Disturbed flow mediated modulation of shear forces on endothelial plane: A proposed model for studying endothelium around atherosclerotic plaques

Abstract

Disturbed fluid flow or modulated shear stress is associated with vascular conditions such as atherosclerosis, thrombosis, and aneurysm. In vitro simulation of the fluid flow around the plaque micro-environment remains a challenging approach. Currently available models have limitations such as complications in protocols, high cost, incompetence of co-culture and not being suitable for massive expression studies. Hence, the present study aimed to develop a simple, versatile model based on Computational Fluid Dynamics (CFD) simulation. Current observations of CFD have shown the regions of modulated shear stress by the disturbed fluid flow. To execute and validate the model in real sense, cell morphology, cytoskeletal arrangement, cell death, reactive oxygen species (ROS) profile, nitric oxide production and disturbed flow markers under the above condition were assessed. Endothelium at disturbed flow region which had been exposed to low shear stress and swirling flow pattern showed morphological and expression similarities with the pathological disturbed flow environment reported previously. Altogether, the proposed model can serve as a platform to simulate the real time micro-environment of disturbed flow associated with eccentric plaque shapes and the possibilities of studying its downstream events.

Figure 1. Results of CFD analysis.

(A) Color contours shows the variations in velocity around the circle block. (B) Velocity profile represented in color contours for laminar flow (C) Path lines image showing the swirling flow and recirculation zone behind the circle block. (D) Wall shear stress around the block calculated from a CFD analysis based on the velocity represented as bar diagram. Graphs quantifying the average shear stress in DS1, DS2, and DS3 regions. The average velocity in region on the front end (DS1) was 0.23 m/s and shear stress was 17 dynes/cm. Region at the other end (DS2) average velocity and shear stress was 0.07 m/s and 5 dynes/cm and 0.46 m/s and 34 dynes/cm² at DS3 region. **P = <0.01 DS1 vs DS2 and DS1 vs DS3.

Figure 2. Pictorial representation of fabrication and working principle of DSSA.

(A) Circular block with a width as that of the gasket placed at the laminar flow path to create disturbed flow around the block. Cartoon explains how circle block placed in PPF system to create disturbed flow (B) Image on the shows the top view of laminar flow; Image at the bottom shows the disturbed flow and regions selected for the study DS1, DS2, and DS3 based on the WSS measurement. (C) Shows the working principle of flow system. Flow setup consists of four main parts 1) Buffer chamber 2) flow controller 3) Parallel plate flow system (PPF) 4) Peristaltic pump. Silicon tubes connect the each part. Media flows through the PPF system collected in the lower reservoir and pumped to the buffer chamber with a use of peristaltic pump. (D) invitro flow apparatus (E) Top and side view of disturbed flow set up.

Figure 3. Morphological changes in endothelial cells exposed to normal and disturbed flow.

(A) Graphical representation of cell morphology under disturbed flow. Cells elongated on the axis of flow in NSS, DS1 and DS3 regions. Cells in DS2 not attained elongated morphology rather maintained the polygonal shape (B) Panels of bright field images taken after 30 min of flow illustrates the cell morphology. Differences in shape are statistically significant (p < 0.05). (C) Graphs represent the cell counting before and after disturbed flow (p < 0.05). (D) Graphs represent the percentage of proapototic and apoptotic cells. Cells were stained with Annexin V and PI for proapototic and apoptotic cells detection. Significant increase in apoptotic cell population observed in DS2 region. *p = 0.01 Static Vs NSS; **p = 0.001 NSS Vs DS2. (E) Fluorescent images showing the highly polarized microfilaments in NSS, DS1 and DS3 cells and thick bands of actin at the periphery of cell in DS2 region (indicated with arrows points). (F) Organization of actin microfilaments under static, NSS and DS conditions. Cells were fixed after 30 min of NSS and DS and stained with phalloidin for actin microfilaments. Actin pattern under disturbed flow dispersed vs concentrated in periphery as whole cell vs membrane in graph. At DS2 region thick actin bands were seen at the periphery whereas centralized pattern seen in NSS and other DS regions. *P = <0.01 Static Vs NSS; NS Vs DS3.

Figure 4. Absence of flow induced migratory structures and shear induced migration in low shear stress region.

