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Diabetic nephropathy (DN) is the major cause of kidney related diseases in patients induced by high glucose (HG) affecting around 40% of type 1 and 2 diabetic patients. It is characterized by excessive inflammation inducing factors, reactive oxygen species (ROS) overproduction, and potential epigenomic related changes. Fucoxanthin (FX), a carotenoid found in brown seaweed, has a structure which includes an allenic bond and a 5,6-monoepoxide in the molecule, with strong antioxidant and anti-inflammatory activity. However, understanding of the impact of FX on DN was lacking. In this study we tested the early effects of high glucose (HG) on mouse mesangial kidney Mes13 cells, a potential in vitro cell culture model of DN. Our results show that HG induced oxidative stress on kidney mesangial Mes13 cells, while FX treatment attenuates the oxidative stress by decreasing the ROS, demonstrated by flow cytometry. Next, we utilized next-generation sequencing (NGS) to profile the HG-induced early epigenomic and transcriptomic changes in this in vitro DN model and the protective effects of FX. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were analyzed using R software in HG and FX treated groups. Differential regulation of signaling pathways was studied using Reactome Pathway Analysis in the comparison. DEG analysis shows that novel biomarkers with specific pathways, including interleukin regulation, Toll-like receptor pathway, and PKA phosphorylation pathways, were found to be modulated by the FX treatment. TGF β 1i1 (TGFB 1i1), MAP-3-kinase-13(MAP3K13) involved in crucial cellular processes including glucose metabolism, phosphodiesterase regulation was methylated in HG, which was demethylated with FX treatment. Integrated transcriptomic and CpG methylome analysis of DEGs and DMRs revealed that genes like adenylate cyclase (Adcy7), calponin 1 (CNN1), potassium voltage-gated channel interacting protein 2 (KCNIP2), phosphatidylinositol-4-phosphate 5-kinase type 1 β (PIP5K1B), and transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), which were modulated by FX in HG-exposed Mes13 cells, potentially modulate ion channel transport and glucose metabolism. In summary, our current study shows that novel early epigenomic and transcriptomic biomarkers were altered during the disease progression of HG-induced DN and that FX modified these alterations potentially contributing to the protective effects of mesangial cells from the HG-induced oxidative stress and damage.

Objective In the past five decades, many small caliber vascular grafts have been developed as bypasses for infrapopliteal or coronary arteries. However, reliable grafts have not been obtained owing to poor patency, mainly caused by early thrombosis or neointimal hyperplasia in the intermediate period after implantation. We developed a novel small caliber heparin-loaded polyethylene terephthalate ultrafine microfiber (HL-PET) graft and evaluated the feasibility to overcome those main causes of graft failure in canine carotid artery implantation. Methods The HL-PET graft with a diameter of 3 mm and length of 30 mm was made with combination of three key technologies: (1) weaving with PET ultrafine microfiber with a high biological porosity allowing for cell ingrowth, (2) heparin loading on microfiber surfaces, and (3) an outer coating with a flexible bioabsorbable polymer for prevention of blood leakage and graft kinking. Kink resistance, water permeability, and loaded heparin were assessed. One HL-PET graft each was implanted into a carotid artery of six animals. Graft patency rate and healing were assessed 24 weeks after implantation. Results Among the six grafts, five were deemed patent (patency rate of >83%), with one occluded 20 weeks after implantation. Histopathology of the patent grafts showed neointima formation with confluent endothelial cell lining (estimated mean endothelial cell coverage area, 89 ± 18%). Intimal hyperplasia at the anastomotic sites and severe chronic inflammatory responses were not observed. Immunohistochemistry with antibodies to endothelial nitric oxide synthase, alpha 2 smooth muscle actin and calponin 1 revealed luminal surface endothelial cell layer with expression of endothelial nitric oxide synthase and vascular smooth muscle cells with contractile phenotype in the subintimal layer. Conclusions The HL-PET graft showed no early postoperative thrombosis and was able to demonstrate a high patency rate with no severe biological response observed after 24 weeks. These results strongly suggest the potential of the HL-PET graft to be used for distal bypasses., Clinical Relevance The HL-PET graft achieved a high patency rate without early postoperative thrombosis or intimal hyperplasia in a small caliber artery implantation study. The histopathologic analysis demonstrated favorable healing property with a high antithrombogenicity. These results strongly suggested the possible use of the HL-PET graft as a bypass for infrapopliteal or coronary arteries.

