Unraveling Immune Cell Interactions and Multicellular Dynamics in GNAQ-Driven Capillary Malformation

Project Summary/Abstract – Capillary malformations (CM) are slow-flow vascular abnormalities present at birth and predominantly manifest as cutaneous lesions. In the context of the rare neurocutaneous disorder Sturge Weber Syndrome, individuals exhibit CM not only on the skin but also within the cerebral and ocular domains. 90% of CM are caused by a somatic activating mutation in GNAQ, the gene encoding the a-subunit of the heterotrimeric G-protein - Gaq. Specifically, the somatic GNAQ mutation involves the substitution of arginine (R) at amino acid position 183 with glutamine (Q) and is notably enriched in endothelial cells (EC) isolated from CM- affected regions in both the skin and the brain. Expansion of isolated EC from patient specimens reveals up to 21% mutant allelic frequency, reflecting the mosaic nature of the mutation. To date, little is known about how the p.R183Q mutation in the Gaq activation domain leads to abnormal capillaries. CM comprises enlarged vessels lined with sprouting EC surrounded by disorganized or a lack of mural cell (pericytes and smooth muscle cells) coverage. Immunohistologic examinations indicate that blood vessels in cutaneous and cerebral lesions of SWS patients exhibit extravascular fibrin and a deficiency of the tight junction protein zona occludens-1, signifying a compromised endothelial barrier in CM. Moreover, CM tissue sections also revealed MRC1pos, LYVE1pos, CD163pos and CD68pos macrophage cells surrounding the CM vessels of the brain and skin suggesting a pro- inflammatory environment. These findings provide a premise for the central hypothesize that the mutant EC recruit macrophages and that this leads to altered and dysfunctional EC-mural cell interaction. Alternative hypothesis is that interaction between mutant EC and neighboring mural cells leads to pro-inflammatory state driving macrophage recruitment and vessel dysfunction. The proposed study will test this hypothesis through the following aims: (1) determine macrophage interaction in mosaic endothelium (2) characterize the interaction among endothelial cells, mural cells, and macrophages in a multicellular spheroid model. Development and characterization of multicellular spheroid model will serve beyond the scope of the proposed work as a predictive platform for drug screening in contrast to traditional 2D cell cultures, effectively mimicking tissue responses to drugs and allowing for the evaluation of drug efficacy and toxicity. Hence, this also meets NIH’s continuing support for non-animal model alternative methods. These studies will be conducted under the supervision of co- mentors, Dr. Joyce Bischoff (vascular biologist) and Dr. Christopher Chen (biomedical engineer). My research advisory committee members and collaborators are leading experts in macrophage biology (Dr. Ruth Franklin), organoids (Dr. Jennifer Lewis and Dr. Alessandro Fiorenzano), neuropathologist (Dr. Sanda Alexandrescu) and bioinformatician (Dr. Vitor Rezende da Costa Aguiar). With additional support from the MOSAIC UE5 awardee sponsored professional development opportunities, continued training in the K99 phase will prepare Dr. Nasim for successful transition to independence.