Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. METHODS AND RESULTS: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. CONCLUSIONS: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Thermal sensitivity of endothelial cells on synthetic vascular graft material.

PURPOSE: The goal is to identify thermal exposures capable of reducing or eliminating cell survival on expanded polytetrafluoroethylene (ePTFE), in an effort to develop a mild hyperthermia treatment of neointimal hyperplasia in ePTFE vascular grafts. MATERIALS AND METHODS: Viable and dead bovine aortic endothelial cells were quantified following different thermal exposure conditions: cells on collagen-coated ePTFE sheets or tissue culture polystyrene dishes were heated at 42° and 45°C to determine their thermal sensitivity on different surfaces, and cells cultured on collagen-coated ePTFE sheets were heated at 43-50°C for various durations, followed by incubation at 37°C for 0 and 20 h, respectively. Significant cell death was set to be 50%. Two types of cell death, apoptosis and necrosis, were distinguished by cell morphology and membrane integrity assessments. RESULTS: The attachment and survival of cells on ePTFE sheets were more sensitive to inhibition by mild heating than those on tissue culture dishes. Exposure to 45°C for 90 min and 50°C for 30 min caused significant necrotic cell death on ePTFE (65% and 75%, respectively). A 37°C/20-h incubation following 30-min exposures at 47° and 50°C increased total cell death (necrosis + apoptosis) from 20% to 50% and 75% to 100%, respectively. CONCLUSION: Cells grown on ePTFE were more susceptible to mild hyperthermia-induced death, compared to those on tissue culture dishes. Significant cell death on ePTFE mainly via apoptosis can be achieved by optimising temperature and duration of exposure.

Modelling ultrasound-induced mild hyperthermia of hyperplasia in vascular grafts.

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) vascular grafts frequently develop occlusive neointimal hyperplasia as a result of myofibroblast over-growth, leading to graft failure. ePTFE exhibits higher ultrasound attenuation than native soft tissues. We modelled the selective absorption of ultrasound by ePTFE, and explored the feasibility of preventing hyperplasia in ePTFE grafts by ultrasound heating. Specifically, we simulated the temperature profiles of implanted grafts and nearby soft tissues and blood under ultrasound exposure. The goal was to determine whether ultrasound exposure of an ePTFE graft can generate temperatures sufficient to prevent cell growth on the graft without damaging nearby soft tissues and blood. METHODS: Ultrasound beams from two transducers (1.5 and 3.2 MHz) were simulated in two graft/tissue models, with and without an intra-graft cellular layer mimicking hyperplasia, using the finite-difference time-domain (FDTD) method. The resulting power deposition patterns were used as a heat source for the Pennes bioheat equation in a COMSOL(®) Multiphysics heat transfer model. 50°C is known to cause cell death and therefore the transducer powers were adjusted to produce a 13°C temperature rise from 37°C in the ePTFE. RESULTS: Simulations showed that both the frequency of the transducers and the presence of hyperplasia significantly affect the power deposition patterns and subsequent temperature profiles on the grafts and nearby tissues. While neither transducer significantly raised the temperature of the blood, the 1.5-MHz transducer was less focused and heated larger volumes of the graft and nearby soft tissues than the 3.2-MHz transducer. The presence of hyperplasia had little effect on the blood's temperature, but further increased the temperature of the graft and nearby soft tissues in response to either transducer. Skin cooling and blood flow play a significant role in preventing overheating of the native tissues. CONCLUSIONS: Modelling shows that ultrasound can selectively heat ePTFE grafts and produce temperatures that cause cell death on the graft. The temperature increase in blood is negligible and that in the adjacent soft tissues may be minimized by skin cooling and using appropriate transducers. Therefore, ultrasound heating may have the potential to reduce neointimal hyperplasia and failure of ePTFE vascular grafts.

Neointimal hyperplasia associated with synthetic hemodialysis grafts.

Stenosis is a major cause of failure of hemodialysis vascular grafts and is primarily caused by neointimal hyperplasia (NH) at the anastomoses. The objective of this article is to provide a scientific review of the biology underlying this disorder and a critical review of the state-of-the-art investigational preventive strategies in order to stimulate further research in this exciting area. The histology of the NH shows myofibroblasts (that are probably derived from adventitial fibroblasts), extracellular matrices, pro-inflammatory cells including foreign-body giant cells, a variety of growth factors and cytokines, and neovasculature. The contributing factors of the pathogenesis of NH include surgical trauma, bioincompatibility of the synthetic graft, and the various mechanical stresses that result from luminal hypertension and compliance mismatch between the vessel wall and graft. These mechanical stimuli are focal in nature and may have a significant influence on the preferential localization of the NH. Novel mechanical graft designs and local drug delivery strategies show promise in animal models in preventing graft NH development. Successful prevention of graft stenosis would provide a superior alternative to the native fistula as hemodialysis vascular access.

Serial analysis of lumen geometry and hemodynamics in human arteriovenous fistula for hemodialysis using magnetic resonance imaging and computational fluid dynamics.

The arteriovenous fistula (AVF) is the preferred form of vascular access for maintenance hemodialysis, but it often fails to mature to become clinically usable, likely due to aberrant hemodynamic forces. A robust pipeline for serial assessment of hemodynamic parameters and subsequent lumen cross-sectional area changes has been developed and applied to a data set from contrast-free MRI of a dialysis patient's AVF collected over a period of months after AVF creation surgery. Black-blood MRI yielded images of AVF lumen geometry, while cine phase-contrast MRI provided volumetric flow rates at the in-flow and out-flow locations. Lumen geometry and flow rates were used as inputs for computational fluid dynamics (CFD) modeling to provide serial wall shear stress (WSS), WSS gradient, and oscillatory shear index (OSI) profiles. The serial AVF lumen geometries were co-registered at 1mm intervals using respective lumen centerlines, with the anastomosis as an anatomical landmark. Lumen enlargement was limited at the vein region near the anastomosis and a downstream vein valve, potentially attributed to the physical inhibition of wall expansion at those sites. This work is the first serial and detail study of lumen and hemodynamic changes in human AVF using MRI and CFD. This novel protocol will be used for a multicenter prospective study to identify critical hemodynamic factors that contribute to AVF maturation failure.

Novel approach for endothelializing vascular devices: understanding and exploiting elastin-endothelial interactions.

Elastin is an essential component of arteries which provides structural integrity and instructs smooth muscle cells to adopt a quiescent state. Despite interaction of endothelial cells with elastin in the internal elastic lamina, the potential for exploiting this interaction therapeutically has not been explored in detail. In this study, we show that tropoelastin (a precursor of elastin) stimulates endothelial cell migration and adhesion more than smooth muscle cells. The biological activity of tropoelastin on endothelial cells is contained in the VGVAPG domain and in the carboxy-terminal 17-amino acids. We show that the effects of the carboxy-terminal 17 amino acids, but not those of VGVAPG, are mediated by integrin α(V)ß(3). We demonstrate that tropoelastin covalently linked to stainless steel disks promotes adhesion of endothelial progenitor cells and endothelial cells to the metal surfaces. The adherent cells on the tropoelastin-coated metal surfaces form monolayers that can withstand and respond to arterial shear stress. Because of the unique effects of tropoelastin on endothelial and smooth muscle cells, coating intravascular devices with tropoelastin may stimulate their endothelialization, inhibit smooth muscle hyperplasia, and improve device performance.