Differentiation Of Vascular Cells Research Articles

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for thetreatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-β1 antagonists. Further, KOS hydrogel increased expression of TGF-β1-associated proteins and modulated the level of free TGF-β1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-β1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Vitamin D Receptor From VSMCs Regulates Vascular Calcification During CKD: A Potential Role for miR-145a.

Vascular calcification (VC) is a highly prevalent complication of chronic kidney disease (CKD) and is associated with the higher morbidity-mortality of patients with CKD. VDR (vitamin D receptor) has been proposed to play a role in the osteoblastic differentiation of vascular smooth muscle cells (VSMCs), but the involvement of vitamin D in VC associated to CKD is controversial. Our aim was to determine the role of local vitamin D signaling in VSMCs during CKD-induced VC. We used epigastric arteries from CKD-affected patients and individuals with normal renal function, alongside an experimental model of CKD-induced VC in mice with conditional deletion of VDR in VSMC. In vitro, experiments in VSMC with or without VDR incubated in calcification media were also used. CKD-affected patients and mice with CKD showed an increase in VC, together with increased arterial expression of VDR compared with controls with normal renal function. Conditional gene silencing of VDR in VSMCs led to a significant decrease of VC in the mouse model of CKD, despite similar levels of renal impairment and serum calcium and phosphate levels. This was accompanied by lower arterial expression of OPN (osteopontin) and lamin A and higher expression of SOST (sclerostin). Furthermore, CKD-affected mice showed a reduction of miR-145a expression in calcified arteries, which was significantly recovered in animals with deletion of VDR in VSMC. In vitro, the absence of VDR prevented VC, inhibited the increase of OPN, and reestablished the expression of miR-145a. Forced expression of miR-145a in vitro in VDRwt VSMCs blunted VC and decreased OPN levels. Our study provides evidence proving that inhibition of local VDR signaling in VSMCs could prevent VC in CKD and indicates a possible role for miR-145a in this process.

Single-cell RNA sequencing analysis of the embryogenic callus clarifies the spatiotemporal developmental trajectories of the early somatic embryo in Dimocarpus longan.

Plant embryogenic calli (ECs) can undergo somatic embryogenesis to regenerate plants. This process is mediated by regulatory factors, such as transcription factors and specifically expressed genes, but the precise molecular mechanisms underlying somatic embryogenesis at the single-cell level remain unclear. In this study, we performed high-resolution single-cell RNA sequencing analysis to determine the cellular changes in the EC of the woody plant species Dimocarpus longan (longan) and clarify the continuous cell differentiation trajectories at the transcriptome level. The highly heterogeneous cells in the EC were divided into 12 putative clusters (e.g., proliferating, meristematic, vascular, and epidermal cell clusters). We determined cluster-enriched expression marker genes and found that overexpression of the epidermal cell marker gene GDSL ESTERASE/LIPASE-1 inhibited the hydrolysis of triacylglycerol. In addition, the stability of autophagy was critical for the somatic embryogenesis of longan. The pseudo-timeline analysis elucidated the continuous cell differentiation trajectories from early embryonic cell division to vascular and epidermal cell differentiation during the somatic embryogenesis of longan. Moreover, key transcriptional regulators associated with cell fates were revealed. We found that ETHYLENE RESPONSIVE FACTOR 6 was characterized as a heat-sensitive factor that negatively regulates longan somatic embryogenesis under high-temperature stress conditions. The results of this study provide new spatiotemporal insights into cell division and differentiation during longan somatic embryogenesis at single-cell resolution.

Chronic Kidney Disease Transdifferentiates Veins into a Specialized Immune-Endocrine Organ with Increased MYCN-AP1 Signaling.