Background In-stent restenosis (ISR) is a complex disease that occurs after coronary stenting procedures. The development of quality materials and improvement of our understanding on significant factors regulating ISR are essential for enhancing prognosis. Vascular smooth muscle cells (VSMCs) are the main constituent cells of blood vessel walls, and dysfunction of VMSCs can exacerbate ISR. Accordingly, in this study, we explored the influence of wrinkled material topography on the biological functions of VSMCs. Methods Polydimethylsiloxane with a wrinkled topography was synthesized using elastomer base and crosslinking and observed by atomic force microscopy. VSMC proliferation, apoptosis, and morphology were determined by Cell Counting Kit-8 assays, fluorescence-assisted cell sorting, and phalloidin staining. α-Smooth muscle actin (α-SMA), major histocompatibility complex (MHC), and calponin 1 (CNN-1) expression levels were measured by quantitative real-time polymerase chain reaction and western blotting. Moreover, p53 and cleaved caspase-3 expression levels were evaluated by western blotting in VSMCs to assess apoptotic induction. Results Surface topographies were not associated with a clear orientation or elongation of VSMCs. The number of cells was increased on wrinkled surfaces (0.7 μm in amplitude, and 3 μm in wavelength [W3]) compared with that on other surfaces, contributing to continuously increased cell proliferation. Moreover, interactions of VSMCs with the W3 surface suppressed phenotypic switching, resulting in ISR via regulation of α-SMA, calponin-1, and SM-MHC expression. The surface with an amplitude of 0.05 μm and a wavelength of 0.5 μm (W0.5) promoted apoptosis by inducing caspase 3 and p53 activities. Conclusion Introduction of aligned topographies on biomaterial scaffolds could provide physical cues to modulate VSMC responses for engineering vascular constructs. Materials with wrinkled topographies could have applications in the development of stents to reduce ISR.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin‐1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD‐1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin‐1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low‐risk) DCIS lesions clustered in a high calponin‐1, T cell low group, whereas the majority of mixed (high‐risk) DCIS lesions clustered in a low calponin‐1, T cell high group, specifically with CD8+ and PD‐1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin‐1 and immune signature as the majority of mixed DCIS lesions, which have low calponin‐1 and T cell enrichment—raising the possibility that these pure DCIS lesions might be at a high risk for progression.

Objectives: Breast cancer has been the second most prevalent and fatal malignancy due to its frequent metastasis to other organs. We aim to study the effects of a key miRNA-mRNA signaling in breast cancer. Results: CNN1 was identified as the key gene in breast cancer by the bioinformatics analysis, and the downregulation of CNN1 in breast cancer tissues and cell lines was observed. Upregulating CNN1 inhibited cell survival, migration, invasion, and adhesion, but enhanced cell apoptosis. miR-106b-5p not only bound to CNN1 mRNA 3’UTR, but also promoted lung metastasis in vivo. Besides, the miR-106b-5p mimic enhanced breast cancer canceration by targeting CNN1 and activating Rho/ROCK1 signaling pathway. Conclusion: Overall, our results proved that miR-106b-5p promoted the metastasis of breast cancer by suppressing CNN1 and activating Rho/ROCK1 pathway. Methods: Bioinformatics analysis was performed to select the key gene in breast cancer. The overexpression and knockdown of Calponin 1 (CNN1) in breast cancer cell lines were performed to conduct cell viability, migrating, invasion, proliferation, adhesion, and apoptosis experiments. To identify the role of miR-106b-5p and Rho/ROCK1 in CNN1-induced breast cancer, a dual-luciferase assay, tumor lung metastasis assay, transcript half-life assay, and Rho/ROCK1 inhibition assay were performed.

