Jatin Patel

Thyroid hormones and fetal neurological development

The development of fetal thyroid function is dependent on the embryogenesis, differentiation, and maturation of the thyroid gland. This is coupled with evolution of the hypothalamic-pituitary-thyroid axis and thyroid hormone metabolism, resulting in the regulation of thyroid hormone action, production, and secretion. Throughout gestation there is a steady supply of maternal thyroxine (T(4)) which has been observed in embryonic circulation as early as 4 weeks post-implantation. This is essential for normal early fetal neurogenesis. Triiodothyronine concentrations remain very low during gestation due to metabolism via placental and fetal deiodinase type 3. T(4) concentrations are highly regulated to maintain low concentrations, essential for protecting the fetus and reaching key neurological sites such as the cerebral cortex at specific developmental stages. There are many known cell membrane thyroid hormone transporters in fetal brain that play an essential role in regulating thyroid hormone concentrations in key structures. They also provide the route for intracellular thyroid hormone interaction with associated thyroid hormone receptors, which activate their action. There is a growing body of experimental evidence from rats and humans to suggest that even mild maternal hypothyroxinemia may lead to abnormalities in fetal neurological development. Our review will focus on the ontogeny of thyroid hormone in fetal development, with a focus on cell membrane transporters and TR action in the brain.

Regulation of hypoxia inducible factors (HIF) in hypoxia and normoxia during placental development.

During the first trimester of pregnancy the human placenta develops in an hypoxic environment caused by the occlusion of uterine spiral arterioles by extravillous trophoblasts (EVT). This period of low oxygen tension is crucial for successful pregnancy. In low oxygen environments, Hypoxia Inducible Factors (HIF) are the main regulators in the transcription of a number of genes. Target genes can induce anaerobic processes, reducing oxygen consumption, or promote angiogenesis, which establishes and enhances the vascular environment. The HIFs can function throughout all stages of placental differentiation and growth both in normal and pathological pregnancies (compromised by hypoxia/ischemia). Interestingly, HIFs respond to a multitude of changes during pregnancy, including 1) low oxygen, 2) renin-angiotensin system (RAS), 3) cytokines, and 4) growth factors, all of which regulate placental function. This review explores oxygen-dependent and oxygen-independent regulation and the role of HIF in placental development and differentiation.

Delivery of maternal thyroid hormones to the fetus

Thyroid hormones (THs) play an essential role in ensuring normal fetal development, particularly that of the central nervous system. Before 16 weeks gestation, the fetus relies solely on transplacental delivery of maternal T(4), and clinical studies suggest that even mild maternal thyroid hormone deficiency adversely affects the intellectual function of offspring. Maternofetal TH transfer is regulated by trophoblast cell membrane transporters, which mediate influx and efflux of THs, placental deiodinases D3 and D2, which control intraplacental TH levels, and TH-binding proteins (transthyretin), which provide transport roles in the placenta. This review discusses new information about mechanisms of transplacental delivery of T(4) to the fetus, providing insight into complex processes that are vitally important for normal fetal development.

Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells From Human Term Placenta

The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony‐forming cells (ECFCs) from human term placenta and to compare them to the well‐established donor‐matched umbilical cord blood (UCB)‐derived ECFCs. A sorting strategy was devised to enrich for CD45−CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL‐ECFCs) upon culture. UCB‐ECFCs were derived using a well‐described assay. PL‐ECFCs were fetal in origin and expressed the same cell surface markers as UCB‐ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL‐ECFCs and UCB‐ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL‐ECFCs and UCB‐ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL‐ECFCs and UCB‐ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL‐ECFCs have significant bio‐banking and clinical translatability potential.

Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling

Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34− ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self‐renew in stringent single‐cell cultures and gave rise to both CD34 + and CD34− cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin‐dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ‐secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self‐renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self‐renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self‐renewal potential. Stem Cells 2016;34:902–912

Expression and uptake of the thyroxine-binding protein transthyretin is regulated by oxygen in primary trophoblast placental cells

