Paolo Poggio: Proprotein convertase subtilisin kexin type 9 knockout mice are protected from valvular calcification

Paolo Poggio, from the Monzino Cardiology Center in Italy, gave a talk examining the effect of the gene PCSK9 on aortic valvular calcification using a knock out mouse model. Paolo started by giving some background into why they chose Oxford Nanopore Technologies a s a sequencing solution. Having never performed any sequencing before, but having a grant deadline looming, a quick and simple method to go form question to answer was required. After a training session at Oxford Nanopore Technologies, Paolo and his team had sequencing data within two weeks of concept. Moving on he began to describe the disease under study in his presentation.

Aortic valvular calcification manifests as accumulated calcium deposits on the aortic valves of the heart. This results in reduced blood flow and is associated with significant cardiovascular morbidity and mortality. Approximately 3 % of the population are affected by the disease and there are currently no pharmacological interventions with invasive surgery being the only option. In addition, if surgery is not undertaken the life expectancy does not exceed 5 years. Although there is no pharmacological intervention available, there is evidence to suggest that proprotein convertase subtisilin kexin type 9 (PCSK9) inhibitors may reduce disease onset and progression through regulation of calcium deposition.

In terms of experimental design, Paolo described how a knock out mouse model was used to measure the total aortic calcium content and compared the values to the wild type control. There was a significant reduction in total calcium content in the aortic valves (p < 0.001) and specific cells, known as valve interstitial cells, has significantly lower calcification rates (p < 0.001). In addition, calcification was induced chemically in this system to show that these levels could return to just below control levels.

A differential gene expression study was designed in order to determine which genes were up- and down-regulated in the knock out mice when compared with the wild type controls. Using Oxford Nanopore's PCR cDNA kit, more than 3 Gb per run was generated, resulting in the detection of > 200 differentially expressed genes with over a log2 fold change in abundance and corrected p values of < 0.05.  Triplicate samples were multiplexed across single flowcells allowing for large changes in dominant transcripts to be detected.

When performing a functional analysis of these differentially expressed genes, upregulated genes were associated with the p38 cascade and cytoskeletal modification, while downregulated genes regulated apotopic pathways, cell adhesion and oxidative stress responses, a lot of these being related to PCSK9 pathways.

Showing that a link between PCSK9 and aortic valvular calcification in a mouse models using both direct measures of calcification and Oxford Nanopore long read cDNA sequencing, lead Paolo onto obtaining a grant from the European Research Area Network on Cardiovascular Diseases in order to study this process in humans.

Using human cells isolated from aortic valve replacements, Paolo showed that PCSK9 was significantly up regulated in aortic valvular calcification samples when compared with controls. As PCSK9 expression is related to oxidative stress, cells were exposed to an oxidative stress via the addition of H2O2 and calcification rates increased.

Paolo finished by saying that based on the results of these experiments, they now have the opportunity to test if pharmacological inhibition of PCSK9 could reduce or even halt the progression of this wide spread and debilitating disease.