Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Anaphylatoxins C3a and C5a differentially regulate endothelial monoculture dnCI in a dose-dependent manner. ( A , B ) Monitoring of dnCI changes in response to 50 nM, 100 nM and 500 nM of anaphylatoxins C3a and C5a revealed a transient decrease in dnCI by 500 nM C5a after 2 h of treatment in HBMEC. At 24 h after anaphylatoxin stimulation, 500 nM C3a significantly decreased the paracellular resistance in HBMEC compared to the untreated control. ( C , D ) In HREC, we observed no significant alterations in paracellular resistance after 2 h of C3a or C5a treatment. A dose-dependent increase in dnCI occurred 24 h after treatment initiation in HREC treated with 50 nM C3a, 100 nM C3a, 50 nM C5a and 100 nM C5a compared to the untreated control. ( A , C ) ↑ indicates analyses time points at 2 and 24 h. All data were normalized to the respective cell index (CI) value before the addition of treatment and to the untreated control (dnCI). ( A , B ) RTCA graph represents data from n = 10 for untreated and 500 nM C3a, n = 6 for 50 nM C3a and 50, 100 and 500 nM C5a and n = 8 for 100 nM C3a treated cells, respectively. ( C , D ) RTCA graph represents data from n = 9 for untreated, n = 6 for 50 nM C5a and 50, 100 and 500 nM C3a, n = 8 for 100 nM C5a and n = 4 for 500 nM C5a, respectively. ( B , D ) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test was used for datasets including non-parametric data. One-way analysis of variance (ANOVA) with Dunnett’s T3 post hoc test was used for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C3a and C5a do not change CDH5 gene expression but alter VE-cadherin on protein level in HBMEC and HREC. ( A , B ) Treatment with 500 nM C3a or 500 nM C5a did not change CDH5 gene expression in HBMEC and HREC compared with the untreated control. ( C ) Two hours after treatment, HBMEC exhibited a heterogeneous VE-cadherin phenotype in untreated and 500 nM C3a-treated cells. HBMEC treated with 500 nM C5a displayed a more homogeneous distribution of VE-cadherin. VE-cadherin mean fluorescent intensity (mean FI) remained stable regardless of treatment. Two hours after treatment, HREC displayed a homogeneous monolayer in all treatment groups without disruption of the endothelial monolayer. VE-cadherin signal intensity was increased by 500 nM C5a. ( D ) After 24 h, HBMEC displayed a homogeneous phenotype in untreated and 500 nM C5a-treated cells, but they were elongated and showed disrupted VE-cadherin expression in 500 nM C3a-treated cells (white arrowheads). VE-cadherin signal intensity was decreased by 500 nM C3a but was increased by 500 nM C5a. In HREC, the homogenous phenotype remained stable over 24 h. 500 nM C3a increased VE-cadherin signal intensity. ( A , B ) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Control, Disruption

