The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5 dKO ) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5 dKO embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa KO ) thyroids, Smad1/5 dKO thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa KO and Smad1/5 dKO thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5 dKO and Vegfa KO thyroids. Laminin α1, β1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane.

Background Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used to manage cardiopulmonary failure refractory to conventional medical and surgical therapies. Despite advances in ECMO equipment, bleeding and thrombosis remain significant complications. While the flow rate for ECMO support is well recognized, less is known about the minimum-rate requirements and haemostasis. We investigated the relationship between different ECMO flow rates, and their effect on haemolysis and coagulation. Methods Ten ex-vivo ECMO circuits were tested using donated, < 24-h-old human whole blood, with two flow rates: high-flow at 4 L/min (normal adult cardiac output; n = 5) and low-flow at 1.5 L/min (weaning; n = 5). Serial blood samples were taken for analysis of haemolysis, von Willebrand factor (vWF) multimers by immunoblotting, rotational thromboelastometry, platelet aggregometry, flow cytometry and routine coagulation laboratory tests. Results Low-flow rates increased haemolysis after 2 h ( p = 0.02), 4 h ( p = 0.02) and 6 h ( p = 0.02) and the loss of high-molecular-weight vWF multimers ( p = 0.01), while reducing ristocetin-induced platelet aggregation ( p = 0.0002). Additionally, clot formation times were prolonged ( p = 0.006), with a corresponding decrease in maximum clot firmness ( p = 0.006). Conclusions In an ex-vivo model of ECMO, low-flow rate (1.5 L/min) altered haemostatic parameters compared to high-flow (4 L/min). Observed differences in haemolysis, ristocetin-induced platelet aggregation, high-molecular-weight vWF multimers and clot formation time suggest an increased risk of bleeding complications. Since patients are often on ECMO for protracted periods, extended-duration studies are required to characterise long-term ECMO-induced haemostatic changes.

The acute respiratory distress syndrome (ARDS) describes a heterogenous population of patients with acute severe respiratory failure. However, contemporary advances have begun to identify distinct sub‐phenotypes that exist within its broader envelope. These sub‐phenotypes have varied outcomes and respond differently to several previously studied interventions. A more precise understanding of their pathobiology and an ability to prospectively identify them, may allow for the development of precision therapies in ARDS. Historically, animal models have played a key role in translational research, although few studies have so far assessed either the ability of animal models to replicate these sub‐phenotypes or investigated the presence of sub‐phenotypes within animal models. Here, in three ovine models of ARDS, using combinations of oleic acid and intravenous, or intratracheal lipopolysaccharide, we investigated the presence of sub‐phenotypes which qualitatively resemble those found in clinical cohorts. Principal Component Analysis and partitional clustering identified two clusters, differentiated by markers of shock, inflammation, and lung injury. This study provides a first exploration of ARDS phenotypes in preclinical models and suggests a methodology for investigating this phenomenon in future studies.