Luca Bravi

EndMT contributes to the onset and progression of cerebral cavernous malformations

Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-β and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.

KLF4 is a key determinant in the development and progression of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss‐of‐function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial‐to‐mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFβ/BMP‐dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3‐MEK5‐ERK5‐MEF2 signaling axis that induces a strong increase in Kruppel‐like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1‐null ECs. KLF4 promotes TGFβ/BMP signaling through the production of BMP6. Importantly, in endothelial‐specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.

Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice

Significance Cerebral cavernous malformation (CCM) disease can lead to brain hemorrhages, seizures, and paralysis. No pharmacological therapy is currently available. Here we define, to our knowledge for the first time in vivo, the sequence of molecular events that lead to CCM vascular cavernomas. We found that β-catenin activation is the first trigger followed by TGF-β signaling, which, in turn, mediates the progression of the disease. We also show that β-catenin signaling is cell-autonomous and independent of Wnt-receptor activation. Most importantly, these studies prompted us to identify pharmacological agents that, by targeting the altered β-catenin signaling, limit the formation of brain vascular cavernomas in mice with CCM3 ablation in endothelial cells. These drugs are currently used in clinics for different pathologies and may be repurposed for CCM therapy. Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell–selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor–independent stimulation of β-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin–driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Defective autophagy is a key feature of cerebral cavernous malformations

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss‐of‐function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss‐of‐function activates the mTOR‐ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole‐cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2‐silenced human endothelial cells and in both cells and tissues from an endothelial‐specific CCM3‐knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial‐to‐mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

VE-cadherin at a glance.

Although being a monolayer the vascular endothelium controls fundamental vessel functions such as permeability, leukocyte extravasation and angiogenesis. The endothelial selective transmembrane constituent of adherens junctions, Vascular Endothelial- (VE-) cadherin plays a crucial role in the regulation of such activities. The signaling pathways controlled by VE-cadherin as well as the ones that regulate VE-cadherin activity start to be elucidated. This delineates a complex network of molecular and functional interactions that can be altered in pathologies.

Endothelial Cells Lining Sporadic Cerebral Cavernous Malformation Cavernomas Undergo Endothelial-to-Mesenchymal Transition

Background and Purpose— Cerebral cavernous malformation (CCM) is characterized by multiple lumen vascular malformations in the central nervous system that can cause neurological symptoms and brain hemorrhages. About 20% of CCM patients have an inherited form of the disease with ubiquitous loss-of-function mutation in any one of 3 genes CCM1, CCM2, and CCM3. The rest of patients develop sporadic vascular lesions histologically similar to those of the inherited form and likely mediated by a biallelic acquired mutation of CCM genes in the brain vasculature. However, the molecular phenotypic features of endothelial cells in CCM lesions in sporadic patients are still poorly described. This information is crucial for a targeted therapy. Methods— We used immunofluorescence microscopy and immunohistochemistry to analyze the expression of endothelial-to-mesenchymal transition markers in the cavernoma of sporadic CCM patients in parallel with human familial cavernoma as a reference control. Results— We report here that endothelial cells, a cell type critically involved in CCM development, undergo endothelial-to-mesenchymal transition in the lesions of sporadic patients. This switch in endothelial phenotype has been described only in genetic CCM patients and in murine models of the disease. In addition, TGF-&bgr;/p-Smad- and &bgr;-catenin-dependent signaling pathways seem activated in sporadic cavernomas as in familial ones. Conclusions— Our findings support the use of common therapeutic strategies for both sporadic and genetic CCM malformations.

The role of microvascular endothelial WNT signaling the formation of the blood brain barrier

We analyzed the pathological consequences of abnormal Wnt/β-catenin signaling in endothelial cells of brain vessels using a murine model of Cerebral Cavernous Malformation (CCM) disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report increased transcription activity of β-catenin in CCM3-knockout endothelial cells in in-vitro and in-vivo models. Such activation is cell-autonomous, independent of Wnt-receptor stimulation, does not induce canonical Wnt/β-catenin signaling and represents an early response to CCM3 ablation that initiates the expression of EndMT makers before the onset of Tgf-β/BMP signaling which is required for the progression of the pathology, as we have previously described. We also show that the NSAIDs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, significantly reduce the number and dimension of vascular lesions in the central nervous system of mice with endothelial cell CCM3 knockout. These NSAIDs thus represent pharmacological tools for inhibition of the formation of vascular lesions, particularly with a view to patients affected by the genetic variant of CCM, who continue to develop new malformations over time.

Junctional Signaling in Endothelial Cells

Cell-to-cell junctions not only support the crucial function of mutual adhesion among the cells of the endothelial layer but they also harmonize the signaling and transcription program of each individual cell to its adhesive interactions with neighboring cells. Such coordination is achieved through association with cell-to-cell junctions of multiprotein complexes that can start specific signaling programs, depending on the state of cell-to-cell junctions, and that can also modulate the organization of cell-to-cell junctions themselves. Such reciprocity between adhesion and signaling is crucial for the physiological activity of the endothelium.