hpbecs  (Epithelix)

Journal: Molecular Medicine Reports

Article Title: CIP2A promotes bronchiolitis obliterans by activating the NF‑κB pathway

doi: 10.3892/mmr.2025.13473

Figure Lengend Snippet: Inhibiting CIP2A blocks the activation of NF-κB signaling in HPBECs. The expression of CIP2A, and phosphorylation of IKBα and expression of p65 were assessed by western blotting. n=3. CIP2A, cell proliferation regulating inhibitor of protein phosphatase 2A; DA, diacetyl; HPBECs, human primary bronchial epithelial cells; IκBα, inhibitor of NF-κB α; NF-κB, nuclear factor-κB; p-, phosphorylated.

Article Snippet: HPBECs were purchased from iCell Bioscience Inc. and were cultured in the HPBECs cultivation system (iCell Bioscience Inc.).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot

Journal: Tissue & cell

Article Title: Down-regulation of platelet-derived growth factor receptor β in pericytes increases blood-brain barrier permeability and significantly enhances α-synuclein in a Parkinson's Disease 3D cell model in vitro under hyperglycemic condition.

doi: 10.1016/j.tice.2025.102751

Figure Lengend Snippet: Fig. 1. Establishment of MVU, NVU, PD, HM, PD-HM and RPN cell models with four different cell types by RAFT 3D cell culture system. (1) HpBECs were seeded in a hydrogel that formed in the bottom of a 24-well plate, with a thickness of approximately 0.5 mm. (2) After incubation for 30 minutes at 37℃with 1 mL of hpBEC culture medium, 1 mL of hpPs cell stock solution was added to the bottom of the well, and the hpPs were seeded on the surface of the hydrogel. (3) Co- culturing of the hpBECs and hpPs was carried out for three days, followed by adding hpAs hydrogel to 24-well inserts. (4) The hpAs were seeded in the hydro- gel that formed in the bottom of the inserts, with a thickness of about 0.5 mm. 200 μL of hpAs culture medium was added, and the hpBECs, hpPs, and hpAs were co- cultured for an additional four days to form a BBB cell model similar to MVU. (5) Subsequently, 200 μL of dopaminergic neuron (SH-SY5Y) cell stock solution was added to the 24-well insert, and the SH-SY5Y cells were seeded on the surface of the hydrogel. After co-culturing for six days, the hpBECs, hpPs, hpAs, and SH-SY5Y cells formed a three-dimensional (3D) structure similar to the NVU BBB cell model. (6) The cells grew together and influenced each other, forming a tightly connected whole. The density of cells was approximately 1.0 × 106 cells/mL, and the hydrogel and the insert were transparent. (7) This model effectively simulated the sequence of BBB cells in vivo in the order from interior to exterior of hpBECs, hpPs, hpAs, and SH-SY5Y cells. To develop the BBB cell model of PD, SH-SY5Y cells were first exposed to 6-OHDA concentrations (50 μmol/L) for 24 h and then co-cultured with the MVU model (Liebner et al., 2018). NVU cell models were co-cultured for 8 days with a glucose concentration of 30 mmol/L high glucose to construct HM (Antoni et al., 2015) (Fig. S1). RPN cell models were constructed with pericytes containing reduced gene PDGFRβ. RAFT: real architecture for tissue; E: hpBECs; P: hpPs; A: hpAs; S: SH-SY5Y cells; P-K: pericytes with gene PDGFRβ downregulated by 30 %; P-P: pericytes with gene PDGFRβ downregulated by 89 %; hpBECs: human primary brain endothelial cells; hpPs: human primary pericytes; hpAs: human primary astrocytes; BBB: blood brain barrier; MVU: microvascular unit; NVU: neurovascular unit; PD: Parkinson’s disease; HM: hyperglycemic model; PD-HM: Parkinson’s disease complicated with hyperglycemic model; RPN: reduced PDGFRβ NVU.

Article Snippet: Human primary brain endothelial cells (hpBECs) were obtained from Angio-Proteomie in Boston, Massachusetts, USA.

