Abstract P162: In vivo miR-431 Inhibition Protects Against Vascular Damage and Hypertension

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Ku-Geng Huo ◽  
Julio C. Fraulob-Aquino ◽  
Tlili Barhoumi ◽  
Chantal Richer ◽  
Suellen C. Coelho ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Type I Collagen ◽  
Vascular Damage ◽  
Mesenteric Arteries ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Type I ◽  
Ang Ii

Background: Vascular injury is an early manifestation of hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury remains unclear. Using small and total RNA sequencing, we identified in murine mesenteric arteries (MAs) a conserved angiotensin (Ang) II-upregulated Dlk1-Dio3 miRNA miR-431 that correlated with blood pressure (BP), and an Ang II-downregulated BP-correlated conserved putative miR-431 target, the transcriptional factor ETS homologous factor ( Ehf ). miR-431 might be involved in vascular remodeling as Ehf regulates expression of extracellular matrix genes including alpha-1 type I collagen ( Col1a1 ) and of other Dlk1-Dio3 miRNAs. In this study, we proposed to validate the miR-431- Ehf - Col1a1 interaction in vitro and in vivo , and determine whether miR-431 inhibition antagonizes angiotensin (Ang) II-induced hypertension and vascular injury. Methods and Results: Transfection of miR-431 mimics into human aortic smooth muscle cells decreased Ehf expression (0.13±0.05 fold, P <0.001) and increased Col1a1 (1.7±0.5 fold, P <0.01), whereas miR-431 inhibitors increased Ehf (1.5±0.2 fold, P <0.001) and decreased Col1a1 (0.89±0.11 fold, P <0.05). Ehf siRNA transfection increased 1.2±0.1 fold Col1a1 ( P <0.01). Co-transfection of miR-431 mimics with luciferase reporter vectors that contain the wild-type but not mutated miR-431 human EHF 3’ UTR binding site decreased 0.51±0.01 fold ( P <0.05) luciferase expression compared to scrambled mimics. miR-431 inhibitor IV injection in mice at day 0 and 7 of Ang II infusion decreased miR-431 (0.16±0.05 fold, P <0.01), Col1a1 (0.58±0.11 fold, P <0.05), increased Ehf (2.9±0.8 fold, P <0.05) in MAs, delayed BP elevation ( P <0.01), improved endothelium-dependent relaxation (33±8 vs 64±7%, P <0.05) and reduced vascular stiffness (strain at 140mmHg: 0.68±0.02 vs 0.58±0.02 ΔD/D, P <0.01) compared to scrambled mimics-injected Ang II-infused mice. Conclusion and Perspectives: miR-431 and its target Ehf may act as master regulators in the pathophysiology of vascular damage in hypertension. miR-431 inhibition has the potential to serve as a novel therapeutic approach for treatment of vascular damage and hypertension.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms

2009 ◽  
Vol 297 (6) ◽  
pp. C1358-C1367 ◽  
Author(s):  
Gerald J. Atkins ◽  
Katie J. Welldon ◽  
Asiri R. Wijenayaka ◽  
Lynda F. Bonewald ◽  
David M. Findlay
Keyword(s):  
Bone Formation ◽  
Vitamin K ◽  
Type I Collagen ◽  
Vitamin K2 ◽  
Type I ◽  
Vitamin K1 ◽  
Vitamin K3 ◽  
Positive Effect

The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that γ-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-κB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.

Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

2001 ◽  
Vol 204 (3) ◽  
pp. 443-455
Author(s):  
C. Faucheux ◽  
S. Nesbitt ◽  
M. Horton ◽  
J. Price
Keyword(s):  
Type I Collagen ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Cathepsin K ◽  
Collagen Matrix ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels

1984 ◽  
Vol 4 (9) ◽  
pp. 1843-1852
Author(s):  
R J Focht ◽  
S L Adams
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Type I Collagen ◽  
Translational Efficiency ◽  
Type I ◽  
Collagen Gene ◽  
Differentiated Cells ◽  
Collagen Gene Expression

We analyzed the control of type I collagen synthesis in four kinds of differentiated cells from chicken embryos which synthesize very different amounts of the protein. Tendon, skin, and smooth muscle cells were found to have identical amounts of type I collagen RNAs; however, the RNAs had inherently different translatabilities, which were observed both in vivo and in vitro. Chondrocytes also had substantial amounts of type I collagen RNAs, even though they directed no detectable synthesis of the protein either in vivo or in vitro. Type I collagen RNAs in chondrocytes display altered electrophoretic mobilities, suggesting that in these cells the reduction in translational efficiency may be mediated in part by changes in the RNA structure. These data indicate that control of type I collagen gene expression is a complex process which is exerted at both transcriptional and post-transcriptional levels.

