How does SARS CoV-2 infect blood vessels?

People who are severely affected by COVID-19 show symptoms of damage to their blood system, as well as to their lungs. For example, their blood may form lots of clots, which can cause damage to lots of different tissues in the body. Also other tissues than the lungs may be damaged in COVID-19, such as the heart, and it is likely that infection spreads to these tissues through the blood system. It will be important to know how blood vessels are infected by SARS CoV-2, the virus that causes COVID-19, to understand how damage to blood vessels and other tissues occurs. Our research will discover which types of cells in blood vessels become infected with SARS CoV-2, which will help us understand how blood vessels and other tissues become damaged in COVID-19, and suggest which cells to target to prevent this damage. The two most likely cells that might be infected are endothelial cells, which form the inside surface of blood vessels, or pericytes, which form part of the outer wall of very small blood vessels. In our work, we will use a special inactive version of SARS CoV-2 that cannot replicate but will label cells that have taken it up (become infected). We will apply this inactive version of the SARS CoV-2 virus to endothelial cells and pericytes that are grown in a dish, and see which cell type becomes most strongly labelled, indicating that it takes up the virus most strongly.

We then want to find out whether the blood vessels are differently infected in different organs, and whether this could explain some of the non-respiratory symptoms that people suffer from. To investigate this, we will use mice. However, SARS CoV-2 does not infect mouse cells, so we will use two approaches. In one set of experiments, we will use genetically altered mice that express the human version of the protein that binds SARS CoV-2. In another set of experiments we will use normal mice and a version of the inactive virus that has a mutation so it binds to the mouse version of the SARS CoV-2 receptor protein. By injecting the inactive virus into the bloodstream of these mice, we will discover which cells the virus infects in different tissues, including the heart and brain.

Finally, we want to find out what factors affect the severity of infection with SARS CoV-2. We will test whether existing inflammation makes it easier for SARS CoV-2 to infect blood vessels, by injecting mice with a bacterial protein that triggers an inflammatory response, before injecting the inactive SARS CoV-2 virus. We will also test whether a gene called APOE4, that seems to be linked with severe COVID-19 in humans, increases infection of blood vessels in our experimental mice.

Together, our experiments will discover how blood vessels become infected with SARS CoV-2, indicating which cells to target with treatments, and will test whether risk factors for severe COVID-19 illness could act by increasing the ability of SARS CoV-2 to infect cells on blood vessels.

Vascular symptoms including hypercoagulation are a key feature of severe COVID-19. They have been assumed to arise via infection of endothelial cells, however recent RNAseq analyses revealed that instead it is pericytes - vascular mural cells with intimate connections with endothelial cells and several vital roles in vascular function - which express the receptor, ACE2, which allows SARS CoV-2 to enter cells. Determining which cells mediate vascular and therefore extrapulmonary organ infection is vital for selecting optimal therapeutic interventions. Through our groups' expertise in vascular biology, virology and clinical haematology, we will identify whether pericytes or endothelial cells are the primary vascular target of SARS CoV-2.

We will utilise a pseudotyped SARS CoV-2 virus (PV), which undergoes a single round of infection and expresses a reporter protein in target cells. We will first compare the efficiency of infection in cultured human pericytes and endothelial cells, before injecting PV into mice and tracking reporter expression across pericytes and endothelial cells of different organs. As SARS CoV-2 does not bind murine ACE2, parallel experiments will test a PV mutated to bind murine ACE2 as well as mice expressing human ACE2. Because increased inflammation and APOE4 genotype both alter vascular permeability and are associated with severe COVID19, we will repeat these experiments in animals with LPS-induced inflammation or that express human ApoE3 or 4.

By identifying the cells that first become infected with SARS CoV-2 and the conditions that increase infection, this project will enable proper targeting of therapies to the relevant cells.