Techniques: Cell Culture, Incubation, Sequencing, In Vivo, Concentration Assay, Construct

Journal: Tissue & cell

Article Title: Down-regulation of platelet-derived growth factor receptor β in pericytes increases blood-brain barrier permeability and significantly enhances α-synuclein in a Parkinson's Disease 3D cell model in vitro under hyperglycemic condition.

doi: 10.1016/j.tice.2025.102751

Figure Lengend Snippet: Fig. 2. The morphological characteristics of hpBECs, hpPs (with or without reduced gene PDGFRβ), hpAs, and SH-SY5Y cells grown alone on 2D and together through RAFT cell culture system on 3D (MVU, NVU, HM, PD, PD-HM and RPN) observed under microscopy and their cell purity measured by flow cytometry. (A-C) represent hpBECs, hpAs, and SH-SY5Y cells, respectively. (D-F) represent hpPs, hpPs with gene PDGFRβ downregulated by 30 % and pericytes with gene PDGFRβ downregulated by 89 % (RT-PCR); (G) A ‘vortex-like’ structure first developed after three days of co-cultivation with hpBECs and hpPs. (H) Following four days of co-culture using the trans-well model with hpBECs, hpPs, and hpAs, a ‘tube-wall’ like structure (MVU) developed around the "vortex" structure (indicated by purple arrows). (I) Co-culturing hpBECs, hpPs, hpAs, and SH-SY5Y cells for six days (using the RAFT 3D cell model) resulted in the formation of a comparatively complete ‘vascular-like’ structure (indicated by by blue arrows), with SH-SY5Y dopaminergic neurons situated in the center, emulating the NVU. The NVU cell model was co-cultured with a high dose of glucose (30 mmol/L) for 8 days (J, HM), with 6-OHDA (50 µmol/L) for 24 h (K, PD cell model), with 6- OHDA (24 h, 50 µmol/L) and high dose of glucose (8 days, 30 mmol/L) (L, PD and HM); HpBECs, hpAs, and SH-SY5Y cells were co-cultured with pericytes with 30 % (M) or 89 % (N) downregulated PDGFRβ gene expression (RPN cell models). (J-N) The ‘vascular-like’ structures were disrupted (indicated by red arrows). 250 μm at a 10X magnification were represented by the scale bar. (W) represented the relative mRNA level of PDGFRβ tested by qRT-PCR; PDGFRB-1- Gene PDGFRβ of pericyte was downregulated by 1.01–0.71 = 30 (%) with interference No.1 shRNA (P-K); PDGFRB-2- Gene PDGFRβ of pericyte was downregulated by 1.01–0.12 = 89 (%) with interference No.2 shRNA (P-P); In addition, flow cytometry was used to confirm that the expression of the PDGFRβ protein had been downregulated by (98.2–62.6)/98.2 = 36.25 % (<50 %) (S) and (98.2–33.9)/98.2 = 65.48 % (>50 %) (U). (X,Y) represented the expression of the PDGFRβ protein which had been downregulated by (50.5325–27.5699)/50.5325 = 45.44 % (<50 %) and (50.5325–7.2565)/50.5325 = 85.64 % (>50 %) using western-blot. The expression levels of PDGFRβ protein in pericytes exhibited statistically significant differences when compared to those in P-K and P-P. The PDGFRβ protein levels (relative expression to GAPDH controls are expressed as mean ± SEM (sample size, n = 3). GAPDH was used as an internal control for normalization. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparisons. (Z) 95 % confidence interval (CI) and individual data have been also indicated in Table S1. P-value < 0.05 was considered statistically significant. * p < 0.05, * * p < 0.01, * ** p < 0.001. To distinguish between negative and positive cell populations using different colours, we overlapped the dot plots representing the populations of negative and positive cells, respectively. HpBEC, hpA and hpP were stained and labeled by corresponding specific antibodies [CD31(O; red) (Bruggisser et al., 2020), GFAP (P; green) (Brenner, 2014), PDGFRβ (Q; blue), CD13(R, T,V; dark blue)] (Lindahl et al., 1997) and their corresponding isotype controls (black population). E: human primary brain endothelial cells; P: human primary pericytes; A: human primary astrocytes; S: SH-SY5Y cells; P-K: pericytes with gene PDGFRβ downregulated by 30 %; P-P: pericytes with gene PDGFRβ downregulated by 89 %; G: high glucose; 6: 6-OHDA; MVU: microvascular unit; NVU: neurovascular unit; PD: Parkinson’s disease; HM: hyperglycemic model; PD-HM: Parkinson’s disease complicated with hyperglycemic model; RPN: reduced PDGFRβ NVU; RAFT: real architecture for tissue; PLKO.1-carrier name; qRT-PCR-Quantitative Real-time PCR; shRNA: short hairpin RNA; CI: 95 % confidence interval.

