[Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells].

OBJECTIVE: To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell. METHODS: Firstly (Lenti-GFP-CGRP, referred to CGRP (+/+)) MSCs were transfected with high expression lentivirus vector of CGRP (MSCs(CGRP+/+)). Protein secretion in the above-mentioned MSCs(CGRP+/+) supernatant was detected with enzyme-linked immunosorbent assay (ELISA). And then MSCs(CGRP+/+) was co-cultured with VSMCs(RAMP1)(+/+) and VSMCs(RAMP1-/-) respectively. Experimental groups were as follows: MSCs +VSMCs MSCs(CGRP+/+) +VSMCs, MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+) and MSCs(CGRP+/+) + VSMCs (RAMP1-/-). Flow cytometry was applied to detect the cycle variation of smooth muscle cells, thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively. RESULTS: After the transfection of CGRP(+/+), MSCs(CGRP+/+) secreted and expressed CGRP protein, the secretory volume of CGRP protein in MSCs(CGRP+/+) increased significantly compared with the control group (19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001, P < 0.05) . After a 72 h co-culturing with VSMCs, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+)group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070, P < 0.05) and the number of cells staying in G0 phase significantly increased (93.51% ± 0.38% vs 84.48% ± 0.31%, P < 0.05) , the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP(+/+) +VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ± 635, P < 0.05), OPN (26 026 ± 2595 vs 44 201 ± 2811, P < 0.05) }; but compared with MSCs(CGRP+/+)+VSMCs(RAMP1)(+/+)group, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)-/-group significantly increased (0.601 ± 0.04 vs 0.270 ± 0.263, P < 0.05) while the number of cells staying in G0 phase significantly declined (78.57% ± 0.68% vs 93.51% ± 0.38%, P < 0.05) . The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCs(CGRP+/+)+VSMCs(RAMP1)-/-group {α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847, P < 0.05), OPN (53 933 ± 1192 vs 26 026 ± 2595, P < 0.05)}. CONCLUSIONS: CGRP gene-modified MSCs may secrete target protein stably. And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation. It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.

Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes.

The important problem of the fate of glycogen-accumulating clear cells in glycogen nephrosis is still unsettled. In this study, we examine whether apoptosis plays a relevant role in the development of diabetic glycogen nephrosis and explore the involvement of the Fas/Fas-L system and the activation of the caspase cascade. Diabetes was induced in rats by streptozotocin injection. Glycogen-accumulating clear cells were identified in renal tissues of hyperglycemic rats. They were found to be concentrated in the thick ascending limbs and distal tubules. Large cellular glycogen accumulations were confirmed by biochemical assays and enzyme-gold cytochemistry. Clear cells displayed apoptotic features such as Annexin V binding, nuclear TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling), and the simultaneous occurrence of Fas, Annexin V, and TUNEL positivity. Western blot analysis demonstrated enhanced expression of Fas receptor/ligand and the activation of the caspase cascade in these cells because cleaved forms of the caspase-3, -8, and -9 were detected. Furthermore, active caspase-3 was located in nuclei by immunoelectron microscopy. Our results indicate that epithelial cells in thick ascending limbs and distal tubules that develop glycogen nephrosis in response to hyperglycemia undergo Fas/Fas-L mediated cell death. Thus, apoptosis could be playing a significant role in renal epithelial cell deletion during diabetes.