A study of strength and vasoactivity in a tissue engineered vascular media

To be successful a tissue engineered small diameter blood vessel must be non-immunogenic, non-thrombogenic, have mechanical properties similar to native vessel and be vasoactive. The vascular media is responsible for the mechanical properties and the vasoactivity of the vessel. The collagen hydrogel approach has been long used and has many advantages, but has not yet achieved the mechanical integrity needed for implantation. No collagen-based tissue engineered vascular media has been shown to be vasoactive using culture techniques required to achieve the cell numbers needed to make a vascular graft. To study collagen synthesis, two model systems were used. Cells were seeded on top of an adsorbed collagen I or fibrin layer. Alternatively the cells were encapsulated in a collagen or fibrin hydrogel. Collagen I, decorin and biglycan synthesis was affected by both matrix type and presentation. After two weeks in culture the smooth muscle cells produce more type I collagen in the collagen based hydrogels then in the fibrin hydrogels and was used for further studies. The collagen based tissue engineered vascular media produced a consistent vasoactive response between two and eight weeks of culture. The smooth muscle cells have functional endothelin, kinin, adrenergic, serotonergic and purinergic receptors. The application of cyclic strain improves both the tissue strength and the contractile response. Use of transforming growth factor-β improved tissue strength, but reduced the contractile response. Transforming growth factor- β actually promoted a more contractile cell phenotype, but a stronger contractile force was required to overcome the thick compact collagen hydrogel and elicit a measurable contraction. This work adds to what is known about collagen-based tissue engineered vascular medias by identifying means of improving not only strength but vasoactivity. The trade-offs found between these two important characteristics are relevant to all tissue engineered medias.Ph.D.Committee Chair: Nerem, Robert M.; Committee Member: Gleason, Rudolf L.; Committee Member: Taylor, W. Robert; Committee Member: Vito, Raymond P.; Committee Member: Wang, Yadon

Low-risk susceptibility alleles in 40 human breast cancer cell lines

Background: Low-risk breast cancer susceptibility alleles or SNPs confer only modest breast cancer risks ranging from just over 1.0 to 1.3 fold. Yet, they are common among most populations and therefore are involved in the development of essentially all breast cancers. The mechanism by which the low-risk SNPs confer breast cancer risks is currently unclear. The breast cancer association consortium BCAC has hypothesized that the low-risk SNPs modulate expression levels of nearby located genes. Methods: Genotypes of five low-risk SNPs were determined for 40 human breast cancer cell lines, by direct sequencing of PCR-amplified genomic templates. We have analyzed expression of the four genes that are located nearby the low-risk SNPs, by using real-time RT-PCR and Human Exon microarrays. Results: The SNP genotypes and additional phenotypic data on the breast cancer cell lines are presented. We did not detect any effect of the SNP genotypes on expression levels of the nearby-located genes MAP3K1, FGFR2, TNRC9 and LSP1. Conclusion: The SNP genotypes provide a base line for functional studies in a well-characterized cohort of 40 human breast cancer cell lines. Our expression analyses suggest that a putative disease mechanism through gene expression modulation is not operative in breast cancer cell lines

Cyclic Adenosine Monophosphate Eliminates Sex Differences in Estradiol-Induced Elastin Production from Engineered Dermal Substitutes

Lack of adult cells’ ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17β-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-β levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17β-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17β-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17β-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes

Genome-wide association study identifies five new breast cancer susceptibility loci

Breast cancer is the most common cancer in women in developed countries. To identify common breast cancer susceptibility alleles, we conducted a genome-wide association study in which 582,886 SNPs were genotyped in 3,659 cases with a family history of the disease and 4,897 controls. Promising associations were evaluated in a second stage, comprising 12,576 cases and 12,223 controls. We identified five new susceptibility loci, on chromosomes 9, 10 and 11 (P = 4.6 × 10?7 to P = 3.2 × 10?15). We also identified SNPs in the 6q25.1 (rs3757318, P = 2.9 × 10?6), 8q24 (rs1562430, P = 5.8 × 10?7) and LSP1 (rs909116, P = 7.3 × 10?7) regions that showed more significant association with risk than those reported previously. Previously identified breast cancer susceptibility loci were also found to show larger effect sizes in this study of familial breast cancer cases than in previous population-based studies, consistent with polygenic susceptibility to the diseas

Tissue engineering of a collagen-based vascular media: Demonstration of functionality

The property of vasoactivity is important for both resistance vessels and larger arteries. Evaluation of smooth muscle cell phenotype is often done in place of functional testing in engineered tissues, assuming a direct correlation between cell phenotype and tissue contractile force. In this study we look at a large panel of vasoactive agents to determine the functionality of our collagen-based tissue. The engineered vascular media elicited a measurable change in force in response to seven of the nine agents used. As part of this characterization, TGFβ1 and TNFα were used to promote a more contractile and synthetic cell phenotype respectively. Both smooth muscle a-actin and vasoconstriction were evaluated in ring sections. Due to large differences in cell-compaction and cell distribution in the tissues, no correlation was found between a-actin expression and contractile strength. This highlights the need for functional testing of engineered tissue and the importance of cell-matrix interactions in vasoactivity

Gene expression profiling assigns CHEK2 1100delC breast cancers to the luminal intrinsic subtypes

CHEK2 1100delC is a moderate-risk cancer susceptibility allele that confers a high breast cancer risk in a polygenic setting. Gene expression profiling of CHEK2 1100delC breast cancers may reveal clues to the nature of the polygenic CHEK2 model and its genes involved. Here, we report global gene expression profiles of a cohort of 155 familial breast cancers, including 26 CHEK2 1100delC mutant tumors. In line with previous work, all CHEK2 1100delC mutant tumors clustered among the hormone receptor-positive breast cancers. In the hormone receptor-positive subset, a 40-gene CHEK2 signature was subsequently defined that significantly associated with CHEK2 1100delC breast cancers. The identification of a CHEK2 gene signature implies an unexpected biological homogeneity among the CHEK2 1100delC breast cancers. In addition, all 26 CHEK2 1100delC tumors classified as luminal intrinsic subtype breast cancers, with 8 luminal A and 18 luminal B tumors. This biological make-up of CHEK2 1100delC breast cancers suggests that a relatively limited number of additional susceptibility alleles are involved in the polygenic CHEK2 model. Identification of these as-yet-unknown susceptibility alleles should be aided by clues from the 40-gene CHEK2 signatur