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Inherited disfunctions of brain microcirculation

Periodic Reporting for period 2 - V-EPC (Inherited disfunctions of brain microcirculation)

Reporting period: 2019-03-01 to 2020-08-31

The problem: Cerebral cavernous malformation (CCM) is a rare neurovascular disease characterized by enlarged and irregular blood vessels that often lead to cerebral hemorrhages and death. CCM occurs in both sporadic and familial forms.
The loss-of-function mutations in anyone of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10) result in CCM lesion formation.
Why is it important for society?
In many cases patients require continuous assistance. The prevalence of the hereditary form is low (less than 1 out of 10,000) while the sporadic form is quite frequent ( 1 out of 200). There is no pharmacological treatment available for CCM patients and the only possible intervention is surgery. The consequences of the disease are seizures, strong headache, paralysis and others.
What are the overall objectives?
The major objectives of our work are:
-the development of murine models closer to the human disease to be used to understand the evolution of the pathology and to find specific inhibitors
-to identify pharmacological therapeutics able to delay or even induce regression of the vascular malformations.
Through experimental work we found that, in mouse models of CCM disease, there are clusters of endothelial cells expressing markers of cell progenitors. We found that these cells were able to upregulate genes promoting Endothelial-to-Mesenchymal transition (End-MT) through signaling pathways close to those used by cancer stem cells undergoing-EMT (epithelial to mesenchymal transition) (see Malinverno et al. Stem Cells, 2017 and Malinverno et al Nature Commun, 2019). We further demonstrated that these resident endothelial progenitors may undergo clonal expansion in response to inactivation of anyone of the CCM genes. Clonal expansion acts as a trigger for the development and progression of vascular CCM malformations.
Based on these observations, translational studies are under way to identify pharmaceutical agents able to reduce the formation of cavernomas and eventually induce their regression.
We also combined different techniques to comprehensively characterize sub-clusters of endothelial cells in both normal conditions and after deletion of CCM3 at early postnatal stage. We identified a specific cluster of endothelial cells resistant to CCM transformation. Conversely, a subset of mitogenic endothelial progenitors and venous capillary endothelial cells appears to be at the origin of CCM lesions.
In mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium as they express mesenchymal/stem-cell markers that are absent in endothelial cells of the normal vasculature. In our work we could show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that co-express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells that migrate in the malformation promoting its expansion. These endothelial cells also acquire mesenchymal characteristics loosing endothelial markers and becoming migratory in a process called End-MT. Morphologically, endothelial cells undergoing End-MT acquire fibroblastoid feature, increased proliferation and permeability. These studies helped us to develop in vitro assays for large screening of pharmacological compounds of different origin and for further in vivo tests on murine models of CCM.