Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Vessel formation with HBMVECs is enhanced at lower Geltrex TM concentrations and by mixed endothelial‐organoid media in the presence of VEGF. A) Graphical representation of 100 000 HBMVECs encapsulated in different Geltrex TM concentrations and grown for 10 days using different media and VEGF doses. Created with Biorender.com. B) Representative confocal images of Calcein AM‐stained endothelial networks formed in 80%, 60%, and 40% Geltrex TM supplemented with 50 ng mL −1 VEGF for 10 days. Scale bars: 500 µm. (C) Percentage of vessel area (%), total vessel length (mm), and lacunarity values of endothelial networks formed in 80%, 60%, and 40% Geltrex TM , with or without 50 ng mL −1 VEGF. Mean ± SD, N = 6 independent wells, two repeats. Two‐way ANOVA; asterisks showing Tukey's post hoc comparisons; ns = not significant ( p > 0.05); * p < 0.05, *** p < 0.001, **** p < 0.0001. D) Representative confocal images of Calcein AM‐stained endothelial networks grown in 50% Geltrex TM with different media (endothelial cell growth media (EC), organoid maturation media (CO), or mixed media at 1:1, 1:3, and 1:7 EC:CO ratios), all supplemented with 50 ng mL −1 VEGF for 10 days. Scale bars: 500 µm. E) Total vessel length (mm), number of junctions per mm 2 , and end points per mm 2 of endothelial networks grown in different media compositions. Mean ± SD, N = 6 independent wells, two repeats. One‐way ANOVA; asterisks showing Tukey's post hoc comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001. F) Representative confocal images of Calcein AM‐stained endothelial networks formed in 50% Geltrex TM with 1:7 mixed media, supplemented with 25 or 50 ng mL −1 VEGF, with media exchanges every 2 or 4 days for 10 days. Scale bars: 500 µm. G) Percentage of vessel area (%), number of junctions per mm 2 , and lacunarity values of endothelial networks formed at different VEGF concentrations (25, 50 ng mL −1 ) and media exchange frequency (every 2 or 4 days). Mean ± SD, N = 6 independent wells, two repeats. Two‐way ANOVA; asterisks showing Tukey's post hoc comparisons; * p < 0.05.

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Staining

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Promoting vascularization of the organoid accelerates early organoid growth and spherical morphology without impacting on the size or tissue stiffness by day 40. A) Brightfield images of standard COs grown according to the Lancaster protocol [ ⁹ ] (CTRL) compared to COs grown in the modified protocol, where HBMVECs were encapsulated in a Geltrex TM droplet and grown in mixed media supplemented with VEGF (BECs MM+VEGF). Representative images were captured on days 8, 11, 16, 22, and 40. Scale bar: 1 mm. B) Organoid size evolution from encapsulation until day 40 across the six tested conditions: CTRL (red), +VEGF (orange), MM+VEGF (yellow), BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple). Organoid size was measured as the area in the brightfield images (mm 2 ). Mean ± SD, N = 16‐25, five independent batches, two different iPSC lines. Mixed‐effect model, followed by Tukey's post hoc comparisons; on day 16, BECs MM+VEGF were significantly different (&) from CTRL (p = 0.001), MM+VEGF (p = 0.0013), BECs CTRL (p = 0.0002), and BECs +VEGF (p = 0.0085). On day 20, BECs MM+VEGF were significantly different ($) from CTRL (p = 0.0143), +VEGF (p = 0.0128), MM+VEGF (p = 0.0027), BECs CTRL (p = 0.0016) and BECs +VEGF (p = 0.0007). C) Largest longitudinal area (mm 2 ) of day 40 organoids across all conditions (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 19–31, 5 independent batches, two different iPSC lines. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05. D) Organoid circularity at day 40 across all conditions (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 14‐27, five independent batches, two different iPSC lines. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05, ** p < 0.01, **** p < 0.0001. E) Nanoindentation measurements of tissue stiffness (Young modulus, in KPa) of CTRL and BECs MM+VEGF organoids. Mean ± SD, N = 10–13, two independent batches, two different iPSC lines. Unpaired T ‐test; ns = not significant ( p > 0.05). F) Degradation of the Geltrex TM droplet area (mm 2 ) over time (white‐filled plots: absence of HBMVECs; grey‐filled plots: encapsulated HBMVECs; dashed lines represent organoids generated with the A‐iPSC line; uninterrupted lines represent organoids generated with the S‐iPSC line). Mean ± SD, N = 9–15, two or three independent batches per each of the two IPSC lines. G) ELISA quantification of VEGF in culture supernatants from CTRL and BECS MM+VEGF organoids. The red line represents the 50 ng mL −1 VEGF dose added in the MM+VEGF media. Mean ± SD, N = 4, two independent batches for each of the two iPSC lines. Two‐way ANOVA, asterisks showing Sidak's post hoc comparisons; **** p < 0.0001. H) ELISA quantification of matrix metalloproteinase 9 (MMP‐9) in culture supernatants from CTRL and BECS MM+VEGF organoids. Mean ± SD, N = 4, two independent batches for each of the two iPSC lines. Two‐way ANOVA, asterisks showing Sidak's post hoc comparisons; **** p < 0.0001.

