Biology subjects

Turowski, P.

Publications and source records attributed to Turowski, P..

5 recordsLinked to original sources

bioRxiv2023

Targeting NRP1 with EG00229 induces neurovascular permeability

NRP1 is a therapeutic target for inhibiting vascular endothelial growth factor (VEGF)-induced blood vessel dysfunction. The small molecule EG00229 was designed to inhibit VEGF binding to NRP1 and reduce pathological blood vessel growth. However, it is unknown whether EG00229 could also be used to reduce VEGF164-induced vascular leakage, which often exacerbates ischemic diseases due to VEGF upregulation. Here, we show that prior treatment with EG00229 prevents VEGF164-induced vascular permeability signalling, but, unexpectedly, also find that EG00229 increased rather than inhibited vascular leakage. Thus, EG00229 increased vascular leakage either when added alone or concurrently with VEGF164, both in perfused retinal explants and across primary brain EC monolayers. This EG00229-induced vascular leakage was not an off-target effect, because it relied on endothelial NRP1 expression and NRP1s VEGF164 binding pocket, yet was independent of VEGFR1 and VEGFR2. Moreover, EG00229 activated molecular events typical of VEGF164-induced paracellular permeability, including p38 MAP kinase (p38) and SRC family kinase (SFK) phosphorylation as well as CDH5 rearrangement in endothelial junctions. Investigating EG00229-induced signalling therefore helps elucidate NRP1-dependent mechanisms of paracellular permeability induction and might help identifying new approaches to modulate the neurovascular barrier.

physiology↗

bioRxiv2023

Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype that protects them from atherosclerosis. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether this is required for athero-protective signaling is unclear. Here, we show that Caveolin-1-rich microdomains become polarized at the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by higher membrane rigidity, filamentous actin (F-actin) and lipid accumulation. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels, while ubiquitously expressed, mediate localized Ca2+ entry at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal bursts in Ca2+ activate the anti-inflammatory factor endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression. Our work reveals a novel polarized mechanosensitive signaling hub that induces an anti-inflammatory response in arterial ECs exposed to high laminar shear stress.

cell biology↗

bioRxiv2022

SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response

Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Modernas mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m{Psi}), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m{Psi}Cm{Psi}AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m{Psi}Cm{Psi}AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m{Psi}Cm{Psi}AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m{Psi}Cm{Psi}AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

immunology↗

bioRxiv2020

Methamphetamine enhances caveolar transport of therapeutic drugs across the rodent blood-brain barrier

The blood-brain barrier (BBB) is a multifactorial and multicellular vascular interface separating the systemic environment from the central nervous system (CNS). It gates cerebral penetration of circulating molecules and cells and is the principal reason for low accumulation of many therapeutics in the brain. Low dose methamphetamine (METH) induces fluid phase transcytosis across the BBB in vitro and could therefore be used to enhance CNS drug delivery. Here we show, that low dose intravascular METH induced significant leakage exclusively via caveolar transport at the intact BBB in rodents ex vivo. Notably, METH-induced leakage was suppressed at 4{degrees}C and in Caveolin-1 (CAV1) knockout mice. Furthermore, METH strongly enhanced brain penetration of therapeutic molecules, namely doxorubicin (DOX), a small chemotherapeutic agent, and aflibercept (AFL), a ca. 100 kDA recombinant protein. Lastly, METH improved the therapeutic efficacy of DOX in a mouse model of human glioblastoma (GBM), as measured by a 25% increase in median survival time (p = 0.0024). Collectively, our data indicated that METH can facilitate preclinical assessment of novel experimental treatments and has the potential to enhance drug delivery to the diseased CNS.

neuroscience↗

bioRxiv2019

AMP-activated protein kinase is a key regulator of retinal vascular permeability

Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to measure and manipulate acute neurovascular permeability and signal transduction to study the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+ and Ca2+/calmodulin-dependent protein kinase kinase (CAMKK). Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS), which in turn increased VE-cadherin phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinase and HSP27, indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, the ex-vivo retina model was easily accessible and, due to its compatibility with small molecule antagonists/agonists and siRNA, constitutes a reliable tool to study regulators and mechanism of neurovascular permeability.

neuroscience↗