Secco, I. | bioRxiv Science

bioRxiv2022

Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity

The microvasculature plays a key role in tissue perfusion, transport of mediators, and exchange of gases and metabolites to and from tissues. Microvascular dysfunction has emerged as an important contributor to cardiovascular diseases. In this study we used human blood vessel organoids (BVOs) as a model of the microvasculature to delineate the mechanisms of microvascular dysfunction caused by metabolic rewiring. BVOs fully recapitulated key features of the normal human microvasculature, including reliance of mature endothelial cells (ECs) on glycolytic metabolism, as concluded from metabolic flux assays using 13C-glucose labelling and mass spectrometry-based metabolomics. Treatment of BVOs with PFK15, a pharmacological inhibitor of glycolysis, resulted in rapid tissue restructuring, vessel regression with reduced pericyte coverage and alterations in tight junction morphology. Proteomic analysis of the BVO secretome revealed remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovered tight junction formation and increased pericyte coverage in microvessels. Our metabolic and proteomics findings demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

cell biology↗

bioRxiv2021

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet pro-coagulant activity

BackgroundThrombosis of the lung micro-vasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting this activity. As TMEM16F plays an important role in the stimulation of the pro-coagulant activity of platelets, and considering that platelet abnormalities are common in COVID-19 patients, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. MethodsWe produced SARS-CoV-2 Spike or VSV-G protein-pseudotyped virions, or generated cells expressing Spike on their plasma membrane, and tested their effects on platelet adhesion (fluorescence), aggregation (absorbance), exposure of phosphatidylserine (flow cytometry for annexin V binding), calcium flux (flow cytometry for fluo-4 AM), and clot formation and retraction. These experiments were also conducted in the presence of the TMEM16F activity inhibitors Niclosamide and Clofazimine. ResultsHere we show that exposure to SARS-CoV-2 Spike promotes platelet activation, adhesion and spreading, both when present on the envelope of virions or upon expression on the plasma membrane of cells. Spike was effective both as a sole agonist or by enhancing the effect of known platelet activators, such as collagen and collagen-related peptide. In particular, Spike exerted a noticeable effect on the procoagulant phenotype of platelets, by enhancing calcium flux, phosphatidylserine externalisation, and thrombin generation. Eventually, this resulted in a striking increase in thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine almost abolished this Spike-induced pro-coagulant response. ConclusionsTogether, these findings provide a pathogenic mechanism to explain thrombosis associated to COVID-19 lung disease, by which Spike present in SARS-CoV-2 virions or exposed on the surface of infected cells, leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing drugs targeting this protein, such as Niclosamide, for COVID-19 therapy.

pathology↗