The endothelial progenitors, once mutated in CCM upregulate genes promoting Endothelial-to-Mesenchymal transition (End-MT) through signaling pathways used by cancer stem cells undergoing EMT (epithelial to mesenchymal transition) (Malinverno et al. Stem Cells, 2017; Malinverno et al Nature Commun, 2019). These resident endothelial progenitors react to inactivation of the CCM genes undergoing clonal expansion and triggering the development and progression of CCM lesions.
Translational studies are under way to identify pharmaceutical agents able to reduce the formation of cavernomas and eventually induce their regression.
Using scRNA-seq we map the diversity of ECs in vascular cavernomas with specific focus on PDCD10. We identified distinct EC clusters and define their functional roles in CMM development and progression. An important step toward the understanding of CCM and any potential therapy is the identification of the EC populations that trigger and sustain the development of these vascular malformations. Through scRNA-seq, we identified some unique characteristics of ECs that form either normal vessels or cavernomas: brain ECs are heterogeneous population distributed across 13 different clusters on the basis of their gene expression. These cells express highly specialized feature functional to form the BBB vasculature. Remarkably, resident progenitor cells were detected in all of the Pdcd10-wt clusters, although in different proportions. These data are published in eLife (doi: 10.7554/eLife.61413)
The mechanism of EndMT characterizing CCM pathogenesis resembles the EMT process to which undergo cancer stem cells. In cancer, this process of de-differentiation is regulated by Polycomb Repressive Complexes (PRCs), epigenetic machineries that regulate gene expression and differentiation state. Therefore, we hypothesized that these epigenetic machineries may be involved also in CCM formation and development.
Upon the loss of CCM there was an epigenetic rewiring dependent on an aberrant activity of PRCs. These changes correlate over the epigenetic landscape with the expression level of the genes.
The epigenetic machineries regulate the mesenchymal transition of ECs through a downregulation of the expression levels of endothelial genes and an upregulation of the expression levels of the mesenchymal genes.
We profiled the different epigenetic landscape of cerebrum and cerebellum-derived ECs ex vivo using our pre-clinical CCM mouse. A novel technique (Cut & Run) has been set up to study epigenetic modifications using a small number of cells. We profiled the epigenetic landscape of cerebrum and cerebellum derived ECs under both wild-type and CCM-null conditions.
As the process of EndMT is pivotal for CCM pathogenesis, we employed a pharmacological approach to target the altered epigenetic mechanisms involved in the de-differentiation process and in other pathological features characterizing CCM-null ECs. We performed in vitro and in vivo treatments taking advantage of the preclinical CCM mouse model. After the treatment with an epigenetic drug targeting PRCs, we were able to reduce the enlargement of vascular lesions.
For the first time, we demonstrate the implication of epigenetic in CCM pathogenesis both in vitro and ex vivo: these data are included in a manuscript that is in preparation.
The high throughput screening of small molecule inhibitors of EndMT identified several molecules with the ability to revert the EndMT-related phenotype of Ccm3-null cells. These includes 3 kinase inhibitors and propranolol, a well-known non-selective adrenergic beta-receptor inhibitor. We treated endothelial-specific Ccm3-deficient mice with oral propranolol, and we show reductions in the number and size of CCM lesions in both the brain and the retina. Furthermore, the β-blocker propranolol stabilized the vasculature, as shown by the reduced vascular permeability and communicative stability between the endothelial cells and the pericytes. These results are included in doi: 10.1161/STROKEAHA.120.029676
Moreover, the clinical trial (funded by AIFA, Italian Medicines Agency) to test the effect of propanolol on patients with familial CCM has been completed. Propranolol was safe and might be beneficial for reducing the incidence of clinical events in people with symptomatic familial cerebral cavernous malformations, justifying definitive phase 3 trial. The results are included in a paper published on Lancet Neurology (Lanfranconi et al, Lancet Neurology, 2022 Nov;doi: 10.1016/S1474-4422(22)00409-4 ).