Transplacental delivery of maternal thyroid hormones to the fetus, in particular thyroxine (T₄), is critical in ensuring normal fetal neurological development. The fetus relies on maternal T₄ till around 16 weeks gestation, but mechanisms of placental T₄ transport are not yet fully elucidated. Placenta produces, secretes and takes up the thyroid hormone-binding protein transthyretin (TTR). Many placental genes are regulated by oxygen levels, which are relatively low (1%) in the early first trimester, rising to 3% in the mid first trimester and 8% in the early second trimester and thereafter. We examined the expression and uptake of TTR in isolated primary human placental cytotrophoblast cells cultured under different oxygen concentrations (1, 3, 8, 21% O₂ and 200 μM desferrioxamine (DFO)) for 24 h. We observed sevenfold higher expression of TTR mRNA and protein levels at 1% O₂ than at 8 and 21% O₂. Significant increases were observed after culture at 3% O₂ and following DFO treatment. We observed significantly higher uptake of ¹²⁵I-TTR and Alexa-594-TTR when cells were cultured at 1 and 3% O₂ and in the presence of 200 μM DFO than at 8 and 21% O₂. When JEG-3 choriocarcinoma cells were transfected with TTR promoter reporter constructs, increased luciferase activity was measured in cells cultured at 1 and 3% O₂ in comparison to 8 and 21% O₂. We conclude that placental TTR expression and uptake is increased by the relative hypoxia observed in the first trimester of pregnancy, a time when materno-fetal T₄ transfer is the sole source of fetal T₄.

Ontogenic changes in human placental sodium iodide symporter expression

The human fetus requires a maternal supply of iodide to synthesize thyroid hormone from 16 weeks gestation. Placental iodide transport is regulated by the sodium iodide symporter (NIS). We studied the ontogeny of NIS in placentas from surgically terminated pregnancies and from normal term pregnancies. NIS mRNA was low at 6 weeks gestation and peaked at 12 weeks gestation. Placental NIS protein levels are significantly correlated with gestational age during early pregnancy and increase with increased placental vascularization. This would lead to increased iodide supply to meet increased fetal requirements for thyroid hormone synthesis as the pregnancy progresses.

Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process

Background: During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. Methods: Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45– cells. Results: Lineage tracing by using Cdh5cre ERt2/Rosa-YFP reporter strategy demonstrated that the CD31–/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr, and Pdfgr&agr;. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf, and Notch. The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on Sox18CreERt2/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Conclusions: Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.

Oxygen concentration regulates expression and uptake of transthyretin, a thyroxine binding protein, in JEG-3 choriocarcinoma cells

Maternal thyroid hormone is provided to the fetus before the onset of fetal thyroid function (at about 16 weeks) and is essential for normal neurologic development. Mechanisms of transport are uncertain but transthyretin (TTR), a thyroxine binding protein produced by the placenta may be involved. Placental oxygen concentrations in early pregnancy are low, about 1% early in the first trimester and rising to 8% over the next 12 weeks. This study investigated the regulation of TTR expression, secretion and uptake in JEG-3 placental cells cultured at different oxygen concentrations. TTR mRNA and protein expression and (125)I-TTR and Alexa-Fluor594-TTR uptake were significantly higher in cells cultured at 1% and 3% O(2), than at 8% O(2). This suggests that increased carrier mediated T(4) transport by placental TTR may be induced by the low oxygen environment of early pregnancy, a time when the fetus has its highest requirement for transport of maternal T(4).

A Regenerative Antioxidant Protocol of Vitamin E and α-Lipoic Acid Ameliorates Cardiovascular and Metabolic Changes in Fructose-Fed Rats

Type 2 diabetes is a major cause of cardiovascular disease. We have determined whether the metabolic and cardiovascular changes induced by a diet high in fructose in young adult male Wistar rats could be prevented or reversed by chronic intervention with natural antioxidants. We administered a regenerative antioxidant protocol using two natural compounds: α-lipoic acid together with vitamin E (α-tocopherol alone or a tocotrienol-rich fraction), given as either a prevention or reversal protocol in the food. These rats developed glucose intolerance, hypertension, and increased collagen deposition in the heart together with an increased ventricular stiffness. Treatment with a fixed combination of vitamin E (either α-tocopherol or tocotrienol-rich fraction, 0.84 g/kg food) and α-lipoic acid (1.6 g/kg food) normalized glucose tolerance, blood pressure, cardiac collagen deposition, and ventricular stiffness in both prevention and reversal protocols in these fructose-fed rats. These results suggest that adequate antioxidant therapy can both prevent and reverse the metabolic and cardiovascular damage in type 2 diabetes.