Neuregulin 1 signaling attenuates tumor necrosis factor-α induced female rat luteal cell death.

The corpus luteum (CL) is a transient ovarian endocrine structure that maintains pregnancy in primates during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL life span, growth, and differentiation are tightly regulated by survival and cell death signals through luteotrophic and luteolytic factors, including the EGF-like factor family. Neuregulin 1 (NRG1), a member of the EGF family, mediates its effect through ErbB2/3 receptors. However, the functional role of NRG1 in luteal cells (LCs) is unknown. Thus, this study investigated the role of NRG1 and its molecular mechanism of action in rat LC. Our experimental results suggest a strong positive correlation between steroidogenic acute regulatory protein (StAR) and NRG1 expression in mid-CL and serum P4 and estrogen (E2) production. In contrast, there was a decrease in StAR and NRG1 expression and P4 and E2 production with an increase in TNFα expression in regressing CL. Further in vitro studies in LCs showed that the knockdown of endogenous Nrg1 promoted the expression of proinflammatory and proapoptotic factors and decreased prosurvival factors expression. Subsequently, treatment with exogenous TNFα under these experimental conditions profoundly elevated proinflammatory and proapoptotic factors. Further analysis demonstrated that the phosphorylation status of ErbB2/3, PI3K, Akt, and ErK1/2 was significantly inhibited under these experimental conditions, whereas the treatment of TNFα further inhibited the phosphorylation of ErbB2/3, PI3K, Akt, and ErK1/2. Collectively, these studies provide new insights into the NRG1-mediated immunomodulatory and prosurvival role in LCs, which may maintain the function of CL.

Exosomes from adipose-derived stem cells alleviate myocardial infarction via microRNA-31/FIH1/HIF-1α pathway.

Our previous study has revealed that exosomes from adipose-derived stem cells (ASCs) promote angiogenesis in subcutaneously transplanted gels by delivery of microRNA-31 (miR-31) which targets factor inhibiting hypoxia-inducible factor-1 (FIH1) in recipient cells. Here we hypothesized that ASC exosomes alleviate ischemic diseases through miR-31/FIH1/hypoxia-inducible factor-1α (HIF-1α) signaling pathway. Exosomes from ASCs were characterized with nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting analysis for exosomal markers. Results from immunoblotting and laser imaging of ischemic mouse hindlimb revealed that miR-31 enriched ASC exosomes inhibited FIH1 expression and enhanced the blood perfusion, respectively. These effects were impaired when using miR-31-depleted exosomes. Immunohistochemistry analysis showed that administration of exosomes resulted in a higher arteriole density and larger CD31+ area in ischemic hindlimb than miR-31-delpleted exosomes. Similarly, knockdown of miR-31 in exosomes reduced the effects of the exosomes on increasing ventricular fraction shortening and CD31+ area, and on decreasing infarct size. Exosomes promoted endothelial cell migration and tube formation. These changes were attenuated when miR-31 was depleted in the exosomes or when FIH1 was overexpressed in the endothelial cells. Furthermore, the results from immunocytochemistry, co-immunoprecipitation, and luciferase reporter assay demonstrated that the effects of exosomes on nuclear translocation, binding with co-activator p300, and activation of HIF-1α were decreased when miR-31 was depleted in the exosomes or FIH1 was overexpressed. Our findings provide evidence that exosomes from ASCs promote angiogenesis in both mouse ischemic hindlimb and heart through transport of miR-31 which targets FIH1 and therefore triggers HIF-1α transcriptional activation.

Exosomes derived from induced vascular progenitor cells promote angiogenesis in vitro and in an in vivo rat hindlimb ischemia model.

