primary dermal microvascular endothelial cells hdmvecs  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: mPR-Specific Actions Influence Maintenance of the Blood–Brain Barrier (BBB)

doi: 10.3390/ijms23179684

Figure Lengend Snippet: Relationships among key players (CCMs and mPRs) within the CmP network in nPR(−) endothelial cells (ECs). ( A ) Silencing of CCM2 decreases the expression levels of both CCM 1/3 proteins in human brain microvascular endothelial cells (HBMVEC). ( A-1 ) After silencing all three CCMs (1, 2, or 3) for 48 h, the expression levels of all three CCM proteins were efficiently targeted and silenced; however, significantly decreased expression of both CCM1/3 proteins was observed in CCM2-KD HBMVEC cells (left upper and lower panels). The relative expression levels of CCMs (1, 2, or 3) proteins were measured through quantification of band intensities and normalized against α-actinin (ACTN1) followed by SC controls (red line), and illustrated with bar plots where light gray bars represent no change and dark gray bars display decreased relative protein levels (right panel) ( n = 3). ( A-2 ) Significantly increased RNA levels of CCM2 isoforms in both CCM1-KD and CCM3-KD in HBMVEC cells were observed. The relative transcription expression changes in CCM1, CCM3, and 5 isoforms of CCM2 in CCMs KD HBMVEC cells were measured by RT-qPCR (Fold) and illustrated in the bar plots, where light gray bars represent the relative RNA levels of CCM1, dark gray bars display relative RNA levels of CCM2 isoforms, and black bars for the relative RNA levels of CCM3 ( n = 3). ( B ) Impacts of mPR-specific actions on the RNA expression of CCM2 isoforms and mPRs in human microvascular endothelial cells. ( B-1 ) Under mPR-specific PRG actions (PRG + MIF) for 48 h, enhanced RNA expression levels of most CCM2 isoforms were observed in both human dermal microvascular endothelial cells (HDMVEC) and human brain microvascular endothelial cells (HBMVEC) cells, while increased RNA expression levels of CCM1 / 3 were only observed in HBMVECs ( n = 3). ( B-2 ) Significantly increased RNA expression of PAQR5/7/8 and PGRMC1 was observed under mPR-specific PRG actions in HBMVECs and human umbilical vein endothelial cells (HUVECs) for 48 h, while only increased RNA expression of PAQR7/8 was observed in HDMVECs, suggesting RNA expression of most mPRs can be dramatically enhanced under mPR-specific PRG actions ( n = 3). ( C ) Impacts of mPR-specific actions on the protein expression of mPRα (PAQR7) in HDMVECs. After silencing all three CCM genes for 48 h, decreased protein expression levels of PAQR7 were observed for all 3 Ccms -KD conditions ( n = 3). ( D ) Impacts of mPR-specific actions on protein expression levels of CCM1/3 in human microvascular endothelial cells (HDMVECs, HUVECs, and HBMVECs) and rat brain microvascular endothelial cells (RBMVECs), compared to mifepristone only (MIF, 20 µM) or vehicle controls (VEH). The relative RNA expression levels were measured through RT-qPCR from at least three different experiments (triplicates per experiment) and normalized to housekeeping gene (ACTB) and scramble control. The relative protein expression levels were measured through quantification of band intensities of targeted proteins by Western blots, subtracted from the surrounding background and normalized against control housekeeping proteins followed by vehicle controls. In all bar plots, the red line is the control baseline for fold change measurements (−/+). ++, +++ above bar indicates p ≤ 0.001 for paired t -test.

Article Snippet: Human brain microvascular endothelial cells (HBMVEC), human dermal microvascular endothelial cells (HDMVEC), human umbilical vein endothelial cells (HUVEC), and rat brain microvascular endothelial cells (RBMVEC) cells were cultured following manufacturers’ recommendations (ATCC) and as previously described [ , , , , ].

Techniques: Expressing, Quantitative RT-PCR, RNA Expression, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: mPR-Specific Actions Influence Maintenance of the Blood–Brain Barrier (BBB)

