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Increased Complement 3 With Suppression of miR-145 Induces the Synthetic Phenotype in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats.

ABSTRACT: Background We previously reported that vascular smooth muscle cells ( VSMC s) from spontaneously hypertensive rats ( SHR s) show the increased expression of complement 3 (C3) and the synthetic phenotype. We targeted the SHR C3 gene (C3 knockout [C3 KO] SHRs ) by the zinc finger gene editing method. In the current study, we investigated the mechanisms underlying the increased expression of C3 and the role of endogenous C3 in the synthetic phenotype of SHR VSMC s in comparison to cells from Wistar-Kyoto ( WKY) rats and C3 KO SHR s. Methods and Results Nonmuscle myosin heavy chain staining of aortas from SHR s at 1 day after birth was stronger in comparison to WKY rats and C3 KO SHR s. DNA synthesis in VSMC s from SHR s was significantly higher in comparison to WKY rats and C3 KO SHR s. Immunohistochemical staining of renin and liver X receptor ? in VSMC s from SHR s was stronger in comparison to WKY rats and C3 KO SHR s. The expression of renin, Krüppel-like factor 5, and liver X receptor ? proteins in VSMC s from SHR s was significantly higher in comparison to WKY rats and C3 KO SHR s. The expression of synthetic phenotype markers osteopontin, matrix gla, and l-caldesmon, growth factors transforming growth factor-?1 and platelet-derived growth factor-A, transcription factors Krüppel-like factor 5 and liver X receptor ?, and angiotensinogen mRNA s in VSMC s from SHR s was significantly higher in comparison to WKY rats and C3 KO SHR s. The expression of miR-145 mRNA in VSMC s from SHR s was suppressed in comparison to cells from WKY rats. miR-145 inhibitor significantly increased the expression of C3 in VSMC s from WKY rats, but not in cells from SHR s. Conclusions These findings indicate that the increased C3 with the suppression of miR-145 induces the synthetic phenotype through Krüppel-like factor 5 and the activation of the renin-angiotensin system through liver X receptor ? in VSMC s from SHR s.

SUBMITTER: Chen L

PROVIDER: S-EPMC6585333 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:RationaleThe phenotypes of vascular smooth muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood.ObjectiveTo determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs.Methods and resultsExperiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L-) medium. Compared with the L- medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters and were generally attenuated both by the blockade of monocarboxylic acid transporter activity and by transfection with iRNA for NDRG (N-myc downstream regulated gene). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+-cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate-dehydrogenase levels, vSMC proliferation, and monocarboxylic acid transporter and NDRG expression were greater in the ischemic zone than in nonischemic tissues.ConclusionsThese results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Project description:Background: Circular RNAs (circRNAs), as a kind of endogenous non-coding RNA, have been implicated in ischemic heart diseases and vascular diseases. Based on theirs high stability with a closed loop structure, circRNAs function as a sponge and bind specific miRNAs to exert inhibitory effects in heart and vasculature, thereby regulating their target gene and protein expression, via competitive endogenous RNA (ceRNA) mechanism. However, the exact roles and underlying mechanisms of circRNAs in hypertension and related cardiovascular diseases remain largely unknown. Methods and Results: High-throughput RNA sequencing (RNA-seq) was used to analyze the differentially expressed (DE) circRNAs in aortic vascular tissues of spontaneously hypertensive rats (SHR). Compared with the Wistar-Kyoto (WKY) rats, there were marked increases in the levels of systolic blood pressure, diastolic blood pressure and mean blood pressure in SHR under awake conditions via the tail-cuff methodology. Totally, compared with WKY rats, 485 DE circRNAs were found in aortic vascular tissues of SHR with 279 up-regulated circRNAs and 206 down-regulated circRNAs. Furthermore, circRNA-target microRNAs (miRNAs) and the target messenger RNAs (mRNAs) of miRNAs were predicted by the miRanda and Targetscan softwares, respectively. Additionally, real-time RT-PCR analysis verified that downregulation of rno_circRNA_0009197, and upregulation of rno_circRNA_0005818, rno_circRNA_0005304, rno_circRNA_0005506, and rno_circRNA_0009301 were observed in aorta of SHR when compared with that of WKY rats. Then, the potential ceRNA regulatory mechanism was constructed via integrating 5 validated circRNAs, 31 predicted miRNAs, and 266 target mRNAs. More importantly, three hub genes (NOTCH1, FOXO3, and STAT3) were recognized according to PPI network and three promising circRNA-miRNA-mRNA regulatory axes were found in hypertensive rat aorta, including rno_circRNA_0005818/miR-615/NOTCH1, rno_circRNA_0009197/ miR-509-5p/FOXO3, and rno_circRNA_0005818/miR-10b-5p/STAT3, respectively. Conclusions: Our results demonstrated for the first time that circRNAs are expressed aberrantly in aortic vascular tissues of hypertensive rats and may serve as a sponge linking with relevant miRNAs participating in pathogenesis of hypertension and related ischemic heart diseases via the circRNA-miRNA-mRNA ceRNAnetwork mechanism.

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Increased Complement 3 With Suppression of miR-145 Induces the Synthetic Phenotype in Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats.

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