promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Allegheny Health Network

The interaction between adenosine and soluble epoxide hydrolase (sEH) in vascular response is not known. Therefore, we hypothesized that lack of sEH in mice enhances adenosine-induced relaxation through A(2A) adenosine receptors (AR) via CYP-epoxygenases and peroxisome proliferator-activated receptor γ (PPARγ). sEH(-/-) showed an increase in A(2A) AR, CYP2J, and PPARγ by 31%, 65%, and 36%, respectively, and a decrease in A(1)AR and PPARα (30% and 27%, respectively) vs. sEH(+/+). 5'-N-ethylcarboxamidoadenosine (NECA, an adenosine receptor agonist), CGS 21680 (A(2A) AR-agonist), and GW 7647 (PPARα-agonist)-induced responses were tested with nitro-l-arginine methyl ester (l-NAME) (NO-inhibitor; 10(-4) M), ZM-241385, SCH-58261 (A(2A) AR-antagonists; 10(-6) M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10(-5) M), 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; 10 μM) or trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB, sEH-inhibitors; 10(-5) M), and T0070907 (PPARγ-antagonist; 10(-7) M). In sEH(-/-) mice, ACh response was not different from sEH(+/+) (P > 0.05), and l-NAME blocked ACh-responses in both sEH(-/-) and sEH(+/+) mice (P < 0.05). NECA (10(-6) M)-induced relaxation was higher in sEH(-/-) (+12.94 ± 3.2%) vs. sEH(+/+) mice (-5.35 ± 5.2%); however, it was blocked by ZM-241385 (-22.42 ± 1.9%) and SCH-58261(-30.04 ± 4.2%). CGS-21680 (10(-6) M)-induced relaxation was higher in sEH(-/-) (+37.4 ± 5.4%) vs. sEH(+/+) (+2.14 ± 2.8%). l-NAME (sEH(-/-), +30.28 ± 4.8%, P > 0.05) did not block CGS-21680-induced response, whereas 14,15-EEZE (-7.1 ± 3.7%, P < 0.05) did. Also, AUDA and t-AUCB did not change CGS-21680-induced response in sEH(-/-) (P > 0.05), but reversed in sEH(+/+) (from +2.14 ± 2.8% to +45.33 ± 4.1%, and +63.37 ± 7.2, respectively). PPARα-agonist did not relax as CGS 21680 (-2.48 ± 1.1 vs. +37.4 ± 5.4%) in sEH(-/-), and PPARγ-antagonist blocked (from +37.4 ± 5.4% to +9.40 ± 3.1) CGS 21680-induced relaxation in sEH(-/-). Our data suggest that adenosine-induced relaxation in sEH(-/-) may depend on the upregulation of A(2A) AR, CYP2J, and PPARγ, and the downregulation of A(1) AR and PPARα.

Adenosine A2A receptor modulates vascular response in soluble epoxide hydrolase-null mice through CYP-epoxygenases and PPARγ.

High salt (4% NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A(2A) receptor (A(2A)AR). Evidence suggests that A(2A)AR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A(2A)AR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A(2A)AR⁺/⁺, but exaggerates contraction in A(2A)AR⁻/⁻. Organ bath and Western blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A(2A)AR⁺/⁺ and A(2A)AR⁻/⁻ mice aorta. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A(2A)AR⁺/⁺, whereas contraction was observed in A(2A)AR⁻/⁻ mice and this was attenuated by A₁AR antagonist (DPCPX). CGS 21680 (selective A(2A)AR agonist) enhanced relaxation in HS-A(2A)AR⁺/⁺ versus NS-A(2A)AR⁺/⁺, which was blocked by EETs antagonist (14,15-EEZE). Compared with NS, HS significantly upregulated the expression of vasodilators A(2A)AR and cyp2c29, whereas vasoconstrictors A₁AR and cyp4a in A(2A)AR⁺/⁺ were downregulated. In A(2A)AR⁻/⁻ mice, however, HS significantly downregulated the expression of cyp2c29, whereas A₁AR and cyp4a were upregulated compared with A(2A)AR⁺/⁺ mice. Hence, our data suggest that in A(2A)AR⁺/⁺, HS enhances A(2A)AR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A₁AR levels, whereas in A(2A)AR⁻/⁻, HS exaggerates contraction through decreased cyp-epoxygenases and increased A₁AR levels.

High salt diet exacerbates vascular contraction in the absence of adenosine A₂A receptor.

This study aims to investigate the signaling mechanism involved in HS-induced modulation of adenosine-mediated vascular tone in the presence or absence of adenosine A2A receptor (A2AAR). We hypothesized that HS-induced enhanced vascular relaxation through A2AAR and epoxyeicosatrienoic acid (EETs) is dependent on peroxisome proliferator-activated receptor gamma (PPARγ) and ATP-sensitive potassium channels (KATP channels) in A2AAR(+/+) mice, while HS-induced vascular contraction to adenosine is dependent on soluble epoxide hydrolase (sEH) that degrades EETs in A2AAR(-/-) mice. Organ bath and Western blot techniques were conducted in HS (4 % NaCl) and normal salt (NS, 0.45 % NaCl)-fed A2AAR(+/+) and A2AAR(-/-) mouse aorta. We found that enhanced vasodilation to A2AAR agonist, CGS 21680, in HS-fed A2AAR(+/+) mice was blocked by PPARγ antagonist (T0070907) and KATP channel blocker (Glibenclamide). Also, sEH inhibitor (AUDA)-dependent vascular relaxation was mitigated by PPARγ antagonist. PPARγ agonist (Rosiglitazone)-induced relaxation in HS-A2AAR(+/+) mice was attenuated by KATP channel blocker. Conversely, HS-induced contraction in A2AAR(-/-) mice was attenuated by sEH inhibitor. Overall, findings from this study that implicates the contribution of EETs, PPARγ and KATP channels downstream of A2AAR to mediate enhanced vascular relaxation in response to HS diet while, role of sEH in mediating vascular contraction in HS-fed A2AAR(-/-) mice.

High salt diet modulates vascular response in A2AAR (+/+) and A 2AAR (-/-) mice: role of sEH, PPARγ, and K ATP channels.

Herein, we highlight the technical feasibility of generating a functional mini thymus with a novel hydrogel system, based on a peptide-based self-assembly platform that can induce the formation of 3-D thymic epithelial cell (TEC) clusters. Amphiphilic peptide EAK16-II co-assembled with its histidinylated analogue EAKIIH6 into beta-sheet fibrils. When adaptor complexes (recombinant protein A/G molecules loaded with both anti-His and anti-EpCAM IgGs) were added to the mix, TECs were tethered to the hydrogel and formed 3-D mini clusters. TECs bound to the hydrogel composites retained their molecular properties; and when transplanted into athymic nude mice, they supported the development of functional T-cells. These mini thymic units of TECs can be useful in clinical applications to reconstitute T-cell adaptive immunity.

Isha Pradhan

Institute of Cellular Therapeutics

Allegheny Health Network

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Isha Pradhan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Cellular Therapeutics

Allegheny Health Network

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Institute of Cellular Therapeutics