In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.

Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

[Biological function of bladder smooth muscle cells regulated by multi-modal biomimetic stress].

Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H 2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.

Villus myofibroblasts are developmental and adult progenitors of mammalian gut lymphatic musculature.

Inside the finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic capillary, the lacteal, sending fats into the systemic blood circulation for energy production. Despite this vital function, mechanisms of formation, assembly alongside lacteals, and maintenance of villus smooth muscle are unknown. By combining single-cell RNA sequencing and quantitative lineage tracing of the mouse intestine, we identified a local hierarchy of subepithelial fibroblast progenitors that differentiate into mature smooth muscle fibers via intermediate contractile myofibroblasts. This continuum persists as the major mechanism for villus musculature renewal throughout adult life. The NOTCH3-DLL4 signaling axis governs the assembly of smooth muscle fibers alongside their adjacent lacteals and is required for fat absorption. Our studies identify the ontogeny and maintenance of a poorly defined class of intestinal smooth muscle, with implications for accelerated repair and recovery of digestive function following injury.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.

SCUBE1 promotes pulmonary artery smooth muscle cell proliferation and migration in acute pulmonary embolism by modulating BMP7.

Objectives: After an episode of acute pulmonary embolism (APE), activated platelets have the ability to release various bioactive factors that can stimulate both proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). SCUBE1 has been previously reported to engage in platelet-platelet interactions, potentially contributing to the activation of platelets in early onset thrombi. The purpose of this study was to examine the alterations in SCUBE1 expression in PASMCs after APE, as well as understand the mechanism behind these changes. Methods: The platelet-rich plasma samples of both APE patients and healthy individuals were collected. A hyperproliferative model of PASMCs was established by using platelet-derived growth factor (PDGF) as a stimulator and various assays were used to investigate how SCUBE1-mediated BMP7 can regulate PDGF-induced PASMC proliferation and migration. Results: Elevated level of SCUBE1 were observed in platelet-rich plasma from patients with APE and in PASMCs induced by PDGF. SCUBE1 interference ameliorated PDGF-driven cell proliferation and migration, and also downregulated PCNA expression. Additionally, mechanistic studies demonstrated that SCUBE1 could directly bind to bone morphogenetic protein 7 (BMP7) and enhance BMP7 expression, which completely abolished the impact of SCUBE1 silencing on proliferation and migration ability of PASMCs after PDGF treatment. Conclusion: In the PDGF-induced proliferation of PASMCs, the expression of SCUBE1 and BMP7 was upregulated. Silencing of SCUBE1 impeded PDGF-induced proliferation and migration of PASMCs by restraining BMP7.

d-Galactose induces the senescence and phenotype switch of corpus cavernosum smooth muscle cells.

Studies regarding age-related erectile dysfunction (ED) based on naturally aging models are limited by their high costs, especially for the acquisition of primary cells from the corpus cavernosum. Herein, d-galactose ( d-gal) was employed to accelerate cell senescence, and the underlying mechanism was explored. As predominant functional cells involved in the erectile response, corpus cavernosum smooth muscle cells (CCSMCs) were isolated from 2-month-old rats. Following this, d-gal was introduced to induce cell senescence, which was verified via ß-galactosidase staining. The effects of d-gal on CCSMCs were evaluated by terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence staining, flow cytometry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, RNA interference (RNAi) was carried out for rescue experiments. Subsequently, the influence of senescence on the corpus cavernosum was determined via scanning electron microscopy, qRT-PCR, immunohistochemistry, TUNEL, and Masson stainings. The results revealed that the accelerated senescence of CCSMCs was promoted by d-gal. Simultaneously, smooth muscle alpha-actin (alpha-SMA) expression was inhibited, while that of osteopontin (OPN) and Krüppel-like factor 4 (KLF4), as well as fibrotic and apoptotic levels, were elevated. After knocking down KLF4 expression in d-gal-induced CCSMCs by RNAi, the expression level of cellular alpha-SMA increased. Contrastingly, the OPN expression, apoptotic and fibrotic levels declined. In addition, cellular senescence acquired partial remission. Accordingly, in the aged corpus cavernosum, the fibrotic and apoptotic rates were increased, followed by downregulation in the expression of alpha-SMA and the concurrent upregulation in the expression of OPN and KLF4. Overall, our results signaled that d-gal-induced accelerated senescence of CCSMCs could trigger fibrosis, apoptosis and phenotypic switch to the synthetic state, potentially attributed to the upregulation of KLF4 expression, which may be a multipotential therapeutic target of age-related ED.