The aging of the population has led to an annual increase in the incidence of vascular calcification (VC). Specific protein 1 (Sp1) is a transcriptional activator that serves an important role in VC. The deacetylation of transcription factors represses their binding to the promoters of downstream genes, thereby causing their downregulation. The present study aimed to investigate the role of deacetylated Sp1 in the development of VC. In the present study, western blotting and immunoprecipitation (IP) were performed to detect the protein levels of acetylated Sp1. Western blotting and immunofluorescence staining were used to analyze phenotypic switching in vascular smooth muscle cells (VSMCs). Alizarin red S, alkaline phosphatase (ALP) activity and calcium content assays were used to assess calcium deposition in VSMCs. Western blotting, flow cytometry, TUNEL staining and caspase3 activity assay were used to evaluate apoptosis of VSMCs. Chromatin immunoprecipitation (ChIP) assay was used to detect Sp1 binding to the BMP2 promoter. The results indicated that, in a β-glycerophosphate (β-GP)-induced VSMC calcification model, the level of acetylated Sp1 was increased. Western blotting and immunofluorescence staining results showed that, compared with the Sp1 overexpression group (Sp1-WT), deacetylated Sp1 (Sp1-K704A) downregulated the expression of osteogenic markers runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and upregulated the expression of contraction marker α-smooth muscle actin (α-SMA) and calponin 1. In addition, deacetylated Sp1 also reduced the ALP activity and calcium content of calcified VSMCs, and the Alizarin red S assay revealed that the calcium crystallization of Sp1-K704A group was markedly decreased. Western blotting, flow cytometry, TUNEL staining and caspase-3 activity assay were detected to indicate that the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein ratio was increased, and caspase-3 activity and the apoptotic rate of VSMCs were decreased, in the Sp1-K704A group, as compared with the Sp1-WT group. ChIP assay revealed that Sp1 binding to the BMP2 promoter was downregulated in the Sp1-K704A group, compared with that in theSp1-WT group. In conclusion, a deacetylated mutant of Sp1 decreased Sp1 binding to the BMP2 promoter, thus decreasing apoptosis, phenotypic switching and calcium deposition in calcified VSMCs. This finding may indicate potential therapeutic targets for VC.

Deoxynivalenol (DON) is a prevalent Fusarium mycotoxin, occurs predominantly in the global environment, especially in cereals, animal feed and food commodities. The widespread contamination causes a serious risk to human and animal health. DON usually impairs weight gain, which is presumably from its capacity to reduce feed intake by interfering with intestinal motility. To clarify the role of smooth muscle cells (SMCs) contractility intestinal motility and growth inhibition caused by DON, twelve weaned piglets were firstly divided into two groups to feed control or Fusarium mycotoxin-contaminated (MC) diet. Results showed that the final body weight, average daily gain and average daily feed intake were significantly reduced in piglets fed the MC diet. Exposure to the MC diet also significantly decreased the thickness of smooth muscle layer and SMCs contractile markers expression (myosin heavy chain 11, smooth muscle actin gamma 2, transgelin, calponin 1) in jejunum and ileum of piglets. Furthermore, oral DON supplementation (3 mg/kg body weight) to mice in six consecutive days could significantly inhibit the upper intestinal transit, impede normal defecation and downregulate SMCs contractile markers expression in small intestine. Finally, we generated a porcine enteric smooth muscle cell line (PISMC), and found that DON could depress its contractility by decreasing PISMC proliferation, migration and contractile markers expression. In conclusion, these findings in vivo and in vitro suggest that DON, as a common environmental toxin, can not only reduce proliferative and motile phenotype, but also decrease contractile apparatus components (contractile markers expression) in SMCs, which in turn influences SMCs contractility and then interferes with intestinal motility and growth performance. Jiangsu Modern Agricultural (Live Pig) Industry Technology System, China [JATS [2020] 412]; Science and Technology Innovation Team Project of Anhui Academy of Agricultural Sciences, China [2021YL027]; China Scholarship Council, ChinaChina Scholarship Council Published version This study was supported by a grant from the Jiangsu Modern Agricultural (Live Pig) Industry Technology System, China (JATS [2020] 412), and Science and Technology Innovation Team Project of Anhui Academy of Agricultural Sciences, China (2021YL027). The authors also would like to thank the China Scholarship Council, China that offered Xu Ji a scholarship for his studying at Virginia Tech as a visiting scholar.

