Biology subjects

Pumiglia, K.

Publications and source records attributed to Pumiglia, K..

3 recordsLinked to original sources

bioRxiv2026

Endothelial KRAS G12V signaling drives aberrant morphogenesis and establishes an AVM transcriptional identity in primary human endothelial cells

Somatic activating mutations in KRAS are found in the endothelium of the majority of sporadic brain arteriovenous malformations (bAVMs), yet the consequences of oncogenic KRAS signaling in endothelial cells during active vessel morphogenesis remain incompletely characterized. We expressed KRASG12V in primary human umbilical vein endothelial cells using a doxycycline-inducible lentiviral system and examined morphogenic behavior, proliferation, migration, and transcriptional output in a three-dimensional planar co-culture angiogenesis assay. KRASG12V-expressing cells failed to organize into vessel-like networks, instead forming compact sheet-like structures that persisted through day 12. A transient proliferative phase at days 3-5 resolved to control levels by day 12, consistent with preserved sensitivity to contact inhibition rather than unrestricted growth. Enhanced migration at day 5 was accompanied by upregulation of a focal adhesion and matrix remodeling program centered on ITGB3, PLAU, PLAUR, and PIK3CG. Translating ribosome-affinity purification sequencing (TRAP-seq) of the EC-specific translatome across four independent donor pools revealed progressive acquisition of an AVM-associated transcriptional identity by day 12, including upregulation of ACVRL1, ENG, JAG1, NOTCH1, ANGPT2, and TEK, with concordance to human bAVM nidus endothelium at both the gene and pathway level. Pharmacological inhibition with Alpelisib (PI3K), Trametinib (MEK), and Pazopanib (VEGFR2) demonstrated that PI3K is the principal organizer of the morphogenic phenotype. These findings characterize a KRASG12V-driven program in endothelial cells that recapitulates core transcriptional features of bAVM endothelium in a primary cell model.

Cell Biology↗

bioRxiv2025

TRAP seq in 3D Angiogenesis Assays Reveals a Distinct Endothelial Translatome Associated with Early and Late Stages of Morphogenesis.

Endothelial cells (ECs) are often a minority cell type in a tissue, limiting the utility of bulk sequencing approaches. Single cell sequencing lacks sensitivity and requires disruptive tissue digestion techniques. TRAP seq (Translating Ribosome Affinity Purification) or "RiboTag" has been used to overcome these limitations. Multicellular co-culture systems allow primary ECs to differentiate and undergo tubular morphogenesis in cell culture, however similar limitations exist with these in vitro assays, as ECs are under-represented by as much as a factor of 10 in many assays. We sought to use TRAP seq to better understand the gene expression landscapes that drive these morphogenic events. We found TRAP seq selectively enriches for endothelial RNA in two distinct co-culture paradigms, in both the planar and fibrin bead co-culture assays. Intriguingly, the use of this technology in vitro, revealed distinct gene expression changes in blood vessel development and in the mitotic cell cycle, with genes unique to early and late phases of morphogenesis. It is widely accepted that expression of the NOTCH signaling pathway is a regulator of angiogenesis in vivo. Correspondingly, we found that a large number of NOTCH related genes relevant to endothelial cell biology, were changing across this morphogenic process, underpinning the importance and utility of this technology in 3D multicellular cultures that model in vivo environments.

cell biology↗

bioRxiv2019

Alpha 6 Integrins Regulate the Expression of Laminin-511 and CXCR4 to Promote Endothelial Tubular Morphogenesis

During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 are known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays in conjunction with RNAi approaches to demonstrate that depletion of either the 4 chain of laminin-411 or the 5 chain of laminin-511 from endothelial cells inhibits sprouting and tube formation. Depletion of 6 integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or 6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when the 6 integrin expression was inhibited. Additionally, the depletion of 6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that 6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the 6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes. Summary statementEndothelial-secreted laminin-511 and 6 integrins promote endothelial morphogenesis by regulating the expression of the chemokine receptor, CXCR4. The depletion of 6 integrins from established tubes results in the loss of tube integrity and laminin-511.

cell biology↗