The downstream network of STAT6 in promoting vascular smooth muscle cell phenotypic switch and neointimal formation.

Intimal thickening caused by the excessive multiplication of vascular smooth muscle cells (VSMCs) is the pathological process central to cardiovascular diseases, including restenosis. In response to vascular injury, VSMCs would undergo phenotypic switching from a fully differentiated, low proliferative rate phenotype to a more pro-proliferative, promigratory, and incompletely-differentiated state. The lack of a full understanding of the molecular pathways coupling the vascular injury stimuli to VSMCs phenotype switching largely limits the development of medical therapies for treating intima hyperplasia-related diseases. The role of signal transducers and activators of transcription 6 (STAT6) in modulating the proliferation and differentiation of various cell types, especially macrophage, has been well investigated, but little is known about its pathophysiological role and target genes in restenosis after vascular injury. In the present work, Stat6-/- mice were observed to exhibit less severe intimal hyperplasia compared with Stat6+/+ mice after carotid injury. The expression of STAT6 was upregulated in VSMCs located in the injured vascular walls. STAT6 deletion leads to decreased proliferation and migration of VSMCs while STAT6 overexpression enhances the proliferation and migration of VSMCs companies with reduced expression of VSMCs marker genes and organized stress fibers. The effect of STAT6 in mouse VSMCs was conserved in human aortic SMCs. RNA-deep-sequencing and experiments verification revealed LncRNA C7orf69/LOC100996318-miR-370-3p/FOXO1-ER stress signaling as the downstream network mediating the pro-dedifferentiation effect of STAT6 in VSMCs. These findings broaden our understanding of vascular pathological molecules and throw a beam of light on the therapy of a variety of proliferative vascular diseases.

BACH1 deficiency prevents neointima formation and maintains the differentiated phenotype of vascular smooth muscle cells by regulating chromatin accessibility.

The transcription factor BTB and CNC homology 1(BACH1) has been linked to coronary artery disease risk by human genome-wide association studies, but little is known about the role of BACH1 in vascular smooth muscle cell (VSMC) phenotype switching and neointima formation following vascular injury. Therefore, this study aims to explore the role of BACH1 in vascular remodeling and its underlying mechanisms. BACH1 was highly expressed in human atherosclerotic plaques and has high transcriptional factor activity in VSMCs of human atherosclerotic arteries. VSMC-specific loss of Bach1 in mice inhibited the transformation of VSMC from contractile to synthetic phenotype and VSMC proliferation and attenuated the neointimal hyperplasia induced by wire injury. Mechanistically, BACH1 suppressed chromatin accessibility at the promoters of VSMC marker genes via recruiting histone methyltransferase G9a and cofactor YAP and maintaining the H3K9me2 state, thereby repressing VSMC marker genes expression in human aortic smooth muscle cells (HASMCs). BACH1-induced repression of VSMC marker genes was abolished by the silencing of G9a or YAP. Thus, these findings demonstrate a crucial regulatory role of BACH1 in VSMC phenotypic transition and vascular homeostasis and shed light on potential future protective vascular disease intervention via manipulation of BACH1.

Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation.

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cß3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin ß3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia.

NCAPG Promotes Pulmonary Artery Smooth Muscle Cell Proliferation as a Promising Therapeutic Target of Idiopathic Pulmonary Hypertension: Bioinformatics Analysis and Experiment Verification.