IntroductionThe purpose of this study was to explore the regulatory mechanisms of FGF2 on carotid atherosclerotic plaque development using bioinformatics analysis.Material and methodsExpression profiles of 32 atheroma plaque (AP) and 32 paired distant macroscopically intact (DMI) tissues samples in GSE43292 dataset were downloaded from the Gene Expression Omnibus database. Following identification of differential expression genes (DEGs), correlation analysis of fibroblast growth factor 2 (FGF2) and DEGs was conducted. Subsequently, functional enrichment analysis and protein-protein interaction network for FGF2 significantly correlated DEGs were constructed. Then, microRNAs (miRNAs) that regulated FGF2 and regulatory pairs of long noncoding RNA (lncRNA)-miRNA were predicted to construct lncRNA-miRNA-FGF2 network.ResultsA total of 101 DEGs between AP and DMI samples were identified, and 31 DEGs were analyzed to have coexpression relationships with FGF2, including 23 positively correlated and 8 negatively correlated DEGs. VAV3 had the lowest r value among all FGF2 negatively correlated DEGs. FGF2 positively correlated DEGs was closely related to “regulation of smooth muscle contraction” [eg., Calponin 1 (CNN1)], while FGF2 negatively correlated DEGs was significantly associated with “platelet activation” [eg., Vav Guanine Nucleotide Exchange Factor 3 (VAV3)]. In addition, totally 12 miRNAs that regulated FGF2 were predicted, and hsa-miR-15a-5p and hsa-miR-16-5p were highlighted in lncRNA-miRNA-FGF2 regulatory network.ConclusionsCNN1 might cooperate with FGF2 to regulate smooth muscle contractility during CAP formation. VAV3 might cooperate with FGF2 to be responsible for the development of CAP through participating in platelet activation. Hsa-miR-15a-5p and hsa-miR-16-5p might participate in the development of CAP via regulating FGF2.

The ingestion of enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, or Salmonella enterica serovar Typhimurium leads to their colonization of the intestinal lumen, which ultimately causes an array of ailments ranging from diarrhea to bacteremia. Once in the intestines, these microbes generate various actin-rich structures to attach, invade, or move within the host intestinal epithelial cells. Although an assortment of actin-associated proteins has been identified to varying degrees at these structures, the localization of many actin stabilizing proteins have yet to be analyzed. Here, we examined the recruitment of the actin-associated proteins, calponin 1 and 2 at EPEC pedestals, L. monocytogenes actin clouds, comet tails and listeriopods, and S. Typhimurium membrane ruffles. In other systems, calponins are known to bind to and stabilize actin filaments. In EPEC pedestals, calponin 1 was recruited uniformly throughout the structures while calponin 2 was enriched at the apical tip. During L. monocytogenes infections, calponin 1 was found through all the actin-rich structures generated by the bacteria, while calponin 2 was only present within actin-rich structures formed by L. monocytogenes near the host cell membrane. Finally, both calponins were found within S. Typhimurium-generated membrane ruffles. Taken together, we have shown that although calponin 1 is recruited to actin-rich structures formed by the three bacteria, calponin 2 is specifically recruited to only membrane-bound actin-rich structures formed by the bacteria. Thus, our findings suggest that calponin 2 is a novel marker for membrane-bound actin structures formed by pathogenic bacteria. Anat Rec, 301:2103-2111, 2018. © 2018 Wiley Periodicals, Inc.

The present study aimed to investigate the effect of calponin 1 (CNN1) on the invasion and migration of lung squamous cell carcinoma (LUSC) cells and the associations between CNN1, tissue inhibitor of metalloproteinases 2 (TIMP2), Dickkopf-1 (DKK1) and the Wnt/β-catenin/c-myc signaling pathway. The expression levels of CNN1 and TIMP2 in LUSC cells and the association between CNN1 and TIMP2 were predicted using the GEPIA database. The cells were transiently transfected to overexpress CNN1, which resulted in inhibition of DKK1 and TIMP2 expression levels. Wound healing and Transwell assays were used to detect the invasive and migratory abilities of LUSC cells. Reverse transcription-quantitative PCR and western blotting were used to investigate the expression levels of CNN1, MMP2, MMP9, E-cadherin, N-cadherin (N-cad), SLUG, DKK1, β-catenin and c-myc. The expression levels of N-cad were detected using immunofluorescence staining. The results indicated that overexpression of CNN1 inhibited the invasion and migration of NCI-H2170 cells. Inhibition of DKK1 reversed this change and the expression levels of β-catenin and c-myc were upregulated, whereas the expression levels of DKK1 were downregulated with a concomitant inhibition of TIMP2. In summary, these results demonstrated that CNN1 regulated the DKK1/Wnt/β-catenin/c-myc signaling pathway by activating TIMP2 to inhibit the invasion, migration and epithelial-to-mesenchymal transition of LUSC cells.