Most patients with end-stage renal disease (ESRD) and advanced chronic kidney disease (CKD) choose hemodialysis as their treatment of choice. Thus, upper-extremity veins provide a functioning arteriovenous access to reduce dependence on central venous catheters. However, it is unknown whether CKD reprograms the transcriptome of veins and primes them for arteriovenous fistula (AVF) failure. To examine this, we performed transcriptomic analyses of bulk RNA sequencing data of veins isolated from 48 CKD patients and 20 non-CKD controls and made the following findings: (1) CKD converts veins into immune organs by upregulating 13 cytokine and chemokine genes, and over 50 canonical and noncanonical secretome genes; (2) CKD increases innate immune responses by upregulating 12 innate immune response genes and 18 cell membrane protein genes for increased intercellular communication, such as CX3CR1 chemokine signaling; (3) CKD upregulates five endoplasmic reticulum protein-coding genes and three mitochondrial genes, impairing mitochondrial bioenergetics and inducing immunometabolic reprogramming; (4) CKD reprograms fibrogenic processes in veins by upregulating 20 fibroblast genes and 6 fibrogenic factors, priming the vein for AVF failure; (5) CKD reprograms numerous cell death and survival programs; (6) CKD reprograms protein kinase signal transduction pathways and upregulates SRPK3 and CHKB; and (7) CKD reprograms vein transcriptomes and upregulates MYCN, AP1, and 11 other transcription factors for embryonic organ development, positive regulation of developmental growth, and muscle structure development in veins. These results provide novel insights on the roles of veins as immune endocrine organs and the effect of CKD in upregulating secretomes and driving immune and vascular cell differentiation.

Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis.

Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

Mechanism of Notch3 signaling pathway regulating the differentiation of aortic dissection vascular stem cells into smooth muscle cells

To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process. Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting. Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05). Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.

Sprouty1 has a protective role in atherogenesis and modifies the migratory and inflammatory phenotype of vascular smooth muscle cells

Background and aimsSprouty1 (Spry1) regulates the differentiation of vascular smooth muscle cells (VSMC), and our aim was to determine its role in atherogenesis. A significant proportion of cells within atherosclerotic lesions are derived from migration and pathological adaptation of medial VSMC. MethodsWe used global Spry1 null mouse, and Myh11-CreERT2, ROSA26-STOPfl/fl-tdTomato-Spry1fl/fl mice to allow for lineage tracing and conditional Spry1 deletion in VSMC. Atherosclerosis was induced by injection of a mutant form of mPCSK9D377Y-AAV followed by Western diet. Human aortic VSMC (hVSMC) with shRNA targeting of Spry1 were also analyzed. ResultsGlobal loss of Spry1 increased inflammatory markers ICAM1 and Cox2 in VSMC. Conditional deletion of Spry1 in VSMC had no effect on early lesion development, despite increased Sca1high cells. After 26 weeks of Western diet, mice with VSMC deletion of Spry1 had increased plaque burden, with reduced collagen content and smooth muscle alpha actin (SMA) in the fibrous cap. Lineage tracing via tdTomato marking Cre-recombined cells indicated that VSMC with loss of Spry1 had decreased migration into the lesion, noted by decreased proportions of tdTomato+ and tdTomato+/SMA + cells. Loss-of-function of Spry1 in hVSMC increased mesenchymal and activation markers, including KLF4, PDGFRb, ICAM1, and Cox2. Loss of Spry1 enhanced the effects of PDGFBB and TNFa on hVSMC. ConclusionsLoss of Spry1 in VSMC aggravated plaque formation at later stages, and increased markers of instability. Our results indicate that Spry1 suppresses the mesenchymal and inflammatory phenotype of VSMC, and its expression in VSMC is protective against chronic atherosclerotic disease.

Influence of FOSL1 Inhibition on Vascular Calcification and ROS Generation through Ferroptosis via P53-SLC7A11 Axis.

Vascular calcification during aging is highly prevalent in patients with cardiovascular disease; however, there is still no improvement in clarifying the development of vascular calcification. FOSL1 is a transcription regulator belonging to the AP-1 family, which has a unique function in vascular senescence, but its role in vascular calcification needs to be further explored. Primary mouse vascular smooth muscle cells were isolated and used to construct a calcification model in vitro. Seven-week-old male C57BL/6 mice were used to build the vitD3-induced calcification model in vivo. qRT-PCR and western blot were used to verify the expression of FOSL1 and other genes expressed in vascular smooth muscle cells and aortas. The level of calcification was determined by Alizarin Red S (ARS) staining and the calcium content assay. The level of cellular GSH was detected by the GSH assay kit. Here, we report that FOSL1 was up-regulated after high-calcium/phosphate treatment in both the in vivo and in vitro vascular calcification models. Functional studies have shown that the reduction of FOSL1 attenuates ferroptosis and calcification in vascular smooth muscle cells, as indicated by ARS staining, calcium content assay, and western blot. The inhibition of FOSL1 downregulated the expression of bone-related molecules including Msh Homeobox 2 (MSX2) and tumor necrosis factor receptor superfamily, member 11b/osteoprotegerin (OPG), suggesting that FOSL1 promoted osteogenic differentiation of vascular smooth muscle cells. Furthermore, we found that the ferroptosis-inducing drug erastin can significantly accelerate calcification in the aortic ring while Ferrostatin-1 (fer-1), a drug to protect cells from ferroptosis, can alleviate calcification. Further experiments have shown that inhibiting FOSL1 can promote the expression of ferroptosis-related genes and attenuate calcification. Functionally, cellular GSH levels were increased after the reduction of FOSL1. In this study, we observed a significant protective effect when we reduced the expression of FOSL1 during vascular calcification, and this effect might regulate ferroptosis to a great extent.

