Matrix Softening Induces Inflammatory Signals of Endothelial Cells

Vascular diseases such as atherosclerosis involve the change of the rigidity in the blood vessel wall. There is evidence that the changes in the blood vessel rigidity may affect the various functions of the cells in the blood vessel, including endothelial cells (ECs) and the smooth muscle cells. On the other hand, blood vessel-on-a-chip has become an emerging research field for dis-ease modeling. However, the effect of material rigidity on blood vessel re-modeling is not well understood. Hereby, an in vitro culture system with the culture substrates matching the rigidity of vessel wall mimicking the condi-tion of healthy (normal) or lipid deposition (soft) were prepared. The stiffness of the substrates was confirmed by atomic force microscope. Although no significant difference was observed in EC morphology, the expression le-vels of the pro-inflammatory cytokines, including interleukin 6 (IL6), tumor necrosis factor α (TNF-α) and interleukin 1β (IL1β), were dramatically induced by soft substrate. Consistently, the inflammation-related JNK signaling was also activated. In addition, the expression level of microRNA-146a (miR-146a) was significantly decreased. Accordingly, mRNA expression level of TNF receptor associated factor 6 (TRAF6), the direct target of miR-146a, was significantly increased. In summary, these findings provide new insight into the matrix rigidity effect on ECs; while engineering the blood vessel model in vitro, matrix with proper rigidity can be carefully tailor to mimic ECs either in a quiescent or an inflammation state. Matrix Softening Induces Inflammatory Signals

atherosclerosis [2] is regulated by biophysical factors, including flow pattern, strain stress, surface topography [3] [4] [5] and rigidity. Due to the deposition of low density lipoprotein (LDL) [6], the rigidity of the blood vessel wall may decrease to 5.5 ± 3.5 kPa [7]. It was reported that increased rigidity induced the change of cytoskeleton and the activation of signaling pathway of cells [8] [9].
However, whether softening impacts the development of atherosclerosis is still poorly understood.
On the other hand, organs-on-a-chip (OOCs) can be used as an in vitro disease model [10]. For the investigation of vascular inflammation, tissue engineering, drug screening and vascularization, several vessel-on-a-chip system were designed [11] [12] [13]. However, these studies mainly focused on the designing of the physical structure of the systems, with fewer consideration of the rigidity of the materials, which is an indispensable influence factor of vascular cell functions.
In this study, substrates matching the rigidity of softened vessel and normal vessel were fabricated, and how substrate rigidity exerts and impacts the inflammatory response and underlying mechanism was investigated.

Substrate Preparation and Rigidity Measurement
Substrates with soft rigidity (8 kPa) and normal rigidity (20 kPa) were prepared following the method mentioned in a previous research [14]. Specifically, solutions with different proportions components, including acrylamide (Sangon Biotech, China) and bis-acrylamide (Sangon Biotech, China), 10% ammonium persulfate solution and tetramethylethylenediamine (TEMED, Klamar, China) were prepared. Then 160 μL solution was dropped onto the gel slick-treated glass slide. A silanized cover glass was then placed onto the top of the liquid quickly.
After polymerization, the substrate was removed from the glass slide and coated with collagen I (Corning, USA).
The rigidity of polyacrylamide (PA) substrates was detected by atomic force microscope (AFM, Bruker, Germany). The probe used for the AFM measurement was a glass ball probe with a radius of 5 μm. Three random points of each substrate were measured and the results were shown as mean ± SEM.

Cell Imaging and Staining
HUVECs were cultured on different substrates for 2 hours and imaged with a reverted microscope (Leica, German). When reached confluence, the cells were

RNA Extraction and RT-qPCR Analysis
An RNA simple total RNA extraction kit (Tiangen Biotech, China) was used to extract total RNA following the manufacturer's instruction. The genomic DNA was removed by gDNase treatment. After reverse transcription process, the samples were analyzed using a Talent qPCR PreMix kit (Tiangen Biotech, China) by SYBR green method. The internal control used in this study was GAPDH.
All mRNA primers were provided by BioTNT (China) and the sequences were shown in Table 1.

Western Blot Analysis
Whole cell lysates were extracted using RIPA buffer with the addition of phos- using an enhanced chemiluminescence system (Bio-Rad, USA) and semi-quantified with Image J by comparing the intensity of the images (NIH, USA). β-tubulin was used as an internal reference in the western blot assay.

