Study Pinpoints Altered Biological Pathways in Endothelial Cells

Several proteins involved in vascular remodeling, metabolism, and oxidative stress are not properly regulated in endothelial cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH), a study found.

The study, “Protein network analyses of pulmonary endothelial cells in chronic thromboembolic pulmonary hypertension,” was published in the journal Nature Scientific Reports.

A rare form of pulmonary hypertension, CTEPH is characterized by high blood pressure in the blood vessels of the lungs caused by the presence of blood clots.

The inner lining of blood vessels, or the vascular endothelium, is made up of a thin layer of endothelial cells. It has many important functions, such as preventing blood clots from forming, reducing inflammation, and regulating the flow of nutrients.

In CTEPH, the vascular endothelium fails to function properly — a process known as endothelial dysfunction — and its underlying mechanisms are not known.

Now, a team led by researchers from the University of Milan, in Italy, examined protein profiles in endothelial cells from five CTEPH patients. Their goal was to identify the molecular processes underlying endothelial impairment in these patients. Healthy pulmonary artery endothelial cells were analyzed as controls.

Using label-free quantification proteomics — a technique able to determine protein expression in complex biological samples — the team identified proteins in patient cells that had altered expression levels compared with levels in healthy endothelial cells.

Of the 673 proteins that were differently expressed, 82 proteins in particular had higher expression in CTEPH cells compared with healthy cells, and 232 had a lower expression. Of note, proteins with a ratio greater than 1.5 were considered upregulated or overexpressed, whereas a ratio under 1.5 characterized proteins that were downregulated or underexpressed.

Tissue scarring-related proteins such as PLOD2 (ratio of 5.50) and TGLN (ratio of 5.28) were highly overexpressed in CTEPH cells, indicating the occurrence of fibrosis. Endoglin (ratio of 3.05), a protein involved in angiogenesis (the development of new blood vessels), was upregulated while NOS3 (ratio of 0.52), necessary for vascular remodeling, was downregulated.

Computational methods were then used to analyze groups of proteins belonging to the same pathway. For example, groups of proteins responsible for cell movement and angiogenesis were predicted to be decreased with a z score of minus 2.994. Z scores lower than zero indicate downregulation, while those higher indicate upregulation.

White blood cells, or leukocytes, have an important role in dissolving blood clots. The data showed that cell movement of leukocytes signaling had a z score of minus 1.50, indicating a decrease.

Many endothelial functions depend on changes in calcium concentration. The team found a decrease of the calcium signaling pathway (z score of minus 2.23), which was related to the downregulation of the PI3K/AKT, mTOR, NFAT, eiF4, and p70S6K pathways.

The team also evaluated oxidative stress pathways. Oxidative stress occurs when there is an imbalance between the production of specific molecules (called reactive oxygen species) that can damage DNA and proteins, and the organism’s ability to neutralize their effects with antioxidants.

The metabolism of reactive oxygen species was decreased (z score of minus 0.684), which may reflect an inadequate response of CTEPH cells to oxidative stress. Additionally, an important antioxidant called GPX4 (ratio of 0.2) was notably underexpressed in CTEPH cells, “suggesting the dysregulation of oxidative stress/damage response,” the researchers wrote.

The respiratory and oxidative phosphorylation pathways in mitochondria, organelles that produce energy in cells, were found to be downregulated in CTEPH cells. Lipid (fat) metabolism was also shown to be dysfunctional in CTEPH. Cells from CTEPH patients had an increased lipid concentration (z score of 1.846), while fatty acid metabolism (z score of minus 0.933) was decreased.

Inflammation was predicted to be increased (z score of 3.147).

“This is the first study to attempt the description of molecular network in endothelial dysfunction associated with CTEPH using patient-derived pathological ECs [endothelial cells],” researchers wrote. “The advanced analytical approach in combination with the study of dysregulated pathways allowed the identification of several important abnormal regulated functional modules … proving the presence of endothelial dysfunction.”

“In summary, the mechanisms underlying endothelial dysfunction implicates the alterations in metabolism, inflammation and oxidative stress events,” the team concluded.

The researchers noted that the expression of these proteins will have to be validated in clinical samples, such as blood, to find out whether they can be used as potential biomarkers for endothelial impairment in CTEPH.