The development of biologically-based tissue-engineered blood vessels (TEBVs) that combine vascular cells with biocompatible extracellular matrix (ECM) scaffolds shows promise, but is problematic in two key areas, which will be addressed in this application: 1) the absence of elastin and 2) excessive maturation times in vitro. In native blood vessels, the rubber-like protein elastin provides resilience and limits vascular smooth muscle cell (VSMC) proliferation. Unfortunately, adult VSMCs synthesize little or no elastin, hence TEBVs incorporating VSMCs from adult patients (to limit tissue rejection) would be deficient in elastin, which can lead to stenosis and mechanical failure of the graft. The Wight Laboratory has discovered that splice variant 3 of the ECM proteoglycan versican (V3) can induce VSMCs in vitro and in vivo to produce and assemble elastin. In the present application, we propose that purified, recombinant V3 (rV3) can be used to stimulate elastic fiber formation within TEBVs as they mature in vitro. In regard to excessive maturation times, many ECM-based scaffold materials are mechanically weak, which requires that TEBVs mature for months in vitro before they are strong enough to engraft safely. To address this problem, the Vernon Laboratory has developed novel, ECM-based scaffolds (microgrooved collagen membranes - MGCMs) that are mechanically strong and induce seeded cells to align uniaxially on the grooves within 24-48 h. MGCM sheets populated with aligned vascular cells have been successfully converted into tubes. We propose to combine this method of TEBV fabrication with rV3-mediated elastogenesis to create strong, elastic TEBVs that will mature in vitro in a relatively short time. This application has 3 Specific Aims:
In Aim 1, rat rV3 will be produced using an Sf9 insect cell expression system, then purified and tested initially for elastogenic capacity on rat VSMC monolayer cultures. Subsequently, MGCM-scaffolded TEBVs, populated with rat VSMCs (for media) and rat dermal fibroblasts (for adventitia), will be exposed to the rV3 during their maturation in vitro.
In Aim 2, we will evaluate the structural, mechanical, and physiological performance of the TEBVs created in Aim 1 (with native arteries as the """"""""gold standard"""""""" for performance) according to the following criteria: 1) cell orientation and population dynamics;2) composition and organization of the ECM produced by the cells;3) mechanical properties, including stress- strain responses and burst-strength;and 4) vasoresponse. Finally, in Aim 3, robust, candidate TEBVs will be populated with endothelial cells to produce a non-thrombogenic lining and transplanted into rats to evaluate their performance in vivo. Endpoints will include TEBV patency, integrity, mechanical properties, endothelialization, thrombogenicity, vasoresponse, and host immune responses. In summary, the work proposed in this application represents the next stage of our extensive preliminary studies of the elastogenic properties of V3 and of methods to fabricate TEBVs. We believe that this work will make significant progress toward the goal of an engineered vascular replacement that functions like a native blood vessel.
Efforts to create small (<5 mm)-diameter tissue-engineered blood vessel (TEBV) replacements for diseased and injured arteries have met with limited success. Utilizing novel approaches, we propose to combine cells with natural structural and signaling molecules to create small-diameter TEBVs with a strength and elasticity like that of native arteries. With approximately 600,000 coronary bypass operations performed per year in the USA and a need for readily-available vascular shunts for dialysis patients and vascular grafts for limbs, successful development of small-diameter TEBV replacements would have a major impact on public health.
|Reed, May J; Damodarasamy, Mamatha; Pathan, Jasmine L et al. (2018) The Effects of Normal Aging on Regional Accumulation of Hyaluronan and Chondroitin Sulfate Proteoglycans in the Mouse Brain. J Histochem Cytochem 66:697-707|
|Wight, Thomas N (2017) Provisional matrix: A role for versican and hyaluronan. Matrix Biol 60-61:38-56|
|Merrilees, Mervyn J; Falk, Ben A; Zuo, Ning et al. (2017) Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin. J Tissue Eng Regen Med 11:295-305|
|Keire, Paul A; Bressler, Steven L; Mulvihill, Eileen R et al. (2016) Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo. Matrix Biol 50:67-81|
|Merrilees, Mervyn J; Zuo, Ning; Evanko, Stephen P et al. (2016) G1 Domain of Versican Regulates Hyaluronan Organization and the Phenotype of Cultured Human Dermal Fibroblasts. J Histochem Cytochem 64:353-63|
|Evanko, Stephen P; Potter-Perigo, Susan; Petty, Loreen J et al. (2015) Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-?1 Induction of Lung Myofibroblasts. Matrix Biol 42:74-92|
|Otsuka, Fumiyuki; Kramer, Miranda C A; Woudstra, Pier et al. (2015) Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: A pathology study. Atherosclerosis 241:772-82|
|Kang, Inkyung; Barth, Jeremy L; Sproul, Erin P et al. (2015) Expression of V3 Versican by Rat Arterial Smooth Muscle Cells Promotes Differentiated and Anti-inflammatory Phenotypes. J Biol Chem 290:21629-41|
|Damodarasamy, Mamatha; Vernon, Robert B; Chan, Christina K et al. (2015) Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation. In Vitro Cell Dev Biol Anim 51:50-8|
|Hu, Jie Hong; Wei, Hao; Jaffe, Mia et al. (2015) Postnatal Deletion of the Type II Transforming Growth Factor-? Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice. Arterioscler Thromb Vasc Biol 35:2647-56|
Showing the most recent 10 out of 12 publications