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[CRISPR/Cas9-mediated TEAD1 knockout induces phenotypic modulation of corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction].

ABSTRACT:

Objective

To construct a corpus cavemosum smooth muscle cell (CCSMCs) line with TEAD1 knockout from diabetic rats with erectile dysfunction (ED) using CRISPR/Cas9 technology and explore the role of TEAD1 in phenotypic modulation of CCSMCs in diabetic rats with ED.

Objective

Models of diabetic ED were established in male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. CCSMCs from the rat models were primarily cultured and identified with immunofluorescence assay. Three sgRNAs (sgRNA-1, sgRNA-2 and sgRNA-3) were transfected via lentiviral vectors into 293T cells to prepare the sgRNA-Cas9 lentivirus. CCSMCs from diabetic rats with ED were infected by the lentivirus, and the cellular expression of TEAD1 protein was detected using Western blotting. In CCSMCs infected with the sgRNA-Cas9 lentivirus (CCSMCs-sgRNA-2), or the empty lentiviral vector (CCSMCs-sgRNA-NC) and the blank control cells (CCSMCs-CK), the expressions of cellular phenotypic markers SMMHC, calponin and PCNA at the mRNA and protein levels were detected using real-time fluorescence quantitative RT-PCR (qRT-PCR) and Western blotting, respectively.

Objective

The primarily cultured CCSMCs from diabetic rats with ED showed a high α-SMA-positive rate of over 95%. The recombinant lentivirus of TEAD1-sgRNA was successfully packaged, and stable TEAD1-deficient CCSMC lines derived from diabetic rat with ED were obtained. Western blotting confirmed that the protein expression of TEAD1 in TEAD1-sgRNA-2 group was the lowest (P < 0.05), and this cell line was used in subsequent experiment. The results of qRT-PCR and Western blotting showed significantly up-regulated expressions of SMMHC and calponin (all P < 0.05) and down-regulated expression of PCNA (all P < 0.05) at both the mRNA and protein levels in TEAD1-deficient CCSMCs from diabetic rats with ED.

Objective

We successfully constructed a stable CCSMCs line with CRISPR/Cas9-mediated TEAD1 knockout from diabetic rats with ED. TEAD1 gene knockout can induce phenotype transformation of the CCSMCs from diabetic rats with ED from the synthetic to the contractile type.

SUBMITTER: Zhang T

PROVIDER: S-EPMC8110442 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe mechanism of erectile dysfunction (ED) caused by a low androgen level is still not clear.AimTo explore the influence of the low testosterone state on G protein-coupled receptor kinase interactor 1 (GIT1) and its contact to erectile function.MethodsThirty male Sprague-Dawley rats aged 8 weeks were distributed at random into 5 groups: control (sham operated), castration, testosterone supplement after castration, castration + vacant lentiviral transfection, and castration + lentiviral transfection. The testis and epididymis were removed through a scrotal incision to develop castrated rats. Four weeks after castration, a lentivirus carrying the GIT1 gene was injected into the middle of rat penile corpus cavernosum. One week after transfection, maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), serum testosterone, nitric oxide, GIT1, endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), p-eNOS/eNOS, and the interaction between eNOS and GIT1 were assessed in the rats.OutcomesThe levels of GIT1 in the penile cavernous tissue of castrated rats are significantly lower than that of controls.ResultsGIT1 was expressed in the cytoplasm and cell membrane of vascular endothelial cells and smooth muscle cells in rat penile tissue. In comparison with normal rats, the castrated rats showed lower levels of GIT1 expression, GIT1 and eNOS interaction, p-eNOS/eNOS, nitric oxide, and ICPmax/MAP (P < .01). Overexpression of GIT1 can intensively enhance the expression level of GIT1, the interaction between GIT1 and eNOS, p-eNOS/eNOS, nitric oxide, and ICPmax/MAP in rats (P < .01).Clinical translationModulating the interaction between eNOS and GIT1 might be a novel method of treating ED caused by a low androgen level.Strengths and limitationsThe impact of GIT1 phosphorylation on the activity of eNOS and its possible mechanisms affecting erectile function require further study.ConclusionA low testosterone state inhibits erectile function in rats by reducing the expression of GIT1 and the protein interaction between GIT1 and eNOS.

Project description:IntroductionCavernosal nerve (CN) injury is commonly caused by radical prostatectomy surgery, and it might directly lead to erectile dysfunction (ED). Currently, the role of mitogen-activated protein kinase (MAPK) family proteins in phenotypic transformation of corpus cavernosum smooth muscle cell (CCSMC) after CNs injury is poorly understood.AimTo investigate the role of p38 MAPK in hypoxia-induced phenotypic transformation of CCSMCs after CN injury.MethodsIn total, 20 Sprague-Dawley rats (male and 8 weeks of age) were randomly divided into 2 groups, including a sham group and CNCI group. In the sham group, rats were sham-operated by identifying 2 CNs without causing direct damage to the CNs. In the CNCI group, rats were subjected to bilateral CN crush injury. CCSMCs were isolated from the normal corpus cavernosum tissues of the Sprague-Dawley rat and then cultured in 21% or 1% O2 concentration context for 48 hours.Main outcome measuresIntracavernous pressure/mean arterial pressure were analyzed to measure erectile response. The impact of hypoxia on penile pathology, as well as the expression of extracellular signal-regulated kinases, the c-Jun NH2-terminal kinase, and p38 MAPK, were analyzed.ResultsCompared with the sham group, the intracavernous pressure/mean arterial pressure rate and α-smooth muscle actin expression of CNCI group were decreased significantly (P = .0001; P = .016, respectively), but vimentin expression was significantly increased (P = .023). Phosphorylated p38 level in CNCI group was decreased significantly (P = .017; sham: 0.17 ± 0.005; CNCI: 0.14 ± 0.02). The CCSMCs in the normoxia group were long fusiform, whereas the morphology of CCSMCs in the hypoxia group became hypertrophic. After hypoxia for 48 hours, the expression of α-smooth muscle actin and phosphorylated p38 MAPK was decreased significantly (P = .01; P = .024, normoxia: 0.66 ± 0.18, hypoxia: 0.26 ± 0.08, respectively), and the expression of hypoxia-inducible factor-1α and collagen I was increased significantly in hypoxia group (P = .04; P = .012, respectively).ConclusionsHypoxia induced the phenotypic transformation of CCSMCs after CNCI might be associated with the downregulation of phosphorylated p38 MAPK. Chen S, Huang X, Kong X, et al. Hypoxia-Induced Phenotypic Transformation of Corpus Cavernosum Smooth Muscle Cells After Cavernous Nerve Crush Injury by Down-Regulating p38 Mitogen-Activated Protein Kinase Expression. Sex Med 2019;7:433-440.

Dataset Information

[CRISPR/Cas9-mediated TEAD1 knockout induces phenotypic modulation of corpus cavernosum smooth muscle cells in diabetic rats with erectile dysfunction].

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Objective

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