Search

Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangiogenesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2-neuropilin 1 (VEGFR2-NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endometrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angiogenesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/ postpartum repair. [ABSTRACT FROM AUTHOR]

R5339 --> 731.8 --> Background: Small intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5‐trisphosphate (IP3) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown. Aim: To examine duodenal expression and function of IRBIT in regulating bicarbonate transport. Methods: Duodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical‐in or flipped apical‐out enteroids. mRNA expression was evaluated using qPCR and re‐analysis of a duodenal sc‐RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back‐titration. Results: IRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5‐fold higher expression than Calu‐3 airway submucosal gland cells or HEK293 kidney‐derived cells (n=1 each). Analysis of duodenal sc‐RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt‐villus axis, primarily localizing in E‐cadherin positive epithelial cells, but also present in the lamina propria. In patient‐derived duodenal enteroids, IRBIT was expressed in both crypt‐like (undifferentiated) and villus‐like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc‐RNAseq analysis, AHCYL1 and SLC26A3 were co‐expressed in 38% of all villus cells, compared to only 5% co‐expressing AHCYL1 and SLC26A6. CFTR‐independent duodenal bicarbonate secretion stimulated by linaclotide (10‐7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10‐6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh‐A250, 10‐5 M; 68±8%, n=10). Linaclotide stimulation of apical‐out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking. Summary and Conclusions: IRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition. [ABSTRACT FROM AUTHOR]

R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50:3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy. [ABSTRACT FROM AUTHOR]