Targeting serotonin receptor 2B inhibits TGFβ induced differentiation of human vascular smooth muscle cells

Vascular Smooth Muscle Cells (VSMCs) are known to be the key drivers of intimal thickening which contribute to early progression of atherosclerosis. VSMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli they could modify the type of matrix proteins produced. Serotonin receptor 2B (5-HT2B receptor/HTR2B) has been implicated in several chronic fibrotic and vascular diseases. Although studies have successfully demonstrated the efficacy of HTR2B blockade in attenuating fibrotic disease, the role of 5-HT2B receptor in TGFβ mediated VSMC differentiation remain largely unknown. In the present study, we investigated the potential of targeting the 5-HT2B receptor to prevent TGFβ induced VSMCs differentiation. Our results showed that 5-HT2B receptors are expressed in human atherosclerotic lesion and HTR2B expression positively correlated to the VSMCs markers. We show that AM1125, a selective 5-HT2B receptor inhibitor, significantly inhibits TGFβ1 induced production of collagen and CTGF. The investigation of underlying mechanisms indicated that 5-HT2B receptor antagonism blocks phospho-Smad2 mediated downstream signaling of TGFβ1 in vascular smooth muscle cells. Collectively, the HTR2B/TGF-β1/Phospho-Smad2 pathway plays a critical role in the regulation of VSMCs differentiation. Our findings might serve 5-HT2B receptor as a therapeutic target to limit TGF-β1 induced VSMC differentiation.

Role of c-Src and reactive oxygen species in cardiovascular diseases.

Oxidative stress, caused by the over production of oxidants or inactivity of antioxidants, can modulate the redox state of several target proteins such as tyrosine kinases, mitogen-activated protein kinases and tyrosine phosphatases. c-Src is one such non-receptor tyrosine kinase which activates NADPH oxidases (Noxs) in response to various growth factors and shear stress. Interaction between c-Src and Noxs is influenced by cell type and primary messengers such as angiotensin II, which binds to G-protein coupled receptor and activates the intracellular signaling cascade. c-Src stimulated activation of Noxs results in elevated release of intracellular and extracellular reactive oxygen species (ROS). These ROS species disturb vascular homeostasis and cause cardiac hypertrophy, coronary artery disease, atherosclerosis and hypertension. Interaction between c-Src and ROS in the pathobiology of cardiac fibrosis is hypothesized to be influenced by cell type and stimuli. c-Src and ROS have a bidirectional relationship, thus increased ROS levels due to c-Src mediated activation of Noxs can further activate c-Src by promoting the oxidation and sulfenylation of critical cysteine residues. This review highlights the role of c-Src and ROS in mediating downstream signaling pathways underlying cardiovascular diseases. Furthermore, due to the central role of c-Src in activation of various signaling proteins involved in differentiation, migration, proliferation, and cytoskeletal reorganization of vascular cells, it is presented as therapeutic target for treating cardiovascular diseases except cardiac fibrosis.

Role of MEF2C in the Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.

VEGF-Expressing Mesenchymal Stem Cell Therapy for Safe and Effective Treatment of Pain in Parkinson’s Disease

Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that mediates the differentiation and function of vascular endothelial cells. VEGF has been implicated in modulating various pains. However, the effects of VEGF in Parkinson’s disease (PD)-related pain have not been studied. The goal of this study was to understand the effects of VEGF-expressing mesenchymal stem cells (MSCs) on PD-related pain and the involved mechanisms. We used two types of MSCs: hAMSC-Vector-GFP and hAMSC-VEGF189-GFP in PD mice. Then, the expression of VEGF and the viability have been compared between two types of MSCs. To demonstrate the therapeutic effect of hAMSC-VEGF189-GFP, we transplanted each cell line in a PD mouse model. Head mechanical withdrawal thresholds were examined. hAMSC-VEGF189-GFP was associated with significantly increased VEGF expression and slightly increased viability, compared with hAMSC-Vector-GFP. The transplanted hAMSC-VEGF189-GFP significantly improved mechanical allodynia and inhibited transient receptor potential vanilloid 1 (TRPV1) expression in site. And such pain relief effects could be partially blocked by TRPV1 agonist. However, we did not observe tumor generation or neuron degeneration in hAMSC-VEGF189-GFP-transplanted animals. Taken together, our data suggest that hAMSC-VEGF189-GFP is safely therapeutically appropriate for treating PD-related pain. VEGF inhibits TRPV1 expression, which may contribute to its analgesic properties.

