Randall F. Ankeny

Laminar Shear Stress Inhibits Cathepsin L Activity in Endothelial Cells

Objective—The cysteine proteases, cathepsins, have been implicated in vascular remodeling and atherosclerosis, processes known to be regulated by shear stress. It is not known, however, whether shear regulates cathepsins. We examined the hypothesis that shear stress regulates cathepsin activity in endothelial cells. Methods and Results—Mouse aortic endothelial cells (MAECs) exposed to atheroprotective, unidirectional laminar shear (LS) degraded significantly less BODIPY-labeled elastin and gelatin in comparison to static and proatherogenic oscillatory shear (OS). The cathepsin inhibitor E64 also reduced this activity. Gelatin zymography showed that cathepsin activity of MAECs was blunted by LS exposure and by a cathepsin L inhibitor but not by cathepsin B and S inhibitors, whereas a cathepsin K inhibitor had a minor effect. Cathepsin L siRNA knocked down cathepsin L expression, gelatinase, and elastase activity in OS and static MAECs. A partial reduction of cathepsin B protein raised the possibility that the siRNA effect on the matrix protease activity could have been attributable to cathepsin L or B. Cathepsin B activity study using the synthetic peptide showed it was not regulated by shear. Conclusions—These results suggest that cathepsin L is a shear-sensitive matrix protease and that it may play an important role in flow-mediated vascular remodeling and atherogenic responses.

Discovery of shear- and side-specific mRNAs and miRNAs in human aortic valvular endothelial cells.

The role of endothelial cells (ECs) in aortic valve (AV) disease remains relatively unknown; however, disease preferentially occurs in the fibrosa. We hypothesized oscillatory shear (OS) present on the fibrosa stimulates ECs to modify mRNAs and microRNAs (miRNAs) inducing disease. Our goal was to identify mRNAs and miRNAs differentially regulated by OS and laminar shear (LS) in human AVECs (HAVECs) from the fibrosa (fHAVECs) and ventricularis (vHAVECs). HAVECs expressed EC markers as well as some smooth muscle cell markers and functionally aligned with the flow. HAVECs were exposed to OS and LS for 24 h, and total RNA was analyzed by mRNA and miRNA microarrays. We found over 700 and 300 mRNAs down- and upregulated, respectively, by OS; however, there was no side dependency. mRNA microarray results were validated for 26 of 28 tested genes. Ingenuity Pathway Analysis revealed thrombospondin 1 (Thbs1) and NF-κB inhibitor-α (Nfkbia) as highly connected, shear-sensitive genes. miRNA array analysis yielded 30 shear-sensitive miRNAs and 3 side-specific miRNAs. miRNA validation confirmed 4 of 17 shear-sensitive miRNAs and 1 of 3 side-dependent miRNAs. Using miRWalk and several filtering steps, we identified shear-sensitive mRNAs potentially targeted by shear-sensitive miRNAs. These genes and signaling pathways could act as therapeutic targets of AV disease.

Preferential Activation of SMAD1/5/8 on the Fibrosa Endothelium in Calcified Human Aortic Valves - Association with Low BMP Antagonists and SMAD6

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.

The role of demineralized bone particle in a PLGA scaffold designed to create a media equivalent for a tissue engineered blood vessel

Poly(lactic-co-glycolic acid) (PLGA) is a copolymer used for scaffolds in tissue engineering due to its biodegradable and biocompatible properties. Demineralized bone particle (DBP) is a natural material containing bone morphogenic proteins (BMPs). The objective of this study was to determine if DBP induced differentiation of mesenchymal stem cells (MSCs) into a smooth muscle cells (SMCs) phenotype and act in a similar manner when treated with TGF-β1 as SMCs under the same conditions. PLGA scaffolds were fabricated with or without DBP using the solvent casting/salt leaching technique. Proliferation of MSCs and SMCs was measured using the MTT assay, while the expression of the smooth muscle-specific markers α-smooth muscle actin (α-SMA), smooth muscle 22 alpha (SM22α), and calponin was measured using RT-PCR and western blotting. Results showed PLGA with DBP scaffolds increased cell proliferation of SMCs (1 and 3 days) and MSCs (3 and 7 days) compared to the PLGA only scaffolds. Smooth muscle-specific genes were significantly increased in SMCs cultured for 7 days on PLGA with DBP scaffolds compared to PLGA only scaffolds and were also significantly increased when used SMC medium containing TGF-β1. Interestingly, the smooth muscle-specific genes were not altered in MSCs by the addition of TGF-β1 to the PLGA with DBP. However, in the 6-well and PLGA only groups, TGF-β1 addition caused increased levels of SM22α and calponin mRNA. Western blotting results were similar to the mRNA levels. These results suggest that the cell growth and gene expression of SMCs were up-regulated by DBP and TGF-β1 in PLGA scaffold; however, they were inadequate for the shift of MSCs into a SMCs phenotype.

Shear Stress Effects on the Inflammation and Thrombogenic State of Baboon Endothelial Outgrowth Cells

Endothelial progenitor cells (EPCs) are believed to play an important role in the injury response of the human vasculature. Sudies have shown how EPCs are activated and play roles in tumor and hindlimb ischemia nevascularization[1,2]. Furthermore, EPCs are increased after surgical procedures and localized to vascular grafts and denuded arteries[2,3]. EPCs have also garnered immense interest in the field of tissue engineering due to their ease of isolation and their high prolifierative rate[2].Copyright

Discovery of Side- and Shear-Dependent miRNAs and mRNAs in Human Aortic Valvular Endothelial Cells

Aortic valve (AV) disease is diagnosed by severe symptoms, such as calcification, and typically treated by AV replacement and repair surgeries. The mechanism by which AV disease occurs, specifically the role of the endothelium remains relatively unknown. It is known that disease preferentially occurs on the fibrosa, or aortic side, where it is exposed to disturbed, oscillatory flow, whereas the ventricularis, or side facing the left ventricle, experiences pulsatile, laminar shear and remains non-calcified [1, 2]. Research shows that regulation of miRNAs, short nucleotide segments targeting mRNAs, coincides with cardiovascular pathologies [3] though expression profiles of miRNAs and the mRNAs they modulate in human AV endothelial cells (HAVECs) have not been reported. We hypothesize that disturbed flow conditions present on the fibrosa stimulate ECs to modify expression of genes and miRNAs to induce a pro-inflammatory phenotype.Copyright