Project description:Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of β-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-β fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.

Project description:Chronic endotheliitis and various cardiovascular co-morbidities are more likely to develop in patients who are recovering from a post-acute SARS-CoV-2 infection. Despite a growing body of clinical data suggesting that the endothelium could be the cause of both cardiac injury and the multi-organ damage found in COVID-19 patients, there is no clear link between endothelial (EC) dysfunction and increased cardiac risk during long COVID. Here, we studied long COVID-19-associated endotheliitis and its implications on cardiac dysfunction. Thrombotic vascular tissues from long COVID patients were harvested and profiled to identify the different mechanisms of viral-induced EC pathogenesis. Human induced pluripotent stem cell (iPSC)–derived ECs were leveraged to model endotheliitis in-a-dish after exposure to SARS-CoV-2, which showed similar EC dysfunction and upregulation of specific cytokines such as CCL2 and IL6, as seen in the primary ECs of long COVID patients. 3D fabricated cardiac organoids generated from iPSC-ECs and iPSC-derived cardiomyocytes (iPSC-CMs) were utilized to understand the pathological influence of endotheliitis on cardiac dysfunction. Notably, cardiac dysfunction was observed only in cardiac organoids that were fabricated with both iPSC-CMs and iPSC-ECs after exposure to SARS-CoV-2. Simultaneous profiling of chromatin accessibility and gene expression dynamics via integration of ATAC-seq and RNA-seq at a single cell resolution revealed CCL2 as the prime cytokine responsible for the non-endothelial “phenotype switching” and the impending cardiac dysfunction in cardiac organoids. This was further validated by high-throughput proteomics that showed CCL2 to be released only by cardiac organoids that were fabricated with iPSC-CMs and iPSC-ECs after SARS-CoV-2 infection. Lastly, disease modeling of the cardiac organoids as well as exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins uncovered a putative mechanism for the cardiac dysfunction involving posttranslational modification of cardiac proteins driven by oxidative stress and inflammation. These results suggest that EC-released cytokines can contribute to the pathogenesis of long COVID-associated cardiac dysfunction, and thus a thorough clinical profiling of vascular health could help identify early signs of heart disease in COVID-19 patients.

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CCL2 accelerates microglia-mediated Abeta oligomer formation and progression of neurocognitive dysfunction.

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CCL2 accelerates microglia-mediated Abeta oligomer formation and progression of neurocognitive dysfunction.

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