Idiopathic pulmonary arterial hypertension (IPAH) is a disease with complex etiology. Currently, IPAH treatment is limited, and patients' prognosis is poor. This study aimed to explore new therapeutic targets in IPAH through bioinformatics. Two data sets (GSE113439 and GSE130391) meeting the requirements were obtained from the Gene Expression Omnibus (GEO) database. Then, differentially expressed genes (DEGs) were identified and analyzed by NetworkAnalyst platform. By enriching Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), we examined the function of DEGs. A protein-protein interaction (PPI) network was constructed to identify central genes using the CytoNCA plug-in. Finally, four central genes, ASPM, CENPE, NCAPG, and TOP2A, were screened out. We selected NCAPG for protein-level verification. We established an animal model of PAH and found that the expression of NCAPG was significantly increased in the lung tissue of PAH rats. In vitro experiments showed that the expression of NCAPG was significantly increased in proliferative pulmonary arterial smooth muscle cells (PASMCs). When NCAPG of PASMCs was knocked down, the cell proliferation was inhibited, which suggested that NCAPG was related to the proliferation of PASMCs. Therefore, these results may provide new therapeutic targets for IPAH.

BRD7 restrains TNF-α-induced proliferation and migration of airway smooth muscle cells by inhibiting notch signaling.

Objective: Bromodomain-containing protein 7 (BRD7) is a key component of the switch/sucrose non-fermentable complex that participates in chromatin remodeling and transcriptional regulation. Although the emerging role of BRD7 in the pathophysiology of various diseases has been observed, its role in asthma remains unknown. Here, we assessed the function of BRD7 as a mediator of airway remodeling in asthma using an in vitro model. Methods: Airway smooth muscle cells (ASMCs) were challenged with tumor necrosis factor-α (TNF-α) to establish an in vitro airway remodeling model. Protein levels were examined using western blotting. Cell proliferation was measured using the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays. Cell migration was assessed using a transwell migration assay. Results: Exposure to TNF-α dramatically decreased BRD7 levels in ASMCs. BRD7 remarkably decreased TNF-α-induced proliferation and migration of ASMCs. In contrast, ASMCs with BRD7 deficiency were more sensitive to TNF-α-induced pro-proliferative and pro-migratory effects. Mechanistically, BRD7 could repress the expression of Notch1 and block the Notch pathway in TNF-α-challenged cells. Notably, reactivation of Notch signaling substantially reversed the BRD7 overexpression-mediated effects, whereas restraining Notch signaling abolished BRD7-depletion-mediated effects on TNF-α-challenged cells. Conclusions: BRD7 inhibits the proliferation and migration of ASMCs elicited by TNF-α by downregulating the Notch pathway. This study indicates that BRD7 may exert a suppressive effect on airway remodeling during asthma.

NORAD Promotes the Viability, Migration, and Phenotypic Switch of Human Vascular Smooth Muscle Cells during Aortic Dissection via LIN28B-Mediated TGF-ß Promotion and Subsequent Enhanced Glycolysis.

Glucose metabolism reprogramming is an important reason for the functional remodeling, growth, and migration of vascular smooth muscle cells (VSMCs). It is also an important basis for the occurrence and development of aortic dissection (AD), but the specific regulatory factors are not clear. Noncoding RNA activated by DNA damage (NORAD) is dysfunctional in many diseases, but the role of NORAD in AD etiology is unclear. We first established a vascular remodeling cell model of AD, and the expression of NORAD in VSMCs was significantly increased. Functional experiments showed that inhibition of NORAD could downregulate the proliferation and migration of VSMCs. Meanwhile, silencing NORAD could also inhibit the flux of glycolysis, suggesting that NORAD may aggravate AD by promoting glycolysis. In addition, mechanism studies have shown that NORAD can exert VSMCs-regulating function by recruiting LIN28B to bind to TGF-ß mRNA, which subsequently facilitates the expression of TGF-ß1 (transforming growth factor ß1). The recovery experiment also showed that overexpression of TGF-ß could reverse the inhibitory effect of NORAD knockdown on VSMCs in terms of proliferation, migration, and glycolysis. Collectively, these results indicated that the NORAD/LIN28B/TGF-ß axis promoted cell proliferation and migration through regulating aerobic glycolysis in VSMCs. Therefore, NORAD may regulate the occurrence of AD by affecting the reprogramming of glucose metabolism, and NORAD can be recognized as a good target for VSMC phenotypic intervention and AD treatment.

von Willebrand Factor: A Central Regulator of Arteriovenous Fistula Maturation Through Smooth Muscle Cell Proliferation and Outward Remodeling.

Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular-carotid AVFs were created in wild-type and vWF-/- mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachio-basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF-/- mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2 weeks, whereas flow in vWF-/- mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio. Conclusions vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia.

Circ_ARHGAP32 acts as miR-665 sponge to upregulate FGF2 to promote ox-LDL induced vascular smooth muscle cells proliferation and migration.

BACKGROUND: Circular RNA (circRNA) is considered to be an important regulator of human diseases, including atherosclerosis (AS). However, the role of circ_ARHGAP32 in AS formation needs further confirmation. OBJECTIVE: To explore the role of circ_ARHGAP32 in AS formation. METHODS: Oxidized low density lipoprotein (ox-LDL) was used to treat vascular smooth muscle cells (VSMCs) to mimic AS cell models in vitro. The expression of circ_ARHGAP32, microRNA (miR)-665, and fibroblast growth factor 2 (FGF2) was analyzed by quantitative real-time PCR. VSMCs function was measured by EdU assay, cell counting kit 8 assay and transwell assay. Protein expression was determined using western blot analysis. Dual-luciferase reporter assay and RNA pull-down assay were performed to verify RNA interaction. RESULTS: Circ_ARHGAP32 was highly expressed in AS patients and ox-LDL-induced VSMCs. Knockdown of circ_ARHGAP32 repressed ox-LDL-induced proliferation and migration in VSMCs. Circ_ARHGAP32 sponged miR-665 to positively regulate FGF2. MiR-665 inhibitor reversed the regulation of sh-circ_ARHGAP32 on ox-LDL-induced VSMCs proliferation and migration. MiR-665 also had a suppressive effect on the proliferation and migration of ox-LDL-induced VSMCs, and this effect could be reversed by FGF2 overexpression. CONCLUSIONS: Circ_ARHGAP32 might be a potential target for AS treatment, which promoted ox-LDL-induced VSMCs proliferation and migration by regulating miR-665/FGF2 network.

IL-37 Expression in Patients with Abdominal Aortic Aneurysm and Its Role in the Necroptosis of Vascular Smooth Muscle Cells.

Background: Our previous studies have shown that interleukin- (IL-) 37 plays a protective role in patients and animal models with coronary artery disease. However, the role of IL-37 in patients with abdominal aortic aneurysm (AAA), another artery disease, is yet to be elucidated. Methods and Results: AAA tissues and plasma samples were obtained from patients with or without surgical intervention. Normal renal aortic tissues were collected from kidney transplant donors. Our findings established that in AAA, IL-37 was distributed in endothelial cells, macrophages, and vascular smooth muscle cells (VSMCs) and that it was chiefly concentrated in VSMCs. Furthermore, the expression was found to be downregulated compared with that in normal artery tissues. Immunofluorescence showed that, unlike normal arteries, IL-37 was translocated to the nucleus of VSMCs in AAA. Moreover, in patients with AAA, the expressions of IL-37, IL-6, and tumor necrosis factor- (TNF-) α were increased in the plasma in comparison with the healthy controls. Correlation analysis revealed that IL-37 was positively correlated with IL-6, TNF-α, age, aneurysm diameter, and blood pressure. Furthermore, human aortic vascular smooth muscle cells (HASMCs) were stimulated with angiotensin II (AngII) in vitro to simulate smooth muscle cell (SMC) damage in AAA. A decrease in IL-37 expression and an increase in receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression were observed in HASMCs stimulated with AngII. On this basis, inhibition of RIPK3 with GSK'872 significantly attenuated necroptosis. Moreover, the necroptosis rates were significantly lowered in HASMCs treated with recombinant IL-37, whereas the rates were enhanced when the cells were depleted of the interleukin. Immunoblotting results showed that both exogenous and endogenous IL-37 could affect the expressions of RIPK3, NLRP3, and IL-1ß. Also, the phosphorylation of RIPK3 and p65 was affected. Meanwhile, IL-37 promoted the transition of SMC from proliferative type to contractile type. Conclusions: The expression of IL-37 in VSMCs decreases in patients with AAA, whereas IL-37 supplementation suppresses RIPK3-mediated necroptosis and promotes the transition of VSMCs from proliferative to contractile type.

