Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin-1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts However, the participation of histatin-1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin-1 promotes angiogenesis, as shown, using the chick chorioallantoic membrane model, and by an tube formation assay, using both human primary cultured endothelial cells (HUVECs) and the EA.hy926 cell line. Specifically, histatin-1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin-1 increased the recruitment of RIN2, a Rab5-guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin-1-dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin-1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin-1 is a novel proangiogenic factor that may contribute to oral wound healing.-Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina-Berríos, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies; but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA-sequencing to investigate mouse brain endothelial cells with specific Ccm3 gene deletion (Ccm3-iECKO). We found that Ccm3 deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared to wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. Lastly, we created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3, expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCM. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.

Background and Purpose: Cerebral cavernous malformations (CCM) present as mulberry-like malformations of the microvasculature of the central nervous system. Current medical treatment of CCM lesions is limited to surgical removal of the vascular malformations. It is, therefore, important to identify therapeutic drug treatments for patients with CCM. Propranolol has shown great benefit in the treatment of infantile hemangioma. In addition, patients with CCM who receive propranolol have demonstrated a reduction of their lesions. Our investigation set out to provide preclinical data to support propranolol as a therapeutic treatment. Methods: An inducible endothelial-specific Ccm3 knockout murine model (CCM3 iECKO ) was used, with assessment of lesion quantity and size following oral treatment with propranolol. Scanning and transmission electron microscopy were used to characterize the CCM3 iECKO lesions and the effects of propranolol on the disease. Immunofluorescent imaging was used to investigate pericyte coverage in the propranolol-treated CCM3 iECKO mice. Results: With propranolol treatment, the lesion quantity, size, and volume decreased in both the brain and retina in the CCM3 iECKO model. Novel characteristics of the CCM3 iECKO lesions were discovered using electron microscopy, including plasmalemmal pits and thickening of the endothelial-pericyte basal membrane. These characteristics were absent with propranolol treatment. Pericyte coverage of the CCM3 iECKO lesions increased after propranolol treatment, and vascular leakage was reduced. Conclusions: This study supports the concept that propranolol can be used to reduce and stabilize vascular lesions and can, therefore, be suggested as a pharmaceutical treatment for CCM.