**4. Conclusions**

The two-hit hypothesis has been widely accepted for many years as a plausible explanation for the phenotypic variability found in HHT. Here, we have reviewed some of the reported environmental and genetic second-hits (Figure 2). Thus, germline heterozygous mutations in HHT genes (first-hit) result in a monoallelic protein loss in endothelial cells. A subsequent environmental stimulus (second-hit) like inflammation, hypoxia, neoangiogenesis, vascular injury, shear stress, radiation, or trauma, can induce the expression/activation of mediators, which generate a microenvironment where HHT protein levels are below the needed functional threshold. Furthermore, the existence of a genetic second-hit (somatic mutation in the normal HHT allele), combined with an environmental trigger (tertiary-hit), and/or the presence of modifier variants, can set a much lower threshold for disease development. In all cases, the consequence is an impaired endothelial cell function that leads to the generation of telangiectases and AVMs. Overall, the current clinical data, as well as in vivo and in vitro experimental results, support the second-hit hypothesis to explain why certain individuals with HHT genotypes develop earlier and/or more severe clinical phenotypes than other family members. Also, the organ-specific location of telangiectases and AVMs in HHT, and the di ffering age of presentation for lesions at each site, sugges<sup>t</sup> that the required second-hits may be tissue-specific. However, major gaps in our knowledge remain, particularly in delineating the exact role of the di fferent second-hits that lead to the development of clinically relevant symptoms in HHT, and how this information can be applied in the clinical practice. Further studies to better understand the pathological mechanisms of HHT, including identification of novel potential second-hits, are needed, as well as the translation of this knowledge into preventive clinical measures and treatments.

**Author Contributions:** Conceptualization, C.B. andM.L.; formal analysis, C.B., P.B.-T., J.M. andM.L.; resources, C.B.; writing—original draft preparation, C.B.; writing—review and editing, C.B., P.B.-T., J.M., and M.L.; visualization, C.B., P.B.-T., J.M. and M.L.; supervision, C.B., P.B.-T., J.M. and M.L.; project administration, C.B.; funding acquisition, C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Consejo Superior de Investigaciones Científicas of Spain (CSIC; gran<sup>t</sup> 201920E022 to C.B.).

**Acknowledgments:** CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by European Regional Development (FEDER) funds. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

**Conflicts of Interest:** The authors declare no conflict of interest.