Notch signaling is a novel regulator of visceral smooth muscle cell differentiation in the murine ureter.

The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system.

hsa_circWDR37_016 Regulates Hypoxia-Induced Proliferation of Pulmonary Arterial Smooth Muscle Cells.

Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of pulmonary vessel walls caused by excessive pulmonary arterial smooth muscle cell (PASMC) proliferation. Our previous clinical studies have demonstrated the importance of the downregulated circRNA in PAH. However, the role of upregulated circRNAs is still elusive. Here, we identified the upregulated circRNA in PAH patients, hsa_circWDR37_016 (circWDR37), as a key regulator of hypoxic proliferative disorder of pulmonary arterial smooth muscle cells (PASMCs). Quantitative real-time PCR (qRT-PCR) analysis validated that exposure to hypoxia markedly increased the circWDR37 level in cultured human PASMCs. As evidenced by flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, wound healing, and Tunel assay, silencing of endogenous circWDR37 attenuated proliferation and cell-cycle progression in hypoxia-exposed human PASMCs in vitro. Furthermore, bioinformatics and Luciferase assay showed that circWDR37 directly sponged hsa-miR-138-5p (miR-138) and was involved in the immunoregulatory and inflammatory processes of PAH. Together, these studies suggested new insights into circRNA regulated the pathology of PAH, providing a new potential therapeutic target for PAH treatment.

Long non-coding RNA HIF1A-AS2 modulates the proliferation, migration, and phenotypic switch of aortic smooth muscle cells in aortic dissection via sponging microRNA-33b.

Aortic dissection (AD), also known as aortic dissecting aneurysm, is one of the most common and dangerous cardiovascular diseases with high morbidity and mortality. This study was aimed to investigate the functional role of long non-coding RNA Hypoxia-inducible factor 1 alpha-antisense RNA 2 (lncRNA HIF1A-AS2) in AD. An in vitro model of AD was established by platelet-derived growth factor-BB (PDGF-BB)-mediated human aortic Smooth Muscle Cells (SMCs). HIF1A-AS2 expression in human AD tissues was determined by quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) assays, followed by investigation of biological roles of HIF1A-AS2 in AD development by Cell Counting Kit-8 (CCK-8), immunofluorescence, and transwell assays. Additionally, the correlation between HIF1A-AS2, miR-33b, and high mobility group AT-hook2 (HMGA2) were identified by RNA immunoprecipitation (RIP), RNA pull-down and luciferase reporter assays. Results showed that HIF1A-AS2 was obviously increased, while the contractile-phenotype markers of vascular SMCs were significantly decreased in human AD tissues, when compared to normal tissues. Inhibition of HIF1A-AS2 attenuated SMCs proliferation and migration, whereas enhanced the phenotypic switch under the stimulation of PDGF-BB. Results from RIP, RNA pull-down and luciferase reporter assays demonstrated that miR-33b directly bound with HIF1A-AS2, and HIF1A-AS2 silencing suppressed the expression of HMGA2, which was induced by miR-33b inhibitor. In conclusion, knockdown of HIF1A-AS2 suppressed the proliferation and migration, while promoted the phenotypic switching of SMCs through miR-33b/HMGA2 axis, which laid a theoretical foundation for understanding the development of AD and shed light on a potential target for AD treatment.

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells.

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.