RNA Cloning and Visualization in Human Atherosclerosis

The research objectives of this project are as follows:

1. Obtain high-quality human atherosclerotic arterial samples from diseased donors.
2. Perform biochemical analysis to determine the abundance, localization and activity of Dicer and double-stranded RNAs in these diseased tissues.

Study Overview

• Status: Completed
• Conditions: Atherosclerosis

Detailed Description

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.

• Study Type: Observational
• Enrollment (Actual): 22

Contacts and Locations

Study Locations

• United States
  • Kentucky
    • Lexington, Kentucky, United States, 40536
      • University of Kentucky Chandler Medical Center

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

Male and Female patients undergoing carotid endarterectomy

Description

Inclusion Criteria:

• Age of 18 and older
• Undergoing carotid endarterectomy

Exclusion Criteria:

• Under 18 years of age
• Not undergoing carotid endarterectomy

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort
Carotid Endocardectomy Patients; 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?

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

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Dicer dysregulation displays cell type-specific patterns.	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)	1 year

Collaborators and Investigators

• Sponsor: University of Virginia
• Investigators: Principal Investigator: Bradley Gelfand, PhD, University of Virginia, Department of Ophthalmology; Principal Investigator: Bradley Gelfand, PhD, Ophthalmology

Study record dates

Study Major Dates

• Study Start: October 1, 2013
• Primary Completion (Actual): November 1, 2016
• Study Completion (Actual): November 1, 2016

Study Registration Dates

• First Submitted: August 20, 2013
• First Submitted That Met QC Criteria: August 22, 2013
• First Posted (Estimate): August 23, 2013

Study Record Updates

• Last Update Posted (Actual): April 13, 2017
• Last Update Submitted That Met QC Criteria: April 11, 2017
• Last Verified: April 1, 2017

More Information

Terms related to this study

• Keywords: atherosclerosis; carotid endartherectomy
• Additional Relevant MeSH Terms: Cardiovascular Diseases; Vascular Diseases; Arteriosclerosis; Arterial Occlusive Diseases; Atherosclerosis
• Other Study ID Numbers: IRB # 13-0623-F1V

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No