Adipose Stromal Cell-Based Elastogenesis Therapy for Adult and Pediatric Aortic Defects

Aortic aneurysm (AA) is a balloon-like enlargement of the aorta exceeding 1.5-times original diameter and possessing a life-threatening risk of rupture if left untreated, representing the 15th leading cause of death in the United States. Currently, surgical intervention is governed by aortic diameter measurements, recommended after adult AA enlarge beyond a “critical diameter” of 5.5cm and pediatric AA exceed a 0.5cm/year growth rate. AA diameter growth is mediated by inflammatory damage to extracellular matrix protein elastin, responsible for aortic recoil during pulsatile blood flow. Therapeutic options for sub-critical AA are limited to “watchful waiting” imaging every 6-to-12 months to monitor diameter growth, or broad-targeted therapeutics (beta blockers, ACE inhibitors) that do not work to rebuild the aortic wall. Recent work by our lab has shown that delivery of adipose-derived stromal cells (ASCs) can slow AA dilation and preserve elastic fibers, by either suppressing inflammatory elastin breakdown or stimulating new elastin deposition. This dissertation work utilized a versatile, fibrin-based 3D SMC aortic culture platform to test whether paracrine signaling using ASC secreted factors (ASC-SF) could induce new human elastin deposition by three different classes of aortic smooth muscle cells (SMCs): healthy adult SMCs, aneurysmal adult SMCs, and aneurysmal pediatric SMCs. Elastin deposition was evaluated at four different points of interest on the elastogenesis cascade: elastin organizational protein transcription (generating tropoelastin, fibulin-4, and fibulin-5 coacervates/globules) [1-3], elastic fiber organization (through LTBP-4, fibulin-4, and fibulin-5 mediated deposition onto fibrillin-1 microfibrils), cross-linked elastin chemical maturity (mediated by lysyl oxidase or LOX, and lysyl oxidase-like 1 or LOXL-1), and mechanical functionality of the deposited extracellular matrix. Additionally, two methods were explored to maximize clinical translation of ASC-SF therapeutic delivery: potency of ASC-SF-derived exosomes, and a magnetic-guided periadventitial in vivo delivery system

Functional Vascular Tissue Engineering Inspired by Matricellular Proteins

Modern regenerative medicine, and tissue engineering specifically, has benefited from a greater appreciation of the native extracellular matrix (ECM). Fibronectin, collagen, and elastin have entered the tissue engineer's toolkit; however, as fully decellularized biomaterials have come to the forefront in vascular engineering it has become apparent that the ECM is comprised of more than just fibronectin, collagen, and elastin, and that cell-instructive molecules known as matricellular proteins are critical for desired outcomes. In brief, matricellular proteins are ECM constituents that contrast with the canonical structural proteins of the ECM in that their primary role is to interact with the cell. Of late, matricellular genes have been linked to diseases including connective tissue disorders, cardiovascular disease, and cancer. Despite the range of biological activities, this class of biomolecules has not been actively used in the field of regenerative medicine. The intent of this review is to bring matricellular proteins into wider use in the context of vascular tissue engineering. Matricellular proteins orchestrate the formation of new collagen and elastin fibers that have proper mechanical properties—these will be essential components for a fully biological small diameter tissue engineered vascular graft (TEVG). Matricellular proteins also regulate the initiation of thrombosis via fibrin deposition and platelet activation, and the clearance of thrombus when it is no longer needed—proper regulation of thrombosis will be critical for maintaining patency of a TEVG after implantation. Matricellular proteins regulate the adhesion, migration, and proliferation of endothelial cells—all are biological functions that will be critical for formation of a thrombus-resistant endothelium within a TEVG. Lastly, matricellular proteins regulate the adhesion, migration, proliferation, and activation of smooth muscle cells—proper control of these biological activities will be critical for a TEVG that recellularizes and resists neointimal formation/stenosis. We review all of these functions for matricellular proteins here, in addition to reviewing the few studies that have been performed at the intersection of matricellular protein biology and vascular tissue engineering

Molecular Imaging of Experimental Abdominal Aortic Aneurysms

Current laboratory research in the field of abdominal aortic aneurysm (AAA) disease often utilizes small animal experimental models induced by genetic manipulation or chemical application. This has led to the use and development of multiple high-resolution molecular imaging modalities capable of tracking disease progression, quantifying the role of inflammation, and evaluating the effects of potential therapeutics. In vivo imaging reduces the number of research animals used, provides molecular and cellular information, and allows for longitudinal studies, a necessity when tracking vessel expansion in a single animal. This review outlines developments of both established and emerging molecular imaging techniques used to study AAA disease. Beyond the typical modalities used for anatomical imaging, which include ultrasound (US) and computed tomography (CT), previous molecular imaging efforts have used magnetic resonance (MR), near-infrared fluorescence (NIRF), bioluminescence, single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Mouse and rat AAA models will hopefully provide insight into potential disease mechanisms, and the development of advanced molecular imaging techniques, if clinically useful, may have translational potential. These efforts could help improve the management of aneurysms and better evaluate the therapeutic potential of new treatments for human AAA disease

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non-specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti-restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC-SMC co-cultures, we investigated the ability of DS-SILY20 , a decorin mimic, to promote EC health. DS-SILY20 increased EC proliferation and migration by 1.5- and 2-fold, respectively, which corresponded to increased phosphorylation of ERK-1/2. Interestingly, IL-6 secretion and the production of both E-selectin and P-selectin were reduced in the presence of 10 μm DS-SILY20 , even in the presence of the potent pro-inflammatory cytokine platelet-derived growth factor (PDGF). In hyperplastic and quiescent EC-SMC co-cultures, DS-SILY20 treatment reduced the secretion of IFNγ, IL-1β, IL-6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E-selectin and P-selectin expression was further reduced following DS-SILY20 treatment in both co-culture models. These results indicate that DS-SILY20 promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd

Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture

Macromolecular components of the vascular extracellular matrix (ECM), particularly elastic fibers and collagen fibers, are critical for the proper physiological function of arteries. When the unique biomechanical combination of these fibers is disrupted, or in the ultimate extreme where fibers are completely lost, arterial disease can emerge. Bioengineers in the realms of vascular tissue engineering and regenerative medicine must therefore ideally consider how to create tissue engineered vascular grafts containing the right balance of these fibers and how to develop regenerative treatments for situations such as an aneurysm where fibers have been lost. Previous work has demonstrated that the primary cells responsible for vascular ECM production during development, arterial smooth muscle cells (SMCs), can be induced to make new elastic fibers when exposed to secreted factors from adipose-derived stromal cells. To further dissect how this signal is transmitted, in this study, the factors were partitioned into extracellular vesicle (EV)-rich and EV-depleted fractions as well as unseparated controls. EVs were validated using electron microscopy, dynamic light scattering, and protein quantification before testing for biological effects on SMCs. In 2D culture, EVs promoted SMC proliferation and migration. After 30 days of 3D fibrin construct culture, EVs promoted SMC transcription of the elastic microfibril gene FBN1 as well as SMC deposition of insoluble elastin and collagen. Uniaxial biomechanical properties of strand fibrin constructs were no different after 30 days of EV treatment versus controls. In summary, it is apparent that some of the positive effects of adipose-derived stromal cells on SMC elastogenesis are mediated by EVs, indicating a potential use for these EVs in a regenerative therapy to restore the biomechanical function of vascular ECM in arterial disease