MiR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding.

It has been shown that miR-145 is involved in the differentiation of vascular smooth muscle cells (VSMCs) and may regulate vascular remodeling. However, the molecular mechanisms behind these pathological processes in hypertension are not fully elucidated. The present study was to examine whether miR-145 modulates phenotypic transformation of VSMCs under normal state and synthetic state and to explore the possible role of ADAM17-mediated ACE2 shedding and ACE2-Ang-(1-7)-Mas receptor axis. Wistar rats were fed with high-sucrose/high-fat diet for 30 weeks to establish a metabolic hypertension animal model. VSMCs were cultured and treated with Ang II with or without miR-145 mimics or miR-145 inhibitor. Results showed the expression of contractile markers α-SMA and SM22α, miR-145, ACE2, and Mas receptor reduced in the thoracic aorta of metabolic hypertensive rats (MHRs), while that of synthetic marker OPN increased as compared to the control group. In in vitro study, miR-145 inhibitor inhibited the expression of α-SMA, SM22α, ACE2, Mas receptor, and the Ang-(1-7) excretion and induced the expression of synthetic markers OPN, EREG, and MMP2. However, miR-145 mimic produced opposite effects on the VSMCs. In addition, in the synthetic VSMC induced by Ang II, miR-145 inhibitor partially reversed the induced expression of OPN, EREG, and MMP2 by Ang II, while further decreasing the expression of α-SMA and SM22α and ACE2-Ang-(1-7)-Mas receptor. Cotreatment with ADAM17 siRNA partially reversed the inducible effect of miR-145 inhibitor on the EREG and MMP2, induced Ang-(1-7) excretion, and upregulated ACE2 and Mas receptor expression. In conclusion, miR-145 alleviates phenotype transition from contractile to synthetic type via ADAM17-mediated ACE2 shedding in VSMCs and retains the activation of ACE2-Ang-(1-7)-Mas axis, which may benefit the vascular structural remodeling in the metabolic hypertension.

MiR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling

BackgroundAtherosclerosis (AS) is the primary cause of coronary artery disease, which is featured by aberrant proliferation, differentiation, and migration of vascular smooth muscle cells (VSMCs). MicroRNAs play crucial roles in AS, but the function of miR-7-5p in AS remains unclear. Here, we aimed to explore the effect of miR-7-5p on AS and VSMCs in vitro and in vivo. MethodsThe in vivo rat AS model and apoE−/− mouse model were established. The carotid artery injury was checked by immunohistochemistry staining. The RNA levels of miR-7-5p and p65 were measured by qPCR assay. Protein levels were checked by western blotting. Cell apoptosis was evaluated by flow cytometry. Cell migration was checked by Transwell assay and wound healing assay. The potential interaction between miR-7-5p with p65 was checked by luciferase reporter gene assay. ResultsMiR-7-5p was downregulated and NF-κB p65 was upregulated in injured carotid arteries in rat model. The carotid artery injury in the AS rats and the treatment of miR-7-5p attenuated the phenotype in the model. Immunohistochemistry staining and Western blot analysis revealed that PCNA levels were increased in injured carotid arteries of the model rats and miR-7-5p could reverse the levels. The cell viability of VSMCs was induced by PDGF-BB but miR-7-5p blocked the phenotype. PDGF-BB decreased apoptosis of VSMCs, while miR-7-5p was able to restore the cell apoptosis in the model. PDGF-BB-induced migration of VSMCs was attenuated by miR-7-5p. miR-7-5p mimic remarkably repressed the luciferase activity of p65 in VSMCs. The levels of p65 were inhibited by miR-7-5p in the cells. The PDGF-BB-promoted cell viability and migration of VSMCs was repressed by miR-7-5p and p65 overexpression reversed the phenotype. ConclusionWe concluded that miR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling.

Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca2+ Entry.

Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mice lacking Notch3 show deficient renal autoregulation. The aim of the study was to investigate the mechanisms involved in the Notch3-mediated control of renal vascular response. To this end, renal resistance vessels (afferent arterioles) were isolated from Notch3-/- and wild-type littermates (WT) and stimulated with angiotensin II (ANG II). Contractions and intracellular Ca2+ concentrations were blunted in Notch3-/- vessels. ANG II responses in precapillary muscle arterioles were similar between the WT and Notch3-/- mice, suggesting a focal action of Notch3 in renal vasculature. Abolishing stored Ca2+ with thapsigargin reduced Ca2+ responses in the renal vessels of the two strains, signifying intact intracellular Ca2+ mobilization in Notch3-/-. EGTA (Ca2+ chelating agent), nifedipine (L-type channel-blocker), or mibefradil (T-type channel-blocker) strongly reduced contraction and Ca2+ responses in WT mice but had no effect in Notch3-/- mice, indicating defective Ca2+ entry. Notch3-/- vessels responded normally to KCl-induced depolarization, which activates L-type channels directly. Differential transcriptomic analysis showed a major down-regulation of Cacna1h gene expression, coding for the α1H subunit of the T-type Ca2+ channel, in Notch3-/- vessels. In conclusion, renal resistance vessels from Notch3-/- mice display altered vascular reactivity to ANG II due to deficient Ca2+-entry. Consequently, Notch3 is essential for proper excitation-contraction coupling and vascular-tone regulation in the kidney.

Hygiene of housing conditions and proinflammatory signals alter gene expressions in porcine adipose tissues and blood cells.

Adipose tissue is an organ with metabolic, endocrine and immune functions. In this tissue, the expressions of genes associated with several metabolic pathways, including lipid metabolism, have been shown to be affected by genetic selection for feed efficiency, an important trait to consider in livestock. We hypothesized that the stimulation of immune system caused by poor hygiene conditions of housing impacts the molecular and cellular features of adipose tissue and that the impact may differ between pigs that diverge in feed efficiency. At the age of 12 weeks, Large White pigs from two genetic lines divergent for residual feed intake (RFI) were housed in two contrasting hygiene conditions (good vs poor). After six weeks of exposure, pigs were slaughtered (n=36). Samples of blood, subcutaneous (SCAT) and perirenal (PRAT) adipose tissues were collected for cell response and gene expression investigations. The decrease in the relative weight of PRAT was associated with a decline in mRNA levels of FASN, ME, LCN2 and TLR4 (P<0.05) in pigs housed in poor conditions compared with pigs housed in good conditions for both RFI lines. In SCAT, the expressions of only two key genes (PPARG and TLR4) were significantly affected by the hygiene of housing conditions. Besides, the mRNA levels of both LCN2 and GPX3 were influenced by the RFI line (P<0.05). Because we suspected an effect of poor hygiene at the cellular levels, we investigated the differentiation of stromal vascular cells isolated from SCAT in vitro in the absence or presence of a pro-inflammatory cytokine, Tumor Necrosis Factor-α (TNF-α). The ability of these cells to differentiate in the absence or presence of TNF-α did not differ among the four groups of animals (P>0.05). We also investigated the expressions of genes involved in the immune response and lipid metabolism in whole blood cells cultured in the absence and presence of LPS. The hygiene conditions had no effect but, the relative expression of the GPX3 gene was higher (P<0.001) in high RFI than in low RFI pigs while the expressions of IL-10 (P=0.027), TGFβ1 (P=0.023) and ADIPOR2 (P=0.05) genes were lower in high RFI than in low RFI pigs. Overall, the current study indicates that the hygiene of housing had similar effects on both RFI lines on the expression of genes in adipose tissues and on the features of SCAT adipose cells and whole blood cells in response to TNF-α and LPS. It further demonstrates that the number of genes with expression impacted by housing conditions was higher in PRAT than in SCAT. It suggests a depot-specific response of adipose tissue to the current challenge.

