Impact of Cyclic Strain on Elastin Synthesis in a 3D Human Myometrial Culture Model.

The synthesis and assembly of mature, organized elastic fibers remains a limitation to the clinical use of many engineered tissue replacements. There is a critical need for a more in-depth understanding of elastogenesis regulation for the advancement of methods to induce and guide production of elastic matrix structures in engineered tissues that meet the structural and functional requirements of native tissue. The dramatic increase in elastic fibers through normal pregnancy has led us to explore the potential role of mechanical stretch in combination with pregnancy levels of the steroid hormones 17ß-estradiol and progesterone on elastic fiber production by human uterine myometrial smooth muscle cells in a three-dimensional (3D) culture model. Opposed to a single strain regimen, we sought to better understand how the amplitude and frequency parameters of cyclic strain influence elastic fiber production in these myometrial tissue constructs (MTC). Mechanical stretch was applied to MTC at a range of strain amplitudes (5%, 10%, and 15% at 0.5 Hz frequency) and frequencies (0.1 Hz, 0.5 Hz, 1 Hz, and constant 0 Hz at 10% amplitude), with and without pregnancy-level hormones, for 6 days. MTC were assessed for cell proliferation, matrix elastin protein content, and expression of the main elastic fiber genes, tropoelastin (ELN) and fibrillin-1 (FBN1). Significant increases in elastin protein and ELN and FBN1 mRNA were produced from samples subjected to a 0.5 Hz, 10% strain regimen, as well as samples stretched at higher amplitude (15%, 0.5 Hz) and higher frequency (1 Hz, 10%); however, no significant effects because of third-trimester mimetic hormone treatment were determined. These results establish that a minimum level of strain is required to stimulate the synthesis of elastic fiber components in our culture model and show this response can be similarly enhanced by increasing either the amplitude or frequency parameter of applied strain. Further, our results demonstrate strain alone is sufficient to stimulate elastic fiber production and suggest hormones may not be a significant factor in regulating elastin synthesis. This 3D culture model will provide a useful tool to further investigate mechanisms underlying pregnancy-induced de novo elastic fiber synthesis and assembly by uterine smooth muscle cells.

Uterine fibroid cell cytoskeletal organization is affected by altered G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase signaling.

OBJECTIVE: To determine whether cyclic strain affects fibroid cell cytoskeletal organization, proliferation, and collagen synthesis differently than myometrial cells. DESIGN: A basic science study using primary cultures of patient-matched myometrial and fibroid cells. SETTING: Academic laboratory. PATIENT(S): Premenopausal women undergoing myomectomy or hysterectomy for the treatment of symptomatic uterine fibroids. INTERVENTION(S): Application of uniaxial strain patterns mimicking periovulation, menses, or dysmenorrhea using the Flexcell tension system or static control. Secondarily, inhibition of G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase. MAIN OUTCOME MEASURE(S): Cell alignment, cell number, and collagen content. RESULT(S): Menses-strained cells demonstrated the most variation in cell alignment, cell proliferation, and procollagen content between myometrial and fibroid cells. Procollagen content decreased in myometrial cells with increasing strain amplitude and decreasing frequency. G protein-coupled estrogen receptor-1 inhibition decreases cellular alignment in the presence of strain. CONCLUSION(S): Mechanotransduction affecting cytoskeletal arrangement through the G protein-coupled estrogen receptor-1-phosphatidylinositol 3-kinase pathway is altered in fibroid cells. These results highlight the importance of incorporating mechanical stimulation into the in vitro study of fibroid pathology.

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 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-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.