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Anaphylatoxin treatment caused increased C3 expression in HREC and increased C3AR1 expression in HBMEC. ( A , B ) qRT-PCR analysis revealed no effect of anaphylatoxin treatment on C3 expression in HBMEC but disclosed an increase in C3 expression in 500 nM C5a-treated HREC 2 h post-treatment and a 500 nM C3a and 500 nM C5a mediated increase in C3 gene expression 24 h post-treatment in HREC. ( C , D ) Two hours’ exposure to 500 nM C5a increased C3AR1 expression in HBMEC, while anaphylatoxin treatment had no significant effect on C3AR1 expression in HREC. ( A – D ) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C3a presence increased in HREC after exposure to C3a and C5a. ( A ) After 2 h, the C3 protein signal intensity increased in C3a- and C5a-treated HBMEC. The C3a signal in untreated HBMEC exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution. In HREC, C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups after 2 h, but the signal intensity increased in C3a- and C5a-treated HREC. HBMEC and HREC C3a signal intensity remained stable across treatment groups and exhibited a spot-wise pattern across all treatment groups after 2 h. ( B ) After 24 h, the C3 signal decreased in C3a-treated HBMEC and returned to baseline in C5a-treated cells. In untreated HBMEC, C3a exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution 24 h post-treatment initiation. The localization of C3a transitioned from spot-wise distribution to a more diffuse cellular distribution in HBMEC treated with 500 nM C3a and 500 nM C5a 24 h after treatment initiation. HREC C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups. C3 signal intensity increased in C3a- and C5a-treated HREC after 24 h. The spot-wise C3a distribution pattern seen after 2 h persisted in untreated HREC 24 h after treatment but expanded to a broader, cell-covering signal in cells treated with 500 nM C3a and 500 nMC5a. C3a signal intensity increased in C3a- and C5a-treated HREC 24 h after treatment initiation. ( A , B ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C5 gene and protein expression are elevated in HREC following C5a stimulation, whereas no significant changes are observed in HBMEC. ( A , B ) qRT-PCR indicated no variations in C5 gene expression among treatment groups or analysis time points at 2 and 24 h in HBMEC. Two hours post-treatment, exposure to 500 nM C3a and 500 nM C5a resulted in a significant upregulation in C5 gene expression in HREC. This effect was reversed 24 h post-treatment. ( C , D ) Stimulation with 500 nM C3a and 500 nM C5a provoked no change in C5 protein signal strength in HBMEC. Two hours post-treatment, C5 exhibited a spot-wise expression pattern in both untreated and 500 nM C3a-treated HREC, whereas HREC treated with 500 nM C5a showed a broader distribution of immunofluorescent signals. C5a increased C5 signal intensity compared to the untreated control and C3a-treated HREC. After 24 h, C5 detection signal returned to baseline in C3a- and C5a-treated cells. ( A , B ) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: C5AR1 transcript expression remains unchanged, but protein detection changes under anaphylatoxic stress. ( A , B ) qRT-PCR analysis showed no difference in C5AR1 gene expression between untreated and C3a- or C5a-treated HBMEC and HREC. ( C ) Treatment with 500 nM C3a and 500 nM C5a reduced C5aR1 protein signal in HBMEC after 2 h of treatment. Immunofluorescent staining of C5aR1 in HREC showed a stable and comparable signal after 2 h in all treatment groups. ( D ) After 24 h, the C5aR1 signal was stable between treatment groups in HBMEC. C5aR1 protein signal intensity increased in 500 nM C5a-treated HREC after 24 h. ( A , B ) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. ( C , D ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Adding 50 nM of C5a, but not 100 nM or 500 nM, counteracted the barrier-disruptive effect of 500 nM C3a and improved the HBMEC barrier after 24 h. ( A , B ) Monitoring of combined anaphylatoxin-treated dnCI changes in HBMEC revealed a C5a dependent increase in dnCI, which counteracted the barrier-disruptive effect of C3a. 24 h post-anaphylatoxin stimulation, HBMEC treated with 500 nM C3a + 50 nM C5a reached a significantly higher dnCI compared to 500 nM C3a-treated cells. ( C , D ) RTCA measurement revealed an increase in dnCI in HREC treated with 500 nM C3a + 100 nM C5a 2 h after treatment addition compared to 500 nM C3a-treated cells. This regulation was maintained for 24 h after exposure to treatment. ( E ) Immunocytochemistry revealed a homogeneous endothelial monolayer in all treatment groups in HBMEC, 2 h post-treatment. ( F ) Twenty-four hours post-treatment, 500 nM C3a decreased the VE-cadherin signal intensity, whereas treatment with 500 nM C3a + 50 nM C5a obtained a VE-cadherin signal strength similar to that of the untreated control. ( A , C ) ↑ indicate analyses time points 2 and 24 h. All data were normalized to the respective CI value before addition of treatment and to the untreated control. ( A , B ) RTCA graph represents the mean of data from n = 9 for 500 nM C3a, n = 6 for 500 nM C5a and n = 7 for 500 nM C3a + 50 nM C5a and 500 nM C3a + 100 nM C5a and n = 4 for 500 nM C3a + 500 nM C5a-treated cells, respectively. ( C , D ) RTCA graph represents the mean of data from n = 6 for 500 nM C3a, 500 nM C3a + 50 nM C5a and 500 nM C3a + 100 nM C5a and n = 4 for 500 nM C5a and n = 5 for 500 nM C3a + 500 nM C5a. ( B , D ) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. ( E , F ) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Immunocytochemistry, Control

Journal: International Journal of Molecular Sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells

doi: 10.3390/ijms252011240

Figure Lengend Snippet: 500 nM C3a and C5a increase the transcellular permeability for IgG in HBMEC but not in HREC, reflecting reduced cell–cell contact integrity at 500 nM C3a but not enhanced paracellular resistance at 500 nM C5a in HBMEC. ( A ) Endothelial cells were allowed to settle and form a continuous monolayer 17 h prior to the addition of 500 nM C3a or 500 nM C5a. After 24 h, 100 µg/mL purified IgG was applied luminally, and abluminal supernatants were harvested 2 h later to assess IgG migration through the endothelial barrier using Western blot detection. ( B ) Western blot quantification revealed a significant increase in IgG migration through the endothelial barrier in 500 nM C3a- and 500 nM C5a-treated HBMEC compared to the untreated control. This effect was not observed in HREC, where the IgG migration rate was similar to the untreated control in cells treated with 500 nM C3a and 500 nM C5a. Bar height represents the mean relative protein level for n = 3 for all treatment groups in both cell types. Cell types were analyzed as separate datasets. Uncropped Western blots are shown in . One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Permeability, Purification, Migration, Western Blot, Control

Journal: medRxiv

Article Title: Factor H-related protein 3 (FHR-3) deposition in kidney allografts – localization and correlation with complement activation

doi: 10.1101/2023.10.24.23297478

Figure Lengend Snippet: Representative images from immunofluorescent double staining of factor H-related protein 3 (FHR-3 in red) and complement activation (C3d in green) in tissues obtained from (A) living donor kidneys (n=8), (B) acute tubular necrosis (ATN) (n=6), (C) Banff IA (n=6), (D) Banff IB (n=6), (E) Banff IIA (n=6), (F) Banff IIB (n=6), and (G) chronic rejection (CR) (n=6). Nuclei were counterstained with DAPI.

