Building strong health and career trajectories through translational research.

Translational research (TR) is the movement of fundamental scientific discoveries into healthcare settings and population health policy, and parallels the goals of DOHaD research. Unfortunately, there is little guidance on how to become a translational researcher. To understand the opinions of DOHaD trainees towards TR, we conducted a workshop at the DOHaD World Congress 2022. We found that trainees were enthusiastic for their work to have translational impact, and that they feel that holistic, multidisciplinary solutions may lead to more generalisable research. However, there lacks support for TR career pathways, which may stall the execution of the long-term vision of the DOHaD agenda. We put forward recommendations for trainees to clarify their purpose in pursuing TR and for seeking relevant people and patronages to support their training paths. For mentors, training institutions, and scientific societies, we recommend developing TR-specific programmes, and implementing training opportunities, networking events, and funding to support these endeavours.

Reproductive age-related changes in the blood brain barrier: expression of IgG and tight junction proteins.

We previously demonstrated that there is a significantly greater transfer of intravenously-injected Evan's blue dye into the forebrain of acyclic (reproductive senescent) females compared to young adult females, indicating that blood brain barrier permeability is compromised in the reproductive senescent forebrain. The present study examined brain IgG expression and microvessel tight junction proteins to assess ovarian age-related changes in microvascular permeability, and further compared young and senescent females with age-matched males to distinguish changes attributable to age and reproductive senescence. Blood brain barrier breakdown are often associated with increased extravasation of plasma proteins and high levels of immunoglobulin G (IgG) in brain. In the present study, IgG expression was dramatically increased in the hippocampus and thalamus, but not the hypothalamus of reproductive senescent females compared to young adult females. In males, IgG expression was increased in all these regions in middle-aged animals (aged-matched to senescent females) as compared to young males (age-matched to the young adult females). Furthermore, the proportion of hippocampal microvessels with perivascular IgG immunoreactivity was significantly greater in reproductive senescent females as compared to young adult females, while middle-aged males and young adult males did not differ. The tight junctions between adjacent microvascular endothelial cells regulated by transmembrane proteins such as claudin-5 and occludin play a critical role in maintaining the blood brain barrier integrity. Increased hippocampal IgG expression in senescent females was paralleled by poor junctional localization of the tight junction protein claudin-5 in hippocampal microvessels. However, there was no difference in hippocampal claudin-5 localization between young adult and middle-aged males, indicating that dysregulation of this junctional protein was associated with ovarian aging. Parallel studies in human brain microvessels also revealed age-dependent disruption in claudin-5 distribution in post-menopausal women compared to pre-menopausal women. Collectively, these data support the hypothesis that constitutive loss of barrier integrity in the forebrain during reproductive senescence may be due, in part, to the selective loss of tight junction proteins in endothelial junctions.

Estrogen receptor-alpha overexpression suppresses 17beta-estradiol-mediated vascular endothelial growth factor expression and activation of survival kinases.

Estrogen and its receptors influence growth and differentiation by stimulating the production and secretion of growth factors. Our previous studies indicate an increased expression of estrogen receptor (ER)-alpha and decreased growth factor synthesis in the olfactory bulb of reproductive senescent female rats as compared with young animals. The present study tests the hypothesis that abnormal overexpression of ERalpha contributes to decreased growth factor synthesis. We developed the HeLa-Tet-On cell line stably transfected with ERalpha (HTERalpha) that expresses increasing amounts of ERalpha with increasing doses of doxycycline (Dox). Increasing doses of Dox had no effect on vascular endothelial growth factor (VEGF) secretion in HTERalpha cells. However, in the presence of 40 nm 17beta-estradiol, VEGF secretion increased in low-dose Dox-exposed HTERalpha cultures, which was attenuated by the ERalpha antagonist, 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]1H-pyrazole dihydrochloride. However, at high-dose Dox and, consequently, high ERalpha levels, estradiol failed to increase VEGF. In the HeLa X6 cell line in which the Tet-On construct is upstream of an unrelated gene (Pitx2A), estradiol failed to induce VEGF at any Dox dose. Furthermore, in the HTERalpha cell line, estradiol selectively down-regulates phospho-ERK2 and phospho-Akt at high ERalpha expression. This study clearly demonstrates that the dose of receptor critically mediates estradiol's ability to regulate growth factors and survival kinases. The present data also support the hypothesis that 17beta-estradiol treatment to an ERalpha overexpressing system, such as the senescent brain, could reverse the normally observed beneficial effect of estrogen.

Ethanol regulates angiogenic cytokines during neural development: evidence from an in vitro model of mitogen-withdrawal-induced cerebral cortical neuroepithelial differentiation.

BACKGROUND: Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube. METHODS: We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines. RESULTS: Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion. CONCLUSION: Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.

17beta-estradiol differentially regulates blood-brain barrier permeability in young and aging female rats.

Because both brain and its vasculature are potent targets of estrogen, age-related decline in estrogen levels or alterations in estrogen receptors may disrupt the integrity of the blood-brain barrier, leading to increased influx of toxic products. The present study tested the hypothesis that the blood-brain barrier is more permeable in reproductive senescent animals and will respond differently to estrogen replacement as compared with young adult females. Young adult and reproductive senescent rats were ovariectomized and replaced with an estrogen or control pellet. We found a 2- to 4-fold increase in extravasation of dye in the olfactory bulb and hippocampus of reproductive senescent females compared with young adults. Furthermore, estrogen significantly reduced dye extravasation in both olfactory bulb and hippocampus in young adults compared with age-matched counterparts that received a control pellet. However, estrogen replacement increased dye extravasation in the hippocampus of reproductive senescent females compared with age-matched control-pellet replaced animals, whereas dye extravasation was unchanged by estrogen in the olfactory bulb of senescent females. There were no age- and estrogen-related differences in dye accumulation in the pituitary gland, which is a circumventricular organ. These results support the hypothesis that the hormonal decline that marks reproductive senescence leads to increased permeability of the blood-brain barrier, which is further exacerbated by estrogen treatment in specific regions.