Functional Foods and Problematic Phosphatases

As the burgeoning middle class in India adopts an increasingly Westernized lifestyle, with its attendant consumption of food of questionable nutritional value, lifestyle diseases pose an ever-increasing threat to health. Diabetes is perhaps the most recognisable of these diseases. One of the most common complications of type-2 diabetes is the thickening of the walls of the arteries due to fat deposition, a condition known as atherosclerosis. At the Vascular Biology lab on the fourth floor in the Bhupat and Jyoti Mehta School of Biosciences, work is underway to determine the mechanisms linking diabetes and atherosclerosis, and to identify potential cures. The lab is headed by Dr. Madhulika Dixit, who entered the field when she started working on her PhD in molecular biology from IIT Bombay. She is currently an Assistant Professor at IIT Madras, and was recently given the Young Faculty Recognition Award this year on Teacher’s Day.

Abhiram is one of the research scholars in the lab. Having taught the microscopy portion of the Cell Biology course to third-year undergraduates in the department and having worked as a TA in their labs, he is probably the best-known research scholar among the undergraduates. He begins by explaining to me the structure of an artery. “There are three types of cells which make up blood vessels: endothelial cells, smooth muscle cells, and endothelial progenitor cells,” he says.

A single layer of endothelial cells, which belong to the same tissue type that forms the inner linings of the various organs and also our skin, makes up the innermost layer of an artery. This layer is surrounded by smooth muscle cells, which help in regulating blood flow by contracting or dilating the artery when required. Endothelial progenitor cells, as the name implies, give rise to new endothelial cells when the blood vessel has to grow.

 

Autopsy specimen of aorta opened lengthwise to reveal luminal surface studded with lesions of atherosclerosis.
Autopsy specimen of aorta opened lengthwise to reveal luminal
surface studded with lesions of atherosclerosis.

 

Atherosclerosis begins when the endothelial cells start synthesising proteins called Cellular Adhesion Molecules (CAMs) in larger-than-usual amounts. CAMs provide binding spots for white blood cells, which then migrate through the arterial walls into the smooth muscle layer, where they secrete growth factors which affect the muscle cells.

 

The illustration shows a normal artery with normal blood flow (figure A) and an artery containing plaque buildup (figure B). CREDIT: NHLBI [Public domain], via Wikimedia Commons
The illustration shows a normal artery with normal blood flow (fig. A) and an artery containing plaque buildup (fig. B).
CREDIT: NHLBI [Public domain], via Wikimedia Commons.
 

 

The muscle cells then change into a “synthetic form,” named so due to their propensity for synthesising compounds like collagen which reduce the elasticity, and subsequently the integrity, of the endothelial cell layer. They also begin producing new cells, a phenomenon termed as “proliferation,” which culminates in the formation of an atherosclerotic plaque, or a fat deposit in the artery.

Work on atherosclerosis in the lab is primarily focused on two areas – determining the pathways by which smooth muscles and progenitor cells are activated to cause atherosclerosis in diabetics, and identifying compounds which can potentially inhibit these pathways.

One of the enzymes the team investigated was a protein called SHP2, which is a part of the insulin-response pathway. It belongs to a class of enzymes known as phosphatases. Organisms can turn their proteins “on” or “off” by adding phosphate groups to, or removing phosphate groups from, certain amino acids on the protein. Enzymes which phosphorylate other proteins are termed kinases, while those which de-phosphorylate are named phosphatases. In general, kinases and phosphatases tend to have very specific protein targets.

In endothelial cells, a protein called eNOS synthesises nitric oxide which, apart from being an important signalling molecule, also inhibits the adhesion of white blood cells onto the arterial walls.

SHP2 regulates eNOS activity and thus plays a direct role in controlling the amount of nitric oxide that is produced. To mimic the conditions of high insulin content in the blood of diabetics, Hemant, a research scholar at the lab, grew human endothelial cells (obtained from umbilical cords and called HUVECs) in an insulin-rich medium for two days. He then performed a test known as an adhesion assay to measure the number of white blood cells which adhere to the HUVECs. In an adhesion assay, HUVECs are first grown on a membrane and then treated with insulin. White blood cells labelled with fluorescent dyes are then added to the membrane. Due to the presence of the CAMs, the white blood cells bind to the HUVECs. After washing off the unbound white blood cells, the bound ones can be counted under a microscope. Hemant found that cells which were chronically exposed to high levels of insulin expressed roughly two-and-a-half times as many CAM molecules as cells grown in normal conditions.

Using a technique called Western Blot, the team then measured the amount of SHP2 produced in insulin-exposed cells, and compared them with normal cells to find that cells exposed to insulin begin producing larger amounts of SHP2 on chronic exposure to insulin. This, in turn, reduces the amount of nitric oxide produced, diminishing the endothelial cells natural protection against atherosclerosis.

The Vascular Biology group at IIT-M is one of three Indian research centres which are a part of the Indo–European “Funcfood” (short for functional foods) project, which aims to identify compounds with potential medicinal value in treating age-related illnesses, from plants which have been used in traditional herbal medicine.

The lab’s work as part of the Funcfood project has been quite fruitful, with the identification of two compounds which inhibit the proliferation of smooth muscles, which results in the formation of atherosclerotic plaques. The first is a compound called isovitexin, extracted from the roots of the Gentiana lutea plant, also called the bitterwort. Due to their bitter taste, the extracts of the root have been historically used in Serbian and Peruvian medicine to make tonics to treat indigestion and gastric infections.

Previous research on these roots had suggested that they have beneficial cardiovascular effects, which was why it was selected as a candidate.

To determine whether the root extracts affected the proliferation of muscle cells, Rushendhiran, a research scholar in the lab, cultured smooth muscle cells in a medium rich in the compound PGDF-BB (short for Platelet-Derived Growth Factor BB, one of the growth factors secreted by white blood cells), and found that a large proportion of them had changed from their normal (“quiescent”) phase to the synthetic phase. Cells which had been treated with the root extracts, however, did not enter the synthetic phase, showing that the extracts contained compounds which could inhibit the onset of atherosclerosis. He also found that the production of nitric oxide, a compound involved in many intra-cellular signalling pathways, was reduced in cells treated with the extract, hinting at the biochemistry behind the process.

The other compound the lab has identified is ellagic acid, a chemical found in large quantities in raspberries. Using a technique very similar to that used by Rushendhiran, Uma Rani, another researcher in the lab, determined that ellagic acid inhibits the effects of PGDF-BB on smooth muscle cells. Taking it one step further, the team studied the effects of ellagic acid on atherosclerotic rats. To do this, they first injected rats with the compound streptozotocin, which kills off the beta-cells of the pancreas which are responsible for producing insulin, making the rats diabetic. Among these animals, the team found that those which had been fed ellagic acid regularly had thinner layers of fat deposited on their arteries, compared to their untreated counterparts. Analyses of their smooth muscle cells also showed that ellagic acid reduced the expression of compounds called cylins, which cause muscle cell proliferation, and are expressed in large amounts in the cells of diabetics. As both ellagic acid and isovitexin are found in natural sources, they can be easily adapted to therapeutic uses by advising diabetics to include more fruits such as raspberries in their diet, giving us a pleasant way of managing a disease which otherwise would have been the blight on a person’s golden years.