The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Telmisartan attenuates aortic hypertrophy in hypertensive rats by the modulation of ACE2 and profilin-1 expression

Profilin-1 has recently been linked to vascular hypertrophy and remodeling. Here, we assessed the hypothesis that angiotensin (Ang) II type I receptor antagonist telmisartan improves vascular hypertrophy by modulation of expression of profilin-1 and angiotensin-converting enzyme 2 (ACE2). Ten-week-old male spontaneously hypertensive rats (SHR) were received oral administration of telmisartan (5 or 10 mg/kg; daily) or saline for 10 weeks. Compared with Wistar–Kyoto (WKY) rats, there were marked increases in systolic blood pressure and profilin-1 expression and reduced ACE2 and peroxisome proliferator activated receptor-γ (PPARγ) levels in aorta of SHR, associated with elevated extracellular-signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) phosphorylation signaling and aortic hypertrophy characterized with increased media thickness, which were strikingly reversed by telmisartan. In cultured human umbilical artery smooth muscle cells (HUASMCs), Ang II induced a dose-dependent increase in profilin-1 expression, along with decreased ACE2 protein expression and elevated ERK1/2 and JNK phosphorylation. In addition, blockade of ERK1/2 or JNK by either specific inhibitor was able to abolish Ang II-induced ACE2 downregulation and profilin-1 upregulation in HUASMCs. Importantly, treatment with telmisartan (1 or 10 μM) or recombinant human ACE2 (2 mg/ml) largely ameliorated Ang II-induced profilin-1 expression and ERK1/2 and JNK phosphorylation and augmented PPARγ ?expression in the cultured HUASMCs. In conclusion, telmisartan treatment attenuates vascular hypertrophy in SHR by the modulation of ACE2 and profilin-1 expression with a marked reversal of ERK1/2 and JNK phosphorylation signaling pathways.     

Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Retracted: Downregulated microRNA-224 aggravates vulnerable atherosclerotic plaques and vascular remodeling in acute coronary syndrome through activation of the TGF-β/Smad pathway

Recent studies have shown that circulating microRNAs (miRNA) play a critical role in diagnosing acute coronary syndrome (ACS). This study aims to investigate the effect of miR-224 on atherosclerotic plaques forming and vascular remodeling in ACS and its relationship with TGF-β/Smad pathway. Myocardial infarction (MI) rat model was established and lentivirus vector of miR-224 inhibitor was prepared for investigating the effect of downregulated miR-224 on the contents of nitric oxide (NO) and endothelin-1 (ET-1), blood lipid levels and inflammatory factor levels in serum as well as the TGF-β/Smad pathway. The rats suffering from MI had decreased survival rates and exhibited reduced levels of NO, high-density lipoprotein cholesterol, and lumen diameter, and Smad7 messenger RNA (mRNA) and protein expression; while had significantly increased ratio of heart weight or body weight, levels of ET-1, inflammatory factors, blood lipid indexes, vascular remodeling indexes, collagen volume fraction, vulnerable atherosclerotic plaque area, VCAM-1 and MMP-2 protein expression, TGF-β, Smad2, Smad3, and Smad4 mRNA and protein expression. After inhibiting the TGF-β/Smad pathway, the rats suffering from MI showed notably opposite trend. In conclusion, downregulation of miR-224 expression promotes the formation of vulnerable atherosclerotic plaques and vascular remodeling in ACS through activation of the TGF-β/Smad pathway. Therefore, this study provides a new therapeutic target for ACS.     

Comprehensive analysis of lncRNA–miRNA–mRNA during proliferative phase of rat liver regeneration

This study aims to reveal the regulatory mechanism of lncRNAs–miRNAs–mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA–miRNA–mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.     

T细胞抗原受体的结构与功能研究进展

T细胞抗原受体(T ceIl receptor,TCR)是T细胞表面关键的受体分子。TCR特异性地识别各种多肽抗原并通过胞内区ITAM磷酸化传递抗原刺激信号,进而引发T细胞的免疫效应。TCR的活性异常将会导致自身免疫病和免疫缺陷病的发生。对于TCR结构和功能的深入研究有助于我们更好地理解免疫反应的分子机理,从而为相关免疫疾病的预防和治疗提供重要的理论依据。该文对TCR的分类、基因重排机制、受体组装方式及其结构基础、TCR对抗原的识别以及活化机制等方面的研究成果进行了总结,综述了近几年来的最新研究进展。     

氰酸盐诱导氧化应激促进肾小管上皮细胞上皮–间充质转化

该研究探讨氰酸盐(cyanate)诱导肾小管上皮细胞氧化应激损伤和促进肾纤维化的作用。氰酸盐作用HK-2肾小管上皮细胞后, CCK8法检测其对细胞活力的影响;倒置显微镜观察细胞形态的改变; DCFH-DA法检测细胞ROS水平;细胞免疫荧光和Western blot分别检测E-cadherin、Fibronectin、α-SMA的表达; Western blot检测TGF-β的表达水平。结果显示, 2 mmol/L氰酸盐明显下调HK-2细胞的活力(P<0.05),细胞形态变为长梭形。氰酸盐作用24 h后, HK-2细胞内ROS水平呈浓度依赖性升高。免疫荧光和Western blot结果均显示,氰酸盐作用24 h后, HK-2的Fibronectin、α-SMA表达升高, E-cadherin表达下降; TGF-β的表达水平随氰酸盐浓度升高而上调(P<0.05)。以上结果表明,氰酸盐诱导肾小管上皮细胞产生过量ROS,上调TGF-β水平促进细胞上皮–间充质细胞转化(epithelia-mesenchymal transition, EMT)。