RNA Cloning and Visualization in Human Atherosclerosis

Study Overview
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Brief Summary
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The research objectives of this project are as follows: Obtain high-quality human atherosclerotic arterial samples from diseased donors. Perform biochemical analysis to determine the abundance, localization and activity of Dicer and double-stranded RNAs in these diseased tissues.

Detailed Description
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Participants enrolled in to this pilot study will not be randomized and will not receive any investigation medication. In collaboration with Dr. Sibu Saha, Professor of Surgery, University of Kentucky, the investigator will obtain freshly isolated human atherosclerotic tissue from patients undergoing carotid endarterectomy. The surgical process by which the tissue is obtained is not part of this research project. These tissues are surgically removed from patients as a treatment for atherosclerosis of the carotid arteries and typically sent for pathological examination. After the the clinical pathology has been completed, the investigator will obtain and analyze a small amount of discarded (200mg) tissue. Subsequent analyses will be performed on the basis of the pathological grading provided by UK Pathology (e.g do readouts of the Dicer pathway correlate with pathological plaque characteristics). Tissue will be anonymized, with only information with respect to drug history, age, gender and pathological grade of the tissue. The will extract protein and RNA. Dicer abundance will be assessed by western blot, quantitative RT-PCR and northern blot. Dicer related RNAs will be measured by quantitative RT-PCR and northern blot. The invesigator will inspect the tissue for evidence of altered Dicer activity by measuring the relative levels of abundant canonical Dicer substrates/enzymatic products (i.e. the ratio of pre- to mature micro-RNA) via northern blot. The investigator predicts that Dicer levels and activity are reduced in human atherosclerotic tissue compared to healthy arteries. Next, the investigator will assess whether downstream mediators of Dicer dysregulation are activated in these tissue samples by comparing levels and activation of the inflammasome (caspase-1, NLRP3, ASC), cytokines (IL-1β, IL-18) and signaling intermediates (MyD88, IRAK1/4) between healthy and diseased tissues. The investigator predicts that atherosclerotic plaques will exhibit evidence of Dicer dysregulation. Next, the investigator will co-stain tissue sections with antibodies recognizing Dicer as well as cell-specific markers (CD31 for endothelium, SMA for smooth muscle, MAC-1 for macrophages) to assess whether Dicer dysregulation displays cell type-specific patterns.

Official Title
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RNA Cloning and Visualization in Human Atherosclerosis

Conditions
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Atherosclerosis

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age of 18 and older Undergoing carotid endarterectomy Exclusion Criteria: Under 18 years of age Not undergoing carotid endarterectomy

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Carotid Endocardectomy Patients<br>Subsequent analyses will be performed on the basis of the pathological grading provided by UK Pathology (e.g do readouts of the Dicer pathway correlate with pathological plaque characteristics). | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Altered Dicer Activity | The investigator will measure the relative levels of abundant canonical Dicer substrates/enzymatic products (i.e. the ratio of pre- to mature micro-RNA) via northern blot | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Are downstream mediators of Dicer dysregulation activated | Analysis will compare levels and activation of the inflammasome (caspase-1, NLRP3, ASC), cytokines (IL-1β, IL-18) and signaling intermediates (MyD88, IRAK1/4) between healthy and diseased tissues. | 1 Year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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atherosclerosis, carotid endartherectomy