Preparation and Biocompatibility of Core-Shell Microspheres for Sequential, Sustained Release of BMP-2 and VEGF

Bone defect repair remains a challenge in orthopedics. This study describes the development and potential effectiveness of vascular endothelial growth factor (VEGF)/bone morphogenetic protein-2 (BMP-2) shell-core microspheres for promoting bone regeneration. Poly(L-lactic acid)/polylactic-co-glycolic acid (PLLA/PLGA) core-shell microspheres loaded with VEGF and BMP-2 were prepared by a coaxial electrospray technique, and their surface morphology, core-shell distribution, and particle size were examined. Different groups of microspheres were prepared with different placement of the growth factors, and the encapsulation efficiency and in vitro release curves were measured. Additionally, the effects of the different groups of microspheres on the proliferation and differentiation of osteoblasts and vascular endothelial cells were investigated. The prepared microspheres had a core-shell structure with good homogeneity and dispersion, a clear boundary, and a smooth surface. On scanning electron microscopy, the mean diameter of the microspheres was similar for all six preparations (P > 0.05). During in vitro release, growth factor was initially released via a brief burst release from the outer shell of the microsphere followed by a slower sustained release. The release of growth factors from the inner core remained relatively slow and sustained. Sequential release of different growth factors was achieved through the inconsistent release rates from the microsphere shell and inner core. All groups of microspheres showed no cytotoxicity, good biocompatibility, and the ability to promote osteoblast proliferation. The microspheres loaded with BMP-2 also promoted osteoblast differentiation, and VEGF-loaded microspheres promoted the proliferation and differentiation of vascular endothelial cells. The BMP-2 (core)/VEGF (shell) microsphere group best promoted osteoblast differentiation. The microspheres prepared in this study exhibited slow sequential release of BMP-2 and VEGF and showed good biocompatibility along with the ability to promote osteoblast differentiation and vascular endothelial cell proliferation.

A novel co-culture assay to evaluate the effects of sympathetic innervation on vascular smooth muscle differentiation

Dedifferentiation of vascular smooth muscle cells (VSMCs) from a functional phenotype to an inverse synthetic phenotype is a symptom of cardiovascular disorders, such as atherosclerosis and hypertension. The sympathetic nervous system (SNS) is an essential regulator of the differentiation of vascular smooth muscle cells (VSMCs). In addition, numerous studies suggest that SNS also stimulates VSMCs to retain their contractile phenotype. However, the molecular mechanisms for this stimulation have not been thoroughly studied. In this study, we used a novel in vitro co-culture method to evaluate the effective cellular interactions and stimulatory effects of sympathetic neurons on the differentiation of VSMCs. We co-cultured rat neural-like pheochromocytoma cells (PC12) and rat aortic VSMCs with this method. Expression of VSMCs contractile genes, including smooth muscle actin (acta2), myosin heavy chain (myh11), elastin (eln), and smoothelin (smtn), were determined by quantitative real-time-PCR analysis as an indicator of VSMCs differentiation. Fold changes for specific contractile genes in VSMCs grown in vitro for seven days in the presence (innervated) and absence (non-innervated) of sympathetic neurons were 3.5 for acta2, 6.5 for myh11, 4.19 for eln, and 4 for smtn (normalized to Tata Binding Protein (TBP)). As a result, these data suggest that sympathetic innervation promotes VSMCs' contractile gene expression and also maintains VSMCs' functional phenotype.

Vaspin alleviates the lncRNA LEF1-AS1-induced osteogenic differentiation of vascular smooth muscle cells via the Hippo/YAP signaling pathway

Vascular calcification (VC) is closely related to higher cardiovascular mortality and morbidity, and vascular smooth muscle cell (VSMC) switching to osteogenic-like cells is crucial for VC. LncRNA LEF1-AS1 promotes atherosclerosis and dental pulp stem cells calcification, while its role in VC remains unknown. Visceral adipose tissue-derived serine protease inhibitor (vaspin) is an adipokine regulating bone metabolism. However, the relationship between vaspin and VC is still unclear. We aimed to explore the role of LEF1-AS1 on VSMC osteogenic transition, whether vaspin inhibited LEF1-AS1-mediated osteogenic differentiation of VSMCs, and the responsible mechanism. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis indicated that LEF1-AS1 overexpression significantly upregulated osteogenic marker Runt-related transcription factor-2 (RUNX2) level and downregulated VSMC contractile marker α-smooth muscle actin (α-SMA) level. Alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity assay, and calcium content assay also suggested that LEF1-AS1 overexpression promoted calcium deposition in VSMCs. However, vaspin treatment abolished this phenomenon. Mechanistically, LEF1-AS1 markedly decreased phosphorylated YAP level, while vaspin reversed LEF1-AS1-induced phosphorylated YAP decline. Our results revealed that LEF1-AS1 accelerated the osteogenic differentiation of VSMCs by regulating the Hippo/YAP pathway, while vaspin eliminated the LEF1-AS1-meditated VSMCs osteogenic phenotype switch.