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Modification, Encapsulation, Generated, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

doi: 10.1002/advs.202507256

Figure Lengend Snippet: Abundant multicellular endothelial networks are formed on the surface of COs, with morphological features dependent on media conditions or Geltrex TM concentration. A) Confocal images of A and S‐COs grown under tested vascularization conditions, wholemount stained for CD31 (green) and βIIITUB (magenta). Dashed lines separate the different conditions: media used (CTRL, +VEGF or MM+VEGF, vertical display), presence or absence of encapsulated HBMVECs (horizontal display). Within each condition, a comparison was made between organoids generated with the A‐and S‐iPSCs lines. Scale bars: 500 µm. At the bottom, higher magnification confocal images showing endothelial networks on the surface of BECs MM+VEGF organoids, for both iPSCs lines. Scale bars: 100 µm. B) Percentage of CD31+ surface in A‐COs. Control (red), +VEGF (orange), MM+VEGF (yellow), BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple); no HBMVECs, white filling; +HBMVECs, grey filling. Violin shape represents data distribution, Median (thick line), interquartile range (thin lines), N = 6–12, three independent batches. Brown‐Forsythe and Welch ANOVA test, asterisks showing Games‐Howell's post hoc comparisons; * p < 0.05, ** p < 0.01. C) Percentage of CD31+ surface in S‐COs. Same color system as above. The violin shape represents data distribution, median (thick line), interquartile range (thin lines), N = 6–8, two independent batches. Kruskall‐Wallis test, asterisks showing Dunn's post hoc comparisons; *p < 0.05, ** p < 0.01, *** p < 0.001. D) Quantification of network end points per mm 2 , average vessel length (mm), and junctions per mm 2 of the HBMVECs‐containing organoids, separated by iPSCs origin. BECs CTRL (green), BECs +VEGF (dark blue), and BECs MM+VEGF (purple). The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 5–12, three or two independent batches for each line. Two‐way ANOVA test; media and iPSC line factor significance expressed on top of each graph. Tukey's post hoc comparisons are represented by brackets over relevant bars; ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. E) Degradation rate of different concentrations of Geltrex TM droplets in BECs MM+VEGF conditions, measured by the area change (mm 2 ) over time. Light blue (60%), black (80%), Bordeaux (95%). Mean ± SD, N = 5, one batch of organoids. F) Representative confocal images of BECs MM+VEGF A‐COs grown at different Geltrex TM concentrations (60%, 80% and 95%), wholemount stained for CD31 (green) and βIIITUB (magenta). Scale bars: 500 µm. On the right, higher magnification images of the endothelial networks are shown. Scale bars: 100 µm. G) Vessel diameter (µm) of the superficial endothelial networks formed under different Geltrex TM concentrations. Light blue (60%), black (80%), Bordeaux (95%). Mean ± SD, N = 9–22 (individual organoids) and >5 areas of interest per organoid analyzed, five different batches, same A‐iPSC line. Kruskal‐Wallis test, asterisks showing Dunn's post hoc comparisons; **** p < 0.0001. H) End points per mm 2 of the superficial endothelial networks formed under different Geltrex TM concentrations. Light blue (60%), white (80%), and Bordeaux (95%) colors were used. The violin shape represents the data distribution, median (thick line), interquartile range (thin lines), N = 9–22, five different batches, and the same A‐iPSC line. One‐way ANOVA test, Tukey's post hoc comparisons; not significant ( p > 0.05).

Article Snippet: [ ] Primary HBMVECs, from a pool of multiple donors, were purchased from Innoprot (Cat# P10361 ).

Techniques: Concentration Assay, Staining, Comparison, Generated, Control

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