Periodic Reporting for period 4 - V-EPC (Inherited disfunctions of brain microcirculation)
Reporting period: 2022-03-01 to 2022-08-31
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 major symptoms of the disease are epileptic seizures, strong headache, paralysis, focal neurological disorders and others.
What are the overall objectives?
The major objectives of our work are:
- to define the nature and functions of Vascular-Endothelial Progenitor Cells (V-EPCs) in normal vessels
- to test the capacity of V-EPCs to trigger the formation of CCM cavernomas
- to develop novel pharmacological tools to reduce or prevent the formation of CCM cavernomas
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 major symptoms of the disease are epileptic seizures, strong headache, paralysis, focal neurological disorders and others.
What are the overall objectives?
The major objectives of our work are:
- to define the nature and functions of Vascular-Endothelial Progenitor Cells (V-EPCs) in normal vessels
- to test the capacity of V-EPCs to trigger the formation of CCM cavernomas
- to develop novel pharmacological tools to reduce or prevent the formation of CCM cavernomas
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 ).
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 ).
IIn mouse models and in human patients, ECs lining the lesions have different features from the surrounding endothelium as they express mesenchymal/stem-cell markers that are absent in ECs of the normal vasculature. In our work we showed that cavernomas originate from clonal expansion of few Ccm3-null ECs 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 ECs also acquire mesenchymal characteristics loosing endothelial markers and becoming migratory in a process called End-MT. Morphologically, endothelial cells undergoing EndMT acquire fibroblastoid feature, increased proliferation and permeability.
These features shown by EndMT undergoing ECs, in the context of CCM disease, are shown likewise by cancer initiating stem cells. In cancer, it has been demonstrated that the process of clonal expansion and de-differentiation to which undergo cancer cells is regulated through epigenetic mechanisms and, in particular by the activity of PRCs. For this reason, we evaluated the involvement of PRCs in CCM pathogenesis performing both molecular and pharmacological studies. We discovered that the mesenchymal transition to which undergo CCM-mutated ECs is regulated through epigenetic mechanisms and that the inhibition of these processes ameliorate CCM pathological phenotype.
Importantly, 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.
These features shown by EndMT undergoing ECs, in the context of CCM disease, are shown likewise by cancer initiating stem cells. In cancer, it has been demonstrated that the process of clonal expansion and de-differentiation to which undergo cancer cells is regulated through epigenetic mechanisms and, in particular by the activity of PRCs. For this reason, we evaluated the involvement of PRCs in CCM pathogenesis performing both molecular and pharmacological studies. We discovered that the mesenchymal transition to which undergo CCM-mutated ECs is regulated through epigenetic mechanisms and that the inhibition of these processes ameliorate CCM pathological phenotype.
Importantly, 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.