The influence of the haematocrit on primary haemostasis in vitro

2005 ◽  
Vol 94 (12) ◽  
pp. 1213-1218 ◽  
Author(s):  
Marco Eugster ◽  
Walter H. Reinhart
Keyword(s):  
Platelet Function ◽  
Type I Collagen ◽  
Inverse Correlation ◽  
Type I ◽  
Membrane Pore ◽  
Closure Time ◽  
Function Analyzer ◽  
Platelet Function Analyzer

SummaryPrimary haemostasis consists of platelet adhesion to subendothelial collagen, their activation and aggregation and finally the formation of a platelet plug. Erythrocytes are involved in this process because they flow in the center of the vessel and push platelets towards the site of action on the vessel wall and enhance shear forces, which activate platelets. In the platelet function analyzer PFA-100® (Dade Behring, Düdingen, Switzerland), the in vivo situation is simulated in vitro with blood being aspirated at high shear rates (5000s-1) through a capillary into a membrane pore with a diameter of 150 μm coated with type I collagen and either epinephrine or adenosine diphosphate. Aggregating platelets plug the pore and stop the flow, which is measured as the closure time. We analysed the influence of erythrocytes on platelet function analyzer measurements by systematic variation of the haematocrit (20,30,40,and 50%) at constant platelet counts of 289±61 ×103/μl plasma, or 152±30 ×103/μl blood, 96±9 ×103/μl blood and 54±5 ×103/μl blood, respectively. An inverse correlation was found between haematocrit and closure time under all circumstances. A decrease of the platelet count by 50 ×103 /μl could be compensated for by a 10% increase in haematocrit. The haematocrit must, therefore, be taken into consideration for the correct interpretation of PFA-100® measurements. Our data also provide a pathophysiological rationale to reduce the risk of bleeding in patients with thrombocytopenia and anaemia by normalizing the haematocrit with erythrocyte transfusions.

Overexpression of Long Non-Coding RNA AP001505.9 Inhibits Dedifferentiation in Human Hyaline Chondrocyte

2020 ◽  
Author(s):  
Lin Chen ◽  
Jinying Xu ◽  
Shuang Lv ◽  
Yan Zhao ◽  
Dongjie Sun ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Expression Profiles ◽  
Type I ◽  
Pellet Culture ◽  
Real Time Quantitative Pcr ◽  
Non Coding Rna ◽  
In Situhybridization ◽  
Chondrocyte Implantation

Abstract Background: Autologous chondrocyte implantation (ACI) requires a large number ofhuman hyaline chondrocytes. Unfortunately, human hyaline chondrocytes oftenundergo dedifferentiation in vitro. Long non-coding RNAs (lncRNA) play aregulatory role in gene expression in many pathological and physiological processes.However, their role in human hyaline chondrocyte dedifferentiation remains unclear.This study aimed to investigate the expression profiles of lncRNAs in human hyalinechondrocyte dedifferentiation.Methods: Human hyaline chondrocytes were cultured in vitro and screened for theoccurrence of dedifferentiation using real-time quantitative PCR (qPCR),immunofluorescence, and western blotting. The expression profiles of lncRNAs andmRNAs during dedifferentiation were analyzed by microarray analysis and real-timeqPCR. We used pellet culture to redifferentiate chondrocytes and the expression ofrelated lncRNAs were assessed. The function of lncRNA AP001505.9(ENST00000569966) was determined by overexpression, fluorescence in situhybridization, competing endogenous RNA (ceRNA) analysis, and double luciferaselabeling.Results: We probed human hyaline chondrocytes dedifferentiation and identified 334upregulated and 381 downregulated lncRNAs. The expression of downregulatedlncRNA AP001505.9 in dedifferentiation was reversed by pellet culture. Theoverexpression of AP001505.9 inhibited dedifferentiation by promoting theexpression of SRY-Box transcription factor 9 (SOX-9) and inhibiting the expressionof type I collagen (COL1) both in vitro and in vivo.Conclusion: This study reveals for the first time the expression profiles of lncRNAsin human hyaline chondrocyte dedifferentiation, thereby providing a new perspectivefor exploring the potential mechanism of chondrocyte dedifferentiation.

