Biology subjects

Okumura, S.

Publications and source records attributed to Okumura, S..

3 recordsLinked to original sources

bioRxiv2023

Spatiotemporal EP4-fibulin-1 expression is associated with vascular intimal hyperplasia

AimsCyclooxygenase-2- and microsomal prostaglandin E synthase-1-derived prostaglandin E2 (PGE2) are involved in vascular intimal hyperplasia (IH). Although extensive studies have revealed the roles of PGE2 receptors (EPs) in IH, spatiotemporal EP expressions and downstream targets have not been fully elucidated. In this study, we focused on EP4 and investigated its role in vascular IH. Methods and ResultsWe generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found prominent EP4 expression in the proliferative neointima 2 weeks after femoral artery wire injury. Expression of EP4 were returned to the baseline level 4 weeks after vascular injury (VI). Injury-induced IH was diminished in vascular smooth muscle cell (VSMC)-specific EP4 heterozygous deficient mice (Ptger4fl/+;SM22-Cre) 2 and 4 weeks after VI compared to SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-specific EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). Systemic EP4 antagonist administration reduced VI-induced IH in wild-type mice. We investigated the role of extracellular matrix proteins, as downstream regulated targets of EP4. Stimulation of EP4 increased mRNA and protein levels of fibulin-1 (a multifunctional glycoprotein) in Ptger4-Tg VSMCs. Fibulin-1C or -1D recombinant proteins increased VSMC proliferation, whereas proliferation was decreased in fibulin-1-deficient VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6-8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was upregulated by EP4 stimulation, and fibulin-1 and ECM1 proteins additively enhanced VSMC proliferation. Similar to EP4 expression, both fibulin-1 and ECM1 were abundantly expressed in the neointima 2 weeks after VI. Furthermore, injury-induced IH was attenuated in VSMC-specific fibulin-1 deletion mice (Fbln1fl/fl;SM22-Cre) compared to Fbln1fl/fl. ConclusionsEP4 was upregulated in proliferative IH, and EP4-induced fibulin-1 cooperated with ECM1 to promote IH through VSMC proliferation. The calcium binding EGF-like modules 6-8 of fibulin-1 are indicated to regulate cell proliferation. A Translational PerspectiveRecent advances in drug-eluting stents have significantly contributed to the reduction of vascular IH. However, the detailed mechanism underlying IH after stenting remains to be elucidated. We found that prostaglandin E2-EP4-induced fibulin-1 plays a role in IH through VSMC proliferation. It is well recognized that prostaglandin E2 plays a role in IH, but inhibition of cyclooxygenase-2 has side effects such as thrombogenesis. Because EP4 and fibulin-1 were upregulated specifically in the neointima after vascular injury, oral or local administration of an EP4 antagonist or the downregulation of fibulin-1 would be potential therapeutic strategies to restrain IH.

physiology↗

bioRxiv2022

Silicon as a microfluidic material for imaging and incubation of droplets

Droplet microfluidics has become a powerful tool in life sciences, underlying digital assays, single-cell sequencing or directed evolution, and it is making foray in physical sciences as well. Imaging and incubation of droplets are crucial, yet they are encumbered by the poor optical, thermal and mechanical properties of PDMS - the de facto material for microfluidics. Here we show that silicon is an ideal material for droplet chambers. Si chambers pack droplets in a crystalline and immobile monolayer, are immune to evaporation or sagging, boost the number of collected photons, and tightly control the temperature field sensed by droplets. We use the mechanical and optical benefits of Si chambers to image [~]1 million of droplets from a multiplexed digital assay - with an acquisition rate similar to the best in-line methods. Lastly, we demonstrate their applicability with a demanding assay that maps the thermal dependence of Michaelis-Menten constants with an array of [~]150,000. The design of the Si chambers is streamlined to avoid complicated fabrication and improve reproducibility, which makes Silicon a complementary material to PDMS in the toolbox of droplet microfluidics. Significance StatementAs the technological engine behind single-cell sequencing and digital assays, droplets microfluidics has revolutionized life science and molecular diagnosis, and is making foray into physical sciences as well. Observing droplets in a controlled manner is becoming crucial, but PDMS - the de facto material of microfluidics - hampers imaging and incubation. Here we revisit silicon as a microfluidic material and show that its superior mechanical, optical and thermal performances improve the throughput and operation of droplets assay.

biochemistry↗

bioRxiv2021

Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice

Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 {+/-} 0.5 to 58 {+/-} 1.2 %) and remained low at 4 weeks (57 {+/-} 1.0 %). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

physiology↗