doi: 10.3390/ijms23179684

Figure Lengend Snippet: mPR-specific actions on nPR(+/−) ECs increases microvascular permeability in vitro. Two different EC lines, nPR(+) EAhy926 ECs, derived from HUVECs, and nPR(−) RBMVECs, were used to measure in vitro permeability with the passage of FITC-conjugated dextran under vehicle and various steroid treatments. ( A ). Impact of sex-steroid-induced mPR-specific actions on the permeability of both nPR(+/−) ECs. Both nPR(+/−) ECs were under sex steroid treatments (PRG (20 µM), MIF (20 µM), and PRG + MIF, (20 µM each)) plated on either uncoated (top panels) or collagen-I coated wells (bottom panels). Although increased levels of permeability were initially observed in both ECs (on Collagen-I coated wells, bottom panels), the permeability of nPR(+) EAhy926 ECs was back to normal after 12 h (bottom right panel), while the permeability was continuously enhanced among all sex hormone treatments for RBMVECs (bottom left panel) on Collagen-I coated wells. Interestingly, permeability remained continuously enhanced among all sex hormone treatments for RBMVECs, when cultured in the absence of collagen-I (upper left panel), while the permeability of nPR(+) EAhy926 ECs did not return to normal until after 48 h (upper right panel), suggesting crosstalk between integrin and PRG-receptors-mediated signaling cascades in nPR(+) EAhy926 ECs, but not in nPR(−) RBMVECs. Four treatments are Vehicle, PRG, MIF, and PRG + MIF sequentially. ( B ). Impact of neurosteroids-induced mPR-specific actions on the permeability of both nPR(+/−) ECs. Both nPR(+/−) ECs treated with two common neurosteroids synthesized from PRG (or PRG metabolites), Allopregnanolone (3a-hydroxy-5a-pregnan-20-one, ALLO, 20 µM) and Pregnanolone (3a-hydroxy-5b-pregnan-20-one, P5, 20 µM), were plated on collagen-I coated wells, and the EC permeability was continuously monitored and measured as aforementioned. ( C ). The summarized feedback regulatory mechanism within the CmP signaling network under mPR-specific PRG actions for nPR(−) ECs. Yellow line separates transcriptional and translational levels. The + symbols represent enhancement, and symbols represent inhibition of the expression of targeted genes/proteins. Red-colored symbols/lines represent positive effects of mPR-specific PRG treatment (PRG + MIF), and blue-colored symbols/lines represent negative effects of treatment. Dark-green-colored letters indicate the direct supporting data generated from this work. Arrow indicates effect direction, solid line is the direct impact, and dotted line is indirect effects. The fluorescence intensity of FITC-dextran was measured using a 96-multiwell fluorescent plate reader. In all bar plots, *, **, and *** above any bar graph indicate p ≤ 0.05, 0.01, and 0.001, respectively, using two-way ANOVA with Holm–Sidak’s multiple comparison correction.

Article Snippet: Human brain microvascular endothelial cells (HBMVEC), human dermal microvascular endothelial cells (HDMVEC), human umbilical vein endothelial cells (HUVEC), and rat brain microvascular endothelial cells (RBMVEC) cells were cultured following manufacturers’ recommendations (ATCC) and as previously described [ , , , , ].

Techniques: Permeability, In Vitro, Derivative Assay, Cell Culture, Synthesized, Inhibition, Expressing, Generated, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: mPR-Specific Actions Influence Maintenance of the Blood–Brain Barrier (BBB)

doi: 10.3390/ijms23179684

Figure Lengend Snippet: mPR-specific actions on nPR(−) ECs are sufficient for blood–brain barrier (BBB) disruption and formation of subcutaneous lesions in vivo. Hemizygous Ccms (1, 2, and 3) mutants and WT (C57 BL/6 J) mice were injected with mPR-specific PRG treatment (a cocktail of PRG + MIF, 100 mg/kg body weight) in peanut oil (vehicle), 5 days a week for 30, 60, and 90 days, respectively. ( A ) BBB permeability was significantly increased only in our 90-day treatment groups for all 3 Ccm mutants, demonstrating that increased microvascular permeability in the brain is associated with a combination of chronic exposure to mPR-specific PRG actions and Ccm deficiency. ( B ) Subcutaneous vessel diameters in posterior sections of ears were classified into four subgroups based on the range of the vessel size (diameters): group-I (8–9 µM), group-II (9–10 µM), group-III (10–11 µM), and group-IV (11–12 µM). Significantly decreased percentage of vessels was found in Ccm2 / Ccm3 within group-I (8–9 µM), compared to WT in the 90-day treatment group, suggesting that more vessels in Ccm2 / Ccm3 mutants are distributed in larger diameter groups under mPR-specific PRG actions. Indeed, the significantly increased percentage of larger vessels in Ccm3 mutant was found in group-II (9–10 µM) compared to WT, and Ccm2 is the only mutant to display vessels in the largest size group, group-IV (11–12 µM) under mPR-specific PRG actions. ( C ) Subcutaneous vessel lesions in the anterior side of mice ears in all Ccm (1, 2, and 3) mutant strains can be visually distinguished in the 90-day treatment groups compared to WT, with Ccm3 mutant having the largest number of CCM lesions. For Evans blue assays, statistical analysis was generated using one-way ANOVA with either Kruskal–Wallis test or uncorrected Fisher’s LSD test where appropriate. All treated mice, upon completion of the last injection, were injected with Evan’s blue dye (EBD) (500 µG/25 G mouse) which was allowed to circulate for 3 h. Fluorescence data were then measured from the homogenized brain tissue and converted to µg/mL based on standard curves generated in the extraction buffer, and normalized based off the tissue weight (µg Evans blue/mg tissue) followed by controls. In ear tissues, statistical significance was performed using unpaired Student’s t -test (*, ** and *** above graphs indicate p ≤ 0.05, 0.01, and 0.001, respectively).

Article Snippet: Human brain microvascular endothelial cells (HBMVEC), human dermal microvascular endothelial cells (HDMVEC), human umbilical vein endothelial cells (HUVEC), and rat brain microvascular endothelial cells (RBMVEC) cells were cultured following manufacturers’ recommendations (ATCC) and as previously described [ , , , , ].

Techniques: In Vivo, Injection, Permeability, Mutagenesis, Generated, Fluorescence