Steven P. Wrenn, Ph.D.

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Steven P. Wrenn, Ph.D. Drexel University
Chemical Engineering Bio-Colloids Laboratory
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Drexel University
MOTIVATION

• Cardiovascular disease is the primary cause of
  mortality

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Drexel University

• Normal Coronary Artery
• Severe Coronary Atherosclerosis

The Internet Pathology Laboratory for Medical
Education
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How can you detect cholesterol nucleation?

• Make vesicles with fluorescent analogs of
  cholesterol and fluorescent phospholipids.
• Measure their fluorescence intensities before
  enzymes are added, and after using a steady-state
  fluorescence spectrophotometer.
• What do these analogs look like?

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Crystal Nucleation Assay
Fluorescent Cholesterol
Cholesterol
Ergo
HO
Dehydroergosterol
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Fluorescent Lecithin
Dansyl
Dansyl
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Alleviation of Energy Transfer
Nucleation (bile salt-induced)
Zone III only
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How do enzymes cause aggregation of vesicles?

• The enyzme phospholipase C cleaves the
  hydrophilic head off of the phopholipid, leaving
  only the hydrophobic tails.
• The hydrophobic part of the vesicles are
  attracted to each other, causing aggregation.
• This is what happens to LDL in the arteries. This
  aggregation leads to an inflammatory response,
  the macrophages engulf the LDL and cholesterol
  nucleates causing plaques in the arteries.

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(phospholipase C-induced) vesicle aggregation
PLC
D
First 2 hrs. (PLC Action)
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LDL
LDL particles have an average size of 22nm. The
core contains 1600 molecules of cholesterol
ester. LDL can move freely in and out of the
intima of the artery until the aggregation
caused by phospholipase C and sphingomylinase
prevents it from leaving.. The macrophages
engulf the aggregation, releasing the
cholesterol to deposit on the artery wall.
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Sphingomyelinase

• Is another enzyme that cleaves the head from
  sphingomyelin, a lipid found in the membrane.
• Again the hydrophobic areas of the vesicles are
  attracted to each other and cause aggregation.
• Since the particles are now much larger than
  before the enzyme was introduced, this shows up
  as increased absorbance on the spectrophotometer.

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Enzyme Sphingomyelinase
Hydrophilic
head
Sphingomyelin
Hydrophobic Tails
Hydrophobic Region causes aggregation of vesicles
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Absorbance vs. Concentration
Film 2 sonicated for 15 mins
Film 1 sonicated for 90 mins

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Absorbance vs Concentration of Phospholipase C
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Unit Long Understanding Goals

• What do the components of the cell membrane look
  like?
• What elements make up these components?
• What are the properties and functions of these
  components?

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Unit Long Understanding Goals

• What happens to excess cholesterol in the diet,
  and what are the roles of LDL and HDL?
• What foods will raise HDL levels and lower LDL?
• What enzymes cause nucleation of cholesterol and
  how do they work?
• How does the aggregation of LDL start the
  inflammatory response?

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Introductory Performance

• In groups, using molecule building kits, the
  students will build models of phospholipids
• On line investigation of properties of
  phospholipids and how they form cell membranes
  and vesicles.
• Simple investigation of one enzyme that breaks
  down phospholipids to see if group can
  hypothesize about what is happening.

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Guided Inquiry

• Using phospholipase C and phospholipid
  vesicles,the students will complete a lab using a
  Spec 20 to gather data on absorbance vs.
  concentration and absorbance vs. time to show
  vesicle aggregation
• Complete 1-2 pg report on role of HDL and LDL in
  cholesterol transport
• Keep a journal for a week on what they eat,and
  analyze the saturated fat. In a group, suggest
  ways to reduce levels of LDL and raise levels of
  HDL in their diets.
• Write a one page reaction paper on guest speaker
  from Drexel explaining how the aggregation of LDL
  triggers the inflammatory response that causes
  plaque formation in arteries.

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Culminating Performance

• Students working in pairs or groups, create a
  model, a presentation on PowerPoint, a Web page
  or a pamphlet using Pagemaker to present their
  knowledge to adults/students whom were not in the
  class

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Acknowledgments

• Ph.D. Students
• - Manasa Gudheti (Gallstones)
• - Andrew Guarino (Atherosclerosis)
• - Gregory Troup (Cholesterol Domains)
• Collaborators
• Steve Wrenn
• - Mun Choi
• Funding
• - The National Science Foundation (RET
  program)