Our laboratory previously demonstrated that cytotrophoblasts and syncytiotrophoblasts in human placental tissue contain hCG/LH receptors. From this finding, we postulated that one role of hCG might be to promote the differentiation of cytotrophoblasts into syncytiotrophoblasts. To test this postulate, cytotrophoblasts were isolated from human term pregnancy placentas and cultured with and without increasing concentrations of highly purified hCG. The results showed that hCG had a biphasic effect on 1) the aggregation of cells without intervening plasma membranes; 2) the expression of cadherin, a cell adhesion receptor that facilitates cellular aggregation; 3) the expression of hCG/LH receptor gene; and 4) the expression of three different hormonal markers of differentiation. The hCG effects were time and dose dependent and hormone specific. The addition of excess polyclonal hCG antibody, but not normal rabbit serum or nonspecific antirabbit immunoglobulin G, decreased basal responses as well as those to exogenous hCG. The polyclonal hCG/LH receptor antibody increased differentiation and dramatically stimulated hCG secretion in the presence or absence of exogenous hCG. (Bu)2cAMP mimicked the actions of hCG. H-89, a protein kinase-A inhibitor, decreased basal as well as exogenous hCG responses. Calphostin, a protein kinase-C inhibitor and lavendustin, a tyrosine kinase inhibitor, on the other hand, had no effect. In summary, it is novel that hCG made in human placenta can regulate the differentiation of cytotrophoblasts, which make little hCG, into syncytiotrophoblasts, which make considerable amounts of hCG.

Aims/hypothesis Diabetic retinopathy is a common microvascular complication of diabetes mellitus and is initiated by inflammation and apoptosis-associated retinal endothelial cell damage. Prostaglandin E 2 (PGE 2 ) has emerged as a critical regulator of these biological processes. We hypothesised that modulating PGE 2 and its E-prostanoid receptor (EP 2 R) would prevent diabetes mellitus-induced inflammation and microvascular dysfunction. Methods In a streptozotocin (STZ)-induced rat model of diabetes, rats received intravitreal injection of PGE 2 , butaprost (a PGE 2 / EP 2 R agonist) or AH6809 (an EP 2 R antagonist). Retinal histology, optical coherence tomography, ultrastructure of the retinal vascular and biochemical markers were assessed. Results Intravitreal injection of PGE 2 and butaprost significantly accelerated retinal vascular leakage, leucostasis and endothelial cell apoptosis in STZ-induced diabetic rats. This response was ameliorated in diabetic rats pre-treated with AH6809. In addition, pre-treatment of human retinal microvascular endothelial cells with AH6809 attenuated PGE 2 -and butaprost-induced activation of caspase 1, activation of the complex containing nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment domain (ASC), and activation of the EP 2 R-coupled cAMP/protein kinase A/cAMP response element-binding protein signalling pathway. Conclusions/interpretation The PGE 2 /EP 2 R signalling pathway is involved in STZ-induced diabetic retinopathy and could be considered as a potential target for diabetic retinopathy prevention and treatment.

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One of the major pathological features of early stage diabetic nephropathy (DN) is tubulointerstitial fibrosis on renal proximal tubular cells (RPTCs), which eventually progresses to fibrosis without proper renal function. However, little is known about the factors regulating these processes. Here, we show that mTOR complex 1 and 2 specifically regulate metabolic control processes of type-1 diabetes in kidney. Using conditional mouse genetic approaches to disable subunits of mTORC1 (Rictor) and mTORC2 (Raptor), respectively or both in PRTCs (Pax8cre), we showed that mice lacking Rictor or Raptor/Rictor develop improved renal pathologies in STZ induced type-1 diabetic model, including low levels of proteinuria, reduced matrix protein accumulation/matrix expansion and tubulointerstitial inflammation. However, the mice lacking Raptor alone were still largely susceptible to STZ toxicity. Meanwhile, Nox4 and oxidative stress levels were significantly reduced in RictorcKO diabetic animal, but to a lesser extent in RaptorcKO diabetic mice. Transcriptome analysis of kidney cortex identified unique pathways that play roles in mTORC/Nox4 pathway during type-1 diabetic pathogenesis, including autophagy, metabolism of lipids and fatty acid regulation. These pathways were largely unchanged in RictorcKO mice when they were exposed to STZ induction. Functionally, losing mTORC2 complex resulted in reduced cellular death and oxidative stress levels, and a profound perturbation of the endocytic machinery. Finally, we found that the treatment with pp242, the mTORC1/2 dual inhibitor, greatly ameliorated the renal pathology in OVE26 mice, a spontaneous type-1 diabetic model. Collectively, our findings highlight a novel mTOR-dependent regulatory network for maintaining metabolic homeostasis in RPTCs during the pathogenesis of DN, and further validates the relevance of simultaneously targeting Nox4 and mTORC2 as a potential early therapeutic option for treating DN. Disclosure C. Chang: None. Z. Zhang: None. K. Xu: None. M. Bhat: None. Q. Shi: None. Funding JDRF (1-FAC-2019-858-A-N)