Mammary Microvessels are Sensitive to Menstrual Cycle Sex Hormones.

The mammary gland is a highly vascularized organ influenced by sex hormones including estrogen (E2) and progesterone (P4). Beyond whole-organism studies in rodents or cell monocultures, hormonal effects on the breast microvasculature remain largely understudied. Recent methods to generate 3D microvessels on-chip have enabled direct observation of complex vascular processes; however, these models often use non-tissue-specific cell types, such as human umbilical vein endothelial cells (HUVECs) and fibroblasts from various sources. Here, novel mammary-specific microvessels are generated by coculturing primary breast endothelial cells and fibroblasts under optimized culture conditions. These microvessels are mechanosensitive (to interstitial flow) and require endothelial-stromal interactions to develop fully perfusable vessels. These mammary-specific microvessels are also responsive to exogenous stimulation by sex hormones. When treated with combined E2 and P4, corresponding to the four phases of the menstrual cycle (period, follicular, ovular, and luteal), vascular remodeling and barrier function are altered in a phase-dependent manner. The presence of high E2 (ovulation) promotes vascular growth and remodeling, corresponding to high depletion of proangiogenic factors, whereas high P4 concentrations (luteal) promote vascular regression. The effects of combined E2 and P4 hormones are not only dose-dependent but also tissue-specific, as are shown by similarly treating non-tissue-specific HUVEC microvessels.

Classical and Non-classical Fibrosis Phenotypes Are Revealed by Lung and Cardiac Like Microvascular Tissues On-Chip.

Fibrosis, a hallmark of many cardiac and pulmonary diseases, is characterized by excess deposition of extracellular matrix proteins and increased tissue stiffness. This serious pathologic condition is thought to stem majorly from local stromal cell activation. Most studies have focused on the role of fibroblasts; however, the endothelium has been implicated in fibrosis through direct and indirect contributions. Here, we present a 3D vascular model to investigate vessel-stroma crosstalk in normal conditions and following induced fibrosis. Human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are co-cultured with (and without) primary human cardiac and lung fibroblasts (LFs) in a microfluidic device to generate perfusable microvasculature in cardiac- and pulmonary-like microenvironments. Endothelial barrier function, vascular morphology, and matrix properties (stiffness and diffusivity) are differentially impacted by the presence of stromal cells. These vessels (with and without stromal cells) express inflammatory cytokines, which could induce a wound-healing state. Further treatment with transforming growth factor-ß (TGF-ß) induced varied fibrotic phenotypes on-chip, with LFs resulting in increased stiffness, lower MMP activity, and increased smooth muscle actin expression. Taken together, our work demonstrates the strong impact of stromal-endothelial interactions on vessel formation and extravascular matrix regulation. The role of TGF-ß is shown to affect co-cultured microvessels differentially and has a severe negative impact on the endothelium without stromal cell support. Our human 3D in vitro model has the potential to examine anti-fibrotic therapies on patient-specific hiPSCs in the future.

Microenvironment stiffness requires decellularized cardiac extracellular matrix to promote heart regeneration in the neonatal mouse heart.

The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. STATEMENT OF SIGNIFICANCE: With the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.