Dermal fibroblast aging contributes to aging-associated functional defects in the skin since dermal fibroblasts are important to maintain skin homeostasis by interacting with epidermis and extracellular matrix. Here we identified that puerarin, an isoflavone contained in Pueraria lobata (Kudzu), can prevent the aging-phenotype of human dermal fibroblasts. Puerarin treatment increased in proliferating cells and decreased in senescence-associated beta-galactosidase positive cells in the high-passage culture of dermal fibroblasts. Moreover, puerarin reduced smooth muscle actin-positive myofibroblasts and the expression of a reticular fibroblast marker, calponin 1 (CNN1), which were induced in high-passage fibroblasts. Fulvestrant, an estrogen receptor antagonist, blocked puerarin-mediated downregulation of SMA and CNN1. Our results suggest that puerarin may be a useful food factor that alleviates aging-related functional defects in the skin.

Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.

Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.

Objective— Dysregulated proliferation of vascular smooth muscle cells (VSMC) plays an essential role in neointimal hyperplasia. CD36 functions critically in atherogenesis and thrombosis. We hypothesize that CD36 regulates VSMC proliferation and contributes to the development of obstructive vascular diseases. Approach and Results— We found by immunofluorescent staining that CD36 was highly expressed in human vessels with obstructive diseases. Using guidewire-induced carotid artery injury and shear stress–induced intima thickening models, we compared neointimal hyperplasia in Apoe −/− , Cd36 −/− /Apoe −/− , and CD36 specifically deleted in VSMC (VSMC cd36 −/− ) mice. CD36 deficiency, either global or VSMC-specific, dramatically reduced injury-induced neointimal thickening. Correspondingly, carotid artery blood flow was significantly increased in Cd36 −/− /Apoe −/− compared with Apoe −/− mice. In cultured VSMCs from thoracic aorta of wild-type and Cd36 −/− mice, we found that loss of CD36 significantly decreased serum-stimulated proliferation and increased cell populations in S phase, suggesting that CD36 is necessary for VSMC S/G2-M-phase transition. Treatment of VSMCs with a TSR (thrombospondin type 1 repeat) peptide significantly increased wild-type, but not Cd36 −/− VSMC proliferation. TSR or serum treatment significantly increased cyclin A expression in wild-type, but not in Cd36 −/− VSMCs. STAT3 (signal transducer and activator of transcription), which reportedly enhances both VSMC differentiation and maturation, was higher in Cd36 −/− VSMCs. CD36 deficiency significantly decreased expression of Col1A1 (type 1 collagen A1 chain) and TGF-β1 (transforming growth factor beta 1), and increased expression of contractile proteins, including calponin 1 and smooth muscle α actin, and dramatically increased cell contraction. Conclusions— CD36 promotes VSMC proliferation via upregulation of cyclin A expression that contributes to the development of neointimal hyperplasia, collagen deposition, and obstructive vascular diseases.

Dermal fibroblast aging contributes to aging-associated functional defects in the skin since dermal fibroblasts maintain skin homeostasis by interacting with the epidermis and extracellular matrix. Here, we found that puerarin, an isoflavone present in Pueraria lobata (Kudzu), can prevent the development of the aging-phenotype in human dermal fibroblasts. Normal human dermal fibroblasts (NHDFs) were subcultivated and high-passage cells were selected as senescent cells, whereas low-passage cells were selected as a young cell control. Puerarin treatment increased cell proliferation and decreased the proportion of senescence-associated beta-galactosidase-positive cells in a high-passage culture of NHDFs. Moreover, puerarin treatment reduced the number of smooth muscle actin (SMA)-positive myofibroblasts and the expression of a reticular fibroblast marker, calponin 1 (CNN1), which were induced in high-passage NHDFs. Fulvestrant, an estrogen receptor antagonist, blocked the puerarin-mediated downregulation of SMA and CNN1. Our results suggest that puerarin may be a useful functional food that alleviates aging-related functional defects in dermal fibroblasts.