Statistics
GraphPad Prism 6.0 software was applied for statistical analysis. All data represented the mean of triplicates unless otherwise indicated and error bars signified standard error of the mean (SEM). Unpaired student's t-test was used for the comparison of two means. Significance was considered if p value < 0.05 and marked with *.

Substrate Preparation and Rigidity Measurement
The rigidity of PA substrates can be tuned by adjusting the proportion of acrylamide and bis-acrylamide. Using AFM, the rigidity of different substrates was confirmed. As Figure 1 indicated, the slope of the curves indicated the rigidity of the matrices. The dotted line showed a more inclined slope than the solid line, indicating the higher rigidity of the 20 kPa than 8 kPa. Furthermore, young's modulus of the substrates verified that rigidity of the fabricated substrates was consistent with our expectation. The mean rigidity was 7.37 kPa and 23.2 kPa, respectively ( Table 2). Thus substrates with rigidity of 8 kPa and 20 kPa were fabricated successfully.

Substrate Rigidity Showed No Influence on ECs Morphology
HUVECs were seeded onto the surface of the collagen I coated PA substrates.

Substrate Rigidity Regulates Expression of Pro-Inflammatory Cytokines
After ECs reached complete confluence on different substrates, the total RNA was extracted and analyzed. The expression of IL6, TNF-α and IL1β was dramatically increased in ECs on soft substrate compared to normal substrate ( Figure 3).
Concretely, the expression of IL6, TNF-α and IL1β in ECs on soft substrate was  compared with normal substrate. Therefore, these results indicated that soft substrate induced the pro-inflammatory response of ECs.

Substrate Rigidity Regulates Inflammatory Signaling Pathways in ECs
Since the expression of inflammatory cytokines was changed by substrate rigidity, we then studied whether the inflammation-related signaling cascades were impacted by substrate rigidity. C-Jun N-terminal kinases (JNK) in mitogen-activated protein kinase (MAPK) signaling pathway were shown to regulate the expression of inflammatory cytokines, including IL6, TNF-α and IL1β [15] [16]. Thus, the activation of MAPK signaling pathway was analyzed ( Figure 4). Unexpectedly, the phosphorylation of JNK in ECs on soft substrate was significantly enhanced compared to normal substrate (Figure 4(A) and Figure 4(B)), illustrating strong regulation of JNK signaling pathway by ECM rigidity.
To further explore the reason why JNK signaling pathway was involved in EC inflammatory response induced by substrate rigidity, the expression of TNF receptor associated factor 6 (TRAF6), an important regulator of JNK signaling path way, was examined. Surprisingly, the expression of TRAF6 in soft rigidity group was high than normal rigidity group ( Figure 5(A)), implying activation of   in ECs on soft rigidity substrate compared to 20 kPa group ( Figure 5(B)), indicating inhibition of miR-146a expression by soft ECM rigidity. These results implied that soft substrate promoted the phosphorylation of JNK via reducing miR-146a (targeting TRAF6) expression compared to normal substrate rigidity.

Discussion and Conclusion
It was reported that at the early stage, high fat diet resulted in decrease of vessel stiffness due to deposition of LDL and the lipid rich area of a vessel was 5.5 ± 3.5 kPa [7]. Thus we fabricated substrates with rigidity matching the stiffness of softened vessels and normal vessels. Surprisingly, the results pointed out that soft ECM rigidity greatly elevated the expression of pro-inflammatory cytokines in ECs. Further investigation found that down-regulation of miR-146a induced by soft ECM rigidity led to up-regulation of TRAF6 thus activating the inflammatory JNK signaling pathway.
TRAF6 is an important regulator in the MAPK signaling pathway. It was reported that TRAF6 regulates the expression of TGF-β-associated kinase 1 via regulating TAK-associated binding protein 1, leading to phosphorylation of MAP kinase kinase 7 (MKK7) [20]. Besides, our study has revealed a new possible physiological function of LDL deposition. Although the biochemical properties of LDL on cell functions have been studied intensively [21], whether the physical properties of LDL impacted the functions of cells remained poorly understood. Our study clearly indicated that changes of physical microenvironment of vessel wall, probably resulted from LDL depositing, induced inflammatory response of ECs. In addition, since these results implied strong regulation of substrate rigidity on ECs inflammatory response, substrate rigidity should be taken into serious consideration in the designing of vessel-on-a-chip system.
In conclusion, the results reported here, from a novel point of view, provided useful information for the successful designing of vessel-on-a-chip system as well as for the pathogenesis of atherosclerosis.