Hereditary haemorrhagic telangiectasia (HHT) is characterised by arteriovenous malformations (AVMs). These vascular abnormalities form when arteries and veins directly connect, bypassing the local capillary system. Large AVMs may occur in the lungs, liver and brain, increasing the risk of morbidity and mortality. Smaller AVMs, known as telangiectases, are prevalent on the skin and mucosal lining of the nose, mouth and gastrointestinal tract and are prone to haemorrhage. HHT is primarily associated with a reduction in endoglin (ENG) or ACVRL1 activity due to loss-of-function mutations. ENG and ACVRL1 transmembrane receptors are expressed on endothelial cells (ECs) and bind to circulating ligands BMP9 and BMP10 with high affinity. Ligand binding to the receptor complex leads to activation of the SMAD1/5/8 signalling pathway to regulate downstream gene expression. Various genetic animal models demonstrate that disruption of this pathway in ECs results in AVMs. The vascular abnormalities underlying AVM formation result from abnormal EC responses to angiogenic and haemodynamic cues, and include increased proliferation, reduced migration against the direction of blood flow and an increased EC footprint. There is growing evidence that targeting VEGF signalling has beneficial outcomes in HHT patients and in animal models of this disease. The anti-VEGF inhibitor bevacizumab reduces epistaxis and has a normalising effect on high cardiac output in HHT patients with hepatic AVMs. Blocking VEGF signalling also reduces vascular malformations in mouse models of HHT1 and HHT2. However, VEGF signalling is complex and drives numerous downstream pathways, and it is not yet clear which pathway (or combination of pathways) is critical to target. This review will consider the recent evidence gained from HHT clinical and preclinical studies that are increasing our understanding of HHT pathobiology and informing therapeutic strategies.

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and endothelial cell number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds.

Hereditary Haemorrhagic Telangiectasia (HHT) causes arteriovenous malformations (AVMs) in multiple organs to cause bleeding, neurological and other complications. HHT is caused by mutations in the BMP co-receptor endoglin. We characterised a range of vascular phenotypes in embryonic and adult endoglin mutant zebrafish and the effect of inhibiting different pathways downstream of VEGF signalling. Adult endoglin mutant zebrafish developed skin AVMs, retinal vascular abnormalities, and cardiac enlargement. Embryonic endoglin mutants develop an enlarged basilar artery (similar to the previously described enlarged aorta and cardinal vein) and larger numbers of endothelial membrane cysts (kugeln) on cerebral vessels. VEGF inhibition prevented these embryonic phenotypes, leading us to investigate specific VEGF-signalling pathways. Inhibiting TOR or MEK pathways prevented abnormal trunk and cerebral vasculature phenotypes, while inhibiting NOS or MAPK pathways had no effect. Combined subtherapeutic TOR and MEK inhibition prevented vascular abnormalities, confirming synergy between these pathways in HHT. These results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling. Combined low dose MEK and TOR pathway inhibition may represent a novel therapeutic strategy in HHT.

Rationale: Hereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin (ENG) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling. Objective: We therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling. Methods and Results: We used light sheet fluorescence microscopy to visualise the vasculature in engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial kugeln on cerebral vessels. Adult engmu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways. Conclusions: These results indicate the HHT- like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT.

terising changes in local gene expression in endothelium sampled from control coronary arteries and from advanced coronary atherosclerotic plaques. Methods and Results A protocol was optimised to perform laser capture microdissection for isolation of endothelium in under 20 minutes, from snap-frozen porcine coronary artery cross-sections for total RNA sequencing (n=5 D374Y-PCSK9 hyperlipidaemic minipigs fed on a high fat high cholesterol diet; 10 vessels). Endothelium was sampled from control left anterior descending arteries consisting of no plaque, and from overlying advanced atherosclerotic plaque in stenotic right coronary arteries. Differential gene expression and gene ontology enrichment analyses revealed the upregulation of numerous atheroprone inflammatory genes in diseased endothelium overlying atherosclerotic plaque compared to healthy endothelium (figure 1: p<0.01, FDR<0.05, fold change >2). Biological pathways related to atherosclerosis that the differentially expressed genes are most enriched in are shown (table 1: enrichment score >3). Conclusions We report in vivo changes in gene expression in diseased endothelium overlying advanced coronary atherosclerotic plaques, with upregulation of inflammatory genes. The differentially expressed genes are enriched in processes related to atherosclerosis, suggesting the validity of this approach to study how gene expression changes during coronary atherosclerotic plaque development in vivo. Our model system allows for further studies coupling together these readouts to a fully validated 3D vessel reconstruction method to co-register gene expression profiles to local blood flow data, as a novel methodology for understanding the mechanisms by which local flow disturbances may affect atherogenesis. This will provide new insights into how disturbed flow and coronary atherosclerotic plaque development are causally related.