-15 nm nanoparticles were used as in vivo blood pool contrast agent in a Skyscan 1176 micro-CT at resolution of 18 μmeter voxel size and a mean x-ray dose of 0.5 Gy. For ex vivo imaging, animals were perfused with MicroFil and imaged at 9 μmeter voxel size. Lumen area was evaluated at postoperative days 7, 14, and 28 first by micro-CT followed by histology. Results: In vivo micro-CT and histology revealed lumen loss starting at day 14. The lumen profile highly correlated (r = 0.79, P<0.0001) between this two methods but absolute lumen values obtained by histology were lower than those obtained by micro-CT. Comparison of in vivo and ex vivo micro-CT imaging revealed excellent correlation (r = 0.83, P<0.01). Post mortem micro-CT yielded a higher resolution than in vivo micro-CT but there was no statistical difference of lumen measurements in the partial carotid artery ligation model. Conclusion: These data demonstrate that in vivo micro-CT is a feasible and accurate technique with low animal stress to image remodeling processes in the murine carotid artery.

BACKGROUND: Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries. METHODS: D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-weekold mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model. RESULTS: D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model. CONCLUSIONS: TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.

Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. Two peptides, CLIRRTSIC and CPRRSHPIC, selectively bound to arterial ECs exposed to disturbed flow not only in the partially ligated carotids but also in the lesser curvature and branching point of the aortic arch in mice as well as human pulmonary artery branches. Peptides were conjugated to branched polyethylenimine-polyethylene glycol polymer to generate polyplexes carrying siRNA targeting intercellular adhesion molecule-1 (siICAM-1). In mouse model, CLIRRTSIC polyplexes carrying si-ICAM-1 specifically bound to endothelium in disturbed flow regions, reducing endothelial ICAM-1 expression. Mass spectrometry analysis revealed that non-muscle myosin heavy chain II A (NMHC IIA) is a protein targeted by CLIRRTSIC peptide. Further studies showed that shear stress regulates NMHC IIA expression and localization in ECs. The CLIRRTSIC is a novel peptide that could be used for targeted delivery of therapeutics such as siRNAs to pro-atherogenic endothelium.