Fig 1.
Hemostatic Components Model (HCM).
Peripheral blood cells (platelets and leukocytes) are seeded at their naturally-occurring ratio onto a collagen-coated substrate, which mimics exposed collagen in the injured vascular wall. Cells are cultured in serum-free medium under hypoxia (3%O2) at 37°C, while released/produced protein factors diffuse through a nano-porous filter and are sampled simultaneously within an exogenous fibrin matrix of varying mass (m). Cell-matrix contact is prevented through the filter. The model simulates the hemostatic microenvironment, while enabling assessment of the function of the fibrin matrix as protein factor-carrier, independently of its role as cell-scaffold.
Fig 2.
Hypoxia potentiates coagulation-mediated angiogenic signaling.
A) Plot of the temporal profile of pericellular O2 tension in coagulated blood cultures, carried out in sealed chambers (bottom area~2cm2) at 37°C for 7 days, for four BIV (blood incubation volume) values (day 0 O2 tension corresponds to that in peripheral venous blood). SF-medium was tested as control. Data shown are typical for a young, healthy subject (n = 4). B) Plot of clot supernatant VEGF concentration vs. BIV after 7 days culture in the same setup, *p<0.01 compared to BIV = 1ml (n = 3). C) Plot comparing the normoxia- vs. hypoxia-induced VEGF expression in anticoagulated blood, obtained from male (n = 20) and female (n = 28) subjects, and cultured at 37°C under normoxia or hypoxia (3% O2) for 7 days. Data are presented as % increase in VEGF concentration in hypoxic culture supernatant, relative to the normoxic baseline. Error bars represent s.e.m., *p<0.01, **p<0.001 corresponds to hypoxic vs. normoxic culture mean VEGF concentration (n = 2 per subject, per condition). D) Plot of the temporal profile of clot supernatant cumulative VEGF concentration in coagulated blood cultures with a BIV = 3ml, carried out in 37°C sealed chambers over 5 days, *p<0.001 compared to day 0 levels. Data shown are from two independent experiments, carried out on blood from same subject (n = 3 per exp.). E) Plot of VEGF and TSP1 concentration in releasates of cell-free fibrin clots that were formed and incubated for 24h in plasma derived from anticoagulated blood, after it had been cultured at 37°C, BIV = 3ml for 2, 4, or 8 days, *p<0.05 compared to day 2 levels (n = 3). F) Plot of the ratio of VEGF and PF4 concentration in plasma, derived from 5 day anticoagulated blood cultures carried out on collagen-coated substrates under hypoxia (3% O2) at 37°C, relative to serum concentration, *p<0.05 corresponds to plasma vs. serum concentration (n = 3). Unless otherwise noted, error bars represent s.d.
Fig 3.
The fibrin matrix functions as carrier of pro- and anti-angiogenic factor proteins.
A) Plot of all proteins identified in fibrin matrix releasates, obtained after 7 days HCM culture, in at least one of two biological replicates with at least one unique peptide via mass spectrometry. Proteins are sorted from high (left) to low (right) abundance according to their IBAQ score. Pro-angiogenic proteins are highlighted in green and anti-angiogenic proteins are highlighted in red. B) Tables of pro-angiogenic proteins marked in green and anti-angiogenic proteins marked in red. Under gene names the gene symbol can be found. Under protein identifiers the uniprot identifier of the respective protein is listed. Unique Peptides FibA and FibB indicate the identified peptides of each protein for the two biological replicates (note; this does not indicate relative protein abundance). Table with complete proteomic analysis and unprocessed output files can be found under supplementary data (S2 and S3 Tables). C) Plot showing the profile of angiogenesis-related proteins, as analysed by proteome profiler array, in the same releasates. White, grey and black bars show matrix-remodelling, pro-angiogenic and anti-angiogenic proteins, respectively. Y-axis represents the ratio of sample signal to reference signal, for each protein (n = 3). Error bars represent s.d.
Fig 4.
Differential and mass-dependent binding of VEGF and PF4 by the fibrin matrix.