Increased matrix metalloproteinase (MMP)-2 is implicated in the vascular remodeling of hypertension. Calponin-1 is a contractile protein, and its absence is associated with vascular smooth muscle cell (VSMC) phenotype switch, which leads to migration and remodeling. We evaluated whether increased MMP-2 activity precedes chronic vascular remodeling by decreasing calponin-1 and inducing VSMC proliferation. Sham or two kidney-one clip (2K1C) rats were treated with doxycycline at 30mg/kg/day. Systolic blood pressure was increased in the 2K1C rats after 1 and 2weeks post-surgery, and doxycycline was effective to reduce it only at 2weeks of hypertension (p

Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and multiple types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation.

The switch of vascular smooth muscle cells (SMCs) from the contractile phenotype to proliferative one can make contributions to atherosclerosis and neointima formation. MiR-21 can prevent the rupture of advanced lesion plaques. We previously reported the protection of DNA topoisomerase II (Topo II) inhibitors against atherosclerosis and vascular calcification. However, it remains unknown if Topo II inhibitors can change SMC phenotypes. Herein, we show that teniposide protected SMC phenotype switching during atherosclerosis by enhancing expression of smooth muscle α-actin (SMA) while reducing osteopontin (OPN) expression in aortic lesion plaques. In vitro, teniposide induced expression of smooth muscle protein 22-α and calponin 1, but inhibited expression of OPN and epiregulin in human aortic SMCs (HASMCs). Moreover, teniposide attenuated platelet derived growth factor-BB-induced HASMC proliferation and migration. Mechanistically, the effect of teniposide on SMC phenotypes was completed, at least in part, by activating miR-21 expression. In addition, teniposide ameliorated ligation-induced carotid artery remodeling in C57BL/6J mice by regulating SMA and OPN expression. Taken together, our study demonstrates that teniposide regulates SMC phenotype switching by upregulating expression of contractile genes in a miR-21-dependent manner, and this function is an important anti-atherogenic mechanism of teniposide.

Hypertension is characterized by maladaptive vascular remodeling and enhanced oxidative stress in the vascular wall. Peroxynitrite may directly activate latent matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMC) by its S-glutathiolation. MMP-2 may then proteolyze calponin-1 in aortas from hypertensive animals, which stimulates VSMC proliferation and medial hypertrophy. Calponin-1 is an intracellular protein which helps to maintain VSMC in their differentiated (contractile) phenotype. The present study therefore investigated whether aortic MMP-2 activity is increased by oxidative stress in early hypertension and then contributes to hypertrophic arterial remodeling by reducing the levels of calponin-1. Male Wistar rats were submitted to the two kidney, one clip (2 K-1C) model of hypertension or sham surgery and were treated daily with tempol (18 mg/kg/day) or its vehicle (water) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP) was daily assessed by tail-cuff plethysmography. After one week, aortas were removed to perform morphological analysis with hematoxylin and eosin staining and to analyze reactive oxygen‑nitrogen species levels by dihydroethidium and immunohistochemistry for nitrotyrosine. MMP-2 activity was analyzed by in situ and gelatin zymography and its S-glutathiolation was analyzed by Western blot for MMP-2 of anti-glutathione immunoprecipitates. Calponin-1 levels were identified in aortas by immunofluorescence. SBP increased by approximately 50 mmHg at the first week in 2 K-1C rats which was unaffected by tempol. However, tempol ameliorated the hypertension-induced increase in arterial media-to-lumen ratio and hypertrophic remodeling. Tempol also decreased hypertension-induced aortic oxidative stress and the enhanced MMP-2 activity. S-glutathiolation may be a potential mechanism by which oxidative stress activates MMP-2 in aortas of 2 K-1C rats. Furthermore, calponin-1 was decreased in aortas from 2 K-1C rats and tempol prevented this. In conclusion, oxidative stress may contribute to the increase in aortic MMP-2 activity, possibly by S-glutathiolation, and this may result in calponin-1 loss and maladaptive vascular remodeling in early hypertension.

