Recommended
More Related Content
What's hot
What's hot (17)
Similar to Platelet derived mi r-223 promotes a phenotypicswitch in arterial injury repair
Similar to Platelet derived mi r-223 promotes a phenotypicswitch in arterial injury repair (19)
More from SAIMA BARKI
More from SAIMA BARKI (7)
Recently uploaded
Recently uploaded (20)
Platelet derived mi r-223 promotes a phenotypicswitch in arterial injury repair
- 1. Platelet-Derived miR-223 Promotes a Phenotypic Switch In Arterial Injury Repair SAIMA BARKI QUAID-E-AZAM UNIVERSITY, ISLAMABAD PAKISTAN 19 MARCH, 2019
- 2. Platelets No genomic DNAAnucleate Diverse array of mRNA and miRNA. Low level of protein expression Aps miRNA complexed with Ago2 AgomiR complex horizontal miRNA transfer into VSMCs Receipient cell modulate gene expression and function
- 3. Platelets and Blood Vessel NO & PGI2 → ↑ increases platelet cAMP thus inhibits platelet aggregation. In healthy vasculature, circulating platelets are maintained in an inactive state by: nitric oxide (NO) prostacyclin (PGI2)
- 4. ARTERIAL INJURY AND PLATELETS ACTIVATION Arterial injury Endothelial denudation Platelet activation and thrombosis
- 5. Arterial Injury And Platelets Release bioactive mediators 01 Internalization by VSMCs 02 DM , Reduced miR- 223 expression, intimal hyperplasia 03
- 6. Platelets internalization by VSMCs and induction of VSMCs differentiation
- 9. Reduced Cell Proliferation : CCK8 & Brdu incorporation Assay
- 10. VSMCs Coculturing With CMDA Labeled Platelets In Presence Of Thrombin
- 11. 3D Reconstruction of VSMCs cocultured with platelets • Confocal laser scanning microscopy • CMFDA-labelled platelets(green)
- 12. TEM: Sequential internalization of Platelets And incorporation in VSMCs
- 13. In vivo study: Sections of femoral arteries of PF4- mT/mG and WT Mice
- 14. APs transferred miR-143,-145 and miR-223 into VSMCs and inhibit VSMCs dedifferentiation
- 16. Heatmap analysis of differentially expressed miRNA in VSMCs + with/without APs
- 17. Quantitative analysis of expression of miRNAs at various time points
- 18. To determine incorporated miRNA functionality in VSMCs IP of AgomiR followed by qPCR
- 19. Expression Study of miRNAs after treatment with prostacyclin and RNAses
- 20. Expression of ACTA2 and KLF4 After prostacyclin/RNAseA and Thrombin
- 21. Thrombin Did Not Change The Levels Of miRNAs In VSMCs.
- 22. Schematic representation of PDGFRβ, KLF4, KLF5 3’UTRs showing putative miRNA target site
- 23. Incorporation of platelet-derived miRNAs is required for inhibiting VSMCs dedifferentiation miR-233 modulates PdgfrB and ACTA2 In Femoral arterial injury
- 24. Levels of miRNAs in PLPS in MEG-01 Cells at different conc. Of glucose
- 25. Expression of dedifferentiation markers from PLPs of MEG-01 at different conc. glucose concentration
- 26. Dedifferentiation marker expression in VSMCs transfected with miRNAs mimics
- 27. miR-143, miR-145, and miR- 223 were overexpressed in high glucose treated MEG-01 cells-derived platelet-like particles (HG PLPs) Lipofectamine transfection
- 28. VSMCs cocultured with APs: dedifferentiation, proliferation and miR-233 detection
- 29. miR-223 modulates Pdgfrβ expression and intimal hyperplasia in vivo after femoral arterial injury
- 30. Immunofluorescence of Pdgfrβ in WT and KO-mice
- 31. Quantification of Pdgfrβ in VSMCs in injured femoral arteries
- 33. Morphometric measurements of I/M Ratio in injured femoral arteries
- 35. The level of miR-223 in VSMCs co-cultured with platelets-derived microparticles and activated platelets.
