3D modelling of pulmonary arterial stenosis and endothelial dysfunction in CTEPH

Figures

• 

  Fig. 1. Design and fabrication of pulmonary vascular stenosis models. (A) Design of a 30% stenosis channel, and (B) 3D-printed mould for an 80% stenosis channel created using Fusion 360 (Autodesk). (C) Fabrication process of semi-circular models of vascular stenosis. Computed tomography (CT) scans from patients with CTEPH were used to determine the most commonly found degrees of stenosis. Moulds of stenotic channels were designed using Fusion 360 (Autodesk) and printed with a stereolithography (SLA) 3D printer (Form3, FormLabs). Polydimethylsiloxane (PDMS) was then cast onto the moulds to produce semi-circular replicas of control (straight) and stenotic vessels. Finally, the chambers were bonded to polymer coverslips (ibiTreat coverslips, Ibidi), with blunt needles inserted into the inlets and outlets to serve as connectors for tubing used in flow perfusion experiments.

• 

  Fig. 2. Wall shear stress and velocity streamlines in circular and semi-circular channels simulated with water. Velocity streamlines are shown in (A) semi-circular and (B) fully circular straight channel, whilst (C) and (D) show corresponding wall shear stress (WSS) values, as indicated. (E) Shows velocity streamlines in semi-circular and (F) in fully circular 80% stenosis models, respectively. (G and H) Show WSS changes in semi-circular and fully circular 80% stenosis models, as indicated. All simulations were performed in CFX (Ansys, Inc.), using an inlet velocity of 60.2 mm s−1 (0.5 L h−1) and outlet pressure of 0.097 kPa for the semi-circular channels, and an inlet velocity of 50 mm s−1 (2.26 L h−1) and outlet pressure of 1.60 kPa for the fully circular channels.

• 

  Fig. 3. Wall shear stress in semi-circular channels simulated with water. Computational fluid dynamics (CFD) results showing wall shear stress (WSS) at the top and bottom of the semi-circular in the control (A–C), 30% stenosis (D–F), 60% stenosis (G–I) and 80% stenosis (J–L) models. Colour scales at the top of the images in (A)–(H) represent a WSS range between 0.0–2.0 Pa (0.0–20 dyn cm−2), while in (J and K) the scale ranges from 0.0–18.1 Pa (0.0–181 dyn cm−2). In (C, F, I and L) the x axis indicates the different regions of the channel: upstream (U; 7 mm), stenosis (S; 11 mm), post-stenotic dilatation (PSD; 13.5 mm), downstream 1 (D1; 18 mm) and downstream 2 (D2; 22.5 mm). All simulations were performed in CFX (Ansys, Inc.), using an inlet velocity of 60.2 mm s−1 (0.5 L h−1) and outlet pressure of 0.097 kPa.

• 

  Fig. 4. HPAECs surface coverage and orientation in semi-circular channels after flow perfusion. (A) HPAECs surface coverage in upstream, stenosis, PSD and downstream (Downstr) areas in semi-circular channels in 30%, 60%, and 80% stenosis models after 6 h flow at 0.5 L h−1. Image analysis was carried out with ImageJ (FIJI). N = 3–5 biological repeats. *P < 0.05, comparison with surface coverage in straight channel control; unpaired t-test. (B) HPAECs alignment. Alignment was measured by scoring % of nuclei oriented within 30 degrees of the flow direction in static controls (representing random orientation), straight channels under flow (control flow) and different areas of 30%, 60% and 80% stenosis models, as indicated. Image analysis was carried out with ImageJ (FIJI). N = 3–6 biological repeats. ###P < 0.01, comparison with flow control; *P < 0.05, **P < 0.01, ***P < 0.00, comparisons, as indicated; unpaired t-test. Values are shown as mean ± SEM. (C) Representative images of HPAECs in static culture and different areas of 80% stenosis model under flow, with predicted mean WSS values. In confocal microscopy images nuclei are (blue), F-actin (red) and VE-cadherin (green). White arrows…

• 

  Fig. 5. Transcriptional profile of HPAECs cultured in 30% and 60% stenosis models. (A) Heatmap showing fold change of expression of marker genes of thrombosis, inflammation, angiogenesis, proliferation, and stimulation by flow (named here as “flow responses”) in 30% model under flow, relative HPAECs cultured in the straight, control channel, under static conditions (static control); p < 0.05. (B) Summary diagram illustrating direction of changes in gene expression in HPAECs cultured in the 30% stenosis model under flow (0.5 L h−1, 6 h). (C) Heatmap showing fold changes in marker gene expression in 60% stenosis model. (D) Summary diagram illustrating direction of changes in gene expression in 60% model. Heatmaps in (A) and (C) were generated using R 4.3.3 (The R Project for Statistical Computing, 2024). PSD: post-stenotic dilatation. Diagrams in (B) and (D) were made using PowerPoint (Microsoft) and Fusion360 (Autodesk).

