RNA Cloning and Visualization in Human Atherosclerosis
| Status: | Active, not recruiting |
|---|---|
| Conditions: | Peripheral Vascular Disease, Cardiology |
| Therapuetic Areas: | Cardiology / Vascular Diseases |
| Healthy: | No |
| Age Range: | 18 - Any |
| Updated: | 5/25/2016 |
| Start Date: | October 2013 |
| End Date: | November 2016 |
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.
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.
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.
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.
Inclusion Criteria:
- Age of 18 and older
- Undergoing carotid endarterectomy
Exclusion Criteria:
- Under 18 years of age
- Not undergoing carotid endarterectomy
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