Biology subjects

Sherpa, C.

Publications and source records attributed to Sherpa, C..

5 recordsLinked to original sources

bioRxiv2026

An endothelial-specific TIE2-L914F mouse model reveals cellular mechanisms of venous malformation progression and recapitulates the patient's response to targeted therapy

TIE2 p.L914F is the most common driver mutation of sporadic venous malformation (VM), a disease characterized by massively enlarged veins. The study of the molecular and cellular mechanisms contributing to TIE2-driven VM has been hindered by the lack of a genetic in vivo model. We generated a mouse system in which TIE2L914F is expressed postnatally in endothelial cells (EC). Mutant mice develop visible lesions with dilated veins with irregular smooth muscle cell coverage, histologically mimicking patients VM. With the use of an mCherry reporter we demonstrate that lesions form within the venous vasculature, and mutant ECs undergo clonal expansion fueled by a biphasic mechanism consisting of an early burst of proliferation followed by cell size increase. Targeted treatment with rapamycin partially reduces lesion growth by targeting increased proliferation and prolongs mouse survival, demonstrating this genetic murine model recapitulates the patients therapeutic response. Together, these findings provide new cellular and mechanistic insights into TIE2 p.L914F-driven venous malformation and establish a genetic platform for preclinical therapeutic studies.

cell biology↗

bioRxiv2026

Constitutive, mosaic expression of TIE2 p.L914F during mouse development causes formation of venous malformation

BackgroundThe hyperactivating p.L914F mutation in TIE2, a receptor tyrosine kinase that is essential for vascular development and function, has been found to drive sporadic venous malformation (VM). While germline or early developmental expression of the mutation is thought to be lethal, mosaic or somatic expression is expected to result in VM disease. However, this has never been shown experimentally. Therefore, we utilized a genetic murine model of TIE2 p.L914F to examine the effects of the mutation in the mosaic condition. ResultsUsing an mTmG reporter mouse, we show that the CMV-Cre mouse line drives mosaic Cre recombination during early embryonic development. We then crossed B6-Tg(Rosa26-TIE2L914F)EBos (TIE2L914F) mice to CMV-Cre mice, to drive mosaic expression of TIE2 p.L914F during development. The offspring of these mice did not have the expected Mendelian ratio of mutant to control animals, indicating that mutant mice experienced partial lethality during development. Furthermore, surviving CMV-Cre;TIE2L914F mutant offspring developed a VM phenotype, with the formation of massively enlarged venous/capillary vessels in various tissues. ConclusionsIn this study, we show that mosaic embryonic expression of the VM-causative mutation TIE2 p.L914F causes partial embryonic lethality and the formation of a VM phenotype. This a novel in vivo model of mutant TIE2-driven VM disease and it illustrates that the extent of the mutational event during development will contribute to varying levels of severity in the VM phenotype.

molecular biology↗

bioRxiv2025

Semaphorin 3A and 3F overexpression in TIE2 hyperactive endothelial cells contribute to the pathological lumen expansion in venous malformation

Abstract (formatted)O_ST_ABSBackgroundC_ST_ABSVenous malformation (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as leading driver of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by recruitment of wild-type endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion. MethodsTo investigate the contribution of TIE2-mutant EC and wild-type EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC. To perform longitudinal studies, we employed a three-dimensional (3D) fibrin gel lumen formation assay and a migration assay, both using wild-type EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion we used RNA-sequencing and short interference (sh)RNA in the TIE2-mutant EC. ResultsWe demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and wild-type EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of wild-type EC. ShRNA-mediated silencing of Sema3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, knock-down of Sema3A or 3F in TIE2-mutant EC normalized the blood vessel size in vivo. ConclusionsOur results demonstrate that wild-type EC are not recruited to the aberrant veins suggesting VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and 3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM. Abstract (not formatted)Venous malformations (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as one of the leading drivers of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by the recruitment of wild-type endothelial cells (EC) to the lesion or instead by TIE2-mutant EC clonal expansion. In a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC, we demonstrated that the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, suggesting lesions form by clonal expansion, while wild-type EC were not recruited to the aberrant veins. Functionally, in a three-dimensional cell competition assay, we showed that TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting and ability to form vascular lumens. In line with these findings, TIE2-mutant EC repelled wild-type EC by reversing their migration direction in a cell confrontation assay. In seeking to define the mechanism driving this repulsion phenotype, we detected elevated levels of the chemorepellent Semaphorin 3A (Sema3A) and Sema3F in the TIE2-mutant EC. ShRNA-mediated silencing of Sema3A or Sema3F rescued the chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, Sema3A or 3F knock-down in TIE2-mutant EC normalized the blood vessel morphology and size in vivo. Taken together, these data strongly indicates that Sema3A and 3F are important players in VM pathogenesis.

molecular biology↗

bioRxiv2023

Hypoxia regulate developmental coronary angiogenesis potentially through VEGFR2- and SOX17-mediated signaling

BackgroundCoronary vessels in embryonic mouse heart arises from multiple progenitor population including sinus venosus (SV), endocardium, and proepicardium. ELA/APJ signaling is shown to regulate coronary growth from SV pathway within the subepicardium, whereas VEGF-A/VEGF-R2 pathways is implicated to regulate coronary growth from endocardium pathway. Our previous study show hypoxia as a potential signaling cue to stimulate overall coronary growth and expansion within the myocardium. However, the role of hypoxia and its downstream signaling pathways in the regulation of coronary vessel development is not known. In this study, we investigated the role of hypoxia in coronary vessel development and have identified SOX17- and VEGF-R2-mediated signaling as a potential downstream pathway of hypoxia in the regulation of coronary vessel development. ResultsWe show that hypoxia gain-of-function in the myocardium through upregulation of HIF-1 disrupts the normal pattern of coronary angiogenesis in developing mouse hearts and displays phenotype that is reminiscent of accelerated coronary growth. We show that VEGF-R2 expression is increased in coronary endothelial cells under hypoxia gain-of-function in vivo and in vitro. Furthermore, we show that SOX17 expression is upregulated in developing mouse heart under hypoxia gain-of-function conditions, whereas SOX17 expression is repressed under hypoxia loss-of-function conditions. Furthermore, our results show that SOX17 loss-of-function disrupts normal pattern of coronary growth. ConclusionCollectively, our data provide strong phenotypic evidence to show that hypoxia might regulate coronary growth in the developing mouse heart potentially through VEGF-R2- and SOX17-mediated downstream signaling pathways.

developmental biology↗

bioRxiv2018

Evolution of the HIV-1 RRE during natural infection reveals key nucleotide changes that correlate with altered structure and increased activity over time

AbstractThe HIV-1 Rev Response Element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promotes nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The "early" RRE was less functionally active than the "late" RRE despite differing in sequence by only four nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over six years of untreated infection. RRE sequence diversity varied over the course of infection with evidence of selection pressure that led to sequence convergence as disease progressed. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding. ImportanceHIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev Response Element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

molecular biology↗