Background The concept of tissue engineering is to deliver to the injury site biological scaffolds carrying functional cells that will enhance healing response. The preferred cell source is autologous in order to reduce immune response in the treated individual. However, in elderly patients age-related changes in synthetic activity of the implanted cells and subsequent alterations in tissue protein content may affect therapeutic outcomes. In this study we investigated the effect of donor age on proteome composition of tenocyte-derived tendon tissue-engineered constructs. Results Liquid chromatography tandem mass spectrometry was used to assess the proteome of tissue-engineered constructs derived from young and old equine tenocytes. Ageing was associated with altered extracellular matrix composition, especially accumulation of collagens (type I, III and XIV), and lower cytoskeletal turnover. Proteins involved in cell responsiveness to mechanical stimuli and cell-extracellular matrix interaction (calponin 1, palladin, caldesmon 1, cortactin) were affected. Conclusions This study demonstrated significant changes in proteome of engineered tendon derived from young and old tenocytes, indicating the impact of donor age on composition of autologous constructs. Electronic supplementary material The online version of this article (10.1186/s12896-018-0414-5) contains supplementary material, which is available to authorized users.

To assess the utility of maternal serum calponin 1 level in the prediction of delivery within 7 days among pregnancies complicated with threatened preterm labor.Eligible women who presented at 24-34 weeks of gestation with threatened preterm labor underwent sampling for serum calponin 1 level and cervical length measurement. They were followed up until delivery prospectively and the perinatal outcomes of the patients were recorded.Of 73 women included in the study, 36 women delivered within 7 days and 37 women delivered beyond 7 days after admission. The maternal serum calponin 1 level was significantly high in women who delivered within 7 days (p: 0.031). The threshold value of 2 ng/mL for maternal serum calponin 1 predicted delivery within 7 days with 61.1% sensitivity and 62.2 specificity (area under curve, 0.658, confidence interval 0.53-0.79). The general accuracy values for maternal cervical length measurement (≤25 mm), serum calponin 1 level (2 ng/mL) and the combination of two tests to predict delivery within 7 days was found to be 64.4%, 61.6% and 72.1%, respectively.The maternal serum calponin 1 level may be a useful biomarker in short-term prediction of preterm birth among pregnancies complicated with threatened preterm labor, in addition to cervical length measurement.

Previous studies have shown that the loss of contractility in aortas from lipopolysaccharide (LPS)-treated rats is related to intracellular activation of matrix metalloproteinase (MMPs). However, the role of MMPs in the vascular refractoriness to vasoconstrictors has not been investigated in a model of polymicrobial sepsis. We evaluated the effects of the oral administration of the MMP inhibitors doxycycline or ONO-4817 in the in vitro vascular reactivity of aortic rings from rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both doxycycline and ONO-4817 did not change vascular responses in sham-operated rats, but fully prevented hyporeactivity to KCl, phenylephrine and angiotensin II in vessels from CLP rats. This protective effect was not associated with changes in hematological parameters or blood nitrate and nitrite. The refractoriness to contractile agents was accompanied by enhanced activity of MMP-2 in aorta from CLP rats, which was abrogated by MMP inhibitors. CLP-induced sepsis did not impair the levels of MMP-2 in aorta, but significantly reduced calponin-1, a regulatory protein of vascular contraction. In addition, augmented levels of TIMP-1 were found in vessels from CLP rats. All these differences were prevented by either doxycycline or ONO-4817. Our study shows, for the first time in the CLP rat model of sepsis, that the vascular refractoriness to different contractile agents induced by polymicrobial sepsis is associated with increased activity of MMP-2 and reduced amounts of calponin-1 in the aorta. These findings reinforce the importance of the enhanced activity of MMPs for vascular failure in septic shock.

Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, calponin 1 is smooth muscle specific and calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of calponin 1 and calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that calponin 1 and calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of calponin 1 was found in smooth muscle of Cnn2(−/−) mice but not vice versa. Cnn1(−/−) and Cnn2(−/−) double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of calponin 1 and calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

International audience; The vascular smooth muscle cell (VSMC) phenotypic switch is a key pathophysiological change in various cardiovascular diseases, such as aortic dissection (AD), with a high morbidity. Polycystin-1 (PC1) is significantly downregulated in the VSMCs of AD patients. PC1 is an integral membrane glycoprotein and kinase that regulates different biological processes, including cell proliferation, apoptosis, and cell polarity. However, the role of PC1 in intracellular signaling pathways remains poorly understood. In this study, PC1 downregulation in VSMCs promoted the expression of SM22α, ACTA2 and calponin 1 (CNN1) proteins. Furthermore, PC1 downregulation in VSMCs upregulated phospho-MEK, phospho-ERK and myc, but did not change phospho-JNK and phospho-p38. These findings suggest that the MEK/ERK/myc signaling pathway is involved in PC1-mediated human VSMC phenotypic switch. Opposite results were observed when an ERK inhibitor was used in VSMCs downregulated by PC1. When the C-terminal domain of PC1 (PC1 C-tail) was overexpressed in VSMCs, the expression levels of phosphor-ERK, myc, SM22α, ACTA2 and CNN1 proteins were downregulated. The group with the overexpressed mutant protein (S4166A) in the PC1 C-tail showed similar results to the group with the downregulated PC1 in VSMCs. These results suggest that the Ser at the 4166 site in PC1 is crucial in the PC1 mediated MEK/ERK/myc signaling pathway, which might be the key pathophysiological cause of AD.

Background & Aims The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene ( Gnas , in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. Methods We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (Gsa SMKO and SM22-CreER T2 , induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas . Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. Results Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene ( Foxf1 ) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. Conclusions Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.

Calponin is an actin filament associated regulatory protein in smooth muscle and non-muscle cells. Calponin-1 is specifically expressed in smooth muscle cells and calponin-2 is expressed in both smooth muscle and multiple non-muscle cell types. The role of calponin-1 in the regulation of smooth muscle contraction has been extensively investigated. Calponin-1 gene knockout mice demonstrated minor but clearly detectable changes in smooth muscle contractility, especially in the urogenital system. Here we compared the impacts of single and double knockout of calponin-1 and calponin-2 genes in mice on smooth muscle contractility. The results showed that either calponin-1 or calponin-2 knockout did not significantly alter the passive tension-active tension relationship in aortic smooth muscle and the mice exhibited normal blood pressure. The double knockout mice survived well into adulthood in the absence of physiological stress. However, the double knockout mice showed higher passive tension and less KCl- and norepinephrine-induced active tension in endothelium-removed aorta rings. The double knockout mice also showed lower systolic and diastolic blood pressures compared to gender-matched wild type controls. The results suggest that calponin-1 and −2 both contribute to the regulation of vascular smooth muscle contractility. The minimized phenotypic effects of calponin-1 and calponin-2 single knockout mice indicate a mutual compensation between the two isoforms in smooth muscle cells. Supporting this notion, analysis of protein expression in various types of smooth muscle showed up-regulations of one isoform when the other was deleted. The results implicate that calponins are molecular targets for modifications of smooth muscle contractility and especially blood pressure. Simultaneously targeting the expression and function of both two calponins may provide a potential therapeutic approach to the development of new treatment of hypertension.

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

Smoothelin-B (SMTL-B) and calponin-1 are important regulators of vascular contraction. SMTL-B contains a calponin-homology domain and is structurally similar to cardiac troponin T. As calponin-1 and troponin T are proteolyzed by intracellular matrix metalloproteinase (MMP)-2 in oxidative stress injury, we hypothesized that SMTL-B is also cleaved by MMP-2 and contributes to lipopolysaccharide (LPS)-induced vascular hypocontractility. Rats received ONO-4817 (an MMP inhibitor) or its vehicle, 2 h prior to being administered lipopolysaccharide (LPS). LPS-induced aorta hypocontractility to potassium chloride or phenylephrine, and reduction of calponin-1 levels, were abolished by ONO-4817 at 6 but not 3 h after LPS. However, the level of SMTL-B was unaltered in LPS aortas and further unaffected by ONO-4817. Despite the importance of SMTL-B in vascular tone, it is not a target of MMP-2 in LPS-induced hypocontractility.