No abstract

Stents are deployed to physically reopen stenotic regions of arteries and to restore blood flow. However, inflammation and localized stent thrombosis remain a risk for all current commercial stent designs. Computational fluid dynamics results predict that nonstreamlined stent struts deployed at the arterial surface in contact with flowing blood, regardless of the strut height, promote the creation of proximal and distal flow conditions that are characterized by flow recirculation, low flow (shear) rates, and prolonged particle residence time. Furthermore, low shear rates yield an environment less conducive for endothelialization, while local flow recirculation zones can serve as micro-reaction chambers where procoagulant and pro-inflammatory elements from the blood and vessel wall accumulate. By merging aerodynamic theory with local hemodynamic conditions we propose a streamlined stent strut design that promotes the development of a local flow field free of recirculation zones, which is predicted to inhibit thrombosis and is more conducive for endothelialization.

Rationale Hemodynamic disturbed flow is associated with susceptibility to atherosclerosis. Endothelial KLF4 is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of disturbed flow. Objective The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue. Methods and Results To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells (HAEC) were subjected to pulsatile undisturbed flow (UF) or oscillatory disturbed flow (DF) containing a flow-reversing phase. Differential CpG site methylation was measured by methylation specific PCR, bisulfite pyrosequencing and restriction enzyme-PCR. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knock-down of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7 fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a MEF2 binding site in the KLF4 promoter near the TSS. DF also induced DNMT-sensitive pro-pathological expression of downstream KLF4 transcription targets NOS3, thrombomodulin (THBD) and MCP-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and NOS3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated MEF2 binding site. Conclusions Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/MEF2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.

Results are presented on the flow field downstream of a body of revolution for Reynolds numbers based on a model length ranging from 1.1 × 106 to 67 × 106. The maximum Reynolds number is more than an order of magnitude larger than that obtained in previous laboratory wake studies. Measurements are taken in the intermediate wake at locations 3, 6, 9, 12 and 15 diameters downstream from the stern in the midline plane. The model is based on an idealized submarine shape (DARPA SUBOFF), and it is mounted in a wind tunnel on a support shaped like a semi-infinite sail. The mean velocity distributions on the side opposite the support demonstrate self-similarity at all locations and Reynolds numbers, whereas the mean velocity distribution on the side of the support displays significant effects of the support wake. None of the Reynolds stress distributions of the flow attain self-similarity, and for all except the lowest Reynolds number, the support introduces a significant asymmetry into the wake which results in a decrease in the radial and streamwise turbulence intensities on the support side. The distributions continue to evolve with downstream position and Reynolds number, although a slow approach to the expected asymptotic behaviour is observed with increasing distance downstream.

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with K i ‫؍‬ 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8-170 ng͞ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

Results are presented on the behavior of the turbulent wake behind a submarine model for a range of Reynolds numbers based on the model length between 0.49×106 and 1.8×106, for test locations between 3 and 9 diameters downstream of the stern. The shape of the model emulates an idealized submarine, and tests were performed with and without stern fins. In the absence of fins, the velocity profile in planes away from the influence of the sail rapidly becomes self-similar and is well described by a function of exponentials. The fins create defects in the velocity profiles in the outer region of the wake, while yielding higher values of turbulence at locations corresponding to the tips of the fins. Measurements conducted in planes away from the midline plane show that the velocity profiles remain self-similar, while the shear stress profiles clearly show the effects of the necklace vortices trailing from the base of the fins.

Drug eluting stents are associated with late stent thrombosis (LST), delayed healing and prolonged exposure of stent struts to blood flow. Using macroscale disturbed and undisturbed fluid flow waveforms, we numerically and experimentally determined the effects of microscale model strut geometries upon the generation of prothrombotic conditions that are mediated by flow perturbations. Rectangular cross-sectional stent strut geometries of varying heights and corresponding streamlined versions were studied in the presence of disturbed and undisturbed bulk fluid flow. Numerical simulations and particle flow visualization experiments demonstrated that the interaction of bulk fluid flow and stent struts regulated the generation, size and dynamics of the peristrut flow recirculation zones. In the absence of endothelial cells, deposition of thrombin-generated fibrin occurred primarily in the recirculation zones. When endothelium was present, peristrut expression of anticoagulant thrombomodulin (TM) was dependent on strut height and geometry. Thinner and streamlined strut geometries reduced peristrut flow recirculation zones decreasing prothrombotic fibrin deposition and increasing endothelial anticoagulant TM expression. The studies define physical and functional consequences of macro-and microscale variables that relate to thrombogenicity associated with the most current stent designs, and particularly to LST.

Conflict of interest: JDW and MLK are coinventors on a patent, PCTUS1932399, entitled "Medical device for the prevention of thrombosis" that is based on the scientific discoveries described in this article. They are stakeholders in a company, Osciflex, that has an option agreement with the University of Pennsylvania to develop a new device to prevent deep venous thrombosis.