(A) Fluorescent image panels show the migratory structures after NSS and Disturbed flow. Arrow indicates the lamellipodia in top panels and filopodia in bottom panels. (B) Graphs represent the filopodia and lamelipodia formation under normal and disturbed flow. Flow induced migratory structures was observed more in NSS and other DS regions but not expressed by DS2 region where the shear stress was low. Lamellipodia formation **P = 0.001 DS1 Vs DS3; ^#p = 0.03 Static Vs NSS; filopoidia **P = 0.001 NSS vs DS2. (C) Ring formation after 30 min of normal and disturbed flow was measured as a normal function of ECs. The number of rings formed at DS2 region immediately after 30 min of disturbed flow as well as after 2 hrs was significantly reduced at DS2 region where as NSS control and DS1 and DS3 regions rings formation increased. Ring **P = 0.001 NSS vs D2 2 hrs; ^#p = 0.001 NS Vs DS3 2 hrs; Static Vs NSS 30 min. Endothelial cells subjected to normal and disturbed flow were analyzed for migratory capacity using (D) Scratch wound heling assay and (E) Bright field images show the comparison of reduction in wound area in Static, NSS, DS1, DS2, and DS3 region in scratch wound healing assay. (F) Trans-well migration assay. Decreased wound healing and the fewer migrated cells observed in DS2 region where the velocity of flow significantly low. Trans-well migration assay *p = 0.01 NSS vs DS3 6 hrs, and DS2 6 hrs, ^#p = 0.05 Static Vs NSS 6 hrs. Wound healing. **P ≤ 0.001 NSS Vs DS2, NSS Vs DS1. ^#p = 0.05 Static Vs NSS.

Figure 5. ROS validation under normal and disturbed flow.

(A) Total ROS estimation was done using DCF fluorescent probe. Both normal and disturbed flow significantly increased the ROS production. ^#p = 0.01 NSS Vs Static *P = 0.001 static Vs DS1; ^$p = 0.01 NSS vs DS2. (B) NO estimation was done using DAR 4 M AM fluorescent probe. Cells in DS2 region where low shear stress observed NO production was low compared to the other DS regions. ^#p = 0.05 NSS vs DS2; **P = 0.001 NSS Vs DS2. (C) Peroxynitrite measurement was done using DHR fluorescent probe. Significant increase in preoxynitrite production was observed in DS 2 region. ^#p = 0.05 NSS vs DS2. (D) Measurement of Superoxide was done using Nitro blue tetrazolium. Superoxide levels were significantly reduced under disturbed flow and increased in NSS control compared to static control. ^#P ≤ 0.005, *p = 0.01 Static Vs DS1, DS2, DS3; ^$p = 0.01 Static Vs NSS. (E) Amplex red used for estimation of hydrogen peroxide. Both normal and disturbed flow significantly increased the H₂ O₂ production. P = 0.001 Static Vs DS3; P = 0.01 Static Vs DS2, DS1 Vs DS2. (F) SOD activity was measured using Pyrogallal assay. SOD activity increased in NSS, DS1 and DS3 region cells compared to static but only slight elevation seen in DS2 region cells compared to static. ^#p = 0.01 Static Vs NSS; **P = 0.001 NSS Vs DS2.

Figure 6. Semi quantitative PCR analysis of disturbed flow markers.

(A) The mRNA expression levels of VEGFR2, PECAM-1, ICAM-1, HIF1 Alpha, MMP2 and MCP-1 were quantified using RT PCR and represented in panels. (B) The mRNA expression levels of VEGFR2, PECAM-1, ICAM-1, HIF1 Alpha, MMP2 and MCP-1 were measured by densitometry and presented as relative expression to housekeeping gene GAPDH in the bar graph. The expression levels seen under normal flow were normalized to a value of 1 as the standard for each factor. Differences in expression are statistically significant (p = <0.05).

Figure 7. Adhesion of RBCs under disturbed flow.

(A) Bright field images showing the RBC (indicated with black arrow points) adhered to ECs (white arrows). (B) Graphs represent the RBC adhesion to sensitized ECs. (C) ECs and RBCs stained with Hematoxylin and Eosin staining. Arrows points indicate RBCs adhered to ECs. **p = <0.05 DS1 Vs DS2 and DS3.

Figure 8. Future Scope of this model for studying the influence of plaque shape in plaque development, growth, and rupture.

(A) Velocity magnitude and flow pattern gained from CFD analysis for different shapes. Low velocity and low shear stress observed in microgrooves of irregular shape (indicated using arrows). (B) Cartoon signifies the influence of shape on atherosclerotic plaque formation and rupture. Stagnation points at the latter plaque stages can be responsible for the thin cap formation and rupture. (C) Cell number was counted after placing different shapes in the DSSA. There was reduction in cell number observed when irregular shape placed (p = <0.05 before Vs after DS). The micro grooves in irregular shape were indicated using arrows. (D) Graph quantifies the cell detachment after 30 min of disturbed flow with different shape.