Induced vascular progenitor cells (iVPCs) were created as an ideal cell type for regenerative medicine and have been reported to positively promote collateral blood flow and improve cardiac function in a rat model of myocardial ischemia. Exosomes have emerged as a novel biomedicine that mimics the function of the donor cells. We investigated the angiogenic activity of exosomes from iPVCs (iVPC-Exo) as a cell-free therapeutic approach for ischemia. Exosomes from iVPCs and rat aortic endothelial cells (RAECs) were isolated using a combination of ultrafiltration and size-exclusion chromatography. Nanoparticle tracking analysis revealed that exosome isolates fell within the exosomal diameter (<150 nm). These exosomes contained known markers Alix and TSG101, and their morphology was validated using transmission electron microscopy. When compared with RAECs, iVPCs significantly increased the secretion of exosomes. Cardiac microvascular endothelial cells and aortic ring explants were pretreated with RAEC-Exo or iVPC-Exo, and basal medium was used as a control. iVPC-Exo exerted an in vitro angiogenic effect on the proliferation, tube formation, and migration of endothelial cells and stimulated microvessel sprouting in an ex vivo aortic ring assay. Additionally, iVPC-Exo increased blood perfusion in a hindlimb ischemia model. Proangiogenic proteins (pentraxin-3 and insulin-like growth factor-binding protein-3) and microRNAs (-143-3p, -291b, and -20b-5p) were found to be enriched in iVPC-Exo, which may mediate iVPC-Exo induced vascular growth. Our findings demonstrate that treatment with iVPC-Exo promotes angiogenesis in vitro, ex vivo, and in vivo. Collectively, these findings indicate a novel cell-free approach for therapeutic angiogenesis.NEW & NOTEWORTHY The results of this work demonstrate exosomes as a novel physiological mechanism by which induced vascular progenitor cells exert their angiogenic effect. Moreover, angiogenic cargo of proteins and microRNAs may define the biological contributors in activating endothelial cells to form a new capillary plexus for ischemic vascular diseases.

Serum and glucocorticoid-regulated kinase 1 promotes vascular smooth muscle cell proliferation via regulation of ß-catenin dynamics.

In response to arterial intimal injury vascular smooth muscle cells (VSMCs) within the vessel wall proliferate upon exposure to growth factors, accumulate, and form a neointima that can occlude the vessel lumen. Serum and glucocorticoid inducible kinase 1 (SGK1) is a growth factor-responsive kinase; however its role in VSMC proliferation is not fully understood. Here, we examined growth factor-dependent regulation of SGK1 and defined a molecular role for SGK1 in stimulation of VSMC proliferation. We found that stimulation of VSMCs with the pro-proliferative growth factor, platelet-derived growth factor BB (PDGF) significantly increased SGK1 mRNA, protein, and kinase activity in aortic VSMCs in vitro. To test the hypothesis that activation of SGK1 activity promotes VSMC proliferation, we examined the effects of stable expression of constitutively active (S422D) and kinase-defective (S422A) mutants of SGK1 on VSMC growth. We found that activation of SGK1 increased, whereas interference of SGK1 signaling inhibited VSMC growth in vitro. Consistent with these findings, expression of the S422D mutant augmented both basal and PDGF-induced BrdU uptake in VSMCs. Conversely, PDGF-induced BrdU uptake was attenuated in VSMCs expressing S422A. Furthermore, we determined that activated SGK1 enhanced basal and PDGF-dependent G1→S cell cycle transition, whereas dominant-negative SGK1 abrogated G1→S cell cycle transition under similar conditions. Downstream signaling by active SGK1 induced basal and PDGF-induced phosphorylation of glycogen synthase kinase 3ß, an effect which was attenuated when SGK1 activity was blocked by expression of the kinase-defective mutant, S422A. We also found that transfection of S422D enhanced ß-catenin-nuclear localization and activation of the TOP/Flash and cyclin D1 transcriptional reporters. These effects were significantly blunted in VSMCs transfected with the S422A mutant. Our results provide compelling evidence of a role for SGK1 in stimulation of arterial VSMC growth via regulation of ß-catenin dynamics and implicate SGK1 in the progression of intimal narrowing following arterial injury. Hence, the findings presented here point to inhibition of SGK1 activity as a novel therapeutic approach for the treatment of occlusive vascular diseases.