Biology subjects

Ejazi, S. A.

Publications and source records attributed to Ejazi, S. A..

3 recordsLinked to original sources

bioRxiv2026

Extracellular matrix proteins modulate lymphatic endothelial cell junction morphology and barrier function.

The extracellular matrix (ECM) plays a pivotal role in lymphatic vasculature physiology, yet the specific contribution of individual ECM components to lymphatic endothelial permeability remains poorly understood, limiting the development of physiologically relevant in vitro models for lymphatic disease research and therapeutic development. Here, we used an in vitro transwell platform to systematically investigate how four clinically relevant ECM proteins, collagen I, fibronectin, fibrin, and laminin, regulate human lymphatic endothelial cell (LEC) barrier function and junctional integrity. Fibrin and collagen I substrates enhanced barrier integrity, demonstrating 80% and 67% increases in transendothelial electrical resistance (TEER), respectively, compared to uncoated controls. FITC-dextran transport assays confirmed these findings, with fibrin and collagen I reducing permeability by 20% and 10%, respectively. Immunofluorescence analysis revealed elevated ZO-1 expression on fibrin, fibronectin, and laminin matrices, while VE-cadherin levels remained unchanged across conditions. Quantitative junctional analysis demonstrated that fibrin increased ZO-1 junction continuity by [~]35%, while collagen I and fibronectin enhanced continuity by [~]22%, with all ECM coatings reducing discontinuous junctions by 60-80%. Mechanistically, RhoA expression was reduced in LECs cultured on fibrin, suggesting decreased stress fiber formation contributes to enhanced barrier function, though overall actin cytoskeletal anisotropy remained unchanged. These findings demonstrate that ECM composition modulates LEC junctional organization and barrier integrity, with fibrin and collagen I exerting the most pronounced barrier-enhancing effects. This engineered platform provides a foundation for developing next-generation in vitro models of lymphatic vasculature that more accurately recapitulate physiological conditions, with applications in lymphedema research, cancer metastasis studies, and immune cell trafficking investigations.

bioengineering↗

bioRxiv2025

Fibroblasts regulate lymphatic barrier functions in a tissue dependent manner

Lymphatic dysfunction has been linked to several pathological conditions, including edema, inflammation, and cancer metastasis. The tight and adherens junction proteins between lymphatic endothelial cells (LECs) are important for preserving lymphatic vascular integrity. Despite the known role of fibroblasts in lymphangiogenesis, the direct impact of fibroblasts on LEC barrier function remains poorly understood. Here, normal human dermal fibroblast (NHDF) secretomes and non-contact co-culture models were used to examine how fibroblasts regulate human lymphatic endothelial cells (hLECs). Co-culture with dermal fibroblasts increased transendothelial electrical resistance (TEER) by > 110% and increased ZO-1 expression by >2-fold. VE-cadherin expression increased by at least 1.3-fold compared to controls. Furthermore, 4kDa dextran transport was markedly reduced, confirming an overall reduced hLEC permeability. Junction morphology analysis further showed a marked shift from discontinuous punctate and perpendicular junctions toward continuous ZO-1-rich borders, while actin anisotropy was reduced with redistribution away from aligned central stress fibers toward a more junction-supportive cytoskeletal architecture. Thrombin challenge revealed that fibroblasts also shape endothelial responses to inflammatory stress, indicating that stromal-endothelial crosstalk regulates both basal and stress-induced barrier behavior. Transcriptomic analysis identified two fibroblast-driven endothelial programs that converged on junctional remodeling but diverged in broader physiological features: activated NHDFs promoted lymphatic identity, junction stabilization, and a more quiescent endothelial state, whereas inactivated NHDFs favored extracellular matrix remodeling and partial EndoMT-like signaling while retaining some junction-supportive features. In contrast, lung fibroblast secretomes weakened barrier function, supporting the idea that stromal regulation of lymphatic permeability is tissue specific. Together, these findings identify fibroblasts as active regulators of lymphatic barrier physiology and reveal stromal heterogeneity as an important determinant of endothelial junction organization, transport, and tissue-specific lymphatic function. NEW AND NOTEWORTHYThis exciting work demonstrates that fibroblasts are active regulators of lymphatic endothelial barrier physiology during homeostasis. Dermal fibroblasts strengthen barrier function and reduce paracellular transport by promoting continuous junction organization, cytoskeletal remodeling, and extracellular matrix reprograming, while fibroblast state and tissue origin produce distinct endothelial programs.

bioengineering↗

bioRxiv2025

Structure and Activity of the Essential UCH Family Deubiquitinase DUB16 from Leishmania donovani

In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. Together these results suggest on-target activity and that DUB16 may be a druggable target to develop new anti-leishmania compounds.

biochemistry↗