Article Snippet: In the experiments, we used recombinant human C3a (Hycult Biotech), recombinant human C5a (Hycult Biotech), and a combination of the two to stimulate the podocytes.

Techniques: Double Staining, Activation Assay

Journal: medRxiv

Article Title: Factor H-related protein 3 (FHR-3) deposition in kidney allografts – localization and correlation with complement activation

doi: 10.1101/2023.10.24.23297478

Figure Lengend Snippet: Different staining patterns of factor H-related protein 3 (FHR-3 in red) and complement activation (C3d in green) in transplanted kidneys. Nuclei were counterstained with DAPI. Representative images of (A) tubular FHR-3 staining (arrows) in acute tubular necrosis (ATN), (B) vascular FHR-3 staining (arrows) in T-cell-mediated rejection with severe tubulitis (Banff IB), and (C) low-intensity FHR-3 staining in Bowman’s capsule (arrows) and tubules (arrows) in chronic rejection (CR). Exemplary images of double staining for FHR-3 (red) and C3d (green) in transplanted kidneys revealing (C) predominant positive glomerular staining for FHR-3, (D) glomerular co-localization of FHR-3 and C3d, and (E) predominant positive glomerular staining for C3d.

Article Snippet: In the experiments, we used recombinant human C3a (Hycult Biotech), recombinant human C5a (Hycult Biotech), and a combination of the two to stimulate the podocytes.

Techniques: Staining, Activation Assay, Double Staining

Journal: medRxiv

Article Title: Factor H-related protein 3 (FHR-3) deposition in kidney allografts – localization and correlation with complement activation

doi: 10.1101/2023.10.24.23297478

Figure Lengend Snippet: (A) Quantification of staining intensity of factor H-related protein 3 (FHR-3) and complement activation (C3d) by double immunofluorescent in living donor kidneys, acute tubular necrosis (ATN), Banff IA, Banff IB, Banff IIA, Banff IIB, and chronic rejection (CR). Each biopsy was scored by two independent observers in a blinded fashion. (B) The correlation between the staining intensity of FHR-3 and C3d using the Spearman Rank correlation coefficient (r represents the spearman’s rho). The dashed blue lines show the 95% confidence interval for the regression line (blue).

Article Snippet: In the experiments, we used recombinant human C3a (Hycult Biotech), recombinant human C5a (Hycult Biotech), and a combination of the two to stimulate the podocytes.

Techniques: Staining, Activation Assay

Journal: Journal of Ophthalmology

Article Title: Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells

doi: 10.1155/2022/3259453

Figure Lengend Snippet: PCR primers used in this study.

Article Snippet: Then, the cells were treated with 0.1 μ M or 0.3 μ M recombinant human complement C3a (Sigma-Aldrich, Cat#204881) for 24, 48, and 72 hours.

Techniques:

Journal: Journal of Ophthalmology

Article Title: Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells

doi: 10.1155/2022/3259453

Figure Lengend Snippet: Effect of C3a on mRNA and protein levels of CCN2/CTGF in cultured RPE cells. Total RNA was extracted, and CCN2/CTGF mRNA was evaluated by RT-PCR analysis. The ratio of the abundance of the target mRNA to GAPDH was evaluated by densitometric analysis (a). ∗ P < 0.05, ∗∗ P < 0.01versus negative control cells; # P < 0.05, ## P < 0.01 versus cells with 0.3 μ M C3a for 72-hour time. ELISA analysis to assess the effect of C3a on protein levels of CCN2/CTGF in cultured RPE cells (b). The expression level was normalised to the negative control. ∗ P < 0.05, ∗∗ P < 0.01 versus negative control; # P < 0.05, ## P < 0.01 versus cells with 0.3 μ M C3a for 72-hour time. The data are presented as the mean ± SD ( n = 4).

Article Snippet: Then, the cells were treated with 0.1 μ M or 0.3 μ M recombinant human complement C3a (Sigma-Aldrich, Cat#204881) for 24, 48, and 72 hours.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Ophthalmology

Article Title: Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells

doi: 10.1155/2022/3259453

Figure Lengend Snippet: Effects of C3 siRNA on the mRNA and protein levels of CCN2/CTGF in RPE cells with exogenous C3a treatment. Total RNA was extracted, and CCN2/CTGF mRNA was evaluated by RT-PCR analysis. The ratio of the abundance of the target mRNA to GAPDH was evaluated by a densitometric analysis (a) ∗ P < 0.05, ∗∗ P < 0.01 versus respective non-transfected cells. ELISA analysis of the effect of C3 siRNA on the protein level of CCN2/CTGF in RPE cells with exogenous C3a treatment (b). The expression level was normalised to the negative control. ∗ P < 0.05, ∗∗ P < 0.01 versus respective nontransfected cells. The data are presented as the mean ± SD ( n = 4).

Article Snippet: Then, the cells were treated with 0.1 μ M or 0.3 μ M recombinant human complement C3a (Sigma-Aldrich, Cat#204881) for 24, 48, and 72 hours.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Negative Control