• 

  Fig. 6. Transcriptional profile of 80% stenosis HPAECs and PEA endothelial cells. (A) Heatmap showing fold change of expression of marker genes of thrombosis, inflammation, angiogenesis, proliferation, and stimulation by flow (named here as “flow responses”) in 80% model under flow (0.5 L h−1, 6 h), relative HPAECs cultured in the straight, control channel, under static conditions (static control). p < 0.05. (B) Direction of changes in gene expression in HPAECs cultured in the 80% stenosis model under flow (0.5 L h−1, 6 h). (C) Isolation of endothelial cells from pulmonary endarterectomy (PEA) specimens. (D) Expression of selected gene markers of inflammation, thrombosis, angiogenesis, proliferation and apoptosis, in PEA endothelial cells from 2 donors. *P < 0.05, **P < 0.001, ***P < 0.001, comparison with static HPAEC control, n = 4. Data normalised to β2 microglobulin (B2M). Heatmap in (A) was generated using R 4.3.3 (The R Project for Statistical Computing, 2024). Diagram (B) was made using PowerPoint (Microsoft) and Fusion 360 (Autodesk).

• 

  Fig. 7. Platelet adhesion in collagen-coated stenotic channels perfused with blood. (A) Platelet adhesion (% surface coverage) in collagen-coated semi-circular channels with 30–80% stenosis, perfused with citrated whole blood for 4 min at 0.17 L h−1 (low flow) and (B) corresponding fluorescence microscopy images. (C) Platelet adhesion at high flow of 0.5 L h−1 and (D) corresponding images of platelets in channels perfused at 0.5 L h−1. In (A and C) the left Y axis shows% of surface coverage and the right Y axis indicates the wall shear stress (WSS; lines) at various regions of the channels (upstream, stenosis, PSD and downstream), as indicated. N = 3–5 biological repeats. Unpaired t-tests were performed to compare two individual groups. **P < 0.01, ***P < 0.001, compared to straight control. Bars show means ± SEM. In (B and D) platelets were labelled with 3,3-dihexyloxacarbocyanine iodide (DiOC6) (green fluorescence). Scale bar = 500 μm.

• 

  Fig. 8. Platelet adhesion in collagen-coated channels with 80% stenosis perfused with citrated whole blood at 0.17 L h−1 and 0.50 L h−1. (A) Images of platelet adhesion and aggregation in control (straight) semi-circular channels after perfusion with citrated whole blood for 4 min at 0.17 L h−1 and (B) 0.50 L h−1. (C) and (D) Show platelet adhesion within the region of 80% stenosis at low and high flow, as indicated. (E) Platelet adhesion within 80% stenosis at low flow (0.17 L h−1). (F) Platelet adhesion in 80% stenosis channel in the presence of anti-vWF antibodies. Images were taken under a fluorescence microscope (EVOS M5000, Invitrogen, Thermo Scientific) at the 4 min time point. Platelets were labelled with 3,3-dihexyloxacarbocyanine iodide (DiOC6) (green fluorescence). Arrows indicate the direction of flow. In (A–D) scale bar = 1 mm, in (E and F) bar = 500 μm.

• 

  Fig. 9. Platelet adhesion in endothelium-coated semi-circular channels perfused with citrated whole blood at 0.17 L h−1. Platelet adhesion in semi-circular channels seeded with human pulmonary artery endothelial cells (HPAECs) and perfused with citrated whole blood for 4 min at 0.17 L h−1. N = 3 biological repeats. Endothelial channels were exposed to flow for 6 hours before blood perfusion. *P < 0.05; **P < 0.01, compared to flow control; ∼P < 0.05, comparison, as indicated; unpaired t-test. Bars show means ± SEM.