Browsing by Subject "Proliferation"

The steroid hormone progesterone has complex physiologic effects. In typical development and function, cells respond to progesterone in a dose- and tissue-specific manner. Despite the wide range of physiologic concentrations, canonical effects of progesterone have been characterized in the context of a high physiologic dose (10nM+), relevant during uterine cycling. This narrow focus has produced a gap in knowledge, particularly as it relates to the effects of post-menopausal low concentration progestins (0.1-0.3nM). Given that healthy tissues possess regulatory mechanisms to sense and respond to progesterone in a non-linear dose-specific manner, we hypothesized that breast malignancies would also display discontinuous dose-specific dynamic responses. Our results show that treatment with low dose progestins (0.1-0.3nM) drives proliferation in T47D human breast cancer cells, while high dose progestins (10nM+) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 (cyclin D1) and SGK1 (serum and glucocorticoid related kinase 1), which are required for initiation of the downstream molecular cascade including phosphorylation of retinoblastoma protein (Rb) and expression E2F1. Expression of CCND1 and SGK1 mRNA are proximal responses to low dose progestin treatment, but transcriptional activation is not mediated by canonical progesterone receptor (PR) activity. Future work is needed to identify previously unexplored mechanisms of PR action in the context of low dose progestin treatments. In summary, these results challenge the assumption of dose response linearity to progestins and show unique functional and molecular effects of low dose progestin treatment. Of potential concern, our findings suggest that certain breast cancers, especially those expressing high levels of PR, may be accelerated by normal post-menopausal circulating concentrations of progestins (0.1-0.3nM). However, these findings also offer a sound rationale for the clinical therapeutic use of high dose progestins for patients with PR+ breast cancer.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte proliferation have been identified as potential candidates for functional heart repair following myocardial infarction (MI). Mature adult CMs are highly resistant to mitogenic stimuli, with many mitogens only slightly raising adult CM proliferation rates in vivo. Conversely, immature neonatal CMs are more proliferative, permitting early neonatal rodent hearts to partially regenerate following injury. It is therefore of importance to improve our understanding of the mechanisms behind CM maturation and cell cycle regulation, which could be aided by the development and utilization of more relevant in vitro systems to study CM proliferation. Of particular interest, CMs in vivo undergo polyploidization, an increase in cellular DNA content without division, during postnatal maturation. This has been shown to correlate with a decline in their proliferative capacity and an increase in cell size and maturation. The goals of this dissertation have been to utilize in vitro cardiac tissue models to: 1) identify potent mitogenic stimuli capable of inducing CM proliferation, 2) describe the underlying mechanisms behind how this stimulus promotes CM proliferation, and 3) investigate the link between CM proliferation, maturation, and polyploidy.

First, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues (ECTs) generated from NRVMs, a constitutively active mutant form of the membrane receptor of mitogen activated protein kinase (MAPK) signaling pathway, human Erbb2 (cahErbb2), was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM cell cycle entry and mitosis, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of MEK or ERK signaling. These results suggested transient activation of Erbb2/ERK axis in cardiomyocytes as a potential strategy for regenerative heart repair.

We then explored whether activating ERK via a constitutively active mutant of a more downstream effector of MAPK pathway, BRAF (BRAF-V600E, caBRAF), can induce pro-proliferative effects in NRVM ECTs. Sustained CM-specific caBRAF expression induced chronic ERK activation, significant tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. CaBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a pro-migratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid, precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase was sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.

In the aforementioned in vitro studies, we relied on use of the more mature and less-proliferative NRVM vs. hiPSC ECT model system for identifying cardiac mitogens. Unlike predominantly polyploid adult CMs, immature hiPSC-CMs are primarily mononuclear and diploid, which makes them permissive to proliferation and therefore potentially less suitable for mitogen screens. We therefore studied whether induction of polyploidy alone in hiPSC-CMs would be sufficient to promote their maturation in vitro. Lentiviral overexpression of dominant negative ECT2 (dnECT2), an important cytokinesis gene, was sufficient to induce polyploidy in hiPSC-CMs and NRVMs. Upon screening of small molecules for polyploidy induction, we identified transient small molecule inhibition of AURKB as an even more effective approach than dnECT2 expression in producing polyploid hiPSC-CMs. Compared to diploid hiPSC-CMs, small molecule-induced polyploid hiPSC-CMs exhibited increased cell size, mitochondrial density, and rates of transcription and translation. Polyploid hiPSC-CMs were also less proliferative than diploid hiPSC-CMs, suggesting an improved potential for use in screening for cardiac mitogens in vitro.

In summary, this dissertation describes the effects of MAPK activation on CMs and shows a causative relationship between polyploidy and some aspects of CM maturation. We produced multiple RNAseq datasets of NRVM ECTs overexpressing different constitutively active MAPK genes, which could be of interest to research beyond cardiac biology. Further investigation of MAPK pathway activation in CMs may show that some downstream ERK effectors mediate the pro-proliferative effects in CMs, while others coordinate CM functional changes or changes in cell hypertrophy and maturation. Dissecting these mechanisms could enable the design of more targeted MAPK pathway modifiers with utility in cardiac regenerative biology and anticancer therapeutics.

The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin α5β1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.