eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering.

This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose-derived stem cells (ASC) differentiated into endothelial-like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO-producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non-thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial-like lineage imparts a non-thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

The use of adult adipose-derived stem cells in the creation of a tissue engineered vascular bypass graft

Even with the advent of the emerging field of endovascular surgery to correct complications of peripheral and cardiovascular disease, bypass of occluded vessels remains an important surgical technique. According to the American Heart Association, 448,000 inpatient bypass procedures were performed in the United States. Additionally, 8,000,000 Americans present with peripheral arterial disease. Moreover, by 2020 the AHA reports more than 700,000 Americans will have a need for kidney dialysis with a requirement for percutaneous, arterovenous access. Autologous vascular tissue remains the gold standard conduit for arterial bypass providing an effective and versatile substitute for use in these life enhancing and saving procedures. Unfortunately, it has been reported that up to 40% of patients lack adequate tissue for successful surgery. In the absence of autologous tissue, surgeons rely on alternative conduits such as prosthetic grafts. When compared to autologous tissue, the results of coronary artery bypass, lower extremity bypass, and vascular access using prosthetic grafts yield significantly lower patency rates and increased frequency of infection indicating a clear need for improved alternatives. The leading field of Tissue Engineering and Regenerative Medicine aims to provide the understanding, methods, and technology required to generate a solution to this important concern.^ The goal of this proposal was to design, create, and evaluate a tissue-engineered vascular bypass graft utilizing adipose-derived stem cells (ASC) seeded onto a natural conduit of decellularized venous tissue. Initial data demonstrated that adipose-derived stem cells can be easily obtained in large quantity and when cultured in the presence of endothelial cell growth factor and stimulated with shear stress, express markers of the endothelial phenotype. However, these cells were lost when seeded on the lumen of a decellularized venous scaffold and exposed to physiologic shear stress. To prevent detachment, the luminal surface was coated with fibronectin prior to ASC seeding. The construct was then pre-conditioned to shear stress utilizing a novel method. Linear shear conditioning and subsequent up-regulation of integrin expression resulted in ASC retention on the venous scaffold. A further limitation of the use of ASC was lack of nitric oxide synthase expression. To address this issue, ASC differentiation towards the endothelial phenotype was advanced through adenoviral transfection with endothelial nitric oxide synthase. The transfection of the stem cells elicited the production of significant concentrations of bioactive nitric oxide gas. Finally, to assess the utility of the tissue engineered graft they were implanted as aortic interposition grafts within a rabbit model. Compared with nonseeded decellularized vein grafts, tissue engineered grafts were protected against thrombosis and intimal hyperplasia. Taken together, the results of this investigation present clear success in the pursuit of our goal.

Endothelial Differentiation of Adipose-Derived Stem Cells from Elderly Patients with Cardiovascular Disease

Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50–89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI3K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α5β1 expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering

Linear Shear Conditioning Improves Vascular Graft Retention of Adipose-Derived Stem Cells by Upregulation of the α5β1 Integrin

Use of adult adipose-derived stem cells (ASCs) as endothelial cell substitutes in vascular tissue engineering is attractive because of their availability. However, when seeded onto decellularized vascular scaffolding and exposed to physiological fluid shear force, ASCs are physically separated from the graft lumen. Herein we have investigated methods of increasing initial ASC attachment using luminal precoats and a novel protocol for the gradual introduction of shear stress to optimize ASC retention. Fibronectin coating of the graft lumen increased ASC attachment by nearly sixfold compared with negative controls. Gradual introduction of near physiological fluid shear stress using a novel bioreactor whereby flow rate was increased every second at a rate of 1.5 dynes/cm2 per day resulted in complete luminal coverage compared with near complete cell loss following conventional daily abrupt increases. An upregulation of the α5β1 integrin was evinced following exposure to shear stress, which accounts for the observed increase in ASC retention on the graft lumen. These results indicated a novel method for seeding, conditioning, and retaining of adult stem cells on a decellularized vein scaffold within a high-shear stress microenvironment

Center for Research in Water Resources, The University of Texas at Austin. Proceedings, Symposium on Water Production Engineering in Texas.

University of Texas Department of Mechanical Engineering Nuclear Engineering ProgramCenter for Water and the Environmen