[Effect of human Wharton's Jelly or adipose derived mesenchymal stem cells culture supernatant on endothelial cells angiogenesis].

Objective: To compare the curative effect of mesenchymal stem cells derived from human Wharton's Jelly(WJ-MSC) or adipose(AD-MSC) culture supernatant on endothelial cells angiogenesis. Methods: WJ-MSC and AD-MSC were isolated, identified, and the culture supernatant of stem cells was collected.The WJ-MSC or AD-MSC supernatant co-cultured with the endothelial cells. The expression levels of pro-angiogenic and anti-angiogenic genes of endothelial cells were assessed using qRT-PCR analysis, and the effects of stem cell culture supernatant on angiogenesis were evaluated by performing a tube formation assay in vitro. Results: After adding WJ-MSC and AD-MSC culture supernatant, the expression levels of pro-angiogenic genes in endothelial cells were upregulated, and the expression levels of anti-angiogenic genes were downregulated significantly in both experimental groups compared to the control group (P<0.01), and tube formation of endothelial cells was also significantly increased in both experimental groups as determined by the increase of the tube length ((43.2±9.2) mm vs (94.3±13.2)mm, (86.1±7.2)mm, P<0.01). Conclusion: The results showed that AD-MSC culture supernatant can promote endothelial cells angiogenesis and its curative effect is similar to that of WJ-MSC.

[Regulatory effect of exosomes secreted by vaginal wall fibroblasts on angiogenesis in patients with stress urinary incontinence].

Objective: Exosomes are considered to mediate intercellular communication by delivering biomolecules like mRNA, miRNA into recipient cells. The purpose of this study was to evaluate the effects of exosomes secreted by fibroblasts from women with stress urinary incontinence (SUI-EXO) on endothelial cells angiogenesis. Methods: Primary fibroblasts were acquired from periurethral vaginal wall tissues and exosomes were prepared by ultracentrifugation of fibroblasts cells conditioned medium. The expression levels of pro-angiogenic and anti-angiogenic genes were assessed using qRT-PCR analysis. Migration of endothelial cells was measured by transwell assay, and the effects of SUI-EXO on angiogenesis were evaluated by performing a tube formation assay in vitro. Results: SUI-EXO was successfully isolated from fibroblasts cells conditional medium and transferred to endothelial cells efficiently. When the endothelial cells were treated with SUI-EXO, the expression levels of pro-angiogenic genes in fibroblasts were downregulated, and the expression levels of anti-angiogenic genes were upregulated significantly (P<0.01). Endothelial cells exhibited a decreased migratory capacity after treatment with SUI-EXO compared to exosomes from health women (64.6±8.7 vs 114.5±14.2,P<0.01), and tube formation of endothelial cells was also significantly inhibited in the SUI-EXO treated group as determined by the increase of the tube length (87.6±13.3 vs 168.5±28.3,P<0.01). Conclusion: This study suggests that SUI-EXO plays related roles in regulating endothelial cells angiogenesis and SUI-EXO maybe involve in the pathogenesis of SUI.

Receptor-Galpha fusion proteins as a tool for ligand screening.

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.

[Distribution of muscarinic receptors of different affinities in smooth muscle of human stomach].

Muscarinic receptors of high and low affinity were found in the fundus and the body of human stomach through the contraction experiment combined with ligand method in vitro. The 2 types of muscarinic receptors with different affinity regulated respectively the contractions of the longitudinal and the circular muscle of human gastric fundus and gastric body. However, in the antrum exists only one kind of muscarinic receptors of high affinity, which regulated the contractions of the longitudinal and the circular muscles of human stomach. The contractile force of the longitudinal muscle induced by exogenous ACh in the fundus and that in the body of human stomach were found to be similar to each other. The contractile force of the circular muscle in the body was found to be the strongest, and the contractile force of both longitudinal and circular muscles in antrum was weaker.

Function and localization of high and low affinity binding sites to muscarinic receptors in longitudinal and circular smooth muscles of human stomach.

The contractile response to acetylcholine (ACh) and the binding of [3H]-quinuclidinyl benzilate [( 3H]QNB) to muscarinic receptors in both longitudinal and circular muscles were examined in fundus, body and antrum of human stomach which was obtained by surgical operation for gastric cancer or ulcer. The values of pD2 and pA2 for ACh and atropine, respectively, on longitudinal muscle of fundus were similar to those of body but were larger than those of antrum. On the other hand, the values of pD2 and pA2 on circular muscles were not different among fundus, body, and antrum and were similar to those on longitudinal muscle of antrum. By Scatchard analysis of receptor binding of [3H]QNB in homogenate, at least two subclasses of binding sites, i.e. high and low affinity sites, were observed in fundus and body, while only high affinity binding site was found in antrum. Thus, we conclude that there are at least two subclasses of muscarinic receptors which regulate the contraction of smooth muscle in different regions of human stomach.