- 36. DM Platelets reduced miR- 223 level and increased intimal hyperplasia
- 37. Relative miR- 223 expression in human and mouse
- 38. Sections from injured and injured femoral arteries of PF4-cre:mT/mG STZ DM Mice
- 39. miR-223 expression in injured femoral arteries of PF4-cre mT/mG mice FISH
- 40. Quantification of miR-223 in mGFP positive VSMCs in injured femoral arteries
- 41. Platelet derived miR-223 is responsible for inhibition of neointima formation in diabetic mice after femoral artery wire injury
- 42. Pdgfrβ expression in PF4-cre mT/mG mice HR CLSM
- 43. Quantification of Pdgfrβ in VSMCs in mGFP +ve VSMCS IN injured femoral arteries
- 44. Immunofluorescence of Pdgfrβ AFTER 4 WEEKS
- 45. Quantification of Pdgfrβ expression in VSMCs in injured femoral arteries
- 46. H&E Staining of cross sections after 4 weeks of injury
- 47. Morphometric measurement of I/M ratio in injured femoral sections
Editor's Notes
- The expression of CNN1 is specific to differentiated mature smooth muscle cells, suggesting a role in contractile functions. Calponin 1 is up-regulated in smooth muscle tissues during postnatal development with a higher content in phasic smooth muscle of the digestive tract. OSTEOPONTIN involved in cell attachment and wound healing. The encoded protein is thought to control the G1-to-S transition of the cell cycle following DNA damage by mediating the tumor suppressor gene p53 It may participate in both promoting and suppressing cell proliferation. Expression of this gene may be changed in a variety of different cancers and in cardiovascular disease. The protein encoded by this gene is a transformation and shape-change sensitive actin cross-linking/gelling protein found in fibroblasts and smooth muscle. Its expression is down-regulated in many cell lines, and this down-regulation may be an early and sensitive marker for the onset of transformation
- Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. 5-bromo-2'-deoxyuridine) is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells.
- CMDA=5-chloromethyl fluorescein diacetate (CMFDA) is a fluorescent dye well suited for monitoring cell movement or location. After loading into cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The green excitation/emission spectra are ideal for multiplexing with red fluorescent dyes and proteins. Thrombin is the principal enzyme of hemostasis. It catalyzes the conversion of fibrinogen to fibrin and activates procoagulant factors V, VIII, XI, and XIII. Additionally, when bound to thrombomodulin, it activates protein C, an anticoagulant zymogen
- A heat map (or heatmap) is a graphical representation of data where the individual values contained in a matrix are represented as colors. "Heat map" is a newer term but shading matrices have existed for over a century.
- HUMan aortic smooth muscle cells
- Actin alpha 2 Glyceraldehyde 3-phosphate dehydrogenase (abbreviated as GAPDH or less commonly as G3PDH GAPDH gene is often stably and constitutively expressed at high levels in many tissues and cells, which is considered as a housekeeping gene. The role of prostacyclin in vascular tissue. Prostacyclin(PGI2) generated by the vascular wall is a potent vasodilator, and the most potent endogenous inhibitor of platelet aggregation so far discovered. Prostacyclin inhibits platelet aggregation by increasing cyclic AMP levels. Prostacyclin (prostaglandin I2, PGI2), produced by the blood vessel wall is the most potent known inhibitor of platelet aggregation induced by such stimuli as ADP and thrombin. It binds to a specific platelet receptor and activates adenylate cyclase, raising the cyclic AMP level in platelets
- cells regress from a specialized function to a simpler state reminiscent of stem cellscells regress from a specialized function to a simpler state reminiscent of stem cells
- Studies of physiological mechanisms governing the differentiation of human megakaryocyte and platelet release; the study of the biochemistry (and differentiation) of erythroid and myeloid cells.
- A loading control is a protein used as a control in a Western blotting experiment. Typically, loading controls are proteins with high and ubiquitous expression, such as beta-actin or GADPH. They are used to make sure that the protein has been loaded equally across all wells. miRNA mimics are RNA duplexes that were designed to faithfully mimic the functions of mature miRNAs.
- mmunofluorescence (IF) is a common laboratory technique, which is based on the use of specific antibodies which have been chemically conjugated to fluorescent dyes. These labeled antibodies bind directly or indirectly to cellular antigens
- I/Mild=intima to media ratio
- Streptozotocin
- 4′,6-diamidino-2-phenylindole
- PF4 drives a VSMC inflammatory phenotype including a decline in differentiation markers, increased cytokine production, and cell proliferation. Smooth muscle α-2 actin is found in smooth muscle cells. Smooth muscles line the internal organs, including the blood vessels, stomach, and intestines. Within smooth muscle cells, smooth muscle α-2 actin forms the core of structures called sarcomeres, which are necessary for muscles to contract. Smooth muscles contract and relax as part of their normal function without being consciously controlled. Fluorescence in situ hybridization (FISH) is a molecular cytogenetictechnique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s[1] to detect and localize the presence or absence of specific DNA sequenceson chromosomes.
- ImageJ is a Java-based image processing program developed at the National Institutes of Health an MF1=MEAN FLOURESCNECE INTENSITY
- MFI=MEAN FLOURESECE INTENSITY