Week 4 – Resolvins May Be the Solution

This week I started the proliferation assay for vascular smooth muscle cells. Using Alamarblue and a fluorescence spectrophotometer I counted the initial number of cells in all my wells. I explained how this assay works in more detail in last week’s post. Here are the steps I followed this week: Alamarblue Proliferation Assay Protocol

The initial number of cells in a few of my wells (Day 1)

My cells were living this week so my advisor suggested that it would be a waste to conduct the assay without any drugs as I had initially planned. I am still in the process of figuring out how I will access the drugs currently used in heart stents. Therefore, this week I used a resolvin, RvD1, that was available in the lab to add to the cells. My goal is to be able to compare the efficacy of this resolvent and the drugs, but this is only possible if I can get my hands on the drugs.

A resolvin (RvD1) is a specific lipid structure that is a part of a class collectively termed as specialized pro-resolving mediators (SPMs). These lipid structures originate from the fish oil components (EPA and DPA) that I mentioned in Week 1

RvD1 structure

Next week I will complete the assay and let you know whether this resolvin is effective in reducing the proliferation of the cells.

Many of the current experiments I have followed in the lab involve the use of resolvins because they are a fairly new discovery in restenosis prevention. Drugs are not the only way to combat inflammation. In fact, our body’s innate inflammatory response involves mediators that help resolve or stop inflammation. Specialized pro-resolving mediators (SPMs) are known as resolution agonists. Compared to drugs they are not toxic and are produced locally in our blood. They have many functions that help to down regulate or slow down the process of inflammation.

 

When a blood vessel wall is injured the inflammatory response has many steps (an image below outlines this process). First, the endothelial cell layer is disrupted. Then, the acute inflammation phase is activated and many events take place. Most importantly, neutrophils, a type of leukocyte, are recruited to the site of injury and macrophages form. Smooth muscle cells also begin to proliferate and migrate to the tunica intima. In the resolution phase, macrophages phagocytose excess cells and aid in ending the inflammation process. In the remodeling phase, a neointima forms and homeostasis is reestablished.

Neointima hyperplasia is the proliferation and migration of vascular smooth muscle cells in the tunica intima, resulting in the thickening of arterial walls and the blocking of blood flow.

 

The Entire Inflammation Process in a Vessel Wall

The lab is researching how to increase the volume of SPMs in our blood to slow down acute inflammation and speed up resolution in order for a cell to return to homeostasis. They hope to do this by wrapping a biodegradable wrap that elutes SPMs (RvD1 specifically) around heart stents. SPMs’s functions include: 1. increase macrophage phagocytosis of cellular debris 2. decrease neutrophil activity 3. speed up tissue remodeling 4. reduce vascular smooth muscle cell proliferation 5. lend to a quicker homeostasis return.

A previous experiment in the lab concluded, “RvD1-loaded “wraps” reduced neointimal formation by 59% versus no-wrap controls …” 

The wrap is still in the prototype stage. The shape and thickness of the wrap are still being debated. They are going to place the prototype around a pig artery soon!