Article Snippet: Human primary brain endothelial cells (hpBECs) were obtained from Angio-Proteomie in Boston, Massachusetts, USA.

Techniques: Cell Culture, Microscopy, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Co-Culture Assay, Gene Expression, Quantitative RT-PCR, shRNA, Expressing, Western Blot, Control, Staining, Labeling, Real-time Polymerase Chain Reaction

Journal: Tissue & cell

Article Title: Down-regulation of platelet-derived growth factor receptor β in pericytes increases blood-brain barrier permeability and significantly enhances α-synuclein in a Parkinson's Disease 3D cell model in vitro under hyperglycemic condition.

doi: 10.1016/j.tice.2025.102751

Figure Lengend Snippet: Fig. 3. Changes to FSC and SSC in NVU, HM, PD, PD-HM, and RPN cell models as observed by flow cytometry. (A-L) FSC and SSC for each cell type unstained were shown in both dot plot and contour plot of flow cytometry. (A-F) After hpBECs, hpAs, hpPs, SH-SY5Y, P-K, P-P cells were cultured separately, the FSC-A and SSC-A characteristics were observed by flow cytometry. Purple arrows were used to represent normal cell populations. (E-F) The FSC-A and SSC-A values for pericytes with 30 % and 89 % downregulation of the PDGFRβ gene showed a decline compared to normal pericytes (C). This population of cells was indicated by a blue arrow; (G-J) showed the FSC-A and SSC-A of flow cytometry observations after hpBECs, hpAs, hpPs, and SH-SY5Y cell co-culture with 6-OHDA and/or high glucose; (K-L) showed the FSC-A and SSC-A of flow cytometry observations after hpBECs, hpAs, and SH-SY5Y cells were co-cultured with 30 % downregulated gene PDGFRβ pericytes (K), or with 89 % downregulated gene PDGFRβ pericytes (L); (G) When hpBECs, hpAs, hpPs, and SH-SY5Y cells were co-cultured, a subset of cell pop- ulations had decreased FSC and increased SSC parameters compared to each cell cultured alone. This population of cells was marked with the red arrow. (G-L) Compared to the NVU model, the FSC-A and SSC-A values for the HM, PD, PD-HM, RPN (30 %), and RPN (89 %) cell models decreased. This population of cells was indicated by a blue arrow. (M-N) The FSC-A and SSC-A characteristics of hpBECs, hpAs, hpPs, P-K, P-P, and SH-SY5Y cells were shown in overlapping histograms for cells cultured separately, co-cultured, and with/without 6-OHDA and/or glucose. FSC: forward scatter; SSC: side scatter; E: hpBECs; P: hpPs; A: hpAs; S: SH-SY5Y cells; G: high glucose; 6: 6-OHDA; P-K: pericytes with gene PDGFRβ downregulated by 30 %; P-P: pericytes with gene PDGFRβ downregulated by 89 %; hpBECs: human primary brain endothelial cells; hpPs: human primary pericytes; hpAs: human primary astrocytes; PD: Parkinson’s Disease; HM: hyperglycemic model; PD- HM: Parkinson’s disease complicated with hyperglycemic model; RPN: reduced PDGFRβ NVU; NVU: neurovascular unit.

Article Snippet: Human primary brain endothelial cells (hpBECs) were obtained from Angio-Proteomie in Boston, Massachusetts, USA.

Techniques: Flow Cytometry, Cell Culture, Co-Culture Assay