scholarly journals Evaluation In Vivo and In Vitro of the Antioxidant, Antinociceptive, and Anti-Inflammatory Activities of Biflavonoids From Ouratea hexasperma and O. ferruginea

2019 ◽  
Vol 14 (6) ◽  
pp. 1934578X1985680 ◽  
Author(s):  
Poliana de Araujo Oliveira ◽  
Queli Cristina Fidelis ◽  
Thayane Ferreira da Costa Fernandes ◽  
Milene Conceição de Souza ◽  
Dayane Magalhães Coutinho ◽  
...  
Keyword(s):  
Type I Collagen ◽  
In Vivo Model ◽  
Type I ◽  
Anti Inflammatory ◽  
Vitro Model ◽  
Factor Α ◽  
Necrosis Factor

Ouratea species are used for the treatment of inflammation-related diseases such as rheumatism and arthritic disorders. The Ouratea genus is a rich source of flavonoids and bioflavonoids and for this reason we evaluated the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of complete Freund’s adjuvant (CFA)-induced arthritis and in the in vitro model of oxidative stress and cellular viability. The CFA-induced arthritis model in rats was followed by paw volume, articular incapacitation and Randall-selitto models, as well as quantification of cytokines and serum C-terminal telopeptide of type I collagen levels. OHME and OFME demonstrated antinociceptive and anti-inflammatory activities, as well as improvement in articular incapacity and reduction in levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α, and type 1 collagen, and increased cell viability. No adverse effects were observed. The results suggest that OHME and OFME can reduce inflammation and bone resorption besides their antioxidant action.

scholarly journals Regulation of valve interstitial cell homeostasis by mechanical deformation: implications for heart valve disease and surgical repair

2017 ◽  
Vol 14 (135) ◽  
pp. 20170580 ◽  
Author(s):  
Salma Ayoub ◽  
Chung-Hao Lee ◽  
Kathryn H. Driesbaugh ◽  
Wanda Anselmo ◽  
Connor T. Hughes ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Interstitial Cell ◽  
Smooth Muscle Actin ◽  
Heart Valve Disease ◽  
Type I ◽  
Tissue Remodelling ◽  
Cell Responses ◽  
Stress States

Mechanical stress is one of the major aetiological factors underlying soft-tissue remodelling, especially for the mitral valve (MV). It has been hypothesized that altered MV tissue stress states lead to deviations from cellular homeostasis, resulting in subsequent cellular activation and extracellular matrix (ECM) remodelling. However, a quantitative link between alterations in the organ-level in vivo state and in vitro- based mechanobiology studies has yet to be made. We thus developed an integrated experimental–computational approach to elucidate MV tissue and interstitial cell responses to varying tissue strain levels. Comprehensive results at different length scales revealed that normal responses are observed only within a defined range of tissue deformations, whereas deformations outside of this range lead to hypo- and hyper-synthetic responses, evidenced by changes in α-smooth muscle actin, type I collagen, and other ECM and cell adhesion molecule regulation. We identified MV interstitial cell deformation as a key player in leaflet tissue homeostatic regulation and, as such, used it as the metric that makes the critical link between in vitro responses to simulated equivalent in vivo behaviour. Results indicated that cell responses have a delimited range of in vivo deformations that maintain a homeostatic response, suggesting that deviations from this range may lead to deleterious tissue remodelling and failure.

scholarly journals MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling

2020 ◽  
Vol 295 (52) ◽  
pp. 18051-18064
Author(s):  
Cherry Wongtrakool ◽  
Junsuk Ko ◽  
Andrew J. Jang ◽  
Kora Grooms ◽  
Sarah Chang ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Airway Remodeling ◽  
Luciferase Reporter ◽  
Mouse Lung ◽  
Luciferase Expression ◽  
Nerve Growth ◽  
Pparγ Agonist

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 μg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 μg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo. Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3′UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

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