A) Plot comparing the VEGF and PF4 retention ratio (clot: plasma concentration) of cell-free fibrin clots (v = 1cm3) that were formed and incubated for 24h in plasma obtained from anticoagulated blood, after it had been cultured for 1h on collagen-coated substrates. Collagen matrices of comparable volume and protein density (v = 1cm3, d = 4 mg/cm3) were also tested as control in the same setup, *p<0.01 and **p<0.05 compared to corresponding PF4 value (n = 3). B) Plot comparing the VEGF and PF4 concentration in releasates of cell-free clots (total wet mass m = 300mg), that were formed and incubated for 24h in plasma derived from 7 day hypoxic blood culture. Afterwards clots were incubated in fresh medium and releasates were sampled every 2, 4, 8, 12 and 24h, *p<0.05 compared to 2h level (n = 3). C) Plots of VEGF (left) and PF4 (right) concentration in releasates of cell-free clots of varying total wet mass (M = 100, 200, 300mg). Clots were formed and incubated in plasma derived from 7 day hypoxic blood culture for 2, 4 and 8h, then centrifuged in fresh medium to obtain releasates, *p<0.05 compared to 100mg at 2h, **p<0.05 compared to 100 and 200mg at 2h (n = 3). D) Plot comparing the VEGF concentration in releasates obtained from equal volume (3cm3) fibrin matrices, cellulose-based hydrogels and polyhexanide hydrogels, that were harvested after 7 days HCM culture, *p<0.0001 compared to fibrin (n = 3). E) Plot comparing the VEGF and PF4 concentration in fibrin matrix releasates (concentrations were corrected for matrix volume-related dilution), obtained after 7 days HCM culture, for two fibrin(ogen) mass values (40 vs. 80mg, same matrix density = 0.04 g/cm3), *p<0.01 compared to fibrin 40mg (n = 3). Error bars represent s.d.
Fig 5.
Releasates of cultured clots induce distinct endothelial cell angiogenic responses in vitro.
A) Image panel showing the 16h tube formation by ECs seeded on matrigel, observed in response to releasates obtained from clots that were cultured on collagen matrices for 4 days at 37°C. Bars = 100 μm. B) Plot showing the mean number of tubules and nodes at 16h, *p<0.001 compared to neg.control and cultured clot releasate, **p<0.01 compared to neg.control. C) Image panel showing the 5 day sprout formation (arrowed) in the aortic ring-matrigel assay, observed in response to releasates obtained from cultured clots. Bars = 100 μm. D) Plot showing the mean number of sprouts over 7 days, *p<0.05 compared to neg.control, **p<0.05 compared to neg. and pos. control. SF medium and VEGF-containing SF medium were tested as negative and positive controls, respectively. For all assays n≥4. Error bars represent s.d.
Fig 6.
Changes in fibrin matrix mass differentially influence endothelial cell angiogenic responses.
A) Image panel showing the 24h EC chemotactic migration through matrigel membrane, observed in response to releasates obtained from matrices of varying fibrin(ogen) mass (40 vs. 120mg). Bars = 100 μm. B) Plot showing the ratio of mean fluorescence intensity of migrating ECs at 24h in response to releasates relative to control medium, *p<0.01 compared to control medium. C) Image panel showing the 16h tube formation by ECs seeded on matrigel, observed in response to releasates obtained from matrices of varying fibrin(ogen) mass (40 vs 80 vs 120mg). Bars = 100 μm. D) Plot showing the mean number of tubules and nodes at 16h, *p<0.001 compared to all conditions except fibrin 40mg,**p<0.01 compared to all conditions except pos.control, ***p<0.01 compared to fibrin 80mg, #p<0.05 compared to all conditions except fibrin 80mg. E) Image panel showing the 5 day sprout formation (arrowed) in the aortic ring-matrigel assay, observed in response to releasates obtained from matrices of varying fibrin(ogen) mass (40 vs 80mg). Bars = 100 μm. F) Plot showing the mean number of sprouts per ring over 7 days. G) Plot showing the mean max. sprout length reached by day 5, *p<0.05 compared to pos.control and fibrin 80mg. Releasates were obtained from cell-free matrices that were harvested following 7 days HCM culture. Where indicated, releasates were incubated with anti-PF4 for 12h before testing. SF medium and VEGF-containing SF medium were tested as negative and positive controls, respectively. For all assays n≥4. Error bars represent s.d.
Fig 7.
Proposed mechanism for matrix-dependent biochemical control of wound angiogenesis.
A distinction is made here between the role of the matrix as cell-scaffold and factor-carrier. Through its carrier function, the fibrin matrix determines the balance of pro-and anti-angiogenic factors in the hemostatic microenvironment (light- and dark-grey triangles). Initially, anti-angiogenic factors (-) (e.g. PF4) are released by activated platelets and bound by the matrix at higher concentrations than pro-angiogenic factors (+) (e.g. VEGF). This inhibits neovessel formation around and into the clot, thus preventing clot destabilization and ensuring effective hemostasis. Meanwhile, increasing clot size and/or cell accumulation within the clot, under limited O2 supply (resulting from vascular injury), contributes to the development of local hypoxia. Hypoxia-induced upregulation of pro-angiogenic factor (e.g. VEGF) expression and downregulation of anti-angiogenic factor (e.g. TSP1) expression potentiates coagulation-mediated angiogenic signaling (*). Following completion of the hemostasic phase, the matrix undergoes controlled degradation through fibrinolysis, leading to depletion in the local pool of anti-angiogenic factors, while release of pro-angiogenic factors leads to the formation of chemoattractive gradients that direct endothelial cell (EC) migration towards the injured site. Angiogenic disinhibition enables vascularization of the matrix through fibrinolysis-mediated EC invasion, which facilitates resolution of hypoxia and tissue repair.